Favipiravir antiviral efficacy against SARS-CoV-2 in a hamster model

Despite no or limited pre-clinical evidence, repurposed drugs are massively evaluated in clinical trials to palliate the lack of antiviral molecules against SARS-CoV-2. Here we use a Syrian hamster model to assess the antiviral efficacy of favipiravir, understand its mechanism of action and determine its pharmacokinetics. When treatment is initiated before or simultaneously to infection, favipiravir has a strong dose effect, leading to reduction of infectious titers in lungs and clinical alleviation of the disease. Antiviral effect of favipiravir correlates with incorporation of a large number of mutations into viral genomes and decrease of viral infectivity. Antiviral efficacy is achieved with plasma drug exposure comparable with those previously found during human clinical trials. Notably, the highest dose of favipiravir tested is associated with signs of toxicity in animals. Thereby, pharmacokinetic and tolerance studies are required to determine whether similar effects can be safely achieved in humans.

a Dose-response curves of antiviral activity in VeroE6 cells (n=3 biologically independent samples). 96-well culture plates of VeroE6 cells were infected with a MOI of 0.001 or 0.01. Cells were grown three days with seven 2-fold serial dilutions of favipiravir (from 500µM to 7.8µM; in triplicate). Cytopathic effect (CPE) was measured using a cell viability assay. Percentage of viral inhibition was calculated using data from untreated cells (virus control; see methods section). b Infectious titer reductions (n=3 biologically independent samples). 96-well culture plates of cells (VeroE6 or Caco-2) were infected with a MOI of 0.001 or 0.01. Cells were grown three days (i) with three dilutions of favipiravir (500µM, 250µM and 125µM; in triplicate) or without drug (0µM; in triplicate). Cell supernatant media were collected to assess infectious titers using a TCID50 assay (see methods section). Data represent mean ±SD. Source data are provided as a Source Data file. Groups of 8 hamsters were intranasally infected with 10 6 , 10 5 or 10 4 TCID50 of virus. Viral replication was quantified using an RT-qPCR assay. a Lung viral RNA yields expressed in viral genome copies/copy of ɣ-actine gene for n=2 animals at days 2, 3, 4 and 7 dpi. b Plasma viral loads expressed in viral genome copies/mL of plasma for n=2 animals at days 2, 3, 4 and 7 dpi. c Clinical course of the disease for n=2 hamsters. Normalized weight at day n was calculated as follows: % of initial weight of the animal at day n. Data represent mean ±SD (details in Supplementary Data 1). Source data are provided as a Source Data file. a b c

Supplementary Figure 3: Dose-response curves
Dose-response curves based on lungs infectious titers were established to determine drug 50%, 90% and 99% effective doses, for both treatment strategies (preemptive and preventive). Source data are provided as a Source Data file.

Supplementary Figure 4: Evaluation of the toxicity for animals infected and treated with high doses of favipiravir
Groups of 4 or 6 hamsters were intranasally infected with 10 6 , 10 5 or 10 4 TCID50 of virus. Clinical follow-up with animals uninfected (n=4 animals/group) or infected (n=6 animals/group) (10 6 , 10 5 and 10 4 TCID50 of virus) and untreated or treated with a dose of Favipiravir of 75mg/day TID (preemptive antiviral therapy, see figure 2). Normalized weight at day n was calculated as follows: (% of initial weight of the animal at day n)/(mean % of initial weight for mock-infected animals at day n). Data represent mean ±SD. For treated animals, no significant difference was observed between uninfected and infected animals at 1, 2 and 3 dpi (Two Way ANOVA with post-hoc Dunnett's multiple comparisons test, statistical details in Supplementary Data 6). Source data are provided as a Source Data file.

Supplementary Figure 5: Lung histopathological changes with preemptive favipiravir therapy
Groups of 4 animals were intranasally infected with 10 4 TCID50 of virus and sacrificed at 5 dpi. At day of sacrifice, lungs were collected, fixed and embedded in paraffin. Tissue sections were stained with hematoxylin-eosin (H&E). a Representative images of lung tissue (left lobe) (scale bar: 2.3mm): multifocal and extensive areas of inflammation for untreated animal, multifocal but limited aeras of inflammation for 37.5mg/day treated animal and normal lung for 75mg/day treated animal (n=4 samples/group). b Representative images of bronchial inflammation (scale bar: 100µ): severe peribronchiolar inflammation and bronchiole filled with neutrophilic exsudates for untreated animal, mild peribonchiolar inflammation for 37.5mg/day treated animal and minimal peribronchiolar inflammation for 75mg/day treated animal. c Representative images of alveolar inflammation (scale bar: 100µ): severe infiltration of alveolar walls, alveoli filled with neutrophils/macrophages for untreated animal, moderate infiltration of alveolar walls, some alveoli filled with neutrophils/macrophages for 37.5mg/day treated animal and focal/mild interstitial inflammation for 75mg/day treated animal (n=4 samples/group). d Representative images of vessel inflammation (scale bar: 50µ): arterial marked infiltration of vascular wall with neutrophils/cell debris (arrow) and endothelial leukocytic accumulation (arrowhead) for untreated animal, venular mild leukocytes extravasation with sparse endothelial accumulation for 37.5mg/day treated animal and normal arteriol for 75mg/day treated animal (n=4 samples/group). Clinical courses of the disease are presented in Supplementary Figure 6. Statistical details are presented in Supplementary Data 7 and 8.

Supplementary Figure 6: Clinical courses of the disease for histological analysis
Groups of 4 hamsters were intranasally infected with 10 4 TCID50 of virus, treated with 37.5 or 75mg/day TID following the two different treatment strategies and sacrificed at 3 and 5 dpi. Clinical course of the disease. Normalized weight at day n was calculated as follows: % of initial weight of the animal at day n (Two Way ANOVA with post-hoc Dunnett's multiple comparisons test, statistical details in Supplementary Data 7). Data represent mean ± SD. Source data are provided as a Source Data file.

Supplementary Figure 7: Plasma concentrations of favipiravir after administration of a single dose of favipiravir
Groups of 12 uninfected animals were treated with a single dose of favipiravir (6.25, 12.5 or 25mg). Groups of 3 animals were sacrificed 30 minutes, 1 hour, 5 hours and 8 hours after the administration of the drug. Data represent mean ± SD. Source data are provided as a Source Data file.

Gene Target Primer and probes sequences Amplicon length Reference
Sars-CoV-2 RNAdependent RNA polymerase