Mechanism of activation of beta-D-2'-deoxy-2'-fluoro-2'-c-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymerase

Beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5'-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2'-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 microM and a kcat of 0.007 s-1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 microM. Similar results were obtained with 2'-C-methyladenosine triphosphate (Ki=1.5 microM) and 2'-C-methylcytidine triphosphate (Ki=1.6 microM). NS5B with the S282T mutation, which is known to confer resistance to 2'-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.     

Inhibition of hepatitis C replicon RNA synthesis by beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine: a specific inhibitor of hepatitis C virus replication

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a cytidine analogue with potent and selective anti-hepatitis C virus (HCV) activity in the subgenomic HCV replicon assay, 90% effective concentration (EC90)=4.6 +/- 2.0 microM. The spectrum of activity and cytotoxicity profile of PSI-6130 was evaluated against a diverse panel of viruses and cell types, and against two additional HCV-1b replicons. The S282T mutation, which confers resistance to 2'-C-methyl adenosine and other 2'-methylated nucleosides, showed only a 6.5-fold increase in EC90. When assayed for activity against bovine diarrhoea virus (BVDV), which is typically used as a surrogate assay to identify compounds active against HCV, PSI-6130 showed no anti-BVDV activity. Weak antiviral activity was noted against other flaviviruses, including West Nile virus, Dengue type 2, and yellow fever virus. These results indicate that PSI-6130 is a specific inhibitor of HCV. PSI-6130 showed little or no cytotoxicity against various cell types, including human peripheral blood mononuclear and human bone marrow progenitor cells. No mitochondrial toxicity was observed with PSI-6130. The reduced activity against the RdRp S282T mutant suggests that PSI-6130 is an inhibitor of replicon RNA synthesis. Finally, the no-effect dose for mice treated intraperitoneally with PSI-6130 for six consecutive days was > or =100 mg/kg per day.     

The mechanism of action of beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine involves a second metabolic pathway leading to beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine 5'-triphosphate, a potent inhibitor of the hepatitis C virus RNA-dependent RNA polymerase

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5'-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5'-triphosphate of the uridine congener, beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5'-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.     

Use of 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil as a novel antiviral agent for hepatitis B virus and Epstein-Barr virus.

A novel anti-hepatitis B virus (anti-HBV) agent, 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU), was synthesized and found to be a potent anti-HBV and anti-Epstein-Barr virus agent. Its in vitro potency was evaluated in 2.2.15 and H1 cells for anti-HBV and anti-Epstein-Barr virus activities, respectively. In vitro cytotoxicity in MT2, CEM, 2.2.15, and H1 cells was also assessed, and the results indicated high antiviral selectivities of L-FMAU in these cells.     

Pharmacokinetics of the antiviral agent beta-L-3'-fluoro-2',3'-didehydro-2',3'-dideoxycytidine in rhesus monkeys

beta-L-3'-Fluoro-2',3'-didehydro-2',3'-dideoxycytidine (L-3'-Fd4C) is a potent and selective antiretroviral nucleoside with activity against lamivudine-resistant human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV) in vitro. The pharmacokinetics of L-3'-Fd4C were characterized in three rhesus monkeys given single intravenous and oral doses. A two-compartment open model was fitted to the plasma and urine data. Plasma concentrations declined in a biexponential fashion with an average beta half-life of 12.45 h and central and steady-state volumes of distribution of 0.43 and 1.90 liters/kg, respectively. The average systemic and renal clearance values were 0.23 and 0.08 liters/kg, respectively, and the apparent mean terminal half-life of the oral dose was 12.5 h. The serum concentrations exceeded the 90% effective concentration value for lamivudine-resistant and wild-type HIV-1 after oral administrations. A large variation was observed in the oral bioavailability, which ranged from 15 to 31%. To determine whether the bioavailability may be improved by using a basic buffer solution, the oral dose was repeated to the same animals in a sodium bicarbonate solution. The bioavailability of L-3'-Fd4C administered with sodium bicarbonate was not significantly different from the bioavailability when the oral dose was administered in the absence of buffer (P = 0.49), suggesting that further development of this compound may warrant other approaches, such as development of a prodrug to improve its oral absorption.     

Inhibition of duck hepatitis B virus replication by 2',3'-dideoxy-3'-fluoroguanosine in vitro and in vivo.

