Modeling shows that the NS5A inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis C virus half-life.

The nonstructural 5A (NS5A) protein is a target for drug development against hepatitis C virus (HCV). Interestingly, the NS5A inhibitor daclatasvir (BMS-790052) caused a decrease in serum HCV RNA levels by about two orders of magnitude within 6 h of administration. However, NS5A has no known enzymatic functions, making it difficult to understand daclatasvir's mode of action (MOA) and to estimate its antiviral effectiveness. Modeling viral kinetics during therapy has provided important insights into the MOA and effectiveness of a variety of anti-HCV agents. Here, we show that understanding the effects of daclatasvir in vivo requires a multiscale model that incorporates drug effects on the HCV intracellular lifecycle, and we validated this approach with in vitro HCV infection experiments. The model predicts that daclatasvir efficiently blocks two distinct stages of the viral lifecycle, namely viral RNA synthesis and virion assembly/secretion with mean effectiveness of 99% and 99.8%, respectively, and yields a more precise estimate of the serum HCV half-life, 45 min, i.e., around four times shorter than previous estimates. Intracellular HCV RNA in HCV-infected cells treated with daclatasvir and the HCV polymerase inhibitor NM107 showed a similar pattern of decline. However, daclatasvir treatment led to an immediate and rapid decline of extracellular HCV titers compared to a delayed (6-9 h) and slower decline with NM107, confirming an effect of daclatasvir on both viral replication and assembly/secretion. The multiscale modeling approach, validated with in vitro kinetic experiments, brings a unique conceptual framework for understanding the mechanism of action of a variety of agents in development for the treatment of HCV.

Racial differences in fibrosis progression after HCV-related liver transplantation.

BACKGROUND: Black recipients undergoing liver transplantation (LT) for hepatitis C virus (HCV) have decreased patient and graft survival compared with white recipients, a finding that is primarily limited to black recipients of livers from white donors. The cause(s) for these discrepant outcomes are unclear but may be related to HCV disease recurrence. The rates of HCV-related disease recurrence and liver fibrosis progression among black and white liver transplant recipients have not been investigated. METHODS: In this study, we compared liver fibrosis progression between 105 black and 364 white recipients after HCV-related LT in a multisite cohort study and assessed the impact of donor race. RESULTS: At 6, 12, and 24 months after LT, there was a significantly higher percentage in the black recipient/white donor (B/W) group with severe fibrosis, defined as stage 3 or 4 (F3/F4), compared with all other recipient/donor race combinations. The adjusted odds ratio of developing F3/F4 for the B/W group was 2.54 (1.49-4.69; reference group, white recipient/white donor). Black recipients with black donors demonstrated a similar rate of progression to F3/F4 as white recipients. Patient survival was also decreased in the B/W group compared with other recipient/donor race combinations. CONCLUSION: African American recipients with white donors have more severe fibrosis progression after HCV-related LT. The mechanisms responsible for accelerating fibrosis progression in this high-risk race-mismatched group need to be investigated.

Hepatitis C Viral Kinetics in the Era of Direct Acting Antiviral Agents and IL28B.

In the last decade hepatitis C virus (HCV) kinetics has become an important clinical tool for the optimization of therapy with (pegylated)-interferon-α (IFN) and ribavirin (RBV). Mathematical models have generated important insights into HCV pathogenesis, HCV- host dynamics, and IFN and RBV's modes of action. Clinical trials with direct acting agents (DAAs) against various steps of the HCV life cycle have revealed new viral kinetic patterns that have not been observed with IFN±RBV. Very recently, studies have showed that single nucleotide polymorphisms (SNPs) in the IL28B gene region were associated with race/ethnicity and with response to IFN±RBV. Here we review our current knowledge of HCV kinetics and related mathematical models during IFN±RBV and/or DAA based therapies, HCV pathogenesis, and the role of IL28B polymorphism on early HCV kinetics. Better understanding of the mode of actions of drug(s) and viral kinetics may help to develop, in the near future, new individualized therapeutic regimens that include DAAs in combination with IFN+RBV.

