Peter W Angus

Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase

BACKGROUND & AIMS Adefovir dipivoxil effectively inhibits both hepatitis B virus (HBV) replication and disease activity in patients with chronic hepatitis B. Resistance to treatment was not observed in 2 recent large placebo-controlled 48-week studies with this drug. The aim of this study was to characterize adefovir resistance in a patient who developed clinical and virologic evidence of breakthrough during a 96-week course of treatment. METHODS HBV DNA was PCR amplified and sequenced. Phenotypic studies used patient-derived HBV as well as specific mutations created by site-directed mutagenesis of a HBV/baculovirus recombinant. RESULTS Following the commencement of treatment with adefovir dipivoxil, the patient initially responded with a 2.4 log(10) decrease in serum HBV DNA and normalization of alanine aminotransaminase levels by week 16. During the second year of treatment, however, serum HBV DNA rose progressively, eventually returning to near-pretreatment levels. This increase in viral replication was associated with a marked increase in alanine aminotransferase and mild changes in bilirubin, albumin, and prothrombin time. Comparison of pretreatment and posttreatment HBV DNA by polymerase chain reaction sequencing identified a novel asparagine to threonine mutation at residue rt236 in domain D of the HBV polymerase. In vitro testing of a laboratory strain encoding the rtN236T mutation and testing of patient-derived virus confirmed that the rtN236T substitution caused a marked reduction in susceptibility to adefovir. CONCLUSIONS The development of this novel mutation in the HBV polymerase confers resistance to adefovir dipivoxil. The patient responded to subsequent lamivudine therapy, achieving normalization of alanine aminotransferase and a significant decrease in serum HBV DNA.

Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial

BACKGROUND Present interferon-based standard of care treatment for chronic hepatitis C virus (HCV) infection is limited by both efficacy and tolerability. We assessed the safety, tolerability, and antiviral activity of an all-oral combination treatment with two experimental anti-HCV drugs-RG7128, a nucleoside polymerase inhibitor; and danoprevir, an NS3/4A protease inhibitor-in patients with chronic HCV infection. METHODS Patients from six centres in New Zealand and Australia who were chronically infected with HCV genotype 1 received up to 13 days oral combination treatment with RG7128 (500 mg or 1000 mg twice daily) and danoprevir (100 mg or 200 mg every 8 h or 600 mg or 900 mg twice daily) or placebo. Eligible patients were sequentially enrolled into one of seven treatment cohorts and were randomly assigned by interactive voice or web response system to either active treatment or placebo. Patients were separately randomly assigned within each cohort with a block size that reflected the number of patients in the cohort and the ratio of treatment to placebo. The random allocation schedule was computer generated. Dose escalation was started in HCV treatment-naive patients; standard of care treatment-experienced patients, including previous null responders, were enrolled in higher-dose danoprevir cohorts. Investigators, personnel at the study centre, and patients were masked to treatment allocation. However, the pharmacist who prepared the doses, personnel involved in pharmacokinetic sample analyses, statisticians who prepared data summaries, and the clinical pharmacologists who reviewed the data before deciding to initiate dosing in the next cohort were not masked to treatment allocation. The primary outcome was change in HCV RNA concentration from baseline to day 14 in patients who received 13 days of combination treatment. All patients who completed treatment with the study drugs were included in the analyses. This study is registered with ClinicalTrials.gov, NCT00801255. FINDINGS 88 patients were randomly assigned to a study drug treatment regimen (n=74 over seven treatment groups; 73 received at least one dose of study drug) or to placebo (n=14, all of whom received at least one dose). The median change in HCV RNA concentration from baseline to day 14 ranged from -3·7 to -5·2 log(10) IU/mL in the cohorts that received 13 days of combination treatment. At the highest combination doses tested (1000 mg RG7128 and 900 mg danoprevir twice daily), the median change in HCV RNA concentration from baseline to day 14 was -5·1 log(10) IU/mL (IQR -5·6 to -4·7) in treatment-naive patients and -4·9 log(10) IU/mL in previous standard of care null responders (-5·2 to -4·5) compared with an increase of 0·1 log(10) IU/mL in the placebo group. The combination of RG7128 and danoprevir was well tolerated with no treatment-related serious or severe adverse events, no grade 3 or 4 changes in laboratory parameters, and no safety-related treatment discontinuations. INTERPRETATION This oral combination of a nucleoside analogue polymerase inhibitor and protease inhibitor holds promise as an interferon-free treatment for chronic HCV. FUNDING Roche Palo Alto.

