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Fw: NATAP/Resist Wksp: HCV Polymerase Inhibitor A-848837

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  • Alley Pat
    ... From: nataphcv@natap.org To: nataphcv@natap.org ; nataphcvhiv@natap.org ; natapindustry@natap.org ; natapdoctors@natap.org Sent: Wednesday, June 14, 2006
    Message 1 of 1 , Jun 16, 2006
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      ----- Original Message -----
      From: nataphcv@...
      To: nataphcv@... ; nataphcvhiv@... ; natapindustry@... ; natapdoctors@...
      Sent: Wednesday, June 14, 2006 2:20 PM
      Subject: NATAP/Resist Wksp: HCV Polymerase Inhibitor A-848837


      NATAP http://natap.org/
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      HCV Polymerase Inhibitor A-848837

      Reported by Jules Levin
      15th Intl HIV Drug resistance Workshop
      June 13-17, 2006, Sitges, Spain

      â?oSelection and characterization of mutations conferring resistance to a HCV RNA dependent RNA polymerase inhibitor in vitroâ?

      Akhter Molla from Abbott reported these study data this morning in the first oral session at the Workshop.

      Abstract
      BACKGROUND: A-848837 is a novel specific inhibitor of the HCV RNA-dependent RNA polymerase with excellent PK in animals. Given the high mutation and replication rate of HCV, it is likely that mutant viruses with decreased susceptibility to antiviral regimens will emerge during therapy. Understanding the in vitro resistance profiles of the HCV inhibitors is therefore of considerable interest. (This drug is a prototype for a potential future Abbott polymerase inhibitor drug).

      METHODS: The anti-HCV activity of A-848837 was determined in HCV replicon cells by the reduction of HCV RNA. HCV replicon colonies resistance to the inhibitors were selected by treating the HCV subgenomic 1b-N replicon cells with A-848837 at a concentration 10 times above its corresponding IC50 in the presence of neomycin. Genotypes of the resistant colonies were determined by sequencing the NS5B polymerase gene. Individual mutations were introduced into a luciferase-expressing replicon by site-directed mutagenesis. The susceptibility of the mutants was evaluated by a trans-replication assay.

      RESULTS: A-848837 displayed potent activity in the HCV replicon tissue culture with an IC50 of 2 and 4 nM against genotypes 1a and 1b, respectively (not active against other genotypes). Following selection with A-848837, replicon colonies were found to be approximately 33- to >800-fold resistant to A-848837. Each colony found one to three of the following mutations: G46A, S368T, I392F, M414T, Y448H, Q514R, G554D, D559G, and Y586C within NS5B polymerase gene. Molecular clones containing the above amino acid substitutions displayed 4- to 255-fold reduced susceptibility to A-848837. In contrast, all mutants retained full susceptibility to the polymerase inhibitor thiophene-2-carboxylic acid (Shire polymerase inhibitor), HCV protease inhibitor, and interferon (IFN). Furthermore, combination of A-848837 with IFN or protease inhibitor produced an additive to synergistic effect in replicon cells. All mutants except I392F exhibited reduced replication capacity compared to the wild-type replicon.

      CONCLUSION: This study suggested that single amino acid substitutions at any of several positions could result in reduced susceptibility to A-848837. However, combination of this inhibitor with IFN or protease inhibitor may be synergistic in controlling replication.

      POSTER

      Introduction
      Like HIV, HCV has a high replication rate in vivo and its polymerase also has poor fidelity. Thus it is highly likely that drug-resistant HCV variants will emerge in patients treated with inhibitors of HCV polymerase. HCV replicons resistant to a number of inhibitors of the RNA-dependent RNA polymerase have been selected in vitro, and the specific amino acid substitutions within the NS5B gene associated with resistance to polymerase inhibitors have been identified.

      A-848837 is a novel, specific inhibitor of the HCV polymerase with a serum-adjusted EC50 in the HCV replicon (31 ng.mL) that equate or exceeds that of any compounds known to be in clinical development. A-848837 demonstrates excellent pharmacokinetics in animals, and following oral dosing, achieves high concentrations in liver, the primary site of HCV replication, with liver:plasma ratios ranging from 20:1 u=in rat to 200:1 in monkey. In view of these features, the investigation of the resistance profile of this potent inhibitor is of interest.

