169 - AT-752, an Oral Guanosine Nucleotide Prodrug, Exhibits Potent in Vitro Activity Against Flaviviruses and Prevents Disease Progression in a Dengue Mouse Model

Background: The increasing global prevalence of human Dengue virus infection and the potential for life-threatening sequelae highlight the significance of this unmet medical need.  Here we report the potent in vitro activity of AT-281, the free base form of AT-752, against Dengue virus and other flaviviruses and the in vivo efficacy of AT-752 in a mouse model of Dengue viral disease.

Methods: Antiviral activities of serial dilutions of AT-281 were evaluated in infected Huh-7 cells.  Effective concentrations of AT-281 required to inhibit virus yield reduction by 90% (EC₉₀) and to prevent cytopathic effect by 50% (EC₅₀) were determined, respectively, by visual examination and by neutral red staining, as was cytotoxicity.  AG129 (α-, β- and γ-interferon knock-out) mice received an oral dose of AT‐752 (1000 mg/kg) 4 h before s.c. inoculation with Dengue virus type 2 (strain D2Y98P, 1x10⁵ virus particles) followed by b.i.d. doses (500 mg/kg) for 7 days starting 1 h post‐inoculation (p.i.).  Six groups each (n=5) of treated and control mice were scheduled to be sacrificed on days 4, 6, 7, 8, 10 and 21 p.i. with serum and spleen viral RNA levels determined by plaque assay.  AT-281 efficacy was evaluated based on overall health score, survival, weight loss and viral load in serum and spleen.

Results: In vitro EC₉₀ values for AT-281 against Dengue, West Nile and Yellow Fever viruses ranged from 0.26 to 0.64 µM and EC₅₀ values for Zika and Japanese encephalitis were 0.21 and 0.64 µM, respectively (Table 1).  No toxicity was observed up to the highest concentrations tested (172 µM).  Oral administration of AT-752 to Dengue-infected AG129 mice substantially improved survival, prevented weight loss and lowered viral loads by day 6, with virus being undetectable on day 8 and thereafter (Figure 1).  Serum and spleen viral loads in control mice declined between days 4 and 8 but no control mice survived beyond day 8.  In contrast, AT-752 treated mice survived up to day 19, eventually succumbing to model-induced CNS sequelae.Table 1. Antiviral Activity of AT-281 Against Various Flaviviruses in Huh-7 Cell Cultures
Figure 1. Efficacy of AT-752 in the AG129 mouse model of Dengue infection. Panel a: health score: 1, healthy; 2, coat slightly ruffled; 3, coat ruffled/wet; 4, coat very ruffled, eyes slightly closed/inset; 5, coat very ruffled, eyes closed/inset; 6, coat very ruffled, eyes closed/inset, moribund requiring humane euthanasia; 7, found dead. Panel b: Kaplan-Meier survival plot. Panel c: percent weight loss. Panel d: serum viremia. Panel e: spleen viral load.


Conclusion: The potent activity of AT-281 against Dengue virus in vitro and the efficacy of its salt form, AT-752, in the terminal AG129 mouse model warrant further clinical development of the drug.  Preclinical safety studies are in progress and clinical trials will be initiated thereafter.