Abdesslem Faraj

Antiviral l-Nucleosides Specific for Hepatitis B Virus Infection

ABSTRACT A unique series of simple “unnatural” nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3′-OH group of the β-l-2′-deoxyribose of the β-l-2′-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides β-l-2′-deoxycytidine, β-l-thymidine, and β-l-2′-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (α, β, and γ) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 108 genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

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.

Inhibition of Hepatitis B Virus Replication by Nucleoside Enantiomers of β-2′,3′-Dideoxypurine Analogues

Various purine β-L-2′,3′-dideoxynucleoside analogues with both sugar and base modifications including β-L-ddG, β-L-ddl, β-L-ddA, 2′-azido-β-L-araddA, 2′-amino-β-L-araddA, 2′,5′-anhydro-β-L-araddA, 2′-azido-β-L-ddA, 2′-amino-β-L-ddA, 2′-fluoro-β-L-ddA, 3′-azido-β-L-ddA, 3′-amino-β-L-ddA, 3′-fluoro-β-L-ddA, 2,6-diamino-β-L-2′,3′-dideoxyfuranosylpurine, 6-cyclopropylamino-β-L-ddA, 2′-azido-6-N-triphenylphosphine-β-L-araddA, 2-amino-6-methylamino-β-L-2′,3′-dideoxyfuranosylpurine, 2-amino-6-cyclopropylamino-β-L-2′,3′-dideoxyfuranosylpurine, 2-amino-6-cyclopentylamino-β-L-2′,3′-dideoxyfuranosylpurine, 2′,3′-didehydro-β-L-ddA and 2′,3′-didehydro-6-N-triphenyl phosphine-β-L-ddA were synthesized and evaluated as potential inhibitors of hepatitis B virus (HBV) replication in HBV DNA-transfected human hepatoblastoma-derived Hep-G2 cells (2.2.15 cells). β-L-ddA, 2′-azido-β-L-ddA, 3′-azido-β-L-ddA, 2″,3′-didehydro-β-L-ddA (β-L-D4A) and a modified base of β-L-D4A, inhibited HBV replication in vitro. β-L-D4A was the more potent and selective antiHBV agent with a 50% effective concentration value of 0.1 μM and a selectivity index of 1800. On the basis of this finding, studies are in progress to synthesize new purine derivatives with the β-L unnatural configuration which hopefully will lead to identifying additional potent and highly selective anti-HBV agents.

Antiviral Activities of β-Enantiomers of 3′-Substituted-3′-deoxythymidine Analogs

Abstract Several β-L-3′-substituted-3′-deoxythymidine were stereospecifically synthesized. None of these analogs inhibited HIV-1 nor HBV replication in vitro suggesting that these β-L-pyrimidine derivatives may not be efficiently phosphorylated inside the cells.

Arabinogalactan (9kDa)-9-β-D-arabinofuranosyladenine-5'-monophosphate, a novel liver-targeted conjugate that selectively inhibits hepatitis B virus replication in vitro

Arabinogalactan (9kDa)-9-β-D-arabinofuranosyladenine-5′-monophosphate [AG (9kDa)-araAMP] was 25-fold more active than the parent compound 9-β-D-arabinofuranosyladenine (araA) in decreasing the amount of hepatitis B virus (HBV) DNA intracellular replicative intermediates in HBV-transfected 2.2.15 cells. The putative active 5′-triphosphate metabolite, araATP, exhibited a 50% inhibitory concentration (IC50) of 5 μM toward woodchuck hepatitis virus DNA polymerase, consistent with the anti-HBV activity of these derivatives. AG (9kDa)-araAMP inhibited HepG2 cell proliferation by only 16% at a concentration as high as 822 μM (araA equivalents) and no effects were observed in the presence of 8.22 μM or 82.2 μM of the drug. Lactate production, mitochondrial DNA (mtDNA) content, and mitochondrial morphology were not affected by AG (9kDa)-araAMP at concentrations between 8.22 μM and 822 μM. In contrast, both araA and its in vivo metabolite, 9-β-D-arabinofuranosylhypoxanthine (araH), induced substantial dose-dependent effects on cell growth, lactate production and mtDNA steady-state levels at concentrations of 8.22 μM and 82.2 μM. Some loss of cristae within the mitochondria was also observed in cells incubated with araH. In summary, AG (9kDa)-araAMP exhibits a potent and selective anti-HBV activity and the lack of toxic effects on host cell mitochondrial functions supports its further development.

