journal of clinical and

JOURNAL OF CLINICAL ANDTRANSLATIONAL HEPATOLOGY

CONTENTS 2016 4(4):281–349

Original Articles

Limited Knowledge of Acetaminophen in Patients with Liver DiseaseSammy Saab, Peter G. Konyn, Matthew R. Viramontes, Melissa A. Jimenez, Jonathan F. Grotts,

Wally Hamidzadah, Veronica P. Dang, Negin L. Esmailzadeh, Gina Choi, Francisco A. Durazo,

Mohamed M. El-Kabany, Steven-Huy B. Han and Myron J. Tong. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

Impact of Different Embolic Agents for Transarterial Chemoembolization (TACE) Procedureson Systemic Vascular Endothelial Growth Factor (VEGF) Levels

Andreas Schicho, Claus Hellerbrand, Kristina Krüger, Lukas P. Beyer, Walter Wohlgemuth,

Christoph Niessen, Ernst Hohenstein, Christian Stroszczynski, Philippe L. Pereira and

Philipp Wiggermann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

miR-29a Promotes Lipid Droplet and Triglyceride Formation in HCV Infection by InducingExpression of SREBP-1c and CAV1

Mennatallah Mamdouh Mahdy, Nada Magdy El-Ekiaby, Rana Mahmoud Hashish, Radwa Ayman Salah,

Rasha Sayed Hanafi, Hassan Mohamed El-Said Azzazy and Ahmed Ihab Abdelaziz . . . . . . . . . . . . . . . . . . . 293

miR-34a: Multiple Opposing Targets and One Destiny in Hepatocellular Carcinoma

Radwa Alaa Yacoub, Injie Omar Fawzy, Reem Amr Assal, Karim Adel Hosny,

Abdel-Rahman Nabawy Zekri, Gamal Esmat, Hend Mohamed El Tayebi and Ahmed Ihab Abdelaziz . . . . . . . 300

Comparison of Vitamin D Levels in Naive, Treated, and Inactive Carriers with ChronicHepatitis B Virus

Shahnaz Sali, Soheil Tavakolpour and Baharan Farkhondemehr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

Review Articles

Hepatitis C Virus: A Review of Treatment Guidelines, Cost-effectiveness, andAccess to Therapy

Shaina M. Lynch and George Y. Wu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

Call for papers

JCTH is a new, comprehensive specialist journal focusing on the recent progress in clinical and basicresearch with direct applications to clinical management of liver diseases. The studies published in JCTHwill represent the most current trends in the field of hepatology, highlighting the topically relevantsubjects of nations worldwide. Publications in JCTH will be presented in formats that emphasize clarityof the study’s objectives and implications of its findings, using high quality visual aids to enhance themanuscript’s esthetic appeal as well as its impact. For our upcoming issue, we encourage you and yourgroup to submit original articles that showcase your work in hepatology and topically relevant reviews topromote our readers’ understanding of the field.

Novel Treatment of Hepatitis C Virus Infection for Patients with Renal Impairment

Goki Suda, Koji Ogawa, Megumi Kimura, Masato Nakai, Takuya Sho, Kenichi Morikawa

and Naoya Sakamoto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

Mechanisms of Accelerated Liver Fibrosis Progression during HIV Infection

Jose D. Debes, Paul R. Bohjanen and Andre Boonstra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

Efficacy and Safety of Daclatasvir in Hepatitis C: An Overview

Nesrine Gamal, Stefano Gitto and Pietro Andreone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

Case Report

Massive Hemolysis Causing Renal Failure in Acute Hepatitis E Infection

Pragya Karki, Sarthak Malik, Bipadabhanjan Mallick, Vishal Sharma and Surinder S Rana . . . . . . . . . . . . . . 345

Reviewer Acknowledgement

2016 Reviewer Acknowledgement

Editorial Office of Journal of Clinical and Translational Hepatology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

Original Article

Limited Knowledge of Acetaminophen in Patients withLiver Disease

Sammy Saab*1,2, Peter G. Konyn2, Matthew R. Viramontes2, Melissa A. Jimenez2,Jonathan F. Grotts3, Wally Hamidzadah2, Veronica P. Dang2, Negin L. Esmailzadeh2,Gina Choi1,2, Francisco A. Durazo1,2, Mohamed M. El-Kabany1,2, Steven-Huy B. Han1,2

and Myron J. Tong2,4

1Department of Medicine, the University of California at Los Angeles, Los Angeles, CA, USA; 2Department of Surgery, theUniversity of California at Los Angeles, Los Angeles, CA, USA; 3Department of Biostatistics, the University of California at Los

Angeles, Los Angeles, CA, USA; 4California and Huntington Medical Research Institutes, Pasadena, CA, USA

Abstract

Background and Aims: Unintentional acetaminophen over-dose remains the leading cause of acute liver failure in theUnited States. Patients with underlying liver disease are athigher risk of poor outcomes from acetaminophen overdose.Limited knowledge of acetaminophen may be a preventablecontributor to elevated rates of overdose and thus acute liverfailure. The purpose of this study is to assess knowledge ofacetaminophen dosing and presence of acetaminophen incommon combination products in patients with liver disease.Methods: We performed a cross-sectional study of patientswith liver disease at the Pfleger Liver Institute at the Univer-sity of California, Los Angeles between June 2015 and August2016. Patients completed a demographic questionnaire andan acetaminophen knowledge survey. Additional informationwas obtained from the medical record. Results: Of 401 pa-tients with liver disease, 30 (15.7%) were able to correctlyidentify that people without liver disease can safely take up to4 g/day of acetaminophen. The majority of patients (79.9%–86.8%) did not know that Norco® (hydrocone/acetamino-phen), Vicodin® (hydrocone/acetaminophen) and Percocet®(oxycodone/acetaminophen) contained acetaminophen. Only45.3% of the patients knew that Tylenol® #3 contained acet-aminophen.Conclusions: We conclude that patients with liverdisease have critically low levels of knowledge of acetamino-phen, putting them at risk both of acetaminophen overdose,as well as undermedication, and inadequate management ofchronic pain. We recommend an increase in education effortsregarding acetaminophen dosage and its safety in the settingof liver disease. Increasing education for those at risk of lowacetaminophen knowledge is essential to minimizing acetami-nophen overdose rates and optimizing pain management.

© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

Acetaminophen is the most commonly used analgesic in theUnited States. Its use is indicated in a wide range of ailments,such as osteoarthritis, migraine headaches and fever.1–3 Partof its attractiveness is that it is inexpensive, well tolerated andreadily available in oral formulations.4 In 2008 alone, therewere 24.6 billion doses consumed in the United States.5

Tylenol® can be consumed alone, or as part of an analgesiccombination. While this drug is of benefit to many, it alsocomes with many risks, as acetaminophen is the mostcommon cause of drug-induced liver failure in the UnitedStates.6

Approximately 60,000 people are hospitalized each yearin the United States for acetaminophen overdose complica-tions.7 The percentage of these individuals who receive adiagnosis of acetaminophen-induced liver toxicity has risenfrom 6% in 1998 to 13% in 2011.6 In most individuals, theoverdose is accidental.6,8,9 These individuals unintentionallyconsume excessive acetaminophen due to a lack of knowl-edge of the maximal safe dose of acetaminophen itself, orlack of awareness of the quantity of common analgesics,which contain acetaminophen.10 Certain patient scenariosaccompanying acetaminophen overdose are linked to pooreroutcomes, such as chronic alcohol consumption, uninten-tional overdose, and underlying liver disease.11–16

In patients with chronic liver disease, the prevalence ofunintentional acetaminophen overdose is disproportionatelyhigh, compared to those without known liver disease.12,17 Aspatients with cirrhosis may have impaired hepatic function tometabolize certain drugs,18 the consumption of a dose ofacetaminophen lower than recommended daily maximummight lead to acute liver injury.19 Providing safe and effectiveanalgesia to patients with cirrhosis can be a clinical challenge.Despite the increased risk of overdose toxicity, acetamino-phen is still considered the safest analgesic for patients withliver cirrhosis.20,21 Acetaminophen is preferred over nonster-oidal anti-inflammatory drugs (NSAIDs) in patients with liverdisease because of the risk of nephrotoxicity, gastrointestinal

Journal of Clinical and Translational Hepatology 2016 vol. 4 | 281–287 281

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 Unported License,permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Keywords: Acetaminophen; Pain management; Cirrhosis.Abbreviations:OR, odds ratio; CI, 95% confidence interval; NSAID, nonsteroidalanti-inflammatory drug; FDA, Federal Drug Administration; IQR, interquartilerange; N/A, not answered; PPO, preferred-provider organization; HMO, healthmaintenance organization; GHPP, genetically handicapped person’s program.Received: 30 September 2016; Revised: 02 December 2016; Accepted: 22December 2016qDOI: 10.14218/JCTH.2016.00049.*Correspondence to: Sammy Saab, Pfleger Liver Institute, UCLA Medical Center,200 Medical Plaza, Suite 214, Los Angeles, CA 90095, USA. Tel: +1-310-206-6705, Fax: +1-310-206-4197, E-mail: [email protected]

toxicity and platelet impairment associated with medicationsin that class.20,22–25

While not much is known specifically about the safety ofacetaminophen use in patients with liver disease, expertsrecommend a maximum daily dose of 4 g/day for patientswith hepatic impairment.20,22,26,27 These recommendationsare based on several randomized trials and observationalstudies whose outcome were changes in findings of liver-associated tests and/or worsening liver function.28–33 In2009, the US Food and Drug Administration (FDA) advisorycommittee issued a relabeling of acetaminophen-containingproducts to better inform patients with liver disease about apotential risk of further liver injury.34 However, patients’understanding of acetaminophen dose after relabeling ofacetaminophen containing products has not been studied inpatients with liver disease. Our hypothesis is that patientswith known liver disease have inadequate knowledge aboutdaily acetaminophen dosage recommendations and commonpain medications that contain acetaminophen.

Methods

Study subjects

This observational, cross-sectional study included adultpatients with liver disease who were seen for follow-up at thePfleger Liver Institute, University of California, Los Angeles.The study was conducted from June 2015 to August 2016. Thesurveys and informed consent processes were administered inboth English and Spanish, and translation services wereprovided for patients whose native language was neitherEnglish nor Spanish.

All eligible patients seen in the Pfleger Liver Institute wereinvited to participate in the study by investigators duringtheir visit at the clinic. Following a short verbal explanationof the study, participants were administered questionnairesas described below. Participation in the study was voluntaryand there was no compensation offered. The University ofCalifornia, Los Angeles Institutional Review Board approvedthe study.

The medical records of all study participants were accessedin order to obtain information about the patients’ insurancetype, liver disease categorization, and whether or not theyhave a diagnosis of cirrhosis. Liver disease diagnosis wascategorized as hepatitis B, autoimmune hepatitis, hepatocel-lular cancer, hepatitis C, non-alcoholic fatty liver disease,alcoholic hepatitis, acetaminophen hepatotoxicity, or other.

Questionnaires

Each participant completed one self-administered question-naire, which was separated into a section for demographics,a section for patient preferences for education on medicine,and a section that assessed their knowledge of appropriateTylenol® use for patients with liver disease. Questions werewritten with an 8th grade reading level as the intendedcomplexity.

The demographics section involved 9 questions includinginquiries regarding patient age, gender identity, ethnicity,education, income, employment status, and how long thepatient had known about their liver disease. The knowledgeassessment consisted of 13 questions including whetheror not Norco, Vicodin, Percocet, or Tylenol® #3 have acet-aminophen, the maximum tablets of each strength of the

previously mentioned analgesic that can be safely taken daily,and the maximum daily amount of acetaminophen that can besafely taken by patients with and without liver cirrhosis.

Statistics

Responses to Tylenol® knowledge survey questions wereformatted for statistical analysis as either correct or incorrect.Correct answers to survey questions were based on medi-cation package inserts.35–38 Answers marked as ‘not sure’were coded as incorrect. Answers left blank by the participantwere coded as incorrect. Discrete variables were presented asthe number of participants belonging to that group, followedby the percentage equivalent in parenthesis. Continuous datawere presented as a median with interquartile range (IQR) inparenthesis, unless otherwise specified. A t-test or Wilcoxonrank sum test was used to compare continuous variablesbased on distribution of data and a Fisher’s exact test or chi-square test was used to compare discrete data. All tests weretwo-sided and a p-value below 0.05 was considered statisti-cally significant. Some variables were formatted for the multi-variable model. Age, education and pain medication usewithin 6 months were dichotomized and employment statuswas combined with amount worked. Variables were enteredinto the multivariable model if they were significant at the0.10 level on univariate analysis. The multivariable modelwas a logistic regression.

Results

Demographics

A total of 401 patients with liver disease were enrolled over aperiod of 15 months. The demographic characteristics of thestudy population are represented in Table 1. The majority ofpatients were male (53.4%) and their median (IQR) age was60 (51–67) years. Most patients were non-Hispanic white(39.7%) and had at least college-level education. Mostpatients had yearly income levels of less than $50,000. Pre-ferred Provider Organization was the common insurance uti-lized by the patients.

The patients’ median (IQR) length of time since thediagnosis of liver disease was made was 7 (2–15) years.The most common diagnoses were hepatitis C (42.6%),hepatitis B (23.9%) and non-alcoholic fatty liver disease(15.7%). Approximately two-thirds of patients had under-lying cirrhosis. Less than 20% of patients were new patientsto our Liver Institute, and less than half of all the patients inour study were able to reveal a caregiver.

Patient preferences for health and medicationinformation

The majority (59.6%) of patients had taken pain medicationin the last 6 months. Participant responses to questionsregarding preferences for health and pain medicine useinformation is shown in Table 2. In particular, we queriedboth the source and quality of where patients obtain informa-tion regarding over-the-counter medicines. Moreover, westratified the data according to whether or not the respond-ents were Hispanic.

The majority of patients (60.5%) claimed that they speakwith a physician before choosing an over-the-counter medi-cation. However, Hispanic patients were also less likely to

282 Journal of Clinical and Translational Hepatology 2016 vol. 4 | 281–287

Saab S. et al: Acetaminophen and liver disease

say they speak with a physician before choosing an over-the-counter medication (p = 0.015). Hispanics were lesslikely than the Non-Hispanics to consider their Physician(p = 0.015) or Nurse (p = 0.01) as a valuable and trustworthysource of information about health and medicine use.

Patient knowledge of common prescriptionmedications containing acetaminophen and maximumrecommended daily dose of acetaminophen

Thirty (7.5%) of the participants were able to correctlyidentify that the recommended maximum daily dose ofacetaminophen that a person without liver disease cansafely take is <4 g/day (Fig. 1). Few patients knew that themaximum acetaminophen dose in the setting of cirrhosis wasless than 3 g per day; there were no differences in knowledgebetween cirrhotic and non-cirrhotic patients (Fig. 2). Approx-imately 20% of our study participants felt that no amount ofacetaminophen was safe (Fig. 2).

Most patients were unaware commonly prescribed painmedications contained acetaminophen. Specifically, themajority of patients (79.9%–86.8%) did not know thatNorco® (hydrocodone/acetaminophen), Vicodin® (hydroco-done/acetaminophen) and Percocet® (oxycodone/acetami-nophen) contained acetaminophen. Only 45.3% knew thatTylenol® #3 contained acetaminophen. The independentpredictors for correctly answering if prescription pain medi-cation contained acetaminophen are shown in Table 3. Themost common predictor of correctly identifying the presenceof acetaminophen in the different pain medication combina-tions was the patient use of pain medications in previous6 months.

Discussion

The results of this study indicate that knowledge of acetami-nophen among patients with liver disease is limited, whichmayexplain why unintentional acetaminophen overdose makes upsuch a large portion of cases of acute liver failure.10 Low levelsof acetaminophen knowledge found in this population alsoposes a potential problem of under-medication and inadequatepain management in patients with liver disease. There cur-rently lacks sufficient data describing prevalence and effective-ness in managing pain in patients with liver disease.

Previously established predictors of health literacy includelocation of residence, household income, highest level of

Table 1. Demographic characteristics of 401 patients with liver disease

CharacteristicNumber ofResponses

Gender identity

Female 187 (46.6%)

Male 214 (53.4%)

Ethnicity

Non-Hispanic White 159 (39.7%)

Hispanic White 123 (30.7%)

African American 26 (6.5%)

Asian 64 (16%)

Other 39 (9.7%)

Education

High school or less 135 (33.7%)

Some college or more 266 (66.3%)

Estimated Annual Income

<$50,000 167 (41.6%)

$50,000–100,000 120 (29.9%)

>$100,000 64 (16%)

N/A 50 (12.5%)

Employment Status

Yes 258 (64.3%)

No 143 (35.7%)

If Employed, Hours Worked per Week

1–20 23 (16.2%)

21–40 46 (32.4%)

>40 71 (50%)

N/A 2 (1.4%)

Insurance type

PPO 165 (41.1%)

Free for Service Medical/MediCal HMO 50 (12.5%)

GHPP/Non-Medical HMO 23 (6.7%)

No Insurance/Self-pay 4 (1%)

Medicare 156 (38.9%)

Other 10 (2.5%)

Etiology of Disease

Hepatitis B 96 (23.9%)

Autoimmune Hepatitis 25 (6.2%)

Hepatocellular Cancer 35 (8.7%)

Hepatitis C 171 (42.6%)

Non-Alcoholic FattyLiver Disease

63 (15.7%)

Acetaminophen Hepatotoxicity 0 (0%)

Other 61 (15.2%)

Presence of Cirrhosis

Yes 255 (63.6%)

No 146 (36.4%)

Table 1. (continued )

CharacteristicNumber ofResponses

Does the Patient Have a Caregiver?

Yes 170 (42.4%)

No 231 (57.6%)

Visit Type

New 73 (18.2%)

Follow-up 328 (81.8%)

Abbreviations: N/A, Not answered; PPO, Preferred provider organization; HMO,Health maintenance organization; GHPP, Genetically handicapped personsprogram.



education, ethnic background, and level of English profi-ciency,39 and others have noted a direct correlation betweenhealth literacy with health outcomes.40 Some studies havefound females in a population to exhibit significantly higherhealth literacy than males.41 Previous studies assessing acet-aminophen knowledge have found interesting gender differ-ences as well, including that females are more likely to informtheir doctor of current acetaminophen use and more likely toknow the acetaminophen content of medications and themaximum recommended daily dose of acetaminophen.42,43

Female participants in this study were more likely to knowthat Vicodin® and Percocet® contained acetaminophenthan males, which fits with the general findings that womentend to have higher health literacy thanmen. This observationis important because, despite increased health literacy,women suffer disproportionately low quality of life with livercirrhosis compared to men44 and tend to have their pain takenless seriously by health care professionals.45 Regarding pref-erences for sources of health information, our results are in

concordance with previous studies, which have demonstratedlower levels of trust in health care professionals among His-panic patients compared to non-Hispanic white patients.46

Levels of trust in Hispanic patients have also been shown todiffer based on levels of English proficiency.47 More researchis necessary to better understand how gender and ethnicityinform a patient’s experience with pain and chronic liverdisease and how some of these disparities can be addressed.

There was a common theme that patients believed acet-aminophen to be inherently toxic to the liver, and thereforecontraindicated in liver disease. These perceptions werereflected in the fact that 230 (57.4%) of our participantsbelieved that patients with liver disease could not safely takeany acetaminophen. Furthermore, of the 230 patients thatprovided an answer other than ‘not sure’ for the Tylenol® #3dosing questions, 78% reported believing that the maximumsafe dose of Tylenol® #3 was the same for both the 300 mg/30 mg and the 300 mg/60 mg strength. Indicating a sharedperception that acetaminophen is the component of the

Table 2. Patient preferences for education on medicine

Question

Number of Responses

p-valueAll Participants(n = 401)

Non-Hispanics(n = 278)

Hispanics(n = 123)

Who do you speak with before choosing an over-the-counter medicine?

Pharmacist 146 (36.4%) 93 (33.5%) 53 (43.1%) 0.072

Physician 243 (60.5%) 180 (64.7%) 63 (51.2%) 0.015

Friends/family 73 (18.2%) 51 (18.3%) 22 (17.9%) 1

Other medical personnel 20 (4%) 14 (5%) 6 (4.9%) 1

Rarely or never talk with above peoplebefore choosing an over the countermedicine

46 (11.5%) 35 (12.6%) 11 (8.9%) 0.314

No response 6 (1.5%) 5 (1.8%) 1 (0.8%) 0.671

Who do you feel are valuable and trustworthy sources of information about health and medicine?

Physician 351 (87.6%) 255 (91.7%) 96 (78%) <0.001

Pharmacist 157 (39.2%) 117 (42.1%) 40 (32.5%) 0.076

Nurse 91 (22.7%) 73 (26.3%) 18 (14.6%) 0.01

Other medical personnel 44 (11%) 22 (7.9%) 22 (7.9%) 0.197

Advertising 3 (7.5%) 2 (0.7%) 1 (0.8%) 1

Friends/family 51 (12.8%) 37 (13.3%) 14 (11.4%) 0.63

No response 10 (2.5%) 4 (1.4%) 6 (4.9%) 0.075

Fig. 2. Patients knowledge of maximum safe dose of Tylenol® forpatients with liver cirrhosis.

Fig. 1. Patients knowledge of maximum safe dose of Tylenol® forpatients without liver cirrhosis.



medication that prevents the safe consumption of a higheramount of tablets. Among 23 patients that provided two ormore responses other than ‘not sure’ for the Percocet dosagequestions, 74% of respondents reported believing that themaximum safe dose of different strengths of Percocet wasthe same. In patients with liver disease, overestimation ofthe toxicity of acetaminophen may be beneficial for loweringrates of acetaminophen overdose. However, it may alsoincrease the likelihood that these patients will utilize moreaddictive methods of pain management since pain is such aprevalent and debilitating component of chronic liver disease.48

In a study of over 2000 healthcare providers, 40% ofparticipants reported that they would recommend against anyacetaminophen in patients with compensated cirrhosis.49 Inthe same study, physicians were more likely to recommendNSAID than acetaminophen use in patients with underlyingliver disease (p = 0.001).49 While acetaminophen has beenshown to be safe in moderation in patients with liver cirrhosis,NSAIDs are contraindicated in this population due to anestablished association with nephrotoxicity, gastrointestinaltoxicity, and platelet impairment.22–25

There is substantial variation in patients’ knowledge ofwhat constitutes a safe maximum dose of acetaminophen(Table 4).50–56 The variation may be partially explained bydifferent survey formatting.42,43 Our survey only listedoptions in gram amount and the correct answer (<4 g/day)was the highest of the 5 options provided. Additionally, overhalf our participants responded ‘not sure’ when askedmaximum safe dose of acetaminophen. These responseswere coded as incorrect. Some studies provided a similar‘not sure’ or ‘I don’t know’ option,42,52,54,55 while others didnot,43,53 but there is no identifiable pattern between thesetwo groups of studies which explains the variability seen.Our study boasts a more diverse patient population thanothers, but that does not appear to contribute to the variabil-ity, as studies measuring lower patient knowledge of Tylenol®

were similar in demographics to those which measured higherpatient knowledge in this study.

The results of this study provide precedent for furtherinvestigating pain management in patients with liver disease.One very important study that would lend clinical significanceto these results would be testing acetaminophen knowledgeusing similar questions in patients who overdose on the drug.Measuring pain scores in patients with liver disease along withmedications used and knowledge/perceived safety of acet-aminophen will provide evidence for or against a systemicproblem of inadequate pain management in liver diseasepatients due to insufficient education measures by providersabout safe and effective analgesia.

Our report has a number of potential limitations. Firstly,data was collected from patients at a single center, and whileour study subjects were ethnically diverse, they may not benecessarily representative of the demographics of the nationas a whole. Another potential limitation is that it is difficultto determine whether or not the results of the study aresecondary to the answers given by the subjects or secondaryto issues related to health literacy among a vulnerablepopulation. However, we wrote the questions at an 8thgrade level and tested in a cohort of patients before conduct-ing the survey. The results of our study were consistent withthose of similar studies which used comparable wording fortheir survey but were performed in different populations.51,52

Furthermore, both the research assistant and investigatorwere available if there were questions requiring furtherclarification.

Underlying liver disease appears to lower the safe con-sumption threshold of acetaminophen use.20,22,26,27 Likewise,the use of alcohol may also affect the safe consumptionthreshold.28,29,31,57 In our study, we did not specifically askabout alcohol use. Given that the threshold is similarlydecreased in patients with chronic liver disease and thosewho habitually consume alcohol, we do not believe omission

Table 3. Independent predictors of correctly identifying acetaminophen for different prescription pain medications

Independent Predictor Odds Ratio95% ConfidenceInterval p-value

Does the prescription drug NORCO® contain Tylenol®?

Rarely or never talking with someone beforechoosing an over-the-counter medication

2.757 1.364 to 5.574 0.006

Taking pain medication within the past 6 months 3.968 1.994 to 7.895 <0.001

Does the prescription drug VICODIN®

Age $ 60 0.455 0.26 to 0.797 0.005

Male gender 0.565 0.318 to 1.005 0.049


Does the prescription drug Tylenol® #3 contain Tylenol®?

Age $ 60 0.427 0.243 to 0.751 0.003

Trusting physicians as a source of information onhealth and medicine use

3.135 0.898 to 10.943 0.043


Does the prescription drug PERCOCET® contain Tylenol®?

Age $ 60 0.441 0.25 to 0.781 0.004

Male gender 0.558 0.313 to 0.995 0.045




of alcohol query is a major limitation of our study. Studiesthat specifically focus on acetaminophen use in patients whohabitually consume alcohol are needed.

We conclude that patients with liver disease have criticallylow levels of knowledge of acetaminophen, putting themat risk both of acetaminophen overdose, as well as under-medication and inadequate management of chronic pain. Werecommend an increase in culturally competent educationefforts regarding acetaminophen dosage and its safety inthe setting of liver disease. Increasing education in those atrisk for low acetaminophen knowledge is essential to mini-mizing acetaminophen overdose burden and optimizing painmanagement.

Acknowledgements

The authors thank Alex Farid, Justin Rheem, MD, and YoussefChallita for assistance in study design, patient recruitmentand data analysis.

Conflict of interest

None

Author contributions

Study concept and design (SS, PGK, MRV, MAJ), acquisition ofdata (MRV, WH, VPD, NLE, GC, FAD, MME, SBH, MJT), analysisand interpretation of data (SS, PGK, MRV, MAJ), drafting of themanuscript (SS, PGK, MRV, MAJ), critical revision of themanuscript for important intellectual content (SS, PGK, MRV,MAJ), statistical analysis (JFG), study supervision (SS).

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[11] Myers RP, Shaheen AA, Li B, Dean S, Quan H. Impact of liver disease, alcoholabuse, and unintentional ingestions on the outcomes of acetaminophenoverdose. Clin Gastroenterol Heptol 2008;6:918–925; quiz 837. doi: 10.1016/j.cgh.2008.02.053.