The antiviral activity of 2',3'-dideoxy-3'-fluoroguanosine (FdG) or its triphosphate was evaluated in the duck hepatitis B virus (DHBV) system in vitro and in vivo. In primary DHBV-infected hepatocytes FdG results in a dose-dependent inhibition of viral replication with a nearly complete inhibition at a concentration of 1 microM. Also in vivo, FdG treatment of DHBV-infected ducklings reduces DHBV DNA replication by more than 90%. These data demonstrate that FdG is a strong inhibitor of DHBV replication in vitro and in vivo.     

Antiviral activity of beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine in woodchucks chronically infected with woodchuck hepatitis virus

The L-nucleoside analog beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (beta-L-Fd4C) was first shown to exhibit potent activity against hepatitis B virus (HBV) in tissue culture and then to significantly inhibit viral spread during acute infection in the duck HBV model (F. Le Guerhier et al., Antimicrob. Agents Chemother. 44:111-122, 2000). We have therefore examined its antiviral activity in a mammalian model of chronic HBV infection, the woodchuck chronically infected with woodchuck hepatitis virus (WHV). Side-by-side comparison of beta-L-Fd4C and lamivudine administered intraperitoneally during short-term and long-term protocols demonstrated a more profound inhibition of viremia in beta-L-Fd4C-treated groups. Moreover, beta-L-Fd4C induced a marked inhibition of intrahepatic viral DNA synthesis compared with that induced by lamivudine. Nevertheless, covalently closed circular (CCC) DNA persistence explained the lack of clearance of infected hepatocytes expressing viral antigens and the relapse of WHV replication after drug withdrawal. Liver histology showed a decrease in the inflammatory activity of chronic hepatitis in woodchucks receiving beta-L-Fd4C. An electron microscopy study showed the absence of ultrastructural changes of hepatic mitochondria, biliary canaliculi, and bile ducts. However, a loss of weight was observed in all animals, whatever the treatment, as was a transient skin pigmentation in all woodchucks during beta-L-Fd4C treatment. There was no evidence that lamivudine or beta-L-Fd4C could prevent the development of hepatocellular carcinoma with the protocols used. These results indicate that beta-L-Fd4C exhibits a more potent antiviral effect than lamivudine in the WHV model but was not able to eradicate CCC DNA and infected cells from the liver at the dosage and with the protocol used.     

Pure nucleoside enantiomers of beta-2',3'-dideoxycytidine analogs are selective inhibitors of hepatitis B virus in vitro.

(-)-beta-L-2',3'-Dideoxycytidine (beta-L-DDC), (+)-beta-D-2',3'-dideoxycytidine (beta-D-DDC), (-)-beta-L-2',3'-dideoxy-5-fluorocytidine (beta-L-FDDC), (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (beta-L-FTC), and (+)-beta-D-1,3-dioxolane-5-fluorocytidine (beta-D-FDOC) were evaluated for their anti-hepatitis B virus (anti-HBV) activities in HBV-transfected human liver cells (2.2.15). The order of decreasing potency for the compounds at the 90% effect level was beta-D-FDOC > beta-L-FTC > beta-L-FDDC approximately beta-L-DDC >> beta-D-DDC. Inhibition of HBV in transfected liver cells by the cytosine nucleosides was selective. The beta-L-nucleoside-5'-triphosphates were consistently more potent inhibitors of woodchuck hepatitis virus DNA polymerase than the corresponding natural beta-D-enantiomers.     

Synergistic inhibition of human immunodeficiency virus isolates (including 3'-azido-3'-deoxythymidine-resistant isolates) by foscarnet in combination with 2',3'-dideoxyinosine or 2',3'-dideoxycytidine.

The combinations of foscarnet plus 2',3'-dideoxyinosine and foscarnet plus 2',3'-dideoxycytidine synergistically inhibit the replication of human immunodeficiency virus type 1 isolates, including two 3'-azido-3'-deoxythymidine-resistant isolates. The combination of 2',3'-dideoxyinosine plus 2',3'-dideoxycytidine showed additive inhibition of the majority of the human immunodeficiency virus isolates tested. All three combinations showed pronounced antagonistic cytotoxicity and thus were less toxic to the growth of peripheral blood mononuclear cells than the separate drugs.     

