M. Subramanian

Sofosbuvir and Ribavirin for Hepatitis C Genotype 1 in Patients With Unfavorable Treatment Characteristics: A Randomized Clinical Trial

IMPORTANCE The efficacy of directly acting antiviral agents in interferon-free regimens for the treatment of chronic hepatitis C infections needs to be evaluated in different populations. OBJECTIVE To determine the efficacy and safety of sofosbuvir with weight-based or low-dose ribavirin among a population with unfavorable treatment characteristics. DESIGN, SETTING, AND PATIENTS Single-center, randomized, 2-part, open-label phase 2 study involving 60 treatment-naive patients with hepatitis C virus (HCV) genotype 1 enrolled at the National Institutes of Health (October 2011-April 2012). INTERVENTIONS In the studys first part, 10 participants with early to moderate liver fibrosis were treated with 400 mg/d of sofosbuvir and weight-based ribavirin for 24 weeks. In the second part, 50 participants with all stages of liver fibrosis were randomized 1:1 to receive 400 mg of sofosbuvir with either weight-based or low-dose 600 mg/d of ribavirin for 24 weeks. MAIN OUTCOMES AND MEASURES The primary study end point was the proportion of participants with undetectable HCV viral load 24 weeks after treatment completion (sustained virologic response of 24 weeks [SVR24]). RESULTS In the first part of the study, 9 participants (90%; 95% CI, 55%-100%) achieved SVR24. In the second part, 7 participants (28%) in the weight-based group and 10 (40%) in the low-dose group relapsed after treatment completion leading to SVR24 rates of 68% (95% CI, 46%-85%) in the weight-based group and 48% (95% CI, 28%-69%; P = .20) in the low-dose group. Twenty individuals participated in a pharmacokinetic-viral kinetic substudy, which demonstrated a slower loss rate of infectious virus in relapsers than in participants who achieved SVR (clearance, 3.57/d vs 5.60/d; P = .009). The most frequent adverse events were headache, anemia, fatigue, and nausea. There were 7 grade 3 events including anemia, neutropenia, nausea, hypophosphatemia, and cholelithiasis or pancreatitis. No one discontinued treatment due to adverse events. CONCLUSION AND RELEVANCE In a population of patients with a high prevalence of unfavorable traditional predictors of treatment response, a 24-week regimen of sofosbuvir and weight-based or low-dose ribavirin resulted in SVR24 rates of 68% and 48%, respectively. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01441180.

Bacillus anthracis Edema and Lethal Toxin Have Different Hemodynamic Effects but Function Together to Worsen Shock and Outcome in a Rat Model

INTRODUCTION To better define the contribution of edema toxins (ETx) and lethal toxins (LeTx) to shock with Bacillus anthracis, recombinant preparations of each were investigated alone or together in rats. METHODS AND RESULTS Lethal dose ranges (0%-100% lethality) of ETx (200-800 microg/kg as a 24-h infusion) were higher than those of LeTx (12.5-200 microg/kg) (P<.0001). However, compared with LeTx, similarly lethal ETx doses produced earlier and greater reductions in mean blood pressure (MBP) and increased, rather than decreased, heart rate (HR) (P<.05 for all). Combining either similar weight or lethal doses of ETx and LeTx increased the hazard ratio for death (log +/- standard error) similar to the sum calculated with the toxins effects alone (2.6+/-1.1 observed vs. 2.9+/-1.0 calculated for similar weight and 3.1+/-1.0 vs. 3.9+/-1.5 for similar lethal doses; P=.5 for both). Early (< or =10 h) and late during infusion, ETx and LeTx together also altered MBP and HR in patterns consistent with the sum of their individual effects. CONCLUSIONS ETx was approximately 10 times less lethal than LeTx but produced greater hypotension and added to the latters harmful effects. These findings suggest that it may be appropriate for antitoxin therapies for B. anthracis to target both ETx and LeTx.