Table 4. Patient and provider ability to correctly identify safe daily dose of acetaminophen

Population CohortTotal numberof patients

Percentageidentifying correctsafe daily dose First AuthorRef

Health care providers

Internal medicine familymedicine (Alabama, USA)

76 76% Hornsby50

Patients

Adult general medicine clinic(Michigan, USA)

104 2.0% Stumpf51

Emergency department(Utah, USA)

1009 7% Fosnocht52

Family medicine practice(Illinois, USA)

102 22.5% Herndon53

Emergency department(France)

500 30% Boudjemai54

Outpatient facilities(Alabama, USA)

284 33% Hornsby55

Emergency department(London, UK)

910 53.8% Wood43

Emergency department(New York, USA)

103 54% Chen42

Hepatology clinic(California, USA)

401 9.7% Our Study



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Original Article

Impact of Different Embolic Agents for TransarterialChemoembolization (TACE) Procedures on SystemicVascular Endothelial Growth Factor (VEGF) Levels

Andreas Schicho*1, Claus Hellerbrand2, Kristina Krüger3, Lukas P. Beyer1,Walter Wohlgemuth1, Christoph Niessen1, Ernst Hohenstein3, Christian Stroszczynski1,

Philippe L. Pereira3 and Philipp Wiggermann1

1Department of Radiology, University Hospital Regensburg, Regensburg, Germany; 2Friedrich-Alexander University Erlangen-Nürnberg, Institute of Biochemistry (Emil-Fischer Zentrum), Erlangen, Germany; 3Clinic for Radiology, Minimally-invasive

Therapies and Nuclear Medicine, SLK Kliniken Heilbronn GmbH, Heilbronn, Germany

Abstract

Background and Aims: Intermediate stage hepatocellularcarcinoma (HCC) can be treated by transarterial chemoem-bolization (TACE). However, there appear to be side effects,such as induction of proangiogenic factors, e.g. vascular en-dothelial growth factor (VEGF), which have been shown to beassociated with a poor prognosis. This prospective study wasdesigned to compare serum VEGF level response after TACEwith different embolic agents in patients with HCC. Methods:Patients were assigned to one of three different TACE regi-mens: degradable starch microspheres (DSM) TACE, drug-eluting bead (DEBDOX) TACE or Lipiodol TACE (cTACE). Allpatients received 50 mg doxorubicin/m2 body surface area(BSA) during TACE. Serum VEGF levels were assessed beforeTACE treatment, 24 h post-treatment and 4 weeks later.Results: Twenty-two patients with 30 TACE treatmentswere enrolled. Compared to baseline VEGF levels, a markedincrease was observed for 24 h post-TACE (164% of baselinelevel) and during the 4-week follow-up (170% of baselinelevel) only for the cTACE arm (p < 0.05). In contrast, theincrease of serum VEGF levels were only 114% and 123%for DEBDOX and 121% and 124% for DSM, respectively.Conclusions: Conventional TACE using Lipiodol showsmarkedincrease in blood levels of the proangiogenic factor VEGF,while DEBDOX and DSM TACE induce only a moderate VEGFresponse.© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

Hepatocellular carcinoma (HCC) is one of the most frequentcancers worldwide. Treatment options are sparse and arehighly dependent on the hepatic tumor infiltration. In thetreatment of intermediate HCC Barcelona Clinic Liver Cancer(BCLC) stage B, conventional transarterial chemoemboliza-tion (cTACE) is an accepted and widely used treatmentapproach. This technique yields objective tumor responserates of approximately 60%–70%1,2 and, when comparedto best supportive care, a 20%–25% benefit of intermediate-to long-term survival.3,4 Beyond the different treatmentregimes, Lipiodol is found to be the predominant embolizationcompound,5 followed by drug-eluting beads (DEB)6 anddegradable starch microspheres (DSM),7 all in conjunctionwith doxorubicin, for example, as a DNA-intercalating cyto-toxic agent. TACE thus affects the tumor in parallel, by deliver-ing doxorubicin in a high concentration to the targeted tissueand reducing blood supply by embolization, thus leading toischemia.8 As hypoxia is a definite threat to viable tissue, itaffects a vast majority of intra- and intercellular signaling pro-cesses counteracting and reversing hypoxia.9 In malignanttissues, these biological countermeasures can potentiallyinterfere with the underlying intention of antitumor treatment.

DSM-TACE, DEB-TACE, and cTACE show different emboli-zation characteristics, which are crucial in the understandingof their mechanisms of biological action. While the beads usedin DEB-TACE lead to an irreversible permanent embolizationand ischemia,10 DSM-TACE leads to a transient ischemia withan occlusion half time of 35–50 minutes in vitro and 90–120minutes in vivo in HCC patients (EmboCept® S).11,12 Lipiodol,the predominant chemoembolization agent, used in cTACEprocedures has an ill-defined wide range of occlusion halftime, ranging between 4 to 12 weeks.5

One of the key agents strongly activated by hypoxia, thevascular endothelial growth factor (VEGF), mediates angio-genesis and is thought to play a key role in tumor growth andmetastatic seeding.13–15 Consequently, anti-VEGF therapiesare being investigated as potential anti-cancer treatmentsand supportives. In HCC, it was shown that a temporary over-production of VEGF is caused by a single session of TACE; anincrease of serum VEGF is related with future distant meta-stases, mainly in lungs and bones.16–18 Moreover, the post-TACE peak of serum VEGF is an independent prognostic



Keywords: Transarterial chemoembolization; Vascular endothelial growth factor;Embolic agents; Hepatocellular carcinoma; Angiogenesis.Abbreviations: BSA, body surface area; BCLC, Barcelona Clinic Liver Cancer;cTACE, conventional TACE using Lipiodol; DEBDOX, drug eluting beads (with dox-orubicin); DSM, degradable starch microspheres; HCC, hepatocellular carcinoma;NO, nitric oxide; TACE, transarterial chemoembolization; VEGF, vascular endothe-lial growth factor.Received: 04 November 2016; Revised: 05 December 2016; Accepted: 22December 2016qDOI: 10.14218/JCTH.2016.00058.*Correspondence to: Andreas Schicho, Department of Radiology, UniversityHospital Regensburg, Franz-Josef-Strauß-Allee 11, Regensburg D-93053,Germany. Tel: +49-941-944-7412, Fax: +49-941-944-7469, E-mail: [email protected]

factor of progression-free survival in HCC.15,19 Anti-VEGFtherapies have been reported and established in metastaticcolorectal carcinoma in combination with other chemother-apeutic agents,20 macula degeneration21 and diabetic retin-opathy.22 Consequently, avoidance of the post-TACE VEGFoverproduction is of major interest for the treatment regi-mens of HCC patients, directly affecting their expected lifespan in a palliative situation.

Our prospective pilot-study addressed the hypothesis thatthe prolonged or permanent occlusion, other than the tran-sient occlusion of DSM-TACE, causes a major and sustainedVEGF response, possibly in accordance with the concept ofan ischemia/reperfusion mechanism.

Methods

This two-center investigator-initiated pilot-study was approvedby the local institutional review board. We included patientswith intermediate stage HCC (BCLC stage B), either provennon-invasive by two imaging modalities or histologically. Sub-jects aged over 18, presenting with 4 or more HCC nodules#3 cm or with a single lesion $3 cm, and without portal veininvasion who had no contraindication for TACE were recruitedfor the study. We only included TACE-naïve patients; in casesof prior surgical resection or local ablation, at least 4 weeks hadto pass before inclusion in the study.

Our study was designed to include 12 patients per treat-ment arm by randomization, addressing 3 different treat-ments; DEB-TACE (DEBDOX™; BTG International Ltd.,London, UK), DSM-TACE (EmboCept® S; PharmaCept GmbH,Berlin, Germany), and cTACE (Lipiodol®; Guerbet LLC, Bloo-mington, IN, USA). Dose of doxorubicin was 50 mg/m2 bodysurface area in each treatment arm. All patients includedreceived standard TACE treatment according to a standardoperating procedure as previously reported.23 Of 36 patientsintended to include, 14 were either lost to 4-weeks of follow-up(n = 7), samples having thawed on transport (n = 4), or rele-vant patient data missing (n = 3).

Plasma levels of VEGF were assessed right before trans-arterial therapy as baseline, 24 h after and 4 weeks afterarterial treatment. A period of at least 4 weeks without intra-arterial therapy was observed before further TACE. Peripheralwhole blood samples were acquired according to our standardoperating procedure, centrifuged at 1500 rpm for 5 minutesand plasma samples were stored at −208C until shipping to acentral laboratory on dry ice. VEGF levels in our samples weremeasured using a commercial ELISA kit (Human VEGF Quan-tikine ELISA Kit; R&D Systems Inc., Minneapolis, MN, USA) bya third-party member not involved in patient inclusion, treat-ment or analysis of results. Plasma levels of VEGF right beforeTACE were set as reference (100%).

Patient characteristics were analyzed by using descriptivestatistics. A t-test or a Mann-Whitney U-test was used tocompare metric data. A p-value <0.05 was considered to indi-cate a statistically significant difference. Statistical analyseswere performed by using the JMP statistics software package(SAS Institute, Cary, NC, USA).

Results

Patients characteristics

In our pilot-study, complete data sets of 22 patients wereobtained, represented by 9 for DSM-TACE, 7 for DEB-TACE,

and 6 for cTACE (Table 1). Thereby, 9 TACE cycles were com-pleted for DSM-TACE, 9 for DEB-TACE, and 12 for cTACE.Patients who received DSM-TACE were mean age of 67 ± 7.3years, those who received DEB-TACE were 70.9 ± 7.7 years,and those who received cTACE were 64.5 ± 12.7; there wereno statistically significant differences between the groups.Tumor burden was calculated as 6.1 ± 4.9 cm (DSM-TACE),5.2 ± 4.3 cm (DEB-TACE), and 6.3 ± 5.1 cm (cTACE), withno statistically significant differences between the groups.

VEGF levels before and after differentTACE procedures

Plasma levels of VEGF right before TACE were set as reference(100%). For DSM-TACE, plasma VEGF levels 24 h after TACEand 4 weeks later were elevated to 121 ± 29% and 124 ±43%, respectively. Results for DEB-TACE did not differ signifi-cantly, with 114 ± 31% at 24 h after TACE (p = 0.35; Figs. 1and 3) and 123 ± 55% at 4 weeks later (Fig. 2).

Table 1. Baseline characteristics of patients and tumor burden

DSM-TACE DEB-TACE cTACE

No. of patients 9 7 6

No. of TACE-cycles

9 9 12

Age,mean 6 SD

67 6 7.3 70.9 6 7.7 64.5 6 12.7

Tumor burden,cm 6 SD

6.1 6 4.9 5.2 6 4.3 6.3 6 5.1

Fig. 1. Serum VEGF levels at 24 h after cTACE with standard Lipiodol,DEB, or DSM. Levels in DEB-TACE (n = 7) and DSM-TACE (n = 9) were sig-nificantly lower than in cTACE (n = 6; p = 0.01 and p = 0.04, respectively). Barsshow mean ± SEM.

Abbreviations: cTACE, conventional transarterial chemoembolization; DEB, drugeluting beads; DSM, degradable starch microspheres; VEGF, vascular endothelialgrowth factor.


Schicho A. et al: Impact of TACE embolic agents on serum VEGF levels

In the cTACE group, VEGF plasma levels were elevatedto 164 ± 68% of baseline at 24 h post-TACE (vs. DSM-TACE:p = 0.04 and vs. DEB-TACE: p= 0.01; Fig. 1) and remained athigh levels (170 ± 81%) for at least 4 weeks after the treat-ment (vs. DSM-TACE: p = 0.04 and vs. DEB-TACE: p = 0.03;Fig. 2).

Discussion

VEGF plays a key role in hypoxia signaling.14 Elevated levelsare found in a vast variety of tissues when put at risk oftissue-threatening ischemia, e.g. in heart (myocardial infarc-tion),24 brain (stroke),25 muscle (peripheral arterial occlusivedisease),26 liver (tumor, infarction)27 and kidney (infarc-tion).28 While VEGF causes both long- and short-term tissueprotective effects in healthy tissue, its effects in tumoroustissues include, but are not limited to, faster and more pro-found metastatic seeding, as well as regeneration and growthof malignant tissues.16–18

Since its first publication in the late 1980’s,29 cTACE becamea standard treatment in intermediate stage HCC.30 In themeantime, other technologies have been reported, includingDSM,7 DEB,6 and Lipiodol as the so-called ‘conventional’cTACE.5 Among the factors potentially interfering with itseffectiveness is a potent VEGF-dependent neoangiogenicresponse due to the ischemia (and reperfusion) caused bythe embolization agents, which shows characteristic occlu-sion half-life values of 90–120 minutes for DSM-TACE, 4 to12 weeks in Lipiodol cTACE, and irreversible occlusion in DEB-TACE. Therefore, we set up a pilot-study to assess changesin VEGF plasma levels depending on the used embolicagents.

Our results show a rise of the VEGF plasma levels abovebaseline in all three treatments, as a result of ischemiacaused by embolization. While the permanent embolizationby DEB-TACE and the transient ischemia in DSM-TACE yieldedlevels of 7% and 22% above baseline at 24 h, respectively, thepersistent but non-permanent embolization in cTACE causesa significantly enlarged (1.7–fold) rise in VEGF plasma levels.These elevations essentially remained unchanged for (at least)4 weeks following the TACE procedure in all three studiedsubstances. Thus, Lipiodol cTACE patients are subject to aprofound VEGF release for at least 4 weeks. These post-cTACEresults are in line with prior published results of both humanand animal study protocols; Sergio et al.19 even found afurther increase of VEGF in their follow-up of 4 weeks aftercTACE. According to the data of 8 patients receiving twoTACE treatments in our study, an accumulative effect seemspossible; further studies are needed to clarify the VEGF serumkinetics after TACE.

Potential underlying mechanism

A hypothetic key to understanding the significant changesarising from the different chemoembolization agents are thechemical properties of the oily Lipiodol emulsion, causing an“unstable” ischemia with reperfusion situation in the targetedtissue. While, to the contrary, DSM has a defined occlusiontime of 90 to 120 minutes in vivo, and Lipiodol has an occlu-sion half time ranging between 4 and 12 weeks. It is knownthat sustained ischemia either causes tissue death by bothnecrosis and apoptosis,26,31,32 or in case of survival, regen-eration and adaptation of the tissue. Other than Lipiodol,DEB-TACE does not enable a reperfusion of the embolized,ischemic tissue, which is crucial for the cellular and humoralresponse to hypoxia. DSM-TACE on the other hand seems tonot yield a durable tissue ischemia, long enough to elicit astronger response in VEGF plasma levels. As an oily emulsion,Lipiodol has no defined particle size, enabling a sustainedbut dynamic situation of tissue hypoxia with subsequent,perpetual reperfusion.

Fig. 2. Serum VEGF levels 1 month after cTACE with standard Lipiodol,DEB, or DSM. Levels in DEB-TACE (n = 7) and DSM-TACE (n = 9) were sig-nificantly lower than in cTACE (n = 6; p = 0.03 and p = 0.04, respectively). Barsshow mean ± SEM.

Abbreviations: cTACE, conventional transarterial chemoembolization; DEB, drugeluting beads; DSM, degradable starch microspheres; VEGF, vascular endothelialgrowth factor.

Fig. 3. Serum-VEGF levels at 24 h and 1 month post-treatment comparedto baseline. Bars show mean ± SEM.

Abbreviation: VEGF, vascular endothelial growth factor.



From a completely different set of organs,33,34 as well asliver,35 it is known that reperfusion is an essential componentin eliciting both strong cellular and humoral signaling pro-cesses, including the expression of cytokines, growth factorsand adhesion molecules that are repeatedly reported to fostergrowth of malignant tumors. Microcirculatory dysfunction,as assumed to be predominant in cTACE, has repeatedlybeen found to resemble a source of hypoxia.36 Mechanisti-cally, besides hypoxia-inducible factor (HIF-)1a,37 which actsas a cell survival factor, and Fas–Fas ligand interactions,38

essential in the regulation of both apoptosis and necrosis,the main component reportedly is the up-regulation of VEGFexpression.

VEGF causes tumor angiogenesis, and it was shownthat, especially in the liver, ischemia/reperfusion-triggeredup-regulation of VEGF causes improved tumor vascularization.Moreover, positive-feedback autoregulatory functions of VEGFwere found via a parallel overexpression of VEGF receptors inmalignant cells, including HCC cells. Recent reports addition-ally credit side effects like promotion of cancer cell survival andcancer cell migration to the up-regulation of VEGF and theVEGF receptor.39

Clinical implication

When transferring these reported observations to the clinicalsetting of HCC treatment by TACE, avoiding the use ofLipiodol in favor of DEB- or DSM-TACE could directly affectthe clinical outcome of HCC patients; it was shown that thetemporary overexpression of circulating VEGF caused by asingle session of cTACE can contribute to inducing futuredistant metastatic seeding, mainly pulmonary and osseous.18

The VEGF serum levels, moreover, were shown to be an inde-pendent prognostic factor of progression-free survival in HCCtreated with cTACE. Therefore, it should be of major interestto preserve low VEGF levels in HCC patients. Preventionof ischemia-induced microcirculation with L-arginine (anenhancer of endothelial nitric oxide synthesis) reduces theseeding of micrometastases by minimizing tissue hypoxia,and direct scavenging of free-circulating VEGF by the anti-VEGF monoclonal antibody bevacizumab is approved by theFDA as first-line therapy, e.g. in metastatic colorectal carci-noma, in combination with other chemotherapeutic agentsand, moreover, is used on- and off-label in a wide variety ofoncology and non-oncology indications, including maculardegeneration and diabetic retinopathy.22

While these approaches try to ameliorate an apparentlyTACE-inherent VEGF up-regulation, the results of our pilot-study support the use of DEB- or DSM-TACE instead of Lipiodolas the first causally determined treatment of VEGF over-expression. Additionally, differences in safety and side effectsof the embolic agents have to be observed and taken intoaccount.

Limitations

Since our study was designed as a pilot-study in a proof-of-principle fashion, some inherent limitations have to beconsidered. The small sample size in each treatment armmay limit generalizability of results. Nonetheless, implica-tions derived from our results for clinical treatment needto be tested in a larger cohort, e.g. investigation of potentialdifferences between DEB-TACE and DSM-TACE, or safety andside effects of different embolic agents.

The major physiological transporters of VEGF in blood arecirculating platelets. It is known that VEGF concentrationshighly correlate with platelet counts. Moreover, the plateletload of VEGF in patients with malignant tumors correlatesquantitatively with the expression of VEGF in malignanttissues. Our study design did not cover collection and analysisof platelet counts. Both platelet count changes as well aschanges of VEGF receptor expression should be addressed byfuture studies.

Previous studies have shown that pre-TACE levels per seare correlated with HCC characteristics, including tumor size,vascular invasion and metastasis. Thus, further studiesshould allow for an in-depth analysis of baseline VEGF-levelsin HCC patients.

Conclusions

Among the factors potentially interfering with the intention ofTACE in HCC, post-interventional up-regulation of VEGF is ofmajor concern, since VEGF is known to promote tumor growthvia neoangiogenesis, metastatic seeding, and both cancer cellmigration and survival. The widely used cTACE causes anenlarged VEGF serum response sustaining for at least 28 dayspost-TACE. On contrary, both DEB-TACE and DSM-TACE elicita significantly lower VEGF response, making the preferred useof these chemoembolization agents the first causally deter-mined approach to an ameliorated post-TACE VEGF over-expression. Parallel use of anti-VEGF therapies could be ofvalue in the treatment of HCC.

Acknowledgments

Financial assistance from PharmaCept GmbH, Berlin, Germanyto Stroszczynski C and Wiggermann P.


None


Study design (CS, PLP, PW), patient inclusion and perform-ance of transarterial chemoembolization (WW, EH, CS, PLP,PW), sample preparation and obtainment of patient data(AS, KK, LPB, CN), vascular endothelial growth factor serumlevel analysis (CH), data analysis (AS, LPB, PW), manuscriptdrafting and figure preparation (AS, LPB), discussion andapproval of manuscript (AS, CH, KK, LPB, WW, CN, EH, CS,PLP, PW), supervision (CS, PLP, PW).

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Original Article

miR-29a Promotes Lipid Droplet and TriglycerideFormation in HCV Infection by Inducing

Expression of SREBP-1c and CAV1

Mennatallah Mamdouh Mahdy1, Nada Magdy El-Ekiaby2,6, Rana Mahmoud Hashish3,Radwa Ayman Salah4, Rasha Sayed Hanafi1, Hassan Mohamed El-Said Azzazy5

and Ahmed Ihab Abdelaziz*2,6

1Department of Pharmaceutical Chemistry, German University in Cairo, New Cairo City, Egypt; 2Department of Pharmacologyand Toxicology, German University in Cairo, New Cairo City, Egypt; 3Department of Pharmaceutical Biology, German Universityin Cairo, New Cairo City, Egypt; 4Department of Stem Cells and Regenerative Medicine, Zewail City of Science and Technology,Giza, Egypt; 5Department of Chemistry, Novel Diagnostic & Therapeutics, American University in Cairo, New Cairo City, Egypt;

6School of Medicine, NewGiza University, Cairo, Egypt

Abstract

Aims: To examine the regulation of SREBP-1c and CAV1 bymicroRNA-29a (miR-29a) in cells infected with hepatitis Cvirus (HCV) in an attempt to control HCV-induced non-alcoholic fatty liver disease. Methods: In order to examinethe manipulation of SREBP-1c and CAV1 by miR-29a, oleicacid (OA)-treated JFH-I-infected Huh-7 cells were used. OAwas added 24 h post-transfection and gene expression wasinvestigated by qRT-PCR at 48 h post treatment. The func-tional impact of the observed alteration in SREBP-1c andCAV1 expression was analyzed by examining lipid droplet(LD) and triglyceride (TG) content at 72 h post-OA treatmentusing light microscopy and spectrophotometry, respectively.Viral load was quantified by qRT-PCR at 72 h post-transfection.Results:OA treatment induced the expression ofmiR-29a andSREBP-1c, as compared to untreated cells. Forced miR-29aexpression led to a significant up-regulation of SREBP-1cas well as CAV1 compared to mock untransfected cells.Ectopic expression of miR-29a resulted in a marked increasein LDs and their respective TGs, while miR-29a antagomirsdecreased both the LD and TG content compared to mock un-transfected cells. Moreover, forcing the expression of miR-29ain JFH-1 HCV-infected Huh-7 cells resulted in 53% reductionin viral titers compared to mock untransfected Huh-7 cells.Conclusion: Inducing miR-29a expression significantly indu-ces SREBP-1c and CAV1 expression, thereby increasing LDs aswell as their respective TGs. Nonetheless, forcing the expres-sion of miR-29a resulted in reduction of HCV RNA levels inHuh-7 cells.

© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

Hepatitis C virus (HCV) is a major causative agent of non-alcoholic fatty liver disease (NAFLD), where 40% to 80% ofHCV-infected patients develop steatosis depending on severalfactors, such as presence of diabetes and obesity as well asother risk factors, making the occurrence of steatosis in HCVinfection 2-fold higher than in other chronic liver diseases,including infection with hepatitis B virus.1,2 Although little isknown about the mechanism leading to lipid accumulationin HCV-infected hepatocytes, some studies showed thatseveral HCV proteins, specifically the structural core and thenon-structural protein 5A (NS5A), can lead to hepatic steato-sis.3 In addition, overexpression of the HCV proteins coreand NS4B independently activates miR-27a expression,leading to larger and more abundant lipid droplets (LDs)as the overexpression represses peroxisome proliferator-activated receptor-a (PPAR)-a and angiopoietin-like protein3 (ANGPTL3), known regulators of triglyceride (TG) homeo-stasis in hepatocytes, which is considered a novel mechanismof HCV-induced steatosis.4 It has also been shown thatHuh-7 hepatoma cells infected with a mutant HCV coreprotein that does not target the LDs had extremely loweramounts of intracellular lipids compared to cells infectedwith the wild-type core protein. Thus, HCV core proteinexpression appears to increase the amount of LDs, and anincreased LD content results in increased HCV core proteinexpression, which is favorable for HCV replication.5

LDs are mono-layered organelles with a hydrophobic coreof TG and cholesteryl esters surrounded by phospholipids.Their size ranges from nanometres to several micrometres indiameter. Several proteins associate with the phospholipidmonolayer, including perilipin and RAB family members,which play a role in the structure, formation and function ofLDs.6,7 Other proteins associate with the surface of LDsin special conditions, including the caveolins (CAVs). CAVs con-stitute the structural framework of caveolae, which are small



Keywords: Caveolin-1; HCV; Lipid droplets; MicroRNA-29a; SREBP-1c.Abbreviations: ANGPTL3, angiopoietin-like protein 3; CAVs, Caveolins; HCV,hepatitis C virus; LD, lipid droplet; miRNA, microRNA; miR-29a, microRNA-29a;NAFLD, Non-alcoholic fatty liver disease; NS5A, non-structural protein 5A; ORO,oil-red-O solution; OA, oleic-acid; (PPAR)-a, peroxisome proliferator activatedreceptor-a; SREBP, sterol regulatory element binding protein; TG, triglyceride.Received: 29 August 2016; Revised: 21 November 2016; Accepted: 07 December2016qDOI: 10.14218/JCTH.2016.00046.*Correspondence to: Ahmed Ihab Abdelaziz, Department of Molecular Medicine,School of Medicine, Newgiza University, Cairo 11431, Egypt. Tel: +20-238277847,E-mail: [email protected]

invaginations of the plasma membrane.8,9 The caveolin familyconsists of three members: CAV1, CAV2 and CAV3.10 CAV1, inparticular, has been reported as important for LD stability.11

Among the regulators of LDs are the family of transcrip-tional regulators of the lipid synthetic genes, known as thesterol regulatory element binding proteins (SREBP). To date,three SREBP isoforms have been identified: SREBP-1a,SREBP-1c and SREBP-2.12–15 It was reported that perturbedexpression of SREBP-1c, in particular, leads to an increase inLD proteins in fatty liver dystrophic mice.16 Several studieshave also demonstrated that there is a positive correlationbetween the amount of LDs and SREBP-1c expression.17–19

SREBP-1 is known to bind to the sterol regulatory elements(SREs) in theCAV1genepromoterandtosubsequently regulateCAV1 expression, either positively or negatively, dependingon the cell type as well as the upstream signal.20,21 Manyresearch groups have investigated the relation between CAV1levelsandLDs indifferent cell types, includingadipocytes, fibro-blasts and hepatocytes.11,22–28 In a study of hepatocytes,NAFLD was shown to be associated with up-regulation ofthe hepatic CAV1 gene and protein expression as well as anincrease in its localization on LDs.29,30 Furthermore, CAV1was found to play a role during liver regeneration.31 However,neither its expression nor it’s relationwith SREBP-1c have beeninvestigated in HCV-infected hepatocytes.

