.jpg)
Biotechs at EASL
from Jules Levin of NATAP: in addition to the broad HCV drug development programs at the major companies and they all have multi-drug programs, 2 biotechs reported new data in several posters, Anadys and Idenix. Here are links to Idenix & Anadys EASL reports, these companies are still looking for partners. Of interest is that it appears right now tht differnt types of NNRTI polymerase inhibitors can be combined in regimens since they bind at different spots so you could have a regimen conceivably with 2 NNRTIs and a nucleotide. Pharmasset reported in posters on 2 new nucleotides with favorable resistance profiles, so there are various types of combinations that could emerge. In addition there is Bioles who is developing Lecteron a new interferon administered every 2 weeks, at this time in early development it had less flu-like symtpoms and similr antiviral efficacy but it remains to be seen if it will be useful, look what happened to Albuferon.
EASL 45th Annual Meeting
(European Association for the Study of the Liver)
April 14-18, 2010
Vienna, Austria
Anadys Shares Climb on Updated Look at Hepatitis C Drug Trial
Luke Timmerman 4/15/10
Anadys Pharmaceuticals (NASDAQ: ANDS) is on the roller coaster again. The San Diego-based biotech company?s shares are up 17 percent this morning after it reported on another interim analysis of its hepatitis C clinical trial at a medical meeting in Europe.
Researchers found that 72 percent of patients who got Anadys? experimental drug (ANA598) and standard therapies had undetectable amounts of the virus in their bloodstream after eight weeks, compared with 38 percent who did that well on a placebo in addition to the standard treatments. The drug was considered well-tolerated, researchers said. The findings were presented today at the European Association for the Study of the Liver meeting in Vienna, Austria.
Anadys has been rolling out the data from this trial in bits and pieces. Today?s slice of the data represents how patients did on a 400 milligram dose, while an earlier analysis showed how patients did on a 200 milligram dose. Investors balked at the earlier analysis of the 200 milligram dose, after Anadys saw an unusually high early response rate among patients in the placebo group. The whole clinical trial is designed to enroll 90 patients who are randomly assigned to get the low dose of the Anadys drug, a high dose, or a placebo. The main goal of the study is to show the drug is wiping out the virus for 12 weeks, although patients will also be followed longer to see if they have undetectable amounts of virus in the blood a full 24 weeks after they complete therapy, which is known as a “sustained viral response,” or a clinical cure.
Anadys shares climbed 18 percent to $3.08 at 10:46 am Eastern time today on heavy trading volume. The stock fell 12 percent back on February 25, after the company reported an earlier analysis which showed the high response rate among placebo patients.
Luke Timmerman is the National Biotechnology Editor for Xconomy. You can e-mail him at ltimmerman@xconomy.com, call 206-624-2374, or follow him on Twitter at http://twitter.com/ldtimmerman.
Thursday, April 22, 2010
BMS NS5A HCV Inhibitor
BMS NS5A HCV Inhibitor
"Individuals infected with HCV are at considerable risk of developing liver cirrhosis, end-stage liver disease and hepatocellular carcinoma.....We designed a mechanistically unbiased approach based on chemical genetics to identify chemical starting points for interfering with HCV replication.....we screened over one million compounds from the Bristol-Myers Squibb proprietary collection in high throughput mode.....The results with BMS-790052 provide clinical validation for the first in a new class of HCV inhibitors that target a viral protein with no known enzymatic function and an as yet poorly understood role in viral replication16, 22. The strategy used to identify a lead HCV NS5A inhibitor and to optimize this molecule into a clinical candidate offers a contemporary demonstration of the effectiveness of an approach to drug discovery based on chemical genetics......In a randomized, double-blind, placebo-controlled, single ascending-dose study, BMS-790052 was administered to subjects with genotype 1 chronic HCV at doses of 1, 10 and 100 mg as an oral solution. All subjects were infected with HCV genotype 1a except for two subjects at 10 mg and three subjects at 100 mg who were infected with genotype 1b. BMS-790052 was safe and well tolerated in HCV-infected subjects after single oral doses up to 100 mg.....BMS-790052 had a mean plasma elimination half-life ranging from 10 to 14 h, and plasma drug levels were similar to those in non-HCV-infected subjects. After single oral doses of 10–100 mg BMS-790052, all subjects had 24-h plasma concentrations above the tenfold protein binding-adjusted EC90 for HCV genotypes 1a and 1b, suggesting the possibility for once daily administration.....A single milligram dose of BMS-790052 produced a mean 1.8 log10 reduction (range 0.2–3.0 log10) in HCV viral load measured 24 h after drug administration. Both the 10 and 100 mg doses produced a greater antiviral effect, with mean plasma viral RNA falling by 3.2 log10 (range 2.9–4.0 log10) and 3.3 log10 (range 2.7–3.6 log10), respectively, at 24 h post-dose. Moreover, the 100 mg dose resulted in a mean maximal HCV RNA decline of 3.6 log10 (range 3.0–4.1 log10) and a prolonged antiviral response was observed in two subjects infected with genotype 1b virus, with an HCV RNA measurement that reached the lower limit of quantification (less than 25 IU ml-1) in one subject"
BMS - Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect - pdf of full article attached
Letters to Nature
Nature , | doi:10.1038/nature08960; Received 29 October 2009; Accepted 26 February 2010; Published online 21 April 2010
Min Gao1, Richard E. Nettles2, Makonen Belema3, Lawrence B. Snyder3, Van N. Nguyen3, Robert A. Fridell1, Michael H. Serrano-Wu3, David R. Langley4, Jin-Hua Sun1, Donald R. O’Boyle II1, Julie A. Lemm1, Chunfu Wang1, Jay O. Knipe5, Caly Chien2, Richard J. Colonno1, Dennis M. Grasela2, Nicholas A. Meanwell3 & Lawrence G. Hamann3
1. Department of Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, USA
2. Department of Discovery Medicine and Clinical Pharmacology, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543, USA
3. Department of Discovery Chemistry,
4. Department of Computer-Aided Drug Design,
5. Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, USA
Correspondence to: Nicholas A. Meanwell3 Correspondence and requests for materials should be addressed to N.A.M. (Email: Nicholas.Meanwell@bms.com).
Abstract
The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people1. Current therapy relies upon a combination of pegylated interferon-α and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus2, 3. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B4. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC50) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log10 reduction in mean viral load measured 24 h post-dose that was sustained for an additional 120 h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144 h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.
We designed a mechanistically unbiased approach based on chemical genetics to identify chemical starting points for interfering with HCV replication. Our differentiating strategy centred on the identification of compounds functionally distinct from those acting on the traditional targets of antiviral research in this field, the NS3 protease and the NS5B RNA-dependent RNA polymerase5, 6, 7. Using a Con-1 genotype 1b replicon replicating in Huh-7 liver cells8, we screened over one million compounds from the Bristol-Myers Squibb proprietary collection in high throughput mode. We used a dual assay format that simultaneously evaluated replication of the related flavivirus bovine viral diarrhoea virus (BVDV) and host cell cytotoxicity as an efficient means of preliminarily eliminating compounds without specificity for HCV9. Active inhibitors were further triaged by evaluation in biochemical assays for NS3 protease, NS3 helicase and NS5B polymerase in addition to screening against a panel of unrelated viruses. The iminothiazolidinone BMS-858 (Fig. 1) emerged as a weak but, more importantly, specific inhibitor of HCV RNA replication (EC50 = 0.57 μM, half-maximum cytotoxic concentration (CC50) = >50 μM) for which resistance was mapped to a tyrosine to histidine substitution at residue 93 in the NS5A protein (Y93H or Y2065H in the HCV polyprotein)10. BMS-858 formed the basis of an extensive series of chemical refinements that focused on improving antiviral potency, broadening inhibitory activity to encompass the HCV 1a genotype, and optimizing for oral bioavailability and sustained pharmacokinetic properties. After defining symmetry as an important contributor to antiviral activity10, a discovery that preceded the disclosure of structural information (see below), we subsequently identified BMS-790052 (Fig. 1) as a development candidate for advancement into clinical trials11. (The studies leading to the identification of BMS-790052 and its preclinical profiling will be the subject of a series of separate publications.) This compound is the most potent HCV replication inhibitor reported so far, with mean EC50 values of 50 and 9 pM against genotype 1a and 1b replicons, respectively (data summarized in Table 1). BMS-790052 displays a therapeutic index (CC50/EC50) of at least 100,000 in vitro and is inactive towards a panel of 10 RNA and DNA viruses, with EC50 values greater than 10 μM, which confirms specificity for HCV (Supplementary Tables 1 and 2).
Picture 1.png
Upon further analysis, we determined that the inhibitory activity of BMS-790052 maps to the first 100 amino acids of HCV NS5A (see below). The antiviral activity of the compound towards additional genotypes was assessed by using replication-competent 1a12 or 1b replicons to construct hybrids in which the entire NS5A coding region or the first 100 amino acids of NS5A from genotypes 2a, 3a, 4a and 5a replaced the corresponding sequence of the parent replicon13. This approach has been validated using genotype-specific inhibitors of HCV NS5A10. As summarized in Table 1, BMS-790052 demonstrates potent inhibitory activity towards all genotypes tested, with EC50 values ranging from 9 to 146 pM. In combination studies, BMS-790052 displayed additive-to-synergistic effects with interferon-α/ribavirin, an inhibitor of NS3 protease (ITMN-191), and both nucleoside and allosteric inhibitors of NS5B polymerase, which is indicative of the potential of this molecule as a candidate for combination therapy with other HCV therapeutic agents (data summarized in Table 2) (Supplementary Tables 3–7). More significantly, BMS-790052 is a potent inhibitor of the JFH-1 genotype 2a infectious virus that replicates in cell culture (EC50 = 28 pM), an assay considered to be a more biologically relevant in vitro cell culture system14. In addition, BMS-790052 displayed similar potency in Huh-7, HeLa and HEK293T cells (Supplementary Table 8), demonstrating that the function(s) of NS5A inhibited by BMS-790052 is (are) highly conserved in different cellular environments.
To confirm that NS5A inhibitors bind to the viral protein, we prepared the biotin-tagged derivative 1, with the natural (S)-configuration at both proline stereocentres, and its diastereomer 2 (Fig. 1). Compound 1 inhibited subgenomic viral RNA replication in the Con-1 genotype 1b replicon with an EC50 of 33 nM but was inactive towards a Y93H replicon (EC50 > 10 μM) whereas the diastereomer 2, used as a control, was inactive in vitro (EC50 > 10 μM). After exposure to active inhibitor 1, the replicon lysate was passed over streptavidin agarose beads, separated by SDS–polyacrylamide gel electrophoresis and probed with an antibody specific for HCV NS5A, with a parallel experiment using the inactive diastereomer 2 serving as a control. NS5A could only be pulled down efficiently with active inhibitor 1 but not by the inactive compound 2 (Fig. 2); NS3 and NS5B were not detected in pull-down experiments with 1, suggesting selective binding to NS5A. In a similar experiment conducted with replicons expressing BVDV RNA, compound 1 failed to bind to BVDV NS5A.
Table 3: Resistance profile of genotype 1a and 1b replicons exposed to BMS-790052
Picture 2.png
HCV NS5A is a 447 amino-acid, zinc-binding phosphoprotein that plays a critically important but enigmatic role in the virus life cycle16. The substitutions conferring resistance to BMS-790052 map to domain I of the protein, which incorporates an amphipathic amino (N)-terminal α-helix (residues 5–25) thought to anchor the protein to the membrane16. The solid-state structure of fragments of domain I (residues 36–198 and 33–191) of HCV NS5A reveal dimeric complexes with patterns of interfacial recognition that anticipate an oligomeric form of the protein17, 18, postulated to sequester RNA within the replication complex19. The unique palindromic topology of BMS-790052 complements the dimeric structure of the NS5A protein. The location of the resistant substitutions suggests that the compound binds across the dimer interface, proximal to the N terminus of domain I between the protein and the membrane, and on the face opposite that of the putative RNA binding domain. We speculate that BMS-790052 may interfere with the precision of dimer association, effecting subtle structural distortions that directly or allosterically interfere with protein function. Under these circumstances, the exquisite inhibitory potency exhibited by BMS-790052 in vitro may be related to the disruption of the function of a limited number of NS5A dimers that compromise the activity of an oligomeric complex. However, although this hypothesis allows for an amplification of inhibitory effect, an alternative explanation relies on the inhibition of two or more activities of NS5A that interact synergistically. Further studies with BMS-790052 will be required to define its exact mode of action more fully and, conversely, the compound is a useful tool with which to study aspects of HCV NS5A function in viral replication. These investigations may be aided by compounds with an in vitro profile similar to 1, which is restricted to genotype 1b inhibition10.
BMS-790052 exhibited no significant effects in an extensive battery of in vitro receptor binding and enzymatic assays designed to assess promiscuity. Despite a molecular mass of over 700 daltons, the compound is orally bioavailable in four preclinical species, with plasma levels readily achieved that surpass the in vitro EC50, and it distributes effectively to the liver.
In a randomized, double-blind, placebo-controlled, single ascending-dose study, BMS-790052 was administered at six dose levels to healthy, non-HCV-infected subjects over a range of 1 to 200 mg as an oral solution. The compound was safe and well tolerated up to 200 mg with no clinically relevant adverse effects. After oral administration, BMS-790052 was readily absorbed, with dose-proportional exposures over the studied dose range, and all subjects had drug concentrations greater than the protein-binding-adjusted EC90 for genotypes 1a and 1b, as measured in the replicon assay, at and beyond 24 h post-dose (Fig. 3). (The protein binding-adjusted EC90 figures were derived from an analysis of the effect of the addition of human serum on antiviral activity in replicons. In the presence of 40% human serum, the EC90 for BMS-790052 is 383 pM (0.28 ng ml-1) for the genotype 1a replicon and 49 pM (0.04 ng ml-1) for the genotyope 1b replicon.)
In a randomized, double-blind, placebo-controlled, single ascending-dose study, BMS-790052 was administered to subjects with genotype 1 chronic HCV at doses of 1, 10 and 100 mg as an oral solution. All subjects were infected with HCV genotype 1a except for two subjects at 10 mg and three subjects at 100 mg who were infected with genotype 1b. BMS-790052 was safe and well tolerated in HCV-infected subjects after single oral doses up to 100 mg. Specifically, there were no deaths, serious adverse events, discontinuations due to adverse events or clinically relevant adverse effects. Headache was the most frequent adverse event, reported by four subjects after administration of BMS-790052. In HCV-infected subjects, BMS-790052 had a mean plasma elimination half-life ranging from 10 to 14 h, and plasma drug levels were similar to those in non-HCV-infected subjects. After single oral doses of 10–100 mg BMS-790052, all subjects had 24-h plasma concentrations above the tenfold protein binding-adjusted EC90 for HCV genotypes 1a and 1b, suggesting the possibility for once daily administration. The plasma HCV RNA levels were measured for up to 6 days after administration; the mean decline from the time of administration to 144 h post-dose is shown in Fig. 4. A single milligram dose of BMS-790052 produced a mean 1.8 log10 reduction (range 0.2–3.0 log10) in HCV viral load measured 24 h after drug administration. Both the 10 and 100 mg doses produced a greater antiviral effect, with mean plasma viral RNA falling by 3.2 log10 (range 2.9–4.0 log10) and 3.3 log10 (range 2.7–3.6 log10), respectively, at 24 h post-dose. Moreover, the 100 mg dose resulted in a mean maximal HCV RNA decline of 3.6 log10 (range 3.0–4.1 log10) and a prolonged antiviral response was observed in two subjects infected with genotype 1b virus, with an HCV RNA measurement that reached the lower limit of quantification (less than 25 IU ml-1) in one subject and 35 IU ml-1 in the other measured at hour 144. Genotypic analysis of samples taken at baseline (T0), 24 (T24) and 144 (T144) hours post-dose revealed that, in general, a marked reduction in viral load was required to detect major HCV variants. Substitutions were observed at amino-acid positions identified using the in vitro replicon system (Supplementary Tables 12–14): M28T, Q30H/R and L31M for genotype 1a, and Y93H for genotype 1b, results that suggest the usefulness of the replicon system for assessing resistance in response to inhibitor pressure in vivo. Follow-up samples were available for only one of these subjects, which revealed that HCV RNA had returned to near baseline; however, genotype analysis was not performed on this sample. As would be anticipated based on the in vitro replicon potency of BMS-790052, a greater and more sustained decline in HCV RNA was observed for subjects infected with genotype 1b (mean 3.6 log10 reduction (range 3.1–4.0 log10) and mean 3.1 log10 reduction (range 2.7–3.4 log10) in HCV viral load measured 24 h after a 10 and 100 mg dose, respectively) than for subjects infected with genotype 1a (mean 1.8 log10 reduction (range 0.2–3.0 log10), mean 2.9 log10 reduction (range 2.9–3.0 log10) and mean 3.6 log10 reduction (range 3.5–3.6 log10) in HCV viral load measured 24 h after a 1, 10 and 100 mg dose, respectively). The mean rates of decline for subjects who received 10 and 100 mg doses of BMS-790052 were similar up to 36 h after dosing, after which the mean decline was greater and more sustained in the subjects who received 100 mg. Subjects who received 1 mg of BMS-790052 had a lower mean decline in HCV RNA than subjects treated with 10 and 100 mg (Fig. 4). However, multiple-dose studies are needed to define the optimal dose range for maximal antiviral effect beyond the first phase of viral decline. The relationships between maximum decline from baseline in HCV RNA and drug pharmacokinetics exposures were explored using Pearson’s correlation coefficients. All estimated Pearson’s correlation coefficients were above 0.65, suggesting that the maximum declines in log10 HCV RNA and log pharmacokinetics exposures (BMS-790052 Cmax, AUC(0-T), AUC(INF), C12 and C24) were positively correlated; that is, that the maximum declines in log10 HCV RNA increase with the exposure to BMS-790052.
Individuals infected with HCV are at considerable risk of developing liver cirrhosis, end-stage liver disease and hepatocellular carcinoma20. The current standard of care for the treatment of HCV infection is a combination of weekly subcutaneous injections of pegylated interferon-α in conjunction with ribavirin, administered orally twice a day, for periods ranging from 24 to 72 weeks, depending on genotype2, 3. However, the side effects associated with this therapeutic regimen impose a significant physiological and psychological burden on the patient. Moreover, sustained virological response rates in those infected with genotype 1 are typically less than 50% (refs 2, 3). The direct-acting HCV antiviral drug candidates currently reported to be in late-stage clinical development are inhibitors of NS3 protease and NS5B polymerase, more traditional antiviral targets. Although these compounds are currently being developed as an add-on therapy to the standard of care, there is considerable anticipation for combinations of direct-acting agents, which will require at least two mechanistically distinct inhibitors to suppress the emergence of resistant virus4, 21. The preliminary in vitro and clinical profile of NS5A inhibitors described in this paper makes them a potentially valuable component of any interferon-free treatment regimen.
The results with BMS-790052 provide clinical validation for the first in a new class of HCV inhibitors that target a viral protein with no known enzymatic function and an as yet poorly understood role in viral replication16, 22. The strategy used to identify a lead HCV NS5A inhibitor and to optimize this molecule into a clinical candidate offers a contemporary demonstration of the effectiveness of an approach to drug discovery based on chemical genetics6. Indeed, this methodology is uniquely applicable to targets similar to HCV NS5A for which precise function is enigmatic and the development of biochemical assays could neither be anticipated nor are feasible. As a specific inhibitor of HCV, BMS-790052 exhibits a spectrum of genotype inhibition, in vitro potency, and efficacy and potency after single oral doses to chronically HCV-infected subjects that is collectively and individually unprecedented. Achieving high potency and selectivity against a non-mammalian target, the traditional goal of antiviral medicinal chemistry, has in the past translated into a wider therapeutic index in the clinic. Although preliminary, these data indicate that inhibitors of HCV NS5A offer considerable promise for the treatment of HCV infection. The in vitro data demonstrating additive to synergistic interactions with known HCV inhibitors suggest that combinations of BMS-790052, with either current standard of care or, ultimately, emerging inhibitors of HCV NS3 and NS5B as part of a cocktail of direct-acting antiviral agents, may lead to therapeutic regimens with better tolerability and improved clinical outcomes21.
"Individuals infected with HCV are at considerable risk of developing liver cirrhosis, end-stage liver disease and hepatocellular carcinoma.....We designed a mechanistically unbiased approach based on chemical genetics to identify chemical starting points for interfering with HCV replication.....we screened over one million compounds from the Bristol-Myers Squibb proprietary collection in high throughput mode.....The results with BMS-790052 provide clinical validation for the first in a new class of HCV inhibitors that target a viral protein with no known enzymatic function and an as yet poorly understood role in viral replication16, 22. The strategy used to identify a lead HCV NS5A inhibitor and to optimize this molecule into a clinical candidate offers a contemporary demonstration of the effectiveness of an approach to drug discovery based on chemical genetics......In a randomized, double-blind, placebo-controlled, single ascending-dose study, BMS-790052 was administered to subjects with genotype 1 chronic HCV at doses of 1, 10 and 100 mg as an oral solution. All subjects were infected with HCV genotype 1a except for two subjects at 10 mg and three subjects at 100 mg who were infected with genotype 1b. BMS-790052 was safe and well tolerated in HCV-infected subjects after single oral doses up to 100 mg.....BMS-790052 had a mean plasma elimination half-life ranging from 10 to 14 h, and plasma drug levels were similar to those in non-HCV-infected subjects. After single oral doses of 10–100 mg BMS-790052, all subjects had 24-h plasma concentrations above the tenfold protein binding-adjusted EC90 for HCV genotypes 1a and 1b, suggesting the possibility for once daily administration.....A single milligram dose of BMS-790052 produced a mean 1.8 log10 reduction (range 0.2–3.0 log10) in HCV viral load measured 24 h after drug administration. Both the 10 and 100 mg doses produced a greater antiviral effect, with mean plasma viral RNA falling by 3.2 log10 (range 2.9–4.0 log10) and 3.3 log10 (range 2.7–3.6 log10), respectively, at 24 h post-dose. Moreover, the 100 mg dose resulted in a mean maximal HCV RNA decline of 3.6 log10 (range 3.0–4.1 log10) and a prolonged antiviral response was observed in two subjects infected with genotype 1b virus, with an HCV RNA measurement that reached the lower limit of quantification (less than 25 IU ml-1) in one subject"
BMS - Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect - pdf of full article attached
Letters to Nature
Nature , | doi:10.1038/nature08960; Received 29 October 2009; Accepted 26 February 2010; Published online 21 April 2010
Min Gao1, Richard E. Nettles2, Makonen Belema3, Lawrence B. Snyder3, Van N. Nguyen3, Robert A. Fridell1, Michael H. Serrano-Wu3, David R. Langley4, Jin-Hua Sun1, Donald R. O’Boyle II1, Julie A. Lemm1, Chunfu Wang1, Jay O. Knipe5, Caly Chien2, Richard J. Colonno1, Dennis M. Grasela2, Nicholas A. Meanwell3 & Lawrence G. Hamann3
1. Department of Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, USA
2. Department of Discovery Medicine and Clinical Pharmacology, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543, USA
3. Department of Discovery Chemistry,
4. Department of Computer-Aided Drug Design,
5. Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, USA
Correspondence to: Nicholas A. Meanwell3 Correspondence and requests for materials should be addressed to N.A.M. (Email: Nicholas.Meanwell@bms.com).
Abstract
The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people1. Current therapy relies upon a combination of pegylated interferon-α and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus2, 3. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B4. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC50) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log10 reduction in mean viral load measured 24 h post-dose that was sustained for an additional 120 h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144 h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors.
We designed a mechanistically unbiased approach based on chemical genetics to identify chemical starting points for interfering with HCV replication. Our differentiating strategy centred on the identification of compounds functionally distinct from those acting on the traditional targets of antiviral research in this field, the NS3 protease and the NS5B RNA-dependent RNA polymerase5, 6, 7. Using a Con-1 genotype 1b replicon replicating in Huh-7 liver cells8, we screened over one million compounds from the Bristol-Myers Squibb proprietary collection in high throughput mode. We used a dual assay format that simultaneously evaluated replication of the related flavivirus bovine viral diarrhoea virus (BVDV) and host cell cytotoxicity as an efficient means of preliminarily eliminating compounds without specificity for HCV9. Active inhibitors were further triaged by evaluation in biochemical assays for NS3 protease, NS3 helicase and NS5B polymerase in addition to screening against a panel of unrelated viruses. The iminothiazolidinone BMS-858 (Fig. 1) emerged as a weak but, more importantly, specific inhibitor of HCV RNA replication (EC50 = 0.57 μM, half-maximum cytotoxic concentration (CC50) = >50 μM) for which resistance was mapped to a tyrosine to histidine substitution at residue 93 in the NS5A protein (Y93H or Y2065H in the HCV polyprotein)10. BMS-858 formed the basis of an extensive series of chemical refinements that focused on improving antiviral potency, broadening inhibitory activity to encompass the HCV 1a genotype, and optimizing for oral bioavailability and sustained pharmacokinetic properties. After defining symmetry as an important contributor to antiviral activity10, a discovery that preceded the disclosure of structural information (see below), we subsequently identified BMS-790052 (Fig. 1) as a development candidate for advancement into clinical trials11. (The studies leading to the identification of BMS-790052 and its preclinical profiling will be the subject of a series of separate publications.) This compound is the most potent HCV replication inhibitor reported so far, with mean EC50 values of 50 and 9 pM against genotype 1a and 1b replicons, respectively (data summarized in Table 1). BMS-790052 displays a therapeutic index (CC50/EC50) of at least 100,000 in vitro and is inactive towards a panel of 10 RNA and DNA viruses, with EC50 values greater than 10 μM, which confirms specificity for HCV (Supplementary Tables 1 and 2).
Picture 1.png
Upon further analysis, we determined that the inhibitory activity of BMS-790052 maps to the first 100 amino acids of HCV NS5A (see below). The antiviral activity of the compound towards additional genotypes was assessed by using replication-competent 1a12 or 1b replicons to construct hybrids in which the entire NS5A coding region or the first 100 amino acids of NS5A from genotypes 2a, 3a, 4a and 5a replaced the corresponding sequence of the parent replicon13. This approach has been validated using genotype-specific inhibitors of HCV NS5A10. As summarized in Table 1, BMS-790052 demonstrates potent inhibitory activity towards all genotypes tested, with EC50 values ranging from 9 to 146 pM. In combination studies, BMS-790052 displayed additive-to-synergistic effects with interferon-α/ribavirin, an inhibitor of NS3 protease (ITMN-191), and both nucleoside and allosteric inhibitors of NS5B polymerase, which is indicative of the potential of this molecule as a candidate for combination therapy with other HCV therapeutic agents (data summarized in Table 2) (Supplementary Tables 3–7). More significantly, BMS-790052 is a potent inhibitor of the JFH-1 genotype 2a infectious virus that replicates in cell culture (EC50 = 28 pM), an assay considered to be a more biologically relevant in vitro cell culture system14. In addition, BMS-790052 displayed similar potency in Huh-7, HeLa and HEK293T cells (Supplementary Table 8), demonstrating that the function(s) of NS5A inhibited by BMS-790052 is (are) highly conserved in different cellular environments.
To confirm that NS5A inhibitors bind to the viral protein, we prepared the biotin-tagged derivative 1, with the natural (S)-configuration at both proline stereocentres, and its diastereomer 2 (Fig. 1). Compound 1 inhibited subgenomic viral RNA replication in the Con-1 genotype 1b replicon with an EC50 of 33 nM but was inactive towards a Y93H replicon (EC50 > 10 μM) whereas the diastereomer 2, used as a control, was inactive in vitro (EC50 > 10 μM). After exposure to active inhibitor 1, the replicon lysate was passed over streptavidin agarose beads, separated by SDS–polyacrylamide gel electrophoresis and probed with an antibody specific for HCV NS5A, with a parallel experiment using the inactive diastereomer 2 serving as a control. NS5A could only be pulled down efficiently with active inhibitor 1 but not by the inactive compound 2 (Fig. 2); NS3 and NS5B were not detected in pull-down experiments with 1, suggesting selective binding to NS5A. In a similar experiment conducted with replicons expressing BVDV RNA, compound 1 failed to bind to BVDV NS5A.
