Potency and Resistance Analysis of Hepatitis C Virus NS5B Polymerase Inhibitor BMS-791325 on All Major Genotypes

BMS-791325 is a hepatitis C virus (HCV) inhibitor binding to the thumb domain of the NS5B RNA-dependent RNA polymerase. BMS-791325 is well characterized in genotype 1 (GT1) and exhibits good inhibitory activity (50% effective concentration [EC₅₀], <10 nM) against hybrid replicons containing patient NS5B sequences from GT3a, -4a, and -5a while potency against GT2 is significantly reduced (J. A. Lemm et al., Antimicrob. Agents Chemother. 58:3485–3495, 2014, doi:http://dx.doi.org/10.1128/AAC.02495-13). BMS-791325 potency against GT6a hybrid replicons is more variable, with two of three hybrid clones having EC₅₀s similar to that for GT1 while a third patient clone was ∼10 times less susceptible to BMS-791325. To characterize the resistance profile of BMS-791325 beyond GT1, curing studies were performed across GT1a and -3a to -6a and demonstrated that GT1a has the highest resistance barrier versus BMS-791325 while GT6a has the lowest. Selection of GT3 to -6 NS5B chimeric replicon cells at different concentrations of BMS-791325 revealed substitutions in the thumb domain of NS5B at residues 494 and 495 that conferred different levels of resistance to BMS-791325 but remained susceptible to NS5A or NS3 protease inhibitors. In addition, we demonstrate that the reduced potency of BMS-791325 against one GT6a patient is due to an A494 polymorphism present in ∼21% of sequences in the European HCV database. The results from this report suggest that BMS-791325 is a candidate for combination treatment of HCV GT3 to -6 chronic infections, and the resistance profiles identified will provide useful information for future clinical development.     

Genotypic and Phenotypic Analyses of Hepatitis C Virus Variants Observed in Clinical Studies of VX-222, a Nonnucleoside NS5B Polymerase Inhibitor

VX-222, a thiophene-2-carboxylic acid derivative, is a selective nonnucleoside inhibitor of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In phase 1 and 2 clinical studies, VX-222 demonstrated effective antiviral efficacy, with substantial reductions in plasma HCV RNA in patients chronically infected with genotype 1 HCV. To characterize the potential for selection of VX-222-resistant variants in HCV-infected patients, the HCV NS5B gene was sequenced at baseline and during and after 3 days of VX-222 dosing (monotherapy) in a phase 1 study. Variants with the substitutions L419C/I/M/P/S/V, R422K, M423I/T/V, I482L/N/T, A486S/T/V, and V494A were selected during VX-222 dosing, and their levels declined over time after the end of dosing. Phenotypic analysis of these variants was conducted using HCV replicons carrying site-directed mutations. Of the 17 variants, 14 showed reduced susceptibility to VX-222 compared with the wild type, with the L419C/S and R422K variants having higher levels of resistance (>200-fold) than the rest of the variants (6.8- to 76-fold). The M423I and A486S variants remained susceptible to VX-222. The 50% effective concentration (EC₅₀) for the L419P variant could not be obtained due to the poor replication of this replicon. The majority of the variants (15/17) were less fit than the wild type. A subset of the variants, predominately the L419S and R422K variants, were observed when the efficacy and safety of VX-222- and telaprevir-based regimens given for 12 weeks were investigated in genotype 1 HCV-infected patients in a phase 2 study. The NS3 and NS5B variants selected during the dual combination therapy showed reduced susceptibility to both telaprevir and VX-222 and had a lower replication capacity than the wild type. The phase 1b study has the ClinicalTrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT00911963","term_id":"NCT00911963"}}NCT00911963, and the phase 2a study has ClinicalTrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT01080222","term_id":"NCT01080222"}}NCT01080222.     