Several microRNAs (miRNAs) can inhibit SREBP-1 expres-sion in hepatocytes, including miR-449, miR-122 and miR-613, thus down-regulating its target lipogenic genes andthe related LD content.32–34 Bioinformatics have previouslyshown that miR-29a targets SREBP-1c.35 miR-29a has beenreported to alleviate cholestatic liver injury as well as fibro-sis.36,37 It has also been reported to be down-regulated in theliver of chronic HCV-infected patients as well as in genotype2a HCV-infected cell models.38 Furthermore, overexpressionof miR-29a led to decreased HCV RNA abundance in HCV gen-otype 2a-infected cell models.38 To date, very limited data isavailable concerning the role of miRNAs in controlling LDs inHCV infection. Hence, this study aimed at uncovering the roleof miR-29a in regulating hepatic expression of SREBP-1c andthe subsequent CAV1, in an attempt to control LD formationas well as their respective TGs in HCV infection.

Methods

Cell culture

Adherent human hepatoma cells (Huh-7 cells) were incubatedat 378C and 5% CO2 following culturing in Dulbecco’s modifiedEagle’s medium supplemented with 10% fetal bovine serum,1% penicillin/streptomycin, 4.5gL−1 glucose and L–glutamine.

In vitro transcription and production of HCV cell culture(HCVcc)

HCV construct (pJFH-I) harboring the full-length HCV geno-type 2a (kindly provided by Prof. Wakita, National Institute ofInfectious Diseases, Tokyo, Japan) was linearized using XbaI,and then purified using phenol/chloroform. In vitro transcrip-tion was carried out with 1 mg of the purified DNA using theMEGAscript® T7 Transcription Kit (AM1330: Ambion, Austin,TX, USA). To produce the HCVcc, liposome-mediated trans-fection of 10 mg of the in vitro transcribed HCV genomic RNAinto Huh-7 cells was performed using the Superfect®Transfection Reagent (Qiagen, Hilden, Germany). At 72 hrs

post-transfection, supernatants were collected, filteredusing 0.45-mm filters, and stored at −808C for further infec-tion of naïve Huh-7 cells.

RNA extraction

Total RNA, mRNA and miRNA, was extracted using the BiozolReagent (Bioer Technology, Hangzhou, China). Huh-7 cellswere first lysed in Biozol then chloroform was added on top ofthe cell lysate. Following centrifugation at 12,000 rpm at 48Cfor 15 min, RNA in the aqueous layer was precipitated usingisopropanol. Followed additional centrifugation at 12,000 rpmat 48C for 15min, the precipitated RNAwas then washed using75% ethanol and dissolved in DPEC water.

Reverse transcription and RNA quantification usingqRT-PCR

miR-29a and the housekeeping gene RNU6B were reversetranscribed into complementary DNA (cDNA) using theTaqMan microRNA Reverse Transcription Kit (Applied Biosys-tems, Foster City, CA, USA) according to manufacturer’sinstructions and using TaqManmicroRNA assays (Applied Bio-systems). Total cellular mRNA was reverse transcribed intocDNA using the high-capacity cDNA reverse transcription kit(Qiagen) according to the manufacturer’s instructions.

The mRNA and miRNA expression levels were quantifiedusing StepOne™ real-time quantitative polymerase chain reac-tion (qRT-PCR) (SN: 271000301; Applied Biosystems). Theamount of miR-29a was then calculated relative to the amountof the housekeeping gene RNU6B, and the mRNA expressionlevel of SREBP-1c and CAV1 was then normalized to thehousekeeping gene B2-microglobulin (B2M) using TaqMansterol regulatory element binding factor-1 (SREBF1), CAV1and B2M assays (Applied Biosystems).

Transfection of oligonucleotides

In order to perform mRNA quantification and intracellular LDimaging, Huh-7 cells were transfected with 25 nM of miR-29amimics or antagomirs, or SREBP-1 siRNAs (used as a positivecontrol in this study) in 96-well plates by means of theHiperfect transfection reagent (Qiagen) using reverse trans-fection method according to the manufacturer’s instructions.In order to quantify TGs as well as viral load, Huh-7 cells weretransfected in 24-well plates by the Hiperfect transfectionreagent (Qiagen) using the traditional transfection methodaccording to the manufacturer’s instructions. Cells werecultured under normal conditions (378C and 5% CO2).

Fatty acid treatment

In order to develop a model for hepatic steatosis in Huh-7 cells,induction of LD formation was achieved by incubating cells withOA. The Huh-7 cells were incubated with 200 mM bovine serumalbumin-coupled with OA at 24 hrs post-transfection with oligo-nucleotides, then at 48 hrs post-OA treatment the gene expres-sionprofilingwasperformedusingqRT-PCR.LDvisualizationandTG quantification were performed at 72 hrs post-OA treatment.

Lipid droplet staining and imaging

A stock solution of 0.35% oil-red-O solution (ORO; Serva,Heidelberg, Germany) was prepared and filtered through a0.22-mm filter. A working solution of ORO was freshly prepared


Mahdy M.M. et al: Lipid droplets, miR-29a and HCV

by diluting the stock solution with double-distilled water at aratio of 6:4. The working solution was left to stand for 20 minand filtered through a 0.22-mm filter. Fixation of cultured cellswas performed using 4% paraformaldehyde in phosphatebuffer saline (PBS; pH = 7.4) for 10 min at room temperature.Fixed cells were then washed with 3 changes of PBS, 10 mineach. Permeabilization was performed by incubating the cellswith 0.05% (Tween 20 in PBS) for 15min followed by 3 washeswith PBS then washing once with 60% isopropanol. Permea-bilized cells were then incubated with ORO stain for 10 minfollowed by 6 washes with distilled water, 5 min each. LDs werevisualized using Axiom Zeiss Light microscope with 100xmagnification (SN 3832001601: Carl Zeiss, Jena, Germany)as described in other research papers.39,40 Images were cap-tured using Zen2011 software (Carl Zeiss).

Triglyceride extraction and quantification

Seventy-two hrs after the OA treatment, TGs were extractedfrom the JFH-I-infected Huh-7 cells using a triglyceridecolorimetric assay kit (Item no. 10010303: Cayman Chem-ical, Ann-Arbor, MI, USA) and then quantified using theTriglycerides-LQ Kit (Spinreact, Girona, Spain) according tothe manufacturer’s instructions.

TGs were quantified using a spectrophotometric-basedTG kit, whereby the TGs are chemically derivatized toquinone and measured at 505 nm. Before calculating the TGconcentration, the average of the standard concentrations ofdifferent experiments was calculated. Concentration of theunknown TGs was calculated using the following 1-pointcalibration equation: absorbance of sample/absorbance ofstandard*TG concentration (200 mgdL-1).

Viral RNA extraction and quantification

Seventy-two hrs post-transfection, viral RNA was extractedfrom HCV-infected Huh-7 cells using the InvisorbSpin VirusRNA Mini Kit (Invitek, Hayward, CA, USA) according to themanufacturer’s protocol. The extracted RNA was stored at−808C until further use. Viral nucleic acid was quantified usinga viral nucleic acid detection kit (Genesig; PrimerDesign,Chandler’s Ford, UK), and following the manufacturer’s pro-tocol using real-time PCR.

Statistical analysis

Data were expressed as mean ± standard error of the mean(SEM). Differences between samples were analyzed usingStudent’s t-test. A p-value lower than 0.05 was consideredsignificant. ***p < 0.001, **p < 0.01, *p < 0.05 and nsindicated not statistically significant. All the data were statisti-cally analyzed using GraphPad Prism 5 (GraphPad Software,Inc. La Jolla, CA, USA).

Results

Impact of OA treatment on miR-29a, SREBP-1c andCAV1 mRNA expression and on LD and TG content inJFH-I-infected cells

OA treatment of JFH-I-infected Huh-7 cells resulted in up-regulation ofmiR-29a (p= 0.0002) and SREBP-1c (p=0.0443)mRNA expression, with no significant change in CAV1 mRNAexpression, as compared to untreated JFH-1-infected cells(Fig. 1A, 1B, 1C). LDs were markedly increased in the

Fig. 1. Impact of oleic acid (OA) treatment on miR-29a, SREBP-1c and CAV1 mRNA expression and on lipid droplet (LD) and triglyceride (TG) content inJFH-I-infected Huh-7 cells. The OA treatment led to A) significant up-regulation of miR-29a mRNA expression (17.74 ± 2.162, p = 0.0002***, n = 5), B) significant up-regulation of SREBP-1c mRNA expression (1.591 ± 0.2129, p = 0.0443*, n = 5) and C) no significant change in the mRNA expression of CAV1 (1.153 ± 0.1940, n = 10), ascompared to untreated cells (1.010 ± 0.08429), (1.000 ± 0.009492), (1.003 ± 0.02906), respectively. Results are expressed as a mean ± SEM. OA treatment led toD) marked increase in LDs, as shown by increase in the red coloration due to the oil red O staining, and as compared to untreated cells, n = 3. Scale bars of 10 mm are shownon the images. E) TG concentration is significantly increased upon OA treatment (102.3 ± 3.068, p = 0.0305*, n = 5) compared to untreated cells (84.58 ± 6.573). Resultsare expressed as a mean ± SEM. Student’s t-test was used for the statistical analysis.



OA-treated JFH-I-infected Huh-7 cells, and the intracellularTG content was also significantly increased (p = 0.0305), ascompared to the untreated JFH-1-infected Huh-7 cells(Fig. 1D, 1E).

Impact of manipulating miR-29a on SREBP-1cand CAV1 mRNA expression and on LD andTG content

At 3 days post-infection with JFH-I, miR-29a oligonucleo-tides were transfected into the cells, and at 24 hrs post-transfection the cells were treated with 200 mMOA. Forty-eighthrs following the OA treatment, qRT-PCR was used to assessthe mRNA expression of SREBP-1c and CAV1. Beforeassessing the expression of both genes, it was importantto confirm the efficiency of transfection of the oligonucleo-tides; mimicking of miR-29a significantly increased itsexpression (p = 0.0035), by a mean of 1157.018-fold com-pared to untransfected cells (Fig. 2A), while miR-29a anta-gomirs resulted in significant reduction in its expressionlevel, by a mean of 0.9-fold compared to untransfectedcells. SREBP-1c as well as CAV1 mRNA expression wasinduced (p = 0.0276 and p = 0.0337, respectively) uponmimicking with miR-29a; however, the antagomir did nothave any effect on either SREBP-1c or CAV1 expression

(Fig. 2B, 2C). Seventy-two hrs following the OA treatment,LD content was observed; mimicking with miR-29a mark-edly increased the LD content, while miR-29a antagomirsled to a slight decrease in the amount of LDs (Fig. 2D). Intra-cellular TGs were significantly increased upon mimickingwith miR-29a (p = 0.0038) and markedly decreased uponantagonizing miR-29a (p = 0.0125) (Fig. 2E).

Impact of SREBP-1c siRNA on LD formation and CAV1mRNA expression

To investigate the association between level of SREBP-1c andLD, OA-treated JFH-I-infected Huh-7 cells were transfected withSREBP-1c siRNAs. SREBP-1c level was measured to ensureefficient repression of SREBP-1c mRNA levels (p < 0.0001)(Fig. 3A). LD formation markedly decreased (Fig. 3B). CAV1mRNA expression significantly increased following SREBP-1csiRNA transfection (p = 0.0030) (Fig. 3C).

Impact of miR-29a on viral replication

Mimicking of miR-29a in JFH-I-infected Huh-7 cells resultedin 53% reduction (p = 0.0499) in viral titers compared tountransfected Huh-7 cells (Fig. 4).

Fig. 2. Impact of manipulating miR-29a on SREBP-1c and CAV1 mRNA expression and on cellular lipid droplet (LD) and triglyceride (TG) content in JFH-I-infected Huh-7 cells.miR-29a mimics or antagomirs were transfected into oleic acid-treated JFH-1-infected Huh-7 cells. A) Efficient delivery of miR-29a mimics into Huh-7cells was confirmed by measuring the amount of miRNA in mimicked cells. miR-29a mimic increased miR-29a expression by a mean of 1157.018-fold (p = 0.0035**, n = 4)and the antagomir decreased its expression by amean of 0.9-fold (p= 0.0111*, n= 4) in comparison of transfected versus mock cells. B) Mimicking with miR-29a lead to up-regulation of SREBP-1c mRNA expression (1.369 ± 0.1292, p = 0.0276*, n = 9) with no significant change observed for antagonizing miR-29a expression (1.067 ± 0.1949,n = 9) compared to untransfected cells (1.043 ± 0.07413). C) miR-29a mimics induced the mRNA expression of CAV1 (1.606 ± 0.1704, p = 0.0337*, n = 9), whileantagomirs did not significantly alter CAV1 mRNA expression (1.406 ± 0.1134, n = 9) compared to untransfected cells (1.081 ± 0.1350). D) Mimicking of miR-29a resultedin increased amount of LDs, as shown by increase in the red coloration due to the oil red O staining, while miR-29a antagomir showed a slight decrease in the amount ofLDs compared to untransfected cells, n = 3. Scale bars of 10 mm are shown on the images. E) Mimicking of miR-29a also significantly increased the concentration of TGs(114.8 ± 2.578, p = 0.0038**, n = 6) and antagonizing miR-29a significantly decreased TG concentration (80.73 ± 4.382, p = 0.0125*, n = 2) compared to untransfectedcells (99.87 ± 2.352). All results are expressed as a mean ± SEM. Student’s t-test was used for the statistical analysis.



Discussion

miR-29a is reported to have anti-viral and anti-fibrotic activ-ities. It was shown to decease the HCV RNA abundancein Huh-7.5 cells infected with the J6 strain of genotype 2a(in a HCVcc system).38 miR-29a was also reported to inhibithepatic stellate cells and thus fibrosis.41 The regulation of LDsby miR-29a in HCV infection has not yet been investigated.However, given the aformentioned reports, miR-29a was ofparticular interest in this study. In silico analysis revealedthat miR-29a targets the 3’UTR of SREBP-1c mRNA. Moreover,a recent study supported that inhibition of miR-29a decreasedthe SREBP-1 levels in the livers of chow diet-fed C57BL/6 Jfemale mice.35

SREBP-1c is an important pro-lipogenic transcriptionfactor, which is induced by the NS2 protein of HCV.42

Activation of SREBP-1c by the liver X receptor/retinoid Xreceptor (LXR/RXR) leads to activation of its target lipogenicgenes and thus increases the amount of LDs.43 Since SREBP-1cis the predominant hepatic isoform among the SREBP familyand CAV1 is regulated by SREBP-1 and is one of the LD proteinsresponsible for its stability, they were chosen to be the mainfocus of this study.11,44 In this context, this study aimed at

uncovering the impact of miR-29a on SREBP-1c and its down-stream target, CAV1, in order to better elucidate their roles inLD formation.

In order to simulate fatty liver conditions, OA treatmentwas performed for LD induction in JFH-I-infected Huh-7cells. miR-29a, SREBP-1c and CAV1 expression were thenscreened. Both miR-29a and SREBP-1c showed a significantup-regulation, whereas no significant change in the expres-sion of CAV1 was observed after OA treatment (Fig. 1A, 1B,1C). The LD and TG content also significantly increased fol-lowing LD induction (Fig. 1D, 1E), indicating that induction ofLDs in cells could alter the level of miRNAs and hence theirimpact on their downstream target genes. Thus, the nextexperiments were all carried out in OA-treated JFH-I-infectedHuh-7 cells.

miR-29a expression was manipulated using oligos andits impact on SREBP-1c, CAV1, LDs and TGs was analyzed.SREBP-1c showed a significant increase at the transcriptionallevel following transfection of miR-29a mimics (Fig. 2B).This finding is in line with those reported by Kurtz et al.35

who recently measured mRNA expression of 883 genes infatty liver mouse models that were injected with lockednucleic acids (LNAs) against miR-29a. Among the lipogenicgenes measured was the SREBF-1 gene which was found tobe down-regulated.35 miR-29a mimics also induced CAV1expression, and thus it was important to investigate whetherthis induction was mediated by SREBP-1c, especially sinceSREBP-1 is known to bind to SREs in the CAV1 gene promoter.Binding of SREBP-1c to the SRE subsequently regulates CAV1expression, either positively or negatively, depending on thecell type as well as the upstream signal.20,21 Thus, it wasimportant to investigate this relationship in OA-treated JFH-1-infected Huh-7 cells. SREBP-1c was knocked down usingsiRNAs, which resulted in a significant increase in CAV1mRNA level (Fig. 3C); this result suggests that miR-29amight have mediated its effect on CAV1 indirectly and notthrough SREBP1c. Moreover, forcing miR-29a expression inOA-treated JFH-I-infected cells led to a marked increase inintracellular LD as well as TG content (Fig. 2D, 2E). In accord-ance with our results, it has been recently demonstrated thatmiR-29a LNAs reduced fatty acid production by 70% in Huh-7cells.35 However, it was shown in another study that miR-29amight protect the liver from development of steatosis.45 More-over, the study of Whittaker and colleagues indicated thatmiR-29a decreases LD content in naïve Huh-7 cells, yet this

Fig. 3. Impact of SREBP-1c siRNA on lipid droplet (LD) formation and CAV1 expression. A) SREBP siRNA were efficiently transfected into oleic acid-treated JFH-1-infected (p < 0.0001***, n = 9). B) A significant decrease in the intracellular level of LDs, as shown by decrease in the amount of the red-colored LDs due to the oil red Ostaining (n= 3) compared to mock untransfected controls. C) SREBP siRNA transfection led to significant up-regulation of CAV1 (p= 0.0030**, n = 9). Results are expressedas mean ± SEM. Student’s t-test was used for the statistical analysis.

Fig. 4. Impact of manipulating miR-29a on viral load. Mimicking of miR-29ain JFH-I-infected Huh-7 cells resulted in 53% reduction (153380 ± 8850,p = 0.0499*, n = 4) in viral titers compared to untransfected Huh-7 cells (289005± 30205). Results are expressed as mean ± SEM. Student’s t-test was used for thestatistical analysis.



contradiction with our obtained results might be due to the HCVinfection.46

It was tempting then to investigate whether the impactof miR-29a on LDs was mediated by SREBP-1 or not. Knock-down of SREBP-1c using specific siRNAs resulted in a markeddecrease in LD content, which might confirm that thisdecrease in the LDs was mediated via SREBP-1c (Fig. 3B).

miR-29a inhibitors resulted in a slight decrease in LDformation, even though they had no impact on SREBP1 andCAV1 mRNA levels (Fig. 2D). Similar to our observations, arecent study showed that miR-182 inhibitors can lead to areduction in the viral load without affecting its respectivetarget gene.47 This finding might reflect the fact that the anta-gomirs were not able to completely repress the miRNA levels,and thus the levels of SREBP-1c and CAV1 were not com-pletely suppressed; however, this minor repression of themiRNA levels was sufficient to repress LDs as well as theirrespective TGs.

Finally, it was interesting to study the sole effect of miR-29aon viral replication. Transfection of miR-29a mimics lead to asignificant decrease in viral load by 53% (Fig. 4). This findingis in line with the study of Bandyopadhyay and colleagues,38

who also showed a decrease in viral load of HCV-infected cellsafter mimicking with miR-29a.

Our study shows a controversial role of miR-29a in HCV-induced steatosis, where it was shown to inhibit HCV viralreplication, while promoting LD formation. This dual functionof miRNAs has been previously observed in several cases,such as the liver-specific miR-122, where it resulted in anincrease in HCV viral replication, while it was shown to act as atumor suppressor miRNA in hepatocellular carcinoma, repres-sing liver cancer development and progression.48,49 Nonethe-less, the contradicting role of miR-182 was also recentlyreported by our group; specifically, it was shown to promoteHCV viral replication, while resulting in a significant augmen-tation of primary natural killer cell cytotoxicity.47

Conclusions

In conclusion, although miR-29a has an anti-HCV action,forcing its expression paradoxically induced LD formation, aswell as of its respective TGs, through inducing the expressionof the transcription factor SREBP-1c. Thus, miR-29a can beconsidered to have a potential role in the pathogenesis ofsteatosis as well as the course of HCV infection.

Acknowledgments

We would like to acknowledge Prof. Takaji Wakita (NationalInstitute of Infectious Diseases, Tokyo, Japan) for kindlyproviding us with the pJFH-I construct. We would also like toacknowledge the German University in Cairo for providing uswith funds.


None


Performed experiments (MMM, RAS, RSH), analyzed andinterpreted data (MMM, NME, RMH, AIA), performed statis-tical analysis (MMM), wrote all drafts of the manuscript

(MMM), revised the manuscript (MMM, NME, HMEA, AIA),designed the experiments (NME, RMH, AIA), provided mate-rials (HMEA). All authors read, discussed and approved themanuscript.

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Original Article

miR-34a: Multiple Opposing Targets and One Destinyin Hepatocellular Carcinoma

Radwa Alaa Yacoub1, Injie Omar Fawzy2, Reem Amr Assal2, Karim Adel Hosny3,Abdel-Rahman Nabawy Zekri4, Gamal Esmat5, Hend Mohamed El Tayebi2

and Ahmed Ihab Abdelaziz*2,6

1Pharmaceutical Biology Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt;2Pharmacology and Toxicology Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt;3Department of General Surgery, Faculty of Medicine, Cairo University, Cairo, Egypt; 4Virology and Immunology, Cancer Biology

Department, National Cancer Institute, Cairo University, Cairo, Egypt; 5Department of Endemic Medicine and Hepatology,Cairo University, Cairo, Egypt; 6School of Medicine, New Giza University, Cairo, Egypt

Abstract

Background and Aims: The role of miR-34a in hepatocellularcarcinoma (HCC) is controversial and several unresolvedissues remain, including its expression pattern and relevanceto tumor etiology, tumor stage and prognosis, and finally, itsimpact on apoptosis. Methods: miR-34a expression wasassessed in hepatitis C virus (HCV)-induced non-metastaticHCC tissues by RT-Q-PCR. Huh-7 cells were transfected withmiR-34a mimics and the impact of miR-34a was examined on84 pro-apoptotic/anti-apoptotic genes using PCR array; its neteffect was tested on cell viability via MTTassay. Results: miR-34a expression was up-regulated in HCC tissues. Moreover,miR-34a induced a large set of pro-apoptotic/anti-apoptoticgenes, with a net result of triggering apoptosis and repressingcell viability. Conclusions: HCC-related differential expressionof miR-34a could be etiology-based or stage-specific, and lowexpression ofmiR-34amay predict poor prognosis. This study’sfindings also emphasize the role of miR-34a in apoptosis.© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

microRNA (miR)-34a has emerged as an important miR incomprehensive research studies, and numerous studies haveproposed its considerable impact on cell proliferation andsurvival.1 miR-34a has several experimentally validateddownstream target genes involved in cell cycle regulation,including NMYC,2 CCND1,3 CCNE2, CDK4, CDK6,4 andC-MET.4,5 These genes promote cells to proceed through G1phase into S phase via the Rb-E2F signaling pathway, and

ectopic expression of miR-34a was shown to induce cell cyclearrest in the G1 phase.6 Moreover, it was reported that over-expression of miR-34a could down-regulate CDC25C andphosphorylated CDC2 expression, which in turn inhibits cellproliferation via cell cycle G2 phase arrest.7,8 miR-34a wasreported to directly target YY1, a negative regulator of thetumor suppressor p53,9 and induce apoptosis in neuroblas-toma cell lines.10 Furthermore, miR-34a ectopic expression inbreast cancer cells suppressed cell proliferation and invasion,and induced apoptosis, and miR-34a expression was foundto be inversely correlated to BCL-2 and SIRT1 expression.11

In hepatocellular carcinoma (HCC), two independentstudies reported that miR-34a had no impact on apoptosis.5,12

On the contrary, another research group showed that miR-34aincreased caspase-3 and -7 activity and induced apoptosis.13

Thus, the role of miR-34a in apoptosis in HCC is ambiguous.Another controversial aspect of miR-34a regards its pattern ofexpression and its relevance to the tumor etiology and stage.miR-34a was found to exhibit a different pattern of expressionin hepatitis C virus (HCV)-related HCC14 than in hepatitis Bvirus (HBV)-induced HCC.5,15 In addition, the expressionlevel of miR-34a was found to vary between HCC tissues inclinical TNM stages III and IV as compared to that in stages Iand II.13 It was also reported that metastatic HCC tissuesshowed a significantly different miR-34a expression profilecompared to non-metastatic tissues.5,13 Despite these con-troversies, miR-34a has entered phase I clinical trials forliver cancer.16

Thus, in this study, we first aimed to elucidate thediscrepancies regarding miR-34a expression pattern via inves-tigating miR-34a expression in a cohort of patients with HCV-progressed non-metastatic HCC. Then, we aimed at dissectingthe potentially related apoptotic pathways through investigat-ing the impact of miR-34a on several downstream apoptoticand anti-apoptotic genes in HCC. Finally, we assessed the netimpact of miR-34a on cell viability.

Methods

Study patients

This study comprised 22 HCC patients who underwent livertransplant surgery at the Kasr El Ainy Hospital, Cairo



Keywords: MicroRNA; Hepatocellular carcinoma; Apoptosis; PCR array.Abbreviations: miRNA or miR, microRNA; HCC, hepatocellular carcinoma; HCV,hepatitis C virus; HBV, hepatitis B virus.Received: 24 August 2016; Revised: 29 November 2016; Accepted: 22 December2016qDOI: 10.14218/JCTH.2016.00031.*Correspondence to: Ahmed Ihab Abdelaziz, School of Medicine, Health SciencesResearch Center (HSRC), New Giza University (NGU), Office: 2.26, New Giza km 22Cairo-Alex desert road, Cairo 11835, Egypt. Tel: +20-2-38277847, E-mail: [email protected]

University, Egypt. In addition, 19 healthy liver biopsies wereobtained. The healthy liver tissues were obtained from liverdonors who volunteered for lobar transplantation surgery forHCC patients. Healthy livers represented non-cirrhotic tissues,and all donors were negative for HCV and HBV infection.Written consent, from both the patient and the donor, to takeliver tissues during the transplantation was obtained priorto the surgery. Patients were subjected to clinical assessment(results presented in Table 1). HCC was diagnosed in allpatients by examining the stage and grade of the tumor.Liver function enzymes (aspartate aminotransferase, alanineaminotransferase, and alkaline phosphatase) and albuminlevels in serum were measured. Also the presence of hepato-tropic infections, such as presence of HCV or HBV, waschecked. All patients were confirmed as non-metastatic withno extra-hepatic manifestations and no vascular invasion,in order to comply with the expanded Milan criteria (UCSFcriteria) for liver transplantation (i.e. single tumor of <6.5 cmin diameter, or 2 or 3 tumors, each of #4.5 cm in diameter orwith total tumor diameter #8 cm) as shown in Table 2. Themajority of the patients (63.6%) had more than one focallesion, as indicated in the pathology report. All experimentswere performed in compliance with the guidelines of the Insti-tutional Review Board of Kasr El Ainy Medical School in CairoUniversity and the ethical committee of the German Universityin Cairo and in accordance to the ethical standards of theDeclaration of Helsinki.

