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845 PRECLINICAL PHARMACOKINETIC AND ADME CHARACTERIZATION OF VCH-916, A NOVEL NON-NUCLEOSIDE HCV NS5B POLYMERASE INHIBITOR
05H. VIRAL HEPATITIS – H) HEPATITIS C – CLINICAL (NEW COMPOUNDS, RESISTANCE) 844 GENOTYPIC ANALYSIS OF HCV NS5B VARIANTS SELECTED FROM PATIENTS TREATED WITH VCH-759 O. Nicolas, I. Boivin, C. St-Denis, J. Bedard. Virology, ViroChem Pharma Inc., Laval, Canada E-mail: [email protected] Background: VCH-759 is a novel orally bioavailable non-nucleoside inhibitor of hepatitis C virus RNA-dependent RNA polymerase. In a phase Ib clinical study involving genotype 1a and 1b-infected subjects, VCH-759 has achieved a 2 log10 or greater decline in HCV RNA levels. During the 10-day dosing period, an initial rapid reduction of viral load was observed in all patients dosed with VCH-759. Some patients have experienced a sustained anti-viral response during treatment but viral breakthrough was noticed for others. The aim of this study was to genotypically and phenotypically characterize the selection of resistant variants and to investigate a potential correlation with the viral kinetics observed. Methods: Selected samples collected at day 1 (pre-dosing), day 11 (day after last dose), and days 17 and 24 (short term follow-up) were used for analysis. The entire NS5B gene was amplified by RT-PCR and directly sequenced. In order to determine the prevalence of each mutation within the population, the region corresponding to the compound binding pocket (amino acids 340–539) was also amplified, cloned, and numerous clones sequenced. The phenotypic and the in vitro replication capacity analysis of variants were performed using the HCV replicon system. Results: Analysis of pre-dosing sera has revealed a wild-type genotype for the positions 419, 423, and 482 of the NS5B when compared to the consensus sequence of genotypes 1a and 1b. A mixture of wild-type and M423T/V/I substitution was the common feature found by population sequencing in samples at day 11, 17, and 24. The M423T and M423V mutants were associated with a respective 18 to 21-fold increase in replicon EC50 values when compared to the wild-type. A less predominant mutation namely, L419M was detected by clonal analysis and was found to confer a 23-fold reduction in VCH-759 susceptibility. Overall, the selected mutants were found to have a reduced replication capacity when compared to the wild-type. Conclusions: HCV variants with mutations conferring resistance to VCH759 were selected during the course of a 10-day treatment. This study illustrates that VCH-759 should be used in a combination therapy to maintain viral suppression and prevent emergence of resistance. 845 PRECLINICAL PHARMACOKINETIC AND ADME CHARACTERIZATION OF VCH-916, A NOVEL NON-NUCLEOSIDE HCV NS5B POLYMERASE INHIBITOR N. Chauret, C. Chagnon-Labelle, M. Diallo, J. Laquerre, S. Plante. DMPK, ViroChem Pharma Inc., Laval, Quebec, Canada E-mail: [email protected] Background and Aims: VCH-916 is a novel non-nucleoside HCV RNA polymerase inhibitor. It has demonstrated sub-micromolar IC50s against the HCV replicons of genotype 1a and 1b. It is currently being evaluated for safety/tolerability, pharmacokinetics and antiviral efficacy in chronically infected HCV patient. The objective of these studies was to characterize the pharmacokinetic profile in relevant preclinical models and ADME properties of VCH-916. Methods: Pharmacokinetic studies were conducted following intravenous and oral administration of VCH-916 in rats and dogs. The routes of excretion and distribution of the compound in the liver were determined in rats following oral administration of 14C-VCH-916. The absorption, metabolic stability, covalent binding, protein binding and the potential of VCH-916 to cause P-glycoprotein inhibition, Cytochrome 450 inhibition and induction were evaluated in vitro in systems derived from human and/or preclinical species. Results: VCH-916 was relatively stable in human microsomes and hepatocytes. Both oxidation and glucuronidation pathways were observed.