Ledipasvir and sofosbuvir plus ribavirin in patients with genotype 1 or 4 hepatitis C virus infection and advanced liver disease: a multicentre, open-label, randomised, phase 2 trial

BACKGROUND Treatment options are limited for patients infected by hepatitis C virus (HCV) with advanced liver disease. We assessed the safety and efficacy of ledipasvir, sofosbuvir, and ribavirin in patients with HCV genotype 1 or 4 and advanced liver disease. METHODS We did an open-label study at 34 sites in Europe, Canada, Australia, and New Zealand. Cohort A included patients with Child-Turcotte-Pugh class B (CTP-B) or CTP-C cirrhosis who had not undergone liver transplantation. Cohort B included post-transplantation patients who had either no cirrhosis; CTP-A, CTP-B, or CTP-C cirrhosis; or fibrosing cholestatic hepatitis. Patients in each group were randomly assigned (1:1) using a computer-generated randomisation sequence to receive 12 or 24 weeks of ledipasvir (90 mg) and sofosbuvir (400 mg) once daily (combination tablet), plus ribavirin (600-1200 mg daily). The primary endpoint was the proportion of patients achieving a sustained virological response 12 weeks after treatment (SVR12). All patients who received at least one dose of study drug were included in the safety analysis and all patients who received at least one dose of study drug and did not undergo liver transplantation during treatment were included in the efficacy analyses. Estimates of SVR12 and relapse rates and their two-sided 90% CI (Clopper-Pearson method) were provided. This exploratory phase 2 study was not powered for formal comparisons among treatment groups; no statistical hypothesis testing was planned or conducted. The trial is registered with EudraCT (number 2013-002802-30) and ClinicalTrials.gov (number NCT02010255). FINDINGS Between Jan 14, 2014, and Aug 19, 2014, 398 patients were screened. Of 333 patients who received treatment, 296 had genotype 1 HCV and 37 had genotype 4 HCV. In cohort A, among patients with genotype 1 HCV, SVR12 was achieved by 20 (87%, 90% CI 70-96) of 23 CTP-B patients with 12 weeks of treatment; 22 (96%, 81-100) of 23 CTP-B patients with 24 weeks of treatment; 17 (85%, 66-96) of 20 CTP-C patients (12 weeks treatment); and 18 (78%, 60-91) of 23 CTP-C patients (24 weeks treatment). In cohort B, among patients with genotype 1 HCV, SVR12 was achieved by 42 (93%, 84-98) of 45 patients without cirrhosis (12 weeks treatment); 44 (100%, 93-100) of 44 patients without cirrhosis (24 weeks treatment); 30 (100%, 91-100) of 30 CTP-A patients (12 weeks treatment); 27 (96%, 84-100) of 28 CTP-A patients (24 weeks treatment); 19 (95%, 78-100) of 20 CTP-B patients (12 weeks treatment); 20 (100%, 86-100) of 20 CTP-B patients (24 weeks treatment); one (50%, 3-98) of two CTP-C patients (12 weeks treatment); and four (80%, 34-99) of five CTP-C patients (24 weeks treatment). All five patients with fibrosing cholestatic hepatitis achieved SVR12 (100%, 90% CI 55-100). Among all patients with genotype 4 HCV, SVR12 was achieved by 14 (78%, 56-92) of 18 patients (12 weeks treatment) and 16 (94%, 75-100) of 17 patients (24 weeks treatment). Seven patients (2%) discontinued ledipasvir-sofosbuvir prematurely due to adverse events. 17 patients died, mainly from complications of hepatic decompensation. INTERPRETATION Ledipasvir-sofosbuvir and ribavirin provided high rates of SVR12 for patients with advanced liver disease, including those with decompensated cirrhosis before or after liver transplantation. FUNDING Gilead Sciences.

