770 CHARACTERIZATION OF THE HEPATOSELECTIVE DISTRIBUTION OF ACH-1625, A POTENT, CLINICAL STAGE HCV NS3 PROTEASE INHIBITOR

POSTERS Methods: The antiviral activity and specificity of IDX320 were evaluated in a variety of standard assays utilizing purified proteases, HCV replicons, and an infectious HCV virus. The resistance profile of IDX320 was determined in replicon selection experiments as well as transient transfection assays using site-directed mutant replicons. Results: IDX320 is a potent non-covalent inhibitor of HCV protease enzymes of genotypes 1a, 1b, 2a, and 4a (0.8 to 1.9 nM IC50 ) and genotype 3a (23 nM IC50 ). In surface plasmon resonance studies, IDX320 bound tightly to NS3/4A protease (KD of 0.8 nM) with a dissociation half-life of >9 hours. Nine human cellular proteases were not inhibited by IDX320 (IC50 ≥15 mM). In cellular assays, IDX320 inhibited genotype 1b replicons with subnanomolar potency (EC50 0.5 nM; SI 50,400), genotype 1a replicons (EC50 3.4 nM; SI >22,985) and genotype 2a JFH-1 virus (EC50 4.7 nM; SI 2,568). Treatment of replicon cells for 14 days produced dosedependent reductions in replicon RNA levels, with a maximum reduction of 3.7 log10 at 10 nM IDX320. The NS3 D168V mutation was the signature resistance mutation, selected in all IDX320resistant replicon cell lines. Studies on replicons bearing sitedirected protease resistance mutations indicated that Q80R, R155K, A156T, or D168A/E/V/Y conferred resistance to IDX320, while T54A, R155Q and A156S mutants remained susceptible. Replicons bearing the D168V mutation as well as the IDX320-resistant cell lines remained fully susceptible to IFN plus ribavirin, and direct-acting antivirals of different classes. Conclusion: IDX320 is a potent inhibitor of HCV NS3/4A protease and HCV replication in cell culture with broad genotypic coverage. In vitro selectivity was demonstrated against several human cellular proteases and cell lines. IDX320 bound tightly to HCV protease with a long dissociation half-life. These favorable in vitro characteristics, along with others presented by Good et al.1 , support the evaluation of IDX320 in the clinic. Reference(s) [1] Good et al, “Preclinical pharmacokinetic profile of IDX320, a novel and potent HCV protease inhibitor”; (submitted EASL 2010)

769 A TRIPLE COMBINATION OF DIRECT-ACTING ANTIVIRAL AGENTS DEMONSTRATES ROBUST ANTI-HCV ACTIVITY IN VITRO M. Lacolla, L.B. Lallos, I. Serra, J.P. Bilello, M.A. Soubasakos, A. Bonin, D.N. Standring. Idenix Pharmaceuticals, Inc., Cambridge, MA, USA E-mail: [email protected] Background and Aims: The future approach to HCV treatment will likely be a combination of multiple direct-acting antiviral (DAA) drugs with distinct modes of action. This study evaluated the in vitro antiviral effect of IDX184 (a nucleotide prodrug polymerase inhibitor) in double and triple combinations with IDX375 (a nonnucleoside polymerase inhibitor) or IDX320 (a protease inhibitor) in the HCV replicon system. Methods: Combinations of IDX184 with IDX375 and/or IDX320 were evaluated in 3-day activity assays and the data was analyzed by multiple mathematical models (Bliss independence, Loewe additivity and combination index). These DAA combinations were also evaluated in a 14-day treatment assay with HCV RNA levels and colony formation as endpoints. Results: In standard assays in the genotype 1b HCV replicon system, the EC50 values of the single agents were 106 nM (IDX184), 2.3 nM (IDX375) and 0.5 nM (IDX320). Double combinations of IDX184 with IDX375 or IDX320 showed additive antiviral activity in vitro compared to treatment with single agents. In 14-day treatments of replicon cells, the single agents at concentrations of 450 nM (IDX184), 15 nM (IDX375) and 1 nM (IDX320) generated reductions in HCV replicon RNA between 0.7 and 1.6 log10 . All double combinations resulted in replicon RNA reductions of 2.0 to 2.6 log10 , consistent with an additive antiviral effect. However, 14-

