Claire Pierra

Pharmacology of β-l-Thymidine and β-l-2′-Deoxycytidine in HepG2 Cells and Primary Human Hepatocytes: Relevance to Chemotherapeutic Efficacy against Hepatitis B Virus

ABSTRACT β-l-Thymidine (l-dT) and β-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 β-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.

ANTI-HBV SPECIFIC β-L-2′-DEOXYNUCLEOSIDES

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3′-position (3′-OH) of the β-L-2′-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3′-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

Intracellular Metabolism of β-l-2′,3′-Dideoxyadenosine: Relevance to Its Limited Antiviral Activity

ABSTRACT The intracellular metabolism of the β-l- enantiomer of 2′,3′-dideoxyadenosine (β-l-ddA) was investigated in HepG2 cells, human peripheral blood mononuclear cells (PBMC), and primary cultured human hepatocytes in an effort to understand the metabolic basis of its limited activity on the replication of human immunodeficiency virus and hepatitis B virus. Incubation of cells with 10 μM [2′,3′,8-3H]-β-l-ddA resulted in an increased intracellular concentration of β-l-ddA with time, demonstrating that these cells were able to transport β-l-ddA. However, it did not result in the phosphorylation of β-l-ddA to its pharmacologically active 5′-triphosphate (β-l-ddATP). Five other intracellular metabolites were detected and identified as β-l-2′,3′-dideoxyribonolactone, hypoxanthine, inosine, ADP, and ATP, with the last being the predominant metabolite, reaching levels as high as 5.14 ± 0.95, 8.15 ± 2.64, and 15.60 ± 1.74 pmol/106 cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a β-glucuronic derivative of β-l-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with β-l-ddA in the presence of increasing concentrations of 5′-methylthioadenosine resulted in decreased phosphorolysis of β-l-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of β-l-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of β-l-ddA by methylthioadenosine phosphorylase (EC2.4.2.28).

Synthesis and antiviral activities of enantiomeric 1-[2-(hydroxymethyl) cyclopropyl] methyl nucleosides.

Abstract Cyclopropyl carbocyclic nucleosides have been synthesized from the key intermediate 2 which was converted to the mesylated cyclopropyl methyl alcohol 3. Condensation of compound 3 with various purine and pyrimidine bases gave the desired nucleosides. All synthesized nucleosides were evaluated for antiviral activity and cellular toxicity. Among them adenine 22 and guanine 23 derivatives showed moderate antiviral activity against HIV-1 and HBV. None of the other compounds showed any significant antiviral activities against HIV-1, HBV, HSV-1 and HSV-2 in vitro up to 100μM.

Pharmacokinetics of β-L-thymidine and β-L-2′-deoxycytidine in woodchucks and monkeys

Publisher Summary Pharmacokinetic studies in woodchucks and monkeys indicated that the disposition of β-L-thymidine (L-dT) is comparable to the pharmacokinetic characteristics of other nucleoside analogs after administration to these animals. Woodchuck is a useful animal model for studying anti-hepatitis B virus (HBV) agents because of many similarities between woodchuck hepatitis virus (WHV) and HBV, hence providing important information on the in vivo efficacy of anti-HBV candidates. The compound has good oral bioavailability and is eliminated and unchanged in urine. 3-L-2’-deoxycytidine (L-dC) exhibits lower oral bioavailability in both species. Several prodrugs that improve the oral bioavailability of L-dC are currently under evaluation. Chronic HBV infection is a major global health problem, affecting approximately 5% of the worlds population. Currently, the only approved treatment options include alpha interferon (INF-α) and lamivudine. Potent in vitro and in vivo efficacy coupled with favorable pharmacokinetic disposition make both L-dT and L-dC promising antiviral candidates for the treatment of chronic HBV infection.

Discovery of benzophosphadiazine drug candidate IDX375: A novel hepatitis C allosteric NS5B RdRp inhibitor

Hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells, and as a consequence is an attractive target for selective inhibition. This paper describes the discovery of a novel family of HCV NS5B non-nucleoside inhibitors inspired by the bioisosterism between sulfonamide and phosphonamide. Systematic structural optimization in this new series led to the identification of IDX375, a potent non-nucleoside inhibitor that is selective for genotypes 1a and 1b. The structure and binding domain of IDX375 were confirmed by X-ray co-crystalisation study.

Chapter 21 – Antiviral β-L-nucleosides specific for hepatitis B virus infection

Publisher Summary For the treatment of chronic hepatitis B virus (HBV) infection with the goal of eliminating persistent viral replication and preventing progression to chronic active hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), approved treatments that are used are alpha interferon (IFN- α) and lamivudine. The response rate to IFN is low and drug-associated side effects are significant. Individuals who are immunosuppressed rarely respond to IFN therapy. A complete antiviral response, as assessed by HBe seroconversion, is seen in only a minority of patients after one year of treatment. With long-term survival increasing for human immunodeficiency virus (HIV)-infected patients, chronic HBV infection is becoming an important determinant of disease outcome in the coinfected patient population. Nucleoside analogs currently under evaluation for the treatment of HBV include adefovir dipivoxil that may be beneficial against lamivudine-resistant mutants and emtricitabine that is similar to lamivudine. Development of famciclovir and lobucavir have been discontinued because of limited antiviral activity and adverse side effects, respectively. Two other potent nucleoside analogs—L-FMAU and BMS200-458—are currently in human trials, although clinical data have not yet been reported.