Hepatitis C virus selectively perturbs the distal cholesterol synthesis pathway in a genotype-specific manner

Hepatitis C virus (HCV) subverts host cholesterol metabolism for key processes in its lifecycle. How this interference results in the frequently observed, genotype‐dependent clinical sequelae of hypocholesterolemia, hepatic steatosis, and insulin resistance (IR) remains incompletely understood. Hypocholesterolemia typically resolves after sustained viral response (SVR), implicating viral interference in host lipid metabolism. Using a targeted cholesterol metabolomic platform we evaluated paired HCV genotype 2 (G2) and G3 patient sera for changes in in vivo HCV sterol pathway metabolites. We compared HCV genotypic differences in baseline metabolites and following antiviral treatment to assess whether sterol perturbation resolved after HCV eradication. We linked these metabolites to IR and urine oxidative stress markers. In paired sera from HCV G2 (n = 13) and G3 (n = 20) patients, baseline sterol levels were lower in G3 than G2 for distal metabolites (7‐dehyrocholesterol (7DHC) 0.017 versus 0.023 mg/dL; Padj = 0.0524, cholesterol 140.9 versus 178.7 mg/dL; Padj = 0.0242) but not the proximal metabolite lanosterol. In HCV G3, SVR resulted in increased levels of distal metabolites (cholesterol [Δ55.2 mg/dL; Padj = 0.0015], 7DHC [Δ0.0075 mg/dL; Padj = 0.0026], lathosterol [Δ0.0430 mg/dL Padj = 0.0405]). In contrast, lanosterol was unchanged after SVR (P = 0.9515). Conclusion: HCV G3, but not G2, selectively interferes with the late cholesterol synthesis pathway, evidenced by lower distal sterol metabolites and preserved lanosterol levels. This distal interference resolves with SVR. Normal lanosterol levels provide a signal for the continued proteolysis of 3‐hydroxyl‐3‐methylglutaryl coenzyme A reductase, which may undermine other host responses to increase cholesterol synthesis. These data may provide a hypothesis to explain why hypocholesterolemia persists in chronic HCV infection, particularly in HCV G3, and is not overcome by host cholesterol compensatory mechanisms. (HEPATOLOGY 2012;56:49–56)

Effect of sofosbuvir and ribavirin treatment on peripheral and hepatic lipid metabolism in chronic hepatitis C virus, genotype 1-infected patients.

Hepatitis C virus (HCV) modulates intrahepatic cholesterol biosynthetic pathways to promote viral replication. Chronic HCV infection is associated with altered metabolism, including dyslipidemia and insulin resistance (IR), which contributes to disease progression and influences response to therapy. To further understand the impact of HCV infection on host metabolism, we examined changes in serum lipid profiles and intrahepatic expression of lipid‐related genes during interferon (IFN)‐free treatment of chronic HCV, genotype 1 infection with sofosbuvir and ribavirin (RBV), and explored associations with treatment outcome. Serum lipids (total cholesterol, low‐density lipoprotein [LDL], high‐density lipoprotein [HDL], and triglycerides [TGs]) and hemoglobin A1C (HbA1C) were measured during treatment, while gene expression of lipid‐related genes was assessed using paired pre‐ and end‐of‐treatment (EOT) liver biopsies from 8 patients (n = 7 sustained virologic response [SVR]; n = 1 relapse) and unpaired EOT liver biopsies from 25 patients (n = 17 SVR; n = 8 relapse). Serum LDL concentration and particle size increased early in therapy, whereas TG concentration and very‐low‐density lipoprotein particle size decreased concomitantly, irrespective of treatment outcome. Whereas LDL increased in patients regardless of treatment outcome, average LDL concentration was lower at baseline and post‐treatment in patients who relapsed. Analysis of paired liver biopsies revealed altered expression of genes associated with lipid transport, assembly, and signaling. In unpaired EOT liver biopsies, intrahepatic expression of fatty acid metabolism and lipid transport genes was lower in patients who experienced treatment relapse. Conclusion: Clearance of HCV using an IFN‐free antiviral regimen results in rapid changes in peripheral and intrahepatic metabolic pathways, implicating a direct effect of HCV replication on lipid homeostasis. (Hepatology 2015;61:790–801)