day treatments with triple DAA combinations (IDX184, IDX375 and IDX320) resulted in RNA reductions of almost 4 log10 , substantially higher than the expected additive effect. Outgrowth of these treated cells in the presence of G418 resulted in an average of <5 repliconbearing colonies remaining (frequency, <0.01%), indicating that most of the replicons had been cleared by triple DAA combination treatment. Conclusion: These studies suggest that a triple combination of IDX184 with direct-acting antivirals from different classes, such as IDX375 (NNI) and IDX320 (PI), can lead to significantly enhanced suppression of HCV in vitro compared to single agents or double combinations. This effect, coupled with the different resistance profiles of the three agents, suggests the potential value of triple combination regimens in future HCV clinical studies. 770 CHARACTERIZATION OF THE HEPATOSELECTIVE DISTRIBUTION OF ACH-1625, A POTENT, CLINICAL STAGE HCV NS3 PROTEASE INHIBITOR K. Stauber, C.W. Marlor, G. Mushtaq, V. Gadhachanda, G. Pais, A. Hashimoto, A. Phadke, M. Huang, M. Deshpande. Achillion Pharmaceuticals, New Haven, CT, USA E-mail: [email protected] Background and Aims: HCV NS3 protease is a clinically validated target for treatment of HCV. ACH-1625 demonstrates high potency and selectivity against HCV NS3 protease. In clinical trials, ACH1625 was very well tolerated after administration of single and multiple ascending doses up to 2000 mg in healthy volunteers. Currently, ACH-1625 is being evaluated in chronically infected HCV patients. The aim of this work was to characterize the hepatoselectivity of ACH-1625. Methods: Oral and intravenous pharmacokinetic profiling was conducted in dogs and rats. The concentration of ACH-1625 in plasma and tissue homogenates was analyzed by LC-MS/MS. Whole body tissue distribution was assessed by autoradiography. Cellular uptake was studied using isolated cryopreserved hepatocytes and individual transporter-expressing cell lines. Results: ACH-1625 shows high liver partitioning. In the rat, the rapid disappearance from plasma after intravenous administration corresponded to liver concentrations that were >1000-fold higher than respective plasma concentrations. At 24 hr after an oral dose of 2 to 50 mg/kg, liver concentrations were at least 100-fold higher than corresponding plasma values. Similarly in the dog, liverto-plasma ratios up to 67-fold were achieved after oral dosing. Subsequent tissue distribution studies in the rat with 14 C-ACH1625 confirmed prolonged hepatic distribution while distribution into most other tissues (excluding the GI tract) was low. ACH1625 (1–100 mM) was rapidly taken up into isolated hepatocytes; maximal uptake was attained by 1 min. Specific transport into cell lines containing expressed human organic anion-transporting polypeptide (OATP) uptake transporters OATP1B1, OATP1B3 and OATP2B1 and rat rOatp1a1 indicate a role for active transport in the hepatoselectivity of ACH-1625. Conclusions: ACH-1625 shows high liver partitioning in the rat and the dog. In vitro studies indicate that the observed hepatoselectivity is due, in part, to active uptake through members of the OATP transporter family. These studies suggest that hepatoselective distribution will occur in humans similarly to that seen in nonclinical species. As a consequence of active uptake into hepatocytes, steady-state liver concentrations will most likely be well above plasma values. Overall, ACH-1625 displays a very favorable pharmacokinetic and safety profile for treatment of Hepatitis-C.

Journal of Hepatology 2010 vol. 52 | S183–S317

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