Diagnosis of Wilson's disease: an experience over three decades

BACKGROUND Wilsons disease is a rare but treatable condition that often presents diagnostic dilemmas. These dilemmas have for the most part not been resolved by the identification and cloning of the Wilsons disease gene. AIMS To report our experience over three decades with patients with Wilsons disease in order to illustrate the diverse patterns of presentation and thereby broaden the approach to diagnosis. METHODS Clinical and laboratory findings of 30 patients with Wilsons disease were reviewed. RESULTS Twenty two patients presented with liver manifestations (eight with fulminant hepatic failure and 14 with chronic liver disease), three with neurological disease, and one with haemolysis; four were asymptomatic siblings of patients with Wilsons disease. Seventy per cent were diagnosed within six months of the onset of symptoms, but diagnosis was delayed for up to nine years. Age range at diagnosis was wide (7–58 years) and five patients were over 40. In patients presenting with non-fulminant disease, 18% had neither Kayser-Fleischer rings nor low caeruloplasmin concentrations. Increased liver copper concentrations were found in all but one patient who had undergone six years of penicillamine treatment. In fulminant hepatic failure (n=8) additional features helpful in the diagnosis included evidence of haemolysis, increased urinary copper (range 844–9375 μg/24 h), and a high non-caeruloplasmin copper (range 325–1743 μg/l). CONCLUSIONS The diagnosis of Wilsons disease still depends primarily on the evaluation of clinical and laboratory evidence of abnormal copper metabolism. No one feature is reliable, but the diagnosis can usually be made provided that it is suspected. Wilsons disease should be considered in patients of any age with obscure hepatic or neurological abnormalities.

Tenofovir disoproxil fumarate rescue therapy following failure of both lamivudine and adefovir dipivoxil in chronic hepatitis B

Objective To determine the efficacy of tenofovir disoproxil fumarate (TDF) in adults with chronic hepatitis B virus (HBV) infection who had previously failed lamivudine (LAM) and had significant viral replication (HBV DNA >105 copies/ml if HBeAg positive, >104 copies/ml if HBeAg negative) despite at least 24 weeks of treatment with adefovir dipivoxil (ADV). Design A prospective open-label study of TDF 300 mg daily. Patients receiving combination ADV/LAM prior to baseline were switched to TDF/LAM. Setting Multiple tertiary referral centres. Methods Sixty patients were enrolled. The median age was 48.5 years (range 21–80), 46 (77%) were male and 40 (67%) were HBeAg positive. Thirty-eight patients (63%) were switched from ADV to TDF, the remainder from ADV/LAM to TDF/LAM. At baseline, substitutions conferring resistance to LAM or ADV were present in 20 patients (33%) and 17 patients (28%), respectively. The median baseline viral load was 5.33 log10 IU/ml (range 2.81–8.04). Patients initially treated with TDF monotherapy with persistent viral replication at or after 24 weeks were switched to TDF/LAM. The main outcome measures were change in HBV viral load from baseline and percentage of patients achieving an undetectable viral load (<15 IU/ml). Results Results are reported at 96 weeks of treatment. One patient discontinued TDF at 10 days due to rash. The time-weighted change in viral load from baseline to week 12 was −2.19 log10 IU/ml overall. The median change in HBV DNA from baseline to weeks 12, 24, 48 and 96 was −2.86, −3.23, −3.75 and −4.03 log10 IU/ml, respectively. At 48 and 96 weeks, 27/59 (46%) and 38/59 (64%) patients achieved a HBV DNA <15 IU/ml. The response was independent of baseline LAM therapy or mutations conferring ADV resistance. Conclusions In heavily pretreated patients with a high rate of genotypic resistance, TDF retains significant activity against HBV although this appears diminished in comparison with studies of naïve patients.