Cell culture

Huh-7 cells were maintained in Dulbecco’s modified Eagle’smedium (DMEM) (Lonza, Switzerland) supplemented with4.5 g/L glucose, 4 mmol/L L-glutamine, 10% fetal bovineserum, 1% penicillin/streptomycin and Mycozap (1:500;Lonza) at 378C in 5% CO2 atmosphere.

Transfection of miRNA oligonucleotides

Huh-7 cell lines were transfected with miR-34a mimics(Catalog #MSY0000255; Qiagen, Germany). Transfectionexperiments were carried out in triplicate, using the HiPerfectTransfection Reagent (Qiagen) according to the manufac-turer’s protocol which called for 37.5 ng oligonucleotides forHuh-7 cells transfected in a 24-well plate; the experiment wasrepeated 3 times. Cells that were only exposed to transfectionreagent are designated as Mock cells, while cells transfectedwith miR-34a mimics are designated as miR-34a cells.

mRNA and miRNA extraction

Total cellular mRNA and miRNA were extracted from the liverbiopsies. Fresh liver samples (HCC and healthy tissues) werecollected during surgery and snap-frozen immediately inliquid nitrogen. The specimens were thenmanually pulverizedin liquid nitrogen and approximately 100 mg of tissue powderwas used for large and small RNA extraction with the mirVanamiRNA Isolation Kit (Ambion, USA), according to the manu-facturer’s protocol.

For extraction of total cellular mRNA from cultured cells,Huh-7 cells were harvested 48 hours post-transfection andlysed using Bizol Reagent (Invitrogen). Chloroform was addedand the aqueous phase containing the RNA was collected.RNA was precipitated using isopropyl alcohol. The RNA pellet

Table 1. Clinical characteristics of the 22 hepatocellular carcinoma (HCC)patients

Age: mean 49 6 13.5

range 35.5–62.5

Sex: male/female 21/1

Aspartateaminotransferase (U/L)

100.5 6 65.8

Alanine aminotransferase(U/L)

85.6 6 95.6

Alkaline Phosphatase (U/L) 110.2 6 60.7

Serum albumin (g/dL) 4.6 6 1.5

Serum alpha-fetoprotein(ng/mL)

155.7 6 22.3

HCV statusa

100% (22 HCC patients)

HBV statusb

18.2% (4/22 HCCpatients)

aHCV status was determined using anti-HCV antibody and/or HCV viral RNA

quantification.bHBV status was determined using anti-HBc and anti-HBs antibodies and by

detection of HBsAg.

Table 2. Number/sizes of focal lesions according to expanded Milancriteria

PatientsNumber offocal lesions Size of focal lesions

Patient 1 Unifocal 2.5 cm

Patient 2 3 focal lesions 2 cm, 2.5 cm and 3 cm

Patient 3 3 focal lesions 2 cm, 2 cm and 3.5 cm

Patient 4 Unifocal 1.5 3 2 cm

Patient 5 3 focal lesions 3 3 4 cm, 1 cm and 1 cm

Patient 6 Unifocal 4 cm

Patient 7 3 focal lesions 4 cm, 1 cm and 1 cm

Patient 8 3 focal lesions 1 cm, 1 cm and 1.5 cm


Patient 10 2 focal lesions 1 cm and 1.7 cm

Patient 11 3 focal lesions 1 cm each



Patient 14 3 focal lesions 1 cm, 1 cm and 4 cm

Patient 15 2 focal lesions 3 cm and 1.5 cm

Patient 16 2 focal lesions 1.5 cm and 3 cm

Patient 17 3 focal lesions 2.5 cm, 2.5 cm and 1.5 cm

Patient 18 3 focal lesions 1.5 cm, 1 cm and 1 cm






Yacoub R. et al: miR-34a in HCC

was then washed using 75% ethanol and dissolved in DPEC-treated water.

RNA concentration and integrity

RNA concentration was measured using a NanoDrop spectro-photometer (Thermo Fisher Scientific, USA) and was found tobe >500 ng/mL. In addition, RNA integrity was confirmed by18S rRNA band detection on 1% agarose gel electrophoresis.RNA samples with optical density (260/280) of >2 wereexcluded from the study.

miRNA and mRNA quantification by TaqMan Real-TimeQ-PCR

The miRNA extracted from liver tissues was reverse tran-scribed into single-stranded cDNA using the TaqMan® Micro-RNA Reverse Transcription Kit (ABI, USA) with specific primersfor hsa-miR-34a and RNU6B. Relative expression of miR-34aand RNU6B (internal control for normalization) was quantifiedusing TaqMan Real-Time Q-PCR (ABI Assay IDs: 000426 and001093, respectively) with the StepOne™ Real-Time PCRSystem (ABI). Relative expression was calculated using the2−DDCT method. All PCR reactions, including controls, wererun in duplicate.

PCR array

Total RNA extracted from Huh-7 cells was reverse transcribedinto cDNA using the RT2 First-Strand Kit (SA Biosciences,Germany). The cDNA was then combined with RT2 SYBRGreen qPCR Master Mix (SA Biosciences) and equal aliquotsof this mixture were added to 96-well plates of the HumanApoptosis RT2 Profiler PCR Array (SA Biosciences) that con-tained the gene-specific primers, human ATGs PAHS012z(SA Biosciences). PCR was performed using the 7500 FastReal-Time PCR System (ABI) with the following thermalcycling conditions: 15 min at 958C for 1 cycle, 10s at 958C,and 1 min at 608C for 40 cycles. Expression of 84 key genesinvolved in apoptosis and 5 housekeeping genes (B2M, ACTB,GAPDH, HPRT1 and RPLPO) was profiled. Threshold cycledata obtained was analyzed using the RT2 Profiler Software(version 3.5; SA Biosciences). Relative gene expression wascalculated using the 2−DDCT method.

Cell viability assay (MTT)

For the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide) assay, 160,000 Huh-7 cells were seeded in500 mL of media per well in a 24-well plate and incubated for24 hours prior to transfection with 37.5 ng oligonucleotides(according to the HiPerfect protocol). Forty-eight hours post-transfection, 1 mL of MTT solution (5 mg/mL MTT in 10%PBS) was added to each well. After incubation for 5 hours,formazan (MTT metabolic product) was resuspended in 1 mLof lysis solution (DMSO: absolute ethanol). Then, 200 mL fromeach well was transferred to a 96-well plate and colorimetricmeasurements and absorbances (572 nm) were measuredusing the Wallac 1420 Victor2 Multilabel Counter (PerkinElmer, USA). To eliminate any intensifying absorption thatthe culture medium itself could cause, culture mediumwithoutcells was assayed with the MTT solution and the subsequentreading was subtracted from all other values. The experimentwas performed in triplicate and repeated three times.

Statistical analysis

All data are presented as mean with standard error of themean (SEM). GraphPad Prism 5.0 was used to prepare allfigures. Statistical significance of the data was analyzed usingunpaired Student’s t-test, and determined at a p value of<0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).

Results

miR-34a screening in HCV-induced non-metastaticHCC liver tissues

The pattern of expression of miR-34a was investigated inHCC tissues taken from 22 patients with HCV-induced non-metastatic HCC and in 19 liver tissues obtained from healthydonors undergoing liver transplantation. The relative expres-sion of miR-34a was significantly higher in HCC tissuescompared to healthy tissues (p = 0.02*) (Fig. 1).

Effect of miR-34a on a set of pro- and anti-apoptoticgenes involved in apoptosis

In order to investigate the impact of miR-34a on apoptosis,Huh-7 cells were transfected with miR-34a mimics, and effectof miR-34a ectopic overexpression was observed on theexpression of 84 genes with critical roles in apoptosis. PCRarray revealed that forcing the expression of miR-34a in Huh-7cells resulted in altered gene expression of a set of pro-apoptotic/anti-apoptotic genes. Specifically, it up-regulatedthe expression of 26 pro-apoptotic genes (Fig. 2a) and 10anti-apoptotic genes (Fig. 2c); in addition, it repressed theexpression of 17 pro-apoptotic genes (Fig. 2b) as well as8 anti-apoptotic genes (Fig. 2d), while the remaining geneswere unaffected.

Fig. 1. Expression pattern of miR-34a in HCV-induced non-metastaticHCC tissues. miR-34a expression pattern was investigated using RT-Q-PCR inliver tissues obtained from 22 patients with HCV-induced non-metastatic HCC and19 healthy donors. miR-34a showed significantly increased expression in HCCtissues, compared to healthy liver tissues. Expression of miR-34a was normalized,in each sample, to the housekeeping gene RNU6B. Results were analyzed usingunpaired Student’s t-test where p < 0.05*, p < 0.01**, p < 0.001***.



Fig. 2. Impact of miR-34a on the expression of pro-apoptotic and anti-apoptotic genes. Huh-7 cells were transfected with miR-34a mimics, and the expression ofseveral apoptosis-relevant genes was evaluated using PCR array in transfected cells as compared to Mock (untransfected) cells. (a) Ectopic overexpression of miR-34a up-regulated the expression of 26 pro-apoptotic genes (b) and down-regulated the expression of 17 pro-apoptotic genes. (c) Expression of 10 anti-apoptotic genes was induced,(d) while expression of 8 anti-apoptotic genes was repressed. Experiments were performed in triplicate and repeated 3 times. Expression of each gene was normalizedto 5 housekeeping genes (B2M, ACTB, GAPDH, HPRT1 and RPLPO). Each bar represents the mean ± SEM, and results were analyzed using unpaired Student’s t-test wherep < 0.05*, p < 0.01**, p < 0.001***.



Impact of miR-34a on cell viability

MTT assay was used for investigating cell viability. Forcedexpression of miR-34a in the Huh-7 cell line showed markedrepression of cellular viability (p = 0.0006***) as comparedto the Mock cells (Fig. 3).

Discussion

miR-34a is a crucial miRNA that has been investigated incancers and also subject to clinical trials. miR-34a has beeninvestigated in HCC, yielding controversial results and raisingseveral questions that encompass diverse aspects: first, theexpression pattern of miR-34a and its relevance to the tumoretiology; second, the relevance of its expression pattern tothe tumor stage and prognosis; and finally, its impact onapoptosis.

In order to answer the first question that has been raisedregarding the pattern of miR-34a expression and its associ-ation with the tumor etiology, we focused on HCV-inducedHCC. In this study, miR-34a expression was shown to be up-regulated in HCV-induced HCC tissues (Fig. 1). This findingcoincides with previous studies showing that miR-34a wasup-regulated in HCV-related HCC, compared to normal livertissues.14,17 On the contrary, miR-34a expression was foundto be down-regulated in HBV-positive HCC tissues.5,13,15,18 Inlight of these findings, it may be presumed that the miR-34aexpression pattern might depend on the etiology of HCC.

Another imperative question was the correlation betweenmiR-34a expression pattern and the prognosis of HCC. Inthis study, results showed that miR-34a expression wasup-regulated in HCV-induced non-metastatic HCC tissues(Fig. 1). In a previous study, miR-34a was found to be over-expressed in non-metastatic tissues, compared to metastaticones, and in clinical TNM stages I and II, compared to stagesIII and IV.5,13 Taken together, these findings suggest thatthe miR-34a expression pattern might be stage-specific andmiR-34a low expression may predict poor prognosis.

In an attempt to unveil the role of miR-34a in apoptosis,providing an answer for the third inquiry, the expression ofmiR-34a was forced in Huh-7 cells, and by using PCR array theexpression of a set of 84 pro-apoptotic and anti-apoptoticgenes was investigated. Interestingly, the expression of 26pro-apoptotic genes was found to be up-regulated, whileanother 17 pro-apoptotic genes were found to be repressed(Fig. 2a and 2b). Regarding the anti-apoptotic genes, miR-34a increased the expression level of 10 genes, although itdown-regulated the expression of 8 genes (Fig. 2c and 2d).miR-34a did not show a significant impact on the expressionof the 23 remaining genes. Our results showed that miR-34ainduced the expression of key players involved in the activa-tion of the intrinsic apoptotic pathway, such as BAK (Fig. 2a).BAK triggers mitochondrial outer membrane permeabiliza-tion, which leads to the release of cytochrome C into the cyto-plasm. Then, APAF1 and cytochrome C form the apoptosome,which activates caspase-9 ultimately resulting in apoptoticcell death.19 In our study, miR-34a was found to not onlyincrease BAK expression but also to induce the expressionof the downstream mediators of intrinsic apoptosis cyto-chrome C, APAF1 and caspase-9 (Fig. 2a).

PCR array revealed that miR-34a may have also inducedextrinsic apoptosis via increasing the levels of TNF signalingpathwaymediators (Fig. 2a). TNF-alpha leads to apoptosis viabinding to the TNF receptor (TNFR), which in turn activatesthe Fas-associated protein with death domain (FADD), whichthen binds to caspase-8 to form the apoptotic death-inducingsignaling complex (DISC).20,21 The findings of the currentstudy indicate that the levels of TNF, TNFR, and caspase-8are elevated by miR-34a ectopic expression (Fig. 2a).

Induction of both intrinsic and extrinsic apoptotic path-ways leads to activation of caspases-3, -6 and -7.22 Theseexecutioner caspases mediate the cleavage of several pro-teins critical to cell survival, including actin, lamin, poly(ADP-ribose) polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Cleavage of these proteins leadsto molecular and morphological changes characteristic toapoptosis, including cell shrinking and membrane blebbing,disruption of nuclear membrane integrity, DNA fragmentationand impaired DNA repair.23 Executioner caspases-3, -6 and -7were all found to be induced by miR-34a in the present study(Fig. 2a). This finding corroborates a previous study in whichmiR-34a ectopic overexpression in Hep3B cells and SNU449cells was shown to promote apoptosis and induce caspase-3and -7 activity.13 Nonetheless, these findings challenge pre-vious reports which did not observe any impact on apoptosisupon overexpressing miR-34a in HepG2 cells.5,12

Finally, this study aimed to evaluate the net impact of miR-34a on cell viability using MTT assay. Results showed thatforcing the expression of miR-34a in Huh-7 cells reduced thecell viability significantly (Fig. 3). This finding is in line withother studies reporting that after forcing miR-34a expressionin HCC cells, a decrease in cell viability is observed.13,15 In thecurrent study, the decrease in cell viability could be explainedby the induction of apoptosis, as revealed by the results of thePCR array. In addition, it is possible that the effect of miR-34aon cell viability could also occur via targeting of cell cyclegenes, since previous studies have reported that miR-34ahas validated targets involved in cell cycle regulation, includ-ing NMYC,2 CCND1,3 CCNE2, CDK4 and CDK6.4

Fig. 3. Impact of miR-34a on cell viability (MTT assay). miR-34a mimicsremarkably inhibited cellular growth and viability in Huh-7 cells. Experiments wereperformed in triplicate and repeated 3 times. Absorbance was measured at572 nm. Each bar represents the mean ± SEM, and results were analyzed usingunpaired Student’s t-test where p < 0.05*, p < 0.01**, p < 0.001***.



Conclusions

This study provides possible explanations for the controversialfindings regarding the expression pattern of miR-34a and itsrole in apoptosis in HCC. Together with findings from previousreports, the current data suggests that miR-34a expressionpattern could be etiology-based and stage-specific, and lowexpression of miR-34a may predict poor prognosis. In thisstudy, it was shown that miR-34a activates dozens of pro-apoptotic and anti-apoptotic genes, with a net effect of inducingapoptosis and repressing cell viability and, hence, emphasizingthe role of miR-34a in apoptosis.


None


Contributed in study concept and design, execution of experi-ments, acquisition of data, statistical analysis and interpre-tation of data, and writing of the manuscript (RY), assistedin experiments, interpretation of data, and revision of themanuscript (IF), assisted in experiments and interpretation ofdata (RA), provided tissue samples and clinical data (KH, GE),performed PCR array experiment and contributed in criticalrevision of themanuscript (ARZ), contributed reagents (HET),contributed in study concept and design and study super-vision and provided critical revision of the manuscript forimportant intellectual content (AIA).

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Original Article

Comparison of Vitamin D Levels in Naive, Treated, andInactive Carriers with Chronic Hepatitis B Virus

Shahnaz Sali, Soheil Tavakolpour* and Baharan Farkhondemehr

Infectious Diseases and Tropical Medicine Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Abstract

Background and Aims: During recent years, the relation-ship between vitamin D levels and chronic hepatitis B (CHB)infection has attracted many researchers’ attention. However,the results relating to the association of vitamin D levelsand HBV infection have been conflicting and there remains alack of knowledge about the effects of antiviral treatments onvitamin D level. Methods: Eighty-four patients with CHBwere assessed and divided into three groups: inactive carriers(n = 28), treated (n = 34), and new (treatment-naïve) cases(n = 22). Thirty-two healthy controls (HCs) were included toenable comparison with the CHB groups. The levels of vitaminD3 were measured and statistically compared among thevarious groups. Results: Male subjects had higher levels ofvitamin D3 (41.25 vs 28.85, p < 0.01). No association wasfound among any of the groups when compared with the HCgroup. Despite the significant association, the HCs demon-strated a higher level of vitamin D3, which was lower in thetreated group, the inactive carrier group, and the new casesgroup (new case [29.82] < inactive carrier [32.91] < treated[39.56] < control [44.88]). The HBV DNA levels were notassociated with vitamin D3 levels in the inactive carriers(p = 0.171), the treated groups (p = 0.192), and the newcases (p = 0.369). Moreover, the alanine transaminase andaspartate transaminase levels were not associated withvitamin D3 levels for any of the HBV-infected groups.Conclusions: Vitamin D3 contributes to the clinical statuesof CHB patients. There is also a possible correlation betweenclinically healthy CHB patients and vitamin D3 level.© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

Recently, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the activeform of vitamin D, has been recognized as a new player inmodulation of the immune system. Despite its critical role inthe immune system, however, impaired vitamin D3 statushas been reported amongst various populations.1 As animmunomodulatory factor, vitamin D3 affects various typesof cells involved in a plethora of immune responses, includ-ing monocytes, macrophages, dendritic cells and both theT and B lymphocytes.2 Indeed, a critical role has beendescribed for vitamin D3 in different autoimmune diseases,such as multiple sclerosis, rheumatoid arthritis, psoriasisand autoimmune blistering diseases.3–7 However, there is alack of in-depth research on the roles of vitamin D3 in viralhepatitis. During recent years, some studies have focusedon the effect of vitamin D3 in hepatitis C virus (HCV) infec-tion, as well as in infection with hepatitis B virus (HBV). Sincethe vitamin D receptor (VDR) is necessary for the effects ofvitamin D3, it is expected that polymorphisms of the VDRgenetic sequences may also contribute to the pathogenesisof several diseases.8 Since VDR is expressed on both Tand Bcells,9 it is especially likely that the function of these cellscould be influenced by vitamin D3 level.

Multiple studies have shown that vitamin D3 level is sig-nificantly correlated with the suppressor function of T cells.10

Several in vitro and in vivo studies of murinemodels, as well aspatient-based clinical studies, have indicated involvement ofthe vitamin D signaling pathway (both vitamin D3 level andVDR status) related to the regulatory T cells (Tregs) and theirfunction. It has been concluded that vitamin D3 acts on antigenpresenting cells (APCs) and T cells to induce peripheral toler-ance via promotion of Tregs.11 Prietl et al.12 demonstrated thatvitamin D3 supplementation is associated with a significantincrease in the Tregs population. Thus, vitamin D could inhibitT helper (Th)1 cells and Th17-associated cytokines, while pro-moting Th2 cells and also expanding the Tregs.13–16

Although, in HBV infection immune responses are not themain player; nonetheless, these could lead to developmentof other diseases, to seroclearance or chronicity. Additionally,it seems that immune cell differentiation and activation arestrongly influenced by the disease course. It was recentlyhypothesized that both Tregs and regulatory B cells (Bregs)may function as a barrier in HBV seroclearance.17 However,the presence of Tregs are essential to prevent HBV flare.18 Allthese reported data are in line with the purported crucial roleof Tregs in HBV infection.

Investigation of the beneficial role of vitamin D in HBVinfectionwas first reported by Luong and Nguyen,19 and severalother studies followed thereafter. Some of the subsequent



Keywords: Chronic Hepatitis B; Vitamin D; Hepatitis B virus.Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; VDR, vitamin Dreceptor; Tregs, regulatory T cells; APCs, antigen presenting cells; Th, T helper;Bregs, regulatory B cells; CHB, chronic hepatitis B; LFTs, liver function tests; HCs,healthy controls; ALT, alanine transaminase; AST, aspartate transaminase; PLT,platelets; BMI, body mass index; HBeAg, hepatitis B e antigen; HBeAb, hepatitis Be antibody; IFN-g, interferon gamma; TNF-a, tumor necrosis factor-alpha.Received: 05 September 2016; Revised: 04 December 2016; Accepted: 16December 2016qDOI: 10.14218/JCTH.2016.00037.*Correspondence to: Soheil Tavakolpour, Infectious Diseases and Tropical Medi-cine Research Center, Shahid Beheshti University of Medical Sciences, Tehran19395, Iran. Tel: +98-9125916349, Fax: +98-2122267157, E-mail: [email protected]

studies have showed a lower level of vitamin D3 in patients withchronic HBV (CHB), and these have mainly been presentedduring the most recent years. Indeed, some authors have sug-gested that the decreased level of this vitamin may be thecause of abnormal findings on liver function tests (LFTs) and/or the characteristic elevation in viral load observed in patientswith CHB. However, other authors have reported not finding anycorrelation between viral load and vitamin D3 level in HBV-infected patients.20

Due to the existence of conflicting results from the differ-ent studies, we sought to perform a study in which wemeasured and analyzed the level of vitamin D3 in patientswith three different clinical statuses of HBV infection and tocompare these data with the same from selected healthycontrols (HCs). Additionally, we made efforts to include somecritical parameters, including levels of alanine transaminase(ALT), aspartate transaminase (AST), platelet count (PLT) andHBV DNA. Recently, Mohamadkhani et al.21 showed thatthere is a negative correlation between vitamin D3 levelsand viral load in Iranian patients, with an acceptable popula-tion (n = 173). Distinctively, however, we attempted to eval-uate the association of plasma vitamin D3 and the differentCHB phases amongst an Iranian population in the presentstudy.

Methods

Eighty-four patients with CHB who presented to the ShahidLabafinejad Hospital in Tehran, Iran from May 2015 toFebruary 2016 were included in the study. The patients weredivided among three groups: inactive carriers (n= 28), treated(n= 34), and new (treatment-naïve) cases (n= 22). The exactdefinitions of these groups are shown in Table 1. Additionally,32 HCs were included to compare their vitamin D3 levelsand some other factors with the three HBV-infected patientgroups. The inclusion criteria used for selection of HCs arepresented in Table 1. Since the available first-line treatmentfor HBV patients in Iran was changed to tenofovir duringthe past years, individuals in the treated group only receivedtenofovir for at least 1 year, at a dose of 300 mg daily.Patients with any other treatments were not included in thetreated group.

Because of the influence of 25-hydroxyvitamin D3 fromsunlight during different seasons, all of the samples used in

this study were collected during autumn and winter. Inaddition to the vitamin D3 levels, ALT, AST, PLT, HBV DNAand bodymass index (BMI) were recorded for every individual(Table 2). In this study, HBV DNA was not necessarily meas-ured at the time of sample analysis. In fact, the results ofmeasured HBV DNAwithin the past 3months were consideredas trustworthy to include in the study.

During the patient selection for study, any patient withany other liver diseases that were not associated with HBV,such as bone disorders, autoimmune diseases, cancer, anyco-infections (human immunodeficiency virus, HCV), and anypatient taking vitamin D3 supplements during the past3 months were excluded to minimize the effects of otherparameters. In addition, the number of male patients enrolledin the study was considerably higher than that of femalepatients (63 vs 21), which was due to the general higher pro-portion of infected males.

Table 1. Definition of the different groups included in this study

Group Defining characteristics

Inactivecarrier

Negative HBeAg and positive HBeAb,undetectable or low levels of HBV DNA(<2000 IU/mL) by PCR-based assays,repeatedly normal ALT levels, and nofibrosis.

Treated Receipt of anti-viral therapy (tenofovir) for atleast 1 year, at the dose of 300 mg daily,within at least 6 months before analysis ofthe vitamin D3 levels, and regardless ofHBeAg or HBeAb status.

New case Newly diagnosed patients based onserological tests, HBV DNA >2000 IU/mL, noanti-viral therapy had been initiated at thetime of vitamin D3 measurement.

Control Healthy individuals without any viralhepatitis and liver disease history, noautoimmune diseases or cancer history, novitamin D3 supplementation or taking ofmulti-vitamins during the past 3 months.

Table 2. Comparison of clinical and biochemical features of HBV patients and healthy controls

Inactive carrier(n = 28)

New case(n = 22)

Treated(n = 34)

Control(n = 32) p

Sex F(%) 10(36) 2(10) 10(29) 14(44) 0.047

M(%) 18(64) 20(90) 24(71) 18(56)

Age, years 47.9 6 15.41 43.85 6 13.73 44.25 6 11.11 44.93 6 15.23 0.713

ALT, IU/L 26.00 6 10.03 58.95 6 22.01 30.11 6 11.89 26.87 6 12.61 0

AST, IU/L 25.21 6 6.23 49.36 6 15.74 29.11 6 13.18 25.67 6 12.92 0

PLT, 105 2.92 6 0.84 3.05 6 1.31 3.08 6 1.05 3.34 6 0.71 0.091

Vitamin D3, ng/mL F 31.91 6 35.79 10.00 6 7.07 22.88 6 25.71 33.14 6 31.08 0.127

M 33.44 6 31.60 31.80 6 34.47 45.27 6 33.40 53.94 6 34.79

BMI 27.57 6 2.99 26.46 6 3.96 27.32 6 4.43 26.68 6 4.87 0.796

Viral load, log IU/mL 3.69 6 3.46 6.45 6 6.99 3.70 6 4.30 N/A 0


Sali S. et al: Vitamin D3 level in HBV patients

Laboratory tests

Collected samples were analyzed for ALT, AST, PLTand vitaminD3 levels. ALT and AST were measured using IFCC (G.T kit;Pars Azmoon Company). PLT was measured by counting.Vitamin D3 level measurement was performed using chem-iluminescence (Roche Kit; Elecsis Company). The measuredvitamin D3 levels were categorized as deficient, insufficientor sufficient according to findings of less than 10 ng/mL,between 10 ng/mL and 20 ng/mL, and more than 20 ng/mL,respectively.

Statistically analysis

SPSS version 16 (IBM Corporation, USA) was used for allstatistical analyses. A p-value of less than 0.05 was consid-ered to indicate statistical significant differences betweengroups.

