Nitazoxanide, tizoxanide and other thiazolides are potent inhibitors of hepatitis B virus and hepatitis C virus replication

doi:10.1016/j.antiviral.2007.08.005

Antiviral Research

Volume 77, Issue 1, January 2008, Pages 56-63

https://doi.org/10.1016/j.antiviral.2007.08.005 Get rights and content

Abstract

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.

Introduction

Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) are major public health problems, causing more than an estimated 500 million chronic infections worldwide (Chen and Morgan, 2006, Lavanchy, 2004). Both viruses are a source of significant progressive liver disease, and are the major risk factors for nearly all cases of primary hepatocellular carcinoma (Chen and Morgan, 2006, Lavanchy, 2004, Wong and Lok, 2006). Licensed standards of care for both viral infections, while effective in many cases, are sub-optimal and do not result in virologic or clinical ‘cures’ in most individuals (Wong and Lok, 2006). The development of drug-resistance in HBV, including strains carrying resistance to multiple licensed agents is an emerging clinical problem, and drug-resistance for future HCV therapies is predicted to be a significant clinical issue (Tomei et al., 2005, Tong et al., 2006, Yim et al., 2006).

Nitazoxanide (NTZ) is a thiazolide anti-infective with activity against anaerobic bacteria, protozoa and viruses (Fox and Saravolatz, 2005, Pankuch and Appelbaum, 2006, Rossignol et al., 2006a, Rossignol and El-Gohary, 2006). Originally developed as a treatment of intestinal protozoan infections, the antiviral properties of NTZ were discovered during the course of its development for treating cryptosporidiosis in patients with acquired immune deficiency syndrome (AIDS). NTZ is marketed in the United States for treating diarrhea and enteritis caused by Cryptosporidium spp. or Giardia lamblia in adults and children down to 12 months of age (Alinia^®, Romark Laboratories, Tampa, Florida USA). Clinical trials have demonstrated effectiveness of NTZ in treating diarrhea and enteritis associated with enteric protozoan infections caused by Cryptosporidium spp., G. lamblia, Entamoeba histolytica and Blastocystis hominis (Amadi et al., 2002, Ortiz et al., 2001, Rossignol et al., 2001, Rossignol et al., 2005, Rossignol et al., 2006b). Recent randomized double-blind clinical trials have demonstrated effectiveness of NTZ in treating Clostridium difficile colitis in adults, rotavirus gastroenteritis in young children, and rotavirus and norovirus gastroenteritis in adults (Musher et al., 2006, Rossignol et al., 2006a, Rossignol and El-Gohary, 2006). The mechanism of action of NTZ against anaerobic organisms is attributed to interference with pyruvate:ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reactions, which are essential for anaerobic energy metabolism (Hoffman et al., 2007). Its mechanism of antiviral activity has not been fully elucidated.

Following oral administration of a 500 mg tablet, NTZ is partially absorbed from the gastrointestinal tract and rapidly hydrolyzed in plasma to form its active circulating metabolite, tizoxanide (TIZ). NTZ is not detected in plasma. Maximum serum concentrations of TIZ, reach approximately 10 μg/mL (37 μM) (Stockis et al., 2002) following oral administration of one 500 mg NTZ tablet (Alinia^®) with food. TIZ is glucurono-conjugated in the liver and excreted in urine and bile. Approximately, two-thirds of an oral dose pass through the intestinal tract and is excreted in feces as TIZ (Broekhuysen et al., 2000). The elimination half-life of TIZ from plasma is approximately 1.5 h. TIZ does not inhibit cytochrome P450 enzymes, and therefore, no drug–drug interactions are expected (Broekhuysen et al., 2000, Stockis et al., 2002). The most commonly reported side-effects in clinical trials include mild abdominal pain, headache, diarrhea and nausea, which occur at rates similar to those reported for patients receiving placebo. While most of the clinical experience with NTZ has involved 3–14 days of treatment, continual use of the drug for periods as long as 4 years has been evaluated in patients with AIDS-related cryptosporidiosis without any significant drug-related adverse events (Fox and Saravolatz, 2005, Rossignol, 2006).

In this report, we present results of in vitro studies characterizing the activities of NTZ, TIZ and other new thiazolides against hepatitis B and hepatitis C viruses.

Section snippets

Materials

Lamivudine (LMV) and adefovir dipovoxil (ADV), and 2′-C-methyl cytidine were purchased from Moraveck Biochemicals, Inc. (La Brea, CA, USA). Recombinant human interferon alpha 2b (IFNα) was purchased from PBL Biomedical Laboratories (Piscataway, NJ USA). All other test compounds (Fig. 1) were provided by Romark Laboratories, L.C. (Tampa, FL, USA). Human serum (heat-inactivated, mixed gender, lot BRH125374) was purchased from Bioreclamation, Inc. (Hicksville, NY).

Antiviral assays

HBV antiviral assays were

Activities of compounds and combinations in 2.2.15 cell cultures

NTZ and its active metabolite, TIZ, exhibited selective inhibition of intracellular HBV replication and extracellular virus production by 2.2.15 cells (Table 1). Several other thiazolides (see Table 1) were also effective inhibitors of HBV replication in this assay system. Combinations of NTZ with either of two drugs licensed for anti-HBV therapy, lamivudine and adefovir dipovoxil, demonstrated synergistic interactions when used to treat 2.2.15 cells (Table 1, Fig. 2A and B). The anti-HBV

Discussion

In this report, we present the first in vitro demonstrations of the activity of nitazoxanide and its active circulating metabolite, tizoxanide, against both HBV and HCV replication. Inhibition of both viruses was dose-dependent and selective relative to cytotoxicity under the conditions of the antiviral assays. Concentrations of tizoxanide required to inhibit viral replication, even those observed in the presence of additional human serum, are readily achieved in human plasma following oral

Acknowledgements

This work was supported by NIAID contract NO1-AI-30046 to GUMC. The wild-type HCV replicon-containing cell lines were generously provided by Apath, Inc. The HBV N236T mutant was kindly provided by S. Xiong (Gilead Sciences, Inc.) to BEK. The LMV-resistant mutants were originally provided by Burroughs Wellcome, Inc. We thank P. Cote (GUMC) for assistance with the statistical analyses. VX-950 was a gift of R. Fathi (XTLBio, Inc.). Thiazolide compounds were synthesized by Andrew Stachulski,

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Nitazoxanide, tizoxanide and other thiazolides are potent inhibitors of hepatitis B virus and hepatitis C virus replication

Abstract

Introduction

Section snippets

Materials

Antiviral assays

Activities of compounds and combinations in 2.2.15 cell cultures

Discussion

Acknowledgements

Lancet

Gastroenterology

Antivir. Res.

Antiviral Res.

Antivir. Res.

Antivir. Res.

Antivir. Res.

Lancet

Clin. Gastroenterol. Hepatol.

Clin. Gastroenterol. Hepatol.

Antivir. Res.

Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group

Hepatology

Efficient initiation of HCV RNA replication in cell culture

Science

Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture

J. Virol.

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Int. J. Clin. Pharmacol. Ther.

The natural history of hepatitis C virus (HCV) infection

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Clin. Infect. Dis.