A randomized study of adefovir dipivoxil in place of HBIG in combination with lamivudine as post–liver transplantation hepatitis B prophylaxis†

Prior to effective prophylaxis, liver transplantation for hepatitis B virus (HBV)‐related disease was frequently complicated by recurrence, which could be severe and rapidly progressive. Combination hepatitis B immunoglobulin (HBIG) and lamivudine prophylaxis reduces this rate of recurrence to <5% at 5 years; however, HBIG administration is costly and inconvenient. We conducted a multicenter randomized study of adefovir dipivoxil substitution for low‐dose intramuscular (IM) HBIG in patients without HBV recurrence at least 12 months posttransplantation for HBV‐related disease. Thirty‐four patients were randomized, 16 to adefovir (1 patient withdrew consent at 3 months and is not considered in the results) and 18 to continue HBIG. All continued lamivudine. Groups were well matched by age, sex, and time since transplantation (median, 4.5 years), and background virological risk for HBV recurrence (30% of patients in the adefovir group, 24% in the HBIG group having detectable HBV DNA at transplantation). All patients were alive at study completion without recurrence. One patient in the adefovir group became hepatitis B surface antigen–positive at 5 months but was persistently HBV DNA undetectable via polymerase chain reaction (sensitivity 14 IU/mL) over the following 20 months. Median creatinine was not significantly changed over the course of the study in either group. One patient in the adefovir group with a background of diabetic and hypertensive nephropathy (baseline creatinine 150 μmol/L) developed increased creatinine leading to dose reduction and ultimately cessation of adefovir at 15 months. Yearly cost of combination adefovir/lamivudine prophylaxis was

Hepatitis B and C virus infections and anti-tumor necrosis factor-α therapy: Guidelines for clinical approach

8,290 versus

Zoledronic Acid Prevents Bone Loss after Liver Transplantation: A Randomized, Double-Blind, Placebo-Controlled Trial

13,718 IM HBIG/lamivudine. Conclusion: Compared with combination HBIG plus lamivudine prophylaxis, combination adefovir plus lamivudine provides equivalent protection against recurrent HBV infection but with better tolerability and less cost. (HEPATOLOGY 2008.)

Chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme 2

Anti‐tumor necrosis factor‐α (TNF) therapy has recently been recognized to be associated with activation of hepatitis B virus (HBV) infection, with a potentially fatal outcome, mirroring experience in the setting of immune suppression and subsequent reconstitution in cancer chemotherapy and transplantation. Although there is no current evidence that anti‐TNF therapy influences the natural history of hepatitis C virus (HCV) infection, the involvement of TNF in the pathogenesis of hepatic injury and extrapolation from other clinical situations heighten awareness of a potential conflict. Preventive strategies should be mandatory. These include screening of all patients for HBV and HCV infection prior to commencement of anti‐TNF therapy, and active monitoring of aminotransferases and, for HBV, viral load during and for 3 months after therapy has terminated. Prophylactic or early intervention strategies with nucleoside analogs are recommended for patients with evidence of HBV infection.

Effect of angiotensin II type 1 receptor blockade on experimental hepatic fibrogenesis.

