Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Keywords: SARS-CoV-2; antiviral therapy; favipiravir; hydroxychloroquine; preclinical model.

Fig. 1.

Kinetics of SARS-CoV-2 replication and lung disease in hamsters. (A) Viral RNA levels in the lungs, ileum, and stool of infected Syrian hamsters. At the indicated time intervals pi, viral RNA levels were quantified by RT-qPCR. The bars represent median values. (B) Infectious viral load in the lung expressed as TCID₅₀ per milligram of lung tissue obtained at day 4 pi. The bars represent median values. (C) Weight change as compared to the weight at day 0 in percentage at the indicated time intervals pi. Bars represent means ± SD. (A–C) The data shown are medians plus the individual hamsters represented as separate data points. (D) Representative H&E images of lungs of SARS-CoV-2−infected hamsters at day 0 and day 4 pi. At day 0 (Top), lungs appear normal; black arrows point at a small lymphoid follicle. At day 4 (Bottom), lungs show peribronchial inflammation and bronchopneumonia in the surrounding alveoli. Right Bottom shows a small focus of bronchopneumonia; alveolar lumina surrounding a small bronchus are filled with inflammatory cells. Images on the Right are magnifications of the black boxes shown in the images on the Left. (Scale bars, 1 mm [Left] and 50 µm [Right].) (E) Representative transversal lung micro-CT images of SARS-CoV-2−infected hamsters at baseline (day 0 pi) and day 3 pi. Light blue arrows indicate infiltration by consolidation of lung parenchyma.

Fig. 2.

In vivo testing of favipiravir and HCQ in the SARS-CoV-2 infection model. (A) Setup of the study. (B) Viral RNA levels in the lungs, ileum, and stool of untreated and treated (favipiravir, HCQ, or HCQ + azithromycin) SARS-CoV-2−infected hamsters at day 4 pi. At the indicated time intervals pi, viral RNA levels were quantified by RT-qPCR. The bars represent median values. (C) Infectious viral load in the lung of untreated hamsters and hamsters receiving treatment (favipiravir, HCQ, or HCQ + azithromycin) expressed as TCID₅₀ per milligram of lung tissue obtained at day 4 pi. The bars represent median values. (D) Weight change of individual hamsters (dots) as compared to the weight at day 0 in percentage points at the indicated time intervals pi. Bars represent means ± SD. (E) Cumulative severity score from H&E stained slides of lungs from SARS-CoV-2−infected hamsters that were untreated (UT, gray) or treated with favipiravir 300 mg/kg (FVP 300, yellow), 600 mg/kg (FVP 600, orange), 1,000 mg/kg (FVP 1000, red), HCQ (blue), or HCQ + azithromycin (blue-white). The bars represent median values. (F) Representative H&E images of lungs at day 4 pi of SARS-CoV-2−infected hamsters and treated with favipiravir. At day 4, lungs of untreated hamsters show significant inflammation in bronchial wall (blue arrows) with apoptotic bodies in respiratory epithelium and extension into adjacent alveoli (orange arrow) and inflammatory cells in arterial wall (red arrow). Lungs of infected hamsters treated with 600 mg⋅kg⁻¹⋅d⁻¹ favipiravir still show a few inflammatory cells in the bronchial wall (blue arrows) and a few focal perivascular lymphocytes (red arrow), whereas lungs of hamsters treated with 1,000 mg⋅kg⁻¹⋅d⁻¹ favipiravir contain even less inflammatory cells in the bronchial wall, but no perivascular inflammation. Apoptotic bodies (black circles) were present in bronchial walls of hamsters from all three groups (Scale bars, 100 µm.) Data were analyzed with the Mann−Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.

High dose of favipiravir reduces infection in a direct contact transmission model. (A) Setup of the study. (B) Viral RNA levels in the lungs, ileum, and stool at day 4 pi are expressed as log₁₀ RNA copies per milligram of tissue. Closed dots represent data from index hamsters (n = 5) inoculated with SARS-CoV-2 1 d before cohousing with sentinel animals. Open dots represent data from sentinel hamsters (n = 5 per condition) which were untreated (gray) or treated with either HCQ (blue) or favipiravir 300 mg/kg (yellow), 600 mg/kg (orange), or 1,000 mg/kg (red), starting 1 d before exposure to index animals. The bars represent median values. (C) Infectious viral loads in the lung at day 4 pi/postexposure are expressed as log₁₀ TCID₅₀ per milligram of lung tissue. The bars represent median values. (D) Weight change at day 4 pi in percentage, normalized to the body weight at the day of infection (index) or exposure (sentinel). Bars represent means ± SD. (E) Cumulative severity score from H&E stained slides of lungs from index SARS-CoV-2−infected hamsters and untreated, favipiravir, and HCQ treated sentinel hamsters. The bars represent median values. (F) Representative coronal and transversal lung micro-CT images of favipiravir and HCQ treated sentinel hamsters at day 4 pi. Light blue arrows indicate examples of pulmonary infiltrates seen as consolidation of lung parenchyma. (G) Micro-CT−derived nonaerated lung volume (reflecting the tissue lesion volume) and aerated lung volume relative to total lung volume of index SARS-CoV-2−infected hamsters and untreated, favipiravir (FVP), and HCQ treated sentinel hamsters. HC, healthy controls. Data were analyzed with the Mann−Whitney U test. ***P < 0.001.

Fig. 4.

Pharmacokinetics of favipiravir and HCQ in infected and sentinel hamsters. (A) Individual plasma trough concentrations of favipiravir in hamsters treated with 300, 600, and 1,000 mg⋅kg⁻¹⋅d⁻¹ BID. The bars represent median values. PrEP, preexposure prophylaxis (in sentinel hamsters). (B) Infectious virus titers in lung tissue at day 4 pi to favipiravir (FVP) plasma trough concentrations of individual hamsters. (C) Individual plasma trough concentrations of HCQ in hamsters treated with HCQ or HCQ and azithromycin (AZT) (n = 14). The bars represent median values. (D) Viral RNA levels in lung tissue at day 4 pi to HCQ plasma trough concentrations of individual hamsters. (E) Summary of trough blood and tissue levels of HCQ in hamsters dosed with 50 mg/kg HCQ sulfate and comparison with in vitro EC₅₀ values.