Results

The levels of vitamin D3 were significantly higher in malepatients than in female patients (41.25 vs 28.85, p < 0.01).Comparison of the vitamin D3 levels between the threeHBV-infected groups and the HCs showed no significant asso-ciation. Although there was no significant association foundbetween the vitamin D3 levels in the different groups, themean vitamin D3 levels were different. As expected, themean level of vitamin D3 was highest in the HCs and lowestin the new cases (new case [29.82] < inactive carrier [32.91]< treated [39.56] < HCs [44.88]). Fig. 1 illustrates the meanlevels of vitamin D3 in all four groups. Furthermore, no sig-nificant association was found to exist between the catego-rized levels of vitamin D3 (Fig. 2). Although there was nosignificant association between vitamin D3 levels amongthe different groups, the mean level of vitamin D3 in any ofthe three HBV-infected groups was lower than that in the HCs.It seems that treatment, which clinically improves patientstatus, could also elevate vitamin D3 towards a normal level.Inactive carriers, who have better clinical status compared

with new cases, showed a higher mean level of vitamin D3.These results indicated a possible correlation between theclinical health status of HBV-infected patients and theirvitamin D3 level.

After analyzing the HBV DNA levels and vitamin D3 levelsin the three group of HBV-infected patients, no significantassociation was found between these two parameters, spe-cifically for the inactive carriers (p = 0.171), the treatedpatients (p = 0.192) and the new cases (p = 0.369). BothALT and AST levels were significantly lower in the femalepatients than in the male patients (ALT: 25.03 vs 37.56,p < 0.001; AST: 23.78 vs 34.07, p < 0.004). In contrast,there was no significant association between the ALT or ASTlevels and vitamin D3 in any of the HBV-infected groups.

Discussion

As described earlier, vitamin D3 is strongly associated withimmune responses. Considering the involvement of immuneresponses in determination of clinical status of individualsinfected with HBV, it is expected that the level of this vitaminis varied during the pathogenic course of HBV infection.Several other studies have shown a significant decline ofthis vitamin in CHB patients, which was not confirmed in thepresent study. We found a similar trend, however, but it wasnot significant. This finding may simply reflect the limitednumber of cases in each of our study groups. However, it ispossible that the decreased levels of vitamin D3 detected inour CHB patients, which was similarly reported in previousstudies, may not be related to immune responses directly butit could affect those responses. Generally, it is accepted thatliver disease, such as HBV infection, can cause an impairedabsorption of vitamin D, which is not directly related to theimmune system. In fact, it could possibly be connected toimpaired bile acid production or gut edema associated withportal hypertension.22

Due to the dependence of vitamin D3 level on sunlight, it isgenerally accepted that in autumn and winter, the level ofvitamin D3 in plasma decreases. The level increases in springand summer. Although vitamin D3 levels of the CHB groupsand the HCs were all measured during the same seasons

Fig. 1. Mean vitamin D3 levels of HBV-infected patients, includinginactive carriers, new cases and treated patients, and healthy controls.

Fig. 2. Frequency of each category of vitamin D3 among HBV-infectedpatients, including inactive carriers, new cases and treated patients, andhealthy controls.



(autumn and winter) in this study, the overall results ofmeasurement in the two other seasons that we did notassess may be different. Additionally, amongst the Iranianpopulation, the level of vitamin D3 has been reported to begenerally lower than the mean global value.23,24 Because oursamples were taken from an Iranian population, the resultsmay be influenced by this issue. Moreover, because the HCswere not selected according to a sex-matching approach anda higher proportion of male patients were present in the studygroups (those with higher vitamin D3 level), the mean levelsof vitamin D3 in the HCs may have been governed by thefemale patients, in contrast to the three other groups exam-ined. Accordingly, all of these potentially confounding factorscould have impacted the mean level of vitamin D3 among theHCs so that it was less than its true level.

Considering the fact that vitamin D3 causes inhibition ofTh1 and Th17 cells, which represent two important arms ofimmune responses during HBV infection, and also increasesthe risk of HBV flare related to promoted HBV-specific immuneresponses, it appears that vitamin D3 contributes to regula-tion of immune responses. This could lead to lower liverdamage, because of declined cytotoxic and non-cytotoxicactions of CD4+ and CD8+ cell-associated cytokines. Fromthe Th2 and Tregs promotion, which leads to decreased HBV-specific immune responses (IFN-g and TNF-a), an elevation inviral load could occur. According to our previous studies, highlevels of anti-HBV cytokines or low levels of these cytokinescould lead to HBV flare and HBV reactivation, respectively.18,25

It seems that vitamin D3 contributes to prevention of HBVflare via a mechanism involving increase in Tregs populationand promotion of their functions. Thus, vitamin D3 supple-mentation could represent a therapeutic option for CHBpatients with HBV flare.

We believe that vitamin D3 has other effects on the HBV-specific immune responses, which have not yet been recog-nized. Based on our current knowledge about the effects ofvitamin D3 on T cell function and the critical roles of T cellsduring HBV infection, an increase in vitamin D3 level is likelyto help in reversing HBV flare, whilst its role in elevation inviral load was not confirmed in the current study, neither hasit been confirmed by others.


None


Study concept and design (SS, ST), acquisition of data (SS,BF), analysis and interpretation of data (ST), drafting of themanuscript (ST, BF), critical revision of the manuscript forimportant intellectual content (SS), administrative, technical,and material support (ST), study supervision (SS).

References

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[2] Baeke F, Takiishi T, Korf H, Gysemans C, Mathieu C. Vitamin D: modulator ofthe immune system. Curr Opin Pharmacol 2010;10:482–496. doi: 10.1016/j.coph.2010.04.001.

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[5] Soleymani T, Hung T, Soung J. The role of vitamin D in psoriasis: a review. IntJ Dermatol 2015;54:383–392. doi: 10.1111/ijd.12790.

[6] Zarei M, Javanbakht MH, Chams-Davatchi C, Daneshpazhooh M, EshraghianMR, DE-Rakhshanian H, et al. Evaluation of vitamin D status in newlydiagnosed pemphigus vulgaris patients. Iran J Public Health 2014;43:1544–1549.

[7] Marzano AV, Trevisan V, Eller-Vainicher C, Cairoli E, Marchese L, Morelli V, etal. Evidence for vitamin D deficiency and increased prevalence of fractures inautoimmune bullous skin diseases. Br J Dermatol 2012;167:688–691. doi:10.1111/j.1365-2133.2012.10982.x.

[8] Valdivielso JM, Fernandez E. Vitamin D receptor polymorphisms and dis-eases. Clin Chim Acta 2006;371:1–12. doi: 10.1016/j.cca.2006.02.016.

[9] Provvedini DM, Tsoukas CD, Deftos LJ, Manolagas SC. 1,25-dihydroxyvitaminD3 receptors in human leukocytes. Science 1983;221:1181–1183.

[10] Smolders J, Thewissen M, Peelen E, Menheere P, Tervaert JW, Damoiseaux J,et al. Vitamin D status is positively correlated with regulatory T cell functionin patients with multiple sclerosis. PLoS One 2009;4:e6635. doi: 10.1371/journal.pone.0006635.

[11] Chambers ES, Hawrylowicz CM. The impact of vitamin D on regulatory Tcells.Curr Allergy Asthma Rep 2011;11:29–36. doi: 10.1007/s11882-010-0161-8.

[12] Prietl B, Pilz S, Wolf M, Tomaschitz A, Obermayer-Pietsch B, Graninger W, etal. Vitamin D supplementation and regulatory T cells in apparently healthysubjects: vitamin D treatment for autoimmune diseases? Isr Med Assoc J2010;12:136–139.

[13] Lemire JM, Archer DC, Beck L, Spiegelberg HL. Immunosuppressive actionsof 1,25-dihydroxyvitamin D3: preferential inhibition of Th1 functions. J Nutr1995;125:1704S–1708S.

[14] Boonstra A, Barrat FJ, Crain C, Heath VL, Savelkoul HF, O’Garra A. 1alpha,25-Dihydroxyvitamin d3 has a direct effect on naive CD4(+) T cells to enhancethe development of Th2 cells. J Immunol 2001;167:4974–4980.

[15] Joshi S, Pantalena LC, Liu XK, Gaffen SL, Liu H, Rohowsky-Kochan C, et al.1,25-dihydroxyvitamin D(3) ameliorates Th17 autoimmunity via transcrip-tional modulation of interleukin-17A. Mol Cell Biol 2011;31:3653–3669. doi:10.1128/MCB.05020-11.

[16] Ardalan MR, Maljaei H, Shoja MM, Piri AR, Khosroshahi HT, Noshad H, et al.Calcitriol started in the donor, expands the population of CD4+CD25+ Tcellsin renal transplant recipients. Transplant Proc 2007;39:951–953. doi: 10.1016/j.transproceed.2007.04.012.

[17] Tavakolpour S. Inhibition of regulatory cells as a possible cure of chronicallyhepatitis B virus infected patients. Immunol Lett 2016;171:70–71. doi: 10.1016/j.imlet.2015.12.007.

[18] Tavakolpour S. The new insight into management of hepatitis B virus patientswith flare. Immunol Lett 2016;173:77. doi: 10.1016/j.imlet.2016.02.008.

[19] Lương Kv, Nguyễn LT. Theoretical basis of a beneficial role for vitamin D inviral hepatitis. World J Gastroenterol 2012;18:5338–5350. doi: 10.3748/wjg.v18.i38.5338.

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[22] Nair S. Vitamin d deficiency and liver disease. Gastroenterol Hepatol (N Y)2010;6:491–493.

[23] Ebrahimi M, Khashayar P, Keshtkar A, Etemad K, Dini M, Mohammadi Z, et al.Prevalence of vitamin D deficiency among Iranian adolescents. J PediatrEndocrinol Metab 2014;27:595–602. doi: 10.1515/jpem-2013-0428.

[24] Hovsepian S, Amini M, Aminorroaya A, Amini P, Iraj B. Prevalence of vitaminD deficiency among adult population of Isfahan City, Iran. J Health Popul Nutr2011;29:149–155.

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Review Article

Hepatitis C Virus: A Review of Treatment Guidelines,Cost-effectiveness, and Access to Therapy

Shaina M. Lynch* and George Y. Wu

Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Health Center, Hartford, USA

Abstract

Hepatitis C virus (HCV) infection remains a significant medicalconcern in the United States and around the world. It is stillone of the leading causes of chronic liver disease, and, formorethan 20 years, there has been little progress in the treatment ofHCV infection. The advent of direct-acting antivirals (DAAs)initiated the era of high efficacy and well-toleratedmedicationswith high cure rates. The efficacy of these medications hasprompted many professional societies around the world toupdate their treatment guidelines to include DAAs as first-linetreatment.GuidelinesbytheAmericanAssociation for theStudyof Liver Disease/Infectious Disease Society of America, WorldHealth Organization, Asian-Pacific Association for the Study ofLiverand theEuropeanAssociation for theStudyofLiverhaveallincorporated DAAs into their treatment guidelines. Despite thepromising data supporting these medications, however, theircost represents a limiting factor to their use, even thoughstudies have shown DAAs to be cost-effective. In addition tothe expense of these medications and limited resources, thereare many barriers preventing patients from receiving thispotentially life-saving treatment. In order to overcome thesebarriers, these issues need to be recognized and addressed.© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

Hepatitis C virus (HCV) infection is a significant medicalconcern in the United States and around the world. HCV-related complications cause 350,000 global deaths annually

and HCV infection remains one of the leading causes of chronicliver disease.1,2 Of the over 185 million people chronicallyinfected with HCV, more than 4 million are in the UnitedStates alone, and the majority of these individuals are notaware of the infection.3–6 It is estimated that about one-thirdof those with chronic HCV infection will go on to develop livercirrhosis or hepatocellular carcinoma.1

In 1998, the United States’ Centers for Disease Controlreleased HCV testing recommendations based on demo-graphics, exposures, risky behaviors and certain medicalconditions. Despite these recommendations, however, morethan 50% of HCV infections have remained unidentified.6 Therisk-based HCV testing guidelines were expanded in 2012 toinclude one-time testing for all persons born between 1945and 1965, as this birth cohort (known as the “baby boomers”)accounted for almost three-fourths of all HCV infectionsnationally.6,7 In order to make steps forward to improvetransmission rates and health outcomes, it is first crucial toidentify those with active infection.6,8

Identifying HCV-infected persons has become essentialsince the advent of direct acting antivirals (DAAs), given theirassociation with high cure rates, which is the primary goal oftreatment. Treatment success is measured based on sustainedvirological response (SVR), which is defined as an undetectablelevel of HCV RNA at 12 weeks (SVR12) or 24 weeks (SVR24)after completion of treatment.9 The first approved DAAs, telap-revir and boceprevir, were introduced in 2011 for the treatmentof HCV genotype 1 infection. Use of telaprevir and boceprevir incombination with pegylated interferon (PegIFN)-a and ribavirin(RBV) led to achievement of SVR rates in 65%–75% of patientswith HCV genotype 1. Unfortunately, this improvement in SVRwas associated with an increase in side effects and cost.9

The development of DAAs ushered in the era of highefficacy and well-tolerated medications for HCV infection.However, due to differences in efficacies of currently availablemedications based on viral factors, such as genotype andsubtype, and host factors, such as liver decompensation andrenal excretion issues, can complicate the selection of optimalagents.9 The purpose of this review is to compare variousguidelines for the treatment of HCV infection, to evaluatethe cost-effectiveness of the new treatment options (DAAs),and to identify barriers limiting access to treatment.

Comparison of guidelines and DAAs

Due to differences in endemicity, genotype and access totreatment, there is no worldwide consensus on HCV therapy.However, regional guidelines from four international organ-izations have been updated. We have reviewed the respectiveguidelines from the American Association for the Study of



Keywords: Hepatitis C; HCV; Drug therapy; Health care costs.Abbreviations: HCV, hepatitis C virus; US, United States; DAAs, direct-actingantivirals; AASLD, American Association for the Study of Liver Diseases; IDSA,infectious Disease Society of America; APASL, Asian Pacific Association for theStudy of Liver; EASL, European Association for the Study of Liver; SVR, sustainedvirological response; PegIFN, pegylated interferon; WHO, World Health Organiza-tion; ProD, Paritaprevir/ritonavir/ombitasvir/dasabuvir; PPIs, Proton pump inhib-itors; RAVs, resistance-associated variants; FDA, Food and Drug Administration;RBV, Ribavirin; HIV, human immunodeficiency virus; TDF, tenofovir disoproxilfumarate; TAF, tenofovir alafenamide; CrCl, creatinine clearance; eGFR, estimatedglomerular filtration rate; HD, hemodialysis; LMIC, low- and middle-income coun-tries; GNI, gross national income; CMS, center for Medicaid and Medicare Serv-ices; PBM, pharmacy benefit manager; CEA, cost effectiveness analysis; ICER,incremental cost-effectiveness ratio; QALY, quality-adjusted life years.Received: 12 July 2016; Revised: 03 October 2016; Accepted: 05 October 2016qDOI: 10.14218/JCTH.2016.00027.*Correspondence to: Shaina M. Lynch, Department of Medicine, Saint FrancisHospital and Medical Center, 114 Woodland Street, Hartford, CT 06105, USA. Tel:+1-860-714-7446, Fax: +1-860-714-1508, E-mail: [email protected]

Liver Disease/Infectious Disease Society of America (AASLD/IDSA), World Health Organization (WHO), Asian Pacific Asso-ciation for the Study of the Liver (APASL) and EuropeanAssociation for the Study of the Liver (EASL), and comparedthem in Tables 1–10.

Given the high SVR rates seen with the new DAAs, it is nosurprise that regimens including these drugs are recommendedby the different organizations. With the exception of the EASL,the organizations generally recommend IFN-free regimens asfirst-line treatment options. The EASL report considered differ-ences in per capita incomes and health insurance systemsacross Europe as a reason for including PegIFN-a in recom-mended treatment regimens.9 The APASL guidelines do notinclude IFN based on several factors. A study in Asia showedthat up to 50% of patients with HCV are deemed unsuitable fortreatment with IFN. Additionally, IFN therapy in Asian countriesis often limited to major treatment centers, with limited accessto isolated areas due to the infrastructure needed to ensuresafety and appropriate treatment.10 According to a study byWedemeyer et al.,11 given the nature of IFN-based treatment,treatment response rates have been reported as low as 5%,even in countries with diagnosis rates greater than 50%.10

Genotype 1

For genotype 1a and 1b, the treatment regimens of sofosbu-vir/ledipasvir and paritaprevir/ritonavir/ombitasvir/dasabuvir

(PrOD) are recommended by all four organizations. Additionalregimens including sofosbuvir/daclatasvir, and sofosbuvir/simeprevir are recommended by the AASLD/IDSA, EASL andWHO.10,12,13 The EASL guidelines contain two PegIFN-a-basedregimens, recommending the combination of PegIFN-a/sofos-buvir/RBV and PegIFN-a/simeprevir/RBV for treatment ofgenotype 1. Given that HCV genotype 1b is the predominantsubtype in the Asian-Pacific region (excluding Australia, Iran,New Zealand, Philippines and Thailand), the APASL guidelinesalso recommend the combination of asunaprevir/daclatasvir,for which data shows efficacy against genotype 1b only. TheAPASL guidelines also recommend the combination of grazo-previr/elbasvir for the treatment of HCV genotype 1b, basedon a phase 2 trial showing SVR rates of 90%–97%.6,9,10,12 TheAASLD/IDSA guidelines recommend grazoprevir/elbasvir forgenotypes 1a and 1b13 (Table 1a, 1b).

The choice of treatment regimen is based on a number offactors, with potential drug interactions being an importantconsideration. The sofosbuvir/ledipasvir regimen may inter-act with proton pump inhibitors (PPIs), and in patients usingthis class of medications, one of the other HCV treatmentregimens should be considered. The PrOD regimen is recom-mended for genotype 1 by AASLD/IDSA, EASL and APASL(genotype 1b) and safe in patients taking PPIs, but has thepotential to interact with drugs that interfere with thecytochrome P450 3A4 isoenzyme, specifically salmeterol, along-acting inhaled beta-agonist.14 Patients with HCV

Table 1a. Comparison of treatment guidelines for treatment-naïve patients infected with HCV genotype 1

Genotype AASLD/IDSA EASL WHO#

APASL

1a · Sofosbuvir/ledipasvir (I-A)· Elbasvir/grazoprevir (I-A)· Sofosbuvir/velpatasvir (I-A)· PrOD/RBV

a

(I-A)· Sofosbuvir/simeprevir (I-A)· Sofosbuvir/daclatasvir (I-B)Alternatives· Sofosbuvir/simeprevir 6 RBV

a*

(II-B)· Elbasvir/grazoprevir 6 RBV

e

(IIa-B)· Sofosbuvir/daclatasvir 6 RBV

a

(IIa-B)

· PegIFN-a/RBV/sofosbuvira

(A1)· PegIFN-a/RBV/simeprevir, thenPegIFN-a/RBV(24 weeks total) (B1)

· Sofosbuvir/ledipasvirb

(A1)· PrOD/RBV

ab

(A1)· Sofosbuvir/simepravir

b(B1)

· Sofosbuvir/daclatasvirb

(B1)

· Sofosbuvir/daclatasvir

ab

· Sofosbuvir/ledipasvir

abcd

Alternatives· Simeprevir/sofosbuvir

ab*

· PrOD/RBVa

· Sofosbuvir/ledipasvir (A1)

1b · Elbasvir/grazoprevir (I-A)· Ledipasvir/sofosbuvir (I-A)· PrOD (I-A)· Sofosbuvir/simeprevir (I-A)· Sofosbuvir/velapatasvir (I-A)· Sofosbuvir/daclatasvir (I-B)Alternatives· Sofosbuvir/daclatasvir 6 RBV

a

· Sofosbuvir/simeprevir 6 RBVa

(IIa-B)

· PegIFN-a/RBV/sofosbuvir (A1)· PegIFN-a/RBV/simeprevir, then PegIFN-a/RBV(24 weeks total) (B1)


(A1)· PrOD

b

(A1)· Sofosbuvir/simepravir (A1)

b(B1)

· Sofosbuvir/daciatasvir (A1)b

(B1)

· Sofosbuvir/dalatasvir

ab


abcd

Alternatives· Sofosbuvir/simepravir

ab*

· PrODb


· PrOD (A1)· Grazoprevir/elbasvir (A1)

· Asunaprevir/daclatasvir(24 weeks)(A2)

*Option in patients with a negative test result for the Q80K variant;

#All regimens: strong recommendation, moderate quality of evidence.

a24 weeks in patients with cirrhosis;

b12 weeks with RBV in patients with cirrhosis;

cTreatment may be shortened to 8 weeks in treatment-naïve persons without cirrhosis if baseline HCV RNA is below 6 million IU/mL;

dIf the platelet count is <75 3 103/mL, then 24 weeks of treatment with RBV should be given;

e16 weeks if baseline NS5A RAVs for elbasvir.

Abbreviations: PrOD, Paritaprevir/ritonavir/ombitasvir/dasabuvir; RAVs, Resistance-associated variants; RBV, Ribavirin.All treatment courses are 12 weeks unless indicated otherwise.


Lynch S.M. et al: Hepatitis C treatment, cost and barriers

genotype 1a with the nonstructural protein 3 (NS3) Q80Kpolymorphism and cirrhosis showed lower SVR rates whentreated with sofosbuvir and simeprevir, and one of the othertreatment regimens should be used in these patients.6

In addition, the presence of baseline NS5A resistance-associated variants (RAVs) significantly reduced the rate ofSVR with a 12-week elbasvir/grazoprevir treatment regimen.Therefore, in patients with baseline NS5A RAVs the combina-tion of elbasvir/grazoprevir should be avoided.13

Genotype 2

The combination of sofosbuvir and RBV for 12 weeks isrecommended by EASL, WHO and APASL for the treatmentof HCV genotype 2.6,9,10,12 The EASL guidelines include thecombination of PegIFN-a/RBV/sofosbuvir, which is recom-mended based on SVR data from the LONESTAR study.9 ForRBV-intolerant individuals, sofosbuvir/daclatasvir is given asan option by EASL, WHO and APASL based on studies showingthat daclatasvir is active against genotype 2 in vitro.9,10,12 TheAPASL guidelines also recommend the combination of sofosbu-vir and velpatasvir, which was approved by the United States’Food and Drug Administration (FDA) on June 28th, 2016.15

The AASLD/IDSA guidelines recommend sofosbuvir/velaptas-vir and, as an alternative, sofosbuvir/daclatasvir for bothtreatment-experienced and treatment-naïve patients. Intreatment-experienced patients, RBV is recommended foruse with sofosbuvir/velaptasvir and may be used, if desired,with daclatasvir, regardless of cirrhosis status. In patients

with compensated cirrhosis, sofosbuvir/daclatasvir, given for16 to 24 weeks, is an alternative regimen13 (Table 2a, 2b).

Genotype 3

With current treatment options, HCV genotype 3 is the mostdifficult subtype to treat.10 The recently updated AASLD/IDSAguidelines recommend sofosbuvir/velpatasvir or daclastavir/sofosbuvir for both treatment-experienced and treatment-naïve patients, with or without cirrhosis. In treatment-naïvepatients, RBV is added to either treatment with the exceptionof patients without cirrhosis who had failed PegIFN-a/RBV.13

The EASL guidelines recommend PegIFN-a/sofosbuvir/RBV(also given as an alternative regimen by the WHO) and, inagreement with WHO and APASL, recommend the IFN-freeregimen of sofosbuvir/RBV for 24 weeks.6,9,10,12 The EASL,WHO and APASL guidelines include the option to treat with aregimen of sofosbuvir/daclatasvir with or without RBV9,10,12

(Table 3a, 3b).

Genotype 4

Generally, the treatment guidelines for HCV genotype 4 arecomparable to those for genotype 1. The combination ofledipasvir and sofosbuvir for 12 weeks is recommendedby all four societies.6,9,10,12 The AASLD/IDSA guidelinesrecommend similar treatments for treatment-naïve andtreatment-experienced patients, with and without cirrhosis,with minor additions to the treatment regimen. The supple-mented regimens include paritoprevir/ritonavir/ombitasvir/

Table 1b. Comparison of treatment guidelines for treatment-experienced patients infected with HCV genotype 1

Genotype AASLD/IDSA EASL WHO# APASL

1a · Sofosbuvir/ledipasvir (I-A)· Elbasvir/grazoprevir (I-A)· Sofosbuvir/velpatasvir (I-A)· PrOD/RBV

a

(I-A)· Sofosbuvir/simeprevir (I-A)· Sofosbuvir/daclatasvir (I-B)Alternatives· Sofosbuvir/ledipasvir

a

(I-A)· Elbasvir/grazoprevir 6 RBV

e(I-B)

· Sofosbuvir/daclatasvir 6 RBVa

(IIa-B)· Sofosbuvir/simeprevir 6 RBV

a

(IIa-B)

· PegIFN-a/RBV/sofosbuvir (A1)· PegIFN-a/RBV/simeprevir, thenPegIFN-a/RBV for 36 weeks(48 weeks total) (B1)

· Sofosbuvir/ledipasvirb(A1)

· PrOD/RBVab


b

(B1)· Sofosbuvir/daciatasvir (A1)

b

(B1)


ab


abcd


ab*

· PrOD/RBVa

· Sofosbuvir/ledipasvir for12 weeks (A1)

1b · Elbasvir/grazoprevir (I-A)· Sofosbuvir/ledipasvir (I-A)· PrOD (I-A)· Sofosbuvir/simeprevir (I-A)· Sofosbuvir/velapatasvir (I-A)· Sofosbuvir/daclatasvir (I-B)Alternatives· Sofosbuvir/ledipasvir

a

(I-A)· Sofosbuvir/daclatasvir 6 RBV

a

· Sofosbuvir/simeprevir 6 RBVa

(IIa-B)

· PegIFN-a/RBV/sofosbuvir (A1)· PegIFN-a/RBV/simeprevir, thenPegIFN-a/RBV for 36 weeks (48weeks total) (B1)


(A1)· PrOD

b


b(B1)

· Sofosbuvir/daciatasvir (A1)b

(B1)


ab


abcd

Alternatives· Simeprevir/sofosbuvir

ab*

· PrODb


· PrOD (A1)· Grazoprevir/elbasvir (A1)

· Asunaprevir/daclatasvir(24 weeks) (A2)

*Option in patients with a negative test result for the Q80K variant;




cTreatment may be shortened to 8 weeks in treatment-naïve persons without cirrhosis if baseline HCV RNA is below 6 million IU/mL;

dIf the platelet count is <75 3 103/mL, then 24 weeks of treatment with RBV should be given.

Abbreviations: PegIFN, Pegylated interferon; PrOD, Paritaprevir/ritonavir/ombitasvir/dasabuvir; RBV, Ribavirin.All treatment courses are 12 weeks unless indicated otherwise.




AASLD/IDSA EASL WHO#

APASL

· Sofosbuvir/velapatasvir (I-A)· Sofosbuvir/daclatasvir (IIa-B)

a· PegIFN-a/RBV/sofosbuvir (B1)· Sofosbuvir/RBV

a

(A1)· Sofosbuvir/daclatasvir (B1)

· Sofosbuvir/RBVa

Alternative· Sofosbuvir/daclatasvir

· Sofosbuvir/RBV (A1)· Sofosbuvir/daclatasvir(24 weeks)* (B1)

· Sofosbuvir/ledipasvir* (B1)· Sofosbuvir/velpatasvir* (B1)

*For RBV intolerant patients;

#All regimens: strong recommendation, low quality of evidence.

a16–24 weeks in patients with cirrhosis.