In Vitro and In Vivo Antiviral Activity and Resistance Profile of Ombitasvir,an Inhibitor of Hepatitis C Virus NS5A

Ombitasvir (ABT-267) is a hepatitis C virus (HCV) NS5A inhibitor with picomolar potency, pan-genotypic activity, and 50% effective concentrations (EC₅₀s) of 0.82 to 19.3 pM against HCV genotypes 1 to 5 and 366 pM against genotype 6a. Ombitasvir retained these levels of potency against a panel of 69 genotype 1 to 6 chimeric replicons containing the NS5A gene derived from HCV-infected patients, despite the existence of natural sequence diversity within NS5A. In vitro resistance selection identified variants that conferred resistance to ombitasvir in the HCV NS5A gene at amino acid positions 28, 30, 31, 58, and 93 in genotypes 1 to 6. Ombitasvir was evaluated in vivo in a 3-day monotherapy study in 12 HCV genotype 1-infected patients at 5, 25, 50, or 200 mg dosed once daily. All patients in the study were HCV genotype 1a infected and were without preexisting resistant variants at baseline as determined by clonal sequencing. Decreases in HCV RNA up to 3.1 log₁₀ IU/ml were observed. Resistance-associated variants at position 28, 30, or 93 in NS5A were detected in patient samples 48 hours after the first dose. Clonal sequencing analysis indicated that wild-type virus was largely suppressed by ombitasvir during 3-day monotherapy, and at doses higher than 5 mg, resistant variant M28V was also suppressed. Ombitasvir was well tolerated at all doses, and there were no serious or severe adverse events. These data support clinical development of ombitasvir in combination with inhibitors targeting HCV NS3/4A protease (ABT-450 with ritonavir) and HCV NS5B polymerase (ABT-333, dasabuvir) for the treatment of chronic HCV genotype 1 infection. (Study M12-116 is registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01181427","term_id":"NCT01181427"}}NCT01181427.)     

Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase

BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC₅₀) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC₅₀s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC₅₀, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC₅₀s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC₅₀. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures in vivo in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC₅₀s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.     

Safety,Tolerability, Pharmacokinetics,and Antiviral Activity of GSK2336805, an Inhibitor of Hepatitis C Virus (HCV) NS5A,in Healthy Subjects and Subjects Chronically Infected with HCV Genotype 1

GSK2336805 is an orally bioavailable hepatitis C virus (HCV) inhibitor working through an NS5A-mediated mechanism. This first-time-in-human study was conducted to assess the safety, tolerability, pharmacokinetics, metabolism, and efficacy of GSK2336805 in healthy subjects and subjects infected with HCV genotype 1. We performed a three-part, randomized, double-blind, placebo-controlled study in 46 healthy subjects and 23 HCV-infected subjects. After an overnight fast, healthy subjects received GSK2336805 as 10 mg, 30 mg, 30 mg plus food, and 60 mg in a single dose and 10 mg (7 days), 30 mg (7 days), and 75 mg (14 days) in a once-daily multiple dose. Subjects with HCV received GSK2336805 as a 1- to 120-mg single dose. In subjects with HCV, reductions in HCV RNA were observed within 4 h and a single dose of GSK2336805 of ≥10 mg resulted in a statistically significant ≥2-log reduction in HCV RNA compared with placebo at 24 h postdose. GSK2336805 was readily absorbed in all subjects, and the half-life (t_1/2) was suitable for once-daily dosing. Administration of GSK2336805 with food had no effect on plasma GSK2336805 exposure; however, absorption was delayed, with a median t_max (time to maximum concentration of drug in serum) of 4.5 versus 2.0 h. Twenty subjects who received GSK2336805 experienced mild to moderate adverse events; none were serious. GSK2336805 was well tolerated and exhibited rapid, significant antiviral activity after a single dose in HCV-infected subjects. These results support the conduct of further studies evaluating GSK2336805 administered once daily for longer durations in combination with peginterferon, ribavirin, and other direct-acting antivirals. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01277692","term_id":"NCT01277692"}}NCT01277692.)     

Clinical and In Vitro Resistance to GS-9669, a Thumb Site II Nonnucleoside Inhibitor of the Hepatitis C Virus NS5B Polymerase