Pharmacodynamics of PEG-IFN-[alpha]-2a and HCV response as a function of IL28B polymorphism in HIV/HCV-coinfected patients.

We examined the association between IL28B single-nucleotide polymorphism rs12979860, hepatitis C virus (HCV) kinetic, and pegylated interferon alpha-2a pharmacodynamic parameters in HIV/HCV-coinfected patients from South America. Twenty-six subjects received pegylated interferon alpha-2a + ribavirin. Serum HCV-RNA and interferon concentrations were measured frequently during the first 12 weeks of therapy and analyzed using mathematical models. African Americans and whites had a similar distribution of IL28B genotypes (P = 0.5). The IL28B CC genotype was overrepresented (P = 0.015) in patients infected with HCV genotype-3 compared with genotype-1. In both genotype-1 and genotype-3, the first-phase viral decline and the average pegylated interferon-alpha-2a effectiveness during the first week of therapy were larger (trend P <= 0.12) in genotype-CC compared with genotypes-TC/TT. In genotype-1 patients, the second slower phase of viral decline (days 2-29) and infected cells loss rate, [delta], were larger (P = 0.02 and 0.11, respectively) in genotype-CC than in genotypes-TC/TT. These associations were not observed in genotype-3 patients.

Pharmacodynamics of PEG-IFN-alpha-2a in HIV/HCV co-infected patients: implications for treatment outcomes.

BACKGROUND & AIMS: The pharmacokinetics and pharmacodynamics of pegylated-interferon-alpha-2a (PEG-IFN) have not been described in HCV/HIV co-infected patients. We sought to estimate the pharmacokinetics and pharmacodynamics of PEG-IFN and determine whether these parameters predict treatment outcome. METHODS: Twenty-six HCV/human immunodeficiency virus (HIV)-co-infected patients were treated with a 48-week regimen of PEG-IFN (180 microg/week) plus ribavirin (11 mg/kg/day). HCV RNA and PEG-IFN concentrations were obtained from samples collected until week 12. A modeling framework that includes pharmacokinetic and pharmacodynamic parameters was developed. RESULTS: Five patients discontinued treatment. Seven patients achieved a sustained virological response (SVR). PEG-IFN concentrations at day 8 were similar to steady-state levels (p=0.15) and overall pharmacokinetic parameters were similar in SVRs and non-SVRs. The maximum PEG-IFN effectiveness during the first PEG-IFN dose and the HCV-infected cell loss rate (delta), were significantly higher in SVRs compared to non-SVRs (median 95% vs. 86% [p=0.013], 0.27 vs. 0.11 day(-1) [p=0.006], respectively). Patients infected with HCV genotype 1 had a significantly lower average first-week PEG-IFN effectiveness (median 70% vs. 88% [p=0.043]), however, 4- to 12-week PEG-IFN effectiveness was not significantly different compared to those with genotype 3 (p=0.114). Genotype 1 had a significantly lower delta compared to genotype 3 (median 0.14 vs. 0.23 day(-1) [p=0.021]). The PEG-IFN concentration that decreased HCV production by 50% (EC(50)) was lower in genotype 3 compared to genotype 1 (median 1.3 vs. 3.4 [p=0.034]). CONCLUSIONS: Both the HCV-infected cell loss rate (delta) and the maximum effectiveness of the first dose of PEG-IFN-alpha-2a characterised HIV co-infected patients and were highly predictive of SVR. Further studies are needed to validate these viral kinetic parameters as early on-treatment prognosticators of response in patients with HCV and HIV.

Ethnicity and body mass index are associated with hepatitis C presentation and progression.

