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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 IC 50 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., Competing Interests: Financial support for this study was in part provided by Romark Laboratories LC, the company that owns the intellectual property rights related to nitazoxanide. J-FR is an employee and stockholder of Romark Laboratories, LC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Piacentini, Riccio, Santopolo, Pauciullo, La Frazia, Rossi, Rossignol and Santoro.)

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., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

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., (© 2022. The Author(s).)

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., Competing Interests: JFR is an employee of and owns equity interest in Romark, L.C. MB, JF and DM are employees of Romark, L.C.; CB is an adviser for Romark, L.C.

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.

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.

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.

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., (© 2020 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020. Published by Elsevier B.V.)

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.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 IC 50 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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 IC 50 s 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.

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 EC 50 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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Nitazoxanide (Alinia(®), NTZ) and tizoxanide (TIZ), its active circulating metabolite, belong to a class of agents known as thiazolides (TZD) endowed with broad anti-infective activities. TIZ and RM-4848, the active metabolite of RM-5038, were shown to stimulate innate immunity in vitro. Because natural resistance to HIV-1 infection in HIV-exposed seronegative (HESN) individuals is suggested to be associated with strong innate immune responses, we verified whether TIZ and RM-4848 could reduce the in vitro infectiousness of HIV-1. Peripheral blood mononuclear cells (PBMCs) from 20 healthy donors were infected in vitro with HIV-1BaL in the presence/absence of TIZ or RM4848. HIV-1 p24 were measured at different timepoints. The immunomodulatory abilities of TZD were evaluated by the expression of type I IFN pathway genes and the production of cytokines and chemokines. TZD drastically inhibited in vitro HIV-1 replication (>87%). This was associated with the activation of innate immune responses and with the up-regulation of several interferon-stimulated genes (ISGs), including those involved in cholesterol pathway, particularly the cholesterol-25 hydroxylase (CH25H). TZD inhibition of HIV-1 replication in vitro could be due to their ability to stimulate potent and multifaceted antiviral immune responses. These data warrant the exploration of TZD as preventive/therapeutic agent in HIV infection.

Nitazoxanide is a broad-spectrum antiviral agent undergoing clinical development for treatment of influenza and other viral respiratory infections. Nitazoxanide exhibits in vitro activity against Middle East respiratory syndrome coronavirus (MERS-CoV) and other coronaviruses, inhibiting expression of the viral N protein. Nitazoxanide also suppresses production of pro-inflammatory cytokines in peripheral blood mononuclear cells and suppresses interleukin 6 production in mice. Having been used extensively in clinical trials and in post-marketing experience, nitazoxanide is an attractive drug candidate for treatment of Middle East respiratory syndrome. Future research should include in vitro mechanism studies, animal models of MERS-CoV infection, clinical trials, including dose-ranging trials, and evaluation of combination therapy with other potential MERS-CoV antivirals., (Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.)