Abbreviations: PegIFN, Pegylated interferon; RBV, Ribavirin.All treatment courses are 12 weeks unless indicated otherwise.



APASL

· Sofosbuvir/velapatasvir (1-A)b

· Sofosbuvir/daclatasvira

(IIa-B)c

· PegIFN-a/RBV/sofosbuvir (B1)· Sofosbuvir/RBV

a

(B1)· Sofosbuvir/daclatasvir (B1)

· Sofosbuvir/RBVAlternative· Sofosbuvir/daclatsvir

· Sofosbuvir/RBV (A1)· Sofosbuvir/daclatasvir(24 weeks)* (B1)

· Sofosbuvir/ledipasvir* (B1)· Sofosbuvir/velpatasvir* (B1)

*For RBV-intolerant patients;


a16–24 weeks in patients with cirrhosis;

bWith RBV for 12 weeks (sofosbuvir/RBV TE);

c± RBV for 24 weeks (sofosbuvir/RBV TE).




APASL

· Sofosbuvir/daclatasvir (I-A)· Sofosbuvir/velpatasvir (I-A)

a

(IIa-B)

· PegIFN-a/sofosbuvir/RBV (B1)· Sofosbuvir/RBV (A1)

b

(B1)· Sofosbuvir/daclatasvir (A1)

c

(B1)


· Sofosbuvir/RBVb

Alternative· Sofosbuvir/PegIFN/ribavirin

· Sofosbuvir/RBVb(A1)

d(B2)


(A2)


a± RBV for 24 weeks (cirrhotics);

b24 weeks, option for non-cirrhotics;

c24 weeks in patients with cirrhosis;

d16 weeks in patients with cirrhosis.




APASL

· Sofosbuvir/daclatasvir (I-A)b

· Sofosbuvir/velpatasvir (I-A)e

· PegIFN-a/RBV/sofosbuvir (B1)· Sofosbuvir/RBV (A1)

c

(B1)· Sofosbuvir/daclatasvir (A1)

d(B1)


b

· Sofosbuvir/RBVa

· Sofosbuvir/RBVc

(A1)· Sofosbuvir/daclatasvir (A2)

b

(B2)

*Option for IFN ineligible patients;


aWith PegIFN-a in patients with cirrhosis;

bWith RBV for 24 weeks in patients with cirrhosis;

c24 weeks treatment;

d24 weeks in patients with cirrhosis;

eWith RBV in patients with cirrhosis.




RBV, sofosbuvir/velpatasvir, and elbasvir/grazoprevir.13

Sofosbuvir/daclatasvir with or without RBV is anotheroption recommended by the EASL, WHO and APASL guide-lines.9,10,12 Additional treatment options given by the WHOand EASL guidelines include simeprevir/sofosbuvir with orwithout RBV and ombitasvir/paritaprevir/ritonavir/RBV.9,12

The EASL guidelines recommend the combinations ofPegIFN-a/simeprevir/RBV and PegIFN-a/sofosbuvir/RBV inaddition to the IFN-free regimens9 (Table 4a, 4b).

Genotypes 5 and 6

The treatment guidelines for HCV genotypes 5 and 6 aremostly adapted from studies on other genotypes, since specificdata is lacking. The combination of sofosbuvir/ledispavir for12 weeks is recommended by all four organizations.6,9,10,12

The treatment regimen of PegIFN-a/sofosbuvir/RBV is anadditional treatment regimen recommended by the EASLguidelines and given as an alternative treatment option bythe WHO guidelines.6,9,12 Sofosbuvir/daclatasvir with orwithoutRBVisrecommendedbytheEASLandAPASLguidelines,based on data that daclatasvir acts against genotypes 5 and 6

in vitro. The APASL and AASLD/IDSA guidelines also recom-mend the newly-approved regimen of sofosbuvir/velpatasvirfor treatment of HCV genotypes 5 and 69,10 (Table 5a, 5b).

Special populations

Cirrhotic patients

Treatment regimens for HCV in patients with decompensatedcirrhosis vary among the different societies’ guidelines. TheAASLD/IDSA guidelines recommend a 12-week course of RBVcombined with ledipasvir/sofosbuvir, sofosbuvir/velpatasvir,or daclastavir/sofosbuvir for genotypes 1 and 4. This recom-mendation is for patients with moderate to severe hepaticimpairment, who may or may not be transplant candidates orthose with hepatocellular carcinoma.6 In patients who are RBVineligible, treatment is extended to 24 weeks. In addition, theAASLD/IDSA guidelines recommend ledipasvir/sofosbuvir andsofosbuvir/velpatasvir for patients who failed a sofosbuvir-based regiment with a prolonged 24-week course of treat-ment. The AASLD/IDSA guidelines also strongly recommendthat these patients be referred to a specialist for treatment.13



APASL

· Paritaprevir/ritonavir/ombitasvir/RBV (I-A)

· Sofosbuvir/velpatasvir (I-A)· Elbasvir/grazoprevir (IIa-B)· Sofosbuvir/ledipasvir (IIa-B)

· PegIFN-a/RBV/sofosbuvir (B1)· PegIFN-a/RBV/simeprevir, thenPegIFN-a/RBV (24 weeks total)(B1)

· Sofosbuvir/ledipasvirab(B1)

· Paritaprevir/ritonavir/ombitasvir/RBV (A1)

· Sofosbuvir/simepravirab

(B2)· Sofosbuvir/daciatasvir

ab(B2)

· Sofosbuvir/daclatasvirabc

· Sofosbuvir/ledipasvirab*


ab

· Ombitasvir/paritaprevir/ritonavir/RBV

a

· Sofosbuvir/ledispavira(A1)

· Sofosbuvir/velpatasvir(A1)

a

(B1)· PrOD

a

(B1)· Sofosbuvir/daclatasvir

ab(B2)

*If the platelet count is <75 3 103/mL, then 24 weeks of treatment with RBV should be given;




cCan be used in patients with decompensated cirrhosis.




APASL

· Paritaprevir/ritonavir/ombitasvir/RBV (I-A)

· Sofosbuvir/velapatasvir (I-A)· Elbasvir/grazoprevir

d

(IIa-B)· Sofosbuvir/ledipasvir

ab(IIa-B)

· PegIFN-a/RBV/sofosbuvir (B1)· PegIFN-a/RBV/simeprevir, thenPegIFN-a/RBV for 36 weeks(48 weeks total) (B1)

· Sofosbuvir/ledipasvirab(B1)

· Paritaprevir/ritonavir/ombitasvir/RBV (A1)

a

· Sofosbuvir/simepravirab(B2)

· Sofosbuvir/daclatasvirab(B2)

· Sofosbuvir/daclatasvirabc

· Sofosbuvir/ledipasvirab*

Alternatives· Sofosbuvir/simpepravir

ab

· Paritaprevir/ritonavir/ombitasvir/RBV

a

· Sofosbuvir/ledispavira(A1)

· Sofosbuvir/velpatasvir(A1)

a

(B1)· Paritaprevir/ritonavir/ombitasvir/dasabuvir

a(B1)

· Sofosbuvir/daclatasvirab

(B2)





cCan be used in patients with decompensated cirrhosis;

d16 weeks with RBV if prior PegIFN-a/RBV treatment failed.




The EASL guidelines recommend ledipasvir/sofosbuvir forgenotypes 1, 4, 5 and 6, along with sofosbuvir and RBV for16–20 weeks for genotype 2, and sofosbuvir/daclatasvirwith RBV for 12 weeks for all genotypes.9 The APASL guide-lines are similar, recommending sofosbuvir-based treatment,with or without ledipasvir (genotype 1) or daclatasvir (geno-type 3) for 12–24 weeks.16 According to the WHO guidelines,simeprevir and the combination of ombitasvir/paritaprevir/ritonavir/dasabuvir are not approved for patients with decom-pensated cirrhosis. While the WHO guidelines recognize dacla-tasvir, ledipasvir and sofosbuvir as feasible and effective inpatients with decompensated liver disease, they recommendthat treatment be restricted to specialized centers in order tomanage complications, and preferably those centers whereliver transplantation is available.12

HIV co-infection

HCV-infected patients that are co-infected with human immu-nodeficiency virus (HIV) are generally treated the same asHIV-negative individuals, but there are certain precautionsthat need to be considered. The APSAL guidelines suggestdeferring treatment for HCV infection until the CD4 count is>200 cells/mm3. Across all the guidelines, the main area ofconcern in HCV/HIV co-infected patients is medication inter-actions, and treatment should be coordinated with anexpert.1,9,10,13 Avoidance of tenofovir is recommended whileusing ledipasvir/sofosbuvir, as the latter potentiates the neph-rotoxic effect of tenofovir disoproxil fumarate (TDF), especially

in combination with other antivirals. A possible alternativefor TDF is tenofovir alafenamide (TAF).13 In addition, whenusing PrOD, rilpivirine and efavirenz should not be used, asthe ritonavir potentiates HIV protease inhibitors, leading toan increase in gastrointestinal side effects, neurologic eventsand aminotransferase elevations.6,10

Renal impairment

The AASLD/IDSA guidelines state that DAA’s can be safelydosed in patients with mild to moderate renal impairment(creatinine clearance (CrCl) rate of 30–80 mL/min). Forpatients with severe renal impairment/end-stage renaldisease (CrCl rate of <30 mL/min) in whom treatment hasbeen elected before renal transplant, the AASLD/IDSA guide-lines recommend the following regimens: PrOD for genotype1b and elbasvir/grazoprevir for genotypes 1 and 4. Forgenotypes 2, 3, 5 or 6, PegINF-a with RBV can be used. RBVmay be added to the regimen if the hemoglobin is >10 g/dL,but caution should be used because of the risk of hemolyticanemia.6,13 For patients with an estimated glomerular filtra-tion rate (eGFR) of 30–50 mL/min, RBV can be given at dosesof 200 mg and 400 mg on alternating days.6

The EASL guidelines recommend that hemodialysis (HD)patients who are suitable for transplant be treated withantivirals. Although they do not state a specific agent, theyrecommend IFN-free regimens and, if possible, RBV-free. TheEASL guidelines also recommend PrOD for patients with severerenal impairment and specifically state that sofosbuvir should

Table 5b. Comparison of treatment guidelines for treatment-experienced patients infected with genotype 5 and 6


APASL

· Sofosbuvir/velpatasvir (IIa-B)· Sofosbuvir/ledipasvir(IIa-C)

· PegIFN-a/RBV/sofosbuvir (B1)· Sofosbuvir/ledipasvir

abc(B1)

· Sofosbuvir/daclatasvirab

(B1)

· Sofosbuvir/ledipasvirab

Alternative· PegIFN-a/RBV/sofosbuvir

· Sofosbuvir/velatasvir (A1)in cirrhosis (B1)

· Sofosbuvir/ledipasvira


ab

(B1)


#All regimens: conditional recommendation, very low quality of evidence.



c24 weeks with RBV if negative predictors of response.


Table 5a. Comparison of treatment guidelines for treatment naïve patients infected with HCV genotypes 5 and 6


APASL

· Sofosbuvir/velpatasvir (I-A)· Sofosbuvir/ledipasvir(IIa-B)

· PegIFN-a/RBV/sofosbuvir (B1)· Sofosbuvir/ledipasvir

abc


ab

(B1)

· Sofosbuvir/ledipasvirab

Alternative· PegIFN-a/RBV/sofosbuvir

· Sofosbuvir/velatasvir (A1)in cirrhosis (B1)

· Sofosbuvir/ledipasvira


ab

(B1)


#All regimens: conditional recommendation, very low quality of evidence.



c24 weeks with RBV if negative predictors of response.




not be administered to patients with eGFR of <30 mL/min.9

The APASL guidelines are comparable to the AASLD/IDSAguidelines with regards to patients with mild to moderaterenal impairment (CrCl rate of 30–80 mL/min).6,10 In addition,the APASL guidelines recommend that simeprevir be given atits standard dose for patients with severe renal impairment(CrCl rate of <30 mL/min).

The APSAL guidelines pay special attention to the lack ofaccess to DAAs in many Asian countries. Given the variabilityin access to DAAs, they suggest referring to the 2012 guide-lines for IFN-based treatment regimens for patients withrenal impairment.10 In accordance with the EASL guidelines,the WHO guidelines report that sofosbuvir lacks safety datain patients with renal impairment.9,12 They also comparePegINF-a2a and PegINF-a2b, the latter being excreted viathe kidneys, and state that although there is a theoreticalaccumulation of PegINF-a2b in patients with HD, there wasno difference clinically. In patients with CrCl of 20–40 mL/min, PegINFa-2a should be dosed at 135 mg/week, with areduced RBV dose. In patients with HD, the RBV may beadministered at 200 mg/day or every other day.12

Barriers to treatment

Although DAA agent-only regimens are recommended asfirst-line therapy for the treatment of HCV infection, accessto these medications has proven to be limited by multiplefactors. Even in the era of DAAs, HCV-infected individualsmay not clear the virus. The reasons for this include host andviral factors, as well as compliance and accessibility. Thelatter are usually due to the healthcare system itself.17

Common issues leading to limited access to treatmentinclude insurance concerns, low health literacy, and lack ofconsistent medical care. In addition, patients may have com-peting health concerns, such as mental illness, comorbidities,substance abuse, lack of social support and homelessness.Patients may be unemployed or under-employed, and priorto the advent of DAAs, many patients feared the side effectsof IFN-based therapy.17

In a study by Stepanova et al.,18 the rate of insurancecoverage for HCV patients ranged from 60%–65%, andmany of the individuals were also found to be under-insured. DAAs have proven to be highly effective but at anextremely high cost, which serves as a major barrier tomore widespread treatment access.19 Although cost is acommon barrier to treatment access worldwide, additionalbarriers can be identified, which vary based on a country’seconomic status.

Low- and middle-income countries (LMIC)

LMIC have a high rate of endemicity of chronic HCV infection.Based on the World Bank classification, LMIC are countrieswith a gross national income (GNI) per capita of less than$1,045 for low-income countries and less the $12,735 formiddle-income countries20 (Table 6). For LMIC, in additionto access to medications, testing required for appropriateselection of agents may not be readily available.21 UnlikeHIV infection, for which most of the affected people live onthe African continent, most of the 185 million people withchronic HCV infection live in Asia.21,22 Specifically, the major-ity of HCV-infected individuals live in middle-income countriessuch as China, Pakistan and Nigeria.21 This is an important

distinction because these Asian countries do not attract tradi-tional financial aid when compared to African countries.23

Drug pricing remains the major barrier to treatment withDAAs, and some progress has been made in this area. Forexample, in Egypt, where the government has worked withpharmaceutical companies to reduce prices, the list price for a12-week treatment with sofosbuvir has dropped to $900.21,22

Attempts to make recommended HCV treatment availablehas also been seen in India. A voluntary licensing agreementwas announced by Gilead Sciences, Inc, enabling 11 Indianmanufacturers to make their generics available to 103 LMICs,excluding China.21,22 This allows countries to obtain low-costgenerics which retail at about 1%–2% of the United States’list prices.22 These low prices could make access to HCVtreatment in LMIC a realistic goal.21 Furthermore, thesuccess of global HIV treatment initiatives provides the prece-dent for successful therapeutic intervention for the manage-ment of a chronic viral infection.22

High-income countries

Barriers to accessing DAAs are not limited to LMIC, as DAAsare expensive and resources are limited even in high-incomecountries. High-income countries are defined as countries witha GNI per capita greater then $12,736.20 Although DAAs havebeen shown to be, for the most part, cost-effective, they havenot proven to be affordable.6 Data from the IFN era revealsthat ;16% of HCV-infected individuals were prescribed anti-viral treatment, with only 9% achieving SVR.24 Given the sig-nificant number of undiagnosed patients with chronic HCVinfection, this number is sobering and illustrates that cost isnot the only barrier to DAAs. Certain barriers can be seenworldwide and regardless of income, which include diagnosis,lack of knowledge of treatment options, medical eligibility,infrastructure for vulnerable populations and high cost.24,25

Despite limitations in the diagnosis and treatment ofchronic HCV, the single most important determinant of

Table 6. AASLD-IDSA recommendations for patients who would receivethe most immediate benefits from treatment

Highest-priority for treatmentowing to highest risk forsevere complications

High-priority fortreatment owing to highrisk for complications

Advanced fibrosis (METAVIRF3) or compensated (METAVIRF4)

Fibrosis (METAVIR F2)

Organ transplant recipients HIV-1 coinfection

Type 2 or 3 cryoglobulinemiawith end-organ manifestations(e.g., vasculitis)

Hepatitis B viruscoinfection

Proteinuria, nephroticsyndrome ormembranoproliferativeglomerulonephritis

Other coexistent liverdisease (e.g.,nonalcoholicsteatohepatitis)

Debilitating fatigue

Type 2 Diabetes mellitus(insulin-resistant)

Porphyria cutanea tarda

Abbreviation: HIV, Human immunodeficiency virus.



accessibility to DAAs in the United States’ is cost. Manyinsurance companies and most Medicaid programs in theUnited States currently only approve payment for patientswith advanced liver fibrosis or other high-priority patients asdescribed by the AASLD/IDSA HCV guidance sections19,26,27

(Table 7). Liver fibrosis is measured by the METAVIR fibrosisscore, with scores of F3 and F4 designating a patient as“highest-priority” and a score of F2 designating a patient as“high-priority”.6,28 High-priority patients include those whoare recipients of organ transplant, or have type 2 or 3 cryoglo-bulinemia with end organ manifestation, renal involvement(nephrotic syndrome, membranoproliferative glomeruloneph-ritis), HIV-1 or hepatitis B co-infection, other liver disease,debilitating fatigue, diabetes mellitus type 2 or porphyriacutanea tarda.6 Providers face significant delays, which couldlead to lack of treatment and loss to follow-up.19 In addition, todate, insurance carriers do not have a uniform policy as to whoqualifies for treatment using DAAs, and each state in theUnited States has its own Medicaid program regarding HCVtreatment.19

In a study conducted by Saab et al.,26 among the410 patients prescribed DAAs between October 2014 and July2015, 81% were insurance-approved for therapy. Variousfactors were found to be associated with approval, includingolder age, employment, lack of comorbidities, liver transplan-tation and advanced liver disease. Furthermore, Medicareinsurance, lack of non-liver comorbidities and presence ofadvanced liver disease were found to be independent predictorsof drug approval. Medicaid insurance was also found to be asso-ciated with a high insurance denial rate for treatment withDAAs.26

These results echo some of the results from a study by Doet al.,24 which found that one in four patients were initiallydenied access to sofosbuvir and ledipasvir, although mostwere eventually approved. The initial denial led to delays inthe initiation of treatment, however. Interestingly, this studyalso found that having Medicare/Medicaid coverage resultedin a higher likelihood of approval than private insurance forthe same stages of advanced liver disease. Unfortunately, thisstudy was limited in that it did not distinguish between Med-icare and Medicaid, and was carried out at a date that pre-ceded prior authorization guidelines.24

The denial rate for Medicaid is concerning because as manyas 25% of patients in the United States with chronic HCV whoare hospitalized are covered by Medicaid. There are manystate requirements set forth by Medicaid that further restrictapproval of DAAs for treatment of HCV infection. In a studyconducted by Barua et al.,27 Medicaid programs in 31 stateswere found to designate sofosbuvir as “not preferred”, withfour states being found to require liver biopsy to prove thelevel of fibrosis, rather than allowing less invasive testing.

Seventeen states were found to apply a “preferred” designa-tion to sofosbuvir, and although proving medical necessitywas not required, 15 of these 17 states required “prior author-ization”. There were also 30 states that were found to requiresofosbuvir be prescribed in consultation with a specialist.27,28

It was also reported that many states also require drugscreening, which further delays treatment to patients withchronic HCV and adds to the expense of their management,even though these patients reportedly showed similar rates ofadherence to treatment as the general population. Additionalhurdles include limiting service based on CD4 count.27,28

These restrictions are clearly contrary to Medicaid guide-lines. Effective November 2015, the Centers for Medicare andMedicaid Services (CMS) released guidance in which theyaddressed restrictions on DAA treatments for HCV. It wasnoted that although states have the discretion to establishlimitations on coverage, they must ensure access to clinicallyappropriate treatment.19 Furthermore, limiting treatmentbased on fibrosis, requiring abstinence and limiting the typeof providers able to prescribe HCV treatment were cited asexamples of unreasonable restrictions.19 As noted by Edlin,29

selective denial of medically necessary care for high-cost con-ditions is a discriminator, and illegal under the Affordable CareAct (Table 7).

Finally, another major hurdle to the treatment of chronicHCV infection is the pharmacy benefit manager (PBM). Theseare generally for-profit intermediaries in the pharmacy supplychain, who greatly influence the actual drug cost.6,19 As notedin a review by Rosenthal et al.,30 the pricing between phar-maceutical companies and PBMs are confidential businessdealings that prevent transparency. These transactions canpotentially have negative impacts on drug pricing and freemarket competition. In addition, PBMs often negotiate con-tracts with pharmaceutical companies with exclusivity, creat-ing restrictions on prescription medications.6

Cost-effectiveness of DAAs

Cost-effectiveness analysis (CEA) is a means of evaluatingbudget limitations for healthcare spending and seeks tobalance public health needs with budget constraints.6 Whendiscussing the cost-effectiveness of DAAs, it is important tonote that the “cost” of a publically available drug is the whole-sale acquisition cost. The actual price paid for the medicationby health insurers varies.6,19 CEA is a formal method tocompare the cost and outcomes between two interventions,one being the standard of care. This is measured using theincremental cost-effectiveness ratio (ICER).19,31–33 The ICERin HCV infection treatment is usually measured as a cost perquality-adjusted life years (QALY) gained between two treat-ment strategies. In the United States, the willingness topay threshold is $50,000–$100,000 per QALY gained.6,19,33

DAAs for HCV infection appear to be cost effective when com-pared to prior therapies. However, the benefit to society andthe payer would not be seen until at least a decade later(Table 8). The projected cost of widespread use of DAAs fortreatment of HCV infection in the United States would exceed$300 billion, which is not feasible.6,19,34 The cost-effectivenessof DAAs is currently affected by the drug prices and the patientpopulations selected as noted by Chhatwal et al.33

Many cost-effectiveness studies have examined sofosbuvirand ledipasvir for treatment of genotype 1 infections. Fromthese studies, it was determined that, in certain patients, HCVtreatment with DAAs was cost-effective, with most ICERs

Table 7. Common prior authorization criteria for sofosbuvir prescriptionunder state Medicare fee-for-service programs

Common prior authorization requirements for sofosbuvir

· Abstain from alcohol use before treatment· Abstain from alcohol abuse before treatment· Abstain from drug use before treatment· Abstain from injection drug use before treatment· Minimum METAVIR fibrosis score· Specialist provider



being less than $100,000.6,19 In addition to the cost of thedrug, age and severity of fibrosis were significant influencersof the ICERs. The greatest cost savings were seen in youngerpatients and in patients with advanced fibrosis. Certainexceptions to this were seen, however, including patientswho required 24 weeks of treatment and patients withcirrhosis.6,19,33,35,36 Treatment of genotype 2 or 3 with DAAagent-only regimens is reportedly less cost-effective whencompared to genotype 1, likely due to the fact that mostpatients achieve cure with PegIFN-a based therapy.6,19,32,35

There are limitations encountered when interpreting cost-effectiveness data for the treatment of HCV with DAAs. One ofthe limitations is that the price of the DAAs used is thewholesale acquisition cost, which is generally not the pricepaid by insurers. The actual acquisition cost negotiated byPBMs is not publicly available, however.35 In addition, manystudies have not taken into account indirect costs, non-medical costs, comorbid conditions and other political, societalor ethical priorities.6,19,31,32,35 In a recent systemic review ofanalytic models for determining the cost-effectiveness ofDAAs, it was shown that most modeling studies have usedsimilar structures and have underestimated the value of HCVtreatment.37

Future directions

As we move into the era of pangenotypic, RBV-free DAAregimens, there remain obstacles to broad implementationof these therapies, including screening and disease assess-ment, public health prioritization and drug pricing.22 DAAsare a powerful tool in the fight against chronic HCV. However,an effective treatment is not enough. The eradication ofHCV will require a significant amount of financial investmentfor screening, prevention and treatment. As noted byHesamizadeh et al.,38 the next steps in the eradication ofchronic HCV involves finding and treating patients withchronic HCV in the general population, improving the avail-ability and affordability of treatment (particularly in LMIC),and focusing on certain special populations, such as patientswith HIV co-infection, kidney disease, thalassemia and livertransplant recipients. All of these goals need to be accom-plished while concentrating on education and preventionsimultaneously.38

Globally, pharmaceutical companies will need to continue towork directly with governments to negotiate cheaper genericversions of the medications, while also weighing their need forprofit.21,38 LMICwill need to address issues related to access tohealthcare and health literacy, a potential model for this beingthe one used to implement HIV treatments.39 In the UnitedStates, the process will need to become more transparent, ascost-effectiveness is determined by the cost of the treatment.At the current time, it is unclear if the pharmaceutical industryoperates by the same free market rules seen in other indus-tries, as noted in the review by Rosenthal et al.30,40 In addition,it may be prudent to consider a uniform national policy to iden-tify who qualifies for treatment with DAAs. Taking some ofthese steps will, hopefully, align with the WHO’s goal of erad-ication of HCV by 2030. As noted by Afdhal et al.,17 “in the US,HCV has all the attributes of an eradicable disease except suf-ficient public investment. Delivering care effectively, safely andbroadly to all patient populations in an economically acceptablefashion must be our goal.” In addition, future CEA needs totake into account the benefits of treating HCV, including pre-venting transmission, and performance in LMIC.37

The recent approval by the United States’ FDA of sofosbu-vir/velpatasvir, the first regimen able to treat all 6 HCVgenotypes, opens the door for the next stage in DAA agent-only treatment regimens.15 As reported in the ASTRAL 1–4trials, this combination had excellent SVR in genotypes1–6.41–43 In the study by Hill et al.,44 the cost of large-scaletreatment access programs was analyzed in developing coun-tries, and further research needs to be conducted to ensurepan-genotypic coverage of HCV.

Conclusions

Guidelines around the world have been updated to reflect theefficacy and tolerability of DAAs, while also consideringregional economic differences. The excitement of the IFN-free DAA era, however, has been dampened by the extremelyhigh cost of these medications. Although there are manybarriers to access to these medications, cost remains thesingle biggest barrier to the widespread use of DAAs. Most ofthe new treatment regimens have been shown to be cost-effective. However, given the disease burden, the widespreadimplementation of treatment is not feasible or affordable atthe current time in most of the world.31–35,40 Much work stillneeds to be done in order to make guideline-driven DAA-based treatment universally affordable and accessible, toachieve the ultimate goal of eradication of chronic HCVinfection.


