Combinatorial Therapy with Long-Acting Tenofovir and Tizoxanide Controls Viral Replication and Liver Inflammation in a Murine AAV-HBV Model of Chronic Hepatitis B.

Chronic hepatitis B (CHB) infection is a major risk factor for progressive cirrhosis and hepatocellular carcinoma. Persistent virus-induced inflammation alters liver function, leading to accelerated disease and increased mortality. Although lifelong treatment with nucleos(t)ide analogs (NAs) is highly effective at suppressing viral replication, covalently closed circular DNA (cccDNA) persists in hepatocytes to sustain chronic infection, underscoring the need for better interventions and combination therapies. The durable suppression of viral replication and restoration of immune responses through long-acting (LA) therapies offer a promising strategy for sustained HBV control. We transformed tizoxanide (TIZ), a broad-spectrum anti-infective and immunomodulatory agent, into a LA lipophilic prodrug formulation (NM2TIZ) for intramuscular or subcutaneous administration. NM2TIZ exhibited long-term stability during storage and was evaluated in AAV-HBV mice using monotherapy and combination therapy approaches with an LA tenofovir prodrug formulation (NM5TFV). NM5TFV reduced the HBV DNA levels by >2 log10 fold. Furthermore, coadministration of NM5TFV with M2TIZ reduced the expression of liver inflammasomes and profibrotic markers. The NM5TFV and NM2TIZ combination reduced HBV replication, inflammation, and fibrogenesis in AAV-HBV-transduced mice.