George Kukolj

An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus

Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.

Faldaprevir and Deleobuvir for HCV Genotype 1 Infection

BACKGROUND Interferon-free regimens would be a major advance in the treatment of patients with chronic hepatitis C virus (HCV) infection. METHODS In this phase 2b, randomized, open-label trial of faldaprevir (a protease inhibitor) and deleobuvir (a nonnucleoside polymerase inhibitor), we randomly assigned 362 previously untreated patients with HCV genotype 1 infection to one of five groups: faldaprevir at a dose of 120 mg once daily and deleobuvir at a dose of 600 mg three times daily, plus ribavirin, for 16, 28, or 40 weeks (TID16W, TID28W, or TID40W, respectively); faldaprevir at a dose of 120 mg once daily and deleobuvir at a dose of 600 mg twice daily, plus ribavirin, for 28 weeks (BID28W); or faldaprevir at a dose of 120 mg once daily and deleobuvir at a dose of 600 mg three times daily, without ribavirin, for 28 weeks (TID28W-NR). The primary end point was a sustained virologic response 12 weeks after the completion of therapy. RESULTS The primary end point was met in 59% of patients in the TID16W group, 59% of patients in the TID28W group, 52% of patients in the TID40W group, 69% of patients in the BID28W group, and 39% of patients in the TID28W-NR group. The sustained virologic response 12 weeks after the completion of therapy did not differ significantly according to treatment duration or dosage among ribavirin-containing regimens. This response was significantly higher with TID28W than with TID28W-NR (P=0.03). Rates of a sustained virologic response 12 weeks after the completion of therapy were 56 to 85% among patients with genotype 1b infection versus 11 to 47% among patients with genotype 1a infection and 58 to 84% among patients with IL28B CC versus 33 to 64% with non-CC genotypes. Rash, photosensitivity, nausea, vomiting, and diarrhea were the most common adverse events. CONCLUSIONS The rate of a sustained virologic response 12 weeks after the completion of therapy was 52 to 69% among patients who received interferon-free treatment with faldaprevir in combination with deleobuvir plus ribavirin. (Funded by Boehringer Ingelheim; SOUND-C2 ClinicalTrials.gov number, NCT01132313.).

Identification of a lipid kinase as a host factor involved in hepatitis C virus RNA replication

A functional screen of an adenovirus-delivered shRNA library that targets approximately 4500 host genes was performed to identify cellular factors that regulate hepatitis C virus (HCV) sub-genomic RNA replication. Seventy-three hits were further examined by siRNA oligonucleotide-directed knockdown, and silencing of the PI4KA gene was demonstrated to have a significant effect on the replication of a HCV genotype 1b replicon. Using transient siRNA oligonucleotide transfections and stable shRNA knockdown clones in HuH-7 cells, the PI4KA gene was shown to be essential for the replication of all HCV genotypes tested (1a, 1b and 2a) but not required for bovine viral diarrhea virus (BVDV) RNA replication.

Binding Site Characterization and Resistance to a Class of Non-nucleoside Inhibitors of the Hepatitis C Virus NS5B Polymerase

The virally encoded NS5B RNA-dependent RNA polymerase has emerged as a prime target in the search for specific HCV antivirals. A series of benzimidazole 5-carboxamide compounds inhibit the cellular RNA replication of a HCV subgenomic replicon and we have advanced our understanding of this class of inhibitors through a combination of complementary approaches that include biochemical cross-linking experiments with a photoreactive analogue followed by mass spectrometry analysis of the enzyme. A novel binding site has been localized for these inhibitors at the junction of the thumb domain and the N-terminal finger loop. Furthermore, the isolation and characterization of resistant replicon mutants that co-localize to this region distinguished this class of compounds from other non-nucleoside NS5B inhibitors that bind to distinct allosteric sites. Resistant mutations that emerged with the benzimidazole 5-carboxamide and related compounds were found at three amino acid positions in the thumb domain: Pro495 with substitutions to Ser, Leu, Ala, or Thr; Pro496 substitutions to Ser or Ala; and a V499A substitution. Mutations at each of these positions conferred different levels of resistance to this drug class: the Pro495 changes provided the greatest shifts in compound potency, followed by moderate changes in potency with the Pro496 substitutions, and finally only minor shifts in potency with V499A. Combinations that include the benzimidazole 5-carboxamide polymerase inhibitors and compounds that bind other sites or other HCV targets, including HCV protease inhibitors, are complementary in cell culture models of HCV RNA replication at suppressing the emergence of resistant variants. This novel class of compounds and unique binding site expand the diversity of HCV antivirals currently under development and offer the potential to improve the treatment of chronic HCV infection.