Table 3: Resistance profile of genotype 1a and 1b replicons exposed to BMS-790052
Picture 2.png
HCV NS5A is a 447 amino-acid, zinc-binding phosphoprotein that plays a critically important but enigmatic role in the virus life cycle16. The substitutions conferring resistance to BMS-790052 map to domain I of the protein, which incorporates an amphipathic amino (N)-terminal α-helix (residues 5–25) thought to anchor the protein to the membrane16. The solid-state structure of fragments of domain I (residues 36–198 and 33–191) of HCV NS5A reveal dimeric complexes with patterns of interfacial recognition that anticipate an oligomeric form of the protein17, 18, postulated to sequester RNA within the replication complex19. The unique palindromic topology of BMS-790052 complements the dimeric structure of the NS5A protein. The location of the resistant substitutions suggests that the compound binds across the dimer interface, proximal to the N terminus of domain I between the protein and the membrane, and on the face opposite that of the putative RNA binding domain. We speculate that BMS-790052 may interfere with the precision of dimer association, effecting subtle structural distortions that directly or allosterically interfere with protein function. Under these circumstances, the exquisite inhibitory potency exhibited by BMS-790052 in vitro may be related to the disruption of the function of a limited number of NS5A dimers that compromise the activity of an oligomeric complex. However, although this hypothesis allows for an amplification of inhibitory effect, an alternative explanation relies on the inhibition of two or more activities of NS5A that interact synergistically. Further studies with BMS-790052 will be required to define its exact mode of action more fully and, conversely, the compound is a useful tool with which to study aspects of HCV NS5A function in viral replication. These investigations may be aided by compounds with an in vitro profile similar to 1, which is restricted to genotype 1b inhibition10.
BMS-790052 exhibited no significant effects in an extensive battery of in vitro receptor binding and enzymatic assays designed to assess promiscuity. Despite a molecular mass of over 700 daltons, the compound is orally bioavailable in four preclinical species, with plasma levels readily achieved that surpass the in vitro EC50, and it distributes effectively to the liver.
In a randomized, double-blind, placebo-controlled, single ascending-dose study, BMS-790052 was administered at six dose levels to healthy, non-HCV-infected subjects over a range of 1 to 200 mg as an oral solution. The compound was safe and well tolerated up to 200 mg with no clinically relevant adverse effects. After oral administration, BMS-790052 was readily absorbed, with dose-proportional exposures over the studied dose range, and all subjects had drug concentrations greater than the protein-binding-adjusted EC90 for genotypes 1a and 1b, as measured in the replicon assay, at and beyond 24 h post-dose (Fig. 3). (The protein binding-adjusted EC90 figures were derived from an analysis of the effect of the addition of human serum on antiviral activity in replicons. In the presence of 40% human serum, the EC90 for BMS-790052 is 383 pM (0.28 ng ml-1) for the genotype 1a replicon and 49 pM (0.04 ng ml-1) for the genotyope 1b replicon.)
In a randomized, double-blind, placebo-controlled, single ascending-dose study, BMS-790052 was administered to subjects with genotype 1 chronic HCV at doses of 1, 10 and 100 mg as an oral solution. All subjects were infected with HCV genotype 1a except for two subjects at 10 mg and three subjects at 100 mg who were infected with genotype 1b. BMS-790052 was safe and well tolerated in HCV-infected subjects after single oral doses up to 100 mg. Specifically, there were no deaths, serious adverse events, discontinuations due to adverse events or clinically relevant adverse effects. Headache was the most frequent adverse event, reported by four subjects after administration of BMS-790052. In HCV-infected subjects, BMS-790052 had a mean plasma elimination half-life ranging from 10 to 14 h, and plasma drug levels were similar to those in non-HCV-infected subjects. After single oral doses of 10–100 mg BMS-790052, all subjects had 24-h plasma concentrations above the tenfold protein binding-adjusted EC90 for HCV genotypes 1a and 1b, suggesting the possibility for once daily administration. The plasma HCV RNA levels were measured for up to 6 days after administration; the mean decline from the time of administration to 144 h post-dose is shown in Fig. 4. A single milligram dose of BMS-790052 produced a mean 1.8 log10 reduction (range 0.2–3.0 log10) in HCV viral load measured 24 h after drug administration. Both the 10 and 100 mg doses produced a greater antiviral effect, with mean plasma viral RNA falling by 3.2 log10 (range 2.9–4.0 log10) and 3.3 log10 (range 2.7–3.6 log10), respectively, at 24 h post-dose. Moreover, the 100 mg dose resulted in a mean maximal HCV RNA decline of 3.6 log10 (range 3.0–4.1 log10) and a prolonged antiviral response was observed in two subjects infected with genotype 1b virus, with an HCV RNA measurement that reached the lower limit of quantification (less than 25 IU ml-1) in one subject and 35 IU ml-1 in the other measured at hour 144. Genotypic analysis of samples taken at baseline (T0), 24 (T24) and 144 (T144) hours post-dose revealed that, in general, a marked reduction in viral load was required to detect major HCV variants. Substitutions were observed at amino-acid positions identified using the in vitro replicon system (Supplementary Tables 12–14): M28T, Q30H/R and L31M for genotype 1a, and Y93H for genotype 1b, results that suggest the usefulness of the replicon system for assessing resistance in response to inhibitor pressure in vivo. Follow-up samples were available for only one of these subjects, which revealed that HCV RNA had returned to near baseline; however, genotype analysis was not performed on this sample. As would be anticipated based on the in vitro replicon potency of BMS-790052, a greater and more sustained decline in HCV RNA was observed for subjects infected with genotype 1b (mean 3.6 log10 reduction (range 3.1–4.0 log10) and mean 3.1 log10 reduction (range 2.7–3.4 log10) in HCV viral load measured 24 h after a 10 and 100 mg dose, respectively) than for subjects infected with genotype 1a (mean 1.8 log10 reduction (range 0.2–3.0 log10), mean 2.9 log10 reduction (range 2.9–3.0 log10) and mean 3.6 log10 reduction (range 3.5–3.6 log10) in HCV viral load measured 24 h after a 1, 10 and 100 mg dose, respectively). The mean rates of decline for subjects who received 10 and 100 mg doses of BMS-790052 were similar up to 36 h after dosing, after which the mean decline was greater and more sustained in the subjects who received 100 mg. Subjects who received 1 mg of BMS-790052 had a lower mean decline in HCV RNA than subjects treated with 10 and 100 mg (Fig. 4). However, multiple-dose studies are needed to define the optimal dose range for maximal antiviral effect beyond the first phase of viral decline. The relationships between maximum decline from baseline in HCV RNA and drug pharmacokinetics exposures were explored using Pearson’s correlation coefficients. All estimated Pearson’s correlation coefficients were above 0.65, suggesting that the maximum declines in log10 HCV RNA and log pharmacokinetics exposures (BMS-790052 Cmax, AUC(0-T), AUC(INF), C12 and C24) were positively correlated; that is, that the maximum declines in log10 HCV RNA increase with the exposure to BMS-790052.
Individuals infected with HCV are at considerable risk of developing liver cirrhosis, end-stage liver disease and hepatocellular carcinoma20. The current standard of care for the treatment of HCV infection is a combination of weekly subcutaneous injections of pegylated interferon-α in conjunction with ribavirin, administered orally twice a day, for periods ranging from 24 to 72 weeks, depending on genotype2, 3. However, the side effects associated with this therapeutic regimen impose a significant physiological and psychological burden on the patient. Moreover, sustained virological response rates in those infected with genotype 1 are typically less than 50% (refs 2, 3). The direct-acting HCV antiviral drug candidates currently reported to be in late-stage clinical development are inhibitors of NS3 protease and NS5B polymerase, more traditional antiviral targets. Although these compounds are currently being developed as an add-on therapy to the standard of care, there is considerable anticipation for combinations of direct-acting agents, which will require at least two mechanistically distinct inhibitors to suppress the emergence of resistant virus4, 21. The preliminary in vitro and clinical profile of NS5A inhibitors described in this paper makes them a potentially valuable component of any interferon-free treatment regimen.
The results with BMS-790052 provide clinical validation for the first in a new class of HCV inhibitors that target a viral protein with no known enzymatic function and an as yet poorly understood role in viral replication16, 22. The strategy used to identify a lead HCV NS5A inhibitor and to optimize this molecule into a clinical candidate offers a contemporary demonstration of the effectiveness of an approach to drug discovery based on chemical genetics6. Indeed, this methodology is uniquely applicable to targets similar to HCV NS5A for which precise function is enigmatic and the development of biochemical assays could neither be anticipated nor are feasible. As a specific inhibitor of HCV, BMS-790052 exhibits a spectrum of genotype inhibition, in vitro potency, and efficacy and potency after single oral doses to chronically HCV-infected subjects that is collectively and individually unprecedented. Achieving high potency and selectivity against a non-mammalian target, the traditional goal of antiviral medicinal chemistry, has in the past translated into a wider therapeutic index in the clinic. Although preliminary, these data indicate that inhibitors of HCV NS5A offer considerable promise for the treatment of HCV infection. The in vitro data demonstrating additive to synergistic interactions with known HCV inhibitors suggest that combinations of BMS-790052, with either current standard of care or, ultimately, emerging inhibitors of HCV NS3 and NS5B as part of a cocktail of direct-acting antiviral agents, may lead to therapeutic regimens with better tolerability and improved clinical outcomes21.
Gilead stops hepatitis drug test after ‘adverse events’
Gilead stops hepatitis drug test after ‘adverse events’
Tuesday, April 20, 2010
Citing “significant laboratory abnormalities and adverse events” in patients, Gilead Sciences stopped a clinical test of a hepatitis C drug.
The Foster City company (NASDAQ: GILD) will review data from this Phase II test of GS 9450, a caspase inhibitor. The company has been studying it for treatment of hepatitis C and also for nonalcoholic steatohepatitis.
Gilead licensed the drug from LG Life Sciences Inc. in 2007, paying $20 million upfront for the rights to the drug except in Korea, China and India. The company also agreed to pay for a collaborative research program and to pay milestones of up to $182 million if the drug did well in development.
“Patient safety is Gilead’s top priority,” the business said in a press release.
Gilead didn’t describe the problems in detail.
Tuesday, April 20, 2010
Citing “significant laboratory abnormalities and adverse events” in patients, Gilead Sciences stopped a clinical test of a hepatitis C drug.
The Foster City company (NASDAQ: GILD) will review data from this Phase II test of GS 9450, a caspase inhibitor. The company has been studying it for treatment of hepatitis C and also for nonalcoholic steatohepatitis.
Gilead licensed the drug from LG Life Sciences Inc. in 2007, paying $20 million upfront for the rights to the drug except in Korea, China and India. The company also agreed to pay for a collaborative research program and to pay milestones of up to $182 million if the drug did well in development.
“Patient safety is Gilead’s top priority,” the business said in a press release.
Gilead didn’t describe the problems in detail.
EARLY ON-TREATMENT RESPONSES DURING PEGYLATED INTERFERON PLUS RIBAVIRIN ARE INCREASED FOLLOWING 13 DAYS OF COMBINATION NUCLEOSIDE POLYMERASE
EARLY ON-TREATMENT RESPONSES DURING PEGYLATED INTERFERON PLUS RIBAVIRIN ARE INCREASED FOLLOWING 13 DAYS OF COMBINATION NUCLEOSIDE POLYMERASE (RG7128) AND PROTEASE (RG7227) INHIBITOR THERAPY (INFORM-1) - see attached full poster report of this interesting study
Reported by Jules Levin, EASL Apr 14-18 2010 Vienna Austria
“the profound viral suppression achieved with combination DAA therapy may reduce the high levels of interferon gamma–inducible protein (IP-10 or CXCL10) associated with chronic HCV infection, thereby reducing endogenous interferon activation levels.3 Thus, potent DAA combination regimens have the potential to not only block viral replication by arresting HCV RNA replication, but also to reconstitute innate host immune responses, thereby increasing the potential of long-term cure with finite DAA therapy…….These encouraging results suggest that, when given at optimal doses, a short course of dual combination therapy does enhance on-treatment response rates.”
E. Gane1, S. Roberts2, C. Stedman3, P. Angus4, B. Ritchie5, R. Elston6, D. Ipe6, P. Morcos6, L. Baher6, I. Najera6, T. Chu6, M. Mannino6, M. Berry7, W. Bradford8, M. Laughlin6, N. Shulman6, P. Smith6
1Auckland Clinical Studies, Auckland, New Zealand; 2Gastroenterology, The Alfred Hospital, Melbourne, VIC, Australia; 3Christchurch Clinical Studies, Christchurch, New Zealand; 4Gastroenterology, Austin Hospital, Melbourne, VIC, Australia; 5Gastroenterology, Royal Adelaide Hospital, Adelaide, SA, Australia; 6Roche Palo Alto LLC, Palo Alto, CA, USA; 7Pharmasset Inc., Princeton, NJ, USA; 8InterMune, Inc., Brisbane, CA, USA
Conclusions
The interferon-free dual oral combination regimen of RG7128/RG7227 produced rapid and profound suppression of HCV RNA replication through 13 days of dosing. These initial reductions in circulating HCV RNA prior to initiating treatment with peginterferon alfa-2a (40KD) plus ribavirin enhanced on-treatment virological responses at week 4 and 12 of treatment and end-of-treatment responses.
The 13-day combination regimen contributed to higher end of treatment virological response rates in groups B, C and D compared with the shorter and lower dose combination regimen in group A. The highest end-of-treatment response rate in treatment-naive individuals (100%) was achieved in patients treated with the highest dosage combination of RG7128/RG7227 (1000/900 mg bid). All patients achieved an RVR at week 4 of treatment with the SOC and were assigned to an abbreviated 24-week regimen. These encouraging results suggest that, when given at optimal doses, a short course of dual combination therapy does enhance on-treatment response rates.
Final SVR-24 results in all cohorts are awaited with great interest. Longer-term follow-up for SVR and larger studies will be needed to assess the clinical merits of combination DAA induction therapy prior to treatment with the SOC. However, such an approach is unlikely to benefit patients with contraindications to interferon, those intolerant of interferon, and individuals who have not responded to previous treatment with the SOC. In these populations, longer duration interferon-free combination DAA studies are planned.
Reported by Jules Levin, EASL Apr 14-18 2010 Vienna Austria
“the profound viral suppression achieved with combination DAA therapy may reduce the high levels of interferon gamma–inducible protein (IP-10 or CXCL10) associated with chronic HCV infection, thereby reducing endogenous interferon activation levels.3 Thus, potent DAA combination regimens have the potential to not only block viral replication by arresting HCV RNA replication, but also to reconstitute innate host immune responses, thereby increasing the potential of long-term cure with finite DAA therapy…….These encouraging results suggest that, when given at optimal doses, a short course of dual combination therapy does enhance on-treatment response rates.”
E. Gane1, S. Roberts2, C. Stedman3, P. Angus4, B. Ritchie5, R. Elston6, D. Ipe6, P. Morcos6, L. Baher6, I. Najera6, T. Chu6, M. Mannino6, M. Berry7, W. Bradford8, M. Laughlin6, N. Shulman6, P. Smith6
1Auckland Clinical Studies, Auckland, New Zealand; 2Gastroenterology, The Alfred Hospital, Melbourne, VIC, Australia; 3Christchurch Clinical Studies, Christchurch, New Zealand; 4Gastroenterology, Austin Hospital, Melbourne, VIC, Australia; 5Gastroenterology, Royal Adelaide Hospital, Adelaide, SA, Australia; 6Roche Palo Alto LLC, Palo Alto, CA, USA; 7Pharmasset Inc., Princeton, NJ, USA; 8InterMune, Inc., Brisbane, CA, USA
Conclusions
The interferon-free dual oral combination regimen of RG7128/RG7227 produced rapid and profound suppression of HCV RNA replication through 13 days of dosing. These initial reductions in circulating HCV RNA prior to initiating treatment with peginterferon alfa-2a (40KD) plus ribavirin enhanced on-treatment virological responses at week 4 and 12 of treatment and end-of-treatment responses.
The 13-day combination regimen contributed to higher end of treatment virological response rates in groups B, C and D compared with the shorter and lower dose combination regimen in group A. The highest end-of-treatment response rate in treatment-naive individuals (100%) was achieved in patients treated with the highest dosage combination of RG7128/RG7227 (1000/900 mg bid). All patients achieved an RVR at week 4 of treatment with the SOC and were assigned to an abbreviated 24-week regimen. These encouraging results suggest that, when given at optimal doses, a short course of dual combination therapy does enhance on-treatment response rates.
Final SVR-24 results in all cohorts are awaited with great interest. Longer-term follow-up for SVR and larger studies will be needed to assess the clinical merits of combination DAA induction therapy prior to treatment with the SOC. However, such an approach is unlikely to benefit patients with contraindications to interferon, those intolerant of interferon, and individuals who have not responded to previous treatment with the SOC. In these populations, longer duration interferon-free combination DAA studies are planned.
Human Genome's partner pulls EU hep C drug application
Human Genome's partner pulls EU hep C drug application
Mon Apr 19, 2010 11:51am EDT
"Recall in Phase III trials Joulferon demonstrated non-inferior efficacy, but higher rates of discontinuations and unique side effects,"
* Chronic hep C treatment being developed with Novartis
- Application withdrawn due to EU regulatory feedback
* Focus on lupus drug Benlysta -- analysts
* Shares down more than 1 pct (Adds analyst comment, details, updates share movement)
April 19 (Reuters) - Human Genome Sciences Inc said its partner Novartis AG withdrew the European marketing application for its chronic hepatitis C drug, after the European regulator indicated that it may seek additional new data.
The feedback from the European Medicines Agency included whether the therapeutic benefit offered by the drug, Joulferon, dosed once every two weeks was sufficient relative to risk, the company said in a statement.
"With today's news...we think it's reasonable to wonder if Food and Drug Administration delays might follow," Christopher Raymond, an analyst at Robert W. Baird, said in a research note.
Human Genome is awaiting a U.S. FDA approval decision later this year for the same treatment, known in the United States as Zalbin, that would compete with Roche Holding AG's big-selling Pegasys and Merck & Co's Inc Pegintron.
Joulferon would be the brand name outside the United States.
However, analysts said Monday's news was not a surprise and the focus remained on Human Genome's eagerly anticipated experimental drug Benlysta, which could become the first new lupus drug approved in half a century.
"Recall in Phase III trials Joulferon demonstrated non-inferior efficacy, but higher rates of discontinuations and unique side effects," said Piper Jaffray analyst Ian Somaiya.
RBC Capital Markets analyst Michael Yee said nobody really expects FDA approval for Zalbin, and if the drug was not approved, Human Genome would not have to invest cash and resources in the product.
Piper Jaffray's Somaiya said he believed a once monthly dosing of Joulferon would provide a more compelling benefit risk profile.
Shares of Human Genome were down 1 percent in late morning trade on Nasdaq. They earlier touched a low of $31.95. (Reporting by Shailesh Kuber in Bangalore; Editing by Aradhana Aravindan)
-------------
Human Genome Sciences Announces Withdrawal of European Marketing Authorization Application For JOULFERON(R) (ZALBIN(TM)) For the Treatment of Chronic Hepatitis C
ROCKVILLE, Md., Apr 19, 2010 (BUSINESS WIRE) -- Human Genome Sciences, Inc. today announced that Novartis has withdrawn a Marketing Authorization Application (MAA) to the European Medicines Agency (EMA) for approval to market JOULFERON(R) (albinterferon alfa-2b, known in the United States as ZALBIN(TM)) for the treatment of chronic hepatitis C.
The decision to withdraw the application was based on feedback from European regulatory authorities in preliminary response to the EMA application, indicating that additional new data would be requested which could not reasonably be generated within the timeframe allowed in the European Centralized Procedure. Feedback included whether the therapeutic benefit offered by JOULFERON dosed once every two weeks is sufficient relative to risk.
ZALBIN (JOULFERON) is being developed by HGS and Novartis under an exclusive worldwide co-development and commercialization agreement entered into in 2006. In November 2009, HGS submitted a Biologics License Application (BLA) to the FDA in the United States for ZALBIN dosed every two weeks, which continues under review. HGS and Novartis are also developing ZALBIN dosed every four weeks, and HGS previously reported the positive interim results of a Phase 2b study of this ZALBIN regimen.
About ZALBIN (albinterferon alfa-2b)
ZALBIN (also known as JOULFERON) is a genetic fusion of human albumin and interferon alfa created using proprietary HGS albumin-fusion technology. Human albumin is the most prevalent naturally occurring blood protein in the human circulatory system, persisting in circulation in the body for approximately 19 days. Research has shown that genetic fusion of therapeutic proteins to human albumin decreases clearance and prolongs the half-life of the therapeutic proteins.
Mon Apr 19, 2010 11:51am EDT
"Recall in Phase III trials Joulferon demonstrated non-inferior efficacy, but higher rates of discontinuations and unique side effects,"
* Chronic hep C treatment being developed with Novartis
- Application withdrawn due to EU regulatory feedback
* Focus on lupus drug Benlysta -- analysts
* Shares down more than 1 pct (Adds analyst comment, details, updates share movement)
April 19 (Reuters) - Human Genome Sciences Inc said its partner Novartis AG withdrew the European marketing application for its chronic hepatitis C drug, after the European regulator indicated that it may seek additional new data.
The feedback from the European Medicines Agency included whether the therapeutic benefit offered by the drug, Joulferon, dosed once every two weeks was sufficient relative to risk, the company said in a statement.
"With today's news...we think it's reasonable to wonder if Food and Drug Administration delays might follow," Christopher Raymond, an analyst at Robert W. Baird, said in a research note.
Human Genome is awaiting a U.S. FDA approval decision later this year for the same treatment, known in the United States as Zalbin, that would compete with Roche Holding AG's big-selling Pegasys and Merck & Co's Inc Pegintron.
Joulferon would be the brand name outside the United States.
However, analysts said Monday's news was not a surprise and the focus remained on Human Genome's eagerly anticipated experimental drug Benlysta, which could become the first new lupus drug approved in half a century.
"Recall in Phase III trials Joulferon demonstrated non-inferior efficacy, but higher rates of discontinuations and unique side effects," said Piper Jaffray analyst Ian Somaiya.
RBC Capital Markets analyst Michael Yee said nobody really expects FDA approval for Zalbin, and if the drug was not approved, Human Genome would not have to invest cash and resources in the product.
Piper Jaffray's Somaiya said he believed a once monthly dosing of Joulferon would provide a more compelling benefit risk profile.
Shares of Human Genome were down 1 percent in late morning trade on Nasdaq. They earlier touched a low of $31.95. (Reporting by Shailesh Kuber in Bangalore; Editing by Aradhana Aravindan)
-------------
Human Genome Sciences Announces Withdrawal of European Marketing Authorization Application For JOULFERON(R) (ZALBIN(TM)) For the Treatment of Chronic Hepatitis C
ROCKVILLE, Md., Apr 19, 2010 (BUSINESS WIRE) -- Human Genome Sciences, Inc. today announced that Novartis has withdrawn a Marketing Authorization Application (MAA) to the European Medicines Agency (EMA) for approval to market JOULFERON(R) (albinterferon alfa-2b, known in the United States as ZALBIN(TM)) for the treatment of chronic hepatitis C.
The decision to withdraw the application was based on feedback from European regulatory authorities in preliminary response to the EMA application, indicating that additional new data would be requested which could not reasonably be generated within the timeframe allowed in the European Centralized Procedure. Feedback included whether the therapeutic benefit offered by JOULFERON dosed once every two weeks is sufficient relative to risk.
ZALBIN (JOULFERON) is being developed by HGS and Novartis under an exclusive worldwide co-development and commercialization agreement entered into in 2006. In November 2009, HGS submitted a Biologics License Application (BLA) to the FDA in the United States for ZALBIN dosed every two weeks, which continues under review. HGS and Novartis are also developing ZALBIN dosed every four weeks, and HGS previously reported the positive interim results of a Phase 2b study of this ZALBIN regimen.
About ZALBIN (albinterferon alfa-2b)
ZALBIN (also known as JOULFERON) is a genetic fusion of human albumin and interferon alfa created using proprietary HGS albumin-fusion technology. Human albumin is the most prevalent naturally occurring blood protein in the human circulatory system, persisting in circulation in the body for approximately 19 days. Research has shown that genetic fusion of therapeutic proteins to human albumin decreases clearance and prolongs the half-life of the therapeutic proteins.
Idenix Pharmaceuticals Reports Positive Results With IDX184 nucleoside polymerase
press announcement
Idenix Pharmaceuticals Reports Positive Results With IDX184 nucleoside polymerase From Interim Analysis of Phase IIa Hepatitis C Study
-- IDX184 50 and 100 mg cohorts in combination with pegylated interferon and ribavirin (PegIFN/RBV) demonstrated a favorable safety profile and potent antiviral activity at 14 days
-- Fifty percent of subjects receiving a total daily dose of 100 mg IDX184 achieved undetectable virus levels by Day 14
-- Study continuing with enrollment of 150 mg cohort
CAMBRIDGE, Mass., April 15, 2010 /PRNewswire via COMTEX/ --Idenix Pharmaceuticals, Inc. (Nasdaq: IDIX), a biopharmaceutical company engaged in the discovery and development of drugs for the treatment of human viral diseases, today announced interim data from a 14-day, phase IIa clinical trial evaluating IDX184, a novel liver-targeted nucleotide prodrug of 2'-methyl guanosine monophosphate, in HCV-infected patients. Data are being presented at the 45th annual meeting of the European Association for the Study of the Liver being held in Vienna, Austria.
The phase IIa clinical trial, initiated in the fourth quarter of 2009, is a randomized, double-blind, placebo-controlled, sequential dose-escalation study evaluating the safety, tolerability, pharmacokinetics and antiviral activity of IDX184 in combination with PegIFN/RBV in treatment-naive HCV genotype 1-infected patients. Patients receive a daily dose of IDX184 or placebo, plus pegIFN/RBV for 14 days and then continue on PegIFN/RBV for an additional 14 days. The study is evaluating four dosing regimens of IDX184 ranging from 50 to 200 mg per day. In the 100 mg and 200 mg cohorts, once daily (QD) and twice daily (BID) regimens are compared. Each cohort includes 20 patients randomized 4:1, IDX184:placebo.
In the data presented today, IDX184 demonstrated potent dose-dependent antiviral activity when combined with PegIFN/RBV. At Day 14, mean (+/- standard deviation) viral load reductions were 1.2 (+/- 1.1), 2.7 (+/- 1.3), 4.0 (+/- 1.7) and 4.2 (+/- 1.9) log(10) IU/mL in the placebo (n=8), 50 mg IDX184 QD (n=16), 50 mg IDX184 BID (n=8) and 100 mg IDX184 QD (n=8) cohorts, respectively. Half of the subjects receiving a total daily dose of 100 mg IDX184 achieved undetectable virus levels (< 15 IU/mL) by Day 14. Liver injury parameters (ALT and AST) improved (normalized) with all doses of IDX184. Antiviral activity and safety parameters measured after a total daily dose of 100 mg IDX184 did not differ between a QD or BID dosing regimen. The side effect profile of the three-drug combination was consistent with the known side effect profile of PegIFN/RBV alone. The most common adverse events reported were fatigue, myalgia, headache and nausea. No virologic breakthrough was observed during treatment with IDX184 in combination with PegIFN/RBV.
"We are very encouraged by these interim data for IDX184 combined with pegylated interferon and ribavirin as the viral load reductions for the 100 mg cohorts are on par with the first-generation nucleoside in development but at a fraction of the dose," said Douglas Mayers, M.D., Idenix's executive vice president and chief medical officer. "We are currently enrolling the 150 mg once-daily dose cohort and look forward to reporting full data later this year. We believe that with the favorable antiviral activity, safety and resistance profile seen to date, IDX184 could be a potential component of future direct-acting antiviral combination regimens."
"With a high barrier to resistance and broad genotypic coverage, nucleosides and/or nucleotides could become an important part of future combination therapy for hepatitis C patients," said Dr. Fred Poordad, a principal investigator in the study, Chief of Hepatology at the Liver Disease and Transplant Center at Cedars-Sinai Medical Center in Los Angeles. "The interim 50 and 100 mg cohort data for IDX184 in combination with pegylated interferon and ribavirin have been promising and we look forward to the continued clinical development of this drug candidate."
