The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein.

Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections and life-threatening diseases in a subset of patients. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 µg/mL and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.

Thiazolide Prodrug Esters and Derived Peptides: Synthesis and Activity.

Amino acid ester prodrugs of the thiazolides, introduced to improve the pharmacokinetic parameters of the parent drugs, proved to be stable as their salts but were unstable at pH > 5. Although some of the instability was due to simple hydrolysis, we have found that the main end products of the degradation were peptides formed by rearrangement. These peptides were stable solids: they maintained significant antiviral activity, and in general, they showed improved pharmacokinetics (better solubility and reduced clearance) compared to the parent thiazolides. We describe the preparation and evaluation of these peptides.

Impairment of SARS-CoV-2 spike glycoprotein maturation and fusion activity by nitazoxanide: an effect independent of spike variants emergence.

SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here, we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell-cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections.

A randomized double-blind placebo-controlled clinical trial of nitazoxanide for treatment of mild or moderate COVID-19.

BACKGROUND: There is an urgent need for treatments of mild or moderate COVID-19 in an outpatient setting. METHODS: A randomized double-blind placebo-controlled clinical trial in 36 centers in the U.S. between August 2020 and February 2021 investigated the safety and effectiveness of oral nitazoxanide 600 mg twice daily for five days in outpatients with symptoms of mild or moderate COVID-19 enrolled within 72 h of symptom onset (ClinicalTrials.gov NCT04486313). Efficacy endpoints were time to sustained clinical recovery (TSR, a novel primary endpoint) and proportion of participants progressing to severe illness within 28 days (key secondary). FINDINGS: 1092 participants were enrolled. 379 with laboratory-confirmed SARS-CoV-2 infection were analyzed. In the primary analysis, median (IQR) TSR were 13·3 (6·3, >21) and 12·4 (7·2, >21) days for the nitazoxanide and placebo groups, respectively (p = 0·88). 1 of 184 (0·5%) treated with nitazoxanide progressed to severe illness compared to 7 of 195 (3·6%) treated with placebo (key secondary analysis, odds ratio 5·6 [95% CI 0·7 - 46·1], relative risk reduction 85%, p = 0·07). In the pre-defined stratum with mild illness at baseline, nitazoxanide-treated participants experienced reductions in median TSR (3·1 days, p = 0·09) and usual health (5·2 days, p < 0·01) compared to placebo. Nitazoxanide was safe and well tolerated. INTERPRETATION: Further trials with larger numbers are warranted to evaluate efficacy of nitazoxanide therapy in preventing progression to severe illness in patients at high risk of severe illness and reducing TSR in patients with mild illness.

The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters.

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

Synthesis, antiviral activity, preliminary pharmacokinetics and structural parameters of thiazolide amine salts.

Background: The thiazolides, typified by nitazoxanide, are an important class of anti-infective agents. A significant problem with nitazoxanide and its active circulating metabolite tizoxanide is their poor solubility. Results: We report the preparation and evaluation of a series of amine salts of tizoxanide and the corresponding 5-Cl thiazolide. These salts demonstrated improved aqueous solubility and absorption, as shown by physicochemical and in vivo measurements. They combine antiviral activity against influenza A virus with excellent cell safety indices. We also report the x-ray crystal structural data of the ethanolamine salt. Conclusion: The ethanol salt of thiazolide retains the activity of the parent together with an improved cell safety index, making it a good candidate for further evaluation.

The Modulation of Cholesterol Metabolism Is Involved in the Antiviral Effect of Nitazoxanide.

We previously investigated the role of Nitazoxanide (NTZ), a thiazolide endowed with antiviral and antiparasitic activity, in HIV-1 infection. NTZ treatment in primary isolated PBMCs was able to reduce HIV-1 infection in vitro by inducing the expression of a number of type-I interferon-stimulated genes. Among them, NTZ was able to induce cholesterol-25-hydroxylase (CH25H), which is involved in cholesterol metabolism. In the present study, we wanted to deepen our knowledge about the antiviral mechanism of action of NTZ. Indeed, by inducing CH25H, which catalyzes the formation of 25-hydroxycholesterol from cholesterol, NTZ treatment repressed cholesterol biosynthetic pathways and promoted cholesterol mobilization and efflux from the cell. Such effects were even more pronounced upon stimulation with FLU antigens in combination. It is already well known how lipid metabolism and virus replication are tightly interconnected; thus, it is not surprising that the antiviral immune response employs genes related to cholesterol metabolism. Indeed, NTZ was able to modulate cholesterol metabolism in vitro and, by doing so, enhance the antiviral response. These results give us the chance to speculate about the suitability of NTZ as adjuvant for induction of specific natural immunity. Moreover, the putative application of NTZ to alimentary-related diseases should be investigated.