Efficacy of the Protease Inhibitor BI 201335, Polymerase Inhibitor BI 207127, and Ribavirin in Patients With Chronic HCV Infection

BACKGROUND & AIMS Therapeutic regimens are being developed for patients with hepatitis C virus (HCV) infection that do not include the combination of peginterferon alfa and ribavirin. We investigated the antiviral effect and safety of BI 201335 (an inhibitor of the NS3/4A protease) and BI 207127 (an inhibitor of the NS5B non-nucleoside polymerase) with ribavirin. METHODS Thirty-two treatment-naïve patients with chronic HCV genotype 1 infection were randomly assigned to groups that were given 400 mg or 600 mg BI 207127 3 times daily plus 120 mg BI 201335 once daily and 1000 to 1200 mg/day ribavirin for 4 weeks. The primary efficacy end point was virologic response (HCV RNA level <25 IU/mL at week 4). Thirty-two patients received treatment; 31 completed all 4 weeks of assigned combination therapy. RESULTS In the group given BI 207127 400 mg 3 times daily, the rates of virologic response were 47%, 67%, and 73% at days 15, 22, and 29; a higher rate of response was observed in patients with genotype-1b compared with genotype-1a infections. In the group given BI 207127 600 mg 3 times daily, the rates of virologic response were 82%, 100%, and 100%, respectively, and did not differ among genotypes. One patient in the group given 400 mg 3 times daily had virologic breakthrough (≥1 log(10) rebound in HCV RNA) at day 22. The most frequent adverse events were mild gastrointestinal disorders, rash, and photosensitivity. There were no severe or serious adverse events; no patients discontinued therapy prematurely. CONCLUSIONS The combination of the protease inhibitor BI 201335, the polymerase inhibitor BI 207127, and ribavirin has rapid and strong activity against HCV genotype-1 and did not cause serious or severe adverse events.

Restoration of HCV-specific CD8+ T cell function by interferon-free therapy

BACKGROUND & AIMS Chronic hepatitis C virus (HCV) infection is characterised by a failure of virus-specific CD8+ T cells that is mainly caused by viral escape and T cell exhaustion. Constant antigen stimulation has been suggested to contribute to HCV-specific CD8+ T cell exhaustion. However, IFN-based therapies failed to recover HCV-specific CD8+ T cell function suggesting that the damage to CD8+ T cells may be permanent even after antigen removal. It was therefore the objective of this study to analyse the impact of inhibition of ongoing viral replication by IFN-free therapy with direct acting antivirals (DAA) on the phenotype and function of HCV-specific CD8+ T cells. METHODS Virus-specific CD8+ T cells obtained from a patient cohort of 51 previously untreated chronically infected patients undergoing IFN-free therapy with a combination of faldaprevir (a protease inhibitor) and deleobuvir (a non-nucleoside polymerase inhibitor) with or without ribavirin were analysed ex vivo and after in vitro expansion at baseline, wk4, wk 12, and after treatment. RESULTS Our results show the rapid restoration of proliferative HCV-specific CD8+ T cells in the majority of patients with SVR12 within 4 weeks of therapy suggesting that IFN-free therapy mediated antigen removal may restore CD8+ T cell function. CONCLUSIONS This study indicates a specific restoration of proliferative HCV-specific CD8+ T cells under IFN-free therapy. This is in contrast to PegIFN-based therapies that have been shown not to restore T cell function during and after chronic infection.