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Phenotyping studies indicated that several enzymes were involved in the biotransformations of VCH-916 (CYP2C8, CYP 3A4, UGT1A3, 1A8, 2B7, 2B17). No covalent binding to microsomal proteins upon oxidative bio-activation was noticed. In all species, plasma protein binding was extensive (>98%). VCH-916 demonstrated limited potential to cause human CYP inhibition or CYP induction. The absorption potential of VCH-916 was shown to be high based on its high permeability in Caco-2 cell monolayer and lack of recognition by intestinal efflux proteins. In addition, VCH-916 had no significant effect of digoxin permeability. In all preclinical species, VCH-916 displays low total body clearance with excellent oral bioavailability (greater than 40%). After oral administration of 14C-VCH-916 to rats, recovery of radioactivity was almost complete (80%) 48 hr post-dosing with the majority of the dose excreted into feces. The exposure of 14C VCH-916 in liver was 5-fold higher than in plasma. Conclusion: VCH-916 is orally bioavailable and exhibits excellent ADME behaviours in terms of permeability and metabolic behaviours. VCH916 is neither a CYP inhibitor/inducer nor a Pgp inhibitor reducing the likelihood of VCH-916 to be involved in drug interactions. From these data, a favorable clinical pharmacokinetic profile is expected in humans.
846 NATURALLY OCURRING NS3 PROTEASE-INHIBITOR RESISTANT-MUTANT A156T IN THE LIVER OF AN UNTREATED CHRONIC HEPATITIS C PATIENT M. Cubero1,2,3 , J.I. Esteban1,2,3 , T. Otero1 , S. Sauleda3,4 , M. Bes3,4 , R. Esteban1,2,3 , J. Guardia1,2,3 , J. Quer1,2,3 . 1 Liver Unit, Department of Medicine, Hospital Universitari Vall D’Hebron, Barcelona, 2 Universitat Aut´onoma de Barcelona, UAB.Bellaterra, Barcelona, 3 CIBER de Enfermedades Hep´aticas y Digestivas (CIBERehd) Del Instituto de Salud Carlos III, Barcelona, 4 Banc de Sang i Teixists, Institut Catal`a de La Salut, Barcelona, Spain E-mail: [email protected] Background and Aims: An increasing number of new hepatitis C virus NS3-protease inhibitors are being evaluated for the treatment of chronic hepatitis C. Treatment-induced selection of mutants conferring resistance to protease inhibitors has been shown both in vivo and in vitro. A specific mutation, A156T has been shown to confer high-level resistance to several such agents (BILN2061, VX-950, SCH446211 (SCH6) and SCH503034). Here we report the presence of the A156T mutation in close to 1% of NS3 sequences within the liver quasispecies of a chronic hepatitis C patient never treated with anti-NS3 protease inhibitors Methods: A total of 128 independent clones from the NS3 protease domain (567 nucleotides each) were sequenced and analysed from the liver biopsy of the anti-protease of an untreated patient. Nested-PCR was performed using the high fidelity DNA polymerase Pfu, which, under the conditions used, has a very low error rate (between 0.13−6.5x10−7). Results: The total number of individual mutations was 51 from 72576 nucleotides sequenced, of which 20 were nonsynonymous mutations. At the amino acid level 86% (110/128) of sequences were equal representing a master sequence that coincided with the consensus one and all sequences had mutations P89Q and Q86P. The principal observation was the presence of mutation A156T at a frequency of 0.78% within the liver of an HCVinfected patient who had never been treated with any of these protease inhibitors. In the mutant named B6b-2006 the codon GCC (Ala) changed to ACC (Thr). Conclusions: Pre-existence of such drug-resistance viruses in an untreated patient is a direct consequence of the quasispecies structure of RNA virus with the continuous generation of variants, and represent an important drawback in the treatment of viral diseases. Therefore any combination therapy using only protease inhibitors, is predicted to be inescapably deemed to failure, opening important ethical considerations in monotherapy treatments and suggest that compounds targeting different regions should be used in combination to decreased the likelihood of resistance, or in combination with other antivirals like interferon and ribavirin.