About HCV
Hepatitis C virus is a common blood-borne pathogen infecting three to four million people worldwide annually. Currently, an estimated 170 million people are infected worldwide, representing a nearly 5-fold greater prevalence than human immunodeficiency virus.(1)
About IDX184
IDX184 is a novel, liver-targeted nucleotide prodrug of 2'-methyl guanosine, which includes Idenix's proprietary liver-targeting technology. This technology enables the delivery of nucleoside monophosphate to the liver, leading to the formation of high levels of nucleoside triphosphate, potentially maximizing drug efficacy and limiting systemic side effects with low, once-daily dosing.
About Idenix
Idenix Pharmaceuticals, Inc., headquartered in Cambridge, Massachusetts, is a biopharmaceutical company engaged in the discovery and development of drugs for the treatment of human viral diseases. Idenix's current focus is on the treatment of infections caused by hepatitis C virus. For further information about Idenix, please refer to www.idenix.com.
Idenix Pharmaceuticals Reports Positive Results With IDX184 nucleoside polymerase From Interim Analysis of Phase IIa Hepatitis C Study
-- IDX184 50 and 100 mg cohorts in combination with pegylated interferon and ribavirin (PegIFN/RBV) demonstrated a favorable safety profile and potent antiviral activity at 14 days
-- Fifty percent of subjects receiving a total daily dose of 100 mg IDX184 achieved undetectable virus levels by Day 14
-- Study continuing with enrollment of 150 mg cohort
CAMBRIDGE, Mass., April 15, 2010 /PRNewswire via COMTEX/ --Idenix Pharmaceuticals, Inc. (Nasdaq: IDIX), a biopharmaceutical company engaged in the discovery and development of drugs for the treatment of human viral diseases, today announced interim data from a 14-day, phase IIa clinical trial evaluating IDX184, a novel liver-targeted nucleotide prodrug of 2'-methyl guanosine monophosphate, in HCV-infected patients. Data are being presented at the 45th annual meeting of the European Association for the Study of the Liver being held in Vienna, Austria.
The phase IIa clinical trial, initiated in the fourth quarter of 2009, is a randomized, double-blind, placebo-controlled, sequential dose-escalation study evaluating the safety, tolerability, pharmacokinetics and antiviral activity of IDX184 in combination with PegIFN/RBV in treatment-naive HCV genotype 1-infected patients. Patients receive a daily dose of IDX184 or placebo, plus pegIFN/RBV for 14 days and then continue on PegIFN/RBV for an additional 14 days. The study is evaluating four dosing regimens of IDX184 ranging from 50 to 200 mg per day. In the 100 mg and 200 mg cohorts, once daily (QD) and twice daily (BID) regimens are compared. Each cohort includes 20 patients randomized 4:1, IDX184:placebo.
In the data presented today, IDX184 demonstrated potent dose-dependent antiviral activity when combined with PegIFN/RBV. At Day 14, mean (+/- standard deviation) viral load reductions were 1.2 (+/- 1.1), 2.7 (+/- 1.3), 4.0 (+/- 1.7) and 4.2 (+/- 1.9) log(10) IU/mL in the placebo (n=8), 50 mg IDX184 QD (n=16), 50 mg IDX184 BID (n=8) and 100 mg IDX184 QD (n=8) cohorts, respectively. Half of the subjects receiving a total daily dose of 100 mg IDX184 achieved undetectable virus levels (< 15 IU/mL) by Day 14. Liver injury parameters (ALT and AST) improved (normalized) with all doses of IDX184. Antiviral activity and safety parameters measured after a total daily dose of 100 mg IDX184 did not differ between a QD or BID dosing regimen. The side effect profile of the three-drug combination was consistent with the known side effect profile of PegIFN/RBV alone. The most common adverse events reported were fatigue, myalgia, headache and nausea. No virologic breakthrough was observed during treatment with IDX184 in combination with PegIFN/RBV.
"We are very encouraged by these interim data for IDX184 combined with pegylated interferon and ribavirin as the viral load reductions for the 100 mg cohorts are on par with the first-generation nucleoside in development but at a fraction of the dose," said Douglas Mayers, M.D., Idenix's executive vice president and chief medical officer. "We are currently enrolling the 150 mg once-daily dose cohort and look forward to reporting full data later this year. We believe that with the favorable antiviral activity, safety and resistance profile seen to date, IDX184 could be a potential component of future direct-acting antiviral combination regimens."
"With a high barrier to resistance and broad genotypic coverage, nucleosides and/or nucleotides could become an important part of future combination therapy for hepatitis C patients," said Dr. Fred Poordad, a principal investigator in the study, Chief of Hepatology at the Liver Disease and Transplant Center at Cedars-Sinai Medical Center in Los Angeles. "The interim 50 and 100 mg cohort data for IDX184 in combination with pegylated interferon and ribavirin have been promising and we look forward to the continued clinical development of this drug candidate."
About HCV
Hepatitis C virus is a common blood-borne pathogen infecting three to four million people worldwide annually. Currently, an estimated 170 million people are infected worldwide, representing a nearly 5-fold greater prevalence than human immunodeficiency virus.(1)
About IDX184
IDX184 is a novel, liver-targeted nucleotide prodrug of 2'-methyl guanosine, which includes Idenix's proprietary liver-targeting technology. This technology enables the delivery of nucleoside monophosphate to the liver, leading to the formation of high levels of nucleoside triphosphate, potentially maximizing drug efficacy and limiting systemic side effects with low, once-daily dosing.
About Idenix
Idenix Pharmaceuticals, Inc., headquartered in Cambridge, Massachusetts, is a biopharmaceutical company engaged in the discovery and development of drugs for the treatment of human viral diseases. Idenix's current focus is on the treatment of infections caused by hepatitis C virus. For further information about Idenix, please refer to www.idenix.com.
‘Warehoused’ Hepatitis C Patients May Boost Merck, J&J, Vertex
‘Warehoused’ Hepatitis C Patients May Boost Merck, J&J, Vertex
April 18, 2010, 8:37 PM EDT
April 19 (Bloomberg) -- At Fred Poordad’s bustling hepatitis C clinic in the heart of Los Angeles, one in every five patients receives no treatment. They are waiting for a wave of new drugs, expected in the next 18 months, that may boost their chance at a cure by as much as 10-fold.
The medicines also may bolster the prospects of Merck & Co., Vertex Pharmaceuticals Inc. and Johnson & Johnson, the companies in a race to get the first new treatment to the market in a decade. About half of patients can’t tolerate the side effects of existing therapies, which generate $2 billion annually in sales. The new drugs could expand the market to $10 billion in five years, said Geoff Porges, an analyst for Sanford C. Bernstein & Co. in New York.
They’re just the first among new therapies anticipated in the next five years as companies seek a single pill to cure the infection. Poordad, chief of hepatology at the Liver Disease and Transplant Center at Cedars-Sinai Medical Center in Los Angeles, doesn’t object when his patients elect to wait for the new drugs, a practice known as “warehousing.”
“The warehousing has been going on for the past year or so,” Poordad said. “I think we’ll see a tremendous increase in the volume of patients that are treated. That’s the most exciting thing in the field for a long time.”
The drugs closest to market, Merck’s boceprevir and telaprevir from Vertex and Johnson & Johnson, are protease inhibitors crafted from the technologies that led to discoveries made in the fight against HIV. The new treatments are being tested as additions to current standard treatments. Both drugs work by blocking the action of the protease enzyme the hepatitis virus needs to replicate, directly stopping it from spreading.
Available by 2011
Merck, of Whitehouse Station, New Jersey; Vertex, based in Cambridge, Massachusetts; and its partner Johnson & Johnson, of New Brunswick, New Jersey, have said they expect results from final-stage clinical trials by the second half of 2010, with submission to U.S. regulators by year-end. Patients can expect the drugs to be available by 2011, executives said on April 16 at the annual meeting of the European Society for the Study of the Liver in Vienna.
“We’re on the brink of a revolution,” Porges said in an interview at the conference. “Investors have been waiting for this for six to seven years, and investigators and physicians have been waiting for almost 10 years.” He has an “outperform” recommendation on Vertex and sees the stock gaining 23 percent in the next year.
Vertex shares rose 0.8 percent to $39.99 in Nasdaq Stock Market trading on April 16. The stock has gained 43 percent in the last year.
Scarring Livers
Most of the 170 million people infected worldwide with hepatitis C don’t know they’re sick, while others fail to respond to existing drugs or can’t tolerate their side effects. The virus is spread by contact with infected blood. About 4 million Europeans and 3.6 million Americans have the disease, and only about 2 percent get treatment, according to Merck.
The illness moves slowly, scarring livers over years or decades, giving doctors and patients a window of time. Once the liver is destroyed, however, patients face cancer or organ failure, with few effective treatments other than a transplant.
The current standard of care is a combination of the generic antiviral pill ribavirin and interferon, an injection sold by Merck as Pegintron and Swiss drugmaker Roche Holding AG as Pegasys. The medications beef up the immune system, helping it clear the virus from the patient’s blood. They can also trigger anemia or make patients feel like they have a never- ending flu. The new drugs don’t erase those side effects but can cut treatment time in half, from a year to about six months.
Rush for Treatment
The protease inhibitors may have the biggest impact in those who have failed prior therapy. Five to 9 percent of them are cured by a second course of treatment with existing drugs. Adding a protease inhibitor appears to boost that number 10- fold, said Poordad, who has participated in studies funded by Merck, Vertex and Johnson & Johnson.
The first two or three new drugs will take the lion’s share of the growing market, Porges said, as warehoused patients rush to get treatment and doctors establish a new standard of care.
Vertex and Johnson & Johnson hold an advantage right now, Porges said, because their drug telaprevir doesn’t cause the same levels of anemia seen as a side effect of its competitor from Merck. Vertex also allowed patients who didn’t respond at all to other therapies into its clinical trials, possibly positioning its drug, which has been in development for at least 12 years, for broader use.
“We think that’s going to be an advantage,” said Bob Kauffman, chief medical officer at Vertex.
Billion-Dollar Business
Hepatitis C is already a billion-dollar annual business for Merck and will drive growth, said Patrick Bergstedt, the company’s senior vice president for vaccines and infectious diseases. The drugmaker acquired most of its treatment franchise in the $41 billion purchase last year of Schering-Plough Corp.
The three drugmakers lead a packed field of competitors. A half-dozen other approaches to curing the disease are in development, with about 30 compounds being studied.
At the liver conference in Vienna, the 1,000-seat conference room devoted to new drug development was overflowing. Doctors, researchers and investors lined the walls and sat in aisles as eight investigators presented data about the second wave of therapies following on the new drugs’ heels.
Among the most exciting are polymerase and NS5A inhibitors, compounds that also block viruses from replicating, said Heiner Wedemeyer, leader of the gastroenterology, hepatology and endocrinology department at Hannover Medical School in Germany.
‘Major Breakthrough’
Though the three categories of compounds all prevent viruses from copying themselves, they work in slightly different ways. Doctors hope that using them together could prevent patients from becoming resistant, a common problem with viral treatments, Kauffman said.
“Now there is light on the horizon,” Wedemeyer said, adding that the first studies to combine only pills, without the side-effect-laden injections, are under way. “If this treatment really works, with no resistance emerging, it could be a major breakthrough.”
Investigators aim in five or 10 years to be able to treat hepatitis C with just one or two pills, Bergstedt said. No one company has all the pieces of the puzzle in development yet, he said. They’re looking aggressively for partnerships on experimental compounds.
“We’d be stupid not to,” the Merck executive said. “Everybody’s talking to each other. Nobody really has all the assets at this stage, and everybody’s starting to position and starting to accumulate.”
At the Vanguard
Bristol-Myers Squibb Co. is at the vanguard of the next transformation, said Howard Liang, a Boston-based analyst for Leerink Swan & Co. While the New York-based company’s drugs are still in the early stage of development, it’s taking a daring approach by combining two of its oral medicines in a cocktail without interferon -- potentially leapfrogging competitors whose individual therapies are further along.
Bristol’s lead treatment is one of the first NS5A inhibitors. Roche and Gilead Sciences Inc. are also aiming for oral combination drugs, and it’s essential to be one of the first, he said.
The goal may be to get good enough results by the time the Merck and Vertex drugs are introduced next year to prompt a second wave of patient warehousing, Liang said.
“If you are Bristol or Roche, and you know you are behind, you aren’t going to catch the initial bolus,” he said. “What you try to do is get some data and say, ‘Look, we have something better on the horizon. Don’t treat everybody. If you can wait, wait.’”
--Editors: Phil Serafino, Kristen Hallam
April 18, 2010, 8:37 PM EDT
April 19 (Bloomberg) -- At Fred Poordad’s bustling hepatitis C clinic in the heart of Los Angeles, one in every five patients receives no treatment. They are waiting for a wave of new drugs, expected in the next 18 months, that may boost their chance at a cure by as much as 10-fold.
The medicines also may bolster the prospects of Merck & Co., Vertex Pharmaceuticals Inc. and Johnson & Johnson, the companies in a race to get the first new treatment to the market in a decade. About half of patients can’t tolerate the side effects of existing therapies, which generate $2 billion annually in sales. The new drugs could expand the market to $10 billion in five years, said Geoff Porges, an analyst for Sanford C. Bernstein & Co. in New York.
They’re just the first among new therapies anticipated in the next five years as companies seek a single pill to cure the infection. Poordad, chief of hepatology at the Liver Disease and Transplant Center at Cedars-Sinai Medical Center in Los Angeles, doesn’t object when his patients elect to wait for the new drugs, a practice known as “warehousing.”
“The warehousing has been going on for the past year or so,” Poordad said. “I think we’ll see a tremendous increase in the volume of patients that are treated. That’s the most exciting thing in the field for a long time.”
The drugs closest to market, Merck’s boceprevir and telaprevir from Vertex and Johnson & Johnson, are protease inhibitors crafted from the technologies that led to discoveries made in the fight against HIV. The new treatments are being tested as additions to current standard treatments. Both drugs work by blocking the action of the protease enzyme the hepatitis virus needs to replicate, directly stopping it from spreading.
Available by 2011
Merck, of Whitehouse Station, New Jersey; Vertex, based in Cambridge, Massachusetts; and its partner Johnson & Johnson, of New Brunswick, New Jersey, have said they expect results from final-stage clinical trials by the second half of 2010, with submission to U.S. regulators by year-end. Patients can expect the drugs to be available by 2011, executives said on April 16 at the annual meeting of the European Society for the Study of the Liver in Vienna.
“We’re on the brink of a revolution,” Porges said in an interview at the conference. “Investors have been waiting for this for six to seven years, and investigators and physicians have been waiting for almost 10 years.” He has an “outperform” recommendation on Vertex and sees the stock gaining 23 percent in the next year.
Vertex shares rose 0.8 percent to $39.99 in Nasdaq Stock Market trading on April 16. The stock has gained 43 percent in the last year.
Scarring Livers
Most of the 170 million people infected worldwide with hepatitis C don’t know they’re sick, while others fail to respond to existing drugs or can’t tolerate their side effects. The virus is spread by contact with infected blood. About 4 million Europeans and 3.6 million Americans have the disease, and only about 2 percent get treatment, according to Merck.
The illness moves slowly, scarring livers over years or decades, giving doctors and patients a window of time. Once the liver is destroyed, however, patients face cancer or organ failure, with few effective treatments other than a transplant.
The current standard of care is a combination of the generic antiviral pill ribavirin and interferon, an injection sold by Merck as Pegintron and Swiss drugmaker Roche Holding AG as Pegasys. The medications beef up the immune system, helping it clear the virus from the patient’s blood. They can also trigger anemia or make patients feel like they have a never- ending flu. The new drugs don’t erase those side effects but can cut treatment time in half, from a year to about six months.
Rush for Treatment
The protease inhibitors may have the biggest impact in those who have failed prior therapy. Five to 9 percent of them are cured by a second course of treatment with existing drugs. Adding a protease inhibitor appears to boost that number 10- fold, said Poordad, who has participated in studies funded by Merck, Vertex and Johnson & Johnson.
The first two or three new drugs will take the lion’s share of the growing market, Porges said, as warehoused patients rush to get treatment and doctors establish a new standard of care.
Vertex and Johnson & Johnson hold an advantage right now, Porges said, because their drug telaprevir doesn’t cause the same levels of anemia seen as a side effect of its competitor from Merck. Vertex also allowed patients who didn’t respond at all to other therapies into its clinical trials, possibly positioning its drug, which has been in development for at least 12 years, for broader use.
“We think that’s going to be an advantage,” said Bob Kauffman, chief medical officer at Vertex.
Billion-Dollar Business
Hepatitis C is already a billion-dollar annual business for Merck and will drive growth, said Patrick Bergstedt, the company’s senior vice president for vaccines and infectious diseases. The drugmaker acquired most of its treatment franchise in the $41 billion purchase last year of Schering-Plough Corp.
The three drugmakers lead a packed field of competitors. A half-dozen other approaches to curing the disease are in development, with about 30 compounds being studied.
At the liver conference in Vienna, the 1,000-seat conference room devoted to new drug development was overflowing. Doctors, researchers and investors lined the walls and sat in aisles as eight investigators presented data about the second wave of therapies following on the new drugs’ heels.
Among the most exciting are polymerase and NS5A inhibitors, compounds that also block viruses from replicating, said Heiner Wedemeyer, leader of the gastroenterology, hepatology and endocrinology department at Hannover Medical School in Germany.
‘Major Breakthrough’
Though the three categories of compounds all prevent viruses from copying themselves, they work in slightly different ways. Doctors hope that using them together could prevent patients from becoming resistant, a common problem with viral treatments, Kauffman said.
“Now there is light on the horizon,” Wedemeyer said, adding that the first studies to combine only pills, without the side-effect-laden injections, are under way. “If this treatment really works, with no resistance emerging, it could be a major breakthrough.”
Investigators aim in five or 10 years to be able to treat hepatitis C with just one or two pills, Bergstedt said. No one company has all the pieces of the puzzle in development yet, he said. They’re looking aggressively for partnerships on experimental compounds.
“We’d be stupid not to,” the Merck executive said. “Everybody’s talking to each other. Nobody really has all the assets at this stage, and everybody’s starting to position and starting to accumulate.”
At the Vanguard
Bristol-Myers Squibb Co. is at the vanguard of the next transformation, said Howard Liang, a Boston-based analyst for Leerink Swan & Co. While the New York-based company’s drugs are still in the early stage of development, it’s taking a daring approach by combining two of its oral medicines in a cocktail without interferon -- potentially leapfrogging competitors whose individual therapies are further along.
Bristol’s lead treatment is one of the first NS5A inhibitors. Roche and Gilead Sciences Inc. are also aiming for oral combination drugs, and it’s essential to be one of the first, he said.
The goal may be to get good enough results by the time the Merck and Vertex drugs are introduced next year to prompt a second wave of patient warehousing, Liang said.
“If you are Bristol or Roche, and you know you are behind, you aren’t going to catch the initial bolus,” he said. “What you try to do is get some data and say, ‘Look, we have something better on the horizon. Don’t treat everybody. If you can wait, wait.’”
--Editors: Phil Serafino, Kristen Hallam
New HCV Drugs EASL 2010
New HCV Drugs EASL 2010
ONCE-DAILY NS5A INHIBITOR (BMS-790052) PLUS PEGINTERFERON-ALPHA-2A AND RIBAVIRIN PRODUCES HIGH RATES OF EXTENDED RAPID VIROLOGIC RESPONSE IN TREATMENT-NAIVE HCV-GENOTYPE 1 SUBJECTS: PHASE 2A TRIAL
S. Pol1, G. Everson2, R. Ghalib3, V. Rustgi4, C. Martorell5, H.A. Tatum6, J. Lim7, C. Hezode8, U. Diva9, P.D. Yin9, R. Hindes9 1Hopital Cochin, Paris, France, 2University of Colorado Denver & Hospital, Denver, CO, 3The Liver Institute at Methodist Dallas Medical Center, Dallas, TX, 4Metropolitan Research, Fairfax, VA, 5The Research Institute, Springfield, MA, 6Options Health Research, Tulsa, OK, 7Yale University School of Medicine, New Haven, CT, USA, 8CHU Henri Mondor, Creteil, France, 9Bristol-Myers Squibb Company, Wallingford, CT, USA. *stanislas.pol@cch.aphp.fr
Background: BMS-790052 is a first-in-class, highly potent, once-daily HCV NS5A inhibitor. In Phase I studies in HCV-infected subjects, BMS-790052 was well-tolerated and exhibited potent antiviral activity.
Methods: In this double-blind study, 48 subjects were randomized 1:1:1:1 to receive placebo, 3 mg, 10 mg or 60 mg of BMS-790052, once-daily in combination with peginterferon-alpha-2a and ribavirin (P/R) for 48 weeks in treatment-naive HCV genotype 1-infected subjects. The primary endpoint was the proportion of subjects with extended rapid virologic response (eRVR) defined as HCV RNA < 10 IU/mL at both Weeks 4 and 12.
Results: Subject baseline and demographic characteristics were well-balanced across treatment arms (n = 12/arm), with mean baseline HCV RNA 6.5 log10 IU/mL. The proportion of subjects achieving eRVR was 42%, 83% and 75% in the 3 mg, 10 mg and 60 mg BMS-790052 + P/R arms, respectively, compared to 8% for P/R. Safety was comparable across treatment arms (see table). Adverse events were consistent with those commonly observed with P/R. Confirmed viral breakthrough was not observed in the 10 mg and 60 mg BMS-790052 arms through Week 12.
Conclusions: BMS-790052 is a potent once-daily NS5A inhibitor that yielded higher eRVR, RVR, and cEVR rates when combined with P/R than P/R alone. The addition of BMS-790052 to P/R was well-tolerated with an AE profile comparable to P/R. These Results support further development of BMS-790052 in combination with P/R or other HCV antivirals.
Impact of Low Dose Ritonavir Boosting on the Pharmacokinetics of RG7227 (ITMN-191), a Highly Potent and Selective Inhibitor of the HCV NS3/4A Protease
Authors: J.O. Haznedar1, J. Fretland2, G. Leong2, S. Blotner3, T Hill4, P. Smith1 and J.Q. Tran1
1Roche, Clinical Pharmacology, South San Francisco, CA, USA; 2Roche Palo Alto LLC, Department of Drug Metabolism and Pharmacokinetics, Palo Alto, CA, USA; 3Hoffmann-La Roche Inc., Pharma Development Innovation Biomathematics, Nutley, NJ, USA; 4Hoffmann-La Roche Inc., Clinical Research & Exploratory Development Operations, Nutley, NJ, USA
Background: Low dose ritonavir (RTV), a potent CYP3A inhibitor, is being used to "boost" the pharmacokinetics (PK) of HIV protease inhibitors (PIs) which are CYP3A substrates, enabling regimen simplification while improving treatment response. RG7227, an HCV NS3/4A PI, is a CYP3A substrate and RTV has a potential to enhance its PK. This Phase 1 study investigated the effects of low dose RTV on the single-dose PK of RG7227.
Material & Methods: A single-center, open-label Phase 1 study in 14 healthy males and females between 18 and 45 years of age. Subjects received RTV 100 mg twice-daily (BID) for 10 days. Single oral doses of RG7227 100 mg were administered on 3 separate occasions: alone (2 days before ritonavir dosing), with the first dose of RTV, and on the last day of RTV dosing. Serial PK blood samples for the measurement of RG7227 were collected at pre-dose and up to 48 hours post-dose.
Results: Twelve subjects completed the study. Single doses of RTV 100 mg increased RG7227 AUC0-inf, Cmax and C12h by 5.69-fold (90% CI: 4.06, 7.96), 3.14-fold (90% CI: 1.87, 5.25) and 50.6-fold (90% CI: 25.6, 100), respectively. Multiple doses of RTV 100 mg BID for 10 days had similar effects on RG7227 AUC0-inf (5.46-fold; 90% CI: 4.03, 7.40) and Cmax (3.19-fold; 90% CI: 2.09, 4.85). The multiple-dose effect of ritonavir on RG7227 C12h was less pronounced than the single dose ritonavir effect (26.9-fold; 90% CI: 17.7, 41.0). This is likely due to the mixed inhibition and induction effects of ritonavir on CYP enzymes. No new safety findings were observed.
Conclusion: Low dose RTV significantly enhances RG7227 Cmin, which appears to be the key parameter driving efficacy and prevention of resistance. Less of an effect was observed on AUC and Cmax, which are parameters often attributed to safety. RG7227 dose and dosing frequency may be reduced when co-administered with RTV, to achieve a high Cmin while providing similar or lower AUC and Cmax compared to an unboosted regimen. A Phase 1b study of RTV-boosted RG7227 QD and BID regimens in combination with SOC in HCV-infected patients is being conducted.
Ritonavir Boosting of Low Dose RG7227/ITMN-191, HCV NS3/4A Protease Inhibitor, Results in Robust Reduction in HCV RNA at Lower Exposures Than Provided by Unboosted Regimens
Authors: E. Gane1, R. Rouzier2, C. Stedman3, A. Wiercinska-Drapalo4, A. Horban5, L. Chang6, Y. Zhang6, P. Sampeur6, I. Najera6, N. Shulman6, P. Smith6 and J. Tran6
1. Auckland Clinical Studies, New Zealand
2. CENTRE CAP, France
3. Christchurch Clinical Studies Trust, New Zealand
4. Department of Hepatology and Immunodeficiencies, Warsaw Medical University, Poland
5. Warsaw Medical University &Hospital of Infectious Diseases. Warsaw, Poland
6. Roche, USA
Background: Co-administration of low dose ritonavir resulted in larger increases in RG7227 trough concentration than area under the curve (AUC) and marginal increase in maximum concentration (Cmax), offering the potential to reduce the dose and/or dosing frequency of RG7227 in future Phase 2 studies. The present study evaluated the safety, tolerability, antiviral activity and pharmacokinetics (PK) of once-daily (QD) and twice-daily (BID) ritonavir boosted RG7227 (RG7227/r) in combination with standard of care (SOC) in treatment-naïve HCV genotype 1 patients.
Material & Methods: In a double-blind, placebo-controlled, Phase 1b multiple-ascending dose (MAD) study, treatment-naïve HCV genotype 1 patients were randomized to receive RG7227/r or placebo/r plus SOC for 15 days. RG7227/r regimens were 100/100mg BID (Cohort 1), 200/100mg QD (Cohort 2), and 200/100mg BID (Cohort 3). Blood samples for HCV RNA, PK, and resistance monitoring were collected at baseline and throughout the study.
Results: Cohort 1 and 2 provided blinded data for this abstract. The following table summarizes virologic response of RG7227/r compared to un-boosted 900 mg BID, the highest dose evaluated in a previous MAD study (placebo/r data are not shown due to small N of 2):
RG7227/r AUC and Cmax were approximately 6- to 9-fold and 4- to 10-fold lower, respectively, than previously observed for 900 mg BID un-boosted. Inter-patient PK variability for RG7227/r was also lower than historical un-boosted RG7227. RG7227/r and placebo/r plus SOC were generally well tolerated. Complete data for all 3 cohorts will be presented.
Conclusion: Relative to un-boosted 900 mg BID, ritonavir boosting of low dose RG7227 provides robust and sustained virologic response at significantly lower AUC and Cmax, parameters which are often correlated with safety. These Results indicate that further exploration of ritonavir boosting on the safety and efficacy of RG7227 is warranted.