BACKGROUND & AIMS: Ethnicity and the metabolic syndrome are believed to affect progression of hepatitis C virus (HCV) infection, but the interaction between these factors is unknown. We evaluated the association between elements of the metabolic syndrome and ethnicity in the histologic progression of HCV in a large, diverse cohort. METHODS: We retrospectively evaluated clinical data and liver biopsy samples from 812 patients who had no cause of liver disease other than HCV infection. Liver biopsies were scored for steatosis, necroinflammatory activity, and fibrosis. For each patient with a known risk factor for viral acquisition, fibrosis index was calculated as an indicator of disease progression. RESULTS: Hispanics had significantly higher fibrosis index (0.13 +/- 0.09) than non-Hispanic whites (0.11 +/- 0.07) and African Americans (0.10 +/- 0.06; P = .001). Fibrosis index correlated with body mass index (BMI), older age at infection, ethnicity, and degree of steatosis. Cirrhosis was present in 50% of Hispanics, 38% of non-Hispanic whites, and 24% of African Americans (P < .001). The presence of cirrhosis was associated additionally with older age, longer duration of infection, BMI, alcohol consumption, and diabetes. In multivariate analysis, only BMI and ethnicity were associated with both fibrosis index and presentation with cirrhosis. Patients with higher BMIs, diabetes mellitus, and steatosis had higher degrees of necroinflammation. CONCLUSIONS: Ethnicity and BMI each were associated with the progression of fibrosis and the presence of cirrhosis. Hispanics had the highest fibrosis index and prevalence of cirrhosis, whereas African Americans had the lowest. Ethnic differences in fibrosis index and cirrhosis persisted after controlling for elements of metabolic syndrome.

Modelling hepatitis C virus kinetics: the relationship between the infected cell loss rate and the final slope of viral decay.

BACKGROUND: Patients infected with hepatitis C virus (HCV) who respond to treatment with interferon-alpha plus ribavirin exhibit biphasic or triphasic viral load decreases. While the rapid first phase is indicative of the effectiveness of therapy in blocking viral production (epsilon), the slope of the final phase (lambda), that is, the second phase in biphasic decreases and the third phase in triphasic decreases, depends on the infected cell loss rate (delta). In standard models, lambda is approximately epsilondelta when the viral clearance rate c>>delta, as has been previously estimated. METHODS: The relationship among epsilon, delta, lambda and the baseline fraction of HCV-infected hepatocytes (pi) was investigated in a model that included proliferation of hepatocytes. RESULTS: We found that lambda was not proportional to epsilon, but rather obeyed a complex relationship that could lead to dramatic increases in estimates of delta as epsilon increased. In particular, when epsilon<99%, lambda moderately underestimated delta in patients with a small pi, whereas delta might be up to 10-fold larger than lambda in patients with a large pi. Interestingly, when epsilon>99%, delta approximately lambda regardless of pi. CONCLUSIONS: Our results indicated that in patients undergoing therapy who achieved a 2 log(10) reduction in viral load (epsilon<99%), previously estimated delta values might represent only a minimal estimate of the infected cell loss rate. Moreover, combining interferon-alpha with new antiviral agents to achieve epsilon>99% should allow for a more accurate estimate of delta in HCV RNA kinetic studies. This might be important when using viral kinetics to estimate the effect of the immune response on viral elimination and the attainment of sustained virological response.

Hepatitis B seroprevalence and disease characteristics in an urban Chinatown community.

