Hepatitis C virus is a blood-borne infection and the leading cause of chronic liver disease (including cirrhosis and cancer) and liver transplantation. Since the identification of HCV in 1989, there has been an extensive effort to identify and improve treatment options. An important milestone was reached in 2011 with the approval of the first-generation HCV NS3/4A protease inhibitors. However, new therapies are needed to improve cure rates, shorten treatment duration, and improve tolerability. Here we summarize the extensive medicinal chemistry effort to develop novel P2 cyclopentane macrocyclic inhibitors guided by HCV NS3 protease assays, the cellular replicon system, structure-based design, and a panel of DMPK assays. The selection of compound 29 (simeprevir, TMC435) as clinical candidate was based on its excellent biological, PK, and safety pharmacology profile. Compound 29 has recently been approved for treatment of chronic HCV infection in combination with pegylated interferon-α and ribavirin in Japan, Canada, and USA.

-Azido-2 0 -deoxy-2 0 -methylcytidine (14) is a potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase, displaying an EC50 value of 1.2 lM and showing moderate in vivo bioavailability in rat (F = 14%). Here we describe the synthesis and biological evaluation of 4 0 -azido-2 0 -deoxy-2 0 -methylcytidine and prodrug derivatives thereof.

POSTERSgenotype-1b HCV infected patients given 10 mg GS-5885/placebo.GS-5885 was well tolerated with significant antiviral activity: Figure: HCV RNA Viral Load Reduction.HCV RNA declined sharply with median decrease of -3.1 to -3.3 log 10 IU/mL (Day 2) and up to -2.0 (1a) and -2.8 (1b) log 10 IU/mL (Day 6) at GS-5885 doses ≥3 mg.The major resistance mutants were M28T, Q30R/H, L31M, and Y93C.Blinded preliminary safety data through Day 7 employed for dose escalation decision-making after 3, 10, and 30-mg cohorts of HCV-1a infected patients showed: no serious adverse events (AEs), no discontinuations due to AEs, infrequent AEs occurring in ≥2 patients headache (5 patients), difficult blood draw (3 patients), and frequent urination and upper respiratory infection in 2 patients each.In the 10-and 30-mg GS-5885 HCV 1a dose groups, 2/24 (8%) patients had transient treatment-emergent Grade 3/4 laboratory abnormalities.One with a Grade 3 ALT (10-mg cohort) due to a clinically insignificant 14 U/L increase returning to baseline on drug, and a second with Grade 3 creatine phosphokinase (30-mg cohort) on Day 7 off-drug associated with weight lifting.HCV RNA change from baseline GS-5885 log 10 IU/mL 1 mg (n = 10) 3 mg (n = 10) 10 mg (n = 10) 30 mg (n = 10) Meanx.x -3.1 -2.8 -3.0 Median x.x -3.1 -3.1 -3.2 Range x.x, x.x -3.7, -2.5 -3.7, -1.6 -3.9, -0.9Conclusions: GS-5885 is a potent HCV NS5A inhibitor demonstrating a 1000-fold (3 log 10 ) reduction in HCV RNA after three, once daily, low oral doses.

Background: MP-424 (telaprevir) is a highly selective inhibitor of the hepatitis C virus (HCV) NS3-4A protease which is currently investigated in phase 3 trial in combination with peginterferon and ribavirin.We reported the first clinical study examining telaprevir monotherapy with an extended dosing period of 24 weeks in Japan (Ozeki I, et al: EASL 2009).After completing study drug therapy, patients were treated with peginterferon alfa-2b and ribavirin.Methods: Five naïve patients were treated with telaprevir monotherapy at 750 mg every 8 hours for up to 24 weeks.All patients, 48 to 68 years of age, had a chronic infection with HCV genotype 1b and their baseline serum HCV RNA levels were at least 1×10 5 IU/ml.Patients who met the definition of viral breakthrough (>2-log increase in HCV RNA above nadir) discontinued telaprevir dosing.RNA sequence was analyzed using the clonal sequencing method.After a withdrawal of MP-424, 4 of the 5 patients were enrolled in the off-study treatment with peginterferon alfa-2b and ribavirin within 4 weeks after the last administration of study drug.Results: At the initiation of off-study treatment, major NS3 protease variants in the patients were T54A, T54S+A156T, and A156V+V158I.All patients achieved undetectable HCV RNA levels by 12 weeks regardless of selected telaprevir-resistant variants.Sustained virologic response (SVR) was achieved in 2 patients who completed the assigned treatment for 48 weeks.The other 2 patients are now receiving peginterferon alfa-2b and ribavirin beyond 48 weeks for extended treatment period to 72 weeks.HCV RNA levels in these 2 patients continue to be undetectable.Conclusions: By the off-study treatment of standard peginterferon alfa-2b and ribavirin, all patients achieved complete EVR regardless of selected drug-resistant variants.Two patients who were typically treated for 48 weeks achieved an SVR.Updated results of this study will be presented.

