Therapeutic treatment with an oral prodrug of the remdesivir parental nucleoside is protective against SARS-CoV-2 pathogenesis in mice

Abstract

The coronavirus disease 2019 (COVID-19) pandemic remains uncontrolled despite the rapid rollout of safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. In addition, the emergence of SARS-CoV-2 variants of concern, with their potential to escape neutralization by therapeutic monoclonal antibodies, emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parent nucleoside of remdesivir, which targets the highly conserved virus RNA-dependent RNA polymerase. GS-621763 exhibited antiviral activity against SARS-CoV-2 in lung cell lines and two different human primary lung cell culture systems. GS-621763 was also potently antiviral against a genetically unrelated emerging coronavirus, Middle East respiratory syndrome CoV (MERS-CoV). The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 administration reduced viral load and lung pathology; treatment also improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral that has recently received EUA approval, proved both drugs to be similarly efficacious in mice. These data support the exploration of GS-441524 oral prodrugs for the treatment of COVID-19.

Fig. 1.. The chemical structure and in vitro potency of GS-621763 in comparison to RDV (GS-5734) and GS-441524.

(A to C) The chemical structures of the parent adenosine nucleoside analog GS-441524 (A), the monophosphoramidate prodrug RDV (B), and GS-621763, the tri-isobutyryl ester of GS-441524 (C) are shown. (D) The mean percent inhibition of SARS-CoV-2 replication by GS-621763, in comparison to the prodrug RDV and the parental nucleoside GS-441524 in A459-hACE2 cells is shown (triplicate samples were analyzed). (E) Cytotoxicity was measured in A459-hACE2 cells treated with GS-621763, RDV, and GS-441524 (triplicate samples were analyzed). (F) Inhibition of SARS-CoV-2-fLUC replication by GS-621763, RDV, and the parental nucleoside GS-441524 in normal human bronchial epithelial (NHBE) cultures is shown (duplicate samples were analyzed). (G) In vitro EC₅₀ values are shown for inhibition of viral replication by GS-621763, RDV, and the parental nucleoside GS-441524 in A459-hACE2 and NHBE cells. Data are presented as mean ± SD. For (D to F), the horizontal dashed line indicates 50% inhibition on the X-axis and the dotted line indicates 0% inhibition on the X-axis.

Fig. 2.. GS-621763 confers therapeutic protection against SARS-CoV-2 infection in mice in a dose-dependent manner.

(A) Plasma pharmacokinetics of GS-441524 is shown in uninfected BALB/c mice following a single oral administration of GS-621763 at either 5 or 20 mg/kg. Plasma concentrations of GS-621763 were below the limit of quantification at all time points. (B) Depicted is the percent starting weight in mice treated therapeutically with vehicle (n=19) or 3 mg/kg (n=9), 10 mg/kg (n=10), and 30 mg/kg (n=10) GS-621763 at 8 hpi with 1x10⁴ PFU SARS-CoV-2 MA10. (C) Lung viral titers were measured in mice that were therapeutically treated with vehicle (n=19) or 3 mg/kg (n=9), 10 mg/kg (n=10), and 30 mg/kg (n=10) GS-621763 at 8 hpi with 1x10⁴ PFU SARS-CoV-2 MA10. The dashed line indicates the limit of detection (LoD). (D) Pulmonary function was measured in mice therapeutically treated with vehicle (n=7) or 3 mg/kg (n=4), 10 mg/kg (n=4), and 30 mg/kg (n=4) GS-621763 at 8 hpi with 1x10⁴ PFU SARS-CoV-2 MA10. PenH is a surrogate measure for bronchoconstriction. (E) Lung congestion scores were calculated in mice therapeutically treated with vehicle (n=19) or 3 mg/kg (n=9), 10 mg/kg (n=10), and 30 mg/kg (n=10) GS-621763 at 8 hpi with 1x10⁴ PFU SARS-CoV-2 MA10. (F and G) Lung pathology was quantified in mice therapeutically treated with vehicle (n=19) or 3 mg/kg (n=9), 10 mg/kg (n=10), and 30 mg/kg (n=10) GS-621763 at 8 hpi with 1x10⁴ PFU SARS-CoV-2 MA10. ATS acute lung injury (F) and diffuse alveolar damage (G) were scored in a blinded manner. Data were analyzed using a two-way ANOVA (weight loss and lung function) or a Kruskal-Wallis test (lung titer, congestion score, and pathology scores), *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. Box and whisker plots in (B) and (D) show Minimum (Min) to Maximum (Max) values. Data in (C), (E), (F), and (G) are shown as mean ± SEM.

