Identification of a novel long-acting 4′-modified nucleoside reverse transcriptase inhibitor against HBV

Background & aims: While certain nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are efficacious in treating HBV infection, their effects are yet to be optimized and the emergence of NRTI-resistant HBV variants is an issue because of the requirement for lifelong treatment. The development of agents that more profoundly suppress wild-type and drug-resistant HBVs, and that have a long-acting effect, are crucial to improve patient outcomes.
Methods: Herein, we synthesized a novel long-acting 4′-modified NRTI termed E-CFCP. We tested its anti-HBV activity in vitro, before evaluating its anti-HBV activity in HBV-infected human-liver-chimeric mice (PXB-mice). E-CFCP’s long-acting features and E-CFCP-triphosphate’s interactions with the HBV reverse transcriptase (HBV-RT) were examined.
Results: E-CFCP potently blocked HBV_{WT}^{D1} production (IC_{50}^{qPCR_cell}_{=}1.8 nM) in HepG2.2.15 cells and HBV_{WT}^{C2} (IC_{50}^{SB_cell}=0.7 nM), entecavir (ETV)-resistant HBV_{ETV-R}^{L180M/S202G/M204V} (IC_{50}^{SB_cell}=77.5 nM), and adefovir-resistant HBV_{ADV-R}^{A181T/N236T} production (IC_{50}^{SB_cell}=14.1 nM) in Huh7 cells. E-CFCP profoundly inhibited intracellular HBV DNA production to below the detection limit, but ETV and tenofovir alafenamide (TAF) failed to do so. E-CFCP also showed less toxicity than ETV and TAF. E-CFCP better penetrated hepatocytes and was better tri-phosphorylated; E-CFCP-triphosphate persisted intracellularly for longer than ETV-triphosphate. Once-daily peroral E-CFCP administration over 2 weeks (0.02~0.2 mg/kg/day) reduced HBV_{WT}^{C2}-viremia by 2-3 logs in PXB-mice without significant toxicities and the reduction persisted over 1-3 weeks following treatment cessation, suggesting once-weekly dosing capabilities. E-CFCP also reduced HBV_{ETV-R}^{L180M/S202G/M204V}-viremia by 2 logs over 2 weeks, while ETV completely failed to reduce HBV_{ETV-R}^{L180M/S202G/M204V}-viremia. E-CFCP’s 4′-cyano and fluorine interact with both HBV_{WT}-RT and HBV_{ETV-R}^{L180M/S202G-M204} -RT via Van der Waals and polar forces, being important for E-CFCP-triphosphate’s interactions and anti-HBV potency.
Conclusion: E-CFCP represents the first reported potential long-acting NRTI with potent activity against wild-type and treatment-resistant HBV.
Lay summary: Although there are currently effective treatment options for HBV, treatment-resistant variants and the need for lifelong therapy pose a significant challenge. Therefore, the development of new treatment options is crucial to improve outcomes and quality of life. Herein, we report preclinical evidence showing that the anti-HBV agent, E-CFCP, has potent activity against wild-type and treatment-resistant variants. In addition, once-weekly oral dosing may be possible, which is preferrable to the current daily dosing regimens.