The emergence of drug-resistant influenza A virus (IAV) strains represents a serious threat to global human health and underscores the need for novel approaches to anti-influenza chemotherapy. Combination therapy with drugs affecting different IAV targets represents an attractive option for influenza treatment. We have previously shown that the thiazolide anti-infective nitazoxanide (NTZ) inhibits H1N1 IAV replication by selectively blocking viral hemagglutinin maturation. Herein we investigate the anti-influenza activity of NTZ against a wide range of human and avian IAVs (H1N1, H3N2, H5N9, H7N1), including amantadine-resistant and oseltamivir-resistant strains, in vitro. We also investigate whether therapy with NTZ in combination with the neuraminidase inhibitors oseltamivir and zanamivir exerts synergistic, additive, or antagonistic antiviral effects against influenza viruses. NTZ was effective against all IAVs tested, with 50% inhibitory concentrations (IC50s) ranging from 0.9 to 3.2 μM, and selectivity indexes (SIs) ranging from >50 to >160, depending on the strain and the multiplicity of infection (MOI). Combination therapy studies were performed in cell culture-based assays using A/Puerto Rico/8/1934 (H1N1), A/WSN/1933 (H1N1), or avian A/chicken/Italy/9097/1997 (H5N9) IAVs; dose-effect analysis and synergism/antagonism quantification were performed using isobologram analysis according to the Chou-Talalay method. Combination index (CI) analysis indicated that NTZ and oseltamivir combination treatment was synergistic against A/Puerto Rico/8/1934 (H1N1) and A/WSN/1933 (H1N1) IAVs, with CI values ranging between 0.39 and 0.63, independently of the MOI used. Similar results were obtained when NTZ was administered in combination with zanamivir (CI=0.3 to 0.48). NTZ-oseltamivir combination treatment was synergistic also against the avian A/chicken/Italy/9097/1997 (H5N9) IAV (CI=0.18 to 0.31). Taken together, the results suggest that regimens that combine neuraminidase inhibitors and nitazoxanide exert synergistic anti-influenza effects., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Originally developed and commercialized as an antiprotozoal agent, nitazoxanide was later identified as a first-in-class broad-spectrum antiviral drug and has been repurposed for the treatment of influenza. A Phase 2b/3 clinical trial recently published in The Lancet Infectious Diseases found that oral administration of nitazoxanide 600mg twice daily for five days reduced the duration of clinical symptoms and reduced viral shedding compared to placebo in persons with laboratory-confirmed influenza. The same study also suggested a potential benefit for subjects with influenza-like illness who did not have influenza or other documented respiratory viral infection. From a chemical perspective, nitazoxanide is the scaffold for a new class of drugs called thiazolides. These small-molecule drugs target host-regulated processes involved in viral replication. Nitazoxanide is orally bioavailable and safe with extensive post-marketing experience involving more than 75 million adults and children. A new dosage formulation of nitazoxanide is presently undergoing global Phase 3 clinical development for the treatment of influenza. Nitazoxanide inhibits a broad range of influenza A and B viruses including influenza A(pH1N1) and the avian A(H7N9) as well as viruses that are resistant to neuraminidase inhibitors. It is synergistic with neuraminidase inhibitors, and combination therapy with oseltamivir is being studied in humans as part of ongoing Phase 3 clinical development. Nitazoxanide also inhibits the replication of a broad range of other RNA and DNA viruses including respiratory syncytial virus, parainfluenza, coronavirus, rotavirus, norovirus, hepatitis B, hepatitis C, dengue, yellow fever, Japanese encephalitis virus and human immunodeficiency virus in cell culture assays. Clinical trials have indicated a potential role for thiazolides in treating rotavirus and norovirus gastroenteritis and chronic hepatitis B and chronic hepatitis C. Ongoing and future clinical development is focused on viral respiratory infections, viral gastroenteritis and emerging infections such as dengue fever., (Copyright © 2014 The Author. Published by Elsevier B.V. All rights reserved.)

In this randomized, double-blind, placebo controlled study, the safety of therapeutic (675 mg) and supra-therapeutic (2,700 mg) doses of nitazoxanide was evaluated in accordance with the ICH E14 guidelines. Fifty six (56) subjects participated in four treatment periods and received single doses of nitazoxanide 675 mg, nitazoxanide 2,700 mg, moxifloxacin 400 mg, or placebo. For 675 mg nitazoxanide, the largest change in QTcF from baseline was observed at 12 hours post-dose with a peak value of 1.6 ms (two-sided 90% CI: -0.3, 3.6 ms). The largest negative change in QTcF was observed at 1 hour post-dose (-2.7 ms with two-sided 90% CI: -4.5, -0.8 ms). The largest change in QTcF from baseline for 2,700 mg nitazoxanide was observed at 24 hours post-dose with a peak value of 3.4 ms (two-sided CI: 1.4, 5.4 ms). These findings demonstrate that neither a single dose of 675 mg nor 2,700 mg nitazoxanide prolonged the QT interval in healthy male and female volunteers. The safety results also demonstrate that all four treatments were well-tolerated and the most frequently reported adverse events in the nitazoxanide and moxifloxacin treatment groups were gastrointestinal disorders which were as expected according to the reference safety information., (© 2014, The American College of Clinical Pharmacology.)