Fig. 3.. Therapeutic protection of mice against SARS-CoV-2 infection is conferred by oral administration of GS-621763 at 12 or 24 hpi.

(A) Depicted is the percent starting weight in in mice treated therapeutically with either 30 mg/kg or 60 mg/kg BID or QD GS-621763 at 12 and 24 hpi (n=10 for all treatment groups, except n=8 for 60 mg/kg, 12 hpi QD treatment group). All mice were infected with 1x10⁴ PFU SARS-CoV-2 MA10. (B) Lung viral titers were measured in mice that were therapeutically treated with either 30 mg/kg or 60 mg/kg BID or QD GS-621763 at 12 and 24 hpi (n=10 for all treatment groups, except n=8 for 60 mg/kg, 12 hpi QD treatment group). All mice were infected with 1x10⁴ PFU SARS-CoV-2 MA10. The dashed line indicates the limit of detection (LoD). (C) Viral nucleocapsid RNA was measured in mice therapeutically treated with either 30 mg/kg or 60 mg/kg BID or QD GS-621763 at 12 and 24 hpi (n=10 for all treatment groups, except n=8 for 60 mg/kg, 12 hpi QD treatment group). All mice were infected with 1x10⁴ PFU SARS-CoV-2 MA10. (D) Pulmonary function was measured in mice therapeutically treated with either 30 mg/kg or 60 mg/kg BID or QD GS-621763 at 12 and 24 hpi (n=4 for all treatment groups). All mice were infected with 1x10⁴ PFU SARS-CoV-2 MA10. PenH is a surrogate measure for bronchoconstriction. (E and F) Lung pathology was quantified in mice therapeutically treated with either 30 mg/kg or 60 mg/kg BID or QD GS-621763 at 12 and 24 hpi (n=10 for all treatment groups, except n=8 for 60 mg/kg, 12 hpi QD treatment group, and n=7 for vehicle group). ATS acute lung injury (E) and diffuse alveolar damage (F) were scored in a blinded manner. Data were analyzed using two-way ANOVA (weight loss and lung function) and Kruskal-Wallis test (lung titer, lung viral RNA, and pathology scores), *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. Box and whisker plots in (A) and (D) show Minimum (Min) to Maximum (Max) values. Data in (B), (C), (E), and (F) are shown as mean ± SEM.

Fig. 4.. Evaluation of therapeutic intervention of GS-621763 in comparison to MPV.

(A) Depicted is the percent starting weight in in mice treated therapeutically with either 30 mg/kg or 60 mg/kg GS-621763 or MPV at 12 or 24 hpi (n=10 for all treatment groups). All mice were infected with 1x10⁴ PFU SARS-CoV-2 MA10. (B) Lung viral titers were measured in mice that were therapeutically treated with either 30 mg/kg or 60 mg/kg GS-621763 or MPV at 12 or 24 hpi (n=10 for all treatment groups). All mice were infected with 1x10⁴ PFU SARS-CoV-2 MA10. The dashed line indicates the limit of detection (LoD). (C) Viral Nucleocapsid RNA was measured in mice therapeutically treated with either 30 mg/kg or 60 mg/kg GS-621763 or MPV at 12 or 24 hpi (n=10 for all treatment groups). All mice were infected with 1x10⁴ PFU SARS-CoV-2 MA10. (D) Pulmonary function was measured in mice therapeutically treated with either 30 mg/kg or 60 mg/kg GS-621763 or MPV at 12 or 24 hpi (n=4 for all treatment groups). All mice were infected with 1x10⁴ PFU SARS-CoV-2 MA10. PenH is a surrogate measure for bronchoconstriction. (E and F) Lung pathology was quantified in mice therapeutically treated with either 30 mg/kg or 60 mg/kg GS-621763 or MPV at 12 or 24 hours (n=10 for all treatment groups). All mice were infected with 1x10⁴ PFU SARS-CoV-2 MA10. ATS acute lung injury (E) and diffuse alveolar damage (F) were scored in a blinded manner. Data were analyzed using two-way ANOVA (weight loss and lung function) and Kruskal-Wallis test (lung titer, lung viral RNA, and pathology scores), *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. Box and whisker plots in (A) and (D) show Minimum (Min) to Maximum (Max) values. Data in (B), (C), (E), and (F) are shown as mean ± SEM.