In vitro and in vivo metabolism and pharmacokinetics of bis [(t-butyl)-S-acyl-2-thioethyl]-beta-L-2',3'-dideoxy-5-fluorocytidine monophosphate.

Abstract Exposure to 10 & M L-FddCMP-bisSATE led to formation of intracellular L-FddCTP levels of 410.1± 46.2 and 242.1 ± 13.2 pmol/106 cells in unstimulated and PHAstimulated PBM cells, respectively; whereas, exposure of cells to the parent nucleoside, L-FddC, generated 5-10-fold less L-FddCTP. In Hep-G2 cells and EGF/HGF stimulated and unstimulated primary cultured hepatocytes, the active metabolite reached 113 ± 29, 23.9 ± 15.6, and 20.6 ± 10.5 pmol/106 cells. Three other metabolites, L-FddCMP-monoSATE, L-FddCMP-SH, and M I, were detected intracellularly and extracellularly in all cell types examined. Intravenous administered dose of 3 mg/kg L-FddCMP-bisSATE to rhesus monkeys resulted in plasma concentration levels of 2.06 ± 1.00 and 0.39 ± 0.15 & M of L-FddCMP-monoSATE and L-FddC, respectively, while the prodrug was completely cleared metabolically within 15 min. Following oral administration of an equivalent dose, the absolute oral bioavailability of L-FddC derived from L-FddCMP-bisSATE administration was 65%.

Structural determinants of HIV-1 reverse transcriptase stereoselectivity towards (beta)-L-deoxy- and dideoxy-pyrimidine nucleoside triphosphates: molecular basis for the combination of L-dideoxynucleoside analogs with non-nucleoside inhibitors in anti HIV chemotherapy.

We have compared the HIV-1 RT mutants containing the single substitutions L100I, K103N, V106A, V179D, Y181I and Y188L, known to confer NNI-resistance in treated patients, to HIV-1 RT wt for their sensitivity towards inhibition by D- and L-deoxy- and dideoxy-nucleoside tiphosphates. The results showed a differential effect of the substitutions on the affinity for both D- and L-enantiomers of deoxy- and dideoxy-nucleoside triphosphates and provide a rationale for the utilization of L-dideoxynucleoside analogs with NNI in combination chemotherapy.

Effect of 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)-5-ethyluracil on mitochondrial functions in HepG2 cells

The effects of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil (D-FEAU) on mitochondrial functions were examined in HepG2 cells. D-FEAU between 0.1 and 10 microM had no apparent inhibitory effect on cell proliferation for 2-week period; however, D-FEAU caused a decrease in mitochondrial DNA (mtDNA) content in a dose-dependent manner with an IC50 value of 2.7 microM. A 20.9% of increase in lactic acid production was observed after the cells were incubated with 10 microM of D-FEAU for 4 days without substantial effect being detected at 0.1 and 1 microM. In addition, no significant changes on mitochondrial morphology were observed in the cells treated with 10 microM of D-FEAU for 14 days under the electron microscope.

Toxicological study of the anti-HBV agent β-L-thymidine

Publisher Summary β-L-thymidine (L-dT) is a potent antiviral nucleoside against hepatitis B virus (HBV) replication in cell culture, and it has a favorable in vitro toxicity profile. L-dT is an unsubstituted, unmodified P-L-deoxynucleoside with potent antiviral activity against hepadnaviruses in vitro and in vivo and a favorable pharmacological profile. As part of its development as a potential oral anti-HBV agent, a series of preclinical studies in vitro and in laboratory animals were conducted to delineate the cytotoxic, genotoxic, toxicologic, and toxicokinetic profiles of L-dT. In vivo efficacy studies conducted on the chronically infected woodchucks indicated that L-dT has a potent antihepadnaviral activity, decreasing plasma viral DNA loads by up to 8 logs. L-dT is nontoxic and nonclastogenic at doses much greater than those most likely to be used clinically. The potent antiviral efficacy and low toxicity in vitro and in vivo make L-dT an attractive candidate for further development as an anti-HBV agent.