Background: Influenza is an important cause of morbidity and mortality worldwide. Treatment options are scarce, and new drugs with novel mechanisms of action are needed. We aimed to assess the efficacy and safety of nitazoxanide, a thiazolide anti-infective, for treatment of acute uncomplicated influenza., Methods: We did a double-blind, randomised, placebo-controlled, phase 2b/3 trial in 74 primary care clinics in the USA between Dec 27, 2010, and April 30, 2011. We enrolled participants aged 12-65 years with fever, at least one respiratory symptom, and one constitutional symptom of influenza within 48 h of symptom onset. We randomly assigned participants to receive either nitazoxanide 600 mg, nitazoxanide 300 mg, or placebo twice daily for 5 days, (ratio 1:1:1) and followed them up for 28 days. Randomisation lists were computer generated and done in blocks of three. Sponsor, investigators, study monitors, patients, and laboratory personnel were all masked to treatment allocation in the study. The primary endpoint was the time from first dose to alleviation of symptoms. The primary analysis was by intention-to-treat for participants with influenza infection confirmed by RT-PCR or culture at baseline. This trial is registered with ClinicalTrials.gov, number NCT01227421., Findings: Of 650 participants screened, 624 (96%) were enrolled. Of these, 212 were randomly assigned to receive placebo twice a day, 201 to receive nitazoxanide 300 mg twice a day, and 211 to receive nitazoxanide 600 mg a day. The median duration of symptoms for participants receiving placebo was 116·7 h (95% CI 108·1-122·1) compared with 95·5 h (84·0-108·0; p=0·0084) for those receiving 600 mg nitazoxanide and 109·1 h (96·1-129·5, p=0·52) for those receiving 300 mg nitazoxanide. Adverse events were similar between the three groups, the most common being headache reported by 24 (11%) of 212 patients enrolled in placebo group, 12 (6%) of 201 patients in the low-dose group, and 17 (8%) of 211 patients in the high-dose group, or diarrhoea, reported by seven (3%) patients in the placebo group, four (2%) patients enrolled in the low-dose group, and 17 (8%) patients in the high-dose group., Interpretation: Treatment with nitazoxanide 600 mg twice daily for 5 days was associated with a reduction of the duration of symptoms in participants with acute uncomplicated influenza. Further studies are warranted to confirm these findings and to assess efficacy of the drug alone or in combination with existing drugs in seriously ill patients and those at risk of influenza complications., Funding: Romark Laboratories LC., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC50s) ranging from 0.3 to 2 μg/ml and 50% cytotoxic concentrations (CC50s) higher than 50 μg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis.

Nitazoxanide and three halogeno-thiazolides, RM-4850, RM-4865, and RM-5038, were tested against Cryptosporidium parvum in experimentally infected immunosuppressed Mongolian gerbils. Daily 400-mg/kg doses of the four test drugs for 5 to 8 consecutive days produced similar reductions of oocyst shedding. Using early-infected gerbils, a shorter 4-day treatment with RM-5038 reduced oocyst shedding by 95%, compared to 47% for nitazoxanide (P = 0.02), suggesting that RM-5038 is more effective than nitazoxanide under the experimental conditions used.

We conducted a double-blind, placebo-controlled clinical trial to demonstrate the efficacy of nitazoxanide suspension for the treatment of presumed infectious diarrhea in children. Eligible patients must have had diarrheal illness lasting 3-29 days. Patients were randomized to receive either nitazoxanide or placebo twice daily for three days. The primary endpoint was time from first dose to resolution of symptoms. One hundred children mean age 3.3 years were enrolled. The median time to resolution of symptoms for nitazoxanide treated patients was 23 hours (IQR 4-48 hours) vs 103.5 hours (IQR 63->168 hours) for placebo (p<0.001). An analysis by disease subset indicated nitazoxanide treated patients had statistically shorter durations of diarrheal illness associated with Giardia lamblia (n=32, p<0.001) and those with no identified enteropathogen (n=38, p=0.008), when compared to placebo. The study medication was well tolerated. Overall, nitazoxanide was effective at reducing the duration of diarrheal illness associated with multiple etiologies, including patients with no identified enteropathogen. These results suggest nitazoxanide may be a viable therapeutic option for the empiric treatment of diarrheal illness in children where the etiology is unknown or presumed to be of infectious origin. Clinical trial registry number NCT01326338., (Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

We report the activities of a number of thiazolides [2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis C virus (HCV) genotypes IA and IB, using replicon assays. The structure-activity relationships (SARs) of thiazolides against HCV are less predictable than against hepatitis B virus (HBV), though an electron-withdrawing group at C(5') generally correlates with potency. Among the related salicyloylanilides, the m-fluorophenyl analogue was most promising; niclosamide and close analogues suffered from very low solubility and bioavailability. Nitazoxanide (NTZ) 1 has performed well in clinical trials against HCV. We show here that the 5'-Cl analogue 4 has closely comparable in vitro activity and a good cell safety index. By use of support vector analysis, a quantitative structure-activity relationship (QSAR) model was obtained, showing good predictive models for cell safety. We conclude by updating the mode of action of the thiazolides and explain the candidate selection that has led to compound 4 entering preclinical development.

