Background
Effective treatment of hepatitis B (CHBV) infection requires adherence to long-term daily antiviral therapies. These include oral tenofovir (TFV) prodrugs [TFV disoproxil-/TFV alafenamide-fumarate – TDF/TAF] or entecavir. Treatment cessation leads to HBV reactivation and disease progression. This underscores the need for long-acting formulations to improve treatment outcomes. Considering these needs, we developed a scalable amino acid-free hydrophobic and lipophilic crystalline phosphonate TFV prodrug (M5TFV). Surfactant-stabilized aqueous nanosuspensions (NM5TFV) were produced at a >300 mg/ml drug concentration. The formulation was stable at room temperature. We now report on possible mechanisms by which NM5TFV sustains suppression of HBV replication in hepatocytes. NM5TFV is well tolerated at the injection site. These promising data support further development and translation of NM5TFV formulation.
Methods
Scalable M5TFV was formulated into aqueous nanosuspensions using Tween 20 and PEG 3350 as the stabilizing surfactants. In vivo efficacy was evaluated in transgenic (Tg05) mice administered a single intramuscular injection of NM5TFV at either 200 or 400 mg/kg TFV equivalents. NM1TFV, an injectable TFV prodrug shown to be longer acting than TAF (Sci Adv, 2023), was used as a control. HBV viral load in blood was tested biweekly until rebound. At the time of animal sacrifice, liver tissues were collected to evaluate treatment-mediated activation of interferon (IFN)-stimulated genes (ISG) and inflammasomes. For in vitro studies, HepAD38 cells were treated with NM5TFV for eight hours, followed by incubation in drug-free media for 48 hours. HBV DNA and cccDNA were determined by qPCR.
Results
Treatment of HepAD38 cells with NM5TFV led to a two-fold reduction in HBV DNA and cccDNA levels. In HBV-infected mice, low and high-dose single injections of NM5TFV exhibited comparable and >2.5 logs 10 reductions in HBV DNA, lasting over two months (Fig. A). Similar observations were recorded for NM1TFV. However, unlike NM5TFV, NM1TFV induced the expression of APOBEG3G and activation of inflammasome receptors, including, NLRP3 and AIM2, and IL1β gene, suggesting divergent antiviral mechanisms of action (Fig. B).
Conclusions
A single dose of NM5TFV sustained suppression of HBV DNA in mice for over two months without activating ISGs and inflammasome. These promising findings support further development of NM5TFV as an ultra-long-acting formulation candidate.