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Combination of the NS3/4A Protease Inhibitor ITMN-191 (R7227) with the Active Moiety of the NS5B Inhibitors
R1626 or R7128 Enhances Replicon Clearance and Reduces the Emergence of Drug
Resistant Variants
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From Jules: R1626 was discontinued due to toxicities but today the 3 companies announced the first clinical studies of 2 oral HCV drugs - R7128 plus ITMN191
Reported by Jules Levin
59th Annual Meeting of the American Association for the Study of Liver Diseases October 31-November 4, 2008 San Francisco, USA
Hua Tan1, Sonal Rajyaguru2, Tony Wu2, Matthew McCown2, Samir Ali2, Wen-Rong Jiang2, Michael Otto3, Phillip Furman3, Isabel Najera2, Klaus Klumpp2, Julian Symons2, Nick Cammack2, Lawrence Blatt1, Scott Seiwert1
1InterMune, Inc., Brisbane, CA 2Roche Palo Alto LLC, Palo Alto, CA 3Pharmasset, Inc., Princeton, NJ
AUTHOR SUMMARY and CONCLUSIONS
In vitro combination of the NS3/4A protease inhibitor ITMN-191 with the active moiety of the NS5B inhibitor R7128 or R1626:
-- Reduces compound concentrations required for cellular elimination of HCV replicon
-- Suppresses the emergence of drug resistant HCV replicons
These studies suggest that the combined antiviral effect of ITMN-191 (R7227) and either R7128 or R1626 in chronic HCV patients would be greater than the antiviral effect displayed by these agents when administered as monotherapy
ABSTRACT
Background: ITMN-191 is an inhibitor of HCV NS3/4A protease activity, and R1626 and R7128 are nucleoside inhibitors of the polymerase activity of HCV NS5B. All three compounds promote multi-log10 reductions in circulating HCV RNA in chronic HCV patients when administered for short durations as monotherapy. Here, to support future clinical studies that would combine ITMN-191 with R1626 or R7128, we investigated the combined antiviral effect of these compounds.
Methods: In the HCV clearance assay, cells harboring an HCV genotype 1b replicon were treated for 2 weeks with ITMN-191, the active moiety of R1626 (R1479), the active moiety of R7128 (PSI 6130), or a combination of inhibitors in the absence of G418 selection for replicon retention. Cells were counted and aliquots harvested for RT-PCR-based quantification of replicon RNA levels under G418 selection over 4 subsequent weeks. In the colony formation assay, cells were treated with either one or two compounds at 1X, 10X, or 15X their respective EC50. After 3 weeks in culture with G418, cells were fixed and stained with crystal violet or total cellular RNA extracted. For drug-drug interaction studies, HCV replicon cells were treated for 3 days with a pair of inhibitors in checkerboard dilution and percent reductions of reporter gene activity obtained.
Results: In the HCV clearance assay, 18 nM ITMN-191 and low _M concentrations of the active moiety of R1626 and R7128 (18 _M & 27 _M,
respectively) eliminated HCV replicon in the absence of G418 selective pressure for replicon retention. Addition of the lowest tested concentration of ITMN-191 (6 nM) to the lowest tested concentration of the active moiety of R1626 or R7128 (0.3 _M & 0.45 _M, respectively) resulted in replicon clearance, demonstrating significant combined antiviral effect.
In the colony formation assay in the presence of G418 selective pressure for replicon retention, NS5B inhibitors at 10X or 15X EC50 supported replicon clearance and did not result in drug resistant colonies.
Similar treatment with ITMN-191 selected resistant colonies, but these were suppressed by an NS5B inhibitor at 1X EC50.
Drug-drug interaction studies over a 3 day incubation period demonstrated additive to slightly synergistic interactions between the two inhibitor classes.
Conclusions:
The combination of ITMN-191 with the active moiety of either R1626 or R7128 results in enhanced antiviral activity and suppression of ITMN-191 resistant variants. These findings suggest that the combination of ITMN-191 with R1626 or R7128 may confer significantly greater antiviral activity than has been observed with these agents in previous monotherapy trials.
NS3/4A protease/helicase
NS3 is a bifunctional protein that possesses both serine protease and
nucleic acid unwinding activity
Protease activity is responsible for cleavage of several linkages in the
HCV poly protein and is essential for the viral life cycle
Inhibitors of serine protease activity have shown promise in clinical
studies
Due to the high tolerance for substitutions in the serine protease
substrate binding site, short duration monotherapy with protease
inhibitors that bind to this site has been reported to lead to viral
breakthrough (drug resistance)
NS5B RNA-dependent RNA polymerase
NS5B is the viral polymerase
Polymerase activity is essential for the viral life cycle
NS5B inhibitors can bind either to the polymerase active site or
allosteric sites
Nucleoside analogs which induce premature termination of the RNA
have shown promising activity in clinical studies
The active site of NS5B is relatively intolerant to substitution. Short
duration monotherapy with RNA elongation inhibitors is not associated
with viral breakthrough
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