Nitazoxanide (NTZ) exhibits potent antiviral activity against hepatitis C virus (HCV) in cell culture. Previously, HCV replicon-containing cell lines resistant to NTZ were selected, but transfer the HCV NTZ-resistance phenotype was not observed following transfection of whole cell RNA. To further explore the nature of the resistance of HCV to NTZ, full length HCV replicon sequences were obtained from two NTZ-resistant (NTZ-11, TIZ-9), and the parental (RP7) cell lines. Numerous nucleotide changes were observed in individual HCV genomes relative to the RP7 HCV consensus sequence, but no common mutations in the HCV non-structural genes or 3'-UTR were detected. A cluster of single nucleotide mutations was found within a 5-base portion of the 5'-UTR in 20/21 HCV replicon sequences from both resistant cell lines. Three mutations (5'-UTR G17A, G18A, C20U) were individually inserted into CON1 ('wild-type') HCV replicons, showed reduced replication (5 to 50-fold), but none conferred resistance to NTZ. RP7, NTZ-11, and TIZ-9 were cured of HCV genomes by serial passage under interferon. Transfection of cured NTZ-11 and TIZ-9 with either whole cell RNAs from RP7, NTZ-11, or TIZ-9, 'wild-type' or the 5'-UTR mutation-containing replicon RNAs exhibited an NTZ-resistance phenotype. TIZ (the active metabolite of NTZ) was found to be inactive against the activity of HCV polymerase, protease, and helicase in enzymatic assays. These data confirm previous speculations that HCV resistance to NTZ is not due to mutations in the virus, and demonstrate that HCV resistance and most likely the antiviral activity of TIZ are due to interactions with cellular target(s)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We report the syntheses and activities of a wide range of thiazolides [viz., 2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis B virus replication, with QSAR analysis of our results. The prototypical thiazolide, nitazoxanide [2-hydroxybenzoyl-N-(5-nitrothiazol-2-yl)amide, NTZ] 1 is a broad spectrum antiinfective agent effective against anaerobic bacteria, viruses, and parasites. By contrast, 2-hydroxybenzoyl-N-(5-chlorothiazol-2-yl)amide 3 is a novel, potent, and selective inhibitor of hepatitis B replication (EC(50) = 0.33 μm) but is inactive against anaerobes. Several 4'- and 5'-substituted thiazolides show good activity against HBV; by contrast, some related salicyloylanilides show a narrower spectrum of activity. The ADME properties of 3 are similar to 1; viz., the O-acetate is an effective prodrug, and the O-aryl glucuronide is a major metabolite. The QSAR study shows a good correlation of observed EC(90) for intracellular virions with thiazolide structural parameters. Finally we discuss the mechanism of action of thiazolides in relation to the present results.

Thirty-nine new thiazolide/thiadiazolide compounds were compared with the nitrothiazole nitazoxanide for activity against Cryptosporidium parvum development in HCT-8 cells. Twenty-seven agents exerted > or =90% inhibition. Agents with a lower 50% inhibitory concentration (IC(50)) than nitazoxanide were either NO(2) or halogen 5 substituted on the thiazole moiety. Other 5 substitutions such as methyl, C(3)H(7), C(6)H(11), H, SO(2)CH(3), and SCH(3) negatively impacted activity. Five-substituted deacetylated analogues exhibited higher IC(50)s than their acetylated counterparts. Halogeno-thiazolide/thiadiazolides may provide valuable nitro-free alternatives to nitazoxanide.

Cryptosporidium species and Giardia intestinalis are the most common enteric protozoan pathogens affecting humans worldwide. In recent years, nitazoxanide has been licensed in the United States for the treatment of cryptosporidiosis in non-immunodeficient children and adults, becoming the first drug approved for treating this disease. There is a need for a highly effective treatment for cryptosporidiosis in immunodeficient patients, but the quest for such a drug has proven to be elusive. While not effective against Cryptosporidium, nitroimidazoles such as metronidazole or tinidazole are effective treatments for giardiasis and can be administered as a single dose. Albendazole and nitazoxanide are effective against giardiasis but require multiple doses. Nitazoxanide is the first new drug developed for treating giardiasis in more than 20years. New potentially promising drug targets in Cryptosporidium and Giardia have been identified, but there appears to be little activity toward clinical development of new drugs., (Copyright 2009. Published by Elsevier Inc.)