SVR WITH TELAPREVIR, PEGINTERFERON ALFA-2A AND RIBAVIRIN IN HCV PATIENTS WITH WELL-CHARACTERIZED PRIOR NULL RESPONSE, PARTIAL RESPONSE, VIRAL BREAKTHROUGH OR RELAPSE AFTER PR
T. Berg1, J.G. McHutchison2, N. Adda3, F. Poordad4, M.L. Shiffman5, P. Ferenci6, J. Heathcote7, J.-M. Pawlotsky8, S. Zeuzem9, H. Reesink10, G. Dusheiko11, E. Martin3, D. Alexanderian3, S. George3, A.J. Muir2 1University Clinic, Leipzig, Leipzig, Germany, 2Duke University Medical Center, Durham, NC, 3Vertex Pharmaceuticals Incorporated, Cambridge, MA, 4Cedars-Sinai Medical Center, Los Angeles, CA, 5Bon Secours Health System, Liver Institute of Virginia, Newport News, VA, USA, 6University of Vienna, Vienna, Austria, 7University of Toronto, Toronto, ON, Canada, 8Hopital Henri Mondor, Creteil, France, 9Johann Wolfgang Goethe University Medical Center, Frankfurt/Main, Germany, 10Academisch Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 11Royal Free and University College School of Medicine, London, UK. *thomas.berg@charite.de
Background and aims: Study107 is an open-label rollover study of telaprevir(T) with peginterferon+ribavirin(PR) in genotype-1 HCV patients who did not achieve SVR following PR treatment in telaprevir Phase 2 studies.
Methods: Null responders (< 1-log10 HCV RNA decrease at week-4 or < 2-log10 at week-12), partial responders (≥2-log10 decrease at week-12, detectable at week-24), patients with viral breakthrough and relapsers from PROVE1/2/3 PR arms were eligible for treatment. Initially, all patients received T 750mg q8h plus PR at standard doses for 12 weeks, followed by 12 weeks of PR(T12/PR24). Protocol was amended to allow partial responders, viral breakthroughs and relapsers with undetectable HCV RNA at weeks 4 and 12 (eRVR) to receive T12/PR24. Partial responders, viral breakthroughs and relapsers with detectable HCV RNA at week-4 and/or week-12 and null responders received an additional 24 weeks of PR(T12/PR48).
Results: Of 117 patients included in an ITT analysis, 97(83%) had baseline HCV RNA ≥800,000IU/mL, 69(59%) had genotype subtype 1a, 44(38%) had cirrhosis or bridging fibrosis, and 9(8%) were black. Viral breakthrough and relapse rates occured in 25%, 23% of prior null responders; 10%, 22% of prior partial responders; 13%, 0% of prior viral breakthroughs; and 0%, 4% of prior relapsers.
The most frequent AEs (≥20%) were fatigue, flu-like-syndrome, nausea, diarrhea, pruritus, rash, headache, insomnia and anemia. Grade 3 rash and Grade 3 anemia were observed in 6(5%) and 6(5%) patients, respectively. Ten(9%) patients discontinued due to AEs, 5(4%) due to rash and 2(2%) to anemia.
Conclusions: Patients with prior relapse, breakthrough and partial response exhibited high SVR rates after 24 weeks of telaprevir-based regimen. High SVR rates were also observed in patients with prior null response after 48 weeks of therapy. MK-5172: A NOVEL HCV NS3/4A PROTEASE INHIBITOR WITH POTENT ACTIVITY AGAINST KNOWN RESISTANCE MUTANTS
S. Carroll1, J. McCauley1, S. Ludmerer1, S. Harper2, V. Summa2, M. Rowley2, M. Rudd1, P. Coleman1, N. Liverton1, J. Butcher1, C. Mcintyre1, J. Romano1, K. Bush1, M. Ferrara2, B. Crescenzi2, A. Petrocchi2, M. Difilippo2, C. Burlein1, J. Dimuzio1, D. Graham1, C. Mchale1, M. Stahlhut1, A. Gates1, C. Fandozzi1, N. Trainor1, D. Hazuda1, J. Vacca1, D. Olsen1 1Antiviral Research, 2Medicinal Chemistry, Merck Research Laboratories, West Point, PA, USA, 3Department of Antiviral Research, Instituto di Ricerche di Biologia Molecolare, Pomezia, Italy, 4Drug Metabolism, Merck Research Laboratories, West Point, PA, USA. *nigel_liverton@merck.com
Background and aims: Efforts toward novel HCV treatment options include the development of direct antiviral agents which inhibit key steps in viral replication, such as HCV NS3/4a protease. Several inhibitors of HCV NS3/4A protease activity have demonstrated antiviral efficacy in clinical studies. However viral variants resistant to inhibition by these compounds have been detected in patients. Our interest is in identifying HCV NS3/4a protease inhibitors displaying good activity against known resistant viral variants that will result in low rates of treatment failure due to viral resistance.
Methods: Medicinal chemistry efforts focused on a novel macrocyclic scaffold and were guided by biochemical and cell-based replicon assays that utilized forms of HCV NS3/4a protease that included known resistance mutations R155K, D168V, and A156T. In vivo efficacy was evaluated in chimpanzees harboring chronic HCV infections.
Results: MK-5172 was identified as a potent macrocyclic inhibitor of NS3/4a (genotype 1b enzyme Ki ∼ 0.02 nM, genotype 1b replicon IC50 = 3.5 nM). In both biochemical and cell-based replicon assays, MK-5172 retained significant inhibitory potency against NS3/4A variants R155K (Ki = 0.07 nM), D168V (Ki 0.3 nM), and A156T (Ki = 5 nM). MK-5172 exhibits significant liver exposure in rat, following a 5 mg/kg oral dose ([rat liver]4h = 27 µM) thus demonstrating effective distribution of compound into liver, the site of HCV replication. Three HCV-infected chimpanzees infected with either wild-type or R155K virus were dosed with MK-5172 orally for seven days at 1 mg/kg b.i.d. All demonstrated high concentrations of MK-5172 in the liver; wild-type virus experienced rapid suppression of greater than three logs, while R155K virus experienced an immediate 1-2 log reduction.
Conclusions: MK-5172 is a novel NS3/4a protease inhibitor with an attractive preclinical profile including high in vitro potency, significant liver exposure upon oral dosing, and potent inhibitory activity against known viral variants that are resistant to other protease inhibitors in development. MK-5172 has also demonstrated antiviral efficacy in a chimpanzee model of chronic HCV infection. Clinical studies with MK-5172 are currently in progress.
SAFETY AND ANTIVIRAL ACTIVITY OF THE HCV NON-NUCLEOSIDE POLYMERASE INHIBITOR VX-222 IN TREATMENT-NAïVE GENOTYPE 1 HCV-INFECTED PATIENTS
M. Rodriguez-Torres1, E. Lawitz2, B. Conway3, K. Kaita4, A.M. Sheikh5, R. Ghalib6, R. Adrover7, C. Cooper8, M. Silva7, M. Rosario9, B. Bourgault10, L. Proulx10, J.G. McHutchison11 1Fundacion de Investigacion de Diego, Santurce, PR, 2Alamo Medical Research, San Antonio, TX, USA, 3University of British Columbia, Vancouver, BC, 4University of Manitoba, Winnipeg, MB, Canada, 5GI Specialists of Georgia, Marietta, GA, 6The Liver Institute at Methodist Dallas, Dallas, TX, USA, 7ACLIRES, Buenos Aires, Argentina, 8University of Ottawa, Ottawa, ON, Canada, 9Hospital Universitario Austral, Buenos Aires, Argentina, 10Vertex Pharmaceuticals Incorporated, Cambridge, MA, USA, 11ViroChem Pharma Inc., Laval, QC, Canada, 12Duke University Medical Center, Durham, NC, USA. *rodztorres@coqui.net
Background and aims: VX-222 is a novel non-nucleoside hepatitis C virus (HCV) polymerase inhibitor with potent in vitro activity. Safety, tolerability, pharmacokinetics and antiviral activity of VX-222 were assessed in a phase Ib/IIa multicenter, randomized, double-blinded, placebo-controlled, dose-ascending study in HCV-infected patients.
Methods: Treatment-naïve HCV genotype 1 patients were randomized to receive VX-222 at doses of 250 mg BID, 500 mg BID, 750 mg BID, 1500 mg QD or placebo for 3 days in a treatment:placebo ratio of 6:2 (8 patients/cohort).
Peginterferon-alfa-2a (P) plus ribavirin (R) was offered to patients at end of study (day 4) for up to 48 weeks, as judged appropriate by the investigator. PR treatment was discontinued in patients who had not experienced a ≥2 log10 HCV RNA level decline at week 12. VX-222 plasma levels were assessed at multiple time points over 12 hours, on days 1 and 3.
Results: Twenty-four patients were enrolled in the first three cohorts. VX-222 exposure increased in a dose-related manner. On day 3, the mean AUC0-12h and Cmax values were 19,490 ng*h/mL (CV 41%) and 2,959 ng/mL (CV 29%), 29,848 ng*h/mL (CV 54%) and 5,044 ng/mL (CV 36%), and 62,952 ng*h/mL (CV 112 %) and 10,288 ng/mL (CV 112%) for the 250 mg, 500 mg, and 750 mg BID groups, respectively. The mean HCV RNA decline achieved on day 4 with placebo, 250 mg, 500 mg, and 750 mg VX-222 BID was 0.1 log10 (range: 0.3 increase to 0.5 decline), 3.1 log10 (range: 2.0 to 4.2), 3.4 log10 (range: 3.2 to 3.6), and 3.2 log10 (range: 2.3 to 3.8), respectively. All AEs reported were mild to moderate, and the most frequently reported AEs by patients that received either active drug or placebo were diarrhea (25%), headache (20%) and nausea (12%). No clinically relevant laboratory abnormalities were reported.
Conclusions: VX-222 was well tolerated and a mean HCV RNA decline of >3 log10 at day 4 was observed in each cohort, Results of the fourth cohort evaluating QD dosing will also be presented. These Results support further evaluation of VX-222 in combination with peginterferon and ribavirin in the treatment of HCV.
IDENTIFICATION AND CHARACTERIZATION OF PPI-461, A POTENT AND SELECTIVE HCV NS5A INHIBITOR WITH ACTIVITY AGAINST ALL HCV GENOTYPES
R. Colonno, E. Peng, M. Bencsik, N. Huang, M. Zhong, A. Huq, Q. Huang, J. Williams, L. Li Presidio Pharmaceuticals, San Francisco, CA, USA. *rich@presidiopharma.com
Background: Several mechanistic classes of oral HCV inhibitors are currently undergoing clinical development. Inhibitors of NS5A have distinguished themselves by their picomolar potency and broad genotype spectrum in HCV replicon assays. Here we report the discovery and preclinical profile of PPI-461, a newly discovered NS5A inhibitor being advanced toward clinical studies.
Methods: Antiviral potency, combination and resistance studies utilized standard HCV replicon systems (genotype 1a and 1b). HCV spectrum studies employed stable or transient replicon assays with insertion of NS5A gene segments from other HCV genotypes (2a, 3a, 4a, 5a, 6a and 7a) into an HCV 1b backbone. Pharmacokinetic studies utilized IV and PO administration of PPI-461 in rats, monkeys and dogs. Extensive ADME and toxicology profiling has been performed in vitro and in rats and monkeys.
Results: PPI-461 was identified through an extensive medicinal chemistry effort and exhibits EC50s of 0.2 and 0.01 nM in HCV 1a and 1b replicon assays, respectively. Antiviral activity (EC50s of 0.1-19 nM) is maintained against all other HCV genotypes in transient/stable replicon assays. Cellular cytotoxicity levels (CC50) are >10 uM in several cell lines and no activity is observed against several other viruses (including BVDV) at 10 uM. PPI-461 is additive to synergistic (CIs 0.29-0.89) when combined with IFN, NS3 protease, NS5B nucleoside and non-nucleoside inhibitors. Resistance studies showed that variants with high levels of resistance could be generated, especially with genotype 1a, but required multiple amino acid substitutions within the NS5A gene. PPI-461 shows excellent stability in liver microsomes and no significant inhibition against P450 isozymes at 10 uM. PPI-461 exhibits good oral bioavailability in rats, monkeys and dogs, with enhanced liver concentrations and plasma half-lives suggestive of once daily dosing in humans. Toxicology studies in rats and monkeys showed that PPI-461 is well tolerated at plasma exposure levels >100,000-fold HCV 1a EC50 levels.
Conclusion: HCV NS5A protein plays a critical role in the HCV viral replicative cycle and is an attractive target for antiviral intervention. PPI-461 is a newly discovered selective and broadly potent inhibitor of HCV NS5A protein with a favorable preclinical profile supportive of advancement into clinical trial
ILIBININ AS A RESCUE TREATMENT FOR HCV-INFECTED PATIENTS SHOWING SUBOPTIMAL VIROLOGIC RESPONSE TO STANDARD COMBINATION THERAPY
M. Biermer, L. Stoehr, B. Schlosser, B. Fulop, F. van Bommel, T. Berg Medizinische Klinik mit Schwerpunkt Gastroenterologie und Hepatologie, Charite Campus Virchow, Berlin, Germany. *michael.biermer@charite.de
During standard antiviral treatment a significant proportion of hepatitis C patients show no complete virologic response despite a marked reduction of viral load in early stages of treatment (partial response). This minimal viremia may persist indicating the treatment termination as a consequence of viral nonresponse.
Recently, high-dose intravenous silibinin has demonstrated significant antiviral activity. We were therefore interested whether patients with partial virologic response can be rescued by the on-treatment addition of high-dose intravenous silibinin infusions.
Eleven patients who received different interferon-based antiviral regimens qualified for the rescue strategy and received silibinin infusions between December 2008 and September 2009. All patients had received peginterferon alfa 2a / ribavirin and four patients additionally received an HCV specific protease inhibitor for the first four weeks of treatment. 9 out of 11 patients showed a plateau of HCV RNA levels ranging from detectable but not quantifiable (< 15 IU/mL) to 2015 IU/mL for at least eight weeks on continuous treatment (total treatment duration 16-30 weeks). The remaining two patients suffered a virologic rebound after cessation of the protease inhibitor (HCV RNA 484 IU/ml at week 6 (patient 1) and 6890 IU/ml at week 5 (patient 2). Every patient received silibinin infusions (1400 mg/day - in 500 ml NaCl) on two consecutive days.
Eight out of eleven patients achieved a complete suppression of viral replication below the limit of detection within the first week after silibinin. Three patients did initially not respond to silibinin, two of the responders showed a viral breakthrough (8 and 14 weeks after silibinin administration during continued SOC treatment) and up to now six patients have undetectable HCV-RNA levels eight to 40 weeks after silibinin administration with continued SOC. Silibinin infusions were generally well tolerated. All patients reported a transient sensation of heat, three patients reported painful bowel-movements, two with a single episode of vomiting.
A two days administration of high-dose intravenous silibinin might be an interesting approach to rescue patients with ongoing minimal residual viremia while on interferon-based therapy. Further follow-up is needed to confirm whether the response induced is long lasting and leads to SVR.
RITONAVIR BOOSTING OF LOW DOSE RG7227/ITMN-191, HCV NS3/4A PROTEASE INHIBITOR, Results IN ROBUST REDUCTION IN HCV RNA AT LOWER EXPOSURES THAN PROVIDED BY UNBOOSTED REGIMENS
E. Gane1, R. Rouzier2, C. Stedman3, A. Wiercinska-Drapalo4, A. Horban4, L. Chang5, Y. Zhang5, P. Sampeur6, I. Najera7, N. Shulman7, P. Smith5, J. Tran5 1Auckland Clinical Studies, Auckland, New Zealand, 2Centre CAP, Montpellier, France, 3Christchurch Clinical Studies Trust, Christchurch, New Zealand, 4Department of Hepatology and Immunodeficiencies, Warsaw Medical University, 5Warsaw Medical University & Hospital of Infectious Diseases, Warsaw, Poland, 6Roche, South San Francisco, CA, 7Roche, Nutley, NJ, 8Roche, Palo Alto, CA, USA. *edgane@adhb.govt.nz
Background: Co-administration of low dose ritonavir resulted in larger increases in RG7227 trough concentration than area under the curve (AUC) and marginal increase in maximum concentration (Cmax), offering the opportunity to reduce the dose and/or dosing frequency of RG7227 in future Phase 2 studies. The present study evaluated the safety, tolerability, antiviral activity and pharmacokinetics (PK) of once-daily (QD) and twice-daily (BID) ritonavir boosted RG7227 (RG7227/r) in combination with standard of care (SOC) in treatment-naïve HCV genotype 1 patients.
Material & Methods: In a double-blind, placebo-controlled, Phase 1b multiple-ascending dose (MAD) study, treatment-naïve HCV genotype 1 patients were randomized to receive RG7227/r or placebo/r plus SOC for 15 days. RG7227/r regimens were 100/100mg BID (Cohort 1), 200/100mg QD (Cohort 2), and 200/100mg BID (Cohort 3). Blood samples for HCV RNA, PK, and resistance monitoring were collected at baseline and throughout the study.
Results: Cohort 1 and 2 provided blinded data for this abstract. The following table summarizes virologic response of RG7227/r compared to un-boosted 900 mg BID, the highest dose evaluated in a previous MAD study (placebo/r data are not shown due to small N of 2):
RG7227/r AUC and Cmax were approximately 6- to 9-fold and 4- to 10-fold lower, respectively, than previously observed for 900 mg BID un-boosted. Inter-patient PK variability for RG7227/r was also lower than historical un-boosted RG7227. RG7227/r and placebo/r plus SOC were generally well tolerated. Complete data for all 3 cohorts will be presented.
Conclusion: Relative to un-boosted 900 mg BID, ritonavir boosting of low dose RG7227 provides robust and sustained virologic response at significantly lower AUC and Cmax, parameters which are often correlated with safety. These Results indicate that further exploration of ritonavir boosting on the safety and efficacy of RG7227 is warranted.
VITAMIN D SUPPLEMENT IMPROVE SVR IN CHRONIC HEPATITIS C (GENOTYPE 1) NAïVE PATIENTS TREATED WITH PEG INTERFERON AND RIBAVIRIN
S. Abu Mouch1,2, Z. Fireman1,2, J. Jarchovsky1,2, N. Assy2,3 1Hepatology Unite, 2Internal Medicine B, Hillel Yaffe Medical Center, Hadera, 3Faculty of Medicine, Technion, Haifa, 4Gastroenterology, Hillel Yaffe Medical Center, Hadera, 5Liver Unit, Ziv Medical Center, Safed, Israel. *saif@hy.health.gov.il
Background: The combination therapy of Peg/RBV is considered the standard of care for chronic hepatitis C (HCV). A sustained viral response (SVR) is obtained in 40-50% of naïve HCV patients with genotype 1. Vitamin D is a potent immuno modulator whose impact on SVR of Peg/RBV based treatment of chronic HCV is unknown.
Aim: To assess whether the supplement of vitamin D to the conventional Peg/RBV therapy could improve SVR.
Methods: Fifty-eight patients with chronic HCV infection were randomized into two groups (intent-to-treat population): 27 (treatment group, age 47±11 yrs, body mass index [BMI] 27±4, 50% male) received pegylated-interferon-alpha2b (1.5 µg/kg once weekly) plus ribavirin (1000-1200 mg/daily) together with vitamin D3 (1000-4000 IU/daily, serum level >32 ng/ml), and 31 (controls, age 49±7 years, BMI 24±3, 60% male) received the same therapy without vitamin D. HCV RNA was assessed by RT-PCR (sensitivity, 50 IU/mL). Undetectable HCV RNA at week 12 and at week 24 post treatment (considered as complete EVR and SVR respectively).
Results: Demographics, disease characteristics, ethnicity, baseline biochemical parameters and adherence to treatment were similar in both groups. The treatment group had a higher mean BMI (27±4 vs 24±3; P< 0.01), viral load (68% vs 58%, P< 0.01), and fibrosis (Metavir scores >F2: 55% vs 18%, P< 0.001) than the controls. All but one treated patient (96%) and 48% (15/31) controls were HCV-RNA negative at week 12 (P< 0.0001). At week 24 post treatment (SVR): 86% (13/15) of treated patients and 41% (5/12) of controls were HCVRNA negative (P< 0.001). AEs were mostly mild and typical of Peg/RBV. There were no SAE.
Conclusions: Supplement of Vitamin D to conventional Peg/RBV therapy for naïve, genotype 1 patients with chronic infection significantly improve SVR.
SILEN-C2: EARLY ANTIVIRAL ACTIVITY AND SAFETY OF BI 201335 COMBINED WITH PEGINTERFERON ALFA-2A AND RIBAVIRIN (PEGIFN/RBV) IN CHRONIC HCV GENOTYPE-1 PATIENTS WITH NON-RESPONSE TO PEGIFN/RBV
M. Sulkowski1, M. Bourliere2, J.-P. Bronowicki3, A. Streinu-Cercel4, L. Preotescu4, T. Asselah5, J.-M. Pawlotsky6, S. Shafran7, S. Pol8, F.A. Caruntu4, S. Mauss9, D. Larrey10, C. Hafner11, Y. Datsenko11, J. Stern12, R. Kubiak11, G. Steinmann11 1Department of Viral Hepatitis, Johns Hopkins University, Baltimore, MD, USA, 2Hopital Saint Joseph, Marseille, 3Hopital de Brabois, Vandoeuvre Cedex, France, 4'Prof. Dr. Matei Bals' Institute of Infectious Diseases 1, Bucharest, Romania, 5Hopital Beaujon, Clichy Cedex, 6Hopital Henri Mondor, Creteil, France, 7University of Alberta, Edmonton, AB, Canada, 8Hopital Cochin, Paris, France, 9Center for HIV and Hepatogastroenterology, Dusseldorf, Germany, 10Hopital Saint-Eloi, Montpellier Cedex, France, 11Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach/ Ri, Germany, 12Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, USA. *msulkowski@jhmi.edu
Background and aims: BI 201335 is a potent HCV NS3/4A protease inhibitor being studied in phase IIb trials of chronic HCV genotype-1 (GT1) infection.
Methods: In a double-blind, randomized, parallel group design, HCV GT1 patients with confirmed non-response to at least 12 wks of PegIFN/RBV treatment were randomized 1:2:1 to (1) 240 mg BI 201335 once daily (QD), (2) 240 mg BI 201335 QD after a 3 day lead-in phase (LI) of PegIFN/RBV, and (3) 240 mg BI 201335 twice daily (BID) after a 3 day LI. Relapsers and patients with liver cirrhosis were excluded. In each group, treatment is for 24 wks with a Background of PegIFN (180 µg/wk) and RBV (1000/1200mg/d). Pre-specified Interim analysis after 12 weeks of therapy is reported. Viral rebound is defined as an increase in plasma HCV RNA ≥ 1 log10 on-treatment from nadir or confirmed increase ≥ 100 IU/ml if previously undetectable.
Results: 288 patients were treated (mean age 49 +/- 9 years; mean BMI 26.4 +/- 4.4 kg/m2; mean log10 HCV RNA at baseline 6.6 IU/mL). BI 201335 with PegIFN/RBV was overall well tolerated and demonstrated potent antiviral activity in all dose groups (Table). Mean ALT improved in all groups. 8% of patients prematurely discontinued treatment due to adverse events (AE). Most frequent AE were gastrointestinal disorders, mostly mild jaundice resulting from isolated unconjugated hyperbilirubinemia, (9.2%, 14.1% and 34.3% in groups 1, 2 and 3) and mostly mild to moderate rash or photosensitivity reactions (severe rash in 1.3, 0.7 and 5.7% in groups 1, 2 and 3).
Conclusions: SILEN-C2 confirmed robust antiviral activity with overall good tolerability and safety of BI 201335 especially if given 240mg once daily in combination with PegIFN/RBV in patients with chronic HCV GT-1 infection who did not respond to a previous course of PegIFN/RBV therapy.
ONCE-DAILY NS5A INHIBITOR (BMS-790052) PLUS PEGINTERFERON-ALPHA-2A AND RIBAVIRIN PRODUCES HIGH RATES OF EXTENDED RAPID VIROLOGIC RESPONSE IN TREATMENT-NAIVE HCV-GENOTYPE 1 SUBJECTS: PHASE 2A TRIAL
S. Pol1, G. Everson2, R. Ghalib3, V. Rustgi4, C. Martorell5, H.A. Tatum6, J. Lim7, C. Hezode8, U. Diva9, P.D. Yin9, R. Hindes9 1Hopital Cochin, Paris, France, 2University of Colorado Denver & Hospital, Denver, CO, 3The Liver Institute at Methodist Dallas Medical Center, Dallas, TX, 4Metropolitan Research, Fairfax, VA, 5The Research Institute, Springfield, MA, 6Options Health Research, Tulsa, OK, 7Yale University School of Medicine, New Haven, CT, USA, 8CHU Henri Mondor, Creteil, France, 9Bristol-Myers Squibb Company, Wallingford, CT, USA. *stanislas.pol@cch.aphp.fr
Background: BMS-790052 is a first-in-class, highly potent, once-daily HCV NS5A inhibitor. In Phase I studies in HCV-infected subjects, BMS-790052 was well-tolerated and exhibited potent antiviral activity.
Methods: In this double-blind study, 48 subjects were randomized 1:1:1:1 to receive placebo, 3 mg, 10 mg or 60 mg of BMS-790052, once-daily in combination with peginterferon-alpha-2a and ribavirin (P/R) for 48 weeks in treatment-naive HCV genotype 1-infected subjects. The primary endpoint was the proportion of subjects with extended rapid virologic response (eRVR) defined as HCV RNA < 10 IU/mL at both Weeks 4 and 12.
Results: Subject baseline and demographic characteristics were well-balanced across treatment arms (n = 12/arm), with mean baseline HCV RNA 6.5 log10 IU/mL. The proportion of subjects achieving eRVR was 42%, 83% and 75% in the 3 mg, 10 mg and 60 mg BMS-790052 + P/R arms, respectively, compared to 8% for P/R. Safety was comparable across treatment arms (see table). Adverse events were consistent with those commonly observed with P/R. Confirmed viral breakthrough was not observed in the 10 mg and 60 mg BMS-790052 arms through Week 12.
Conclusions: BMS-790052 is a potent once-daily NS5A inhibitor that yielded higher eRVR, RVR, and cEVR rates when combined with P/R than P/R alone. The addition of BMS-790052 to P/R was well-tolerated with an AE profile comparable to P/R. These Results support further development of BMS-790052 in combination with P/R or other HCV antivirals.
Impact of Low Dose Ritonavir Boosting on the Pharmacokinetics of RG7227 (ITMN-191), a Highly Potent and Selective Inhibitor of the HCV NS3/4A Protease
Authors: J.O. Haznedar1, J. Fretland2, G. Leong2, S. Blotner3, T Hill4, P. Smith1 and J.Q. Tran1
1Roche, Clinical Pharmacology, South San Francisco, CA, USA; 2Roche Palo Alto LLC, Department of Drug Metabolism and Pharmacokinetics, Palo Alto, CA, USA; 3Hoffmann-La Roche Inc., Pharma Development Innovation Biomathematics, Nutley, NJ, USA; 4Hoffmann-La Roche Inc., Clinical Research & Exploratory Development Operations, Nutley, NJ, USA
Background: Low dose ritonavir (RTV), a potent CYP3A inhibitor, is being used to "boost" the pharmacokinetics (PK) of HIV protease inhibitors (PIs) which are CYP3A substrates, enabling regimen simplification while improving treatment response. RG7227, an HCV NS3/4A PI, is a CYP3A substrate and RTV has a potential to enhance its PK. This Phase 1 study investigated the effects of low dose RTV on the single-dose PK of RG7227.
Material & Methods: A single-center, open-label Phase 1 study in 14 healthy males and females between 18 and 45 years of age. Subjects received RTV 100 mg twice-daily (BID) for 10 days. Single oral doses of RG7227 100 mg were administered on 3 separate occasions: alone (2 days before ritonavir dosing), with the first dose of RTV, and on the last day of RTV dosing. Serial PK blood samples for the measurement of RG7227 were collected at pre-dose and up to 48 hours post-dose.
Results: Twelve subjects completed the study. Single doses of RTV 100 mg increased RG7227 AUC0-inf, Cmax and C12h by 5.69-fold (90% CI: 4.06, 7.96), 3.14-fold (90% CI: 1.87, 5.25) and 50.6-fold (90% CI: 25.6, 100), respectively. Multiple doses of RTV 100 mg BID for 10 days had similar effects on RG7227 AUC0-inf (5.46-fold; 90% CI: 4.03, 7.40) and Cmax (3.19-fold; 90% CI: 2.09, 4.85). The multiple-dose effect of ritonavir on RG7227 C12h was less pronounced than the single dose ritonavir effect (26.9-fold; 90% CI: 17.7, 41.0). This is likely due to the mixed inhibition and induction effects of ritonavir on CYP enzymes. No new safety findings were observed.
