icon-    folder.gif   Conference Reports for NATAP  
 
  EASL
43rd Annual Meeting of the European Association For The Study Of The Liver
Milan, Italy
April 23-27, 2008
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Nitazoxanide, tizoxanide and other thiazolides are potent inhibitors of hepatitis B virus and hepatitis C virus replication...
 
 
  There were 4 presentations at EASL. I'll be reporting them to you in detail.
 
Antiviral Research
Volume 77, Issue 1, January 2008, Pages 56-63
 
These are extracts from the publication leaving out Methods.
 
Brent E. Korba a,, Abigail B. Montero a, Kristine Farrar a, Karen Gayea, Sampa Mukerjee a, Marc S. Ayers b, Jean-Francüois Rossignol b,c a Department of Microbiology and Immunology, Georgetown University Medical Center, 3900 Reservoir Rd. Washington, DC20007 USA b The Romark Institute for Medical Research, 3000 Bayport Drive, Tampa, FL 33607 USA c Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, 269 Campus Drive, Stanford, CA 94305, USA
 
"...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.....Nitazoxanide was active against both genotypes 1a and 1b HCV, commonly observed LMV and ADV-resistant HBV mutants, as well as representative HCV mutants resistant to telaprevir.....If TIZ exerts its observed antiviral effects by targeting cellular processes, it is possible that resistance may not be as significant a problem for thiazolides as it is typically for direct-acting antiviral agents. The lack of diminished activity against several clinically relevant drug-resistant HBV and HCV variants presents the potential for use in rescue therapy or in combination with existing agents, and is consistent with an indirect mechanism of antiviral activity.....
 
....Nitazoxanide exhibited synergistic activity in combination with LMV or ADV against HBV, and with IFNa or 2'CmeC against HCV, consistent with a mechanism of action that is different from these antiviral agents. Pre-treatment of HCV replicon cells with nitazoxanide potentiated the effect of subsequent treatment with combinations containing IFNa.....Thiazolides may offer the possibility of new more effective combination treatments for chronic hepatitis C, whether used as additions to current standard-of- care, as replacements for ribavirin, or as part of novel combination regimens with other anti-HCV drugs in development...... Nitazoxanide, tizoxanide and other drugs from the thiazolide class are promising new antiviral agents that may enhance current or future therapies for viral hepatitis caused by HBV or HCV. NTZ is undergoing phase II clinical development for hepatitis C, and NTZ other thiazolides are also being developed for treating either hepatitis B or hepatitis C. The implications of a new class of antiviral drugs with the properties described herein could be significant from a clinical perspective."
 
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.
 
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, 2005, 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 10ug/mL (37uM) (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 drugĞdrug 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 3Ğ14 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).
 
Results
 
Hepatitis B virus (HBV)
 
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 assays were conducted under confluence as this provides the conditions for optimal HBV replication (Sells et al., 1988; Korba and Gerin, 1992). While under the conditions of the antiviral assay NTZ displayed minimal cytotoxicity (>100uM, Table 1), cytotoxicity of NTZ in rapidly dividing cultures of 2.2.15 cells was higher (20±1.3uM). NTZ and RM4850 were effective inhibitors of several HBV LMV-resistant and one ADV-resistant constructs in transient transfection assays in Huh7 cells (Table 2). No significant differences in potency of these thiazolides relative to that observed for wild-type HBV were observed for any of the drug-resistant viruses tested.
 