Evaluation of mitochondrial toxicity in Marmota himalayana treated with metacavir, a novel 2',3'-dideoxyguanosine prodrug for treatment of hepatitis B Virus

Metacavir (PNA) is a novel synthetic nucleoside analogue for the treatment of hepatitis B virus (HBV). Our recent studies showed that PNA, a prodrug of 2',3'-dideoxyguanosine (ddG), exhibited lower mitochondrial toxicity in long-term cultures of HepG2 cells. In the current study, we examined the long-term effects of PNA on mitochondrial toxicity in Marmota himalayana (Himalayan marmot). Himalayan marmots were treated daily with oral PNA (50 or 100 mg/kg), ziduvidine (AZT) (100 mg/kg), or water (control) for 90 days. PNA treatment did not alter the body weight or plasma lactate acid level. In livers from the animals treated with PNA at 100 mg/kg/day, histopathology showed mild steatosis or small focal liver cell necrosis. Electron microscopy also showed minor proliferation and partial mitochondrial swelling with crista reduction. Measurement of respiratory chain complex enzyme activity and mitochondrial DNA (mtDNA) content revealed no significant differences in skeletal muscle, liver, and kidney tissues between animals treated with PNA and controls. In contrast, in Himalayan marmots treated with AZT we observed delayed toxicity, including lactic acidosis, severe hepatic steatosis, obvious mitochondrial damage, and significant decreases in respiratory chain complex enzyme activity and mtDNA content. This is similar to the delayed toxicity syndrome observed previously in animals and humans. In summary, PNA treatment did not alter mitochondrial enzyme activity or mtDNA content. This suggests that PNA could pose a very low risk for adverse mitochondrion-related effects. However, long-term hepatotoxic effects of PNA were observed, and this indicates a need for continued monitoring of PNA-associated hepatotoxicity in clinical trials.     

Pharmacokinetics of 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine in patients with severe human immunodeficiency virus infection

This article describes the pharmacokinetics of 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxyinosine (ddI) as determined during phase I clinical trials in patients with acquired immunodeficiency syndrome and acquired immunodeficiency syndrome-related complex. Drug levels were determined by HPLC in plasma, cerebrospinal fluid, and urine after administration of the drugs either intravenously or as an oral liquid given with antacid. ddA was metabolized rapidly and quantitatively to ddI to such an extent that ddA was undetectable in the plasma even during continuous intravenous administration of ddA. The plasma kinetics of ddI were generally monoexponential and were characterized by a half-life of 38 minutes. This probably does not accurately reflect the kinetics of the active species of ddI, which appears to be 2',3'-dideoxyadenosine triphosphate, formed intracellularly. Oral bioavailability was 38% for oral liquid given with antacid. The total body clearance averaged 1.00 L/kg/hr, with a volume of distribution of 1.01 L/kg. Approximately 36% of the intravenous dose could be recovered unchanged in the urine. The level of ddI in the cerebrospinal fluid 1 hour after drug infusion averaged 21% of that of the simultaneous plasma level. It is concluded that ddI has pharmacokinetic properties that are amenable to its clinical use.     

In vitro susceptibilities of wild-type or drug-resistant hepatitis B virus to (-)-beta-D-2,6-diaminopurine dioxolane and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil

Prolonged treatment of chronic hepatitis B virus (HBV) infection with lamivudine ([-]-beta-L-2',3'-dideoxy-3' thiacytidine) or famciclovir may select for viral mutants that are drug resistant due to point mutations in the polymerase gene. Determining whether such HBV mutants are sensitive to new antiviral agents is therefore important. We used a transient transfection system to compare the sensitivities of wild-type HBV and four lamivudine- and/or famciclovir-resistant HBV mutants to adefovir [9-(2-phosphonyl-methoxyethyl)-adenine; PMEA] and the nucleoside analogues (-)-beta-D-2, 6-diaminopurine dioxolane (DAPD) and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU). The drug-resistant mutants contained amino acid substitutions in the polymerase protein. We found that the M550I and M550V plus L526M substitutions, which confer lamivudine resistance, did not confer cross-resistance to adefovir or DAPD, but conferred cross-resistance to L-FMAU. The M550V substitution in isolation conferred a similar phenotype to M550I, except that it did not confer significant resistance to L-FMAU. The L526M substitution, which is associated with famciclovir resistance, conferred cross-resistance to L-FMAU but not to adefovir or DAPD. Inhibition of HBV secretion by DAPD, L-FMAU, and adefovir did not always correlate with inhibition of the generation of intracellular HBV replicative intermediates, suggesting that these analogs may preferentially inhibit specific stages of the viral replication cycle.