Treatment with GS-9669, a novel nonnucleoside inhibitor (site II) of hepatitis C virus (HCV) nonstructural 5B (NS5B) polymerase, resulted in significant antiviral activity in HCV genotype (GT) 1 patients dosed at 50 and 500 mg once daily (QD) and at 50, 100, and 500 mg twice daily (BID) for 3 days. This report characterizes the virologic resistance to GS-9669 in vitro and in GT1 HCV-infected patients from a phase I clinical study. An in vitro resistance selection study with GS-9669 revealed substitutions at several NS5B residues that conferred resistance. The M423 variants were selected at low drug concentrations (5× the 50% effective concentration [EC₅₀]), and the L419, R422, and I482 variants were selected at higher drug concentrations (20× the EC₅₀). During the phase I clinical study, substitutions at NS5B residues 419, 422, and 486 were the predominant changes associated with GS-9669 monotherapy. Substitutions at position 423 were observed only in GT1a patients in the low-dose groups (50 and 100 mg BID). Interestingly, four HCV patients had substitutions at position 423 at baseline. Consistent with the low resistance level at this position, three patients with M423I or M423V at baseline achieved >2-log₁₀ reductions of HCV RNA when treated with 100 mg BID or with 500 mg QD or BID of GS-9669. The fourth patient, who had the M423V substitution at baseline, had a 4.4-log₁₀ reduction of HCV RNA with 500 mg BID of GS-9669. Phenotypic analyses demonstrated that the viral isolates with multiple GS-9669 resistance-associated variants have reduced susceptibility to GS-9669 and lomibuvir (VX-222) but are not cross-resistant to other classes of HCV inhibitors. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01431898","term_id":"NCT01431898"}}NCT01431898.)     

In Vitro Antiviral Activity and Preclinical and Clinical Resistance Profile of Miravirsen,a Novel Anti-Hepatitis C Virus Therapeutic Targeting the Human Factor miR-122

Miravirsen is a β-d-oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC₅₀) of 0.67 μM. No cytotoxicity was observed up to the highest concentration tested (>320 μM) in different cell culture models, yielding a therapeutic index of ≥297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2′-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5′ untranslated region (UTR) from cells passaged in the presence of up to 20 μM (40-fold the miravirsen EC₅₀ concentration) at day 72 of passage but not at earlier time points (up to 39 days of passage). Likewise, a C3U nucleotide change was observed in the HCV 5′UTR from subjects with viral rebound after the completion of therapy in a miravirsen phase 2 clinical trial. An HCV variant constructed to contain the A4C change was fully susceptible to miravirsen. A C3U HCV variant demonstrated overall reductions in susceptibility to miravirsen but was fully susceptible to all other anti-HCV agents tested. In summary, miravirsen has demonstrated broad antiviral activity and a relatively high genetic barrier to resistance. The identification of nucleotide changes associated with miravirsen resistance should help further elucidate the biology of miR-122 interactions with HCV. (The clinical trial study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01200420","term_id":"NCT01200420"}}NCT01200420).     

In Vitro and In Vivo Antiviral Activity and Resistance Profile of the Hepatitis C Virus NS3/4A Protease Inhibitor ABT-450

The development of direct-acting antiviral agents is a promising therapeutic advance in the treatment of hepatitis C virus (HCV) infection. However, rapid emergence of drug resistance can limit efficacy and lead to cross-resistance among members of the same drug class. ABT-450 is an efficacious inhibitor of HCV NS3/4A protease, with 50% effective concentration values of 1.0, 0.21, 5.3, 19, 0.09, and 0.69 nM against stable HCV replicons with NS3 protease from genotypes 1a, 1b, 2a, 3a, 4a, and 6a, respectively. In vitro, the most common amino acid variants selected by ABT-450 in genotype 1 were located in NS3 at positions 155, 156, and 168, with the D168Y variant conferring the highest level of resistance to ABT-450 in both genotype 1a and 1b replicons (219- and 337-fold, respectively). In a 3-day monotherapy study with HCV genotype 1-infected patients, ABT-450 was coadministered with ritonavir, a cytochrome P450 3A4 inhibitor shown previously to markedly increase peak, trough, and overall drug exposures of ABT-450. A mean maximum HCV RNA decline of 4.02 log₁₀ was observed at the end of the 3-day dosing period across all doses. The most common variants selected in these patients were R155K and D168V in genotype 1a and D168V in genotype 1b. However, selection of resistant variants was significantly reduced at the highest ABT-450 dose compared to lower doses. These findings were informative for the subsequent evaluation of ABT-450 in combination with additional drug classes in clinical trials in HCV-infected patients. (Study M11-602 is registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01074008","term_id":"NCT01074008"}}NCT01074008.)     