Background & Aims: New therapies are needed to treat patients infected with hepatitis C virus (HCV), a major worldwide cause of chronic liver disease. Nitazoxanide (NTZ), originally used to treat cryptosporidiosis infection, recently was shown to have unexpected antiviral activity in the HCV replicon system and in chronically infected patients. A pilot clinical study suggested that NTZ can augment the antiviral effect of interferon (IFN), although the molecular basis for its effect was unknown., Methods: We analyzed the effects of NTZ on the regulation of eukaryotic initiation factor-2alpha (eIF2alpha) and its IFN-induced kinase, protein kinase activated by double-stranded RNA (PKR), in cells that support HCV RNA replication and in vitro biochemical assays., Results: NTZ increased eIF2alpha phosphorylation, a modification known to mediate host cell antiviral defenses. The addition of IFN to cell cultures increased NTZ-induced eIF2alpha phosphorylation. NTZ also increased PKR phosphorylation. In vitro, NTZ promoted PKR autophosphorylation, a key step in activating PKR's kinase activity for eIF2alpha. Finally, NTZ-induced eIF2alpha phosphorylation was reduced in the presence of specific inhibitors of PKR autophosphorylation., Conclusions: An important mechanism of NTZ's action involves activation of PKR, a key kinase that regulates the cell's innate antiviral response. These observations could explain the clinical antiviral effect of NTZ. NTZ might represent a new class of small molecules capable of potentiating and recapitulating important antiviral effects of IFN.

The emergence of highly contagious influenza A virus strains, such as the new H1N1 swine influenza, represents a serious threat to global human health. Efforts to control emerging influenza strains focus on surveillance and early diagnosis, as well as development of effective vaccines and novel antiviral drugs. Herein we document the anti-influenza activity of the anti-infective drug nitazoxanide and its active circulating-metabolite tizoxanide and describe a class of second generation thiazolides effective against influenza A virus. Thiazolides inhibit the replication of H1N1 and different other strains of influenza A virus by a novel mechanism: they act at post-translational level by selectively blocking the maturation of the viral hemagglutinin at a stage preceding resistance to endoglycosidase H digestion, thus impairing hemagglutinin intracellular trafficking and insertion into the host plasma membrane, a key step for correct assembly and exit of the virus from the host cell. Targeting the maturation of the viral glycoprotein offers the opportunity to disrupt the production of infectious viral particles attacking the pathogen at a level different from the currently available anti-influenza drugs. The results indicate that thiazolides may represent a new class of antiviral drugs effective against influenza A infection.

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). The aim of this work was to document inhibitory activities of nitazoxanide (NTZ, [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide]) and new thiazolides/thiadiazolides on S. neurona in vitro development, and investigate their structure-activity relationships. S. neurona was grown in bovine turbinate cell cultures. At concentrations varying from 1.0 to 5.0mg/L, nitazoxanide and 21 of 32 second generation thiazolide/thiadiazolide agents exerted a > or =95% maximum inhibition on S. neurona development. Most active agents were either NO(2) or halogen substituted in position 5 of their thiazole moiety. In contrast, other 5-substitutions such as hydrogen, methyl, SO(2)CH(3), and CH(3) negatively impacted activity. Compared with derivatives with an acetylated benzene moiety, deacetylated compounds which most probably represent primary metabolites exhibited similar inhibitory activities. Present data provide the first evidence of in vitro inhibitory activities of nitazoxanide and new thiazolides/thiadiazolides on S. neurona development. Active halogeno-thiazolide/thiadiazolides may provide a valuable nitro-free alternative to nitazoxanide for EPM treatment depending on further evaluation of their in vivo activities.

Background: Thiazolides have emerged as a new class of broad-spectrum antiviral drugs, and the first thiazolide, nitazoxanide, is in late-stage clinical trials for treating chronic hepatitis C., Objective: To review the chemistry, pharmacology, toxicology and efficacy of thiazolides as antiviral agents with emphasis on clinical development of nitazoxanide in treating chronic hepatitis C., Methods: Literature search, information from Romark Laboratories and my personal experience with the discovery and development of thiazolides serve as the sources for this review., Conclusions: Thiazolides are metabolically stable, highly bound to plasma proteins and are associated with a favorable toxicology profile. Phase II clinical trials have demonstrated efficacy and safety of nitazoxanide added to peginterferon with or without ribavirin in treating patients with chronic hepatitis C. More limited clinical data indicated potential in treating chronic hepatitis B, and three randomized controlled trials have demonstrated efficacy in reducing the duration of viral gastroenteritis. New generation thiazolides with the nitro group of nitazoxanide replaced by a non-reducible group are not active against anaerobes but retain broad-spectrum activity against viruses. Further studies are needed. Research indicates that these drugs may play an important and complementary role in combination with other classes of antiviral drugs.

