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HCV Protease Inhibitors-BILN 2061: a major step toward new therapeutic strategies in hepatitis C
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Jnl of Hepatology
Volume 41, Issue 1, (July 2004)
Tarik Asselah and Patrick Marcellin
Note from Jules Levin: BILN-2061 clinical development has been stopped but the company developing it Boehringer Ingleheim is developing additional HCV drugs.
An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger G, Bonneau P, Bos M, Cameron DR, Cartier M, Cordingley MG, Faucher AM, Goudreau N, Kawai SH, Kukolj G, Lagace L, LaPlante SR, Narjes H, Poupart MA, Rancourt J, Sentjens RE, St George R, Simoneau B, Steinmann G, Thibeault D, Tsantrizos YS, Weldon SM, Yong CL, Llinas-Brunet M.Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.[Abstract reproduced by permission of Nature 2003; 426:186--189].
Introduction
The hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide, with approximately 170 million people infected. The severity of the disease varies widely from mild chronic hepatitis to cirrhosis and hepatocellular carcinoma. The reference treatment is the combination of pegylated interferon and ribavirin. Efficacy of treatment in clinical trials is approximately 80% in patients infected by HCV genotype 2 or 3, whereas in patients infected by HCV genotype 1 response to treatment is less than 50%. Furthermore, current treatment has significant side effects and is poorly tolerated. Therefore, new anti-HCV drugs are needed. In the last years, extensive research has been conducted to elucidate the structure of HCV enzymes in order to produce specific enzyme inhibitors.
NS3 serine protease
The HCV, identified in 1989, is an enveloped Flavivirus with a 9.6 kb single strand RNA genome. The human immunodeficiency virus also has an RNA genome, and discovery of protease inhibitors that reduce HIV replication, has permitted considerable progress in the treatment of HIV infection. The same methodology is used for HCV new drug development studies. The HCV RNA genome serves as a template for viral replication and as a viral messenger RNA for viral production. It is translated into a polyprotein which is cleaved by proteases. All of the HCV enzymes -NS2-3 and NS3-4A proteases, NS3 helicase, and NS5B RdRp- are essential for HCV replication, and are therefore potential drug discovery targets. Target based anti-HCV drugs in development are indicated in Table 1.
Table 1. Targed based anti-HCV drug in development
| Molecules | Mechanism of action | Development status | | | | | BILN 2061 | NS3/4A serine protease inhibitor | Phase 2 | | VX 950 NS3/4A | serine protease inhibitor | Preclinical | | JTK 003 NS5B | RdRP inhibitor | Phase 1/2 | | IRES ribozyme | Ribozyme | Phase 1/2 | | ISIS 14803 | Antisense oligonucleotide (IRES) | Phase 2 |
NS3 serine protease is located in the N-terminal region of NS3. The NS3 serine protease domain associates with the NS4A cofactor to cleave four specific sites. This enzyme has been extensively characterized at the biochemical level and its structure is known. The serine protease activity of NS3 is an attractive target for new drugs that could block viral replication efficiently.
Persistent infections with HCV are likely to depend on inhibition of host defences by the virus. It has been recently shown, that the HCV NS3/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3), a key cellular antiviral signalling molecule. Disruption of NS3/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3 phosphorylation after cellular challenge with an unrelated virus. Furthermore, dominant-negative or constitutively active IRF-3 mutants, respectively, enhanced or suppressed HCV RNA replication in hepatoma cells. Thus, the NS3/4A protease represents a dual therapeutic target, the inhibition of which may both block viral replication and restore IRF-3 control of HCV infection.
Replicon
The absence of cell culture model supporting full replication of HCV, and of convenient animal models, has limited the knowledge of HCV life cycle and the testing for antiviral molecules. The recent development of subgenomic HCV RNA replicons capable of replicating in the human hepatoma cell line, Huh 7, has been a significant advance. These models are improving, even though currently the level of replication is low and not all genotypes can replicate. In addition, virion assembly does not occur and particles release is not observed. However, this model (replicon) is currently the best so far for the study of HCV replication and the testing for antiviral molecules.
Efficiency of NS3 protease inhibitor in patients with chronic hepatitis C
Recently, Lamarre et al. reported the efficacy of BILN 2061, a NS3 protease inhibitor. This molecule, identified in a target-based antiviral drug discovery, has a highly specific and significant activity. The ability of this molecule to inhibit NS3 protease activity has been demonstrated in the subgenomic HCV replicon cell model. The polyprotein processing seems to be inhibited at all NS3-dependent cleavage sites (since the accumulation of NS3-NS5B precursor in the presence of BILN 2061 has been shown). Based on these data, the drug was selected for further evaluation in man.