Susceptibilities of Genotype 1a, 1b,and 3 Hepatitis C Virus Variants to the NS5A Inhibitor Elbasvir

Elbasvir is an investigational NS5A inhibitor with in vitro activity against multiple HCV genotypes. Antiviral activity of elbasvir was measured in replicons derived from wild-type or resistant variants of genotypes 1a, 1b, and 3. The barrier to resistance was assessed by the number of resistant colonies selected by exposure to various elbasvir concentrations. In a phase 1b dose-escalating study, virologic responses were determined in 48 noncirrhotic adult men with chronic genotype 1 or 3 infections randomized to placebo or elbasvir from 5 to 50 mg (genotype 1) or 10 to 100 mg (genotype 3) once daily for 5 days. The NS5A gene was sequenced from plasma specimens obtained before, during, and after treatment. Elbasvir suppressed the emergence of resistance-associated variants (RAVs) in vitro in a dose-dependent manner. Variants selected by exposure to high elbasvir concentrations typically encoded multiple amino acid substitutions (most commonly involving loci 30, 31, and 93), conferring high-level elbasvir resistance. In the monotherapy study, patients with genotype 1b had greater reductions in HCV RNA levels than patients with genotype 1a at all elbasvir doses; responses in patients with genotype 3 were generally less pronounced than for genotype 1, particularly at lower elbasvir doses. M28T, Q30R, L31V, and Y93H in genotype 1a, L31V and Y93H in genotype 1b, and A30K, L31F, and Y93H in genotype 3 were the predominant RAVs selected by elbasvir monotherapy. Virologic findings in patients were consistent with the preclinical observations. NS5A-RAVs emerged most often at amino acid positions 28, 30, 31, and 93 in both the laboratory and clinical trial. (The MK-8742 P002 trial has been registered at ClinicalTrials.gov under identifier {"type":"clinical-trial","attrs":{"text":"NCT01532973","term_id":"NCT01532973"}}NCT01532973.)     

Model-Based Phase 3 Dose Selection for HIV-1 Attachment Inhibitor Prodrug BMS-663068 in HIV-1-Infected Patients: Population Pharmacokinetics/Pharmacodynamics of the Active Moiety,BMS-626529

BMS-663068 is an oral prodrug of the HIV-1 attachment inhibitor BMS-626529, which prevents viral attachment to host CD4⁺ T cells by binding to HIV-1 gp120. To guide dose selection for the phase 3 program, pharmacokinetic/pharmacodynamic modeling was performed using data from two phase 2 studies with HIV-1-infected subjects (n = 244). BMS-626529 population pharmacokinetics were described by a two-compartment model with first-order elimination from the central compartment, zero-order release of prodrug from the extended-release formulation into a hypothetical absorption compartment, and first-order absorption into the central compartment. The covariates of BMS-663068 formulation type, lean body mass, baseline CD8⁺ T-cell percentage, and ritonavir coadministration were found to be significant contributors to intersubject variability. Exposure-response analyses showed a relationship between the log_e-transformed concentration at the end of a dosing interval (C_tau) normalized for the protein binding-adjusted BMS-626529 half-maximal (50%) inhibitory concentration (PBAIC₅₀) and the change in the HIV-1 RNA level from the baseline level after 7 days of BMS-663068 monotherapy. The probability of achieving a decline in HIV-1 RNA level of >0.5 or >1.0 log₁₀ copies/ml as a function of the log_e-transformed PBAIC₅₀-adjusted C_tau after 7 days of monotherapy was 99 to 100% and 57 to 73%, respectively, for proposed BMS-663068 doses of 400 mg twice daily (BID), 600 mg BID (not studied in the phase 2b study), 800 mg BID, 600 mg once daily (QD), and 1,200 mg QD. On the basis of a slight advantage in efficacy of BID dosing over QD dosing, similar responses for the 600- and 800-mg BID doses, and prior clinical observations, BMS-663068 at 600 mg BID was predicted to have the optimal benefit-risk profile and selected for further clinical investigation. (The phase 2a proof-of-concept study AI438006 and the phase 2b study AI438011 are registered at ClinicalTrials.gov under numbers {"type":"clinical-trial","attrs":{"text":"NCT01009814","term_id":"NCT01009814"}}NCT01009814 and {"type":"clinical-trial","attrs":{"text":"NCT01384734","term_id":"NCT01384734"}}NCT01384734, respectively.)     