Conclusion: Low dose RTV significantly enhances RG7227 Cmin, which appears to be the key parameter driving efficacy and prevention of resistance. Less of an effect was observed on AUC and Cmax, which are parameters often attributed to safety. RG7227 dose and dosing frequency may be reduced when co-administered with RTV, to achieve a high Cmin while providing similar or lower AUC and Cmax compared to an unboosted regimen. A Phase 1b study of RTV-boosted RG7227 QD and BID regimens in combination with SOC in HCV-infected patients is being conducted.
Ritonavir Boosting of Low Dose RG7227/ITMN-191, HCV NS3/4A Protease Inhibitor, Results in Robust Reduction in HCV RNA at Lower Exposures Than Provided by Unboosted Regimens
Authors: E. Gane1, R. Rouzier2, C. Stedman3, A. Wiercinska-Drapalo4, A. Horban5, L. Chang6, Y. Zhang6, P. Sampeur6, I. Najera6, N. Shulman6, P. Smith6 and J. Tran6
1. Auckland Clinical Studies, New Zealand
2. CENTRE CAP, France
3. Christchurch Clinical Studies Trust, New Zealand
4. Department of Hepatology and Immunodeficiencies, Warsaw Medical University, Poland
5. Warsaw Medical University &Hospital of Infectious Diseases. Warsaw, Poland
6. Roche, USA
Background: Co-administration of low dose ritonavir resulted in larger increases in RG7227 trough concentration than area under the curve (AUC) and marginal increase in maximum concentration (Cmax), offering the potential to reduce the dose and/or dosing frequency of RG7227 in future Phase 2 studies. The present study evaluated the safety, tolerability, antiviral activity and pharmacokinetics (PK) of once-daily (QD) and twice-daily (BID) ritonavir boosted RG7227 (RG7227/r) in combination with standard of care (SOC) in treatment-naïve HCV genotype 1 patients.
Material & Methods: In a double-blind, placebo-controlled, Phase 1b multiple-ascending dose (MAD) study, treatment-naïve HCV genotype 1 patients were randomized to receive RG7227/r or placebo/r plus SOC for 15 days. RG7227/r regimens were 100/100mg BID (Cohort 1), 200/100mg QD (Cohort 2), and 200/100mg BID (Cohort 3). Blood samples for HCV RNA, PK, and resistance monitoring were collected at baseline and throughout the study.
Results: Cohort 1 and 2 provided blinded data for this abstract. The following table summarizes virologic response of RG7227/r compared to un-boosted 900 mg BID, the highest dose evaluated in a previous MAD study (placebo/r data are not shown due to small N of 2):
RG7227/r AUC and Cmax were approximately 6- to 9-fold and 4- to 10-fold lower, respectively, than previously observed for 900 mg BID un-boosted. Inter-patient PK variability for RG7227/r was also lower than historical un-boosted RG7227. RG7227/r and placebo/r plus SOC were generally well tolerated. Complete data for all 3 cohorts will be presented.
Conclusion: Relative to un-boosted 900 mg BID, ritonavir boosting of low dose RG7227 provides robust and sustained virologic response at significantly lower AUC and Cmax, parameters which are often correlated with safety. These Results indicate that further exploration of ritonavir boosting on the safety and efficacy of RG7227 is warranted.
SVR WITH TELAPREVIR, PEGINTERFERON ALFA-2A AND RIBAVIRIN IN HCV PATIENTS WITH WELL-CHARACTERIZED PRIOR NULL RESPONSE, PARTIAL RESPONSE, VIRAL BREAKTHROUGH OR RELAPSE AFTER PR
T. Berg1, J.G. McHutchison2, N. Adda3, F. Poordad4, M.L. Shiffman5, P. Ferenci6, J. Heathcote7, J.-M. Pawlotsky8, S. Zeuzem9, H. Reesink10, G. Dusheiko11, E. Martin3, D. Alexanderian3, S. George3, A.J. Muir2 1University Clinic, Leipzig, Leipzig, Germany, 2Duke University Medical Center, Durham, NC, 3Vertex Pharmaceuticals Incorporated, Cambridge, MA, 4Cedars-Sinai Medical Center, Los Angeles, CA, 5Bon Secours Health System, Liver Institute of Virginia, Newport News, VA, USA, 6University of Vienna, Vienna, Austria, 7University of Toronto, Toronto, ON, Canada, 8Hopital Henri Mondor, Creteil, France, 9Johann Wolfgang Goethe University Medical Center, Frankfurt/Main, Germany, 10Academisch Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 11Royal Free and University College School of Medicine, London, UK. *thomas.berg@charite.de
Background and aims: Study107 is an open-label rollover study of telaprevir(T) with peginterferon+ribavirin(PR) in genotype-1 HCV patients who did not achieve SVR following PR treatment in telaprevir Phase 2 studies.
Methods: Null responders (< 1-log10 HCV RNA decrease at week-4 or < 2-log10 at week-12), partial responders (≥2-log10 decrease at week-12, detectable at week-24), patients with viral breakthrough and relapsers from PROVE1/2/3 PR arms were eligible for treatment. Initially, all patients received T 750mg q8h plus PR at standard doses for 12 weeks, followed by 12 weeks of PR(T12/PR24). Protocol was amended to allow partial responders, viral breakthroughs and relapsers with undetectable HCV RNA at weeks 4 and 12 (eRVR) to receive T12/PR24. Partial responders, viral breakthroughs and relapsers with detectable HCV RNA at week-4 and/or week-12 and null responders received an additional 24 weeks of PR(T12/PR48).
Results: Of 117 patients included in an ITT analysis, 97(83%) had baseline HCV RNA ≥800,000IU/mL, 69(59%) had genotype subtype 1a, 44(38%) had cirrhosis or bridging fibrosis, and 9(8%) were black. Viral breakthrough and relapse rates occured in 25%, 23% of prior null responders; 10%, 22% of prior partial responders; 13%, 0% of prior viral breakthroughs; and 0%, 4% of prior relapsers.
The most frequent AEs (≥20%) were fatigue, flu-like-syndrome, nausea, diarrhea, pruritus, rash, headache, insomnia and anemia. Grade 3 rash and Grade 3 anemia were observed in 6(5%) and 6(5%) patients, respectively. Ten(9%) patients discontinued due to AEs, 5(4%) due to rash and 2(2%) to anemia.
Conclusions: Patients with prior relapse, breakthrough and partial response exhibited high SVR rates after 24 weeks of telaprevir-based regimen. High SVR rates were also observed in patients with prior null response after 48 weeks of therapy. MK-5172: A NOVEL HCV NS3/4A PROTEASE INHIBITOR WITH POTENT ACTIVITY AGAINST KNOWN RESISTANCE MUTANTS
S. Carroll1, J. McCauley1, S. Ludmerer1, S. Harper2, V. Summa2, M. Rowley2, M. Rudd1, P. Coleman1, N. Liverton1, J. Butcher1, C. Mcintyre1, J. Romano1, K. Bush1, M. Ferrara2, B. Crescenzi2, A. Petrocchi2, M. Difilippo2, C. Burlein1, J. Dimuzio1, D. Graham1, C. Mchale1, M. Stahlhut1, A. Gates1, C. Fandozzi1, N. Trainor1, D. Hazuda1, J. Vacca1, D. Olsen1 1Antiviral Research, 2Medicinal Chemistry, Merck Research Laboratories, West Point, PA, USA, 3Department of Antiviral Research, Instituto di Ricerche di Biologia Molecolare, Pomezia, Italy, 4Drug Metabolism, Merck Research Laboratories, West Point, PA, USA. *nigel_liverton@merck.com
Background and aims: Efforts toward novel HCV treatment options include the development of direct antiviral agents which inhibit key steps in viral replication, such as HCV NS3/4a protease. Several inhibitors of HCV NS3/4A protease activity have demonstrated antiviral efficacy in clinical studies. However viral variants resistant to inhibition by these compounds have been detected in patients. Our interest is in identifying HCV NS3/4a protease inhibitors displaying good activity against known resistant viral variants that will result in low rates of treatment failure due to viral resistance.
Methods: Medicinal chemistry efforts focused on a novel macrocyclic scaffold and were guided by biochemical and cell-based replicon assays that utilized forms of HCV NS3/4a protease that included known resistance mutations R155K, D168V, and A156T. In vivo efficacy was evaluated in chimpanzees harboring chronic HCV infections.
Results: MK-5172 was identified as a potent macrocyclic inhibitor of NS3/4a (genotype 1b enzyme Ki ∼ 0.02 nM, genotype 1b replicon IC50 = 3.5 nM). In both biochemical and cell-based replicon assays, MK-5172 retained significant inhibitory potency against NS3/4A variants R155K (Ki = 0.07 nM), D168V (Ki 0.3 nM), and A156T (Ki = 5 nM). MK-5172 exhibits significant liver exposure in rat, following a 5 mg/kg oral dose ([rat liver]4h = 27 µM) thus demonstrating effective distribution of compound into liver, the site of HCV replication. Three HCV-infected chimpanzees infected with either wild-type or R155K virus were dosed with MK-5172 orally for seven days at 1 mg/kg b.i.d. All demonstrated high concentrations of MK-5172 in the liver; wild-type virus experienced rapid suppression of greater than three logs, while R155K virus experienced an immediate 1-2 log reduction.
Conclusions: MK-5172 is a novel NS3/4a protease inhibitor with an attractive preclinical profile including high in vitro potency, significant liver exposure upon oral dosing, and potent inhibitory activity against known viral variants that are resistant to other protease inhibitors in development. MK-5172 has also demonstrated antiviral efficacy in a chimpanzee model of chronic HCV infection. Clinical studies with MK-5172 are currently in progress.
SAFETY AND ANTIVIRAL ACTIVITY OF THE HCV NON-NUCLEOSIDE POLYMERASE INHIBITOR VX-222 IN TREATMENT-NAïVE GENOTYPE 1 HCV-INFECTED PATIENTS
M. Rodriguez-Torres1, E. Lawitz2, B. Conway3, K. Kaita4, A.M. Sheikh5, R. Ghalib6, R. Adrover7, C. Cooper8, M. Silva7, M. Rosario9, B. Bourgault10, L. Proulx10, J.G. McHutchison11 1Fundacion de Investigacion de Diego, Santurce, PR, 2Alamo Medical Research, San Antonio, TX, USA, 3University of British Columbia, Vancouver, BC, 4University of Manitoba, Winnipeg, MB, Canada, 5GI Specialists of Georgia, Marietta, GA, 6The Liver Institute at Methodist Dallas, Dallas, TX, USA, 7ACLIRES, Buenos Aires, Argentina, 8University of Ottawa, Ottawa, ON, Canada, 9Hospital Universitario Austral, Buenos Aires, Argentina, 10Vertex Pharmaceuticals Incorporated, Cambridge, MA, USA, 11ViroChem Pharma Inc., Laval, QC, Canada, 12Duke University Medical Center, Durham, NC, USA. *rodztorres@coqui.net
Background and aims: VX-222 is a novel non-nucleoside hepatitis C virus (HCV) polymerase inhibitor with potent in vitro activity. Safety, tolerability, pharmacokinetics and antiviral activity of VX-222 were assessed in a phase Ib/IIa multicenter, randomized, double-blinded, placebo-controlled, dose-ascending study in HCV-infected patients.
Methods: Treatment-naïve HCV genotype 1 patients were randomized to receive VX-222 at doses of 250 mg BID, 500 mg BID, 750 mg BID, 1500 mg QD or placebo for 3 days in a treatment:placebo ratio of 6:2 (8 patients/cohort).
Peginterferon-alfa-2a (P) plus ribavirin (R) was offered to patients at end of study (day 4) for up to 48 weeks, as judged appropriate by the investigator. PR treatment was discontinued in patients who had not experienced a ≥2 log10 HCV RNA level decline at week 12. VX-222 plasma levels were assessed at multiple time points over 12 hours, on days 1 and 3.
Results: Twenty-four patients were enrolled in the first three cohorts. VX-222 exposure increased in a dose-related manner. On day 3, the mean AUC0-12h and Cmax values were 19,490 ng*h/mL (CV 41%) and 2,959 ng/mL (CV 29%), 29,848 ng*h/mL (CV 54%) and 5,044 ng/mL (CV 36%), and 62,952 ng*h/mL (CV 112 %) and 10,288 ng/mL (CV 112%) for the 250 mg, 500 mg, and 750 mg BID groups, respectively. The mean HCV RNA decline achieved on day 4 with placebo, 250 mg, 500 mg, and 750 mg VX-222 BID was 0.1 log10 (range: 0.3 increase to 0.5 decline), 3.1 log10 (range: 2.0 to 4.2), 3.4 log10 (range: 3.2 to 3.6), and 3.2 log10 (range: 2.3 to 3.8), respectively. All AEs reported were mild to moderate, and the most frequently reported AEs by patients that received either active drug or placebo were diarrhea (25%), headache (20%) and nausea (12%). No clinically relevant laboratory abnormalities were reported.
Conclusions: VX-222 was well tolerated and a mean HCV RNA decline of >3 log10 at day 4 was observed in each cohort, Results of the fourth cohort evaluating QD dosing will also be presented. These Results support further evaluation of VX-222 in combination with peginterferon and ribavirin in the treatment of HCV.
IDENTIFICATION AND CHARACTERIZATION OF PPI-461, A POTENT AND SELECTIVE HCV NS5A INHIBITOR WITH ACTIVITY AGAINST ALL HCV GENOTYPES
R. Colonno, E. Peng, M. Bencsik, N. Huang, M. Zhong, A. Huq, Q. Huang, J. Williams, L. Li Presidio Pharmaceuticals, San Francisco, CA, USA. *rich@presidiopharma.com
Background: Several mechanistic classes of oral HCV inhibitors are currently undergoing clinical development. Inhibitors of NS5A have distinguished themselves by their picomolar potency and broad genotype spectrum in HCV replicon assays. Here we report the discovery and preclinical profile of PPI-461, a newly discovered NS5A inhibitor being advanced toward clinical studies.
Methods: Antiviral potency, combination and resistance studies utilized standard HCV replicon systems (genotype 1a and 1b). HCV spectrum studies employed stable or transient replicon assays with insertion of NS5A gene segments from other HCV genotypes (2a, 3a, 4a, 5a, 6a and 7a) into an HCV 1b backbone. Pharmacokinetic studies utilized IV and PO administration of PPI-461 in rats, monkeys and dogs. Extensive ADME and toxicology profiling has been performed in vitro and in rats and monkeys.
Results: PPI-461 was identified through an extensive medicinal chemistry effort and exhibits EC50s of 0.2 and 0.01 nM in HCV 1a and 1b replicon assays, respectively. Antiviral activity (EC50s of 0.1-19 nM) is maintained against all other HCV genotypes in transient/stable replicon assays. Cellular cytotoxicity levels (CC50) are >10 uM in several cell lines and no activity is observed against several other viruses (including BVDV) at 10 uM. PPI-461 is additive to synergistic (CIs 0.29-0.89) when combined with IFN, NS3 protease, NS5B nucleoside and non-nucleoside inhibitors. Resistance studies showed that variants with high levels of resistance could be generated, especially with genotype 1a, but required multiple amino acid substitutions within the NS5A gene. PPI-461 shows excellent stability in liver microsomes and no significant inhibition against P450 isozymes at 10 uM. PPI-461 exhibits good oral bioavailability in rats, monkeys and dogs, with enhanced liver concentrations and plasma half-lives suggestive of once daily dosing in humans. Toxicology studies in rats and monkeys showed that PPI-461 is well tolerated at plasma exposure levels >100,000-fold HCV 1a EC50 levels.
Conclusion: HCV NS5A protein plays a critical role in the HCV viral replicative cycle and is an attractive target for antiviral intervention. PPI-461 is a newly discovered selective and broadly potent inhibitor of HCV NS5A protein with a favorable preclinical profile supportive of advancement into clinical trial
ILIBININ AS A RESCUE TREATMENT FOR HCV-INFECTED PATIENTS SHOWING SUBOPTIMAL VIROLOGIC RESPONSE TO STANDARD COMBINATION THERAPY
M. Biermer, L. Stoehr, B. Schlosser, B. Fulop, F. van Bommel, T. Berg Medizinische Klinik mit Schwerpunkt Gastroenterologie und Hepatologie, Charite Campus Virchow, Berlin, Germany. *michael.biermer@charite.de
During standard antiviral treatment a significant proportion of hepatitis C patients show no complete virologic response despite a marked reduction of viral load in early stages of treatment (partial response). This minimal viremia may persist indicating the treatment termination as a consequence of viral nonresponse.
Recently, high-dose intravenous silibinin has demonstrated significant antiviral activity. We were therefore interested whether patients with partial virologic response can be rescued by the on-treatment addition of high-dose intravenous silibinin infusions.
Eleven patients who received different interferon-based antiviral regimens qualified for the rescue strategy and received silibinin infusions between December 2008 and September 2009. All patients had received peginterferon alfa 2a / ribavirin and four patients additionally received an HCV specific protease inhibitor for the first four weeks of treatment. 9 out of 11 patients showed a plateau of HCV RNA levels ranging from detectable but not quantifiable (< 15 IU/mL) to 2015 IU/mL for at least eight weeks on continuous treatment (total treatment duration 16-30 weeks). The remaining two patients suffered a virologic rebound after cessation of the protease inhibitor (HCV RNA 484 IU/ml at week 6 (patient 1) and 6890 IU/ml at week 5 (patient 2). Every patient received silibinin infusions (1400 mg/day - in 500 ml NaCl) on two consecutive days.
Eight out of eleven patients achieved a complete suppression of viral replication below the limit of detection within the first week after silibinin. Three patients did initially not respond to silibinin, two of the responders showed a viral breakthrough (8 and 14 weeks after silibinin administration during continued SOC treatment) and up to now six patients have undetectable HCV-RNA levels eight to 40 weeks after silibinin administration with continued SOC. Silibinin infusions were generally well tolerated. All patients reported a transient sensation of heat, three patients reported painful bowel-movements, two with a single episode of vomiting.
A two days administration of high-dose intravenous silibinin might be an interesting approach to rescue patients with ongoing minimal residual viremia while on interferon-based therapy. Further follow-up is needed to confirm whether the response induced is long lasting and leads to SVR.
RITONAVIR BOOSTING OF LOW DOSE RG7227/ITMN-191, HCV NS3/4A PROTEASE INHIBITOR, Results IN ROBUST REDUCTION IN HCV RNA AT LOWER EXPOSURES THAN PROVIDED BY UNBOOSTED REGIMENS
E. Gane1, R. Rouzier2, C. Stedman3, A. Wiercinska-Drapalo4, A. Horban4, L. Chang5, Y. Zhang5, P. Sampeur6, I. Najera7, N. Shulman7, P. Smith5, J. Tran5 1Auckland Clinical Studies, Auckland, New Zealand, 2Centre CAP, Montpellier, France, 3Christchurch Clinical Studies Trust, Christchurch, New Zealand, 4Department of Hepatology and Immunodeficiencies, Warsaw Medical University, 5Warsaw Medical University & Hospital of Infectious Diseases, Warsaw, Poland, 6Roche, South San Francisco, CA, 7Roche, Nutley, NJ, 8Roche, Palo Alto, CA, USA. *edgane@adhb.govt.nz
Background: Co-administration of low dose ritonavir resulted in larger increases in RG7227 trough concentration than area under the curve (AUC) and marginal increase in maximum concentration (Cmax), offering the opportunity to reduce the dose and/or dosing frequency of RG7227 in future Phase 2 studies. The present study evaluated the safety, tolerability, antiviral activity and pharmacokinetics (PK) of once-daily (QD) and twice-daily (BID) ritonavir boosted RG7227 (RG7227/r) in combination with standard of care (SOC) in treatment-naïve HCV genotype 1 patients.
Material & Methods: In a double-blind, placebo-controlled, Phase 1b multiple-ascending dose (MAD) study, treatment-naïve HCV genotype 1 patients were randomized to receive RG7227/r or placebo/r plus SOC for 15 days. RG7227/r regimens were 100/100mg BID (Cohort 1), 200/100mg QD (Cohort 2), and 200/100mg BID (Cohort 3). Blood samples for HCV RNA, PK, and resistance monitoring were collected at baseline and throughout the study.
Results: Cohort 1 and 2 provided blinded data for this abstract. The following table summarizes virologic response of RG7227/r compared to un-boosted 900 mg BID, the highest dose evaluated in a previous MAD study (placebo/r data are not shown due to small N of 2):
RG7227/r AUC and Cmax were approximately 6- to 9-fold and 4- to 10-fold lower, respectively, than previously observed for 900 mg BID un-boosted. Inter-patient PK variability for RG7227/r was also lower than historical un-boosted RG7227. RG7227/r and placebo/r plus SOC were generally well tolerated. Complete data for all 3 cohorts will be presented.
Conclusion: Relative to un-boosted 900 mg BID, ritonavir boosting of low dose RG7227 provides robust and sustained virologic response at significantly lower AUC and Cmax, parameters which are often correlated with safety. These Results indicate that further exploration of ritonavir boosting on the safety and efficacy of RG7227 is warranted.
VITAMIN D SUPPLEMENT IMPROVE SVR IN CHRONIC HEPATITIS C (GENOTYPE 1) NAïVE PATIENTS TREATED WITH PEG INTERFERON AND RIBAVIRIN
S. Abu Mouch1,2, Z. Fireman1,2, J. Jarchovsky1,2, N. Assy2,3 1Hepatology Unite, 2Internal Medicine B, Hillel Yaffe Medical Center, Hadera, 3Faculty of Medicine, Technion, Haifa, 4Gastroenterology, Hillel Yaffe Medical Center, Hadera, 5Liver Unit, Ziv Medical Center, Safed, Israel. *saif@hy.health.gov.il
Background: The combination therapy of Peg/RBV is considered the standard of care for chronic hepatitis C (HCV). A sustained viral response (SVR) is obtained in 40-50% of naïve HCV patients with genotype 1. Vitamin D is a potent immuno modulator whose impact on SVR of Peg/RBV based treatment of chronic HCV is unknown.
Aim: To assess whether the supplement of vitamin D to the conventional Peg/RBV therapy could improve SVR.
Methods: Fifty-eight patients with chronic HCV infection were randomized into two groups (intent-to-treat population): 27 (treatment group, age 47±11 yrs, body mass index [BMI] 27±4, 50% male) received pegylated-interferon-alpha2b (1.5 µg/kg once weekly) plus ribavirin (1000-1200 mg/daily) together with vitamin D3 (1000-4000 IU/daily, serum level >32 ng/ml), and 31 (controls, age 49±7 years, BMI 24±3, 60% male) received the same therapy without vitamin D. HCV RNA was assessed by RT-PCR (sensitivity, 50 IU/mL). Undetectable HCV RNA at week 12 and at week 24 post treatment (considered as complete EVR and SVR respectively).
Results: Demographics, disease characteristics, ethnicity, baseline biochemical parameters and adherence to treatment were similar in both groups. The treatment group had a higher mean BMI (27±4 vs 24±3; P< 0.01), viral load (68% vs 58%, P< 0.01), and fibrosis (Metavir scores >F2: 55% vs 18%, P< 0.001) than the controls. All but one treated patient (96%) and 48% (15/31) controls were HCV-RNA negative at week 12 (P< 0.0001). At week 24 post treatment (SVR): 86% (13/15) of treated patients and 41% (5/12) of controls were HCVRNA negative (P< 0.001). AEs were mostly mild and typical of Peg/RBV. There were no SAE.
Conclusions: Supplement of Vitamin D to conventional Peg/RBV therapy for naïve, genotype 1 patients with chronic infection significantly improve SVR.
SILEN-C2: EARLY ANTIVIRAL ACTIVITY AND SAFETY OF BI 201335 COMBINED WITH PEGINTERFERON ALFA-2A AND RIBAVIRIN (PEGIFN/RBV) IN CHRONIC HCV GENOTYPE-1 PATIENTS WITH NON-RESPONSE TO PEGIFN/RBV
M. Sulkowski1, M. Bourliere2, J.-P. Bronowicki3, A. Streinu-Cercel4, L. Preotescu4, T. Asselah5, J.-M. Pawlotsky6, S. Shafran7, S. Pol8, F.A. Caruntu4, S. Mauss9, D. Larrey10, C. Hafner11, Y. Datsenko11, J. Stern12, R. Kubiak11, G. Steinmann11 1Department of Viral Hepatitis, Johns Hopkins University, Baltimore, MD, USA, 2Hopital Saint Joseph, Marseille, 3Hopital de Brabois, Vandoeuvre Cedex, France, 4'Prof. Dr. Matei Bals' Institute of Infectious Diseases 1, Bucharest, Romania, 5Hopital Beaujon, Clichy Cedex, 6Hopital Henri Mondor, Creteil, France, 7University of Alberta, Edmonton, AB, Canada, 8Hopital Cochin, Paris, France, 9Center for HIV and Hepatogastroenterology, Dusseldorf, Germany, 10Hopital Saint-Eloi, Montpellier Cedex, France, 11Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach/ Ri, Germany, 12Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, USA. *msulkowski@jhmi.edu
Background and aims: BI 201335 is a potent HCV NS3/4A protease inhibitor being studied in phase IIb trials of chronic HCV genotype-1 (GT1) infection.
Methods: In a double-blind, randomized, parallel group design, HCV GT1 patients with confirmed non-response to at least 12 wks of PegIFN/RBV treatment were randomized 1:2:1 to (1) 240 mg BI 201335 once daily (QD), (2) 240 mg BI 201335 QD after a 3 day lead-in phase (LI) of PegIFN/RBV, and (3) 240 mg BI 201335 twice daily (BID) after a 3 day LI. Relapsers and patients with liver cirrhosis were excluded. In each group, treatment is for 24 wks with a Background of PegIFN (180 µg/wk) and RBV (1000/1200mg/d). Pre-specified Interim analysis after 12 weeks of therapy is reported. Viral rebound is defined as an increase in plasma HCV RNA ≥ 1 log10 on-treatment from nadir or confirmed increase ≥ 100 IU/ml if previously undetectable.
Results: 288 patients were treated (mean age 49 +/- 9 years; mean BMI 26.4 +/- 4.4 kg/m2; mean log10 HCV RNA at baseline 6.6 IU/mL). BI 201335 with PegIFN/RBV was overall well tolerated and demonstrated potent antiviral activity in all dose groups (Table). Mean ALT improved in all groups. 8% of patients prematurely discontinued treatment due to adverse events (AE). Most frequent AE were gastrointestinal disorders, mostly mild jaundice resulting from isolated unconjugated hyperbilirubinemia, (9.2%, 14.1% and 34.3% in groups 1, 2 and 3) and mostly mild to moderate rash or photosensitivity reactions (severe rash in 1.3, 0.7 and 5.7% in groups 1, 2 and 3).
Conclusions: SILEN-C2 confirmed robust antiviral activity with overall good tolerability and safety of BI 201335 especially if given 240mg once daily in combination with PegIFN/RBV in patients with chronic HCV GT-1 infection who did not respond to a previous course of PegIFN/RBV therapy.
Saturday, April 17, 2010
72% OF PATIENTS RECEIVING ANA598 IN PHASE II COMBINATION STUDY WITH INTERFERON AND RIBAVIRIN ACHIEVE UNDETECTABLE LEVELS OF VIRUS AT WEEK EIGHT
72% OF PATIENTS RECEIVING ANA598 IN PHASE II COMBINATION STUDY WITH INTERFERON AND RIBAVIRIN ACHIEVE UNDETECTABLE LEVELS OF VIRUS AT WEEK EIGHT
Absence of Viral Breakthrough and Favorable Safety Profile Confirm Previous Results
Data Being Presented Today at the 45th Annual EASL Meeting
LINKS: Anadys at the EASL Annual Meeting, Vienna, Austria, April 14-18, 2010
(not a webcast event)
* Key Data Slides from ANA598 Late-Breaker Poster
* Late-breaker poster presentation on ANA598 Phase II data: April 15, 2010
* Poster presentation on ANA598 preclinical data: April 16, 2010
SAN DIEGO, April 15, 2010 -- Anadys Pharmaceuticals, Inc. announced today that 72% of hepatitis C patients receiving ANA598 400 mg twice daily (bid) plus standard of care (SOC) achieved undetectable levels of virus at week eight in an ongoing Phase II study, compared to 38% of patients receiving placebo plus SOC.
