High doses of favipiravir counter SARS-CoV-2 in hamsters, Hydroxychloroquine has no significant effect: Study

Researchers report that treatment with high doses of favipiravir, an antiviral drug that has been used to treat pandemic influenza in Japan, significantly reduced SARS-CoV-2 levels in the lungs, improved lung pathology, and reduced virus transmission by direct contact in a hamster model, whereas treatment with the antimalarial drug hydroxychloroquine had no significant effect on virus levels or transmission, suggesting that favipiravir, but not hydroxychloroquine, merits further study for COVID-19 treatment in humans, according to the authors.

The study led by Suzanne J. F. Kaptein, Sofie Jacobs, ana Langendries, Laura Seldeslachts among a team of researchers was published in the peer previewed journal PNAS (Proceedings of the National Academy of Sciences of the United States of America).

The previous lack of consensus around the use of hydroxychloroquine for COVID-19 patients underlines the need to thoroughly assess the in vivo efficacy of drugs against SARS-CoV-2. Small mal infection models, such as the hamster model, have a pivotal place herein. We here show in vivo preclinical results with favipiravir which indicate that antiviral efficacy against SARS-CoV-2 might only be achieved with a very high dose. Hydroxychloroquine, on the other hand, completely lacks antiviral activity, thus providing no scientific basis for its further use in COVID-19 patients. With this study on two key antiviral candidates, we establish the baseline for SARS-CoV-2 antiviral treatment, which will allow us to identify superior antiviral candidates in the near future.

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 mal 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 modelling 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 mal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

* antiviral therapy

* SARS-CoV-2

* preclinical model

* favipiravir

* hydroxychloroquine

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) first emerged in Wuhan, China, in December 2019. From there, the virus rapidly spread around the globe, infecting more than 21 million people so far. SARS-CoV-2 is the causative agent of COVID-19. Common clinical mfestations of COVID-19 are fever, dry cough, paired in a minority of patients with difficulty breathing, muscle and/or joint pain, headache/dizziness, decreased sense of taste and smell, diarrhoea, and nausea. A small subset of patients will develop to acute respiratory distress syndrome, characterized by difficult breathing and low blood oxygen levels, which may directly result in respiratory failure.

In addition, an overreaction of the host's immune and inflammatory responses can result in a vast release of cytokines ("cytokine storm"), inducing sepsis and multiorgan damage, which may lead to organ failure. To date, more than 750,000 patients worldwide have already succumbed to COVID-19. Hence, in response to the ongoing pandemic, there is a desperate need for therapeutic and prophylactic options.

At present, no specific antiviral drugs have been developed and approved to treat infections with human coronaviruses. Nonetheless, antiviral drugs could fulfil an important role in the treatment of COVID-19 patients.

Slowing down the replication of SARS-CoV-2 by the antiviral treatment could be beneficial and prevent or alleviate symptoms. In addition, antiviral drugs could be used as prophylaxis to protect health care workers and high-risk groups. However, a specific, highly potent antiviral drug for SARS-CoV-2 will take years to develop and evaluate in clinical studies. Therefore, the main focus for COVID-19 treatment on the short term is on the repurposing of drugs that have been approved for other diseases.

Repurposed drugs can, however, not be expected to be highly potent inhibitors of SARS-CoV-2, since these were not developed and optimized specifically against this virus. In cell culture, several repurposed drugs inhibit SARS-CoV-2 replication. Although preclinical in vivo evidence evaluating the efficacy of some of these repurposed drugs for COVID-19 treatment is lacking, clinical trials have already been conducted or are currently ongoing. Two such drugs are hydroxychloroquine (HCQ) and favipiravir.