Viral Resistance in Hepatitis C Virus Genotype 1-Infected Patients Receiving the NS3 Protease Inhibitor Faldaprevir (BI 201335) in a Phase 1b Multiple-Rising-Dose Study

Faldaprevir (BI 201335) is a selective NS3/4A protease inhibitor under development for the treatment of chronic hepatitis C virus (HCV) infection. NS3/4A genotyping and NS3 protease phenotyping analyses were performed to monitor the emergence of resistance in patients with HCV genotype 1 infection receiving faldaprevir alone or combined with pegylated interferon alfa 2a and ribavirin (PegIFN-RBV) during a phase 1b study. Among all baseline variants, a maximum 7-fold reduction in in vitro sensitivity to faldaprevir was observed for a rare NS3 (V/I)170T polymorphism. During faldaprevir monotherapy in treatment-naive patients, virologic breakthrough was common (77%, 20/26) and was associated with the emergence of resistance mutations predominantly carrying NS3 substitutions R155K in GT1a and D168V in GT1b. D168V conferred a greater reduction in faldaprevir sensitivity (1,800-fold) than R155K (330-fold); however, D168V was generally less fit than R155K in the absence of selective drug pressure. Treatment-experienced patients treated with faldaprevir-PegIFN-RBV triple therapy showed higher viral load reductions, lower rates of breakthrough (8%, 5/62), and less frequent emergence of resistance-associated variants compared with faldaprevir monotherapy. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00793793","term_id":"NCT00793793"}}NCT00793793.)     

Ultradeep Sequencing Study of Chronic Hepatitis C Virus Genotype 1 Infection in Patients Treated with Daclatasvir,Peginterferon, and Ribavirin

Direct-acting antivirals (DAAs) are either part of the current standard of care or are in advanced clinical development for the treatment of patients chronically infected with hepatitis C virus (HCV) genotype 1, but concern exists with respect to the patients who fail these regimens with emergent drug-resistant variants. In the present study, ultradeep sequencing was performed to analyze resistance to daclatasvir (DCV), which is a highly selective nonstructural protein 5A (NS5A) inhibitor. Eight patients with HCV genotype 1b, who were either treatment naive or prior nonresponders to pegylated interferon plus ribavirin (Rebetol; Schering-Plough) (PEG-IFN/RBV) therapy, were treated with DCV combined with PEG-IFN alpha-2b (Pegintron; Schering-Plough, Kenilworth, NJ) and RBV. To identify the cause of viral breakthrough, the preexistence and emergence of DCV-resistant variants at NS5A amino acids were analyzed by ultradeep sequencing. Sustained virological response (SVR) was achieved in 6 of 8 patients (75%), with viral breakthrough occurring in the other 2 patients (25%). DCV-resistant variant Y93H preexisted as a minor population at higher frequencies (0.1% to 0.5%) in patients who achieved SVR. In patients with viral breakthrough, DCV-resistant variant mixtures emerged at NS5A-31 over time that persisted posttreatment with Y93H. Although enrichment of DCV-resistant variants was detected, the preexistence of a minor population of the variant did not appear to be associated with virologic response in patients treated with DCV/PEG-IFN/RBV. Ultradeep sequencing results shed light on the complexity of DCV-resistant quasispecies emerging over time, suggesting that multiple resistance pathways are possible within a patient who does not rapidly respond to a DCV-containing regimen. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01016912","term_id":"NCT01016912"}}NCT01016912.)     

Efficacy and Safety of Danoprevir-Ritonavir plus Peginterferon Alfa-2a–Ribavirin in Hepatitis C Virus Genotype 1 Prior Null Responders

