The triple combination of Remdesivir (GS-441524), Molnupiravir and Ribavirin is highly efficient in inhibiting coronavirus replication in human nasal airway epithelial cell cultures and in a hamster infection model

Abstract

The use of fixed dose-combinations of antivirals with different mechanisms of action has proven a key in the successful treatment of infections with HIV and HCV. For the treatment of infections with SARS-CoV-2 and possible future epi-/pandemic coronaviruses, it will be important to explore the efficacy of combinations of different drugs, in particular to avoid resistance development, such as in patients with immunodeficiencies. As a first effort, we studied the antiviral potency of combinations of antivirals. To that end, we made use of primary human airway epithelial cell (HAEC) cultures grown at the air-liquid interface that were infected with the beta coronavirus OC43. We found that the triple combination of GS-441524 (parent nucleoside of remdesivir), molnupiravir, and ribavirin resulted in a more pronounced antiviral efficacy than what could be expected from a purely additive antiviral effect. The potency of this triple combination was next tested in SARS-CoV-2 infected hamsters. To that end, for each of the drugs, intentionally suboptimal or even ineffective doses were selected. Yet, in the lungs of all hamsters that received triple prophylactic therapy with suboptimal/inactive doses of GS-441524, molnupiravir, and ribavirin, no infectious virus was detectable. Our finding indicate that co-administration of approved drugs for the treatment of coronavirus infections should be further explored but also against other families of viruses with epidemic and pandemic potential for which no effective antiviral treatment is available.

Figure 1.. Antiviral activity of GS-441524 and molnupiravir against HCoV-OC43 in HAEC cultures.

Compounds were added to the basal medium at different concentrations 1 hour prior to infection with HCoV-OC43 at 34°C. Basal medium, with or without compounds, was refreshed every other day from day 0 to day 6. Viral RNA in apical washes were quantified by RT-qPCR. Dose-response and time-dependent activity of GS-441524 (A) and molnupiravir (B). All data are mean ± SD of at least three replicates. Grey box indicates time of treatment. LLOQ presents lower limit of quantification.

Figure 2.. Comparing the antiviral activity of combinations of GS-441524, molnupiravir (MOV), and ribavirin (RBV) on HCoV-OC43 replication in nasal human airway epithelial cell (HAEC) cultures.

Compounds were added to the basal medium starting 1 hour before infection and treatment continued for 6 days. Nasal HAEC were infected with HCoV-OC43 at 3×10⁵ copies/insert and incubated at 34°C. Viral RNA in apical washes was quantified by RT-qPCR. (A) Kinetics of HCoV-OC43 replication with or without different monotherapies and combinations of inhibitors. (B) Summaries of the antiviral activities of each treatment on day 6, 8, and 10 were analysed. Two independent experiments with nasal HAEC from a pool of donor were performed (n=3–6). LLOQ represents the lower limit of quantification. Statistical significance between infected untreated control and other groups was calculated by one-way ANOVA with two-sided Dunn's post hoc test. Data are mean ± SD of at least three biological replicates.

Figure 3.. Antiviral effect of combinations of GS-441524, molnupiravir (MOV), and ribavirin (RBV) on SARS-CoV-2 infection in hamsters

(Top) Design of the study. (Bottom) Box plots, with whiskers indicating min to max, representing body weight change (left), and infectious viral titers in the lungs (right). Weight change shown as percentage of change on total weight between day 0 and day 4 p.i.. Infectious virus titers in the lungs at day 4 p.i. as determined as log₁₀ TCID₅₀ per mg lung tissue. Statistical analysis was performed with the Kruskal-Wallis test with Dunn’s comparison. Data are pool of three independent experiments with n=12 for vehicle and the triple therapy-treated group, n=10 for the monotherapy-treated groups and n=4 or 6 for the dual therapy groups.