Purpose: The effects of nitazoxanide on warfarin pharmacokinetics and pharmacodynamics are examined., Methods: This was a Phase I, single-center, open-label, randomized, two-way, crossover study. Secondary endpoints included an evaluation of the effect of nitazoxanide on coagulation parameters observed after a single dose of warfarin and an assessment of the overall tolerability of study treatments. Fourteen healthy men were selected for the study. The study consisted of two treatment periods (Treatment A and Treatment B), each lasting 6 days, with a washout period of at least 21 days between both warfarin intakes. All subjects were scheduled to receive both Treatment A and Treatment B, according to the randomization list. Treatment A consisted of a single oral dose of 25 mg warfarin sodium (five 5-mg tablets). Treatment B consisted of a single oral intake of 25 mg warfarin sodium (five 5-mg tablets) and one 500-mg tablet of nitazoxanide (with nitazoxanide 500 mg continued twice daily for up to 6 days)., Results: All 14 subjects received Treatment B, and 13 of the 14 subjects received Treatment A. Pharmacokinetic results were similar in both treatments, and pharmacodynamic parameters were similar in both treatments. Fourteen adverse events occurred in eight subjects after administration of at least one dose of the study drug. Eleven adverse events occurred in six subjects after treatment with warfarin and nitazoxanide, and three adverse events occurred in two subjects after treatment with warfarin alone. At discharge, a high hemoglobin level and a low total bilirubin level were reported in both groups., Conclusion: Coadministration of nitazoxanide twice daily for six days did not affect the pharmacokinetic or pharmacodynamic properties of a single 25-mg dose of warfarin sodium. Administration of a single dose of warfarin or combined administration of a single dose of warfarin and multiple doses of nitazoxanide appeared safe and well tolerated.

Nitazoxanide, the first thiazolide, was originally developed for the treatment of Cryptosporidium parvum. More recently, antiviral activity of nitazoxanide against hepatitis B virus (HBV) and hepatitis C virus was recognized in in vitro systems. These basic studies led to phase II clinical trials that demonstrated the safety and efficacy of nitazoxanide in combination with peginterferon, with or without ribavirin, in the treatment of chronic hepatitis C genotype 4. The sustained virologic response rate was 79% and 80% in two studies, which was higher than the response rate of 50% with the standard of care with peginterferon plus ribavirin. In very preliminary studies of patients with chronic hepatitis B, nitazoxanide suppressed serum HBV DNA and led to loss of hepatitis B e antigen in the majority of patients and hepatitis B surface antigen in approximately a quarter of patients. Randomized controlled studies of naive and nonresponder patients with chronic hepatitis C genotype 1 are underway, new second generation and controlled release thiazolides are being developed, and future studies of patients with chronic hepatitis B are planned.

Background & Aims: Sustained virologic response (SVR) rates of 50%-60% have been achieved in patients with chronic hepatitis C genotype 4 treated with peginterferon plus ribavirin. The safety and efficacy of nitazoxanide plus peginterferon alfa-2a, with or without ribavirin, were evaluated in a randomized controlled trial at 2 centers in Egypt., Methods: Previously untreated patients with chronic hepatitis C and genotype 4 infection were assigned randomly to groups that were given standard of care (peginterferon alfa-2a and ribavirin for 48 weeks, n = 40), nitazoxanide monotherapy for 12 weeks followed by nitazoxanide plus peginterferon alfa-2a for 36 weeks (n = 28), or nitazoxanide monotherapy for 12 weeks followed by nitazoxanide plus peginterferon alfa-2a and ribavirin for 36 weeks (n = 28). Therapeutics included nitazoxanide (500 mg) twice daily, peginterferon alfa-2a (180 microg) once weekly, and weight-based ribavirin (1000-1200 mg/day)., Results: The percentages of rapid virologic response (RVR), defined as undetectable HCV RNA at week 4 of combination therapy, and SVR were significantly higher in patients given the triple therapy compared with the standard of care (64% vs 38%, P = .048; and 79% vs 50%, P = .023; respectively). Patients given nitazoxanide plus peginterferon alfa-2a had intermediate rates of RVR (54%) and SVR (61%). Adverse events were similar across treatment groups except for higher rates of anemia in the groups receiving ribavirin., Conclusions: The combination of nitazoxanide, peginterferon alfa-2a, and ribavirin increased the percentages of patients with RVR and SVR, compared with patients given peginterferon plus ribavirin, without an increase in adverse events.