Danoprevir (DNV) is a hepatitis C virus (HCV) protease inhibitor that achieves high sustained virologic response (SVR) rates in combination with peginterferon alfa-2a–ribavirin in treatment-naive HCV genotype 1 (G1)-infected patients. This study explored the efficacy and safety of ritonavir-boosted DNV (DNVr) plus peginterferon alfa-2a–ribavirin in G1-infected prior peginterferon-ribavirin null responders. Null responders (<2-log₁₀ reduction in HCV RNA level at week 12) were given an open-label combination of 100 mg of ritonavir and 100 mg of DNV (100/100 mg DNVr) every 12 h (q12h) plus peginterferon alfa-2a–ribavirin for 12 weeks. All patients achieving an early virologic response (EVR; ≥2-log₁₀ decrease in HCV RNA by week 12) continued treatment with peginterferon alfa-2a–ribavirin; those without an EVR discontinued all study drugs. Twenty-four prior null responders were enrolled; 16 patients (67%) were infected with HCV G1b, and 8 (33%) were infected with G1a. Ninety-six percent of patients had an IL28B non-CC genotype. A sustained virologic response at 24 weeks posttreatment (SVR24) was achieved in 67% of patients, with a higher rate in G1b-infected (88%) than G1a-infected (25%) patients. Resistance-related breakthrough occurred in 4/8 G1a and 1/16 G1b patients through the DNV resistance-associated variant (RAV) NS3 R155K. NS3 R155K was also detected in 2/2 G1a patients who relapsed. Treatment was well tolerated. Two patients withdrew prematurely from study medications due to adverse events. Two serious adverse events were reported; both occurred after completion of DNVr therapy and were considered unrelated to treatment. No grade 3 or 4 alanine aminotransferase (ALT) elevations were observed. DNVr plus peginterferon alfa-2a–ribavirin demonstrated high SVR24 rates in HCV G1b-infected prior null responders and was well tolerated. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01185860","term_id":"NCT01185860"}}NCT01185860.)     

Characterization of Hepatitis C Virus Resistance from a Multiple-Dose Clinical Trial of the Novel NS5A Inhibitor GS-5885

GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log₁₀ HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at ≥3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30- and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number {"type":"clinical-trial","attrs":{"text":"NCT01193478","term_id":"NCT01193478"}}NCT01193478.)     

Pharmacokinetics,Safety, and Tolerability of Faldaprevir in Patients with Renal Impairment

Faldaprevir is a potent hepatitis C virus (HCV) NS3/4A protease inhibitor with negligible urinary excretion. We assessed the pharmacokinetics and safety of a single oral dose of faldaprevir (480 mg) in 32 HCV-negative subjects with renal impairment or normal renal function. Compared with subjects with normal renal function, the adjusted geometric mean ratios (90% confidence intervals in parentheses) for overall exposure area under the concentration-time curve from zero to infinity (AUC_0–∞) were 113.6% (41.6 to 310.2%), 178.3% (85.2 to 373.0%), and 169.2% (73.2 to 391.2%) for subjects with mild, moderate, and severe renal impairment, respectively. Overall, 5/8 (63%) subjects with normal renal function and 20/24 (83%) subjects with renal impairment reported adverse events, with gastrointestinal events being the most common. No severe or serious adverse events or deaths were reported. These results suggest that moderate or severe renal impairment can result in a modest increase in faldaprevir exposure. The increase in exposure may be related to decrease in the activity of the liver uptake transporter OATP1B1 as a result of renal impairment. Given this relatively slight increase in exposure, a dose adjustment in HCV patients with renal impairment is not warranted. (This study has been registered at ClinicalTrials.gov under registration number {"type":"clinical-trial","attrs":{"text":"NCT01957657","term_id":"NCT01957657"}}NCT01957657.)     

Endogenous intrahepatic IFNs and association with IFN-free HCV treatment outcome

BACKGROUND. Hepatitis C virus (HCV) infects approximately 170 million people worldwide and may lead to cirrhosis and hepatocellular carcinoma in chronically infected individuals. Treatment is rapidly evolving from IFN-α–based therapies to IFN-α–free regimens that consist of directly acting antiviral agents (DAAs), which demonstrate improved efficacy and tolerability in clinical trials. Virologic relapse after DAA therapy is a common cause of treatment failure; however, it is not clear why relapse occurs or whether certain individuals are more prone to recurrent viremia.METHODS. We conducted a clinical trial using the DAA sofosbuvir plus ribavirin (SOF/RBV) and performed detailed mRNA expression analysis in liver and peripheral blood from patients who achieved either a sustained virologic response (SVR) or relapsed.RESULTS. On-treatment viral clearance was accompanied by rapid downregulation of IFN-stimulated genes (ISGs) in liver and blood, regardless of treatment outcome. Analysis of paired pretreatment and end of treatment (EOT) liver biopsies from SVR patients showed that viral clearance was accompanied by decreased expression of type II and III IFNs, but unexpectedly increased expression of the type I IFN IFNA2. mRNA expression of ISGs was higher in EOT liver biopsies of patients who achieved SVR than in patients who later relapsed.CONCLUSION. These results suggest that restoration of type I intrahepatic IFN signaling by EOT may facilitate HCV eradication and prevention of relapse upon withdrawal of SOF/RBV.TRIAL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01441180","term_id":"NCT01441180"}}NCT01441180.FUNDING. Intramural Programs of the National Institute of Allergy and Infectious Diseases, National Institutes of Health Clinical Center, and National Cancer Institute; German Research Foundation.     

