There is a global aim to reduce the burden of chronic hepatitis B (CHB) infection and prevent the
development of HBV-associated end-stage liver disease and cancer. The improvement of existing therapeutics
is expected to help achieve this goal. Specifically, the usage of once two-month injectable nucleos(t)ide
analogs in combination with immunomodulating antiviral compounds instead of life-long daily pills has the
strong potential to help to achieve a functional cure for CHB. To this end, we propose to transform water-soluble
antiviral drugs, first-line drugs tenofovir (TFV) and entecavir (ETV), and immunomodulating drug tizoxanide
(TIZ) into hydrophobic lipophilic crystalline prodrugs. We will formulate them as nanosuspensions suitable for
intramuscular injection. The efficient optimization of physicochemical properties of nanocrystals is expected to
improve their pharmacokinetics (PK) and pharmacodynamics (PD) profiles. This optimization will enhance
uptake of the prodrug nanocrystals by liver macrophages and hepatocytes to ensure a slow release and
sustained therapeutic drug concentrations at the site of hepatitis B viral replication.
The treatment with long-acting TFV, ETV, and TIZ is expected to decrease dosing frequency, limit toxicity,
and facilitate sustained viral suppression and treatment. A functional cure for HBV is expected to be achieved
via multifactorial mechanisms, including inhibition of viral polymerase, prevention of cccDNA formation, and the
clearance of HBV micro-chromosomes via stimulation of host innate immunity by TIZ. Thus, the overall
objective of this proposal is to develop clinically translatable, long-acting, injectable, antiviral drug
nanoformulations to increase adherence and enhance drug delivery to sites of persistent HBV infection,
thereby facilitating sustained viral suppression and finite cure. To this end, three specific aims are proposed:
Aim 1: Develop long-acting anti-HBV prodrug nanoformulations and evaluate the drug efficacy. Here, we will
apply pronucleotide (ProTide) and a modified HepDirect prodrug technology to transform existing drugs into
hydrophobic prodrugs suitable for formulation as nanosuspensions to achieve prolonged therapeutic active
drug concentrations in hepatocyte. This is expected to improve drug biodistribution to infected hepatocytes
without compromising drug potency and safety profile. The prodrug formulations will be screened in vitro in
human macrophages as a potential drug depot and in infected hepatocytes as final targets. The anti-HBV
activity of prodrug nanocrystals will be examined in vitro and in vivo using HBV-infected humanized mice. Aim
2: To develop long-acting TIZ nanoformulation and evaluate the mechanisms by which TIZ suppresses HBV
replication in infected hepatocytes. Aim 3: To evaluate the synergistic efficacy of the selected long-acting TIZ
and NUC formulations and evaluate the ability of this combination to eliminate HBV cccDNA from hepatocytes
and significantly reduce the concentration of HBsAg.