BILN 2061 is biologically available through oral ingestion. The tolerability and pharmacokinetics of this drug after oral single doses (5 to 2400 mg) has been evaluated in healthy male subjects. The study evaluated increasing doses of the drug in groups of 8 men (6 active drug and 2 placebo) and no serious adverse events were observed. The maximum tolerated dose was 2000 mg since the higher dose was limited by minor intestinal adverse events. The medication was administered in a PEG ethanol solution and demonstrated pharmacokinetic evidence to support twice daily dosing of up to 200 mg.
Then, BILN 2061 was evaluated in two phase one studies in patients infected with HCV. In the first study, 31 patients with HCV genotype 1 infection and mild liver fibrosis were evaluated. Subjects were treated with placebo, 25, 200 or 500 mg of BILN 2061 given orally twice daily for two days with 10--14 days of follow-up after treatment. Among them, 15/31 (48%) were HCV treatment naive. The serum HCV RNA level decreased by greater than one (1.0) log10 in almost all patients: 7 of 9 at the 25 mg dose, and 8 of 8 patients at the 200 and 500 mg dose. No differences were noted in the responsiveness of treatment naive patients and those who had previously failed interferon based therapy. HCV RNA levels returned to baseline within 1--7 days after stopping the drug. Tolerability was good and no safety issues were identified among the 25 active drug recipients.
In the second study, safety, tolerability and antiviral effect of BILN 2061 were reported in patients with HCV genotype 1 and significant liver fibrosis (Ishak stage 3 and 4 but no cirrhosis). In a randomized, double-blind group comparison, 10 patients were treated with BILN 2061, 200 mg orally twice daily or placebo for 2 days (8 active drug and 2 placebo). Fifty percent of the patients were HCV treatment naive. All 8 patients treated with BILN 2061 demonstrated at least a 2log10 decrease in serum HCV RNA level at the end of the two day dosing period and 2 patients had a decrease of greater than 3log10. After stopping therapy, HCV RNA level returned to baseline in all subjects. No safety issues were identified.
Potential limitations of protease inhibitorsReturn to Article Outline
First, strong specificity of these inhibitors may be associated with limitation of efficiency in different genotypes. In 10 patients with chronic hepatitis C (9 of them naïve for treatment), 3 with genotype 2 and 7 with genotype 3, the antiviral activity of BILN 2061 was not uniform and less pronounced compared to the previous results reported in patients infected with genotype 1.
Secondly, since HCV has a high mutation replication rate, resistance is suspected. Recently, in vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor, has been reported. Moreover, in vitro resistance studies using a sub-genomic replicon system compared VX-950 (a potent NS34A protease inhibitor) to BILN 2061. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. Interestingly, the major BILN 2061 resistant mutations at Asp (168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. In the future, resistance to one drug might be sensitive to another one, and as in HIV, combination treatment (NS34A protease inhibitor and NS5 polymerase inhibitor for example) might be more efficient.
Conclusion
The article by Lamarre et al., describes very preliminary results and there are important potential limitations for the use of protease inhibitors in the treatment of chronic hepatitis C. Strong specificity of these inhibitors might be associated with limitation of efficiency in different genotypes. The administration of such drugs may induce viral resistance on a short or medium term. Furthermore, the absence of toxicity of long-term administration needs to be demonstrated. Indeed, concern regarding BILN 2061 has been raised recently with relevant side effects (cardiotoxicity) of high, supra-therapeutic doses in animals. Boehringer Ingelheim is currently studying the available pre-clinical data in order to decide on their impact on the clinical development of this substance. There are currently no trials ongoing with BILN 2061 and decisions about future trials will be made after thorough evaluation of toxicity findings in animal studies.
However, the results from Lamarre et al. constitute a major step in the field of HCV drug development since it is the first clinical evidence of an antiviral effect of an enzyme inhibitor (NS3/4A protease inhibitor) in patients with chronic hepatitis C. Indeed, this is the first example of an enzyme inhibitor identified in a target-based antiviral drug discovery with a highly specific and significant activity in the subgenomic HCV replicon cell model, and an antiviral effect confirmed in patients. The efficiency showed in human is a demonstration that NS34A protease is a good target for drug development and opens the way towards a new exciting field for therapy of chronic hepatitis C. Many other enzyme inhibitors, in particular protease inhibitors, have been produced and are currently in preclinical phase or in phase I clinical trials and more are coming. Hopefully, some of these drugs will demonstrate their efficacy and safety and will be good candidates for improving, by using them in combinations with or without interferon, the efficacy of treatment of patients with chronic hepatitis C.
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