The preliminary analysis of results through eight weeks also showed that ANA598 400 mg bid plus SOC was well tolerated, with an adverse event profile comparable to SOC alone. As seen before at 200 mg bid, no patient experienced viral breakthrough on ANA598.
“The data for ANA598 400 mg bid through eight weeks demonstrates potent antiviral activity and a favorable safety profile, as was seen previously at 200 mg bid,” said Steve Worland, Ph.D., President and CEO of Anadys. “The absence of viral breakthrough in either cohort to date demonstrates that non-nucleosides with superior pharmacokinetics, such as ANA598, can provide a substantial pharmacological barrier to resistance. Coupled with preclinical results that strongly support combinations with other direct antivirals of diverse mechanisms, we are very pleased that the clinical profile to date establishes ANA598 as an attractive agent to advance into Phase IIb development.”
In previously released results for the group receiving ANA598 200 mg bid plus SOC in this study, the high percentage of patients who achieved undetectable levels of virus at week eight (69%) was maintained through week 12, when 73% of patients achieved a complete Early Virological Response, or cEVR, as undetectable level of virus is referred to at 12 weeks. Dosing through 12 weeks in the 400 mg bid group and the control group is currently concluding, and Anadys expects to release antiviral response and safety results through week 12 for these groups in the latter half of May.
Preliminary Antiviral Response Assessment
No patient receiving ANA598 400 mg bid has experienced viral breakthrough (defined as a confirmed increase of >1 log from any prior measurement) as of the latest data available.
Preliminary Safety Assessment at 400 mg bid
Safety information is available as of a data snapshot that was taken once the last enrolled patient had received eight weeks of treatment. ANA598 400 mg bid demonstrated a favorable safety and tolerability profile through eight weeks, although conclusions regarding safety and tolerability cannot be made until results in more patients and potentially over longer duration are known.
The incidence of all adverse events was similar between the active and control groups, with reported adverse events being typical for patients treated with interferon and ribavirin. The incidence of rash was comparable between the ANA598 dose groups and also consistent with
historical reports of rash incidence due to interferon and ribavirin. Through eight weeks, 32% of patients (11/34) receiving ANA598 400 mg bid plus SOC developed rash, compared to 21% of patients at week four and 41% at week 12 for patients who received ANA598 200 mg bid plus SOC. At 400 mg bid, one patient discontinued ANA598 and SOC due to a grade 3 rash and one patient with a grade 1 rash discontinued ANA598. Safety laboratory values were comparable between the ANA598 and control arms.
EASL Presentations on ANA598
The data from the ongoing Phase II study is being presented today in a late-breaker poster
presentation titled “Safety and Antiviral Activity of ANA598 in Combination with Pegylated
Interferon alpha-2A Plus Ribavirin in Treatment-Naïve Genotype 1 Chronic HCV Patients” at the 45th Annual Meeting of the European Association for the Study of the Liver (EASL) in Vienna, Austria. Data through eight weeks is being presented for the group receiving ANA598 400 mg bid plus SOC as well as for the group receiving placebo plus SOC. Data through the entire 12 weeks of ANA598 dosing is being presented for the group that received ANA598 200 mg bid plus SOC.
The late breaker poster and slides of key data excerpted from the poster can be accessed on the Company’s website at www.anadyspharma.com.
In addition to the data from the Phase II combination study, Anadys will also present data on the preclinical profile of ANA598 at the EASL meeting on Friday, April 16, 2010. In a poster titled “Enhanced In Vitro Antiviral Activity of ANA598 in Combination with Other Anti-HCV Agents Support Combination Treatment”, Anadys will present preclinical data showing enhanced antiviral activity and suppression of resistance when ANA598 is combined in vitro with other anti-HCV agents that act through diverse mechanisms, including protease inhibition, polymerase inhibition (both nucleoside and non-nucleoside inhibitors) and inhibition of host functions. As of April 16, 2010, the poster will be accessible on the Company’s website at
www.anadyspharma.com.
Percentage of Patients with Undetectable Levels of Virus by Week
Percentage of Patients with Undetectable Virus (<15 IU/mL) by Study Week
Summary of Reported AEs from Ongoing Phase II Study*
Phase II Combination Study
In the ongoing Phase II study, a total of approximately 90 treatment-naïve genotype 1 patients are to receive ANA598 or placebo in combination with Pegasys® (peginterferon alfa-2a) and Copegus® (ribavirin, USP) for 12 weeks at dose levels of 200 mg bid or 400 mg bid, each with a loading dose of 800 mg bid on day one. After week 12, patients are to continue receiving SOC.
Patients who achieve undetectable levels of virus at weeks 4 and 12 will be randomized to stop all treatment at week 24 or 48. The primary endpoint of the study is the proportion of patients who achieve undetectable levels of virus at week 12 (defined as complete Early Virological Response, or cEVR). Additional endpoints include safety and tolerability as well as the proportion of patients with undetectable levels of virus at week 4 (defined as Rapid Virological Response, or RVR). Patients will be followed for 24 weeks after stopping therapy to determine the rate of
Sustained Virological Response, or SVR. Approximately 90 patients have been enrolled in this study – with approximately 30 patients receiving ANA598 plus SOC at each dose level and 30 patients receiving placebo plus SOC. The study is being managed by the Duke Clinical Research Institute (DCRI) under the leadership of John McHutchison, M.D. and is being conducted at a number of clinical sites in the United States.
About ANA598
ANA598 is a non-nucleoside inhibitor of the HCV RNA polymerase and is wholly owned by
Anadys. Anadys has completed three Phase I clinical studies of ANA598 that have demonstrated potent antiviral activity and good tolerability. In a monotherapy study in treatment-naïve genotype 1 patients, treatment with ANA598 for three days led to median end-of-treatment declines in viral load ranging from 2.4 to 2.9 log10 in three separate dose groups. No patient at any dose level showed evidence of viral breakthrough while on ANA598, and there were no serious adverse events. Those patients from the monotherapy study who subsequently received pegylated interferon and ribavirin all exhibited further viral load decline, demonstrating that viral variants revealed by brief treatment with ANA598 remain susceptible to current SOC, consistent with prior in vitro results.
Anadys has completed two long-term chronic toxicology studies of ANA598 (26 weeks duration in rats and 39 weeks duration in monkeys). The No Observed Adverse Effect Level, or NOAEL, is 1000 mg/kg, the highest dose tested, in both the rat and monkey. The completed toxicology studies support the ongoing Phase II clinical study as well as future clinical studies of longer duration.
Anadys has presented in vitro data supporting the use of ANA598 in combination with interferonalpha as well as with direct antivirals currently in development. In particular, data has shown that ANA598 is synergistic in vitro with interferon-alpha as well as representative HCV protease and polymerase inhibitors. In vitro combination treatment at clinically relevant concentrations of interferon-alpha and ANA598 results in clearance of HCV RNA from cells rather than selection of resistant isolates. Furthermore, ANA598 retains full activity in vitro against mutations conferring resistance to protease inhibitors, nucleoside polymerase inhibitors and non-nucleoside polymerase inhibitors that act at binding sites distinct from that of ANA598, while protease and nucleoside polymerase inhibitors retain full activity in vitro against mutations conferring resistance to ANA598.
ANA598 has received Fast Track Status from the FDA for the treatment of chronic hepatitis C.
Absence of Viral Breakthrough and Favorable Safety Profile Confirm Previous Results
Data Being Presented Today at the 45th Annual EASL Meeting
LINKS: Anadys at the EASL Annual Meeting, Vienna, Austria, April 14-18, 2010
(not a webcast event)
* Key Data Slides from ANA598 Late-Breaker Poster
* Late-breaker poster presentation on ANA598 Phase II data: April 15, 2010
* Poster presentation on ANA598 preclinical data: April 16, 2010
SAN DIEGO, April 15, 2010 -- Anadys Pharmaceuticals, Inc. announced today that 72% of hepatitis C patients receiving ANA598 400 mg twice daily (bid) plus standard of care (SOC) achieved undetectable levels of virus at week eight in an ongoing Phase II study, compared to 38% of patients receiving placebo plus SOC.
The preliminary analysis of results through eight weeks also showed that ANA598 400 mg bid plus SOC was well tolerated, with an adverse event profile comparable to SOC alone. As seen before at 200 mg bid, no patient experienced viral breakthrough on ANA598.
“The data for ANA598 400 mg bid through eight weeks demonstrates potent antiviral activity and a favorable safety profile, as was seen previously at 200 mg bid,” said Steve Worland, Ph.D., President and CEO of Anadys. “The absence of viral breakthrough in either cohort to date demonstrates that non-nucleosides with superior pharmacokinetics, such as ANA598, can provide a substantial pharmacological barrier to resistance. Coupled with preclinical results that strongly support combinations with other direct antivirals of diverse mechanisms, we are very pleased that the clinical profile to date establishes ANA598 as an attractive agent to advance into Phase IIb development.”
In previously released results for the group receiving ANA598 200 mg bid plus SOC in this study, the high percentage of patients who achieved undetectable levels of virus at week eight (69%) was maintained through week 12, when 73% of patients achieved a complete Early Virological Response, or cEVR, as undetectable level of virus is referred to at 12 weeks. Dosing through 12 weeks in the 400 mg bid group and the control group is currently concluding, and Anadys expects to release antiviral response and safety results through week 12 for these groups in the latter half of May.
Preliminary Antiviral Response Assessment
No patient receiving ANA598 400 mg bid has experienced viral breakthrough (defined as a confirmed increase of >1 log from any prior measurement) as of the latest data available.
Preliminary Safety Assessment at 400 mg bid
Safety information is available as of a data snapshot that was taken once the last enrolled patient had received eight weeks of treatment. ANA598 400 mg bid demonstrated a favorable safety and tolerability profile through eight weeks, although conclusions regarding safety and tolerability cannot be made until results in more patients and potentially over longer duration are known.
The incidence of all adverse events was similar between the active and control groups, with reported adverse events being typical for patients treated with interferon and ribavirin. The incidence of rash was comparable between the ANA598 dose groups and also consistent with
historical reports of rash incidence due to interferon and ribavirin. Through eight weeks, 32% of patients (11/34) receiving ANA598 400 mg bid plus SOC developed rash, compared to 21% of patients at week four and 41% at week 12 for patients who received ANA598 200 mg bid plus SOC. At 400 mg bid, one patient discontinued ANA598 and SOC due to a grade 3 rash and one patient with a grade 1 rash discontinued ANA598. Safety laboratory values were comparable between the ANA598 and control arms.
EASL Presentations on ANA598
The data from the ongoing Phase II study is being presented today in a late-breaker poster
presentation titled “Safety and Antiviral Activity of ANA598 in Combination with Pegylated
Interferon alpha-2A Plus Ribavirin in Treatment-Naïve Genotype 1 Chronic HCV Patients” at the 45th Annual Meeting of the European Association for the Study of the Liver (EASL) in Vienna, Austria. Data through eight weeks is being presented for the group receiving ANA598 400 mg bid plus SOC as well as for the group receiving placebo plus SOC. Data through the entire 12 weeks of ANA598 dosing is being presented for the group that received ANA598 200 mg bid plus SOC.
The late breaker poster and slides of key data excerpted from the poster can be accessed on the Company’s website at www.anadyspharma.com.
In addition to the data from the Phase II combination study, Anadys will also present data on the preclinical profile of ANA598 at the EASL meeting on Friday, April 16, 2010. In a poster titled “Enhanced In Vitro Antiviral Activity of ANA598 in Combination with Other Anti-HCV Agents Support Combination Treatment”, Anadys will present preclinical data showing enhanced antiviral activity and suppression of resistance when ANA598 is combined in vitro with other anti-HCV agents that act through diverse mechanisms, including protease inhibition, polymerase inhibition (both nucleoside and non-nucleoside inhibitors) and inhibition of host functions. As of April 16, 2010, the poster will be accessible on the Company’s website at
www.anadyspharma.com.
Percentage of Patients with Undetectable Levels of Virus by Week
Percentage of Patients with Undetectable Virus (<15 IU/mL) by Study Week
Summary of Reported AEs from Ongoing Phase II Study*
Phase II Combination Study
In the ongoing Phase II study, a total of approximately 90 treatment-naïve genotype 1 patients are to receive ANA598 or placebo in combination with Pegasys® (peginterferon alfa-2a) and Copegus® (ribavirin, USP) for 12 weeks at dose levels of 200 mg bid or 400 mg bid, each with a loading dose of 800 mg bid on day one. After week 12, patients are to continue receiving SOC.
Patients who achieve undetectable levels of virus at weeks 4 and 12 will be randomized to stop all treatment at week 24 or 48. The primary endpoint of the study is the proportion of patients who achieve undetectable levels of virus at week 12 (defined as complete Early Virological Response, or cEVR). Additional endpoints include safety and tolerability as well as the proportion of patients with undetectable levels of virus at week 4 (defined as Rapid Virological Response, or RVR). Patients will be followed for 24 weeks after stopping therapy to determine the rate of
Sustained Virological Response, or SVR. Approximately 90 patients have been enrolled in this study – with approximately 30 patients receiving ANA598 plus SOC at each dose level and 30 patients receiving placebo plus SOC. The study is being managed by the Duke Clinical Research Institute (DCRI) under the leadership of John McHutchison, M.D. and is being conducted at a number of clinical sites in the United States.
About ANA598
ANA598 is a non-nucleoside inhibitor of the HCV RNA polymerase and is wholly owned by
Anadys. Anadys has completed three Phase I clinical studies of ANA598 that have demonstrated potent antiviral activity and good tolerability. In a monotherapy study in treatment-naïve genotype 1 patients, treatment with ANA598 for three days led to median end-of-treatment declines in viral load ranging from 2.4 to 2.9 log10 in three separate dose groups. No patient at any dose level showed evidence of viral breakthrough while on ANA598, and there were no serious adverse events. Those patients from the monotherapy study who subsequently received pegylated interferon and ribavirin all exhibited further viral load decline, demonstrating that viral variants revealed by brief treatment with ANA598 remain susceptible to current SOC, consistent with prior in vitro results.
Anadys has completed two long-term chronic toxicology studies of ANA598 (26 weeks duration in rats and 39 weeks duration in monkeys). The No Observed Adverse Effect Level, or NOAEL, is 1000 mg/kg, the highest dose tested, in both the rat and monkey. The completed toxicology studies support the ongoing Phase II clinical study as well as future clinical studies of longer duration.
Anadys has presented in vitro data supporting the use of ANA598 in combination with interferonalpha as well as with direct antivirals currently in development. In particular, data has shown that ANA598 is synergistic in vitro with interferon-alpha as well as representative HCV protease and polymerase inhibitors. In vitro combination treatment at clinically relevant concentrations of interferon-alpha and ANA598 results in clearance of HCV RNA from cells rather than selection of resistant isolates. Furthermore, ANA598 retains full activity in vitro against mutations conferring resistance to protease inhibitors, nucleoside polymerase inhibitors and non-nucleoside polymerase inhibitors that act at binding sites distinct from that of ANA598, while protease and nucleoside polymerase inhibitors retain full activity in vitro against mutations conferring resistance to ANA598.
ANA598 has received Fast Track Status from the FDA for the treatment of chronic hepatitis C.
59 Percent of Patients Overall Achieved SVR with Telaprevir-Based Regimens in Study 107 After Not Achieving SVR with at Least One try
59 Percent of Patients Overall Achieved SVR with Telaprevir-Based Regimens in Study 107 After Not Achieving SVR with at Least One try
Posted on: Thu, 15 Apr 2010 09:45:00 EDT
VIENNA, Apr 15, 2010 (BUSINESS WIRE) --
---97% of prior treatment relapsers and 55% of prior treatment partial responders achieved SVR with 24-week or 48-week telaprevir-based regimens-
In conjunction with an oral presentation at the 45th Annual Meeting of the European Association for the Study of the Liver (EASL) in Vienna, Vertex Pharmaceuticals Incorporated (Nasdaq: VRTX | Quote | Chart | News | PowerRating) today announced that 59 percent of patients overall who received a telaprevir-based combination regimen in Study 107 achieved a sustained viral response (SVR) after failing to achieve SVR with a least one prior course of treatment for hepatitis C virus (HCV) infection. Specifically, 56% of prior treatment null responders (n=27) achieved SVR after treatment with a 48-week telaprevir-based combination regimen, and 97% of prior treatment relapsers (n=29) and 55% of prior treatment partial responders (n=29) achieved SVR after treatment with a 24-week or 48-week telaprevir-based combination regimen. Ten patients (9%, n=117) discontinued all therapy due to adverse events, with rash being the most common reason for discontinuation.
Study 107 was an open-label Phase 2 rollover study of patients who did not achieve SVR after receiving pegylated interferon (Peg-IFN) and ribavirin (RBV) in the control arms of the Phase 2 PROVE trials of telaprevir. Telaprevir is an investigational oral HCV protease inhibitor being developed by Vertex in collaboration with Tibotec and Mitsubishi Tanabe Pharma. A Phase 3 registration program for telaprevir is nearing completion, in both treatment-naive and treatment-failure HCV patients. The Phase 3 REALIZE trial is evaluating a 48-week telaprevir-based treatment regimen in treatment-failure patients, including null responder patients. In the second half of 2010, Vertex plans to submit a New Drug Application to the U.S. Food and Drug Administration (FDA) for telaprevir for both treatment-naive and treatment-failure patients.
"The majority of genotype 1 patients who undergo their first regimen of pegylated-interferon and ribavirin fail to achieve SVR and are left with few options for subsequent re-treatment of their disease," said Thomas Berg, M.D., Medical Development, Hepatology Section, University Clinic, Leipzig, Germany. "Treatment with telaprevir-based regimens in Study 107 resulted in an overall SVR rate of 59 percent across all patients enrolled in the study, with 56 percent of the most difficult-to-treat null responder patients achieving SVR with a 48-week telaprevir-based regimen."
"Study 107 provided important insight into the potential future use of telaprevir-based regimens in the treatment of patients who failed to respond to currently approved therapies," said Robert Kauffman, M.D., Ph.D., Vertex's Senior Vice President, Clinical Development and Chief Medical Officer. "Based on information generated in Study 107, as well as data from the PROVE 3 clinical trial, we believe that a 48-week treatment regimen may increase the likelihood that certain treatment-failure patients are able to achieve SVR. In our Phase 3 REALIZE trial in treatment-failure patients, we are evaluating a 48-week treatment regimen and are currently awaiting final SVR results, which we expect in the third quarter."
Study 107 Design and Results
Study 107 was an open-label, Phase 2 rollover study of telaprevir in combination with Peg-IFN and RBV in patients who had previously received treatment with Peg-IFN and RBV in the control arms of either of the PROVE 1, PROVE 2 or PROVE 3 trials, and did not achieve SVR. Patients in Study 107 were well-characterized as null responders, partial responders, relapsers or breakthroughs, based on their antiviral response documented as a result of their participation in the control arms of the PROVE clinical trials.
When Study 107 began, all patients were to receive 12 weeks of telaprevir in combination with Peg-IFN and RBV followed by an additional 12 weeks of Peg-IFN and RBV, for a total of 24 weeks of therapy. Stopping rules required any patient who did not achieve HCV RNA of 25 IU/mL or less by week 4 to stop all treatment. In 2008, Study 107 was amended and underwent several changes, most notably to the duration of treatment. The changes to treatment duration in Study 107 were aimed at providing patients with a higher likelihood of achieving SVR. Following the amendments, only patients who did not achieve HCV RNA of 100 IU/mL or less at week 4 were required to stop therapy. In addition, prior treatment null responder patients were to receive a 48-week telaprevir-based treatment regimen. Patients with prior treatment relapse, prior treatment viral breakthrough and prior treatment partial response were eligible to rece ive a response-guided 24-week telaprevir-based treatment regimen if they achieved undetectable HCV RNA at week 4 and 12, otherwise these patients would receive a 48-week regimen.
A total of 117 patients enrolled in Study 107, including 51 patients with prior treatment null response, 29 patients with prior treatment partial response, 8 patients with prior treatment viral breakthrough, and 29 patients with prior treatment relapse.
An overall SVR rate of 59 percent and an overall relapse rate of 16 percent were observed in Study 107. Sustained viral response rates for each arm of Study 107 are as follows:
see link to study above with all the data presented.
Study 107 Safety and Tolerability
Adverse events reported in Study 107 were similar to those reported in prior Phase 2 trials of telaprevir. The most common adverse events reported were rash (all types), fatigue, pruritus, and headache. Discontinuation of all therapy due to adverse events occurred in 10 patients (9%; n=117), with rash being the most common reason for discontinuation.
About Telaprevir
Telaprevir is an investigational, oral inhibitor of HCV protease, an enzyme essential for viral replication, and is being evaluated as part of a global Phase 3 registration program in more than 2,200 treatment-naive and treatment-failure patients. Vertex is collaborating with Tibotec and Mitsubishi Tanabe Pharma to develop telaprevir. Vertex retains commercial rights to telaprevir in North America. Tibotec has rights to commercialize telaprevir in Europe, South America, Australia, the Middle East and other countries. Mitsubishi Tanabe Pharma has rights to commercialize telaprevir in Japan and certain Far East countries.
Posted on: Thu, 15 Apr 2010 09:45:00 EDT
VIENNA, Apr 15, 2010 (BUSINESS WIRE) --
---97% of prior treatment relapsers and 55% of prior treatment partial responders achieved SVR with 24-week or 48-week telaprevir-based regimens-
In conjunction with an oral presentation at the 45th Annual Meeting of the European Association for the Study of the Liver (EASL) in Vienna, Vertex Pharmaceuticals Incorporated (Nasdaq: VRTX | Quote | Chart | News | PowerRating) today announced that 59 percent of patients overall who received a telaprevir-based combination regimen in Study 107 achieved a sustained viral response (SVR) after failing to achieve SVR with a least one prior course of treatment for hepatitis C virus (HCV) infection. Specifically, 56% of prior treatment null responders (n=27) achieved SVR after treatment with a 48-week telaprevir-based combination regimen, and 97% of prior treatment relapsers (n=29) and 55% of prior treatment partial responders (n=29) achieved SVR after treatment with a 24-week or 48-week telaprevir-based combination regimen. Ten patients (9%, n=117) discontinued all therapy due to adverse events, with rash being the most common reason for discontinuation.
Study 107 was an open-label Phase 2 rollover study of patients who did not achieve SVR after receiving pegylated interferon (Peg-IFN) and ribavirin (RBV) in the control arms of the Phase 2 PROVE trials of telaprevir. Telaprevir is an investigational oral HCV protease inhibitor being developed by Vertex in collaboration with Tibotec and Mitsubishi Tanabe Pharma. A Phase 3 registration program for telaprevir is nearing completion, in both treatment-naive and treatment-failure HCV patients. The Phase 3 REALIZE trial is evaluating a 48-week telaprevir-based treatment regimen in treatment-failure patients, including null responder patients. In the second half of 2010, Vertex plans to submit a New Drug Application to the U.S. Food and Drug Administration (FDA) for telaprevir for both treatment-naive and treatment-failure patients.
"The majority of genotype 1 patients who undergo their first regimen of pegylated-interferon and ribavirin fail to achieve SVR and are left with few options for subsequent re-treatment of their disease," said Thomas Berg, M.D., Medical Development, Hepatology Section, University Clinic, Leipzig, Germany. "Treatment with telaprevir-based regimens in Study 107 resulted in an overall SVR rate of 59 percent across all patients enrolled in the study, with 56 percent of the most difficult-to-treat null responder patients achieving SVR with a 48-week telaprevir-based regimen."
"Study 107 provided important insight into the potential future use of telaprevir-based regimens in the treatment of patients who failed to respond to currently approved therapies," said Robert Kauffman, M.D., Ph.D., Vertex's Senior Vice President, Clinical Development and Chief Medical Officer. "Based on information generated in Study 107, as well as data from the PROVE 3 clinical trial, we believe that a 48-week treatment regimen may increase the likelihood that certain treatment-failure patients are able to achieve SVR. In our Phase 3 REALIZE trial in treatment-failure patients, we are evaluating a 48-week treatment regimen and are currently awaiting final SVR results, which we expect in the third quarter."
Study 107 Design and Results
Study 107 was an open-label, Phase 2 rollover study of telaprevir in combination with Peg-IFN and RBV in patients who had previously received treatment with Peg-IFN and RBV in the control arms of either of the PROVE 1, PROVE 2 or PROVE 3 trials, and did not achieve SVR. Patients in Study 107 were well-characterized as null responders, partial responders, relapsers or breakthroughs, based on their antiviral response documented as a result of their participation in the control arms of the PROVE clinical trials.
When Study 107 began, all patients were to receive 12 weeks of telaprevir in combination with Peg-IFN and RBV followed by an additional 12 weeks of Peg-IFN and RBV, for a total of 24 weeks of therapy. Stopping rules required any patient who did not achieve HCV RNA of 25 IU/mL or less by week 4 to stop all treatment. In 2008, Study 107 was amended and underwent several changes, most notably to the duration of treatment. The changes to treatment duration in Study 107 were aimed at providing patients with a higher likelihood of achieving SVR. Following the amendments, only patients who did not achieve HCV RNA of 100 IU/mL or less at week 4 were required to stop therapy. In addition, prior treatment null responder patients were to receive a 48-week telaprevir-based treatment regimen. Patients with prior treatment relapse, prior treatment viral breakthrough and prior treatment partial response were eligible to rece ive a response-guided 24-week telaprevir-based treatment regimen if they achieved undetectable HCV RNA at week 4 and 12, otherwise these patients would receive a 48-week regimen.
A total of 117 patients enrolled in Study 107, including 51 patients with prior treatment null response, 29 patients with prior treatment partial response, 8 patients with prior treatment viral breakthrough, and 29 patients with prior treatment relapse.
An overall SVR rate of 59 percent and an overall relapse rate of 16 percent were observed in Study 107. Sustained viral response rates for each arm of Study 107 are as follows:
see link to study above with all the data presented.
Study 107 Safety and Tolerability
Adverse events reported in Study 107 were similar to those reported in prior Phase 2 trials of telaprevir. The most common adverse events reported were rash (all types), fatigue, pruritus, and headache. Discontinuation of all therapy due to adverse events occurred in 10 patients (9%; n=117), with rash being the most common reason for discontinuation.
About Telaprevir
Telaprevir is an investigational, oral inhibitor of HCV protease, an enzyme essential for viral replication, and is being evaluated as part of a global Phase 3 registration program in more than 2,200 treatment-naive and treatment-failure patients. Vertex is collaborating with Tibotec and Mitsubishi Tanabe Pharma to develop telaprevir. Vertex retains commercial rights to telaprevir in North America. Tibotec has rights to commercialize telaprevir in Europe, South America, Australia, the Middle East and other countries. Mitsubishi Tanabe Pharma has rights to commercialize telaprevir in Japan and certain Far East countries.