HCQ is an antimalaria drug that has been widely used to treat patients with malaria, rheumatoid arthritis, and systemic lupus erythematosus. This drug is also able to inhibit a broad range of viruses from different virus families in cell culture, including coronaviruses (SARS-CoV-1, Middle East respiratory syndrome-CoV). Favipiravir is a broad-spectrum antiviral drug that has been approved in Japan since 2014 to treat pandemic influenza virus infections. Both drugs have shown antiviral efficacy against SARS-CoV-2 in Vero E6 cells, albeit modestly for favipiravir. Enzymatic assays with the SARS-CoV-2 RNA-dependent RNA polymerase demonstrated that favipiravir acts as a nucleotide analogue via a combination of chain termination, slowed viral RNA synthesis, and lethal mutagenesis. However, proof of efficacy in mal models is still lacking for both drugs.

Nevertheless, clinical trials were initiated early on in the pandemic to assess the efficacy of HCQ and favipiravir to treat COVID-19 patients. For HCQ, these trials were small anecdotal studies or inconclusive small randomized trials and thus did not lead to conclusive results. Despite the lack of clear evidence, HCQ has been widely used for the treatment of COVID-19, often in combination with a second-generation macrolide such as azithromycin. Results from mal models and rigorous randomised controlled trials are thus required to clarify the efficacy of HCQ and favipiravir in the treatment of COVID-19 patients.

Infection models in small mals are crucial for the evaluation and development of antiviral drugs. Although rhesus and cynomolgus macaques seem to be relevant models for studying the early stages of COVID-19 in humans, preclinical models using smaller mals are essential to ensure efficient and ethical allocation of resources toward designing (relevant) preclinical and clinical efficacy studies. Syrian hamsters are permissive to SARS-CoV-2 and develop mild lung disease similar to the disease observed in early-stage COVID-19 patients. Nevertheless, evidence of antiviral efficacy of repurposed drugs in small mal models is lacking to date. In this work, we characterize Syrian hamsters as a model for the evaluation of antiviral drugs in therapeutic and prophylactic settings against SARS-CoV-2. We then use this model to evaluate the antiviral efficacy of HCQ and favipiravir against SARS-CoV-2 in infected hamsters and in a transmission setting.

Results

Characterisation of Hamster Model for Antiviral Drug Evaluation. We further characterise SARS-CoV-2 infection and readouts of disease in hamsters to be able to use this model for the evaluation and development of antiviral drugs. To investigate SARS-CoV-2 replication and shedding, the lung, ileum, and stool of infected hamsters were harvested at different time points postinfection (pi) for viral RNA quantification by RT-qPCR. Infectious virus titers were additionally determined in lung samples. SARS-CoV-2 efficiently replicates in the lungs of the hamsters, with viral RNA being detected in the lungs from day 1 pi and reaching a maximum level of ~7 log10 RNA copies per mg of tissue at 4 d pi. (A similar kinetic profile was found in the ileum and stool samples, albeit at lower levels of 2 to 3 log10 RNA copies per mg of tissue. Titrations of homogenized lung tissue contained infectious particles from 1 d pi and reached levels of ~5 log10 50 per cent tissue culture infectious dose (TCID50)/mg tissue from day 2 pi onward which is in line with the viral RNA levels. Infected mals displayed a slight weight loss of about 5 per cent by day 2 pi, which was completely resolved by day 4 pi. No other signs of disease or distress were observed in the hamsters at any time point pi.

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 um [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 the consolidation of lung parenchyma.

Akin to what is currently done in clinical practice, we evaluated the development of lung disease in a non-invasive way by microcomputed tomography (micro-CT) scanning the infected mals under isoflurane gas anaesthesia. Dense lung infiltrations and bronchial dilation were simultaneously present from day 3 pi onward, becoming more pronounced at day 4 pi. Longitudinal follow-up of radiological pathology showed signs of multifocal pulmonary infiltrates and lung consolidation on day 3 pi. (Analysis by hematoxylin/eosin (H&E) staining of lungs of infected hamsters at day 4 pi showed signs of bronchopneumonia and peribronchial inflammation, which were not present at the day of inoculation.

( With inputs from ANI )