      Objectives
      o To characterize the development of resistance in response to in vitro selection with the HCV polymerase inhibitor A-848837
      o to identify the specific mutations associated with in vitro resistance
      o to define the replication capacities of the resistant mutants.

      RESULTS
      A-848837 is a highly potent inhibitor of HCV genotypes 1a and 1vb. The highest levels of resistance were observed with the G554D single mutant in genotype 1b and C316Y together with I539M/I in genotype 1a. Other colonies were 33- to 168-fold resistant to A-848837. Purified recombinant enzymes containing the single mutations G554D or D559G exhibited 1300- to 2400-fold resistance to A-848837. Recombinant single mutants G554D (genotype 1b replicon) and C316Y (genotype 1a replicon) exhibited 255- to 2888-fold reduced susceptibility to A-848837 in a transient transfection assay. Mutants resistant to A-848837 remain susceptible to IFN, BILN2061, HCV protease inhibitor), and a thiophene HCv polymerase inhibitor.

      The replication capacities of all mutant replicons with the exception of !392F were substantially impaired compared with the wild-type replicon. Resistance mutations occur in or close to the inhibitor-binding region and directly affect the enzyme-inhibitor interaction.

      Treatment of the 1b replicon with IFN at 20 times EC50 produced <1.5 log HCV RNA decline at passage 2, whereas treatment with A-848837 at 20 times EC50 produced approximately 3.0 log RNA decline.

      Y448H, G554D, D559G mutations were detected at passage 2 during the treatment with A-848837.

      The combination of 20 times EC50 IFN plus 20 times EC50 A-848837 displayed an apparent synergistic effect. Simultaneous treatment with both drugs was able to reduce RNA to an undetectable level after passage 4.

      No resistant colonies were identified when neomycin was added at passage 6, indicating that replicon cells were cured by the combination treatment.

      CONCLUSIONS
      Multiple mutational patterns were observed in response to in vitro selection with A-848837 and several single mutations were shown to confer intermediate to high levels of resistance to the inhibitor.

      The dominant resistance mutations selected in NS5B were C316Y and M414T in genotype 1a and M414T, Y448H, A553T, G554D, and D559G in genotype 1b. These mutations occur in or close to the inhibitor-binding region and directly affect the enzyme-inhibitor interaction.

      Recombinant polymerases and/or replicons containing each of these six mutations substantially decreased the inhibitory activity of A-848837 in both biochemical assays and replicon tissue culture (23-fold up to 2800-fold).

      The replication capacities of G554D, S368T, Y448H, D559G, A553T, and M414T were substantially lower than that of the corresponding wild-type replicon.

      Treatment with IFN alone or A-848837 at 20 times EC50 reduced HCV RNA levels 2-3 log copies whereas the combination of 20 times EC50 of both inhibitors together reduced HCV RNA over 7 logs, leading to undetectable HCV RNA within 12 days of treatment.


      Methods
      HCV polymerase biochemical IC50 values were determined against purified recombinant wild-type or mutant enzymes from genotype 1b-BK using a 2.1 kilobase truncated HCV RNA as a template.

      The antiviral activity of HCV inhibitors was determined by monitoring the levels of HCV RNA and/or SEAP activity using HCV subgenomic replicons.

      HCV resistant replicon variants were selected by culturing the HCV 1b-N strain in the presence of A-848837 at a concentration 10 times its EC50 in the presence of G418.

      Following in vitro passage, the NS5B polymerase gene was PCR-amplified from each resistant colony and population sequenced.

      Mutations in the NS5B gene were introduced individually into an HCV 1b-N shuttle vector by site-directed mutagenesis.

      Inhibitor susceptibilities of the site-directed mutants were determined using a transient transfection assay by measurement of luciferase activity at day 4. Replicon capacities were obtained by comparison of luciferase activity between mutant clones and the wild-type replicon.

      Combination studies were performed by incubating replicon cells in the presence of single compound or combination of two compounds without G418. Cells were passaged every 4 days and HCV RNA copy number was determined at each passage.



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