Safety and efficacy of ledipasvir/sofosbuvir for the treatment of genotype 1 hepatitis C in subjects aged 65 years or older

Elderly subjects have been historically underrepresented in clinical trials involving antiviral hepatitis C therapies. The aim of this analysis was to retrospectively evaluate the safety and efficacy of ledipasvir/sofosbuvir (LDV/SOF) by age groups of <65 years versus ≥65 years among subjects enrolled in phase 3 trials. Four open‐label phase 3 clinical trials evaluated the safety and efficacy of LDV/SOF with or without ribavirin (RBV) for the treatment of genotype 1 chronic hepatitis C virus. Sustained virological response at 12 weeks, treatment‐emergent adverse events (AEs), and graded laboratory abnormalities were analyzed according to age group. Of the 2293 subjects enrolled in four phase 3 trials, 264 (12%) were ≥65 years of age, of whom 24 were aged ≥75 years. Sustained virological response at 12 weeks was achieved by 97% (1965/2029) of subjects aged <65 years and 98% (258/264) of subjects aged ≥65 years. The most common AEs in both LDV/SOF groups that occurred in ≥10% of subjects were headache and fatigue. The rate of study discontinuation due to AEs was similar in the two age cohorts. The use of RBV in 1042 (45%) subjects increased the number of AEs, treatment‐related AEs, and AEs leading to study drug modification/interruption, particularly among elderly subjects. Conclusions: LDV/SOF with or without RBV was highly effective for treatment of genotype 1 chronic hepatitis C virusin subjects aged 65 and older. Addition of RBV did not increase sustained virological response at 12 weeks rates but led to higher rates of AEs, especially in elderly subjects. (Hepatology 2016;63:1112–1119)

Simtuzumab treatment of advanced liver fibrosis in HIV and HCV-infected adults: results of a 6-month open-label safety trial

Chronic liver injury can result in fibrosis that may progress over years to end‐stage liver disease. The most effective anti‐fibrotic therapy is treatment of the underlying disease, however when not possible, interventions to reverse or slow fibrosis progression are needed.

Fluid support worsens outcome and negates the benefit of protective antigen-directed monoclonal antibody in a lethal toxin-infused rat Bacillus anthracis shock model.

Objective:The aim of this study was to test the effects of normal saline treatment either alone or in combination with protective antigen-directed monoclonal antibody in a lethal toxin-infused rat model of anthrax sepsis. Design:Prospective controlled animal study. Setting:Animal research laboratory. Subjects:Sprague-Dawley rats (n = 539). Interventions:We initially tested the efficacy of three normal saline doses (5, 10, or 20 mL/kg/hr intravenously for 24 hrs) or none (controls) started when rats were treated with either lethal toxin (24-hr infusion) or, for comparison, lipopolysaccharide (24-hr infusion) or Escherichia coli (intravenous bolus). We then investigated delaying normal saline for 6 hrs or combining it with protective antigen-directed monoclonal antibody following lethal toxin challenge. Measurements and Main Results:Dose did not alter the effects of normal saline with any challenge (p not significant for all) or when combined with protective antigen-directed monoclonal antibody, so this variable was averaged in analysis. In initial studies, normal saline decreased mortality (mean hazards ratio of survival ± se) significantly with E. coli challenge (−0.66 ± 0.25, p = .009 averaged over normal saline dose) but not lipopolysaccharide (−0.17 ± 0.20). In contrast, normal saline increased mortality significantly with lethal toxin (0.69 ± 0.20, p = .001) in a pattern different from E. coli and lipopolysaccharide (p ≤ .002 for each). In subsequent studies, normal saline alone once again increased mortality (0.8 ± 0.3, p = .006), protective antigen-directed monoclonal antibody alone reduced it (−1.7 ± 0.8, p = .03), and the combination had intermediate effects that were not significant (0.04 ± 0.3). Conclusions:These findings raise the possibility that normal saline treatment may actually worsen outcome with anthrax lethal toxin. Furthermore, lethal toxin-directed therapies may not be as beneficial when used in combination with this type of fluid support.

