- Email: [email protected]
778 PRECLINICAL PROPERTIES OF THE NOVEL HCV NS3 PROTEASE INHIBITOR GS-9451
POSTERS apoptosis in human hepatoma cell line Huh7.5 and promoting insulin resistance in patients infected by genotype 1 (Pazienza et al. Hepatology 2007). The aims were: a. to analyze if the use of rapamycin in patients with recurrent chronic hepatitis C after liver transplantation improved sustained viral response (SVR) when receiving SOC; b. to know the effect of rapamycin in Huh7.5 cells infected with the JFH-1 replicon on mTOR and IRS-1 gene expression. Patients and Methods: A cohort study of 43 patients with recurrent chronic hepatitis C after liver transplantation, genotype 1 were treated with peginterferon 180 mcg/week and ribavirin (1000– 1200 mg/d), of whom 29 received tacrolimus (controls) and 14 received rapamycin at its usual schedule of immunosuppression after transplantation. Both groups were were controlled by fibrosis, baseline viral load, age and BMI. Huh7.5 cells were cultured in supplemented DMEM medium and incubated at 37°C, 5% CO2 . Cells were transfected with the JFH-1 replicon (genotype 2a). Rapamycin was added (10 nM) to cell cultures. Gene expression was analyzed by semi-quantitative PCR in real time. Results: Patients treated with rapamycin showed SVR in 2 out of 14 (14%) versus 8 out of 29 (28%) in patients receiving tacrolimus; p = ns. In Huh7.5 cells culture, rapamycin significantly increased gene expression in comparison with control of mTOR in both uninfected (1.8 fold) and HCV-infected cells (4.5 fold). Rapamycin also induced the expression of PI3K in HCV-infected cells (3 fold). Rapamycin did not alter cell growth rate at the concentration assayed. Conclusion: Rapamycin treated patients with recurrent chronic hepatitis C after liver transplantation did not obtain improvement in SVR compared to the group of patients receiving tacrolimus. In an in vitro system of HCV infection, rapamycin enhanced mTOR and PI3K gene expression, showing an interaction with the insulin signaling pathway. 778 PRECLINICAL PROPERTIES OF THE NOVEL HCV NS3 PROTEASE INHIBITOR GS-9451 A. Corsa, M. Robinson, H. Yang, B. Peng, G. Cheng, B. Schultz, O. Barauskas, M. Hung, X. Liu, C. Yang, Y. Wang, G. Rhodes, R. Pakdaman, M. Shen, C. Sheng, C. Kim, W. Delaney. Gilead Sciences, Foster City, CA, USA E-mail: [email protected] Background: GS-9451 is a HCV specific NS3 protease inhibitor being developed for the treatment of genotype 1 (GT1) HCV infection. Here we profile key preclinical properties of GS9451 including in vitro potency, selectivity, cross-resistance and combination activity as well as pharmacokinetic properties in preclinical species. Methods: Potency and selectivity of GS-9451 were measured biochemically using GT1b NS3 protease and multiple mammalian proteases. Cell-based potency (including drug combination studies) and cytotoxicity were measured in GT1a, GT1b and GT2a HCV replicon cell lines. Cross-resistance was evaluated by transient HCV replicon transfection. Metabolic stability was assessed in rat, dog and human microsomes and cryopreserved primary hepatocytes. Plasma pharmacokinetics were assessed in SpragueDawley rats and Cynomolgus monkeys after oral and intravenous administration. Results: GS-9451 demonstrated potent and selective inhibition of NS3 protease in biochemical assays (Ki = 420 pM for NS3 with >50,000 fold selectivity against all tested mammalian proteases). In multiple genotype 1a and 1b replicon cell lines, GS-9451 had median EC50 values of 9.3 nM and 7.3 nM, respectively, with minimal cytotoxicity (CC50 values ≥40,000 nM). GS-9451 was less active in GT2a replicon cells (EC50 = 316 nM). Additive to synergistic in vitro antiviral activity was observed when GS-9451 was combined
