Development of Novel Agents Active against Hepatitis B Virus

Division Of Basic Sciences - Nci

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Abstract

cccDNA persistence in HBV-harboring hepatocytes prevents the cure of chronic hepatitis B (CHB). This study aimed to determine whether E-CFCP, a novel long-acting nucleoside analog (NA), can reduce cccDNA levels and prevent the emergence of HBV variants resistant to E-CFCP. Here, HepG2.2.15 cells and the HBV-infected uPA-SCID-humanized-liver mouse (PXB-mouse) model were used to quantify intrahepatocellular cccDNA levels. The genetic barrier of E-CFCP was also assessed. E-CFCP's EC50 and EC50 for blocking HBV-DNA synthesis in HepG2.2.15 cells were 0.0045 microM and 0.4 microM, respectively, indicating potent chain-termination during early-phase HBV reverse transcription. At concentrations of greater than 1 microM, E-CFCP completely inhibited HBV-DNA synthesis, whereas other NAs only partially inhibited synthesis; only ETV achieved 100% inhibition at 10 microM. Subculturing HepG2.2.15 cells with E-CFCP (10 microM) over 10-39 days resulted in a 72-82% decrease in cccDNA levels, as quantified by droplet-digital PCR, compared to untreated cells. No significant reduction was observed with ETV or 3TC. When a clinical isolate mixture with NA resistance (n=9) was passaged in PXB mice treated with E-CFCP, I91F and S223A substitutions emerged. However, recombinant HBV carrying I91F+S223A remained sensitive to E-CFCP, ETV, and TAF. ETV-resistant HBV with I91F+S223A showed high resistance to ETV (EC50=208 microM), but remained sensitive to E-CFCP (EC50=0.248 microM). Structural studies carried out using the Maestro suite from Schrodinger indicated that the I91F/S223 substitutions only moderately weakened E-CFCP interactions with HBV-RT. When we asked whether E-CFCP reduced the cccDNA copy numbers when chronically HBV-infected PXB mice were continuously treated with long-term (up to 9 weeks) oral E-CFCP QD administration, both E-CFCP and ETV reduced the HBV viremia as we previously demonstrated. As we expected, E-CFCP significantly lessened the copy numbers of cccDNA when examined after 9 weeks of administration (0.92 copies/cell), while 2.1 cccDNA copies/cell were seen in vehicle-receiving mice (n=6 for each; p=0.0043). The failure of E-CFCP to completely "knock-down" the cccDNA copy number would be explained by at least a few limiting natures in the PXB mouse models employed, which differ from what would occur in human bodies. (i) Because of the severe immunodeficiency nature of PXB mice, long-term experimental observation using such mice is often hampered and limited due to the development of malignancies and other complications. (ii) Dilution effects that are brought about by continuous recruit of uninfected hepatocytes occurring from hepatocytic stem cells are unlikely to take place in PXB mice since the transplanted hepatocytes are improbable to contain stem-cell-level hepatocytes. (iii) The hepatocytes transplanted to immunodeficient mice are likely terminally-differentiated cells and the decrease and clearance of cccDNA copies through mitosis are also limited. Due to these limitations, it is possible that the reduction levels in cccDNA observed in the present study were limited, despite the high magnitudes of HBV-DNA synthesis inhibition by E-CFCP. In conclusion, E-CFCP effectively blocks HBV-DNA synthesis, significantly reduces intrahepatocellular cccDNA levels, and possesses a high genetic barrier, making it a promising candidate for contributing to CHB cure.