Systemic efficacy on Cryptosporidium parvum infection of aminoxanide (RM-5061), a new amino-acid ester thiazolide prodrug of tizoxanide.

Cryptosporidiosis is a gastrointestinal illness with profuse diarrhoea. Although there are no other Food and Drug Administration (FDA)-approved alternatives for the treatment of cryptosporidiosis, nitazoxanide (NTZ) can be qualified as partially effective. In immunosuppressed conditions, severe and/or disseminated cryptosporidiosis may occur and patients should be treated parenterally. To achieve the goal of developing parenteral treatment for cryptosporidiosis, the current study was undertaken to investigate the in vitro and in vivo anticryptosporidial activity of aminoxanide. This new l-tert-leucyl thiazolide is a soluble prodrug of tizoxanide (TIZ), the main metabolite of NTZ. Confirming the good efficacy of aminoxanide in Cryptosporidium parvum-infected HCT-8 cells with a 50% inhibitory concentration of 1.55 µm (±0.21), in immunosuppressed C. parvum-infected Mongolian gerbils (Meriones unguiculatus), a 5-day treatment with a daily intramuscular dose of 100 mg kg−1 aminoxanide resulted in a 72.5% oocyst excretion inhibition, statistically equivalent to 75.5% in gerbils treated with a 4-fold lower oral dose of NTZ. Cryptosporidium parvum-induced intestinal pathology and inflammation were improved. Aminoxanide provides an injectable form of TIZ that NTZ was unable to do and is a promising drug for which optimization of the formulation should be further explored. These results represent a first promising step towards the goal of developing a parenteral treatment for cryptosporidiosis.

Viral burden, inflammatory milieu and CD8+ T-cell responses to influenza virus in a second-generation thiazolide (RM-5061) and oseltamivir combination therapy study.

BACKGROUND: Influenza viruses cause significant morbidity and mortality, especially in young children, elderly, pregnant women and individuals with co-morbidities. Patients with severe influenza disease are typically treated with one neuraminidase inhibitor, oseltamivir or zanamivir. These antivirals need to be taken early to be most effective and often lead to the emergence of drug resistance and/or decreased drug susceptibility. Combining oseltamivir with another antiviral with an alternative mode of action has the potential to improve clinical effectiveness and reduce drug resistance. METHODS: In this study, we utilized a host-targeting molecule RM-5061, a second-generation thiazolide, in combination with oseltamivir to determine whether these compounds could reduce viral burden and understand their effects on the immune response to influenza virus infection in mice, compared with either monotherapy or placebo. RESULTS: The combination of RM-5061 and OST administered for 5 days after influenza infection reduced viral burden at day 5 post-infection, when compared to placebo and RM-5061 monotherapy, but was not significantly different from oseltamivir monotherapy. The inflammatory cytokine milieu was also reduced in animals which received a combination therapy when compared to RM-5061 and placebo-treated animals. Antiviral treatment in all groups led to a reduction in CD8+ T-cell responses in the BAL when compared to placebo. CONCLUSIONS: To our knowledge, this is the first time a combination of a host-targeting compound, RM-5061, and neuraminidase inhibitor, OST, has been tested in vivo. This antiviral combination was safe in mice and led to reduced inflammatory responses following viral infection when compared to untreated animals.

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.

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.

Prophylaxis of ferrets with nitazoxanide and oseltamivir combinations is more effective at reducing the impact of influenza a virus infection compared to oseltamivir monotherapy.

Combination therapy is an alternative approach to reduce viral shedding and improve clinical outcomes following influenza virus infections. In this study we used oseltamivir (OST), a neuraminidase inhibitor and nitazoxanide (NTZ), a host directed drug, and found in vitro that the combination of these two antivirals have a synergistic relationship. Using the ferret model of (A/Perth/265/2009, (H1N1)pdm09), virus infections, we found that the combination of NTZ and OST was more effective than either NTZ or OST independently in preventing infection and reducing duration of viral shedding. However, these benefits were only seen if treatment was administered prophylactically, as opposed to therapeutically. We also found that if prophylactically treated ferrets that had detectable virus in the upper respiratory tract, no virus was detected in the lower respiratory tract. This benefit was not observed with NTZ or OST alone. The combination of NTZ and OST enhances the antiviral effect of OST, which is the standard of care in most settings.