with other agents including IFN-a, RBV, the polymerase inhibitor GS-9190 and the NS5A inhibitor GS-5885. GS-9451 retained wildtype activity against multiple classes of NS5B inhibitor resistance mutations as well as NS5A inhibitor resistance mutations. GS-9451 was stable in the microsomes and hepatocytes of all tested species, including human. GS-9451 showed good oral bioavailability in both rats (62%) and monkeys (49%) with intravenous plasma elimination half-lives of 0.6 hours and 3.9 hours, respectively; GS-9451 was eliminated through biliary excretion. Conclusions: GS-9451 is a potent and selective inhibitor of genotype 1 HCV NS3 with favorable preclinical pharmacokinetic properties. These results are consistent with the potent antiviral activity observed in a recent Phase 1b study (Lawitz et al. Hepatology 2010 V52, No 4, 714A). Results of in vitro crossresistance and combination antiviral assays support the ongoing development of GS-9451 in combination with GS-9190 and other anti-HCV agents. 779 PRECLINICAL CHARACTERIZATION OF THE NOVEL HCV NS3 PROTEASE INHIBITOR GS-9256 H. Yang, C. Yang, Y. Wang, G. Rhodes, M. Robinson, B. Schultz, O. Barauskas, B. Peng, G. Cheng, R. Wang, X. Liu, R. Pakdaman, C. Sheng, C. Kim, W. Delaney. Gilead Sciences, Foster City, CA, USA E-mail: [email protected] Background: GS-9256 is a HCV specific NS3 protease inhibitor currently in phase 2 development for the treatment of genotype 1 (GT1) HCV infection. Here we characterize the in vitro potency, selectivity, cross-resistance and combination activity of GS-9256 and describe its pharmacokinetic properties in non-human species. Methods: Potency and selectivity of GS-9256 were measured using GT1b NS3 protease and several mammalian proteases. Cell-based potency and cytotoxicity (including drug combination studies) were measured in GT1a, GT1b and GT2a HCV replicon cell lines. Metabolic stability was assessed in rat, dog and human microsomes and primary hepatocytes. Plasma pharmacokinetics were assessed in mice, rats and dogs after oral and intravenous administration. Results: GS-9256 demonstrated potent and selective inhibition of NS3 protease in biochemical assays (Ki = 90 pM for GT1 NS3 with >10,000-fold selectivity over all tested mammalian proteases). In multiple GT1a and 1b replicon cell lines, GS-9256 had median EC50 values of 30 nM and 99 nM, respectively. CC50 values ranged between 19,000–45,000 nM. Compared to GT1, GS9256 had less activity against GT2 (EC50 =951 nM) in replicon assays. Additive to synergistic in vitro antiviral activity was observed when GS-9256 was combined with other agents including IFN-a, RBV, the NS5B inhibitor GS-9190 and the NS5A inhibitor GS-5885. GS-9256 retained wild-type activity against all tested NS5B and NS5A inhibitor resistance mutations. GS-9256 was stable in the microsomes and hepatocytes of all tested species, including human. GS-9256 had complete bioavailability in mice and moderate bioavailability in rats (14%) and dogs (21%). Intravenous plasma elimination half-lives were approximately 2 hours in mice, 0.6 hours in rats and 5 hours in dogs; GS-9256 was eliminated without metabolism through biliary excretion. Conclusions: GS-9256 is a potent and selective inhibitor of genotype 1 NS3 protease with a favorable pharmacokinetic profile in animal models. These preclinical results are consistent with the promising antiviral potency, safety and pharmacokinetic profile recently established in phase 1 studies of GS-9256 in HCV-infected patients (Lawitz et al. J.Hep 2010 S1 V52 S466). Results of in vitro cross-resistance and combination antiviral assays support the ongoing development of GS-9256 in combination with GS-9190 and other anti-HCV agents.
