Preclinical Antiviral and Safety Profiling of the HBV RNA Destabilizer AB-161

Abstract

HBV RNA destabilizers are a class of small-molecule compounds that target the noncanonical poly(A) RNA polymerases PAPD5 and PAPD7, resulting in HBV RNA degradation and the suppression of viral proteins including the hepatitis B surface antigen (HBsAg). AB-161 is a next-generation HBV RNA destabilizer with potent antiviral activity, inhibiting HBsAg expressed from cccDNA and integrated HBV DNA in HBV cell-based models. AB-161 exhibits broad HBV genotype coverage, maintains activity against variants resistant to nucleoside analogs, and shows additive effects on HBV replication when combined with other classes of HBV inhibitors. In AAV-HBV-transduced mice, the dose-dependent reduction of HBsAg correlated with concentrations of AB-161 in the liver reaching above its effective concentration mediating 90% inhibition (EC₉₀), compared to concentrations in plasma which were substantially below its EC₉₀, indicating that high liver exposure drives antiviral activities. In preclinical 13-week safety studies, minor non-adverse delays in sensory nerve conductance velocity were noted in the high-dose groups in rats and dogs. However, all nerve conduction metrics remained within physiologically normal ranges, with no neurobehavioral or histopathological findings. Despite the improved neurotoxicity profile, microscopic findings associated with male reproductive toxicity were detected in dogs, which subsequently led to the discontinuation of AB-161's clinical development.

Keywords: HBV RNA; HBV RNA destabilizer; PAPD5; PAPD7.

Figure 1

Chemical structures of AB-452 and AB-161.

Figure 2

AB-161 inhibits PAPD5 and PAPD7 enzymatic activity. (A) Recombinant human PAPD5 or (B) PAPD7 was incubated with increasing concentrations of AB-161 and the inhibitory effect against ATP incorporation was measured. Half maximal inhibition (IC₅₀) was determined based on the dose–response curves. Mean IC₅₀ values were determined from 3 independent experiments.

Figure 3

AB-161 destabilized intracellular HBV RNA and suppressed viral protein production and replication. (A) HepG2.2.15 cells were treated with DMSO or AB-161 at increasing concentrations (0.3 to 1700 nM) for 48 h. The relative intensities of HBV pgRNA (3.5 kb) and preS/S RNA (2.4/2.1 kb) in each sample are expressed as percentages of those from untreated cells and are indicated below each lane. (B) Time course analysis of HBV pgRNA and preS/S RNA from HepAD38 cells treated with GLS4 in the presence or absence of AB-161 (100 nM). Total RNA was extracted before the addition of AB-161 (0 h) or at 2, 4, 8, and 16 h post-addition of AB-161. Ribosomal 28S and 18S rRNA were included as loading controls. (C) Interference of HBV life cycle by AB-161. HepG2.2.15 cells were treated with DMSO, ETV (1 μM), GLS4 (1 μM), or AB-161 (100 nM). Intracellular HBV pgRNA and preS/S RNA were analyzed via Northern blot, HBV core via Western blot (β-actin as loading control), HBV capsid and encapsidated DNA via particle gel assay, HBV rcDNA and ssDNA via Southern blot.

Figure 4

AB-161 mediates reduction in HBsAg in an AAV-HBV mouse model. (A) AAV-HBV mice received once-daily oral doses of AB-161 or vehicle control from Days 0 to 13. Data represent serum HBsAg levels relative to pre-dose baseline levels of each animal. HBsAg was assessed via ELISA. Data represent group mean (n = 6–9) ± standard deviation. (B) AB-161 unbound concentrations in liver and (C) plasma at 24 h post-last dose (C_24h). Unbound concentrations were calculated by applying a fraction-unbound factor of 0.024 to total C_24h liver concentrations measured by LC-MS. Fraction-unbound factor represents 2.4% of AB-161, estimated to be free based on 97.6% mouse plasma protein binding.

Figure 5

Nerve conduction velocity for the peroneal nerve among rats administered either vehicle or AB-161 (30 mg/kg, 120 mg/kg, 500 mg/kg). Individual female (red) and male (blue) rats were monitored for nerve conduction velocity (m/s) at (A) baseline, (B) near end of treatment (day 70 ± 3), and (C) end of recovery (day 119 ± 3). Normal physiological range (dotted reference line) for rat is >30 m/s. Mean for each group was determined and expressed as a horizontal line. Number of animals (n) are indicated underneath each group. * Indicates statistically significant difference (p < 0.05).

Figure 6

Nerve conduction velocity for the peroneal nerve among dogs administered either vehicle or AB-161 (10 mg/kg, 30 mg/kg, 100 mg/kg). Individual female (red) and male (blue) dogs were monitored for nerve conduction velocity (m/s) at (A) baseline, (B) near end of treatment (day 70 ± 3), and (C) end of recovery (day 119 ± 3). Normal physiological range (dotted reference line) for dog is >40 m/s. Mean for each group was determined and expressed as a horizontal line. Number of animals (n) are indicated underneath each group. * Indicates statistically significant difference (p < 0.05).