A Pilot Clinical Trial of Nitazoxanide in the Treatment of Chronic Hepatitis B.

Chronic infection by the hepatitis B virus (HBV) has remained a major public health problem. To achieve an HBV cure, we will likely need to combine antivirals with different viral targets as well as immunotherapy. Here, we report data from a pilot proof-of-concept clinical trial of nitazoxanide in treating chronic hepatitis B. Conclusion: Nitazoxanide offers novel mechanisms of antiviral activity, and it would be interesting to evaluate the potential of combining nitazoxanide with oral nucleos(t)ide analogues.

Nitazoxanide inhibits paramyxovirus replication by targeting the Fusion protein folding: role of glycoprotein-specific thiol oxidoreductase ERp57.

Paramyxoviridae, a large family of enveloped viruses harboring a nonsegmented negative-sense RNA genome, include important human pathogens as measles, mumps, respiratory syncytial virus (RSV), parainfluenza viruses, and henipaviruses, which cause some of the deadliest emerging zoonoses. There is no effective antiviral chemotherapy for most of these pathogens. Paramyxoviruses evolved a sophisticated membrane-fusion machine consisting of receptor-binding proteins and the fusion F-protein, critical for virus infectivity. Herein we identify the antiprotozoal/antimicrobial nitazoxanide as a potential anti-paramyxovirus drug targeting the F-protein. We show that nitazoxanide and its circulating-metabolite tizoxanide act at post-entry level by provoking Sendai virus and RSV F-protein aggregate formation, halting F-trafficking to the host plasma membrane. F-protein folding depends on ER-resident glycoprotein-specific thiol-oxidoreductase ERp57 for correct disulfide-bond architecture. We found that tizoxanide behaves as an ERp57 non-competitive inhibitor; the putative drug binding-site was located at the ERp57-b/b' non-catalytic domains interface. ERp57-silencing mimicked thiazolide-induced F-protein alterations, suggesting an important role of this foldase in thiazolides anti-paramyxovirus activity. Nitazoxanide is used in the clinic as a safe and effective antiprotozoal/antimicrobial drug; its antiviral activity was shown in patients infected with hepatitis-C virus, rotavirus and influenza viruses. Our results now suggest that nitazoxanide may be effective also against paramyxovirus infection.

The second-generation thiazolide haloxanide is a potent inhibitor of avian influenza virus replication.

The emergence of new avian influenza virus (AIV) strains able to infect humans represents a serious threat to global human health. In addition to surveillance and vaccine development, antiviral therapy remains crucial for AIV control; however, the increase in drug-resistant AIV strains underscores the need for novel approaches to anti-influenza chemotherapy. We have previously shown that the thiazolide anti-infective nitazoxanide (NTZ) inhibits influenza A/PuertoRico/8/1934(H1N1) virus replication, and this effect was associated with inhibition of viral hemagglutinin (HA) maturation. Herein we investigated the activity of the second-generation thiazolide haloxanide (HLN) against H5N9, H7N1 and H1N1 AIV infection in vitro, and explored the mechanism of the antiviral action. Using the A/chicken/Italy/9097/1997(H5N9) AIV as a model, we show that HLN and its precursor p-haloxanide are more effective than NTZ against AIV, with IC50 ranging from 0.03 to 0.1 µg/ml, and SI ranging from 200 to >700, depending on the multiplicity of infection. Haloxanide did not affect AIV entry into target cells and did not cause a general inhibition of viral protein expression, whereas it acted at post-translational level by inhibiting HA maturation at a stage preceding resistance to endoglycosidase-H digestion. Importantly, this effect was independent of the AIV-HA subtype and the host cell. Immunomicroscopy and receptor-binding studies confirmed that HLN-induced alterations impair AIV-HA trafficking to the host cell plasma membrane, a key step for viral morphogenesis. The results indicate that haloxanide could provide a new tool for treatment of avian influenza virus infections.

Second-generation nitazoxanide derivatives: thiazolides are effective inhibitors of the influenza A virus.

AIM: The only small molecule drugs currently available for treatment of influenza A virus (IAV) are M2 ion channel blockers and sialidase inhibitors. The prototype thiazolide, nitazoxanide, has successfully completed Phase III clinical trials against acute uncomplicated influenza. RESULTS: We report the activity of seventeen thiazolide analogs against A/PuertoRico/8/1934(H1N1), a laboratory-adapted strain of the H1N1 subtype of IAV, in a cell culture-based assay. A total of eight analogs showed IC50s in the range of 0.14-5.0 µM. Additionally a quantitative structure-property relationship study showed high correlation between experimental and predicted activity based on a molecular descriptor set. CONCLUSION: A range of thiazolides show useful activity against an H1N1 strain of IAV. Further evaluation of these molecules as potential new small molecule therapies is justified.