Effect of NTZ on production of HBV proteins
 
Unlike most viruses (including HCV), HBV RNA transcription and protein production are effectively separated from viral genome replication due to the presence of a long-lived population of covalently closed viral template genomes in the host cell nucleus (cccDNA) (see Locarnini, 2004 for a review). Intracellular HBV replication takes place in viral nucleocapsids located in the cytoplasm. As a result, most compounds that inhibit HBV DNA replication (e.g. nucleoside analogues), do not typically alter HBV protein production, especially in cell culture. Suspecting a novel mechanism of action of NTZ against HBV, we conducted studies to determine if the drug inhibited the production of major HBV proteins. As assessed by semiquantitative EIA, NTZ reduced the levels of extracellular HBV surface and e antigens (HBsAg, HBeAg), as well as the levels of intracellular HBV nucleocapsid core antigen (HBcAg) in a dose-dependent manner (Table 3, Fig. 3). The potency of NTZ against HBsAg and HBeAg was similar to that observed against HBV virion production in the same experiment. The relative potency of NTZ against intracellular HBcAg was similar to that observed for the inhibition of intracellular HBV DNA replication. No quantitative interference with the ability of the EIAs to detect HBV proteins was observed in samples from control cultures to which 10 uMNTZ was added (data not shown).
 
NTZ did not induce a reduction in intracellular HBV RNA as assessed by Northern blot hybridization (Table 3, Fig. 3). In the same experiment, LMV did not affect the levels of HBV proteins or HBV RNA despite inducing significant reductions in HBV virion production and intracellular HBV DNA replication (Table 3).
 
Hepatitis C virus (HCV)
 
Activities of compounds and combinations in HCV replicon cell cultures
 
NTZ and TIZ selectively reduced intracellular HCV replication in AVA5 cells
(Table 4). Combinations of NTZ or TIZ with either recombinant human interferon alpha 2b (IFNa), or an NS5B (HCV polymerase) inhibitor, 2'-C-methyl cytidine (2'CmeC, Pierra et al., 2005), exhibited synergistic interactions against HCV replication (Table 4, Fig. 2C and D). Only two of the other thiazolides, RM4832 and RM4863, exhibited activity against HCV (Table 4). Antiviral activities of NTZ, TIZ and RM4832 against a full-length genotype 1a replicon (Blight et al., 2003) were equivalent to that observed for AVA5 cells (genotype 1b) (Table 4).
 
NTZ was an effective inhibitor of an NS5B and two NS3 drug-resistant mutants in Huh7 cells (Table 5). No significant differences in potency of NTZ relative to that observed for 'wild type' HCV (in AVA5 cells) was observed for any of the drug-resistant mutants tested.
 
Although the mechanism of action of NTZ is not known, the divergence of the intracellular protozoa and viruses inhibited suggests that a cellular, rather than common viral function is being affected. It is conceivable that changes in the intracellular environment induced by NTZ may alter the effect of subsequent treatment with other anti-HCV agents that also act though the induction of cellular pathways, particularly IFNa. As a preliminary investigation into this issue, we evaluated the effect of a regimen consisting of 3 days treatment with NTZ followed by 3 days of a combination of NTZ plus IFNa. Pre-treatment with NTZ monotherapy further improved the potency of combination treatment with NTZ plus IFNa by approximately three-fold (p < 0.02, Table 6). This staggered combination regimen was also modestly synergistic when compared to the simultaneous NTZ plus IFNa treatment combination
(Fig. 2, panels E and F). Pre-treatment did not, however, affect the potency of combination treatment with 2'CmeC (Table 6).
 
Effect of human serum on anti-HCV potency and
cytotoxicity of TIZ in AVA5 cells

 
NTZ and its circulating metabolite, TIZ, are highly bound (>99%) to plasma proteins in human serum (Broekhuysen et al., 2000; Stockis et al., 2002). To evaluate the effect of human serum on the anti-HCV potency and cytotoxicity of TIZ, human serumwas added to the culture medium at various concentrations (Table 7). The CC50, as well as the EC50, and EC90 of TIZ increased with increasing concentrations of human serum up to 20%. The EC50 and EC90 in the presence of 30% human serum were similar to those at 20% human serum (a plateau effect) suggesting that maximum extent of protein binding had been reached. The levels of HCV and B-actin RNA in untreated cultures were similar at different concentrations of human serum up to 30% (data not shown). Higher concentrations of human serum significantly lowered cell viability (data not shown). The maximum EC50 and EC90 observed for NTZ in the presence of serum in these studies were at or below the average peak blood levels of 37uM observed in patients given the prescribed dose of this drug (Stockis et al., 2002).
 
