Orally delivered MK-4482 inhibits SARS-CoV-2 replication in the Syrian hamster model

Abstract

The COVID-19 pandemic progresses unabated in many regions of the world. An effective antiviral against SARS-CoV-2 that could be administered orally for use following high-risk exposure would be of substantial benefit in controlling the COVID-19 pandemic. Herein, we show that MK-4482, an orally administered nucleoside analog, inhibits SARS-CoV-2 replication in the Syrian hamster model. The inhibitory effect of MK-4482 on SARS-CoV-2 replication is observed in animals when the drug is administered either beginning 12 h before or 12 h following infection in a high-risk exposure model. These data support the potential utility of MK-4482 to control SARS-CoV-2 infection in humans following high-risk exposure as well as for treatment of COVID-19 patients.

Fig. 1. EIDD-1931 inhibits SARS-CoV-2 replication in human lung epithelial Calu-3 cells.

Cells were pretreated for 1 h with differing EIDD-1931 concentrations, followed by infection with SARS-CoV-2 at a MOI of 0.01 for 1 h. After 1 h, media was replaced, and cells were cultured in the presence of drug for 24 h at 37 °C in a 5% CO₂ incubator. a Virus yield in the cell supernatant was measured by quantitative RT-PCR of clarified culture supernatant by using primer and probe sets to quantify total viral RNA (N gene; genomic and subgenomic RNA). b DMSO control versus media. Replicates were analyzed in duplicate (mean ± SD are shown). c IC₅₀ values were determined using results from the RT-PCR following log-based transformation of drug concentrations and normalization to percentage inhibition based on diluent alone controls by fitting to drug-dose response curves using Prism software. d Absence of toxicity (>90% viability; shown by dotted line) at highest EIDD-1931 concentration used for analysis of SARS-CoV-2 replication (40μM) was confirmed using CellTiter-Glo® 2.0 Assay (Promega, Corp., Madison, WI, USA) as per the manufacturer’s protocol. Individual sample values of replicates are shown.

Fig. 2. Syrian hamster model—study design, viral shedding, viral load, infectious titers, and viral antigen.

a Hamsters were intranasally infected with SARS-CoV-2. MK-4482 was administered pre-infection at 12 and 2 h before infection, or post-infection starting 12 h post-infection. Treatment was continued in both groups every 12 h for 3 consecutive days. Animals were euthanized on day 4 and lungs were harvested. T = treatment (red: pre-infection and black: post-infection treatments); I = infection; S = swab samples and N = necropsy. b Oral swab samples (N = 6 per group) were collected on days 2 and 4 post-infection and viral shedding determined by RT-PCR (p Value Vehicle vs Pre-treatment = >0.9999, p Value Vehicle vs Post-treatment = >0.9999, One-way ANOVA, Kruskal–Wallis test). c Oral swab samples (N = 6 per group) were titered for infectious virus (TCID₅₀) on Vero E6 cells (p Value Vehicle vs Pre-treatment = 0.5701, p Value Vehicle vs Post-treatment = >0.9999, One-way ANOVA, Kruskal–Wallis test). d Lung viral loads (N = 6 per group) were determined by using RT-PCR (p Value Vehicle vs Pre-treatment = 0.0189, p Value Vehicle vs Post-treatment = 0.1032, One-way ANOVA, Kruskal–Wallis test). e Lung samples (N = 6 per group) were homogenized and titered for infectious virus (TCID₅₀) on Vero E6 cells. Two independent lung samples were measured from each animal (N = 12 per group) (p Value Vehicle vs Pre-treatment = 0.0091, p Value Vehicle vs Post-treatment = 0.0102, One-way ANOVA, Kruskal–Wallis test). b–e Blue circle, vehicle control; red square, pre-infection treatment; green triangle, post-infection treatment. Summary of Results: b, c No statistical significance in virus shedding (RT-PCR or TCID₅₀) between either of the two MK-4482 treatment groups and vehicle controls. d Significant difference in lung viral loads (RT-PCR) between pre-infection group compared to the vehicle control. Although post-infection group trended towards lower levels, no significant difference between this group and vehicle control. e Infectious titers in the lungs (TCID₅₀) were significantly different between both pre-infection and post-infection groups, compared to vehicle control group, but no significance was found between treatment groups from each other. One-way ANOVA followed by Kruskal–Wallis analysis and a pairwise Wilcox test was used to analyze differences among groups. *p < 0.05, **p < 0.008.

Fig. 3. Pathological analysis of the lung tissue.

Hematoxylin and eosin (H&E) staining was used on lung sections to examine lung pathology post-inoculation. Immunohistochemistry (IHC) was used to detect viral antigen in the same lung sections from each animal (N = 6 per group). a, d, g Untreated vehicle control, (b, e, h) pre-infection treatment with antiviral drug MK-4482 and (c, f, i) post-infection treatment with MK-4482. (a–f) H&E stain (g, h, i) IHC for SARS-CoV-2 nucleocapsid antibody. a Lung 20X: multifocal, moderate broncho-interstitial pneumonia. b, c Lung 20X: minimal peribronchial interstitial pneumonia. d Lung 200X epithelial cell necrosis (arrow), edema (asterisk), interstitial pneumonia (arrowhead). e, f peribronchial and interstitial infiltrates (arrow). g Lung 20X; insert 200X: numerous immunoreactive bronchiolar epithelial cells, type I and II pneumocytes and fewer macrophages. h, i Lung 20X; insert 200X: scattered to moderate numbers of immunoreactive bronchiolar epithelial cells, type I and II pneumocytes and macrophages. Pictures were taken in RGB color space (sRGB IEC61966-2.1) with a threshold set to 128. a–c, g–i Scale bar is 200μm. d–f Scale bar is 20μm.

Fig. 4. Morphometric analysis of viral antigen and drug concentration in the lungs.

a A longitudinal cross section of the right lung of each animal (N = 6 per group) was stained for viral antigen and scanned to measure the total amount of viral antigen present in the lung section. b EIDD-1931 concentrations in the lungs. a, b Blue circle, vehicle control; red square, pre-infection treatment; green triangle, post-infection treatment. Summary of results. a The area of lung staining positive for viral antigen showed a statistically significant difference between both of the MK-4482 treatment groups, compared to vehicle controls. No difference between individual treatment groups was present. (One-way ANOVA followed by Kruskal–Wallis analysis and a pairwise Wilcox test was used to analyze differences among groups. **p < 0.008).