Background: Vancomycin is the only US Food and Drug Administration-approved drug for treatment of Clostridium difficile infection (CDI). Metronidazole has been widely used for this purpose but may be inferior to vancomycin, especially for hospitalized patients with severe disease. We report a prospective, double-blind, randomized controlled trial comparing nitazoxanide with vancomycin for treatment of CDI., Methods: Fifty patients with CDI were randomized to receive vancomycin or nitazoxanide for 10 days. An initial response was considered to be the absence of all CDI symptoms between days 11 and 13, and a final response was considered to be lack of symptom recurrence by day 31., Results: One patient fulfilled an exclusion criterion and was removed from the study. Twenty-seven patients received vancomycin, and 23 received nitazoxanide; 23 and 18 patients, respectively, completed the full course of treatment. Initial responses occurred in 20 (74%) of 27 patients treated with vancomycin and in 17 (77%) of 22 patients treated with nitazoxanide (95% confidence interval, -24% to +28%). In those who completed therapy, response rates were 87% (20 of 23 patients) in the vancomycin group and 94% (17 of 18 patients) in the nitazoxanide group (95% confidence interval, -18% to +30%). Times to complete resolution of symptoms were similar in the 2 groups (P = .55). Two patients in the vancomycin group and 1 patient in the nitazoxanide group experienced relapse within 31 days after beginning treatment. Sustained response rates were 78% (18 of 23 patients) for the vancomycin group, and 89% (16 of 18 patients) for the nitazoxanide group (95% confidence interval, -18% to +35%)., Conclusions: The small sample precludes conclusions about noninferiority of nitazoxanide to vancomycin. Nevertheless, this is the first recent randomized controlled trial to compare any antimicrobial agent other than metronidazole with vancomycin. Results suggest that nitazoxanide may be as effective as vancomycin in treating CDI., Trial Registration: ClinicalTrials.gov identifier: NCT00384527.

Nitazoxanide and its primary metabolite, tizoxanide, inhibit hepatitis C virus (HCV) replication in HCV replicon systems. To study the potential for resistance, we subjected Huh7 cells harboring HCV replicons to serial passage in 250 muM G418 and increasing concentrations of nitazoxanide or tizoxanide. Passage of the replicon-containing cell lines in either compound resulted in increases in the 50% effective concentrations (EC(50)s) (7- to 13-fold), EC(90)s (14- to 36-fold), and 50% cytotoxic concentrations (2- to 4-fold) of both compounds. Serial passage in either compound did not alter the susceptibility of HCV replicons to ribavirin or 2'-C-methylcytidine. Interestingly, serial passage in nitazoxanide or tizoxanide resulted in increased sensitivity to alpha interferon 2b: EC(50)s and EC(90)s were reduced three- and eightfold, respectively. Replicons isolated from these cell lines had no greater ability to confer tizoxanide resistance, or increased susceptibility to alpha interferon, than replicons isolated from the parental cell line that had not previously been exposed to nitazoxanide or tizoxanide. These findings are indicative of a cell-mediated activity differing from that of other anti-HCV drugs but complementary with interferon and are consistent with the enhanced response rates observed clinically when nitazoxanide is combined with pegylated interferon therapy. Finally, unlike data for other compounds in advanced clinical development for HCV, these data are consistent with resistance in HCV replicon-containing cell lines conferred by changes in the host and not by mutations in the virus.

Nitazoxanide, the first thiazolide, was originally developed for the treatment of Cryptosporidium parvum. The antiviral activity of nitazoxanide was discovered by serendipity in patients with AIDS who were treated for cryptosporidial diarrhea and had HBV or HCV co-infection. In preliminary open-label studies of patients with chronic hepatitis B, nitazoxanide suppressed serum HBV DNA and led to loss or seroconversion of hepatitis B e antigen in the majority of patients, as well as hepatitis B surface antigen in approximately a quarter of patients. In Phase II studies of patients with chronic hepatitis C genotype 4, nitazoxanide combined with peginterferon alfa-2a, with or without ribavirin, increased the sustained virologic response rate to 79-80 versus 50% with peginterferon plus ribavirin standard of care. Randomized, controlled studies of naive and nonresponder patients with chronic hepatitis C genotype 1 and patients with chronic hepatitis B are underway, and new second generation thiazolides are being developed.