BACKGROUND & AIMS: Chronic HBV infection is prevalent among Asian immigrants and is an important cause of cirrhosis and hepatocellular carcinoma. The aim of this study was to evaluate the HBsAg seroprevalence and to characterize hepatitis B in persons who presented to an urban Chinatown internal medicine practice. METHODS: Records were reviewed retrospectively from 4671 adult patients who had at least 1 office visit during a 2-year period. Demographic information and laboratory data were collected. An elevated ALT level was defined as >19 IU/mL for women and >30 IU/mL for men. RESULTS: All patients were ethnically Chinese, and 97% were born in Asia. HBsAg testing was available in 64% (3012/4671) of cases. The HBsAg seroprevalence rate was 11.1% (335/3012) overall and 14.9% in persons aged 30-39 years. HBeAg testing was available for 75% (250/335) of HBsAg+ cases. Seventy-five percent (188/250) were HBeAg-. Sixty percent (26/43) of HBeAg+ patients with available data had HBV DNA >10(5) copies/mL and an elevated ALT level. Sixteen percent (21/128) of HBeAg- patients with available data had HBV DNA >10(4) copies/mL and an elevated ALT level. CONCLUSIONS: The HBsAg seroprevalence was high (11.1%) in Chinese immigrants presenting for general medical care. A majority (75%) of HBsAg+ patients were HBeAg-. Sixty percent of HBeAg+ cases and 16% of HBeAg- patients with available data had both HBV DNA and ALT levels that would prompt consideration of antiviral therapy. These findings highlight the importance of testing and medical management of hepatitis B in Chinese Americans.

A mathematical model of hepatitis C virus dynamics in patients with high baseline viral loads or advanced liver disease.

BACKGROUND & AIMS: Patients with baseline hepatitis C virus-RNA levels (bHCV-RNA)>6 log IU/mL or cirrhosis have a reduced probability of a sustained-virologic response (SVR). We examined the relation between bHCV-RNA, cirrhosis, and SVR with a mathematical model that includes the critical-drug efficacy (epsilonc; the efficacy required for a drug to clear HCV), the infection-rate constant (beta), and the percentage of HCV-infected hepatocytes (pi). METHODS: The relation between baseline factors and SVR was evaluated in 1000 in silico HCV-infected patients, generated by random assignment of realistic host and viral kinetic parameters. Model predictions were compared with clinical data from 170 noncirrhotic and 75 cirrhotic patients. RESULTS: The ranges chosen for beta and the viral production rate (p) resulted in bHCV-RNA levels that were in agreement with the distribution observed in US patients. With these beta and p values, higher bHCV-RNA levels led to higher epsilonc, resulting in lower SVR rates. However, higher beta values resulted in lower bHCV-RNA levels but higher pi and (epsilonc), predicting lower rates of SVR. Cirrhotic patients had lower bHCV-RNA levels than noncirrhotic patients (P=.013), and more had bHCV-RNA levels<6 log IU/mL (P<.001). Even cirrhotic patients with lower bHCV-RNA levels had lower SVR rates. An increase in beta could explain the results observed in cirrhotic patients. CONCLUSIONS: Our model predicts that higher bHCV-RNA levels lead to higher epsilonc, reducing the chance of achieving SVR; cirrhotic patients have lower SVR rates because of large pi values, caused by increased rates of hepatocyte infection.

The histologic spectrum of liver disease in African-American, non-Hispanic white, and Hispanic obesity surgery patients.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is a prominent cause of chronic liver disease in African Americans, non-Hispanic whites, and Hispanics. The aim of this study was to evaluate ethnic differences in the prevalence of NAFLD and non-alcoholic steatohepatitis (NASH) and to compare the severity of histologic features of NASH in obesity surgery patients. METHODS: Subjects consisted of 238 patients who had a routine liver biopsy at the time of obesity surgery. Demographic and clinical variables pertaining to the metabolic syndrome were collected retrospectively. Liver biopsies were evaluated according to the scoring system proposed by the Nonalcoholic Steatohepatitis Clinical Research Network and NASH was defined as a NASH activity score > or =5. RESULTS: African Americans had lower rates of steatosis than non-Hispanic whites (P<0.001) and Hispanics (P=0.03). Among patients with steatosis, African Americans had lower rates of NASH than non-Hispanic whites (P=0.05) and Hispanics (P=0.02) and lower rates of fibrosis score > or =F2 than non-Hispanic whites (P=0.002) and Hispanics (P=0.04). Ethnic differences in rates of NAFLD, NASH, and fibrosis > or =F2 persisted when controlling for demographic variables and features of the metabolic syndrome in logistic regression analysis. There were no significant differences in steatosis, NASH, or fibrosis > or =F2 between non-Hispanic whites and Hispanics. CONCLUSIONS: African-American obesity surgery patients have a lower rate of NAFLD, NASH, and less severe fibrosis than non-Hispanic whites and Hispanics.

