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

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.

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.

E-CFCP potently blocked HBV_WT^D1 production (IC₅₀^qPCR_cell₌1.8 nM) in HepG2.2.15 cells and HBV_WT^C2 (IC₅₀^SB_cell=0.7 nM), entecavir (ETV)-resistant HBV_ETV-R^{L180M/S202G/M204V} (IC₅₀^SB_cell=77.5 nM), and adefovir-resistant HBV_ADV-R^A181T/N236T production (IC₅₀^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.

E-CFCP represents the first reported potential long-acting NRTI with potent activity against wild-type and treatment-resistant HBV.

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.