Background: TMC435 is an NS3/NS4A protease inhibitor in development for treatment of HCV infection.Methods: OPERA-1 is an ongoing double blind, placebo-controlled Phase IIa trial to assess the antiviral activity, safety and pharmacokinetics of once-daily (QD) regimens of TMC435 in HCV genotype 1 treatmentnaive and treatment-experienced patients.Interim 4-week results of three dose cohorts (25, 75 or 200 mg QD) in treatment-naive patients are reported here.Patients were randomized to receive either 7 days of monotherapy of TMC435 or placebo followed by 21 days triple therapy with TMC435 or placebo, PegIFNa-2a and ribavirin (RBV) (Panel A); or 28 days of triple therapy with TMC435 or placebo, and PegIFNa-2a/RBV (Panel B).Thereafter, patients continued on PegIFNa-2a/RBV (Standard Of Care).Stopping rules were included.Results: There were no TMC435-related treatment discontinuations, Grade 3 or 4 adverse events (AEs) or serious AEs.AEs were generally mild to moderate.Hepatic AST and ALT values improved during therapy.There were no clinically relevant changes in other laboratory parameters, ECGs, QTc, and vital signs.All three TMC435 doses in combination with PegIFN/RBV showed antiviral activity superior to PegIFN/RBV alone.During 7-day monotherapy, an antiviral dose-relationship was observed for TMC435.On triple therapy, a similar antiviral activity was noted between the 75 mg and 200 mg doses.In the 25, 75, 200 mg 4-week triple therapy arms, 6/9, 9/9 and 10/10 patients had HCV RNA concentrations below the lower limit of quantification (<25 IU/mL) and 3/9, 8/9 and 7/10 had undetectable HCV RNA (<10 IU/mL) at day 28, respectively.At week 12, 6/9 patients of the 25 mg and all patients of the 75 mg triple arm had undetectable HCV RNA.Viral breakthroughs during TMC435 treatment were only observed in Panel A and were associated with mutations conferring reduced susceptibility to TMC435 in vitro.Conclusion: TMC435 at doses of 25, 75 or 200 mg QD administered for 4 weeks was well tolerated and demonstrated potent antiviral activity.The trial is ongoing for treatment-experienced patients.

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ABSTRACT More than 100 transcripts of various abundances and kinetic classes are expressed during phases of productive and latent infections by herpes simplex virus (HSV) type 1. To carry out rapid global analysis of variations in such patterns as a function of perturbation of viral regulatory genes and cell differentiation, we have made DNA microchips containing sets of 75-mer oligonucleotides specific for individual viral transcripts. About half of these are unique for single transcripts, while others function for overlapping ones. We have also included probes for 57 human genes known to be involved in some aspect of stress response. The chips efficiently detect all viral transcripts, and analysis of those abundant under various conditions of infection demonstrates excellent correlation with known kinetics of mRNA accumulation. Further, quantitative sensitivity is high. We have further applied global analysis of transcription to an investigation of mRNA populations in cells infected with a mutant virus in which the essential immediate-early α27 (UL54) gene has been functionally deleted. Transcripts expressed at 6 h following infection with this mutant can be classified into three groups: those whose abundance is augmented (mainly immediate-early transcripts) or unaltered, those whose abundance is somewhat reduced, and those where there is a significant reduction in transcript levels. These do not conform to any particular kinetic class. Interestingly, levels of many cellular transcripts surveyed are increased. The high proportion of such transcripts suggests that the α27 gene plays a major role in the early decline in cellular gene expression so characteristic of HSV infection.