Improvement of hepatic fibrosis and patient-reported outcomes in non-alcoholic steatohepatitis treated with selonsertib

Patient‐reported outcomes (PROs) represent patients’ perspective about their well‐being.

Identification of a Novel Hepatitis C Virus Genotype From Punjab, India: Expanding Classification of Hepatitis C Virus Into 8 Genotypes

Background Hepatitis C virus (HCV) exhibits great genetic diversity and is classified into 7 genotypes (GTs), with varied geographic prevalence. Until the recent development of pangenotypic direct-acting antiviral regimens, the determination of HCV GT was necessary to inform optimal treatment. Methods Plasma samples with unresolved GT using standard commercial genotyping methods were subjected to HCV full-genome sequencing, and phylogenetic analysis was performed to assign GT. Results Four patients, previously classified as GT5 by LiPA or Abbott RealTime polymerase chain reaction assays, were identified as infected with a novel HCV GT. This novel HCV GT, GT8, is genetically distinct from previously identified HCV GT1-7 with >30% nucleotide sequence divergence to the established HCV subtypes. All 4 patients were originally from Punjab, India, but now reside in Canada and are epidemiologically unlinked. Despite presence of baseline resistance-associated substitutions within the GT8 virus of all 4 patients (NS3: V36L, Q80K/R; NS5A: Q30S, Y93S), all patients achieved a sustained virologic response; 2 treated with sofosbuvir/velpatasvir/voxilaprevir for 8 weeks, 1 with sofosbuvir/ledipasvir plus ribavirin for 24 weeks and 1 with sofosbuvir plus daclatasvir for 12 weeks. Conclusions The discovery of a novel HCV GT8 confirms the circulation of this newly identified lineage in the human population.

Retreatment of HCV Genotype-1 with Sofosbuvir and Ledipasvir after Relapse with Sofosbuvir and Ribavirin: A Pilot Study