Context Rapid bone loss occurs in the first few months after liver transplantation and may be associated with fractures. Contribution This randomized, double-blind trial found that infusing zoledronic acid within 1 week of liver transplantation and again 1, 3, 6, and 9 months after transplantation prevented bone loss more effectively than did placebo infusions. Differences between groups lessened over time as some patients receiving placebo regained bone after several months. Zoledronic acid sometimes caused postinfusion hypocalcemia and temporary secondary hyperparathyroidism. Cautions The trial was not powered to assess differences in fracture outcomes. Implications Zoledronic acid infusions can prevent bone loss after liver transplantation. The Editors Osteoporosis is a frequent complication of end-stage liver disease of various causes. Initially described in patients with cholestatic liver disease (1-4), it also occurs in association with cirrhosis secondary to hepatitis B and C virus infection and alcohol abuse (5-8). Although contributing factors that are common to postmenopausal osteoporosis have been identified, such as age, gonadal status, and vitamin D deficiency, cirrhosis is an independent risk factor for osteoporosis (8, 9). Some patients with cirrhosis eventually need liver transplantation, a procedure that is associated with accelerated bone loss, particularly within the first 3 to 6 months (10-13). Investigators report fractures rates of 16% to 40% during this period, predominantly of the vertebrae and ribs (12-16). Pain and restricted mobility after a fracture delay rehabilitation of patients and are severe enough at times to require hospitalization. It is not clear whether bisphosphonate drugs, which inhibit osteoclast-mediated bone resorption, reverse bone loss after transplantation. One uncontrolled study reported variable results (17). A recent controlled study, however, did not find any benefit of intravenous pamidronate in liver transplant recipients who had unusually little acute post-transplantation bone loss and few fractures in both the treatment and control groups (18). In our experience, bone disease and fractures after transplantation are major problems. Thus, we performed a randomized, double-blind, placebo-controlled trial of a potent intravenous bisphosphonate, zoledronic acid, in patients undergoing liver transplantation. The primary end point was bone density change at 3 months after transplantation; secondary end points were bone density change at 12 months and biochemical variables of bone turnover. Methods Setting and Patients From July 2000 to July 2003, we recruited adults undergoing transplantation for chronic liver disease from 2 large transplantation centers within the Australian and New Zealand Liver Transplant program. We based recruitment primarily on convenience for individuals, including whether they resided close to the transplantation center and were not already taking part in other research studies. We excluded patients if they had a serum creatinine level greater than 1.5 times the upper limit of normal (for example, >165 mol/L [>1.86 mg/dL]), had had treatment within the previous 12 months with agents known to affect bone metabolism (bisphosphonates, calcitriol, or sex hormones), or had hypocalcemia (corrected serum calcium level <2.2 mmol/L [<8.8 mg/dL]). We discussed the study and obtained written informed consent from patients at their pretransplantation assessment. After transplantation, just before the first infusion, we asked the patients to verbally re-consent. Recruited patients were similar to adult patients in the Australian Liver Transplant Database (www.cs.nsw.gov.au/gastro/livertransplant) with respect to age, sex, ChildPugh score, and cause of cirrhosis. The ethics review committees at both institutions approved the study protocol. Randomization and Interventions As soon as was feasible after liver transplantation, patients were randomly assigned centrally to receive zoledronic acid or placebo. Random assignment was done by using the method of stratified minimization (19) on the basis of the following variables: age, sex, baseline bone mineral density (BMD), and primary immunosuppressive therapy (cyclosporine or tacrolimus). Of note, all but 4 patients were recruited from 1 center; these 14 patients were randomly allocated to 2 per treatment group. Hospital pharmacists prepared infusions and coded them to maintain blinding. Five infusions of zoledronic acid (4 mg in 100 mL of normal saline, administered over 15 minutes) or saline were given over a 12-month period. The first infusion was administered within 7 days of liver transplantation and then again at months 1, 3, 6, and 9 after transplantation. All patients received calcium carbonate, 600 mg/d, and vitamin D supplementation (ergocalciferol, 1000 U/d) from the time of transplantation listing and throughout the study. The standard immunosuppression regimen after transplantation at both