Host-targeted nitazoxanide has a high barrier to resistance but does not reduce the emergence or proliferation of oseltamivir-resistant influenza viruses in vitro or in vivo when used in combination with oseltamivir

Highlights

• Serial passaging was used to determine the propensity for influenza viruses to develop resistance to tizoxanide.

• Tizoxanide selective pressure up to 20 μM did not result in virus populations with altered drug susceptibility.

• Host-targeted Nitazoxanide has a high barrier to antiviral resistance.

• Tizoxanide/oseltamivir combination therapy did not prevent the emergence or selection of oseltamivir resistant virus.

Abstract

A major limitation of the currently available influenza antivirals is the potential development of drug resistance. The adamantanes, neuraminidase inhibitors, and more recently polymerase inhibitors, have all been associated with the emergence of viral resistance in preclinical, clinical studies or in clinical use. As a result, host-targeted drugs that act on cellular proteins or functions have become an attractive option for influenza treatment as they are less likely to select for resistance. Nitazoxanide (NTZ) is a host-targeted antiviral that is currently in Phase III clinical trials for the treatment of influenza. In this study, we investigated the propensity for circulating influenza viruses to develop resistance to nitazoxanide in vitro by serially passaging viruses under selective pressure. Phenotypic and genotypic analysis of viruses passaged ten times in the presence of up to 20 μM tizoxanide (TIZ; the active metabolite of nitazoxanide) showed that none had a significant change in TIZ susceptibility, and amino acid substitutions arising that were unique to TIZ passaged viruses, did not alter TIZ susceptibility.

Combination therapy, particularly utilising drugs with different mechanisms of action, is another option for combatting antiviral resistance, and while combination therapy has been shown to improve antiviral effects, the effect of reducing the emergence and selection of drug-resistant virus has been less widely investigated. Here we examined the use of TIZ in combination with oseltamivir, both in vitro and using the ferret model for influenza infection and found that the combination of the two drugs did not provide significant benefit in reducing the emergence or selection of oseltamivir-resistant virus.

These in vitro findings suggest that clinical use of NTZ may be significantly less likely to select for resistance in circulating influenza viruses compared to virus-targeted antivirals, and although the combination of NTZ with oseltamivir did not reduce the emergence of oseltamivir-resistant virus in vitro or in vivo, combination therapy with NTZ and other newer classes of influenza antiviral drugs should be considered due to NTZ's higher host-based barrier to resistance.

Introduction

Antiviral drugs provide an option to vaccination for the control of influenza virus infections, particularly in pandemic situations when suitable vaccines are unlikely to be available for the first 6–9 months (Koonin and Patel, 2018). However, the successful treatment of influenza using currently available antivirals would be limited if the pandemic virus was initially resistant or if antiviral resistance emerged when the antivirals were widely used (Dunning et al., 2014; Lina et al., 2018). Prior to 2018, two classes of antiviral drugs have been widely approved for the treatment of influenza, the adamantanes which target the virus M2 ion channel protein, and the neuraminidase inhibitors (NAIs). Adamantanes are no longer used due to widespread resistance amongst circulating influenza strains (Nelson et al., 2009), and while the current levels of circulating NAI resistant viruses are low (approx. 0.5%), they are still detected each year (Lackenby et al., 2018).

Recently, components of the influenza virus polymerase have been targeted by several drugs in clinical development. Favipiravir, which induces lethal mutagenesis (Baranovich et al., 2013), baloxavir marboxil, and pimodivir, target the PB1, PA, and PB2 protein subunits of the influenza polymerase respectively and all three inhibit M2 protein resistant, and NAI resistant viruses (Mifsud et al., 2019). Baloxavir recently received approval in Japan and the USA in 2018 for the treatment of acute and uncomplicated influenza (Heo, 2018; Mullard, 2018). However, baloxavir, and pimodivir have been associated with the emergence of viral resistance in a clinical setting (Finberg et al., 2018; Hayden et al., 2018; Noshi et al., 2018), while resistance to favipiravir has been described in vitro (Goldhill et al., 2018).

Historically, many viruses including influenza, HIV and Hepatitis C viruses have evaded virus-targeted antivirals during treatment (Baumert et al., 2019; Clutter et al., 2016). High virus mutation rates (particularly in RNA viruses), high rates of virus replication, and large viral loads, all contribute to the development of viral resistance under selective pressure of direct-acting antiviral drugs (Mason et al., 2018).

However, antiviral drugs acting on host cellular proteins or functions are less likely to select for resistant viruses, and therefore are attractive as treatment options (Loregian et al., 2014; van de Wakker et al., 2017). Nitazoxanide is a host-targeted antiviral that is currently in Phase III clinical trials for the treatment of influenza (Rossignol, 2014), and produces its antiviral effect by blocking the maturation of the viral glycoprotein HA (Rossignol et al., 2009). Tizoxanide (TIZ), the active metabolite of NTZ, has been shown to have potent in vitro activity against influenza A and B viruses, including those resistant to neuraminidase inhibitors (Tilmanis et al., 2017).

Combination therapy has been shown to be advantageous over antiviral monotherapy in the treatment of influenza, with pre-clinical studies reporting beneficial effects that include synergistic drug interactions (Belardo et al., 2015; Byrn et al., 2015; Fukao et al., 2018; Mifsud et al., 2020; Tarbet et al., 2014), extending treatment windows (Marathe et al., 2016), and increasing survival rates of influenza infected mice (Tarbet et al., 2012). Clinical studies are ongoing to substantiate clinical benefits of combining different classes of influenza antiviral drugs, particularly polymerase inhibitors and NAIs (Beigel et al., 2017; Dunning et al., 2014; Finberg et al., 2018; Hayden et al., 2018).

The effect of combination therapy on reducing the emergence of drug-resistant virus has been less widely investigated, with only a small number of studies to date focussing on combinations of virus-targeted influenza antiviral drugs (Baz et al., 2018; Ilyushina et al., 2006; Kiso et al., 2018; Pires de Mello et al., 2018).

The first objective of this study was to evaluate the propensity for influenza viruses to develop resistance to the host-targeted TIZ in vitro, by serially passaging different viruses under drug selective pressure. The second objective was to investigate whether TIZ, in combination with oseltamivir, impedes the emergence or selection of oseltamivir resistance in seasonal influenza viruses, both in vitro and in vivo using the ferret model of influenza infection.