Context The treatment of hepatitis C virus is changing rapidly with the introduction of interferon-free regimens that include direct-acting antiviral drugs, such as sofosbuvir. Although treatment generally results in sustained viral response, some patients have relapse after initial treatment. Contribution In a small, nonrandomized study, 14 patients who had relapse after treatment with sofosbuvir plus ribavirin were re-treated with sofosbuvir plus ledipasvir for 12 weeks. All patients, including those with advanced liver disease, achieved a sustained virologic response. The combination was generally well-tolerated. Implication Treatment with sofosbuvir plus ledipasvir may be efficacious in patients who have relapse after initial therapy for hepatitis C virus. Larger randomized studies of this promising combination are warranted. The Editors Chronic hepatitis C virus (HCV) infection is a major cause of chronic liver disease and hepatocellular carcinoma and the leading indication for liver transplantation in Western countries (1, 2). Until recently, HCV treatment consisted of combination therapy with pegylated interferon (IFN) and ribavirin, with the addition of an HCV protease inhibitor (boceprevir or telaprevir) in recent years. In 2013, the U.S. Food and Drug Administration approved sofosbuvir plus ribavirin as the first IFN-free treatment of HCV genotype 2 or 3 and for patients with genotype 1 (GT-1) who are IFN-ineligible (37). Although sofosbuvir (an NS5B inhibitor) plus ribavirin is well-tolerated and effective with sustained virologic response (SVR) rates from 68% to 76% in patients with HCV GT-1 (8, 9), modest rates of relapse in patients with advanced liver disease and transplant recipients have been seen (8, 10, 11). Recent studies using sofosbuvir combined with ledipasvir (an NS5A inhibitor) for 12 weeks demonstrated high SVR rates (95% to 99%) in treatment-naive patients with HCV GT-1 (1215). Ledipasvir has potent antiviral activity against the S282T resistance-associated variant, known to reduce susceptibility to sofosbuvir in vitro (16). As previously reported, 17 of 54 patients treated with sofosbuvir plus ribavirin for 24 weeks in the National Institute of Allergy and Infectious Diseases (NIAID) SPARE study had relapse after treatment (8). We hypothesized that the combination of a second potent direct-acting antiviral agent (DAA) with sofosbuvir could result in SVR, even in patients with relapse after sofosbuvir plus ribavirin therapy and who may harbor the S282T mutation. To test this hypothesis, we re-treated those patients with sofosbuvir plus ledipasvir for 12 weeks. Methods Design Overview All patients who had relapse after 24 weeks of treatment with sofosbuvir plus ribavirin in the NIAID SPARE study (8) were offered re-treatment with sofosbuvir plus ledipasvir in the ongoing, phase 2a, open-label NIAID SYNERGY study (ClinicalTrials.gov: NCT01805882). Of the 17 eligible patients, 3 did not participate in the study. Fourteen patients enrolled and were treated with 400 mg of sofosbuvir and 90 mg of ledipasvir, administered daily as a single combination tablet, for 12 weeks. Setting and Patients The trial was conducted at the National Institutes of Health (NIH) in Bethesda, Maryland, and at community clinics that are part of the District of Columbia Partnership for HIV/AIDS Progress in Washington, DC. Written informed consent, which was approved by the NIAID Institutional Review Board, was obtained from all study patients. Eligibility criteria included documented HCV GT-1 infection and relapse in the NIAID SPARE study (8). Liver fibrosis stage was determined by biopsy within 3 years of enrollment in the NIAID SPARE study. The study was approved by the NIAID Institutional Review Board. It was conducted in adherence to the Good Clinical Practice guidelines and with regulatory requirements consistent with the Declaration of Helsinki. Outcomes and Follow-up Efficacy Assessments Plasma HCV RNA levels were measured using the Abbott RealTime HCV assay, with a lower limit of quantification of 12 IU/mL and a lower limit of detection of 3 IU/mL, or the Roche Cobas TaqMan HCV assay, version 2.0, with a lower limit of quantification of 43 IU/mL and a lower limit of detection of 15 IU/mL. Safety Assessments Patients were closely monitored for adverse events. Clinical laboratory results were assessed while receiving therapy (4, 8, and 12 weeks) and afterward (2, 4, 8, and 12 weeks after treatment). Adverse events were graded from 1 (mild) to 4 (severe), according to the NIAID Division of AIDS Toxicity Table (version 1.0). Interleukin-28B Genotyping Interleukin-28B genotype (rs12979860) was determined as previously described (8). Clinical End Points The primary end point was the proportion of patients with unquantifiable plasma HCV viral load 12 weeks after treatment completion. Safety end points included frequency and severity of adverse events, discontinuations due to adverse events, and safety laboratory changes. Statistical Analysis Primary safety and efficacy data were analyzed by intention-to-treat (all patients who initiated study medication). Missing virologic data were imputed if data from preceding and