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with other agents including IFN-a, RBV, the polymerase inhibitor GS-9190 and the NS5A inhibitor GS-5885. GS-9451 retained wildtype activity against multiple classes of NS5B inhibitor resistance mutations as well as NS5A inhibitor resistance mutations. GS-9451 was stable in the microsomes and hepatocytes of all tested species, including human. GS-9451 showed good oral bioavailability in both rats (62%) and monkeys (49%) with intravenous plasma elimination half-lives of 0.6 hours and 3.9 hours, respectively; GS-9451 was eliminated through biliary excretion. Conclusions: GS-9451 is a potent and selective inhibitor of genotype 1 HCV NS3 with favorable preclinical pharmacokinetic properties. These results are consistent with the potent antiviral activity observed in a recent Phase 1b study (Lawitz et al. Hepatology 2010 V52, No 4, 714A). Results of in vitro crossresistance and combination antiviral assays support the ongoing development of GS-9451 in combination with GS-9190 and other anti-HCV agents. 779 PRECLINICAL CHARACTERIZATION OF THE NOVEL HCV NS3 PROTEASE INHIBITOR GS-9256 H. Yang, C. Yang, Y. Wang, G. Rhodes, M. Robinson, B. Schultz, O. Barauskas, B. Peng, G. Cheng, R. Wang, X. Liu, R. Pakdaman, C. Sheng, C. Kim, W. Delaney. Gilead Sciences, Foster City, CA, USA E-mail: [email protected] Background: GS-9256 is a HCV specific NS3 protease inhibitor currently in phase 2 development for the treatment of genotype 1 (GT1) HCV infection. Here we characterize the in vitro potency, selectivity, cross-resistance and combination activity of GS-9256 and describe its pharmacokinetic properties in non-human species. Methods: Potency and selectivity of GS-9256 were measured using GT1b NS3 protease and several mammalian proteases. Cell-based potency and cytotoxicity (including drug combination studies) were measured in GT1a, GT1b and GT2a HCV replicon cell lines. Metabolic stability was assessed in rat, dog and human microsomes and primary hepatocytes. Plasma pharmacokinetics were assessed in mice, rats and dogs after oral and intravenous administration. Results: GS-9256 demonstrated potent and selective inhibition of NS3 protease in biochemical assays (Ki = 90 pM for GT1 NS3 with >10,000-fold selectivity over all tested mammalian proteases). In multiple GT1a and 1b replicon cell lines, GS-9256 had median EC50 values of 30 nM and 99 nM, respectively. CC50 values ranged between 19,000–45,000 nM. Compared to GT1, GS9256 had less activity against GT2 (EC50 =951 nM) in replicon assays. Additive to synergistic in vitro antiviral activity was observed when GS-9256 was combined with other agents including IFN-a, RBV, the NS5B inhibitor GS-9190 and the NS5A inhibitor GS-5885. GS-9256 retained wild-type activity against all tested NS5B and NS5A inhibitor resistance mutations. GS-9256 was stable in the microsomes and hepatocytes of all tested species, including human. GS-9256 had complete bioavailability in mice and moderate bioavailability in rats (14%) and dogs (21%). Intravenous plasma elimination half-lives were approximately 2 hours in mice, 0.6 hours in rats and 5 hours in dogs; GS-9256 was eliminated without metabolism through biliary excretion. Conclusions: GS-9256 is a potent and selective inhibitor of genotype 1 NS3 protease with a favorable pharmacokinetic profile in animal models. These preclinical results are consistent with the promising antiviral potency, safety and pharmacokinetic profile recently established in phase 1 studies of GS-9256 in HCV-infected patients (Lawitz et al. J.Hep 2010 S1 V52 S466). Results of in vitro cross-resistance and combination antiviral assays support the ongoing development of GS-9256 in combination with GS-9190 and other anti-HCV agents.
Journal of Hepatology 2011 vol. 54 | S209–S361
S313