Severe mammarenaviral disease in guinea pigs effectively treated by an orally bioavailable fusion inhibitor, alone or in combination with favipiravir

Abstract

Infections by pathogenic New World mammarenaviruses (NWM)s, including Junín virus (JUNV), can result in a severe life-threatening viral hemorrhagic fever syndrome. In the absence of FDA-licensed vaccines or antivirals, these viruses are considered high priority pathogens. The mammarenavirus envelope glycoprotein complex (GPC) mediates pH-dependent fusion between viral and cellular membranes, which is essential to viral entry and may be vulnerable to small-molecule inhibitors that disrupt this process. ARN-75039 is a potent fusion inhibitor of a broad spectrum of pseudotyped and native mammarenaviruses in cell culture and Tacaribe virus infection in mice. In the present study, we evaluated ARN-75039 against pathogenic JUNV in the rigorous guinea pig infection model. The compound was well-tolerated and had favorable pharmacokinetics supporting once-per-day oral dosing in guinea pigs. Importantly, significant protection against JUNV challenge was observed even when ARN-75039 was withheld until 6 days after the viral challenge when clinical signs of disease are starting to develop. We also show that ARN-75039 combination treatment with favipiravir, a viral polymerase inhibitor, results in synergistic activity in vitro and improves survival outcomes in JUNV-challenged guinea pigs. Our findings support the continued development of ARN-75039 as an attractive therapeutic candidate for treating mammarenaviral hemorrhagic fevers, including those associated with NWM infection.

Keywords: ARN-75039; Antiviral; Arenavirus; Favipiravir; Junin virus; Mammarenavirus.

Fig. 1.

In vitro inhibition of JUNV Romero by increasing concentrations ARN-75039 and favipiravir. Virus titers (mean ± standard deviation; n = 4) were determined by endpoint titration of Vero cells lysates collected 4 days after treatment and infection with JUNV (MOI of 0.002). The EC₉₀ values were determined by linear regression analysis. There was no cytotoxicity at the highest doses of ARN-75039 (200 nM) or favipiravir (100 μM) in uninfected cells treated in parallel for 4 days.

Fig. 2.

Oral PK of ARN-75039 in Hartley guinea pigs. Males (n = 2) and females (n = 2) were dose by oral instillation of 30 mg/kg of ARN-75039 formulated in carrot baby food or carrot baby food containing 2% lecithin and 23% water. Plasma concentrations are shown over 24 h and each data point represents the mean and standard deviation.

Fig. 3.

Once daily oral ARN-75039 treatment protects guinea pigs against lethal JUNV challenge. Animals in each group (n = 7) were treated QD with 60 mg/kg ARN-75039 or placebo for 14 days beginning 2, 4, or 6 days (d) post-challenge with JUNV. A) Survival outcome and B) clinical parameter data are shown. Change in body weight is represented as the group mean and standard error of the surviving guinea pigs relative to their starting weights on the day of challenge (day 0). Body temperature data are represented as the group mean and standard error. Body weight and temperature data from sham-infected normal control animals (n = 2) are included for comparison. *** p < 0.001, * p < 0.05, compared to guinea pigs treated with the baby carrot food placebo.

Fig. 4.

Impact of oral ARN-75039 treatment on viremia in JUNV-infected guinea pigs. Animals in each group were treated as described in Fig. 3. Blood was collected on day 12 p.i. and serum infectious JUNV titers were determined by end point titration as described in the Materials and Methods. *** p < 0.001 compared to animals treated with the carrot baby food placebo.

Fig. 5.

Twice daily treatment does not increase the efficacy of oral ARN-75039 in guinea pigs challenged with JUNV. Animals in each group (n = 7) were treated BID with 60 mg/kg ARN-75039 (divided into two 30 mg/kg doses) or placebo for 14 days beginning 4 or 6 days (d) p.i. with JUNV. A) Survival outcome and B) clinical parameter data are shown. Change in body weight is represented as the group mean and standard error of the surviving guinea pigs relative to their starting weights on the day of challenge (day 0). Body temperature data are represented as the group mean and standard error. Body weight and temperature data from sham-infected normal control animals (n = 3) are included for comparison. *** p < 0.001, ** p < 0.01, compared to guinea pigs treated with the baby carrot food placebo.

Fig. 6.

Impact of oral BID ARN-75039 treatment on viremia in JUNV-infected guinea pigs. Animals in each group were treated as described in Fig. 5. Blood was collected on day 12 p.i. and serum infectious JUNV titers were determined by end point titration as described in the Materials and Methods. *** p < 0.001 compared to animals treated with the carrot baby food placebo.

Fig. 7.

Evaluation of combined inhibitory activities of ARN-75039 and favipiravir. A) Percent inhibition dose response heat map of ARN-75039 and favipiravir combination treatment matrix. The larger number represents the percent inhibition, and the smaller number reflects the standard deviation. B) Bliss two-dimensional topography synergy map of ARN-75039 and favipiravir interaction. The Bliss synergy score was calculated to be 9.3 ± 3.0. A Bliss synergy score of > 10 indicates synergistic activity. Data are from 3 independent experiments.

Fig. 8.

Therapeutic efficacy of combined oral ARN-75039 and favipiravir in the guinea pig JUNV infection model. Animals in each group (n = 7) were treated BID with 60 mg/kg ARN-75039 or 250 mg/kg favipiravir, alone or in combination, for 14 days beginning on day 5 post-challenge with JUNV. A) Survival outcome and B) clinical parameter data are shown. Change in body weight is represented as the group mean and standard error of the surviving guinea pigs relative to their starting weights on the day of challenge (day 0). Body temperature data are represented as the group mean and standard error. Body weight and temperature data from sham-infected normal control animals (n = 2) are included for comparison. *** p < 0.001, *** p < 0.01, * p < 0.05, compared to guinea pigs treated with the carrot baby food placebo. ^^ p < 0.01 compared to animals treated with favipiravir alone.