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 was observed in animals when the drug was administered either beginning 12 hours before or 12 hours 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.

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

Cells were pretreated for 1 hour with differing EIDD-1931 concentrations, followed by infection with SARS-CoV-2 at a MOI of 0.01 for 1 hour. After 1 hour, media was replaced, and cells were cultured in the presence of drug for 24 hours 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) 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. (C) 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 manufacturer’s protocol. For A to C, means are shown ± standard deviation.

Figure 2:. Syrian hamster model - Study design, viral shedding, viral load, infectious titers and viral antigen.

(A) Study design. Hamsters were infected with SARS-CoV-2 by the intranasal route. MK-4482 was administered either pre-infection at 12 and 2 hours prior to infection, or post-infection with treatment started 12 hours post-infection. Treatment was then continued in both treatment groups every 12 hours for 3 consecutive days until end of the experiment. Animals were euthanized on day 4 and lungs were harvested for pathology and virology. ‘T’ denotes treatment (red: pre-infection and black: post-infection treatments); ‘I’ denotes infection; ‘S’ denotes swab samples and ‘N’ indicates necropsy. (B) Viral shedding. Oral swabs were collected on days 2 and 4 post-infection to measure viral shedding, determined by RT-PCR (N gene: genomic and subgenomic) (C) Viral load in lung tissue. Lung viral loads based on RT-PCR (N gene: genomic and subgenomic) were determined as a correlate for lower respiratory tract infection. (D) Infectious virus in lung tissue. Lung samples were homogenized and titered for infectious on Vero E6 cells. Infectious titers were determined as TCID₅₀ equivalents using the Reed-Muench method. Two independent lung samples were measured from each animal. (B-D) Blue circle, vehicle control; red square, pre-infection treatment; green triangle, post-infection treatment. Summary of Results: (B) No statistical significance in virus shedding was found between either of the two MK-4482 treatment groups and vehicle controls. (C) There was a significant difference in lung viral loads between the pre-infection group compared to the vehicle control. Although the post-infection group trended towards lower levels, there was no significant difference between this group and vehicle control. (D) Infectious titers in the lungs 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. For B to D geometric means are shown. ANOVA followed by Kruskal-Wallis analysis and a pairwise Wilcox test was used to analyze differences among groups. *p<0.05, **p<0.008

Figure 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. (A, D and G) untreated vehicle control, (B, E and H) pre-infection treatment with antiviral drug MK-4482 and (C, F and I) post-infection treatment with MK-4482. (A-F) H&E stain (G, H and I) IHC for SARS-CoV-2 nucleocapsid antibody. (A) Lung 20X: multifocal, moderate broncho-interstitial pneumonia. (B and C) Lung 20X: minimal peribronchial interstitial pneumonia. (D) Lung 200X epithelial cell necrosis (arrow), edema (asterisk), interstitial pneumonia (arrowhead). (E and 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 and I) Lung 20X; insert 200X: scattered to moderate numbers of immunoreactive bronchiolar epithelial cells, type I and II pneumocytes and macrophages.

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

(A) A longitudinal cross section of the right lung 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 and 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. For A and B, means are shown. ANOVA followed by Kruskal-Wallis analysis and a pairwise Wilcox test was used to analyze differences among groups. **p<0.008