Vitamin D is helpful with Liver Fibrosis
"We have shown that the biochemical profile of G1 CHC patients is characterized by lower-than-normal serum 25(OH)D levels, and that a low 25(OH)D level is independently related to severe fibrosis and a low likelihood of SVR after standard-of-care antiviral therapy.....Our study shows that low 25(OH)D levels are independently associated with female sex and with severity of necroinflammatory activity....This study also offers the first evidence that low 25(OH)D serum levels, together with known risk factors for fibrosis severity, such as older age, low cholesterol levels, and high necroinflammatory activity,[26] are independently associated with the presence of severe fibrosis......It is conceivable that a reduced 25(OH)D level might by itself favor progression of fibrosis. Different experimental models showed that vitamin D, via interaction with vitamin D receptor, protects against oxidative stress production,[29] can influence the migration, proliferation, and gene expression of fibroblasts,[30][31] and reduces the inflammatory and fibrogenic activity of liver stellate cells.....However further prospective cohort studies will be needed to determine the causal association between vitamin D deficiency and fibrosis in patients with CHC....Another interesting finding of this study is the evidence that lower 25(OH)D serum levels were an independent negative risk factor for SVR....this observation will require further validation in different, large cohorts of patients, but is supported by experimental data that suggest a role of vitamin D in the modulation of the immune response,[32][33] and by recent clinical data[36] reporting higher early virological response rate in CHC treated with standard of care plus vitamin D, compared with those treated with standard of care only"
Low vitamin D serum level is related to severe fibrosis and low responsiveness to interferon-based therapy in genotype 1 chronic hepatitis C
Hepatology April 2010
Salvatore Petta 1 2 *, Calogero Cammà 1 3, Concetta Scazzone 4, Claudio Tripodo 5, Vito Di Marco 1, Antonino Bono 4, Daniela Cabibi 5, Giusalba Licata 1, Rossana Porcasi 5, Giulio Marchesini 6, Antonio Craxí 1
1Cattedra ed Unità Operativa di Gastroenterologia, DiBiMIS, University of Palermo, Italy
2Dipartimento di Biopatologia e Metodologie Biomediche, University of Palermo, Italy
3IBIM Consiglio Nazionale delle Ricerche, Palermo, Italy
4Dipartimento di Biotecnologie Mediche e Medicina Legale Sezione Chimica e Biochimica Medica, University of Palermo, Italy
5Cattedra di Anatomia Patologica, University of Palermo, Italy
6Dipartimento di Medicina e Gastroenterologia, Alma Mater Studiorum, University of Bologna, Italy
email: Salvatore Petta (petsa@inwind.it)
*Correspondence to Salvatore Petta, Gastroenterologia & Epatologia, Piazza delle Cliniche, 2, 90127 Palermo, Italy
Abstract
25-Hydroxyvitamin D (25[OH]D) can potentially interfere with inflammatory response and fibrogenesis. Its role in disease progression in chronic hepatitis C (CHC) and its relation with histological and sustained virological response (SVR) to therapy are unknown. One hundred ninety-seven patients with biopsy-proven genotype 1 (G1) CHC and 49 healthy subjects matched by age and sex were consecutively evaluated. One hundred sixty-seven patients underwent antiviral therapy with pegylated interferon plus ribavirin. The 25(OH)D serum levels were measured by high-pressure liquid chromatography. Tissue expression of cytochrome (CY) P27A1 and CYP2R1, liver 25-hydroxylating enzymes, were assessed by immunochemistry in 34 patients with CHC, and in eight controls.
The 25(OH)D serum levels were significantly lower in CHC than in controls (25.07 ± 9.92 g/L versus 43.06 ± 10.19; P < 0.001). Lower levels of 25(OH)D were independently linked to female sex (P = 0.007) and necroinflammation (P = 0.04) by linear regression analysis. CYP27A1, but not CYP2R1, was directly related to 25(OH)D levels (P = 0.01), and inversely to necroinflammation (P = 0.01). Low 25(OH)D (odds ratio [OR], 0.942; 95% confidence interval [CI], 0.893-0.994) and cholesterol (OR, 0.981; 95%CI, 0.969-0.992) levels, older age (OR, 1.043; 95%CI, 1.002-1.085), high ferritin (OR, 1.003; 95%CI, 1.001-1.005), and necroinflammation (OR, 2.235; 95%CI, 1.014-4.929) were independently associated with severe fibrosis (F3-F4) by multivariate logistic analysis. Seventy patients (41%) achieved SVR. By multivariate analysis, hepatic steatosis (OR, 0.971; 95%CI, 0.944-0.999), lower cholesterol (OR, 1.009; 95% CI, 1.000-1.018), and 25(OH)D levels (OR, 1.039; 95%CI, 1.002-1.077) were independently associated with no SVR. Conclusion: G1 CHC patients had low 25(OH)D serum levels, possibly because of reduced CYP27A1 expression. Low vitamin D is linked to severe fibrosis and low SVR on interferon (IFN)-based therapy.
the activated hormonal form of vitamin D, 1-25-dihydroxyvitamin D, is essential for calcium and bone homeostasis.[1][2] Vitamin D 25 and 1alpha-hydroxylation occurs in the liver and in the kidney, respectively, involving different isoforms of cytochrome P450 (CYP), namely CYP2R1, CYP27A1, and others in the liver, and CYP27B1 in the kidney.[3]
Vitamin D deficiency is associated with many common and serious pathological conditions, including cancer, autoimmune disease, cardiovascular disease, insulin resistance (IR), and diabetes.[1][4][5] There is also an association between vitamin D status and both cholestatic and noncholestatic chronic liver diseases.[6-10] In patients with noncholestatic chronic hepatitis and cirrhosis, some studies[8-10] have reported normal serum levels of 25-hydroxyvitamin D (25[OH]D), the liver-hydroxylated form of vitamin D and the best estimate of overall vitamin D status.[1][2] Conversely, other studies have found low serum 25(OH)D levels in patients with chronic hepatitis and cirrhosis of different origins.[11-13] Low 25(OH)D levels have been related to poor liver function because of the association between vitamin D status and hepatic function indexes[11] or the stage of cirrhosis.[11][14][15] In keeping with these studies, several reports describe reduced bone mineral density in patients with chronic liver disease[8][9][13][15][17][18] and cirrhosis.[9][10][12][13][19] Recently, Targher et al.[18] observed lower 25(OH)D serum levels in patients with biopsy-proven nonalcoholic fatty liver disease, identifying an independent association between the histological characteristics of nonalcoholic fatty liver disease and low 25(OH)D levels. Experimental evidence also suggested the potential ability of vitamin D, through interaction with its nuclear receptor (vitamin D receptor), to interfere with inflammatory response and fibrogenesis.[4]
The aim of our study was to evaluate serum levels of 25(OH)D in a cohort of patients with biopsy-proven genotype 1 (G1) chronic hepatitis C (CHC), and to investigate the potential relationships between 25(OH)D, the histological features of disease, and the response to antiviral therapy.
Abbreviations
25(OH)D, 25-hydroxyvitamin D; ALT, alanine aminotransferase; AUC, area under the curve; CHC, chronic hepatitis C; CI, confidence interval; CY, cytochrome; G1, genotype 1; GGT, gamma-glutamyltransferase; HCV, hepatitis C virus; IR, insulin response; OR, odds ratio; SVR, sustained virological response.
Discussion
We have shown that the biochemical profile of G1 CHC patients is characterized by lower-than-normal serum 25(OH)D levels, and that a low 25(OH)D level is independently related to severe fibrosis and a low likelihood of SVR after standard-of-care antiviral therapy.
Lower levels of serum 25(OH)D have been previously reported in populations heterogeneous for cause and severity of chronic liver disease.[11][19] We confirmed a 25(OH)D reduction in a homogeneous cohort of patients with G1 CHC, at low prevalence of F4 fibrosis. Although a significant trend in 25(OH)D levels reduction was observed with increasing stage of fibrosis, a significant reduction was also observed in the subgroup of patients with mild fibrosis (F1), making it unlikely that low 25(OH)D levels could be entirely explained by reduced liver function.
Our study shows that low 25(OH)D levels are independently associated with female sex and with severity of necroinflammatory activity. Although the study was not designed to clarify the correlation between female sex and lower 25(OH)D levels, because of the observed reduction in women older than 55 years, but not in men of the same age range, and because of the significant interaction between sex and age, we can speculate that hormonal alterations in postmenopausal women likely modulate the vitamin D status. Our results also underline an inverse relationship between 25(OH)D and the severity of necroinflammatory activity. The cross-sectional design of our study is unable to dissect the temporal relation between changes in 25(OH)D and necroinflammation. However, CYP27A1 liver expression was directly related to serum 25(OH)D levels, and inversely associated with the severity of necroinflammatory activity. Therefore, the hepatic necroinflammatory activity caused by the HCV infection could be responsible for 25 (OH)D levels reduction by different mechanisms, such as a selectively reduced liver expression of enzymes involved in liver hydroxylation of vitamin D3.
This study also offers the first evidence that low 25(OH)D serum levels, together with known risk factors for fibrosis severity, such as older age, low cholesterol levels, and high necroinflammatory activity,[26] are independently associated with the presence of severe fibrosis. We were not able to confirm IR as a risk factor for fibrosis severity, as reported by others.[26][27] The lack of this association could be attributable to differences in the mean age, alcohol use, and prevalence of obesity and diabetes. In any case we confirmed the important effect of metabolic alterations in fibrosis severity, providing evidence for an association between severe fibrosis and high ferritin levels, a surrogate marker of IR and metabolic syndrome.[28]
Interestingly, considering only noninvasive parameters, the AUC of the model that includes vitamin D levels to predict severe fibrosis remains good. This suggests the potential use of serum 25(OH)D levels as a noninvasive marker of liver fibrosis, a use that needs to be tested and validated in large prospective cohort studies in patients with CHC of all genotypes, and in chronic liver disease of other origins.
It is conceivable that a reduced 25(OH)D level might by itself favor progression of fibrosis. Different experimental models showed that vitamin D, via interaction with vitamin D receptor, protects against oxidative stress production,[29] can influence the migration, proliferation, and gene expression of fibroblasts,[30][31] and reduces the inflammatory and fibrogenic activity of liver stellate cells.[32][33] However further prospective cohort studies will be needed to determine the causal association between vitamin D deficiency and fibrosis in patients with CHC.
Another interesting finding of this study is the evidence that lower 25(OH)D serum levels were an independent negative risk factor for SVR. Again, this observation will require further validation in different, large cohorts of patients, but is supported by experimental data that suggest a role of vitamin D in the modulation of the immune response,[32][33] and by recent clinical data[36] reporting higher early virological response rate in CHC treated with standard of care plus vitamin D, compared with those treated with standard of care only. Finally, in line with data from the literature, we found that steatosis[35] and lower cholesterol levels, a known surrogate marker of fibrosis severity,[26] were independently associated with lower SVR rate. We did not find any association between IR and SVR, in keeping the conflicting data reported in the literature on the role of IR as a predictor of SVR.[36]
The main limitation of this study lies in its cross-sectional nature and its inability to dissect the temporal relation between 25(OH)D and fibrosis. A further methodological drawback is the potentially limited external validity of the results for different populations and settings. Another limitation of this study is the lack of data on the potential confounders that may influence the levels of vitamin D, such as exposure to sunshine, dietary intake, and the prevalence of osteoporosis. However, all of the subjects involved in this study lived in Sicily, where sunshine is abundant, even if we cannot rule out differences in sun exposure between healthy controls and CHC patients. However, data on the prevalence of osteoporosis were not available, nor was the dietary intake of vitamin D, which, however, remains a rather crude measure of vitamin D status because of recall bias. The lack of these data in both cases and controls makes a systematic error very unlikely. Also, data on osteoporosis were not available in both groups. Lack of data on polymorphisms of vitamin D hydroxylating enzymes, and on other variables involved in vitamin D metabolism, such as parathyroid hormone, and in vitamin D signaling regulation also could affect the interpretation of our findings.
In conclusion, this study, showing low levels of serum 25(OH)D and of their liver hydroxylating enzymes in G1 CHC patients, and suggesting a relation of vitamin D status with the severity of liver disease and response to therapy, opens a new area of research on the potential use of vitamin D in patients with CHC.
Results
Patient Features and Histology.
The baseline features of the 197 patients are shown in Table 1. Most of our patients were in the overweight to obesity range. One patient in four had fibrosis of at least 3 by Scheuer score, with a high prevalence of moderate/severe necroinflammation (grading 2-3). Half of the cases had histological evidence of steatosis, though of moderate/severe grade in only 23 cases (11.7%).
The control subjects (25 women and 24 men, mean age of 53.7 ± 12.8 years) were comparable for body mass index with the HCV population (26.1 ± 3.5 kg/m2). Six had arterial hypertension.
Serum 25(OH)D Levels.
Mean serum values of 25(OH)D in G1 CHC patients were significantly lower than in controls (25.1 ± 9.9 ug/L versus 43.1 ± 10.2 ug/L; P < 0.0001; Fig. 1). Accordingly, 25(OH)D serum levels of less than 30 ug/L were found in 144 (73%) G1 CHC patients and in only three control subjects (6%, P < 0.001). Advanced age (P = 0.004), female sex (P < 0.001), high waist circumference (P < 0.06), high ALT (P = 0.09), and low high-density lipoprotein levels (P = 0.01), the severity of necroinflammatory activity (P = 0.01), and the severity of fibrosis (P < 0.001) were associated with lower 25(OH)D levels in G1 CHC, though only female sex (P = 0.007), the severity of necroinflammatory activity (P = 0.04), and the severity of fibrosis (P = 0.009) were independent factors in multiple linear regression analysis (Table 2). Figure 1 also shows the distribution of serum 25(OH)D levels in relation to sex. A significant difference was observed in CHC patients (27.60 ± 9.39 ug/L in men versus 22.23 ± 9.77 ug/L in women; P = 0.0001) (Fig. 1). Figure 2 shows the distribution of 25(OH)D according to necroinflammatory activity.
Interestingly, women 55 years of age or older had lower 25(OH)D serum levels than their counterparts of younger than 55 years (20.2 ± 9.5 versus 24.5 ± 9.7; P = 0.03). Conversely, in men we observed no difference in 25(OH)D serum levels between patients 55 or older and younger than 55 years of age (26.72 ± 9.08 versus 28.52 ± 9.72; P = 0.33). To account for possible interaction between sex and age, a term for the product of the two variables was included in the linear multivariate model, and showed that the interaction between the two risk factors was significant (P = 0.002).
Considering 25(OH)D as a categorical variable, low vitamin D levels (<30 ug/L) were independently associated with high necroinflammatory activity (odds ratio [OR], 1.99; 95% confidence interval [CI], 1.16-3.42, P = 0.01) and with the interaction term between sex and age (OR, 1.015; 95%CI, 1.005-1.026, P = 0.005).
CYP27A1 and CYP2R1 Liver Evaluation.
In a random sample of 34 patients (19 men [55%]; mean age, 50 ± 12.7 years; 13 (38%) with severe fibrosis; 23 (67%) with moderate-severe necroinflammatory activity; mean 25(OH)D levels 25.96 ± 9.90 ug/L), with baseline features not significantly different from the entire group (data not shown), we evaluated the immunohistochemical expression of CYP27A1 and CYP2R1 with a four-grade semiquantitative scoring system. The same analysis was performed in eight control samples from subjects, without liver disease, who underwent cholecystectomy.
CYP27A1 was expressed, with a score of 3 in 75% (6/8) of controls versus 0% (0/34) of cases (P < 0.001), with a score of 2 in 25% (2/8) of controls versus 12% (4/34) of cases (P = 0.42), with a score of 1 in 0% (0/8) of controls versus 25% (12/34) of cases (P = 0.10), and with a score of 0 in 0% (0/8) of controls versus 53% (18/34) of cases (P = 0.04). Similarly, CYP2R1 was expressed with a score of 3 in 50% (4/8) of controls versus 0% (0/34) of cases (P < 0.001), with a score of 2 in 50% (4/8) of controls versus 15% (5/34) of cases (P = 0.10), with a score of 1 in 0% (0/8) of controls versus 50% (17/34) of cases (P = 0.05), and with a score of 0 in 0% (0/8) of controls versus 35% (12/34) of cases (P = 0.10). According to these data, the overall expression of both CYP27A1 and CYP2R1 was significantly down-modulated (P = 0.0001 for both CYP27A1 and CYP2R1) in G1 CHC samples (Supporting Document 1).
The degree of expression of CYP2R1 proved to be neither significantly related to 25(OH)D serum levels nor associated with biochemical, anthropometric, and histological features. Conversely, a significant association was found between a decreased expression of CYP27A1 and low 25(OH)D serum levels (P = 0.01) (Fig. 3A). Moreover, CYP27A1 expression was negatively associated with the degree of necroinflammatory activity (P = 0.031) (Fig. 3B). No significant associations were found between CYP27A1 expression and the various biochemical, anthropometric, and histological features, other than inflammation grade (data not shown).
Case and control samples showed an overlapping picture because CYP450 proved to be diffusely expressed with a moderate intensity in both groups (Supporting Document 2). This suggests a maintained expression of cytochromes other than those involved in vitamin D metabolism in G1 CHC samples.
Variables Related to Severe Fibrosis.
The univariate and multivariate comparison of variables between patients with and without severe fibrosis (F3-F4) are reported in Table 3. Older age, male sex, low platelet count, high baseline values of ALT, high GGT, low cholesterol, high ferritin, low 25(OH)D, steatosis, and high necroinflammatory activity were associated with severe fibrosis (P < 0.10). Multivariate logistic regression analysis showed that the following features were independently linked to severe fibrosis (Scheuer score >/=3): older age (OR, 1.043; 95% CI, 1.002-1.085, P = 0.03), low cholesterol (OR, 0.981; 95%CI, 0.969-0.992, P = 0.001), high ferritin (OR, 1.003; 95%CI, 1.001-1.005, P = 0.007), low 25(OH)D (OR, 0.942; 95%CI, 0.893-0.994, P = 0.02), and high necroinflammatory activity (grading) (OR, 2.235; 95%CI, 1.014-4.929; P = 0.04). The overall area under the curve (AUC) of this model was good (AUC, 0.870). Figure 4, showing the distribution of 25(OH)D serum levels according to the stage of fibrosis, highlighted a significant trend in 25(OH)D reduction among the four fibrosis stages (P < 0.0001). However, even patients with fibrosis F1 had mean serum 25(OH)D levels significantly lower than control subjects (29.5 ± 10.9 g/L versus 43.1 ± 10.2 ug/L; P < 0.0001).
Excluding steatosis and grading from the model, older age (OR, 1.043; 95%CI, 1.004-1.083; P = 0.03), cholesterol (OR, 0.980; 95%CI, 0.968-0.991; P = 0.001), and ferritin (OR, 1.003; 95%CI, 1.001-1.005; P = 0.004) were the noninvasive predictors of severe fibrosis. The overall AUC of this model was similarly good (AUC, 0.854).
Comparing patients with significant fibrosis (F2-F4) with subjects with no significant fibrosis (F1), we confirmed that low serum 25(OH)D levels were independently related to significant fibrosis (data not shown).
The model having fibrosis as an ordinal dependent variable by multiple logistic regression analysis included older age (P = 0.001), low platelets (P = 0.03), low cholesterol (P = 0.001), high ferritin (P = 0.007), low 25(OH)D (P = 0.0006), and high necroinflammatory activity (=P = 0.0002).
Factors Associated with SVR.
One hundred sixty-seven patients underwent and completed the antiviral treatment program. SVR was achieved in 70 individuals (41.9%). Among 97 patients (58.1%) who did not achieve SVR, nine were lost to follow-up, and 14 withdrew from antiviral therapy because of side effects. High GGT, low cholesterol, low 25(OH)D, greater steatosis, and severe necroinflammatory activity were associated with lack of SVR (P < 0.10) (Table 4). By logistic regression, low cholesterol (OR, 1.009; 95%CI, 1.000-1.018; P = 0.04), low 25(OH)D levels (OR, 1.039; 95%CI, 1.002-1.077; P = 0.03), and greater steatosis (OR, 0.971; 95%CI, 0.944-0.999; P = 0.04) were the only independent negative predictors of SVR.
A per protocol analysis of 144 patients confirmed that low cholesterol (OR, 1.012; 95% CI 1.002-1.022; p=0.02) and low 25(OH)D levels (OR, 1.048; 95%CI, 1.008-1.080; P = 0.02), as well as greater steatosis (OR, 0.970; 95%CI, 0.941-1.000; P = 0.04), were negative independent predictors of SVR.
Patients and Methods
Patients.
From January 2007 to December 2008, a total of 197 consecutive patients with G1 CHC, resident in Sicily and recruited at the Gastrointestinal and Liver Unit at the University Hospital in Palermo, fulfilling all inclusion and exclusion criteria detailed later, were assessed. Patients were included if they had a histological diagnosis of CHC (any degree of fibrosis, including cirrhosis) on a liver biopsy performed within 6 months before enrollment. G1 CHC patients were characterized by the presence of anti-hepatitis C virus (HCV) and HCV RNA, with persistently abnormal alanine aminotransferase (ALT), and by alcohol consumption of less than 20 g/day in the last year or more, evaluated by a specific questionnaire. Exclusion criteria were (1) advanced cirrhosis (Child-Pugh B and C); (2) hepatocellular carcinoma; (3) other causes of liver disease or mixed causes (excessive alcohol consumption, hepatitis B, autoimmune liver disease, Wilson's disease, hemochromatosis, a1-antitrypsin deficiency); (4) cancer or chronic intestinal diseases; (5) human immunodeficiency virus infection; (6) therapy with medications known to affect vitamin D3 metabolism, including vitamin/mineral supplements; (7) previous treatment with antiviral therapy, immunosuppressive drug, or regular use of steatosis-inducing drugs; and (8) active intravenous drug addiction.
Forty-nine randomly-selected, nondiabetic, healthy blood donors of the same ethnic group as CHC patients and living in Sicily, recruited from January 2008 to December 2008, matched for age and sex, were enrolled as controls. Alcohol consumption of more than 20 g/day during the previous year or therapy with medications known to affect vitamin D3 metabolism (calcium, vitamin D supplementation, hormonal therapy, alendronate) were additional exclusion criteria. All had normal ALT values (<30 UI/L), and no evidence of viral infection (anti-HCV, anti-human immunodeficiency virus, and hepatitis B surface antigen negative) or steatosis, verified by ultrasound scan.
The study was performed in accordance with the principles of the Declaration of Helsinki and its appendices. Approval was obtained from the hospital's Institutional Review Board and Ethics Committee, and written informed consent was obtained from all cases and controls.
Clinical and Laboratory Assessment.
Clinical and anthropometric data were collected at the time of liver biopsy. Body mass index was calculated on the basis of weight in kilograms and height (in meters). Waist circumference was measured at the midpoint between the lower border of the rib cage and the iliac crest. The diagnosis of arterial hypertension was based on the following criteria: systolic blood pressure at least 135 mm Hg or diastolic blood pressure 85 mmHg or more (measured three times within 30 minutes, in the sitting position and using a brachial sphygmomanometer), or use of blood-pressure-lowering agents. The diagnosis of type 2 diabetes was based on the revised criteria of the American Diabetes Association, using a value of fasting blood glucose at least 126 mg/dL on at least two occasions.[20] In patients with a previous diagnosis of type 2 diabetes, current therapy with insulin or oral hypoglycemic agents was documented.
A 12-hour overnight fasting blood sample was drawn at the time of biopsy to determine serum levels of ALT, gamma-glutamyltransferase (GGT), total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, ferritin, plasma glucose concentration, and platelet count. Serum insulin was determined by a two-site enzyme enzyme-linked immunosorbent assay (Mercodia Insulin ELISA, Arnika). IR was determined with the homeostasis model assessment method.[21]
The analysis of serum 25(OH) D was performed using a Chromosystem reagent kit and a chromatographic system equipped with a Waters 1525 Binary high-pressure liquid chromatography pump connected to a photo diode array detector, and detection was carried out at 265 nm. In accordance with the kit's instructions, a serum 25(OH)D concentration of 30 ug/L was considered the threshold value for identifying low levels of vitamin D.
All patients were tested at the time of biopsy for HCV-RNA by qualitative polymerase chain reaction (Cobas Amplicor HCV Test version 2.0; limit of detection: 50 IU/mL). HCV RNA positive samples were quantified by Versant HCV RNA 3.0 bDNA (Bayer Co. Tarrytown, NY) expressed in IU/mL. Genotyping was performed by INNO-LiPA, HCV II, Bayer.
Histology.
Slides were coded and read by one pathologist (D.C.) who was unaware of the patient's identity and history. A minimum length of 15 mm of biopsy specimen or the presence of at least 10 complete portal tracts was required.[22] Biopsy specimens were classified according to the Scheuer numerical scoring system.[23] The percentage of hepatocytes containing macrovescicular fat was determined for each 10× field. An average percentage of steatosis was then determined for the entire specimen. Steatosis was assessed as the percentage of hepatocytes containing fat droplets (minimum 5%) and evaluated as a continuous variable. Steatosis was classified as mild at 5% to 30% or moderate-severe at 30% or more.
CYP27A1 and CYP2R1 Liver Immunohistochemical Evaluation.
Immunohistochemistry was performed on liver biopsy tissue sections by means of the streptavidin-biotin-peroxidase method. All samples were fixed for 24 hours with 10% buffered formalin, paraffin-embedded, and cut in serial sections of 3 ug. Tissue morphology was evaluated by hematoxylin-eosin staining.
Immunohistochemical detection of CYP2R1 and CYP27A1 was performed using anti-human CYP2R1 (C-15) and CYP27A1 (P-17) (goat polyclonal antibody, Santa Cruz Biotechonology, Inc.). After dewaxing, the endogenous peroxidase activity was inhibited by the pretreatment of the tissue section with 3% hydrogen peroxide before incubation with the primary antibody. The sections were washed twice, after all incubation steps, in phosphate-buffered saline for 5 minutes. Then slides were microwave-oven heated three times for 5 minutes in Tris/ethylenediaminetetra-acetic acid pH 9.0 buffer (heat-induced epitope retrieval), and washed with phosphate-buffered saline. Sections were subsequentially incubated in the presence of the primary antibody overnight at -4°C. The specimens were then incubated with the LSAB HRP detection kit (Universal DakoCytomation LSAB+ System HRP) at room temperature, according to the manufacturer's instructions. As a chromogen, 3-amino-9-ethylcarbazole was used for 5 minutes at room temperature with subsequent nuclear counterstaining with hematoxylin. Normal mouse serum was used instead of primary antibodies as a negative control.
A four-grade semiquantitative scoring system (in other words, score 0-3), performed by one pathologist (C.T.) unaware of other variables, was adopted for the evaluation of CYP2R1 and CYP27A1 immunohistochemical expression. The score was graded according to the intensity of the staining: score 0 was defined as the absence of significant reactivity, scores 1 and 2 as slight and moderate reactivity, respectively, and score 3 as intense reactivity. Because a slight degree of variation could be observed in the immunohistochemical expression of CYP2R1 and CYP27A1 among different areas of the same sample, the most intense reactivity observed in the biopsy specimen was recorded as the summary score.
Immunohistochemical analysis of CYP450 was performed for control purposes using a specific primary monoclonal antibody (clone 1A2, Abcam, MA) and evaluated by the same four-grade semiquantitative scoring system adopted for CYP27A1 and CYP2R1 assessment.
Antiviral Treatment Schedule and Outcomes.
Patients were treated with pegylated interferon a-2a (Pegasys, Roche, Basel, Switzerland) 180 ug /week or pegylated interferon 2b (Peg-Intron, Schering) 1.5 ug/kg/week plus ribavirin at a dosage of 1000 or 1200 mg/day according to body weight (1000 mg/day for a body weight of <75 kg, 1200 mg/day for a body weight of >75 kg) for 48 weeks. Patients were withdrawn from treatment if they did not achieve a virological response, defined as undetectable serum HCV RNA by polymerase chain reaction, within 24 weeks after start of treatment.[24] Sustained virological response (SVR) was defined as negative serum HCV RNA at polymerase chain reaction 6 months after stopping antiviral therapy.
Statistics.
Continuous variables were summarized as mean ± standard deviation, and categorical variables as frequency and percentage. The Student t test and analysis of variance were used when appropriate. Multiple linear regression analysis was performed to identify independent predictors of 25(OH)D serum levels as a continuous dependent variable. As candidate risk factors for low serum levels of 25(OH)D, we selected age, sex, body mass index, waist circumference, baseline ALT, platelet count, GGT, ferritin, total cholesterol, high-density lipoprotein cholesterol, triglycerides, blood glucose, insulin, homeostasis model assessment score, diabetes, arterial hypertension, HCV-RNA levels, steatosis, and activity score.
Multiple logistic regression models were used to assess the relationship of both fibrosis and SVR to the demographic, metabolic, and histological characteristics of patients. In the first model, the dependent variable was severe fibrosis coded as 1 = F3 to F4 in the fibrosis score versus 0 = F1 to F2. Because fibrosis grade is nonlinear, we also performed ordinal logistic regression with fibrosis F0 to F4 as the dependent variable. In the second model, the dependent variable was SVR coded 1 = present versus 0 = absent. As candidate risk factors we selected the same independent variables included in the 25(OH)D model and added 25(OH)D serum levels as an additional independent variable. In this model, we included all patients who received at least one dose of pegylated interferon (intention-to-treat analysis).