succeeding time points were obtained. Baseline demographics were described using frequency statistics. Comparisons were calculated using either nonparametric tests or t tests with GraphPad Prism, version 6.0 (GraphPad Software). Role of the Funding Source This study was funded in part by the National Cancer Institute, the intramural programs of the NIH Clinical Center and NIAID, and a Collaborative Research and Development Agreement between the NIH and Gilead Sciences. The study sponsor was the Regulatory Compliance and Human Participants Protection Branch of the NIAID. The sponsor reviewed the study and provided oversight. However, it did not play a role in designing the study; collecting, analyzing, or interpreting the data; or the preparation, review, approval, or submission of the manuscript for publication. All study medications were provided by Gilead Sciences, which did not have a role in the design or conduct of the study, writing of the manuscript, or the decision to submit the manuscript for publication. Results Baseline Characteristics of Patients Most study patients were black men with an unfavorable interleukin-28B non-CC genotype (Table 1). There was a high representation of patients with HCV GT-1a infection, increased baseline HCV viral load (>800000 IU/mL), increased body mass index (>30 kg/m2), and advanced liver disease (Knodell Histology Activity Index score of 3 or 4) (17). Table 1. Baseline Characteristics of Study Patients Presence of S282T Mutation At the time of relapse after sofosbuvir plus ribavirin treatment in the SPARE trial, 13 of 14 patients had wild-type virus by population sequencing (Supplement). The 1 exception achieved unquantifiable HCV RNA levels by week 2 of therapy in SPARE and the virus remained undetectable through the end of treatment. He missed his visit at 26 weeks (2 weeks after treatment), but at 28 weeks (4 weeks after treatment), his HCV viral load was 374 IU/mL and the S282T mutation was readily detected by population sequencing. By 36 weeks (12 weeks after treatment), his HCV viral load was 81286 IU/mL and the S282T mutation was no longer detectable. He initiated sofosbuvir plus ledipasvir treatment 50 weeks after relapse. Supplement. HCV Resistance Mutations Virologic Response Each of the 14 patients treated with sofosbuvir plus ledipasvir had HCV RNA levels below the lower limit of quantification by 4 weeks (Roche Cobas TaqMan HCV Assay, version 2.0), which was maintained through the end of treatment (12 weeks). All patients achieved SVR 12 weeks after completion of treatment. Changes in Hemoglobin Levels No significant change in hemoglobin levels during sofosbuvir plus ledipasvir treatment was seen, in contrast to the decrease seen with sofosbuvir plus ribavirin. The mean hemoglobin level change was 0.07 g/L versus 11.70 g/L at 4 weeks (P=0.01), 0.5 g/L versus 12.6 g/L at 8 weeks (P=0.01), and 3.1 g/L versus 12.1 g/L at 12 weeks (P=0.06) with sofosbuvir plus ledipasvir and sofosbuvir plus ribavirin, respectively. No participant had a decrease in hemoglobin level of 15.0 g/L or greater with sofosbuvir plus ledipasvir compared with 8 of 14 (57%) with sofosbuvir plus ribavirin. Changes in Renal Function Renal variables did not change significantly over the course of treatment, although a single participant with grade 2 renal insufficiency at baseline (estimated glomerular filtration rate, 54 mL/min/1.73 m2) developed a grade 3 event. After 6 weeks of receiving study medications, he had received amoxicillin in the context of a dental procedure and continued preenrollment medications (benazepril, telmisartan, and simvastatin). In this context, his estimated glomerular filtration rate decreased to 29 mL/min/1.73 m2 (grade 3 toxicity); however, it improved (grade 2) within 1 week of withdrawing amoxicillin, returned to baseline within 3 weeks, and remained stable thereafter. He received study drugs without interruption. Safety All patients completed treatment, and no grade 4 adverse events or laboratory abnormalities occurred. The most common adverse events were myalgia and hypophosphatemia, and most adverse events were mild (Table 2). Four grade 3 events occurred (increased creatinine levels described above [n=1], hypercholesterolemia [n=1], and hypophosphatemia [n=2]). Table 2. Treatment Discontinuations, Adverse Events, and Laboratory Abnormalities Discussion In this study, we demonstrate that patients with HCV GT-1 who have viral relapse after sofosbuvir plus ribavirin therapy can be successfully re-treated with sofosbuvir plus ledipasvir. Seven (50%) of the patients in this study had advanced liver disease, which has been shown to be associated with relapse after sofosbuvir plus ribavirin therapy (8, 10, 11). In addition to having a high prevalencBackground The interferon (IFN)-free regimen of sofosbuvir and ribavirin for 24 weeks was recently approved to treat chronic hepatitis C virus (HCV), genotype-1 (GT-1) infection for interferon-ineligible patients . However, sofosbuvir/ribavirin therapy is associated with treatment relapse in 15-30% of HCV GT-1 study subjects. Neither the mechanism of relapse nor the optimal retreatment strategy for these subjects is defined.