In a recent study posted to the bioRxiv* preprint server, researchers presented a complete pre-clinical evaluation of the antiviral activity of nitazoxanide against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

They first confirmed the antiviral efficacy of nitazoxanide (NTZ) and tizoxanide (TIZ) (its active metabolite) in vitro before investigating their activity against SARS-CoV-2 using reconstituted human airway epithelium and a Syrian hamster model.

Since there were no approved small molecules to target coronavirus viral replication, multiple human clinical trials were rapidly initiated to address the emergence of SARS-CoV-2, including those involving an oral treatment by nitazoxanide with no or limited pre-clinical evidence of antiviral efficacy. Drug repurposing has also been considered as an interesting strategy to find an active antiviral therapy against SARS-CoV-2.

The study

In this work, the researchers first evaluated the in vitro efficacy of NTZ and TIZ against SARS-CoV-2, using two different cell lines, the VeroE6, and Caco-2 cells. Then, they investigated the ex vivo efficacy of NTZ using a model of reconstituted human airway epithelial of bronchial origin.

Further investigation to determine the potential antiviral activity of NTZ in vivo using a hamster model of SARS-CoV-2 infection was also performed. To assess other administration routes for NTZ, the researchers explored the antiviral efficacy of an intranasal NTZ emulsion. They also characterized the pharmacokinetic profile of TIZ in hamsters after the administration of the NTZ suspension or TIZ formulated in 10% [Tween 80, 80% EtOH (70:30 v/v)] and 90% distilled water, homogenous opaque suspension.

Despite promising in vitro results and new hypotheses on its antiviral mechanism, NTZ failed to reduce the severity of SARS-CoV-2 infection in vivo in the Syrian hamster model.”

Results

The researchers found that NTZ possesses EC50 under 5µM in two different cell lines. Additionally, they demonstrated that NTZ was active in bronchial human airway epithelia, which largely resembles the structural, functional, and innate immune features of the human respiratory epithelium; However, at lower potency as compared to Remdesivir, the positive control in this assay.

The study data showed that the active metabolite TIZ is indeed active against SARS-CoV-2 in vitro with an EC50 of 7.48µM, strengthening the potential use of NTZ in coronavirus disease 2019 (COVID-19) management. Previous studies have reported that the TMEM16 inhibitor, NTZ protects against cell fusion induced by SARS-CoV-2 spike protein in cell culture. Although NTZ showed promising in vitro results and new hypotheses on its antiviral mechanism, it failed to reduce the severity of SARS-CoV-2 infection in vivo in the Syrian hamster model.

When using two different dosing regimens of NTZ, no significant improvement in terms of the clinical course of the disease, viral replication, and/or histopathological damages in the lungs was observed. These findings were demonstrated by the insufficient pulmonary diffusion of TIZ since peak concentrations in lungs (1-hour post-treatment) that never exceeded its in vitro or ex vivo EC50. They were also confirmed by the low accumulation of TIZ over time in the lungs and similar trough concentrations after three days of multiple doses of NTZ and those found four hours post-treatment in the single-dose model.

The pharmacokinetic (PK) modeling and simulations confirmed the lack of NTZ efficacy in the in vivo hamster model of SARS-CoV-2 infection. Simulations depicted that the dose of 500mg/kg/day BID (found ineffective in this study) was sufficient to achieve C_max and AUC above those observed in humans at the usual dose of 1000mg/kg/day, but not sufficient to reach trough concentrations (C_min) observed in humans at this same dose.

The study findings suggested that at the usual dose of 1000mg/kg/day in humans, NTZ would not affect SARS-CoV-2 replication. The researchers enhanced the pulmonary diffusion and explored the possible antiviral activity of TIZ within the upper respiratory tract by treating hamsters with an intranasal NTZ emulsion formulation. Since no significant improvement in any of the disease endpoints was observed, this alternative route of administration proved ineffective in the model.

The TIZ trough concentration measured in lungs after three days of intranasal NTZ administration was very low which partly explains the lack of antiviral efficacy. The pharmacokinetic data revealed that the use of NTZ as an antiviral against SARS-CoV-2 is inappropriate at the current standard formulation and dosage. The low pulmonary bioavailability of NTZ is the major challenge that needs to be addressed to properly evaluate the potential antiviral effect of NTZ in an animal or human model.

In conclusion, the optimization of the NTZ formulation may allow the review of the potential use of the drug for the treatment of SARS-CoV-2 infection.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Saurabh Chaturvedi

Saurabh Chaturvedi is a freelance writer from Jaipur, India. He is a gold medalist in Masters in Pharmaceutical Chemistry and has extensive experience in medical writing. He is passionate about reading and enjoys watching sci-fi movies.