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 administration of nitazoxanide (Broekhuysen et al., 2000; Stockis et al., 2002). Four of six other thiazolides tested exhibited selective activity against HBV replication, and two exhibited selective activity against HCV replication.
 
Nitazoxanide was active against both genotypes 1a and 1b HCV, commonly observed LMV and ADV-resistant HBV mutants, as well as representative HCV mutants resistant to telaprevir and 2'CmeC. Activity against genotype 2a HCV as well as additional drug-resistant HCV remains to be evaluated. If TIZ exerts its observed antiviral effects by targeting cellular processes, it is possible that resistance may not be as significant a problem for thiazolides as it is typically for direct-acting antiviral agents. The lack of diminished activity against several clinically relevant drug-resistant HBV and HCV variants presents the potential for use in rescue therapy or in combination with existing agents, and is consistent with an indirect mechanism of antiviral activity.
 
Nitazoxanide exhibited synergistic activity in combination with LMV or ADV against HBV, and with IFNa or 2'CmeC against HCV, consistent with a mechanism of action that is different from these antiviral agents. Pre-treatment of HCV replicon cells with nitazoxanide potentiated the effect of subsequent treatment with combinations containing IFNa, but not 2'CmeC, suggesting an interesting complementary activity with IFNa. Thiazolides may offer the possibility of new more effective combination treatments for chronic hepatitis C, whether used as additions to current standard-of- care, as replacements for ribavirin, or as part of novel combination regimens with other anti-HCV drugs in development. The activity of nitazoxanide against HBV proteins could prove to be advantageous in treating chronic hepatitis B. Reductions in HBV protein levels, especially HBsAg and HBeAg, are of critical importance in elucidating long term antiviral responses in vivo (Menne et al., 2002; Korba et al., 2004;Wong and Lok, 2006).
 
While the mechanism of antiviral activity of nitazoxanide, tizoxanide, and the other thiazolides has not been fully elucidated, our findings are indicative of a mechanism that differs from direct-acting antiviral drugs and involves cellular processes
.
 
It is not at all apparent if the mechanisms of antiviral effectiveness and cytotoxicity involve similar cellular targets. The lack of an effect on levels of HBV RNA transcription indicates a post-transcriptional (possibly post-translational) mechanism. We propose that thiazolides may alter cellular processes required for virus protein production/maturation and/or assembly. Consistent with this hypothesis is the observation that the relative potencies of NTZ against secreted and intracellular HBV proteins are similar to that against secreted HBV virions and intracellular HBV RI, respectively. Given the substantial differences in the roles of these viral proteins, effects on more than one cell process are likely involved. Further studies are ongoing in our laboratories to more fully characterize the mechanism of activity of these drugs against HBV and HCV replication, and the potential for development of resistance.
 
The limited panel of thiazolides examined provides some initial clues into which components of TIZ are important for its anti-HBV and anti-HCV activities. The observation that the dual anti-hepatitis activity of TIZ can be separated into compounds that possess only anti-HBV or anti HCV activity has the potential to further elucidate the mechanism of action and identify the critical cellular targets involved. There will be a need to examine additional thiazolides for antiviral and cytotoxicity profiles.
 
Nitazoxanide, tizoxanide and other drugs from the thiazolide class are promising new antiviral agents that may enhance current or future therapies for viral hepatitis caused by HBV or HCV. NTZ is undergoing phase II clinical development for hepatitis C, and NTZ other thiazolides are also being developed for treating either hepatitis B or hepatitis C. The implications of a new class of antiviral drugs with the properties described herein could be significant from a clinical perspective.