Variables associated with the dependent variable at univariate analyses (probability threshold, P
Low vitamin D serum level is related to severe fibrosis and low responsiveness to interferon-based therapy in genotype 1 chronic hepatitis C
Hepatology April 2010
Salvatore Petta 1 2 *, Calogero Cammà 1 3, Concetta Scazzone 4, Claudio Tripodo 5, Vito Di Marco 1, Antonino Bono 4, Daniela Cabibi 5, Giusalba Licata 1, Rossana Porcasi 5, Giulio Marchesini 6, Antonio Craxí 1
1Cattedra ed Unità Operativa di Gastroenterologia, DiBiMIS, University of Palermo, Italy
2Dipartimento di Biopatologia e Metodologie Biomediche, University of Palermo, Italy
3IBIM Consiglio Nazionale delle Ricerche, Palermo, Italy
4Dipartimento di Biotecnologie Mediche e Medicina Legale Sezione Chimica e Biochimica Medica, University of Palermo, Italy
5Cattedra di Anatomia Patologica, University of Palermo, Italy
6Dipartimento di Medicina e Gastroenterologia, Alma Mater Studiorum, University of Bologna, Italy
email: Salvatore Petta (petsa@inwind.it)
*Correspondence to Salvatore Petta, Gastroenterologia & Epatologia, Piazza delle Cliniche, 2, 90127 Palermo, Italy
Abstract
25-Hydroxyvitamin D (25[OH]D) can potentially interfere with inflammatory response and fibrogenesis. Its role in disease progression in chronic hepatitis C (CHC) and its relation with histological and sustained virological response (SVR) to therapy are unknown. One hundred ninety-seven patients with biopsy-proven genotype 1 (G1) CHC and 49 healthy subjects matched by age and sex were consecutively evaluated. One hundred sixty-seven patients underwent antiviral therapy with pegylated interferon plus ribavirin. The 25(OH)D serum levels were measured by high-pressure liquid chromatography. Tissue expression of cytochrome (CY) P27A1 and CYP2R1, liver 25-hydroxylating enzymes, were assessed by immunochemistry in 34 patients with CHC, and in eight controls.
The 25(OH)D serum levels were significantly lower in CHC than in controls (25.07 ± 9.92 g/L versus 43.06 ± 10.19; P < 0.001). Lower levels of 25(OH)D were independently linked to female sex (P = 0.007) and necroinflammation (P = 0.04) by linear regression analysis. CYP27A1, but not CYP2R1, was directly related to 25(OH)D levels (P = 0.01), and inversely to necroinflammation (P = 0.01). Low 25(OH)D (odds ratio [OR], 0.942; 95% confidence interval [CI], 0.893-0.994) and cholesterol (OR, 0.981; 95%CI, 0.969-0.992) levels, older age (OR, 1.043; 95%CI, 1.002-1.085), high ferritin (OR, 1.003; 95%CI, 1.001-1.005), and necroinflammation (OR, 2.235; 95%CI, 1.014-4.929) were independently associated with severe fibrosis (F3-F4) by multivariate logistic analysis. Seventy patients (41%) achieved SVR. By multivariate analysis, hepatic steatosis (OR, 0.971; 95%CI, 0.944-0.999), lower cholesterol (OR, 1.009; 95% CI, 1.000-1.018), and 25(OH)D levels (OR, 1.039; 95%CI, 1.002-1.077) were independently associated with no SVR. Conclusion: G1 CHC patients had low 25(OH)D serum levels, possibly because of reduced CYP27A1 expression. Low vitamin D is linked to severe fibrosis and low SVR on interferon (IFN)-based therapy.
the activated hormonal form of vitamin D, 1-25-dihydroxyvitamin D, is essential for calcium and bone homeostasis.[1][2] Vitamin D 25 and 1alpha-hydroxylation occurs in the liver and in the kidney, respectively, involving different isoforms of cytochrome P450 (CYP), namely CYP2R1, CYP27A1, and others in the liver, and CYP27B1 in the kidney.[3]
Vitamin D deficiency is associated with many common and serious pathological conditions, including cancer, autoimmune disease, cardiovascular disease, insulin resistance (IR), and diabetes.[1][4][5] There is also an association between vitamin D status and both cholestatic and noncholestatic chronic liver diseases.[6-10] In patients with noncholestatic chronic hepatitis and cirrhosis, some studies[8-10] have reported normal serum levels of 25-hydroxyvitamin D (25[OH]D), the liver-hydroxylated form of vitamin D and the best estimate of overall vitamin D status.[1][2] Conversely, other studies have found low serum 25(OH)D levels in patients with chronic hepatitis and cirrhosis of different origins.[11-13] Low 25(OH)D levels have been related to poor liver function because of the association between vitamin D status and hepatic function indexes[11] or the stage of cirrhosis.[11][14][15] In keeping with these studies, several reports describe reduced bone mineral density in patients with chronic liver disease[8][9][13][15][17][18] and cirrhosis.[9][10][12][13][19] Recently, Targher et al.[18] observed lower 25(OH)D serum levels in patients with biopsy-proven nonalcoholic fatty liver disease, identifying an independent association between the histological characteristics of nonalcoholic fatty liver disease and low 25(OH)D levels. Experimental evidence also suggested the potential ability of vitamin D, through interaction with its nuclear receptor (vitamin D receptor), to interfere with inflammatory response and fibrogenesis.[4]
The aim of our study was to evaluate serum levels of 25(OH)D in a cohort of patients with biopsy-proven genotype 1 (G1) chronic hepatitis C (CHC), and to investigate the potential relationships between 25(OH)D, the histological features of disease, and the response to antiviral therapy.
Abbreviations
25(OH)D, 25-hydroxyvitamin D; ALT, alanine aminotransferase; AUC, area under the curve; CHC, chronic hepatitis C; CI, confidence interval; CY, cytochrome; G1, genotype 1; GGT, gamma-glutamyltransferase; HCV, hepatitis C virus; IR, insulin response; OR, odds ratio; SVR, sustained virological response.
Discussion
We have shown that the biochemical profile of G1 CHC patients is characterized by lower-than-normal serum 25(OH)D levels, and that a low 25(OH)D level is independently related to severe fibrosis and a low likelihood of SVR after standard-of-care antiviral therapy.
Lower levels of serum 25(OH)D have been previously reported in populations heterogeneous for cause and severity of chronic liver disease.[11][19] We confirmed a 25(OH)D reduction in a homogeneous cohort of patients with G1 CHC, at low prevalence of F4 fibrosis. Although a significant trend in 25(OH)D levels reduction was observed with increasing stage of fibrosis, a significant reduction was also observed in the subgroup of patients with mild fibrosis (F1), making it unlikely that low 25(OH)D levels could be entirely explained by reduced liver function.
Our study shows that low 25(OH)D levels are independently associated with female sex and with severity of necroinflammatory activity. Although the study was not designed to clarify the correlation between female sex and lower 25(OH)D levels, because of the observed reduction in women older than 55 years, but not in men of the same age range, and because of the significant interaction between sex and age, we can speculate that hormonal alterations in postmenopausal women likely modulate the vitamin D status. Our results also underline an inverse relationship between 25(OH)D and the severity of necroinflammatory activity. The cross-sectional design of our study is unable to dissect the temporal relation between changes in 25(OH)D and necroinflammation. However, CYP27A1 liver expression was directly related to serum 25(OH)D levels, and inversely associated with the severity of necroinflammatory activity. Therefore, the hepatic necroinflammatory activity caused by the HCV infection could be responsible for 25 (OH)D levels reduction by different mechanisms, such as a selectively reduced liver expression of enzymes involved in liver hydroxylation of vitamin D3.
This study also offers the first evidence that low 25(OH)D serum levels, together with known risk factors for fibrosis severity, such as older age, low cholesterol levels, and high necroinflammatory activity,[26] are independently associated with the presence of severe fibrosis. We were not able to confirm IR as a risk factor for fibrosis severity, as reported by others.[26][27] The lack of this association could be attributable to differences in the mean age, alcohol use, and prevalence of obesity and diabetes. In any case we confirmed the important effect of metabolic alterations in fibrosis severity, providing evidence for an association between severe fibrosis and high ferritin levels, a surrogate marker of IR and metabolic syndrome.[28]
Interestingly, considering only noninvasive parameters, the AUC of the model that includes vitamin D levels to predict severe fibrosis remains good. This suggests the potential use of serum 25(OH)D levels as a noninvasive marker of liver fibrosis, a use that needs to be tested and validated in large prospective cohort studies in patients with CHC of all genotypes, and in chronic liver disease of other origins.
It is conceivable that a reduced 25(OH)D level might by itself favor progression of fibrosis. Different experimental models showed that vitamin D, via interaction with vitamin D receptor, protects against oxidative stress production,[29] can influence the migration, proliferation, and gene expression of fibroblasts,[30][31] and reduces the inflammatory and fibrogenic activity of liver stellate cells.[32][33] However further prospective cohort studies will be needed to determine the causal association between vitamin D deficiency and fibrosis in patients with CHC.
Another interesting finding of this study is the evidence that lower 25(OH)D serum levels were an independent negative risk factor for SVR. Again, this observation will require further validation in different, large cohorts of patients, but is supported by experimental data that suggest a role of vitamin D in the modulation of the immune response,[32][33] and by recent clinical data[36] reporting higher early virological response rate in CHC treated with standard of care plus vitamin D, compared with those treated with standard of care only. Finally, in line with data from the literature, we found that steatosis[35] and lower cholesterol levels, a known surrogate marker of fibrosis severity,[26] were independently associated with lower SVR rate. We did not find any association between IR and SVR, in keeping the conflicting data reported in the literature on the role of IR as a predictor of SVR.[36]
The main limitation of this study lies in its cross-sectional nature and its inability to dissect the temporal relation between 25(OH)D and fibrosis. A further methodological drawback is the potentially limited external validity of the results for different populations and settings. Another limitation of this study is the lack of data on the potential confounders that may influence the levels of vitamin D, such as exposure to sunshine, dietary intake, and the prevalence of osteoporosis. However, all of the subjects involved in this study lived in Sicily, where sunshine is abundant, even if we cannot rule out differences in sun exposure between healthy controls and CHC patients. However, data on the prevalence of osteoporosis were not available, nor was the dietary intake of vitamin D, which, however, remains a rather crude measure of vitamin D status because of recall bias. The lack of these data in both cases and controls makes a systematic error very unlikely. Also, data on osteoporosis were not available in both groups. Lack of data on polymorphisms of vitamin D hydroxylating enzymes, and on other variables involved in vitamin D metabolism, such as parathyroid hormone, and in vitamin D signaling regulation also could affect the interpretation of our findings.
In conclusion, this study, showing low levels of serum 25(OH)D and of their liver hydroxylating enzymes in G1 CHC patients, and suggesting a relation of vitamin D status with the severity of liver disease and response to therapy, opens a new area of research on the potential use of vitamin D in patients with CHC.
Results
Patient Features and Histology.
The baseline features of the 197 patients are shown in Table 1. Most of our patients were in the overweight to obesity range. One patient in four had fibrosis of at least 3 by Scheuer score, with a high prevalence of moderate/severe necroinflammation (grading 2-3). Half of the cases had histological evidence of steatosis, though of moderate/severe grade in only 23 cases (11.7%).
The control subjects (25 women and 24 men, mean age of 53.7 ± 12.8 years) were comparable for body mass index with the HCV population (26.1 ± 3.5 kg/m2). Six had arterial hypertension.
Serum 25(OH)D Levels.
Mean serum values of 25(OH)D in G1 CHC patients were significantly lower than in controls (25.1 ± 9.9 ug/L versus 43.1 ± 10.2 ug/L; P < 0.0001; Fig. 1). Accordingly, 25(OH)D serum levels of less than 30 ug/L were found in 144 (73%) G1 CHC patients and in only three control subjects (6%, P < 0.001). Advanced age (P = 0.004), female sex (P < 0.001), high waist circumference (P < 0.06), high ALT (P = 0.09), and low high-density lipoprotein levels (P = 0.01), the severity of necroinflammatory activity (P = 0.01), and the severity of fibrosis (P < 0.001) were associated with lower 25(OH)D levels in G1 CHC, though only female sex (P = 0.007), the severity of necroinflammatory activity (P = 0.04), and the severity of fibrosis (P = 0.009) were independent factors in multiple linear regression analysis (Table 2). Figure 1 also shows the distribution of serum 25(OH)D levels in relation to sex. A significant difference was observed in CHC patients (27.60 ± 9.39 ug/L in men versus 22.23 ± 9.77 ug/L in women; P = 0.0001) (Fig. 1). Figure 2 shows the distribution of 25(OH)D according to necroinflammatory activity.
Interestingly, women 55 years of age or older had lower 25(OH)D serum levels than their counterparts of younger than 55 years (20.2 ± 9.5 versus 24.5 ± 9.7; P = 0.03). Conversely, in men we observed no difference in 25(OH)D serum levels between patients 55 or older and younger than 55 years of age (26.72 ± 9.08 versus 28.52 ± 9.72; P = 0.33). To account for possible interaction between sex and age, a term for the product of the two variables was included in the linear multivariate model, and showed that the interaction between the two risk factors was significant (P = 0.002).
Considering 25(OH)D as a categorical variable, low vitamin D levels (<30 ug/L) were independently associated with high necroinflammatory activity (odds ratio [OR], 1.99; 95% confidence interval [CI], 1.16-3.42, P = 0.01) and with the interaction term between sex and age (OR, 1.015; 95%CI, 1.005-1.026, P = 0.005).
CYP27A1 and CYP2R1 Liver Evaluation.
In a random sample of 34 patients (19 men [55%]; mean age, 50 ± 12.7 years; 13 (38%) with severe fibrosis; 23 (67%) with moderate-severe necroinflammatory activity; mean 25(OH)D levels 25.96 ± 9.90 ug/L), with baseline features not significantly different from the entire group (data not shown), we evaluated the immunohistochemical expression of CYP27A1 and CYP2R1 with a four-grade semiquantitative scoring system. The same analysis was performed in eight control samples from subjects, without liver disease, who underwent cholecystectomy.
CYP27A1 was expressed, with a score of 3 in 75% (6/8) of controls versus 0% (0/34) of cases (P < 0.001), with a score of 2 in 25% (2/8) of controls versus 12% (4/34) of cases (P = 0.42), with a score of 1 in 0% (0/8) of controls versus 25% (12/34) of cases (P = 0.10), and with a score of 0 in 0% (0/8) of controls versus 53% (18/34) of cases (P = 0.04). Similarly, CYP2R1 was expressed with a score of 3 in 50% (4/8) of controls versus 0% (0/34) of cases (P < 0.001), with a score of 2 in 50% (4/8) of controls versus 15% (5/34) of cases (P = 0.10), with a score of 1 in 0% (0/8) of controls versus 50% (17/34) of cases (P = 0.05), and with a score of 0 in 0% (0/8) of controls versus 35% (12/34) of cases (P = 0.10). According to these data, the overall expression of both CYP27A1 and CYP2R1 was significantly down-modulated (P = 0.0001 for both CYP27A1 and CYP2R1) in G1 CHC samples (Supporting Document 1).
The degree of expression of CYP2R1 proved to be neither significantly related to 25(OH)D serum levels nor associated with biochemical, anthropometric, and histological features. Conversely, a significant association was found between a decreased expression of CYP27A1 and low 25(OH)D serum levels (P = 0.01) (Fig. 3A). Moreover, CYP27A1 expression was negatively associated with the degree of necroinflammatory activity (P = 0.031) (Fig. 3B). No significant associations were found between CYP27A1 expression and the various biochemical, anthropometric, and histological features, other than inflammation grade (data not shown).
Case and control samples showed an overlapping picture because CYP450 proved to be diffusely expressed with a moderate intensity in both groups (Supporting Document 2). This suggests a maintained expression of cytochromes other than those involved in vitamin D metabolism in G1 CHC samples.
Variables Related to Severe Fibrosis.
The univariate and multivariate comparison of variables between patients with and without severe fibrosis (F3-F4) are reported in Table 3. Older age, male sex, low platelet count, high baseline values of ALT, high GGT, low cholesterol, high ferritin, low 25(OH)D, steatosis, and high necroinflammatory activity were associated with severe fibrosis (P < 0.10). Multivariate logistic regression analysis showed that the following features were independently linked to severe fibrosis (Scheuer score >/=3): older age (OR, 1.043; 95% CI, 1.002-1.085, P = 0.03), low cholesterol (OR, 0.981; 95%CI, 0.969-0.992, P = 0.001), high ferritin (OR, 1.003; 95%CI, 1.001-1.005, P = 0.007), low 25(OH)D (OR, 0.942; 95%CI, 0.893-0.994, P = 0.02), and high necroinflammatory activity (grading) (OR, 2.235; 95%CI, 1.014-4.929; P = 0.04). The overall area under the curve (AUC) of this model was good (AUC, 0.870). Figure 4, showing the distribution of 25(OH)D serum levels according to the stage of fibrosis, highlighted a significant trend in 25(OH)D reduction among the four fibrosis stages (P < 0.0001). However, even patients with fibrosis F1 had mean serum 25(OH)D levels significantly lower than control subjects (29.5 ± 10.9 g/L versus 43.1 ± 10.2 ug/L; P < 0.0001).
Excluding steatosis and grading from the model, older age (OR, 1.043; 95%CI, 1.004-1.083; P = 0.03), cholesterol (OR, 0.980; 95%CI, 0.968-0.991; P = 0.001), and ferritin (OR, 1.003; 95%CI, 1.001-1.005; P = 0.004) were the noninvasive predictors of severe fibrosis. The overall AUC of this model was similarly good (AUC, 0.854).
Comparing patients with significant fibrosis (F2-F4) with subjects with no significant fibrosis (F1), we confirmed that low serum 25(OH)D levels were independently related to significant fibrosis (data not shown).
The model having fibrosis as an ordinal dependent variable by multiple logistic regression analysis included older age (P = 0.001), low platelets (P = 0.03), low cholesterol (P = 0.001), high ferritin (P = 0.007), low 25(OH)D (P = 0.0006), and high necroinflammatory activity (=P = 0.0002).
Factors Associated with SVR.
One hundred sixty-seven patients underwent and completed the antiviral treatment program. SVR was achieved in 70 individuals (41.9%). Among 97 patients (58.1%) who did not achieve SVR, nine were lost to follow-up, and 14 withdrew from antiviral therapy because of side effects. High GGT, low cholesterol, low 25(OH)D, greater steatosis, and severe necroinflammatory activity were associated with lack of SVR (P < 0.10) (Table 4). By logistic regression, low cholesterol (OR, 1.009; 95%CI, 1.000-1.018; P = 0.04), low 25(OH)D levels (OR, 1.039; 95%CI, 1.002-1.077; P = 0.03), and greater steatosis (OR, 0.971; 95%CI, 0.944-0.999; P = 0.04) were the only independent negative predictors of SVR.
A per protocol analysis of 144 patients confirmed that low cholesterol (OR, 1.012; 95% CI 1.002-1.022; p=0.02) and low 25(OH)D levels (OR, 1.048; 95%CI, 1.008-1.080; P = 0.02), as well as greater steatosis (OR, 0.970; 95%CI, 0.941-1.000; P = 0.04), were negative independent predictors of SVR.
Patients and Methods
Patients.
From January 2007 to December 2008, a total of 197 consecutive patients with G1 CHC, resident in Sicily and recruited at the Gastrointestinal and Liver Unit at the University Hospital in Palermo, fulfilling all inclusion and exclusion criteria detailed later, were assessed. Patients were included if they had a histological diagnosis of CHC (any degree of fibrosis, including cirrhosis) on a liver biopsy performed within 6 months before enrollment. G1 CHC patients were characterized by the presence of anti-hepatitis C virus (HCV) and HCV RNA, with persistently abnormal alanine aminotransferase (ALT), and by alcohol consumption of less than 20 g/day in the last year or more, evaluated by a specific questionnaire. Exclusion criteria were (1) advanced cirrhosis (Child-Pugh B and C); (2) hepatocellular carcinoma; (3) other causes of liver disease or mixed causes (excessive alcohol consumption, hepatitis B, autoimmune liver disease, Wilson's disease, hemochromatosis, a1-antitrypsin deficiency); (4) cancer or chronic intestinal diseases; (5) human immunodeficiency virus infection; (6) therapy with medications known to affect vitamin D3 metabolism, including vitamin/mineral supplements; (7) previous treatment with antiviral therapy, immunosuppressive drug, or regular use of steatosis-inducing drugs; and (8) active intravenous drug addiction.
Forty-nine randomly-selected, nondiabetic, healthy blood donors of the same ethnic group as CHC patients and living in Sicily, recruited from January 2008 to December 2008, matched for age and sex, were enrolled as controls. Alcohol consumption of more than 20 g/day during the previous year or therapy with medications known to affect vitamin D3 metabolism (calcium, vitamin D supplementation, hormonal therapy, alendronate) were additional exclusion criteria. All had normal ALT values (<30 UI/L), and no evidence of viral infection (anti-HCV, anti-human immunodeficiency virus, and hepatitis B surface antigen negative) or steatosis, verified by ultrasound scan.
The study was performed in accordance with the principles of the Declaration of Helsinki and its appendices. Approval was obtained from the hospital's Institutional Review Board and Ethics Committee, and written informed consent was obtained from all cases and controls.
Clinical and Laboratory Assessment.
Clinical and anthropometric data were collected at the time of liver biopsy. Body mass index was calculated on the basis of weight in kilograms and height (in meters). Waist circumference was measured at the midpoint between the lower border of the rib cage and the iliac crest. The diagnosis of arterial hypertension was based on the following criteria: systolic blood pressure at least 135 mm Hg or diastolic blood pressure 85 mmHg or more (measured three times within 30 minutes, in the sitting position and using a brachial sphygmomanometer), or use of blood-pressure-lowering agents. The diagnosis of type 2 diabetes was based on the revised criteria of the American Diabetes Association, using a value of fasting blood glucose at least 126 mg/dL on at least two occasions.[20] In patients with a previous diagnosis of type 2 diabetes, current therapy with insulin or oral hypoglycemic agents was documented.
A 12-hour overnight fasting blood sample was drawn at the time of biopsy to determine serum levels of ALT, gamma-glutamyltransferase (GGT), total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, ferritin, plasma glucose concentration, and platelet count. Serum insulin was determined by a two-site enzyme enzyme-linked immunosorbent assay (Mercodia Insulin ELISA, Arnika). IR was determined with the homeostasis model assessment method.[21]
The analysis of serum 25(OH) D was performed using a Chromosystem reagent kit and a chromatographic system equipped with a Waters 1525 Binary high-pressure liquid chromatography pump connected to a photo diode array detector, and detection was carried out at 265 nm. In accordance with the kit's instructions, a serum 25(OH)D concentration of 30 ug/L was considered the threshold value for identifying low levels of vitamin D.
All patients were tested at the time of biopsy for HCV-RNA by qualitative polymerase chain reaction (Cobas Amplicor HCV Test version 2.0; limit of detection: 50 IU/mL). HCV RNA positive samples were quantified by Versant HCV RNA 3.0 bDNA (Bayer Co. Tarrytown, NY) expressed in IU/mL. Genotyping was performed by INNO-LiPA, HCV II, Bayer.
Histology.
Slides were coded and read by one pathologist (D.C.) who was unaware of the patient's identity and history. A minimum length of 15 mm of biopsy specimen or the presence of at least 10 complete portal tracts was required.[22] Biopsy specimens were classified according to the Scheuer numerical scoring system.[23] The percentage of hepatocytes containing macrovescicular fat was determined for each 10× field. An average percentage of steatosis was then determined for the entire specimen. Steatosis was assessed as the percentage of hepatocytes containing fat droplets (minimum 5%) and evaluated as a continuous variable. Steatosis was classified as mild at 5% to 30% or moderate-severe at 30% or more.
CYP27A1 and CYP2R1 Liver Immunohistochemical Evaluation.
Immunohistochemistry was performed on liver biopsy tissue sections by means of the streptavidin-biotin-peroxidase method. All samples were fixed for 24 hours with 10% buffered formalin, paraffin-embedded, and cut in serial sections of 3 ug. Tissue morphology was evaluated by hematoxylin-eosin staining.
Immunohistochemical detection of CYP2R1 and CYP27A1 was performed using anti-human CYP2R1 (C-15) and CYP27A1 (P-17) (goat polyclonal antibody, Santa Cruz Biotechonology, Inc.). After dewaxing, the endogenous peroxidase activity was inhibited by the pretreatment of the tissue section with 3% hydrogen peroxide before incubation with the primary antibody. The sections were washed twice, after all incubation steps, in phosphate-buffered saline for 5 minutes. Then slides were microwave-oven heated three times for 5 minutes in Tris/ethylenediaminetetra-acetic acid pH 9.0 buffer (heat-induced epitope retrieval), and washed with phosphate-buffered saline. Sections were subsequentially incubated in the presence of the primary antibody overnight at -4°C. The specimens were then incubated with the LSAB HRP detection kit (Universal DakoCytomation LSAB+ System HRP) at room temperature, according to the manufacturer's instructions. As a chromogen, 3-amino-9-ethylcarbazole was used for 5 minutes at room temperature with subsequent nuclear counterstaining with hematoxylin. Normal mouse serum was used instead of primary antibodies as a negative control.
A four-grade semiquantitative scoring system (in other words, score 0-3), performed by one pathologist (C.T.) unaware of other variables, was adopted for the evaluation of CYP2R1 and CYP27A1 immunohistochemical expression. The score was graded according to the intensity of the staining: score 0 was defined as the absence of significant reactivity, scores 1 and 2 as slight and moderate reactivity, respectively, and score 3 as intense reactivity. Because a slight degree of variation could be observed in the immunohistochemical expression of CYP2R1 and CYP27A1 among different areas of the same sample, the most intense reactivity observed in the biopsy specimen was recorded as the summary score.
Immunohistochemical analysis of CYP450 was performed for control purposes using a specific primary monoclonal antibody (clone 1A2, Abcam, MA) and evaluated by the same four-grade semiquantitative scoring system adopted for CYP27A1 and CYP2R1 assessment.
Antiviral Treatment Schedule and Outcomes.
Patients were treated with pegylated interferon a-2a (Pegasys, Roche, Basel, Switzerland) 180 ug /week or pegylated interferon 2b (Peg-Intron, Schering) 1.5 ug/kg/week plus ribavirin at a dosage of 1000 or 1200 mg/day according to body weight (1000 mg/day for a body weight of <75 kg, 1200 mg/day for a body weight of >75 kg) for 48 weeks. Patients were withdrawn from treatment if they did not achieve a virological response, defined as undetectable serum HCV RNA by polymerase chain reaction, within 24 weeks after start of treatment.[24] Sustained virological response (SVR) was defined as negative serum HCV RNA at polymerase chain reaction 6 months after stopping antiviral therapy.
Statistics.
Continuous variables were summarized as mean ± standard deviation, and categorical variables as frequency and percentage. The Student t test and analysis of variance were used when appropriate. Multiple linear regression analysis was performed to identify independent predictors of 25(OH)D serum levels as a continuous dependent variable. As candidate risk factors for low serum levels of 25(OH)D, we selected age, sex, body mass index, waist circumference, baseline ALT, platelet count, GGT, ferritin, total cholesterol, high-density lipoprotein cholesterol, triglycerides, blood glucose, insulin, homeostasis model assessment score, diabetes, arterial hypertension, HCV-RNA levels, steatosis, and activity score.
Multiple logistic regression models were used to assess the relationship of both fibrosis and SVR to the demographic, metabolic, and histological characteristics of patients. In the first model, the dependent variable was severe fibrosis coded as 1 = F3 to F4 in the fibrosis score versus 0 = F1 to F2. Because fibrosis grade is nonlinear, we also performed ordinal logistic regression with fibrosis F0 to F4 as the dependent variable. In the second model, the dependent variable was SVR coded 1 = present versus 0 = absent. As candidate risk factors we selected the same independent variables included in the 25(OH)D model and added 25(OH)D serum levels as an additional independent variable. In this model, we included all patients who received at least one dose of pegylated interferon (intention-to-treat analysis).
Variables associated with the dependent variable at univariate analyses (probability threshold, P
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