Abstract Body

Tenofovir (TFV) prodrugs (TFV alafenamide, TAF and TFV disoproxil fumarate, TDF) are recommended for the treatment of chronic hepatitis B (HBV) in patients with HIV co-infection. However, TAF and TDF exhibit short half-lives and therefore require frequent administration. Consequently, this has resulted in treatment failures mainly due to patient non-adherence. To this end, we transformed TFV into a long-acting prodrug formulation (NM1TFV) and demonstrated sustained active diphosphate metabolite (TFV-DP) levels in key HIV and HBV target cells and tissues of Sprague Dawley rats for up to two months [Nat Commun 12,5458(2021)]. We now demonstrate that a single intramuscular dose of NM1TFV provides sustained efficacy in HBV-infected chimeric liver humanized mice and HBV transgenic Tg05 mice.

A lipophilic TFV prodrug, M1TFV, was synthesized and nanoformulated into stable poloxamer 407 stabilized aqueous nanocrystals (NM1TFV) by high-pressure homogenization. Solid drug nanocrystals of TAF (NTAF) were produced and used as controls. Formulation efficacy was evaluated in two mouse models (HBV-infected humanized liver TK-NOG mice and HBV transgenic Tg05 mice) following a single intramuscular injection of 168 mg/kg TFV equivalents of either NM1TFV or NTAF. HBV DNA levels in peripheral blood were assessed biweekly for 12 weeks. HBV markers HBcAg and HBsAg were evaluated on stained liver sections of TK-NOG mice. Drug levels were quantified by mass spectrometry.

NM1TFV suppressed HBV DNA in blood to undetectable levels in all the infected humanized mice over twelve weeks with stable human albumin level (Figure 1). High drug concentrations were recorded at 12 weeks in livers and muscle injection sites of animals treated with NM1TFV. In contrast, NTAF exhibited a limited inhibitory effect on HBV DNA replication levels, consistent with undetectable drug concentrations recorded in the liver at 12 weeks. Notably, the expression of HBcAg and HBsAg was significantly decreased in NM1TFV treated animals compared to NTAF. Enhanced sustained efficacy is also demonstrated for NM1TFV in HBV transgenic mice.

Collectively, this work demonstrates that a single intramuscular injection of NM1TFV into infected humanized mice suppresses HBV replication over three months with no notable adverse events.