Liver Injury with Features Mimicking Autoimmune Hepatitis following the Use of Black Cohosh.

There are a growing number of cases detailing acute hepatic necrosis in patients taking black cohosh (Cimicifuga racemosa), an over-the-counter herbal supplement for management of menopausal symptoms. Our aim is to illustrate two cases of liver injury following the use of black cohosh characterized by histopathological features mimicking autoimmune hepatitis. Both patients reported black cohosh use for at least six months and had no evidence of another cause of liver disease. Their liver biopsies showed a component of centrilobular necrosis consistent with severe drug-induced liver injury. In addition, the biopsies showed characteristics of autoimmune-like liver injury with an interface hepatitis dominated by plasma cells. Although serum markers for autoimmune hepatitis were not particularly elevated, both patients responded to corticosteroids, supporting an immune-mediated component to the liver injury. Liver injury following the use of black cohosh should be included in the list of differential diagnoses for chronic hepatitis with features mimicking autoimmune hepatitis.

Does nonalcoholic fatty liver disease predispose patients to hepatocellular carcinoma in the absence of cirrhosis?

CONTEXT: Hepatocellular carcinoma (HCC) is recognized as a complication of cirrhosis related to nonalcoholic fatty liver disease (NAFLD). Diabetes and the metabolic syndrome are also associated with HCC. However, it is not clear whether NAFLD predisposes patients to HCC in the absence of cirrhosis. OBJECTIVE: To seek evidence that HCC can develop in NAFLD unaccompanied by cirrhosis. DESIGN: Retrospective case study was performed on cases from 2004 to 2007 at the University of Illinois at Chicago Medical Center, using the key words hepatocellular carcinoma, liver explant, and liver resection. The diagnosis of HCC was identified and confirmed by hematoxylin-eosin-stained slides in 50 cases. Cause of liver disease was determined by review of liver histology, clinical history, and laboratory data. RESULTS: Three patients presented with advanced HCC with features of metabolic syndrome, including an elevated body mass index. Each patient had bland steatosis on liver biopsy, without fibrosis or cirrhosis. None of the 3 patients had evidence of any cause for liver disease other than NAFLD. CONCLUSIONS: The cases presented here suggest that NAFLD may predispose patients to HCC in the absence of cirrhosis. Further studies are needed to confirm this potentially important observation.

Hepatitis C Viral Kinetics in Special Populations.

Mathematical models of hepatitis C viral (HCV) kinetics provide a means of estimating the antiviral effectiveness of therapy, the rate of virion clearance and the rate of loss of HCV-infected cells. They have also proved useful in evaluating the extrahepatic contribution to HCV plasma viremia and they have suggested mechanisms of action for both interferon-α and ribavirin. Viral kinetic models can explain the observed HCV RNA profiles under treatment, e.g., flat partial response, biphasic and triphasic viral decay and viral rebound. Current therapy with (pegylated) interferon-α and ribavirin has a poorer success in patients having insulin resistance, hepatic fibrosis, African American ethnicity, HCV/HIV-coinfection, HCV genotype-1 and high baseline viral load. The use of mathematical modeling and statistical analysis of experimental data have been useful in understanding some of these treatment obstacles.

Immunohistochemical expression of components of the Akt-mTORC1 pathway is associated with hepatocellular carcinoma in patients with chronic liver disease.