Efficacy of a Novel Sublingual Spray Formulation of Artemether in African Children with Plasmodium falciparum Malaria

The efficacy of sublingual artemether (ArTiMist) was investigated in two studies. In study 1, 31 children were randomized to sublingual artemether (n = 16) or intravenous (i.v.) quinine (n = 15). In study 2, 151 children were randomized to sublingual artemether (n = 77) or i.v. quinine (n = 74). For both studies, patients weighed between 5 and 15 kg and had either severe or complicated malaria based on WHO criteria, or they had uncomplicated malaria but were unable to tolerate oral medication as a result of nausea, vomiting, or diarrhea. Patients received either 3 mg/kg of body weight of sublingual artemether or a loading dose of 20 mg/kg of i.v. quinine followed by 10 mg/kg every 8 h i.v. thereafter. The primary endpoint was parasitological success, defined as a reduction in parasite count of ≥90% of that at baseline at 24 h after the first dose. Other endpoints based on parasite clearance and clinical response were evaluated. In study 1, there were parasitological success rates of 93.3% (14/15) and 66.7% (10/15) for the sublingual artemether and quinine treatments, respectively. In study 2, 94.3% (66/70) of the ArTiMist-treated patients and 39.4% (28/71) of the quinine-treated patients had parasitological success (P < 0.0001). Indicators of parasite clearance (parasite clearance time [PCT], time for parasite count to fall by 50% [PCT₅₀], time for parasite count to fall by 90% [PCT₉₀], and percent reduction in parasitemia from baseline at 24 h [PRR₂₄]) were significantly superior for children treated with sublingual artemether compared to those treated with i.v. quinine. There were no differences between treatments for the clinical endpoints, such as fever clearance time. The local tolerability of sublingual artemether was good. Sublingual artemether leads to rapid parasite clearance and clinical recovery. (Studies 1 and 2 are registered at ClinicalTrials.gov under registration numbers {"type":"clinical-trial","attrs":{"text":"NCT01047436","term_id":"NCT01047436"}}NCT01047436 and {"type":"clinical-trial","attrs":{"text":"NCT01258049","term_id":"NCT01258049"}}NCT01258049, respectively.)     

Resistance Analysis of Baseline and Treatment-Emergent Variants in Hepatitis C Virus Genotype 1 in the AVIATOR Study with Paritaprevir-Ritonavir,Ombitasvir, and Dasabuvir

AVIATOR, a phase 2 clinical trial, evaluated ritonavir-boosted paritaprevir (a protease inhibitor), ombitasvir (an NS5A inhibitor), and dasabuvir (a nonnucleoside polymerase inhibitor) (the three-drug [3D] regimen) with or without ribavirin (RBV) for 8, 12, or 24 weeks in 406 HCV genotype 1 (GT1)-infected patients. The rate of sustained virologic response 24 weeks after treatment ranged from 88% to 100% across the arms of the 3D regimen with or without RBV; 20 GT1a-infected patients and 1 GT1b-infected patient experienced virologic failure (5.2%). Baseline resistance-conferring variants in NS3 were rare. M28V in GT1a and Y93H in GT1b were the most prevalent preexisting variants in NS5A, and C316N in GT1b and S556G in both GT1a and GT1b were the most prevalent variants in NS5B. Interestingly, all the GT1a sequences encoding M28V in NS5A were from the United States, while GT1b sequences encoding C316N and S556G in NS5B were predominant in the European Union. Variants preexisting at baseline had no significant impact on treatment outcome. The most prevalent treatment-emergent resistance-associated variants (RAVs) in GT1a were R155K and D168V in NS3, M28T and Q30R in NS5A, and S556G in NS5B. The single GT1b-infected patient experiencing virologic failure had no RAVs in any target. A paritaprevir-ritonavir dose of 150/100 mg was more efficacious in suppressing R155K in NS3 than a 100/100-mg dose. In patients who failed after receiving 12 or more weeks of treatment, RAVs were selected in all 3 targets, while most patients who relapsed after 8 weeks of treatment did so without any detectable RAVs. Results from this study guided the selection of the optimal treatment regimen, treatment duration, and paritaprevir dose for further development of the 3D regimen. (This study has been registered at ClinicalTrials.gov under registration number {"type":"clinical-trial","attrs":{"text":"NCT01464827","term_id":"NCT01464827"}}NCT01464827.)     