Potency, safety, and pharmacokinetics of the NS3/4A protease inhibitor BI201335 in patients with chronic HCV genotype-1 infection

BACKGROUND & AIMS BI201335 is a highly specific and potent HCV protease inhibitor. This multiple rising dose trial evaluated antiviral activity and safety in chronic HCV genotype-1 patients. METHODS Thirty-four treatment-naïve patients were randomized to monotherapy with placebo or BI201335 at 20-240 mg once-daily for 14 days, followed by combination with pegylated interferon alfa/ribavirin (PegIFN/RBV) through Day 28. Nineteen treatment-experienced patients received 48-240 mg BI201335 once-daily with PegIFN/RBV for 28 days. HCV-RNA was measured with Roche COBAS TaqMan. RESULTS In treatment-naïve patients, median maximal viral load (VL) reductions during 14-day monotherapy were -3.0, -3.6, -3.7, and -4.2 log(10) for the 20, 48, 120, and 240 mg groups. VL breakthroughs (≥1 log(10) from nadir) were seen in most patients on monotherapy and were caused by NS3/4A variants (R155K, D168V) conferring in vitro resistance to BI201335. Adding PegIFN/RBV at Days 15-28 led to continuous viral load reductions in most patients. In treatment-experienced patients, treatment with BI201335 and PegIFN/RBV achieved VL<25 IU/ml at Day 28 in 3/6, 4/7, and 5/6 patients in the 48, 120, and 240 mg dose groups. VL breakthroughs were observed during triple combination in only 3/19 patients. BI201335 was generally well tolerated. Mild rash or photosensitivity was detected in four patients. Mild unconjugated hyperbilirubinemia was the only dose-dependent laboratory abnormality of BI201335. BI201335 elimination half-life supports once-daily dosing. CONCLUSIONS BI201335 combined with PegIFN/RBV was well tolerated and induced strong antiviral responses. These results support further development of BI201335 in HCV genotype-1 patients.

Preclinical characterization of BI 201335, a C-terminal carboxylic acid inhibitor of the hepatitis C virus NS3-NS4A protease.

ABSTRACT BI 201335 is a hepatitis C virus (HCV) NS3-NS4A (NS3 coexpressed with NS4A) protease inhibitor that has been shown to have potent clinical antiviral activity. It is a highly optimized noncovalent competitive inhibitor of full-length NS3-NS4A proteases of HCV genotypes 1a and 1b with Ki values of 2.6 and 2.0 nM, respectively. Ki values of 2 to 230 nM were measured against the NS3-NS4A proteases of HCV genotypes 2 to 6, whereas it was a very weak inhibitor of cathepsin B and showed no measurable inhibition of human leukocyte elastase. BI 201335 was also shown to be a potent inhibitor of HCV RNA replication in vitro with 50% effective concentrations (EC50s) of 6.5 and 3.1 nM obtained in genotype 1a and 1b replicon assays. Combinations of BI 201335 with either interferon or ribavirin had additive effects in replicon assays. BI 201335 had good permeability in Caco-2 cell assays and high metabolic stability after incubation with human, rat, monkey, and dog liver microsomes. Its good absorption, distribution, metabolism, and excretion (ADME) profile in vitro, as well as in rat, monkey, and dog, predicted good pharmacokinetics (PK) in humans. Furthermore, drug levels were significantly higher in rat liver than in plasma, suggesting that distribution to the target organ may be especially favorable. BI 201335 is a highly potent and selective NS3-NS4A protease inhibitor with good in vitro and animal ADME properties, consistent with its good human PK profile, and shows great promise as a treatment for HCV infection.