Genetic and immune determinants of immune activation in HIV-exposed seronegative individuals and their role in protection against HIV infection.

Soon thereafter infection is established, hosts strive for an efficient eradication of microorganisms, with as limited tissue damage as possible, and durable immunological protection against re-infection. On the other hand, pathogens have developed countermeasures to escape host surveillance and to warrant diffusion to other hosts. In this molecular arms race the final results relies on multiple variables, including the genetic and immunologic e correlates of protection available for the host. In the field of HIV-infection, natural protection has been repeatedly associated to the presence of an immune activation state, at least in some cohorts of HESN (HIV-exposed seronegative). Indeed, these subjects, who naturally resist HIV-infection despite repeated exposure to the virus, are characterized by an increased expression of activation markers on circulating cells and greater production of immunological effector molecules both in basal condition and upon specific-stimulation. Although these results are not univocally shared, several publications emphasize the existence of a correlation between polymorphisms in genes associated with increased immune activation and the HESN phenotype. In this review, we will describe some of the genetic variants associated with protection against HIV infection. Understanding the basis of HIV resistance in HESN is mandatory to develop new preventative and therapeutic interventions.

The susceptibility of circulating human influenza viruses to tizoxanide, the active metabolite of nitazoxanide.

Nitazoxanide is a thiazolide compound that was originally developed as an anti-parasitic agent, but has recently been repurposed for the treatment of influenza virus infections. Thought to exert its anti-influenza activity via the inhibition of hemagglutinin maturation and intracellular trafficking in infected cells, the effectiveness of nitazoxanide in treating patients with non-complicated influenza is currently being assessed in phase III clinical trials. Here, we describe the susceptibility of 210 seasonal influenza viruses to tizoxanide, the active circulating metabolite of nitazoxanide. An optimised cell culture-based focus reduction assay was used to determine the susceptibility of A(H1N1)pdm09, A(H3N2), and influenza B viruses circulating in the southern hemisphere from the period March 2014 to August 2016. Tizoxanide showed potent in vitro antiviral activity against all influenza viruses tested, including neuraminidase inhibitor-resistant viruses, allowing the establishment of a baseline level of susceptibility for each subtype. Median EC50 values (±IQR) of 0.48 µM (0.33-0.71), 0.62 µM (0.56-0.75), 0.66 µM (0.62-0.69), and 0.60 µM (0.51-0.67) were obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses respectively. There was no significant difference in the median baseline tizoxanide susceptibility for each influenza subtype tested. This is the first report on the susceptibility of circulating viruses to tizoxanide. The focus reduction assay format described is sensitive, robust, and less laborious than traditional cell based antiviral assays, making it highly suitable for the surveillance of tizoxanide susceptibility in circulating seasonal influenza viruses.

Synthesis and pre-clinical studies of new amino-acid ester thiazolide prodrugs.

Thiazolides are polypharmacology agents with at least three mechanisms of action against a broad spectrum of parasites, bacteria and viruses. In respiratory viruses they inhibit the replication of orthomyxoviridae and paramyxoviridae at a post-translational level. Nitazoxanide 1a, the prototype thiazolide, was originally developed as an antiparasitic agent and later repurposed for the treatment of viral respiratory infections. The second generation thiazolides following nitazoxanide, such as the 5-chloro analogue RM-5038 2a, are also broad-spectrum antiviral agents as we have reported. Both 1a and its effective circulating metabolite, tizoxanide 1b, are 5-nitrothiazole derivatives, while RM-5038 2a and its de-acetyl derivative RM-4848 2b are the corresponding 5-chloro derivatives. Recently 1a has completed phase II-III clinical trials in the United States, Canada, Australia and New Zealand in a total of 2865 adults and adolescents of at least 12 months of age with viral acute respiratory illness. Since its biodisposition is primarily seen in the gastro-intestinal tract, its efficacy in systemic viral diseases requires relatively high oral doses. The chemical synthesis of new derivatives with a better systemic absorption was therefore urgently needed. In order to improve their systemic absorption, new amino-ester prodrug derivatives of 1b and RM4848 2b were prepared and tested for their animal pharmacology, pharmacokinetics and toxicology. RM-5061 8a in rats showed 7-fold higher blood concentration compared to 1a: absolute bioavailability increased from 3 to 20%, with a good safety profile in animal safety pharmacology and toxicology.