None


Drafted the manuscript (SML), critically revised themanuscript (GYW).

Acknowledgments

Support from the Herman Lopata Chair in Hepatitis Researchand a research grant from Alexion Corp. is gratefullyacknowledged.

Table 8. Incremental cost-effectiveness ratio (ICER) per quality-adjusted life year (QALY) based on HCV genotype

Hepatitis C virus(HCV) genotype ICERs per QALY

1

1 Treatment-naïve: less than $0 to$31,452

2

Interferon-experienced patients:$84,744 to $178,295

2 US$ 35,500 to US$238,0002,3

3 US$ 410,5182

4 US$ 34,349 to US$ 80,7932

1When the ICER is determined, it is compared to the willingness to pay threshold,

which is typically considered $50,000 to $100,000/QALY;2Depending on presence or absence of cirrhosis;

3In treatment-naïve patients without cirrhosis, the manufactures price for

sofosbuvir led to ICERs above the willingness to pay threshold. Negotiating lowercost can lead to ICERs dropping to acceptable levels.



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Review Article

Novel Treatment of Hepatitis C Virus Infection for Patientswith Renal Impairment

Goki Suda*, Koji Ogawa, Megumi Kimura, Masato Nakai, Takuya Sho,Kenichi Morikawa and Naoya Sakamoto

Department of Gastroenterology and Hepatology, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan

Abstract

Prevalence of hepatitis C virus (HCV) infection is high inpatients with end-stage renal dysfunction, including patientsundergoing hemodialysis (HD). The HCV infection itself cancause glomerulonephritis and puts individuals at increasedrisk of developing end-stage renal disease; fortunately, suc-cessful HCV eradication sometimes restore HCV-related renaldysfunction. Moreover, the prognosis of dialysis patientsinfected with HCV is significantly worse and the renal allograftsurvival in HCV-infected patients is also worse than in dialysispatients without HCV infection. If life prognosis is favorable,therefore, anti-HCV therapy is strongly recommended forHCV-infected patients with severe renal dysfunction. Thestandard therapy for HCV-infected patients with severe renaldysfunction has historically been interferon-based therapy.However, this therapy remains ineffective in achieving high,sustained viral response rates and the rate of adverse eventsand treatment discontinuation due to treatment-inducedadverse events continues to be high in patients with severerenal dysfunction. Safe and effective anti-HCV therapies areurgently needed, and crucial, for patients with severe renaldysfunction. Recently, direct-acting antivirals (DAAs) thatspecifically target viral proteins have been developed, andthese targets include the NS3, NS5A, and NS5B of HCV. Clinicaltrials have revealed high efficacy and safety of the DAA-basedtherapies, but patients with severe renal dysfunction werenot included in the majority of these trials. However, severalrecent reports have shown high efficacy and safety for someregimens of DAA combination therapy for HCV-infectedpatients with severe renal dysfunction. In this review, wediscuss novel treatments for HCV-infected patients with severerenal dysfunction and the pharmacokinetics of these drugs.© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

Hepatitis C virus (HCV) infection affects approximately130–150 million people worldwide and is one of the primaryfactors of liver cirrhosis and hepatocellular carcinoma.1–3 Therate of HCV infection is generally high in patients with end-stage renal dysfunction, including patients on hemodialysis(HD).4,5 However, the reported prevalence of HCV infectionamong HD patients varies from 5% to approximately 60% indifferent countries.6–9

HCV infection sometimes causes extrahepatic disorders,including lymphoma, lichen planus, diabetes mellitus andrenal dysfunction.10 In addition, HCV infection is the primarycause of mixed cryoglobulinemia, which is known to inducemembrane-proliferative glomerulonephritis,11 and causesincreased risk of developing end-stage renal disease.12

Successful HCVeradication restores the HCV-related renal dys-function,13,14 as has been confirmed by a large cohort study.Hsu et al.15 reported that the 8-year cumulative incidence ofend-stage renal disease was significantly lower in HCV-infectedpatients treated with anti-HCV therapy than in the untreatedcontrol group (0.15% vs. 1.32%); in addition, the anti-HCVtherapy was also found to significantly suppress acute coro-nary syndrome and ischemic stroke.

The prognosis of HCV-infected patients on HD is alsosignificantly worse than that of non-HCV-infected dialysispatients.16–18 A recent meta-analysis of seven studies involv-ing 11,589 HD patients showed that HCV infection wasan independent risk factor of mortality in HD patients.19 Inaddition, anti-HCV is mandatory in HCV-infected patientswho are candidates for kidney transplantation because thechance of renal allograft survival is worse in HCV-infectedpatients than in non-HCV-infected patients.20 Thus, HCV-infected patients with chronic renal dysfunction have an addi-tional indication for HCV eradication therapy. The KidneyDisease Improvement Global Outcome (KIDIGO) andJapanese Society for Dialysis Therapy (JSDT) highly recom-mend anti-HCV therapy for dialysis patients with an HCVinfection, if life prognosis is favorable.21,22 Moreover, thedemographic profile of HCV-infected patients has shown atrend in increasing age, year by year,23,24 and the numberof cases with renal dysfunction are expected to increaseover time due to age-related decline in the renal function.25

The standard therapy for HCV-infected patients with severerenal dysfunction has historically been interferon (IFN)-basedtherapy. However, this therapy remains incapable of achievinga high rate of sustained viral response (SVR), even for patientswith normal renal function.26–28 Host factors, such as the



Keywords: DAAs; HCV; Hemodialysis; CKD.Abbreviations: DAAs, direct-acting antivirals; HD, hemodialysis; HCV, hepatitisC virus; IFN, interferon; PEG-IFN, pegylated-interferon; SOF, sofosbuvir; RBV,ribavirin; SMV, simeprevir; LDV, ledipasvir; eGFR, estimated glomerular filtrationrate; GT, genotype; SVR, sustained viral response; SAE, serious adverse event;PTV, paritaprevir; OBV, ombitasvir; DSV, dasabuvir.Received: 29 August 2016; Revised: 21 November 2016; Accepted: 07 December2016qDOI: 10.14218/JCTH.2016.00032.*Correspondence to: GokiSuda,DepartmentofGastroenterologyandHepatology,Graduate School of Medicine, Hokkaido University, North 15, West 7, Kita-ku,Sapporo, Hokkaido 060-8638, Japan. Tel: +81-11-716-1161, Fax: +81-11-706-7867, E-mail: [email protected]

IL-28B genotype29 or hepatitis viral protein-induced IFN signal-ing impairment, are considered responsible for this failure.30,31

In addition, IFN-based therapies are associated with adverseevents(AEs)andoftreatmentdiscontinuationduetotreatment-induced AEs, with especially high rates in patients with severerenal dysfunction, as compared to patients with normal renalfunction. Therefore, safe and effective anti-HCV therapies areurgently needed and critical for patients with severe renaldysfunction, including dialysis patients.

Bartenschlager et al.32 developed an HCV replicon systemin 1999, which along with the advancements in the structuralanalyses of HCV proteins, led to rapid progress in the develop-ment of direct-acting antivirals (DAAs). DAAs directly targetviral proteins and, as such, mainly consist of three classesfor combating HCV infection: those that inhibit the HCV NS3protein, which has protease activity; those that inhibit theHCV NS5A protein; and those that inhibit the NS5B protein,which has polymerase activity.

A number of clinical trials have revealed that the IFN-freeDAA combination therapies lead to significant improvementsin SVR rates and safety;33–37 unfortunately, however, patientswith severe renal dysfunction were excluded from the majorityof these trials. Additionally, in DAA combination therapy, DAAs-resistant cases,38,39 drug-drug interaction and drug excretion(Fig. 1) should be carefully monitored. Several recent studieshave shown efficacy and safety for the IFN-free DAA combina-tion therapy when used in HCV-infected patients with severerenal dysfunction40–42 (Table 1). According to these data, thestandard therapy for HCV-infected patients with severe renaldysfunction could change from the traditional IFN-based strat-egy to the IFN-free DAAs strategy, similar to that used inpatients with normal renal function.

In this review, we discuss the traditional and novel treat-ments for HCV-infected patients with severe renal dysfunctionand the pharmacokinetics of these drugs.

IFN-based therapy

Outline of IFN-based therapy for patients with renaldysfunction

Before the development of DAAs, pegylated (PEG)-IFN mono-therapy or in combination with ribavirin (RBV) was the stand-ard therapy for chronic HCV infection. IFN-based therapy wasalso the standard therapy for patients with renal dysfunc-tion.22 In patients with severe renal dysfunction, the clearanceof IFN and RBV is reduced, because these drugs are mainlyexcreted renally.43 In addition, because of the high molecularweight of INF, HD is unable to remove significant amountsof it. Moreover, patients with renal dysfunction often haveanemia or other complications, and INF- or RBV-induced AEsbecome more problematic.

Efficacy and safety of IFN monotherapy for patientswith renal dysfunction

IFN monotherapy

In 2008, Gordon et al.27 reported a meta-analysis of clinicaltrials using INF monotherapy for hepatitis C treatment inpatients on chronic HD. A total of 20 clinical studies between1966 and 2007 were analyzed. The SVR rates ranged from19% to 71% and the overall SVR rate was 41% [95% confi-dence interval (CI): 33–49]. In addition, the overall treatmentdiscontinuation rate was relatively high, at 26% (95% CI:20–34).

PEG-INF monotherapy

In 2015, Fabrizi et al.44 reported a meta-analysis of clinicaltrials using PEG-INF monotherapy for hepatitis C treatment in

Fig. 1. Anti-HCV drugs and excretion.


Suda G. et al: Novel therapy for HCV in renal impairment

patients on chronic HD. They analyzed 744 patients from24 clinical studies, including 5 randomized control studiesperformed between 2006 and 2014. Overall, the estimated

SVR was 40% (95% CI: 35–46). The dropout rate was 14%(95% CI: 9–20). The most frequent AEs requiring discontin-uation of treatment were hematological and gastrointestinal

Table 1. Overview of the efficacy and safety of IFN-free DAAs combination therapies

Treatment regimen(treatment duration) Patients HCV GT Number

SVR rate (%)(ITT) SAE (%)

Treatmentdiscontinuedrate (%) Special notes

Grazoprevir/elbasvir (12 weeks)

(Roth et al.)40 CKD 4/5includingHD

GT1 122 94.2(115/122)

14.4(16/111)

0 Adverseeffects werecomparable toplacebocontrol group

Paritaprevir/ritonavir/ombitasvir, and dasabuvir with or without RBV (12 weeks)

(Pockros et al.)41 CKD 4/5includingHD

GT1 20 90 (18/20) 20 (4/20) 0 RBV add-on inpatients withGT1a HCVinfection

Daclatasvir/asunaprevir (24 weeks)

(Suda et al.)42 HD GT1 21 95.5 (20/21) 5 (1/21) 5 (1/21)

(Toyoda et al.)72 HD GT1b 28 100 (28/28) 0 3.6 (1/28)

(Kawakami et al.)68 HD GT1 18 100 (18/18) 5.5 (1/18) 0

Sofosbuvir-based therapy

SOF/PEG-IFN/RBV,SOF/RBV,SOF/SMV,SOF/SMV/RBV(Saxena et al.)79

eGFR < 45 GT1-6 73 83 (53/64) 22 (16/73) 7 (5/73) Patients withreduced renalfunctionexperiencedmorefrequently,worsening ofthe renalfunction andseriousadverseeffects

eGFR < 30 GT1-3 17 88 (15/17) 18 (3/17) 6 (1/17)

SOF/SMV (12 weeks)(Nazario et al.)80

eGFR < 30includingHD

GT1 17 100 (17/17) 0 0

SOF/SMV (12 weeks)

SOF/LDV (12 weeks)

(Singh et al.)83 HD GT1,3,4 8 87.5 (7/8) 0 0

SOF/PEG-IFN/RBV,SOF/RBV,SOF/SMV,SOF/SMV/RBV(Beinhardt et al.)84

HD GT1,3,4 10 90 (9/10) 50 (5/10) 0

SOF/LDVSOF/SMVSOF/DCVSOF/RBV(12–24 weeks)(Desnoyer et al.)74

HD GT1,2 12 83% (10/12) 0 0

Abbreviations: DAAs, direct-acting antivirals; HD, hemodialysis; HCV, hepatitis C virus; IFN, interferon; PEG-IFN, pegylated-interferon; SOF, sofosbuvir; RBV, ribavirin; SMV,simeprevir; LDV, ledipasvir; eGFR, estimated glomerular filtration rate; GT, genotype; SVR, sustained viral response; SAE, serious adverse event.



problems. The authors concluded that the efficacy and safetyof PEG-INF monotherapy for dialysis patients with severerenal dysfunction was unsatisfactory.

Efficacy and safety of IFN and RBV for patients withrenal dysfunction

In 2014, Fabrizi et al.45 reported ameta-analysis of clinical trialsusing PEG-INF and RBV for hepatitis C treatment in patients onchronic HD. They analyzed 287 patients from11 clinical studies,including 2 control studies, performed between 1998 and2013. Overall, the estimated SVR was 60% (95% CI: 47–71).The dropout rate was 18% (95% CI: 8–35). The most frequentAEs requiring discontinuation of treatment were anemia(11/46, 23%) and infections (6/46, 13%).

However, in some countries, RBV administration is contra-indicated in patients with severe renal dysfunction, becauseRBV is eliminated through the kidney and cannot be elimi-nated by dialysis.21,43

IFN-based therapy with HCV NS3 protease inhibitorsfor patients with renal dysfunction

The first-generation protease inhibitors, including telaprevir,boceprevir and the PEG-IFN and RBV combination therapy,could achieve an SVR rate of 75% to 85% in patients withnormal renal function.46–48 However, severe AEs, includingcutaneous rash,49 anemia and renal impairment,50 werereported. The data regarding the triple antiviral therapy forHCV-infected patients with renal dysfunction are limited,51–55

but the reported SVR rates have varied between 17% and 86%and the dropout rates have varied between 10% and 20%.

IFN-free DAA combination therapy

In the case of DAA administration to patients on HD, specialattention should be paid to drug-drug interaction, becausethese patients usually receive various drugs. Caution is neces-sary, especially for ritonavir administration, which inhibitsCYP3A4.

Grazoprevir and elbasvir combination therapy for HCV-infected patients with renal dysfunction

Grazoprevir is an HCV NS3 protease inhibitor with broadin vitro activity against multiple HCV genotypes and resistancevariants.56 Grazoprevir is administered at a dose of 100 mgonce a day and is a substrate of CYP3A4, P-gp and OATP. Thisdrug is eliminated mostly through liver, with less than 1%excreted renally.57 Elbasvir is an HCV NS5A inhibitor withpotent multiple genotypic antiviral activity in vitro.58 Elbasviris administered at a dose of 50 mg once a day and it is also asubstrate of CYP3A4, P-gp and OATP.57 Similar to grazoprevir,elbasvir is mainly metabolized through the liver, with less than1% eliminated renally.57

The pharmacokinetics of elbasvir were studied in non-HCV-infected dialysis subjects and subjects with severe renaldysfunction, and then compared with healthy controls.57

The area under the curve (AUC) was 25% higher for the HDsubjects and 46% higher for the subjects with severe renaldysfunction, compared with the controls. In the pharmacoki-netic analysis of grazoprevir in HCV-infected patients, theAUC was 10% higher for the HD patients and 40% higherfor the patients with severe renal impairment, compared to

the controls. In addition, elbasvir and grazoprevir are notremoved by HD.

Several trials showed high efficacy and safety of elbasvirand grazoprevir for HCV-infected patients with various com-plications.40,59–62 In a phase 3 trial for patients with genotype1 or 4HCVandHIV co-infection (C-EDGECO-INFECTION),whenthis combination therapy was administered for 12 weeks anSVR rate of 96%was achieved (210/218).63

The C-SURFER is a phase 3 randomized study designed toevaluate the safety and efficacy of grazoprevir and elbasvircombination therapy for genotype 1 HCV-infected patients withsevere renal dysfunction (stage 4–5 chronic kidney disease(CKD), including HD patients).40 Two-hundred-and-twenty-fourpatients with severe renal dysfunction were randomly assignedto receive grazoprevir and elbasvir for 12 weeks (n = 111) orplacebo (deferred treatment group, n = 113). In addition, 11patients were not randomized and received the dual therapy,and they underwent intensive pharmacokinetic evaluation. Ofthese 235 patients, 179 (76%) needed HD, 122 (52%) hadHCV genotype 1a infection, 14 (6%) were cirrhotic, 80 (34%)had diabetesmellitus and 108 (46%) were African American. Inpatients treated with grazoprevir and elbasvir, the SVR12 rate(ITT analysis) was 94.3% (115/122). And, in the modified fullanalysis set (excluding patients who failed to receive one ormore doses of drug due to issues unrelated to the hepatitis Ctreatment), the SVR12 rate was 99% (115/116). In the safetyanalysis, no patients in the grazoprevir and elbasvir therapygroup were found to have discontinued because of an AE. Onthe contrary, in the control group, 5 (4%) patients discontinuedthe placebo treatment due to AEs. In the grazoprevir and elbas-vir therapy group, AEs were reported for 76% of the cases;however, these results were comparable with the placebogroup (84%). Thus, the C-SURFER study indicated that grazo-previr and elbasvir for 12 weeks was safe and effective, even inpatients infected with HCV genotype 1 and stage 4–5 CKD.

Paritaprevir (PTV)/ritonavir, ombitasvir (OBV), anddasabuvir (DSV) with or without RBV combinationtherapy for HCV-infected patients with renaldysfunction

OBV is an HCV NS5A inhibitor, PTV is a second-generationNS3 protease inhibitor, ritonavir is the pharmacokineticenhancer that is a CYP3A inhibitor, andDSV is a non-nucleosideNS5B polymerase inhibitor. PTV, ritonavir and OBV are admin-istered at a dose of 150 mg, 100 mg and 25 mg once a day,respectively.DSV,however, isadministeredatadoseof250mgtwice a day. PTV is a substrate of CYP3A4/5, P-gp, OATP1B1and OATP1B3 and is metabolized mainly through the liver.64

The single-dose pharmacokinetics of PTV were studied in non-HCV-infected subjects with severe renal dysfunction, and com-pared with subjects with normal renal function. The AUC valuesincreased by 45% in subjects with severe renal dysfunction,compared with the controls.64 Ritonavir is administered toinhibit CYP3A4, resulting in enhancement of the PTV effect. Thesingle-dose pharmacokinetics of ritonavir were studied andthe AUC values increased by 114% in subjects with severerenal dysfunction, compared with the control subjects. OBV ismetabolized mainly by amide hydrolysis and oxidative metabo-lism, and biliary excretion is the major elimination route. Theresult of single-dose pharmacokinetic study of OBV indicatedthat the AUC values were similar between subjects with severerenal dysfunction and controls. DSV is a substrate of CYP3A4,P-gp, BCRP and organic cation transporter 1, and is mainly



metabolized throughthe liver.Thesingle-dosepharmacokineticsof DSV were studied and the AUC values increased by 50% insubjects with severe renal dysfunction, compared with thecontrol subjects.64

In clinical trials, this regimen showed a high rate ofSVR12.

36,65,66 However, patients with severe renal dysfunc-tion were not included. Recently, the RUBY-I study investi-gated the safety and efficacy of these combination therapyin patients with stage 4 or 5 CKD.41 This prospective multi-center study included 20 treatment-naive patients with HCVgenotype 1 infection and without cirrhosis. Six patients withCKD stage 4 and 14 patients with CKD stage 5 or who were onHD were included. Fourteen patients (70%) had the IL-28Bnon-CC genotype and 4 patients (20%) had F3 liver fibrosis.Thirty-three patients with HCV genotype 1a were treated withthis combination therapy and RBV and 7 patients with HCVgenotype 1b infection were treated with the combinationtherapy alone. Overall, 90% (18/20) of the patients achievedSVR12 (95% CI: 69.9–97.2). One patient died after finishingthe treatment, due to issues unrelated to the treatment,and 1 patient experienced virological relapse. In the safetyanalysis, all 20 patients were found to have completed the12 weeks of treatment. Ninety-five percent of the enrolledpatients experienced AEs; however, they were mostly mildor moderate and no patient discontinued treatment due toAEs. A common AE was anemia in the RBV add-on group,and RBV therapy was interrupted in 9 patients due to anemia.

Asunaprevir (ASV) and daclatasvir (DCV) combinationtherapy for HCV infected patients with renaldysfunction

DCV is a first-in-class NS5A inhibitor and has potent pan-genotypicantiviralactivity invitro.67ASV isasecond-generationNS3 protease inhibitor and has antiviral activity againstmultiple HCV genotypes in vitro. ASV is administered at adose of 100 mg twice a day, and is metabolized by CYP3Aand eliminated mostly in the feces.37 The pharmacokineticsof ASV were studied in non-HCV-infected dialysis subjectsand compared with healthy controls.37 The Cmax was 28.6%higher and the AUCwas 10.1% lower in dialysis subjects, com-pared with the controls. Recently, Kawakami et al.68 reportedthe ASV pharmacokinetics determined in HCV-infected dialy-sis patients and compared with HCV-infected patients withnormal renal function. The AUC from 0 to 6 h (AUC 0–6 h) ofASV was significantly lower in HD patients than in the controls(1345 ± 741 lgh/mL vs. 4769 ± 1964 lgh/mL). DCV is admin-istered at a dose of 60 mg once a day, and is metabolized byCYP3A and eliminated mostly in the feces (88%).69 The phar-macokinetics of DCV were also studied in non-HCV-infecteddialysis subjects and compared with healthy controls.69 TheAUC was 26.9% higher in dialysis subjects, compared withthe controls.

In phase 3 trials for patients with genotype 1b infection,the DCV/ASV combination therapy for 24 weeks achieved ahigh SVR rate (82–95%).39,70,71 However, data of efficacyand safety for HCV-infected patients with renal dysfunctionwere not obtained in these clinical trials. This regimen hasbeen approved in several countries and the real-world out-comes have already been reported, including the efficacyand safety of this combination therapy for HD patients.42,68,72

We reported the efficacy and safety of DCV/ASV combinationtherapy from a study of 21 HCV-infected dialysis patients.42

A total of 95.5% (20/21) of the patients achieved SVR12. Of

the 21 patients on dialysis, 3 had NS5A RAVs-Y93H and all ofthe patients with NS5A RAVs achieved SVR12. On the otherhand, 1 patient with NS3 D168E RAVs at baseline experiencedrelapse at 4 weeks post-treatment. In the safety analysis,95.5% of the enrolled patients completed the 24 weeks oftherapy and no patient had lethal AEs. One patient discontin-ued treatment due to an elevated alanine aminotransferase(ALT) level. ALT elevations were observed in 14.3% of thepatients, and this result is comparable with a phase 3 studyconducted in Japan.39

Toyoda et al.72 used propensity score matching to comparethe efficacy and safety of the DCV/ASV combination therapyin 28 patients on HD with those of 56 patients without renaldysfunction. They showed that the rate of SVR12 was 100%(28/28) for the dialysis patients (94.6% for the patients withnormal renal function) and that serum HCV RNA disappearedsignificantly earlier in the HD patients. In addition, they showedthat treatment-related AEs were comparable between the twogroups. Additionally, Kawakami et al.68 analyzed the pharma-cokinetics of DCV and ASV in the dialysis setting and showeda high efficacy of this combination therapy for dialysis patients,with an SVR rate of 100% (18/18).

Sofosbuvir-based therapy for HCV-infected patientswith renal dysfunction

Sofosbuvir is a potent nucleoside NS5B polymerase inhibitorthat has high genetic barrier and high efficacy. Sofosbuviris usually administered at a dose of 400 mg once a daycombined with other DAAs, such as the NS5A inhibitorsledipasvir or DCV, and the NS3 protease inhibitor simepreviror RBV. Sofosbuvir is firstly metabolized to a pharmacologi-cally active nucleoside analog triphosphate GS-461203.73

Subsequently, GS-461203 is metabolized to the inactivemetabolite GS-331007. Importantly, this GS-331007 ismainly excreted through the kidney. The single-dose pharma-cokinetics of sofosbuvir were studied in non-HCV subjectswith moderate (estimated glomerular filtration rate (eGFR)between 30–50 mL/min/1.73 m2) or severe renal impairment(eGFR less than 30 mL/min/1.73 m2) and in HD subjects, andcompared with subjects with normal renal function. Thesofosbuvir AUC was 107% and 171% higher in the patientswith moderate and severe renal impairment, compared withthe control subjects. The GS-331007 AUC was 88% and451% higher in the patients with moderate and severe renalimpairment, respectively. In the dialysis subjects, the sofos-buvir AUC was 28−60% higher, compared to the control sub-jects, when sofosbuvir was administered before or after HD.The GS-331007 AUC in the dialysis subjects was 1280% or2070% higher than in the control subjects when sofosbuvirwas administered before or after HD.73 Thus, exposure ofsofosbuvir and of the metabolite GS-331007 are consideredto be quite increased in patients with renal dysfunction. Quiterecently, Desnoyer et al.74 reported the pharmacokinetics andsafety of a sofosbuvir-containing regime in dialysis patients.They showed that sofosbuvir plasma concentrations werenever detectable before and after the HD and, on the contraryto previously reported results in patientswithnormal renal func-tion, higher GS-331007 plasma concentrations were observed.However, no GS-331007 accumulation was observed, and theregimen was well tolerated generally. Nevertheless, until nowsofosbuvir has not been recommended for patients with renaldysfunction, and in some countries it is even contraindicated.75

Further large-number studies are necessary.



Several studies about sofosbuvir-based therapy for patientswith renal dysfunction have been recently reported.76–79

Saxena et al.79 reported the outcomes of sofosbuvir-basedtherapy for patients with renal dysfunction by using the HCV-TARGET database, which is a multicenter, real-world cohort. Ofthe 1789 enrolled patients, 73 had eGFR of less than 45 mL/min/1.73 m2 (18 patients with eGFR # 30 mL/min/1.73 m2

and 5 patients on dialysis). These patients were compared to1716 patients with eGFR > 45 mL/min/1.73 m2. The includedtreatment regimen was sofosbuvir/simeprevir at 40%,sofosbuvir/RBV at 30%, sofosbuvir/PEG-INF/RBV at 18% andsofosbuvir/simeprevir/RBV at 11%; all patients with eGFR# 45 mL/min/1.73 m2 were treated with sofosbuvir 400 mgonce a day. Patients with baseline eGFR # 45 mL/min/1.73 m2 had a significantly higher rate of cirrhosis (73%) ascompared with the control group (24%). SVR12 was achieved in53 of the 64 (83%) patients with eGFR < 45 mL/min/1.73 m2;this was comparable to patients with eGFR > 45 mL/min/1.73 m2. In addition, 15 of the 17 (88%) patients with eGFR# 30 mL/min/1.73 m2 and all 5 patients on HD at baselineachieved SVR12. However, in the safety analysis, the patientswith eGFR # 45 mL/min/1.73 m2 were found to have experi-enced significantly higher rates of anemia (31%), worseningof the renal function (10%), and any serious AEs (18%). Theauthors concluded that patients with renal impairment needclose expert monitoring.