UNLABELLED: Activation of the Akt-mTORC1 signaling pathway was evaluated in premalignant and hepatocellular carcinoma (HCC) lesions by assessing the expression of pS6, an Akt effector, and PTEN, an Akt suppressor. METHODS: Immunohistochemical staining for pS6 and PTEN was performed on liver tissue from 52 patients with cirrhosis, with and without HCC. Two pathologists independently evaluated pS6 staining on a semiquantitative scale and categorized PTEN staining as present or absent. RESULTS: In the HCC group, pS6 staining was greatest in HCC, followed by dysplasia, and benign cirrhotic tissue (P < 0.001). pS6 staining was greater in cirrhotic tissue from patients with HCC compared to cirrhosis in patients without HCC (P = 0.03). PTEN staining in tumor was absent in 8/33 (24%) cases. Loss of PTEN expression was more common in patients with higher tumor stage, compared to those with stage 1 tumors (P = 0.04). CONCLUSION: Immunohistochemical evidence of activation of the Akt-mTORC1 pathway is associated with HCC.

Diabetes and hepatic oxidative damage are associated with hepatitis C progression after liver transplantation.

BACKGROUND: Posttransplant diabetes mellitus (PTDM) is common after liver transplantation and was recently identified as a risk factor for hepatitis C progression. Increased levels of oxidative stress have been identified in diabetes and hepatitis C. The aim of this study was to evaluate the relationship among PTDM, oxidative damage in liver biopsy specimens, and fibrosis progression posttransplant. METHODS: Subjects consisted of 27 hepatitis C-infected liver transplant recipients who had liver biopsy specimens available from 49 protocol liver biopsies. Paraffin embedded liver tissue sections were stained for 8-hydroxy-2' deoxyguanosine (8-OHdG), an indicator of hydroxyl radical mediated tissue damage. The percentage of cells staining for 8-OHdG in a histologic section was categorized as high (>66%) versus low score (< or =66%). Fibrosis index was calculated as fibrosis score (0-4)/ years posttransplant. Time to bridging fibrosis or cirrhosis (F3-4) was compared as a function of PTDM and 8-OHdG score. RESULTS: Considering all 49 biopsies, fibrosis index was higher in cases with PTDM (P<0.001) and high 8-OHdG score (P=0.004). High 8-OHdG score was associated with PTDM (P=0.012). In time to event analyses, time to F3-4 was more rapid in patients with PTDM (P=0.02) and in those with high 8-OHdG scores (P<0.001). CONCLUSIONS: This study confirmed a relationship between PTDM and hepatitis C fibrosis progression and found that oxidative damage in liver biopsy specimens was associated with PTDM and more rapid development of advanced fibrosis.

A 7 gene signature identifies the risk of developing cirrhosis in patients with chronic hepatitis C.

UNLABELLED: Clinical factors such as age, gender, alcohol use, and age-at-infection influence the progression to cirrhosis but cannot accurately predict the risk of developing cirrhosis in patients with chronic hepatitis C (CHC). The aim of this study was to develop a predictive signature for cirrhosis in Caucasian patients. All patients had well-characterized liver histology and clinical factors; DNA was extracted from whole blood for genotyping. We validated all significant markers from a genome scan in the training cohort, and selected 361 markers for the signature building. Using a "machine learning" approach, a signature consisting of markers most predictive for cirrhosis risk in Caucasian patients was developed in the training set (N = 420). The Cirrhosis Risk Score (CRS) was calculated to estimate the risk of developing cirrhosis for each patient. The CRS performance was then tested in an independently enrolled validation cohort of 154 Caucasian patients. A CRS signature consisting of 7 markers was developed for Caucasian patients. The area-under-the-ROC curves (AUC) of the CRS was 0.75 in the training cohort. In the validation cohort, AUC was only 0.53 for clinical factors, increased to 0.73 for CRS, and 0.76 when CRS and clinical factors were combined. A low CRS cutoff of <0.50 to identify low-risk patients would misclassify only 10.3% of high-risk patients, while a high cutoff of >0.70 to identify high-risk patients would misclassify 22.3% of low-risk patients. CONCLUSION: CRS is a better predictor than clinical factors in differentiating high-risk versus low-risk for cirrhosis in Caucasian CHC patients. Prospective studies should be conducted to further validate these findings.