Background: Nitazoxanide, licensed in the US for treatment of Cryptosporidium parvum and Giardia lamblia, inhibits hepatitis C virus replication in replicon systems., Aim: To evaluate the safety and efficacy of nitazoxanide monotherapy for the treatment of chronic hepatitis C., Methods: This multicentre, randomized, double-blind, placebo-controlled study randomized 50 adult patients with chronic hepatitis C genotype 4 at three centres in Egypt to nitazoxanide 500 mg tablet or placebo twice daily for 24 weeks. Patients were followed up every 4 weeks during treatment and for 24 weeks after therapy., Results: Seven of 23 patients (30.4%) in the nitazoxanide group achieved undetectable serum HCV RNA compared to 0 of 24 in the placebo group during therapy (P = 0.004). Each of the seven responders had baseline HCV RNA levels < or =400 000 IU/mL. Six of the seven virological responders were followed up for 24 weeks after the end of treatment, and four patients (17.4% of 23 treated) had a sustained virological response. Adverse events were similar in the nitazoxanide and placebo groups., Conclusion: Nitazoxanide monotherapy is safe and effective in achieving sustained virological response in a modest number of patients with chronic hepatitis C genotype 4, particularly in patients with low baseline serum HCV RNA levels.

Nitazoxanide (NTZ), a thiazolide anti-infective, is active against anaerobic bacteria, protozoa, and a range of viruses in cell culture models, and is currently in phase II clinical development for treating chronic hepatitis C. In this report, we characterize the activities of NTZ and its active metabolite, tizoxanide (TIZ), along with other thiazolides against hepatitis B virus (HBV) and hepatitis C virus (HCV) replication in standard antiviral assays. NTZ and TIZ exhibited potent inhibition of both HBV and HCV replication. NTZ was equally effective at inhibiting replication of lamivudine (LMV) and adefovir dipovoxil (ADV)-resistant HBV mutants and against 2'-C-methyl cytidine (2'CmeC) and telaprevir (VX-950)-resistant HCV mutants. NTZ displayed synergistic interactions with LMV or ADV against HBV, and with recombinant interferon alpha-2b (IFN) or 2'CmeC against HCV. Pre-treatment of HCV replicon-containing cells with NTZ potentiated the effect of subsequent treatment with NTZ plus IFN, but not NTZ plus 2'CmeC. NTZ induced reductions in several HBV proteins (HBsAg, HBeAg, HBcAg) produced by 2.2.15 cells, but did not affect HBV RNA transcription. NTZ, TIZ, and other thiazolides are promising new antiviral agents that may enhance current or future anti-hepatitis therapies.

Amoebiasis is a significant cause of morbidity worldwide and is the third leading cause of death from parasitic diseases. This study evaluated nitazoxanide, a thiazolide anti-infective, in the treatment of intestinal and hepatic amoebiasis. Prospective, randomised, double-blind, placebo-controlled studies were conducted in outpatients with intestinal amoebiasis from the Nile Delta of Egypt. Nitazoxanide was administered twice daily for 3 days at doses of 500mg (age > or =12 years), 200mg (age 4-11 years) or 100mg (age 1-3 years). Seventeen adults hospitalised with hepatic amoebiasis were treated with 500mg nitazoxanide twice daily for 10 days. Four days after completion of therapy, 32 (94%) of 34 nitazoxanide-treated patients with intestinal amoebiasis resolved symptoms compared with 15 (50%) of 30 patients who received placebo (P<0.001). Thirty-two (94%) of 34 nitazoxanide-treated patients were free of Entamoeba histolytica in two post-treatment stool specimens compared with only 13 (43%) of 30 patients receiving placebo (P<0.0001). All patients with hepatic amoebiasis responded to nitazoxanide therapy. Nitazoxanide is effective in treating invasive intestinal amoebiasis and in eliminating E. histolytica colonisation of the intestinal tract. Further studies are warranted in patients with hepatic amoebiasis.

Thiol-olefin co-oxygenation (TOCO) of substituted allylic alcohols generates beta-hydroxy peroxides that can be condensed in situ with various ketones, to afford a series of functionalised 1,2,4-trioxepanes in good yields. Manipulation of the phenylsulfenyl group in 8a-8c allows for convenient modification to the spiro-trioxepane substituents. Surprisingly, and in contrast to the 1,2,4-trioxanes examined, 1,2,4-trioxepanes are inactive as antimalarials up to 1000 nM and we rationalize this observation based on the inherent stability of these systems to ferrous mediated degradation. FMO calculations clearly show that the sigma* orbital of the peroxide moiety of 1,2,4-trioxane derivatives 4a and 14b are lower in energy and more accessible to attack by Fe(II) compared to their trioxepane analogues 8b and 9b.