centers consisted of tacrolimus or cyclosporine, azathioprine, and methylprednisolone at 500 mg on day 1, decreasing to 20 mg by day 12. Alternative immunosuppressive therapy included the use of mycophenolate instead of azathioprine in some patients. Biopsy-proven cellular rejection was treated with pulses of methylprednisolone, 1000 mg/d for 3 days, followed by reduced doses of prednisone similar to those used after transplantation, aiming to reach a prednisone dosage of 20 mg/d by day 14. Assessments and Follow-up Before transplantation, baseline data, including the ChildPugh score, were re-collected every 6 months (designated as time 0 months). Bone mineral density at L2 to L4 of the lumbar spine, femoral neck, and total hip was measured by using dual x-ray absorptiometry on a Prodigy densitometer (Lunar, Madison, Wisconsin) in both centers at baseline and at months 3, 6, and 12 after transplantation. In vitro and in vivo coefficients of variation for BMD at the lumbar spine, hip, spine, and total body were less than 1.0 and less than 2.0%, respectively. The BMD data were expressed as T-scores (that is, SDs removed from the average BMD of young healthy controls) using the North American reference ranges provided by the densitometer manufacturer. Radiographs of the thoracolumbar spine were taken at the baseline assessment and at 12 months after transplantation. New vertebral fractures were identified by radiology report. Blood and urine samples were collected in the morning at the pretransplantation assessment and were subsequently taken every 6 months until liver transplantation and at months 1, 3, 6, 9, and 12 months after transplantation. Serum testosterone levels (in men), parathyroid hormone (PTH) levels, and urinary free deoxypyridinoline levels, corrected for creatinine, were measured on the Immulite 2000 autoanalyzer (Diagnostic Products Corp., Los Angeles, California). Both 25- and 1,25-hydroxyvitamin D levels were measured by radioimmunoassay (Diasorin, Stillwater, Minnesota) after an initial extraction from serum with acetonitrile and a second solvent column extraction step for 1,25-hydroxyvitamin D. Serum levels of insulin-like growth factor I were measured by using a double antibody radioimmunoassay after acidethanol extraction (Bioclone, New South Wales, Australia). The coefficients of variation, measured at 2 levels, were less than 8% for total testosterone, intact PTH, and 25- and 1,25-hydroxyvitamin D; less than 11.0% for insulin-like growth factor I; and 17.7% and 6.9% for urinary deoxypyridinoline at 27 nmol/L and 107 nmol/L, respectively. Bone-specific alkaline phosphatase (ALP) was measured by using competitive immunocapture assay on an Access analyzer (Beckman Coulter, Inc., Fullerton, California). Adverse events were assessed at each follow-up point by open-ended questions asked by the treating physician and by review of charts by the research nurse. Physicians and nurses were blinded to treatment group. Physicians rated events as expected or unexpected and assessed potential attribution to therapy. Fractures or renal impairment and serum creatinine levels greater than 165 mol/L (>1.86 mg/dL) resulted in discontinuation of therapy. Hypocalcemia (corrected serum calcium) was graded according to the following criteria: grade 1, 1.90 to 2.14 mmol/L (7.6 to 8.6 mg/dL); grade 2, 1.70 to 1.89 mmol/L (6.8 to 7.6 mg/dL); grade 3, 1.50 to 1.69 mmol/L (6.0 to 6.8 mg/dL); and grade 4, less than 1.50 mmol/L (<6.0 mg/dL). Statistical Analysis The primary end points of change in BMD at 3 months for the hip and the lumbar spine were used as the basis for a priori sample size calculations. On the basis of a 10% difference in BMD change, a 2-tailed P value of 0.05, and 80% power, 34 patients per group were required. Anticipating a 10% noncompletion rate, we estimated that 75 patients would need to be recruited. Linear mixed-effects models were fitted to BMD at the lumbar spine, femoral neck, and total hip and to biochemical data. Treatment (zoledronic acid or placebo), time, and their 2-way interaction were considered as fixed effects, as were the baseline body weight, serum PTH level, and testosterone level (in men). These baseline variables showed some imbalance between treatment groups. Differences between treatments were estimated as both unadjusted and adjusted for potential confounding variables, baseline weight, and serum PTH level. Because estimates of treatment differences in men further adjusted for testosterone changed by less than 10%, this covariate was not included in any final models. All tabulated results refer to men and women combined. The unadjusted treatment differences are based on intention-to-treat analyses of all 62 patients. Because 10 patients had missing baseline weight or serum PTH measurements, the adjusted analyses represent modified intention-to-treat analyses of the remaining 52 patients. Unadjusted and adjusted treatment differences for the percentage change in BMD fro