Faldaprevir combined with pegylated interferon alfa-2a and ribavirin in treatment-naïve patients with chronic genotype1 HCV: SILEN-C1 trial†‡

Faldaprevir (BI 201335) is a potent, hepatitis C virus (HCV) NS3/4A protease inhibitor with pharmacokinetic properties supportive of once‐daily (QD) dosing. Four hundred and twenty‐nine HCV genotype (GT)‐1 treatment‐naïve patients without cirrhosis were randomized 1:1:2:2 to receive 24 weeks of pegylated interferon alfa‐2a and ribavirin (PegIFN/RBV) in combination with placebo, faldaprevir 120 mg QD with 3 days of PegIFN/RBV lead‐in (LI), 240 mg QD with LI, or 240 mg QD without LI, followed by an additional 24 weeks of PegIFN/RBV. Patients in the 240 mg QD groups achieving maintained rapid virologic response (mRVR; viral load [VL] <25 IU/mL at week 4 and undetectable at weeks 8‐20) were rerandomized to cease all treatment at week 24 or continue receiving PegIFN/RBV up to week 48. VL was measured by Roche TaqMan. Sustained virologic response (SVR) rates were 56%, 72%, 72%, and 84% in the placebo, faldaprevir 120 mg QD/LI, 240 mg QD/LI, and 240 mg QD groups. Ninety‐two percent of mRVR patients treated with faldaprevir 240 mg QD achieved SVR, irrespective of PegIFN/RBV treatment duration. Eighty‐two percent of GT‐1a patients who received faldaprevir 240 mg QD achieved SVR versus 47% with placebo. Mild gastrointestinal disorders, jaundice resulting from isolated unconjugated hyperbilirubinemia, and rash or photosensitivity were more common in the active groups than with placebo. Discontinuations resulting from adverse events occurred in 4%, 11%, and 5% of patients treated with 120 mg QD/LI, 240 mg QD/LI, and 240 mg QD of faldaprevir versus 1% with placebo. Conclusion: Faldaprevir QD with PegIFN/RBV achieved consistently high SVR rates with acceptable tolerability and safety at all dose levels. The 120 and 240 mg QD doses are currently undergoing phase 3 evaluation. (HEPATOLOGY 2013;57:2143–2154)

Hepatitis C Virus Subgenomic Replicons in the Human Embryonic Kidney 293 Cell Line

ABSTRACT Hepatitis C virus (HCV) infects liver cells and its replication in other cells is incompletely defined. Human hepatoma Huh-7 cells harboring subgenomic HCV replicons were used in somatic cell fusion experiments with human embryonic kidney 293 cells as a means of examining the permissiveness of 293 cells for HCV subgenomic RNA replication. 293 cells were generally not permissive for replication of Huh-7 cell-adapted replicons. However, upon coculturing of the two cell lines, we selected rare replicon-containing cells, termed 293Rep cells, that resembled parental 293 cells. Direct metabolic labeling of cells with 33P in the presence of actinomycin D and Northern blotting to detect the negative strand of the replicon demonstrated functional RNA replicons in 293Rep cells. Furthermore, Western blots revealed that 293Rep cells expressed the HCV nonstructural proteins as well as markers of the naïve 293 cells but not Huh-7 cells. Propidium iodide staining and fluorescence-activated cell sorting analysis of 293Rep cells revealed that clone 293Rep17 closely resembled naïve 293 cells. Transfection of total RNA from 293Rep17 into naïve 293 cells produced replicon-containing 293 cell lines with characteristics distinct from those of Huh-7-derived replicon cell lines. Relative to Huh-7 replicons, the 293 cell replicons were less sensitive to inhibition by alpha interferon and substantially more sensitive to inhibition by poly(I)-poly(C) double-stranded RNA. This study established HCV subgenomic replicons in nonhepatic 293 cells and demonstrated their utility in expanding the study of cellular HCV RNA replication.