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Phenotyping studies indicated that several enzymes were involved in the biotransformations of VCH-916 (CYP2C8, CYP 3A4, UGT1A3, 1A8, 2B7, 2B17). No covalent binding to microsomal proteins upon oxidative bio-activation was noticed. In all species, plasma protein binding was extensive (>98%). VCH-916 demonstrated limited potential to cause human CYP inhibition or CYP induction. The absorption potential of VCH-916 was shown to be high based on its high permeability in Caco-2 cell monolayer and lack of recognition by intestinal efflux proteins. In addition, VCH-916 had no significant effect of digoxin permeability. In all preclinical species, VCH-916 displays low total body clearance with excellent oral bioavailability (greater than 40%). After oral administration of 14C-VCH-916 to rats, recovery of radioactivity was almost complete (80%) 48 hr post-dosing with the majority of the dose excreted into feces. The exposure of 14C VCH-916 in liver was 5-fold higher than in plasma. Conclusion: VCH-916 is orally bioavailable and exhibits excellent ADME behaviours in terms of permeability and metabolic behaviours. VCH916 is neither a CYP inhibitor/inducer nor a Pgp inhibitor reducing the likelihood of VCH-916 to be involved in drug interactions. From these data, a favorable clinical pharmacokinetic profile is expected in humans.
846 NATURALLY OCURRING NS3 PROTEASE-INHIBITOR RESISTANT-MUTANT A156T IN THE LIVER OF AN UNTREATED CHRONIC HEPATITIS C PATIENT M. Cubero1,2,3 , J.I. Esteban1,2,3 , T. Otero1 , S. Sauleda3,4 , M. Bes3,4 , R. Esteban1,2,3 , J. Guardia1,2,3 , J. Quer1,2,3 . 1 Liver Unit, Department of Medicine, Hospital Universitari Vall D’Hebron, Barcelona, 2 Universitat Aut´onoma de Barcelona, UAB.Bellaterra, Barcelona, 3 CIBER de Enfermedades Hep´aticas y Digestivas (CIBERehd) Del Instituto de Salud Carlos III, Barcelona, 4 Banc de Sang i Teixists, Institut Catal`a de La Salut, Barcelona, Spain E-mail: [email protected] Background and Aims: An increasing number of new hepatitis C virus NS3-protease inhibitors are being evaluated for the treatment of chronic hepatitis C. Treatment-induced selection of mutants conferring resistance to protease inhibitors has been shown both in vivo and in vitro. A specific mutation, A156T has been shown to confer high-level resistance to several such agents (BILN2061, VX-950, SCH446211 (SCH6) and SCH503034). Here we report the presence of the A156T mutation in close to 1% of NS3 sequences within the liver quasispecies of a chronic hepatitis C patient never treated with anti-NS3 protease inhibitors Methods: A total of 128 independent clones from the NS3 protease domain (567 nucleotides each) were sequenced and analysed from the liver biopsy of the anti-protease of an untreated patient. Nested-PCR was performed using the high fidelity DNA polymerase Pfu, which, under the conditions used, has a very low error rate (between 0.13−6.5x10−7). Results: The total number of individual mutations was 51 from 72576 nucleotides sequenced, of which 20 were nonsynonymous mutations. At the amino acid level 86% (110/128) of sequences were equal representing a master sequence that coincided with the consensus one and all sequences had mutations P89Q and Q86P. The principal observation was the presence of mutation A156T at a frequency of 0.78% within the liver of an HCVinfected patient who had never been treated with any of these protease inhibitors. In the mutant named B6b-2006 the codon GCC (Ala) changed to ACC (Thr). Conclusions: Pre-existence of such drug-resistance viruses in an untreated patient is a direct consequence of the quasispecies structure of RNA virus with the continuous generation of variants, and represent an important drawback in the treatment of viral diseases. Therefore any combination therapy using only protease inhibitors, is predicted to be inescapably deemed to failure, opening important ethical considerations in monotherapy treatments and suggest that compounds targeting different regions should be used in combination to decreased the likelihood of resistance, or in combination with other antivirals like interferon and ribavirin.