SILEN-C3, a Phase 2 Randomized Trial with Faldaprevir plus Pegylated Interferon α-2a and Ribavirin in Treatment-Naive Hepatitis C Virus Genotype 1-Infected Patients

Faldaprevir is an investigational hepatitis C virus (HCV) NS3/4A protease inhibitor which, when administered for 24 weeks in combination with pegylated interferon α-2a and ribavirin (PegIFN/RBV) in treatment-naive patients in a prior study (SILEN-C1; M. S. Sulkowski et al., Hepatology 57:2143–2154, 2013, doi:10.1002/hep.26276), achieved sustained virologic response (SVR) rates of 72 to 84%. The current randomized, open-label, parallel-group study compared the efficacy and safety of 12 versus 24 weeks of 120 mg faldaprevir administered once daily, combined with 24 or 48 weeks of PegIFN/RBV, in 160 treatment-naive HCV genotype 1 patients. Patients with maintained rapid virologic response (HCV RNA of <25 IU/ml at week 4 and undetectable at weeks 8 and 12) stopped all treatment at week 24, otherwise they continued PegIFN/RBV to week 48. SVR was achieved by 67% and 74% of patients in the 12-week and 24-week groups, respectively. Virologic response rates were lower in the 12-week group from weeks 2 to 12, during which both groups received identical treatment. SVR rates were similar in both groups for patients achieving undetectable HCV RNA. Most adverse events were mild or moderate, and 6% of patients in each treatment group discontinued treatment due to adverse events. Once-daily faldaprevir at 120 mg for 12 or 24 weeks with PegIFN/RBV resulted in high SVR rates, and the regimen was well tolerated. Differences in the overall SVR rates between the 12-week and 24-week groups were not statistically significant and possibly were due to IL28B genotype imbalances; IL28B genotype was not tested, as its significance was not known at the time of the study. These results supported phase 3 evaluation. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00984620","term_id":"NCT00984620"}}NCT00984620).     

Effect of a High-Calorie,High-Fat Meal on the Bioavailability and Pharmacokinetics of PA-824 in Healthy Adult Subjects

PA-824 is a novel nitroimidazo-oxazine being developed as an antituberculosis agent. Two randomized studies evaluated the pharmacokinetics and safety of a single oral dose of PA-824 administered to healthy adult subjects 30 min after a high-calorie, high-fat meal (fed state) versus after a minimum 10-h fast (fasted state). A total of 48 subjects were dosed in the two studies in a randomized crossover design with PA-824 at dose levels of 50, 200, or 1,000 mg in the fed state or fasted state. After the administration of PA-824, the geometric mean ratios of C_max and AUC_0–∞ revealed an increase in exposure with the addition of a high-calorie, high-fat meal compared to the fasted state by 140 and 145% at 50 mg, 176 and 188% at 200 mg, and 450 and 473% at 50, 200, and 1,000 mg, respectively. The median T_max in the fed state was 4 h for the 50-mg dose and 5 h for the 200- and 1,000-mg doses. In the fasted state, the median T_max was 4 h for the 50- and 200-mg doses and 6.5 h for the 1,000-mg dose. All doses were well tolerated, and no serious adverse events occurred in either study. (This study has been registered at ClinicalTrials.gov under registration numbers {"type":"clinical-trial","attrs":{"text":"NCT01828827","term_id":"NCT01828827"}}NCT01828827 and {"type":"clinical-trial","attrs":{"text":"NCT01830439","term_id":"NCT01830439"}}NCT01830439.)