On the other hand, Nazario et al.80 reported that 15 dialysispatients and 2 patients with severe renal dysfunction allachieved SVR12, with only mild AEs experienced by patientson the sofosbuvir and simeprevir combination therapy. Singhet al.83 reported that 8 dialysis patients received sofosbuvir-based therapy (4 patients with ledipasvir and 4 patients withsimeprevir) and 7 (88%) achieved SVR12. In addition, somestudies on sofosbuvir plus RBV or ledipasvir/sofosbuvir inadults with HCV infection and renal insufficiency are stillongoing (NCT01958281). Thus, the results of the clinicaltrials are expected.

Conclusion

In this review, we described the efficacy, safety and pharma-cokinetics of novel anti-HCV drugs in patients with severerenal dysfunction. These data reveal that grazoprevir/elbasvircombination therapy, PTV/ritonavir/OBV with or without DSVtherapy, and DCV/ASV combination therapy are highly effec-tive and safe for patients with severe renal dysfunction.Sofosbuvir is highly effective and has high genetic barrier,thus representing one of the key drugs for anti-HCV therapy;however, data on sofosbuvir treatment for patients withsevere renal dysfunction are still pending. The results ofongoing clinical trials are expected. Therefore, the use ofsofosbuvir to treat HCV-infected patients with eGFR < 30 mL/min/1.73 m2 or on-dialysis remains off-label and should beleft to experienced physicians or centers, and administeredwith full consent of the treated patients.

Timing of HCV treatment in a kidney transplant candidatemight depend on the waiting time for the kidney transplant. Ifthe waiting time is lengthy, prompt DAAs therapy is necessary,because of several treatment benefits, including restored liverfunction.42 On the other hand, if the waiting time is short,because favorable outcomes of either pre- or post-kidneytransplant DAAs treatment have been reported,40–42,72,81

both timing could be selected.

According to the emerging data, the standard therapy forHCV-infected patients with severe renal dysfunction will likelychange from IFN-based therapy to IFN-free DAAs-basedtherapy. However, the data to date have been mainly limitedto genotype 1-infected patients with severe renal dysfunc-tion. Therefore, if a good prognosis is expected, dialysispatients with genotype 1 HCV infection should be consideredfor DAAs therapy. In dialysis patients with HCV infection otherthan genotype 1, except for dialysis patients for whom prompttreatment would be required, waiting for the next-generationDAAs would be the optimal treatment option; this is becauseclinical trials on the next-generation pan-genotypic DAAstherapy, which are expected to be possible to administer inpatients with renal dysfunction, are underway.82 In addition,real world data are still limited. Further investigations are stillnecessary.

Acknowledgements

This study was supported in part by grants from the Ministryof Education, Culture, Sports, Science and Technologyof Japan and the Japan Agency for Medical Research andDevelopment.


Professor Naoya Sakamoto received lecture fees from BristolMyers Squibb and Pharmaceutical K.K, grants and endow-ments from MSD K.K and Chugai Pharmaceutical Co., Ltd, anda research grant from Gilead Sciences, Inc. Dr. Goki Sudareceived research grants from Bristol Myers Squibb. The otherauthors have nothing to disclose.


Conceived the topic of the review article (GS), collected andreviewed articles (GS, KO, MK, KM, NS), wrote themanuscriptand reviewed the final version (GS, KO, MK, KM, NS),collected the data and created the figure and table (GS, MK,MN, TS).

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Review Article

Mechanisms of Accelerated Liver Fibrosis Progressionduring HIV Infection

Jose D. Debes*1, Paul R. Bohjanen1 and Andre Boonstra2

1Department of Medicine, Division of Infectious Disease and International Medicine, University of Minnesota,Minneapolis, MN, USA; 2Department of Gastroenterology and Hepatology, Erasmus MC, Rotterdam, The Netherlands

Abstract

With the introduction of antiretroviral therapy (ART), adramatic reduction in HIV-related morbidity and mortalityhas been observed. However, it is now becoming increasinglyclear that liver-related complications, particularly rapid fib-rosis development from ART as well as from the chronic HIVinfection itself, are of serious concern to HIV patients. Thepathophysiology of liver fibrosis in patients with HIV is amultifactorial process whereby persistent viral replication,and bacterial translocation lead to chronic immune activationand inflammation, which ART is unable to fully suppress,promoting production of fibrinogenic mediators and fibrosis.In addition, mitochondrial toxicity, triggered by both ART andHIV, contributes to intrahepatic damage, which is even moresevere in patients co-infected with viral hepatitis. In recentyears, new insights into the mechanisms of acceleratedfibrosis and liver disease progression in HIV has beenobtained, and these are detailed and discussed in this review.© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

Liver disease is amajor cause of morbidity and themain causeof mortality, independent of acquired immunodeficiency syn-drome (AIDS), in persons infected with the human immu-nodeficiency virus (HIV), with liver fibrosis being a highlysignificant contributor.1 Although HIV co-infection with hepa-titis Bvirus (HBV)orhepatitisCvirus (HCV) is frequent, there ismountingevidenceof an increased risk in liver-relatedmorbid-ity andmortality in the absence of viral hepatitis.2,3 Cross-sec-

tional studies using liver stiffness measurement (LSM) bytransient elastography have shown a significant degree of fib-rosis amongHIV patients, from 17% in one study to a stagger-ing 41% in a recent study that used lower cutoff values of LSM,although both studies involved patients without viral hepati-tis.4,5 Moreover, liver fibrosis progression is accelerated duringHIV and HCV co-infection. An analysis using paired liver biop-sies showed progression of at least one fibrosis stage(METAVIR) in 34% of HIV/HCV co-infected individuals over a2.5-year period.6

Hepatic fibrosis is a dynamic process initiated by liver injurythat results in increased deposition of extracellular matrixproteins in the space of Disse, the area in between thehepatocytes and the liver sinusoids, which is mainly inhabitedby hepatic stellate cells (HSCs).7,8 Accumulation of extracellu-lar matrix proteins and their decreased removal by matrixmetalloproteinases results in a progressive replacement ofthe liver parenchyma by scar tissue, leading to liver fibrosisand its complications.9 Activation of HSCs is a key event inthe process leading to excessive deposition of extracellularmatrix proteins and the subsequent fibrosis. This activation ofHSCs is triggered by numerous events, such as the release ofcellular components by injured hepatocytes, lipid accumula-tion, the secretion of reactive oxygen species (ROS) producedbymacrophages, andexposure to cytokinesproducedby intra-hepatic macrophages, lymphocytes and endothelial cells.10 Inthis review, we summarize and comment on the differentpotential mechanisms and multiple factors related to liver fib-rosis during HIV infection (Fig. 1). These include: the effects ofantiretroviral therapy (ART), persistent HIV infection-inducedimmune activation, inflammation due to bacterial transloca-tion from the gastrointestinal tract into the portal circulation,and insulin resistance.Wealsodescribemechanisms related toco-infection with viral hepatitis, but we have not extended onthis topic since there are multiple comprehensive reviewsabout this subject in the literature.11,12

Our search strategy included search of the PubMed data-base from 1980 until 2016. We used multiple search terms,including: HIV, liver fibrosis, inflammation, mitochondrialoxidation, etc. We included primarily research articles, aswell as review articles for general relevant and not contro-versial data.

Metabolic dysfunction during HIV infection

Prior to the availability of effective ART, patients with HIVinfection exhibit progressive impairment of their immunesystems, leading to AIDS and death. With effective ART, thedevelopment of AIDS can be prevented and people with HIV



Keywords: HIV; Liver fibrosis; Mitochondrial toxicity; Bacterial translocation.Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; AIDS, acquiredimmunodeficiency virus; LSM, liver stiffness measurement; HSC, hepatic stellatecell; ROS, reactive oxygen species; ART, antiretroviral therapy; NAFLD, non-alcoholic fatty liver disease; HALS, HIV-associated lipodystrophy syndrome;CTGF, connective tissue growth factor; MCP-1, chemokine monocyte chemoat-tractant protein 1; TIMP, tissue inhibitor metalloproteinases; TRAIL, TNF-relatedapoptosis inducing ligand receptor; TGF-b, tumor growth factor-beta; LPS, lipo-polysaccharide; NK, natural killer; NRTI, nucleoside reverse transcriptase inhib-itor; d4T, stavudine; AZT, Zidovudine; DAA, direct acting antiviral.Received: 30 August 2016; Revised: 14 October 2016; Accepted: 21 October2016qDOI: 10.14218/JCTH.2016.00034.*Correspondence to: Jose D. Debes, Department of Medicine, Division of Infec-tious Disease and International Medicine, University of Minnesota, 2001 6th StreetSE, Minneapolis, MN 55455, USA. Tel: +1-612-624-6353, Fax: +1-612-301-1292, E-mail: [email protected]

infection on successful ART have almost the same life expect-ancy as HIV-uninfected persons (although in countries suchas the United States, these elite responders to ART representless than 50% of the HIV population).13 As HIV-infectedpatients age, they develop increased abdominal obesity andexhibit an increased incidence of non-alcoholic fatty liverdisease (NAFLD), with 30-40% of HIV-infected patientsshowing evidence of NAFLD versus 15-20% of HIV-uninfectedindividuals.14,15 Moreover, a recent study shows that HIV-positive individuals with NAFLD have almost double therates of steatohepatitis (lobular inflammation and elevatedlevels of aspartate aminotransferase/alanine aminotransfer-ase) compared to age/sex-matched HIV-negative controls.16

Since excessive lipid accumulation in the liver leads to HSCactivation, NAFLD and steatohepatitis enhance the risk ofdeveloping liver fibrosis. A study in a Canadian populationshowed that the mean BMI among HIV-infected patients withliver histology compatible with NAFLD was 26, compared to amean BMI of 30 among non-infected patients, suggesting that

HIV patients have a lower threshold for developing fattyinfiltration of the liver.17 A cross-sectional study examininghepatic steatosis in HIV patients receiving ART suggestedthat certain medications included in ART represented inde-pendent risk factors for NAFLD and subsequent fibrosis devel-opment.18 However, that study analyzed patients that werealready on treatment and did not prospectively address theeffect of ART treatment itself on NAFLD.

A recent study by our group that evaluated Hispanics withHIV before starting ART found that 66% of HIV-infectedsubjects had some degree of fatty liver.19 In addition, HIVpatients can present with HIV-associated lipodystrophy syn-drome (HALS). HALS is associated with increased fatty aciddeposition in hepatocytes secondary to elevated adiponectinlevels associated to lipodystrophy. Also, HALS can lead toinsulin resistance, with an increase in plasma glucose.20 Highlevels of insulin and glucose eventually stimulate HSC prolifer-ation and increase expression of connective tissue growthfactor (CTGF) which promotes liver fibrosis progression.21,22

Fig. 1. Factors affecting liver fibrosis during human immunodeficiency virus (HIV) infection. HIV can induce a direct effect on hepatic stellate cells (HSCs), affect Tcells and Kupffer cells (KCs), affect hepatocytes through co-receptors, such as CCR5 and CXCR4, and affect mitochondrial DNA. HIV can also increase gut permeabilitythrough depletion of intestinal CD4+ cells, increasing bacterial translocation. Antiretroviral therapy (ART) can induce insulin resistance and mitochondrial toxicity in the liver.Other factors like hepatitis B (HBV), hepatitis C (HCV) and alcohol consumption can affect hepatocytes’ worsening liver fibrosis.


Debes J.D. et al: Liver fibrosis in HIV

Although the mechanisms are not entirely clear, it seemsevident that HIV patients present higher degrees of fat infiltra-tion in the liver in a BMI-related or -unrelated manner, whichworsens the direct and immune-related effects of the virus inliver fibrosis.

Role of persistent HIV replication and chronicimmune activation

Although ART blocks HIV replication, this effect is not com-plete and persistent viral replication occurs, even in cases ofeffective ART outcome.23,24 HIV does not replicate in hepato-cytes, but the HIV co-receptors CXCR4 and CCR5 areexpressed on the hepatocyte surface, and the HIV proteingp120 can induce cell signaling in the liver through theseco-receptors.25,26 In hepatocytes, signals triggered throughactivation of CXCR4 and CCR5 increase expression of pro-collagen alpha-1, a component of type I collagen found inthe extracellular matrix that is characteristic of advanced fib-rosis. Although the studies that yielded these findings wereperformed using hepatic cell lines, it is likely that such effectstranslate to physiologic conditions and contribute to liverfibrosis.27

In contrast to hepatocytes, HIV directly infects HSCs, andthis infection has been shown to promote HSC collagenI expression and secretion of the proinflammatory chemokinemonocyte chemoattractant protein-1 (MCP-1).28,29 The HIVgp120protein also induces activation of tissue-inhibitormetal-loproteinase (TIMP). The two proteins, MCP-1 and TIMP, areimportant for chemotaxis of leukocytes, and these mediatorspromote liver inflammation and fibrogenesis.28,30 HIV rendershepatocytes sensitive to the TNF-related apoptosis-inducingligand receptor (TRAIL), which can lead to hepatocyte deathand subsequent liver fibrosis.31,32 HIV can also infect livermacrophages, known as Kupffer cells.11,33 Although the con-sequences of HIV expression in Kupffer cells remain unclear,these cells are known to playa key role in hepatocyteapoptosisand to be involved in the induction of steatosis.34 Moreover, arecent study showed that HIV-infected macrophage/mono-cytic cells secrete high levels of tumor growth factor-b (TGF-b), which in turn activates HSC to promote fibrosis.35

Interestingly, HIV infection leads to a decreased level ofIL-17-producing CD4+ T cells, a subset of CD4+ cells involvedin liver fibrosis andmodulation of HSCs.36 Moreover, the intra-hepatic compartment in patients with chronic HCV containsmore IL-17-expressing T cells, and neutrophils represent animportant source of IL-17 in the human liver, particularly inlate fibrosis stages.37,38 The interaction between HIV andIL-17 likely plays a role during liver fibrosis, but yet no directcorrelation between HIV, IL-17 and liver fibrosis has beenexposed.

HIV-specific T cells may also play roles in promoting liverfibrosis, but evaluating intrahepatic virus-specific T cellresponses is difficult due to the low frequency of these cellsin the liver and limited availability of liver biopsy materialfrom patients.39 A study performed in HIV/HCV co-infectedpatients demonstrated the presence of both HCV-specificand HIV-specific T cells in the liver.40 Interestingly, thatstudy also showed that HIV-specific T cells were more func-tional than the HCV-specific T cells, and therefore more wellequipped to promote HSC activation.

Thus, persistent HIV infection can promote inflammatoryprocesses in the liver that may stimulate the development offibrosis. The specific interactions of HIV with Kupffer cells, as

well as intrahepatic T cell alterations in HIV, deserve furtherresearch to better understand their role and potential ther-apeutic targeting for liver fibrosis.

Microbial translocation and inflammation during HIVinfection

In the last decade it has become increasingly clear thatmicrobial translocation is an important determinant of clinicalmanifestations and disease progression of HIV.41 Damage tothe intestinal mucosa in patients with HIV leads to disruptionof the gut epithelial barrier, facilitating leakage of microbialproducts from the gastrointestinal tract, with bacterial trans-location into the portal and systemic circulation.42 This isthought to be the consequence of virus-induced depletion ofCD4+ Tcells in the gut and exhaustion of the intestinal macro-phage phagocytic function, both of which lead to enterocyteapoptosis and disruption of tight junctions with loss of integ-rity of the gut mucosa.43,44

Increased microbial translocation results in elevated circu-lating levels of lipopolysaccharide (LPS), LPS-binding protein,soluble CD14 and other mediators, which remain elevatedduring effective ART.41,45 When bacterial translocation occurs,Kupffer cells in the liver become activated through toll-likereceptor (TLR)4 and other pathogen recognition receptorsacting as the first line of defense by clearing bacteria fromthe liver through phagocytosis. In this process, Kupffer cellsare induced to produce cytokines, such as tumor necrosisfactor (TNF), interleukin (IL)-1 and IL-6, which are pro-inflam-matory mediators that promote liver fibrosis by directly acti-vating HSCs or by priming and recruiting other leukocytepopulations.46,47 Similar to infection of CD4+ T cells, HIV canalso directly infect Kupffer cells and promote release of pro-fibrotic mediators.48 However, one study reported that HIVreduces the number of Kupffer cells in the liver via unknownmechanisms.49 This HIV-induced loss of Kupffer cells couldlimit the ability of these cells to directly activate HSCs, butlikely leads to higher levels of circulating microbial products,which in turn can affect immune responses, promotinginflammation.

One of the most well-known pro-fibrogenic mediators isTGF-b, which is produced by Kupffer cells in response to TLRligation upon exposure to microbial compounds.50 Indeed,many studies have reported a positive correlation betweenliver fibrosis and serum TGF-b concentrations, intrahepaticTGF-b mRNA levels as well as strong immunohistochemicalstaining for TGF-b in liver tissue.51,52 TGF-b directly activatesHSC to promote fibrosis, but also plays a homeostatic role toprevent excessive damage by potently suppressing the func-tion of natural killer (NK) cells and Tcells, leading to decreasedhepatocyte apoptosis and release of HSC-activating media-tors.53 Studies showing a clear impact of bacterial translo-cation on liver fibrosis have been performed in HIV/HCVco-infected patients.54 A recent study in HIV mono-infectedindividuals showed increased levels of soluble CD14 correlat-ing with fibrosis, suggesting activation of monocytes inresponse to translocation; however, more studies are neces-sary to further clarify this concept.5

Mitochondrial dysfunction and toxicity

HIV itself can induce mitochondrial toxicity. Studies haveshown that ART-naïve patients exhibit depletion of mitochon-drial DNA in CD8+ as well as B cells, and in CD4+ cells to a



lesser extent (Fig. 2).55,56 Because these lymphocyte subsetsare known to be hyperactivated in persons with chronic HIVinfection, it is possible that persistent activation and high cellturnover causes mitochondrial DNA loss. This mitochondrialdysfunction can induce apoptosis in CD4+ and CD8+ cells andcontribute to fat accumulation in the liver with eventualfibrosis.56,57 Abnormal mitochondrial function induces the

production of ROS, which induces oxidative stress with liverinjury and decreases beta-oxidation of fatty acids, leading toaccumulation of fat in the cytosol.58 These events are keytriggers of NAFLD and steatohepatitis that will lead to thedevelopment of liver fibrosis in this setting.58,59

Interestingly, adipose tissue of untreated HIV-infectedpatients shows an increase in mitochondrial DNA content.55

Fig. 2. Effects of human immunodeficiency virus (HIV) and antiretroviral therapy (ART) on mitochondrial toxicity. HIV virus can decrease mitochondrial DNAin CD4+ and CD8+ Tcells. Different ARTcomponents can inhibit pol-gamma, acetylate kinase and RNA polymerase related to mitochondria, thereby inhibiting mitochondrialDNA. Decrease in mitochondrial DNA leads to less mitochondria and less beta-oxidation of fatty acids, with an increase in free fatty acids and their deposition in liverparenchyma leading to steatosis. Fat also accumulates in peripheral tissues augmenting insulin resistance (IR), which can also be affected by ART (through inhibition oflipolysis), all of which contributes to steatosis and liver fibrosis.



This could represent a compensatory mechanism to counter-act HIV-directed mitochondrial toxicity or HIV-related oxida-tive stress, but could also alter lipid metabolism and promotefat accumulation in the liver. ART, particularly that withnucleoside reverse transcriptase inhibitors (NRTIs), candirectly induce mitochondrial toxicity.56,59 This is of impor-tance since it is estimated that half of the 35 million peopleinfected with HIV worldwide are on ART.60 The specific mech-anisms by which NRTIs induce mitochondrial toxicity are notentirely clear and differ from drug to drug.61 However, mostNRTIs inhibit pol-gamma, a DNA polymerase critical for thereplication of mitochondrial DNA.59 This inhibition leads tomitochondrial dysfunction due to DNA depletion and micro-vesicular steatosis.58

Other mechanisms unrelated to mitochondrial DNA havebeen proposed. Some NRTIs, such as stavudine (d4T), caninhibit mitochondrial RNA in cell lines. For d4T, this finding islikely related to its ability to induce stress on the mitochon-drial RNA polymerase, which may occur in addition toinduction of mitochondrial DNA damage.62 Zidovudine (AZT)inhibits the mitochondrial adenylate kinase and adenosinenucleotide translocator in isolated mitochondria, bothevents leading to mitochondrial dysfunction.61 The majorityof studies related to ART and mechanisms of mitochondrialtoxicity have been performed in vitro, and the mostcommon indicator for toxicity is measurement of mitochon-drial DNA that, although generally accepted, is a rather con-troversial measurement of mitochondrial dysfunction.63

Overall, NRTI-containing regimens present the highest riskfor liver fibrosis, and no ARTregimen is considered completelysafe.60 This is of importance, since although most ART regi-mens applied in the developed world do not include NRTIs, thegreat majority of HIV-infected patients are treated in thedeveloping world (such as Africa) with regimens in whichNRTIs are part of most first line therapies for ART.64,65 Inter-estingly, a recent study suggested that exposure to didano-sine (even if later replaced) is a risk factor for liver fibrosis inHIV, although the mechanisms behind this effect areunclear.66 HIV-infected patients receiving protease inhibitorsfrequently experience lipodystrophy (known as HALS asdescribed above). Protease inhibitors can decrease peripherallipolysis through inhibition of GLUT-4 activity, thereby increas-ing adipocyte size.67 These hypertrophic adipocytes in theperipheral tissue and abdomen lose functional activity andbecome resistant to insulin. Consequently, insulin-resistant

adipocytes secrete less adiponectin, which in turn increasesbody fat, worsening deposition of liver fat and fibrosis.68

Newer protease inhibitors exhibit less incidence of HALS;however, data is emerging on the direct role of HIV-mediatedinflammation promoting endothelial lipase and phospholipaseA2 and thus inducing HALS.69,70

Finally, some HIV-infected populations have a higheralcohol consumption behavior.71 It is well known that alcoholis metabolized in the liver, with acetaldehyde promoting gluta-thione depletion and lipid peroxidation, which leads to mito-chondrial damage exacerbating hepatic steatosis andfibrosis.72 Recent data suggests that alcohol binge drinkingcan increase bacterial translocation from the gut micro-biome.73 This effect could be exacerbated in HIV-infected indi-viduals, leading to a higher degree of alcoholic steatohepatitisand liver fibrosis. Nonetheless, it remains unclear whether theoverall effects of alcohol consumption are accentuated orworsened during HIV infection.74,75

Co-infection with viral hepatitis

Approximately 5 million persons are co-infected with HIV andHCVworldwide.76 Patients co-infectedwithHIVandHCVexpe-rience a more rapid progression to liver fibrosis (Table 1) anddevelop hepatocellular carcinoma at a younger age than thoseinfected with either virus alone (52 vs. 64 years, respec-tively).77–79While the exactmechanisms for advanced fibrosisin co-infection are not entirely clear, several studies have shedlight on this interaction. Both HIVandHCV infection can inducehepatocyte apoptosis via activation of the TRAIL receptors,and increaseTRAIL expression by hepatocytes.32,80 HIV infec-tion renders hepatocytes sensitive to TRAIL with increasedTRAIL-R2 expression. It is possible that upon subsequent chal-lenge of the liver with HCV, TRAIL production increases, result-ing in hepatocyte death with liver fibrosis.31

HCV/HIV co-infected persons also have increased plasmalevels of LPS.81,82 In these co-infected patients with advancedliver disease, bacterial clearance from the liver is impaired,whichmay establish an enhanced state of intrahepatic inflam-mation and create an environment for more rapid progressionof liver fibrosis.12,83 Moreover, the HIV gp120 protein canenhance HCV-induced regulation of TGF-b. Besides the pro-fibrinogenic effects described above, TGF-b can also increaseHCV RNA replication in cell cultures, which is furtherenhanced by HIV, suggesting that HIV augments liver

Table 1. Studies addressing liver fibrosis progression in HIV/HCV using biopsies

Nameref YearN ofpatients Findings Additional comments

Konerman et al.6 2014 282 Accelerated fibrosis progression Association with AST/ALT andfibrosis

Leite et al.79 2015 30 Accelerated fibrosis progression Association with AST/ALT andfibrosis

Schmid et al.96 2015 42* 25% experienced fibrosis progression *N of patients with 2 liver biopsies

Macias et al.83 2009 135 Fibrosis progression; Decreasedprogression by ART

Interval between biopsies of 3.2years

Schiavini et al.78 2011 58 Reduced fibrosis progression with ART Correlation between fibrosis andCD4 count

Sterling et al.97 2010 59* Similar rate of fibrosis in HIV/HCV than inHCV

*59 HIV/HCV vs 59 HCV; 5-yearinterval



damage pathways already established by HCV as well asincreases HCV RNA replication.84,85 With the arrival of newdirect acting antiviral (DAA) medications for treatment ofHCV, it is likely that the fibrinogenic effects related to activeHCV replication in HIV-infected patients will be dramaticallyreduced.86

Interestingly, despite the increasing understanding ofmechanisms by which HCV and HIV interact to cause liverdisease, relatively little is known about viral interactionsbetween HBV and HIV. Moreover, most studies about thenatural history of HBV in HIV have been conducted in areas ofthe world where HBV is acquired in adulthood, leading to thequestion of whether the effects of HIV are the same when HIVis acquired following establishment of chronic HBV, as occursin countries with high HBV endemicity.

Approximately 8% of HIV-infected patients are co-infectedwith HBV, although this rate varies depending on the geo-graphic location. Similar to its interaction with HCV, HIVaccelerates the course of liver disease caused by HBV.87,88

Moreover, HBV patients co-infected with HIV are less likelyto become negative for hepatitis B e antigen, increasing therisk of active hepatitis and liver damage compared to cases ofHBV mono-infection.89 LPS levels are elevated in patientsco-infected with HBV and HIV, likely predisposing to similarintrahepatic inflammation and fibrosis as seen with HIV/HCVco-infection.90 Interestingly, HBV has been shown to suppressTLR-mediated innate immune responses, eliciting activationand expression of pro-inflammatory cytokines.91 This couldhave implications in the development of liver fibrosis duringHIV co-infection, but to date there are no studies addressingthis issue.

TGF-b expression has also been associated to liverfibrosis in HBV-infected patients, but a role in HIV co-infected patients has not been demonstrated.92 Differingfrom HCV/HIV co-infections, liver fibrosis mediated throughapoptosis-related receptors does not seem to play a majorrole in HBV/HIV co-infection. It has also been hypothesizedthat HIV modulation of the HBV-specific immune responsealters the hepatic cytokine environment, affecting liver fib-rosis and disease progression, but this hypothesis has notbeen thoroughly tested. 93

Conclusions

With the introduction of ART, a dramatic reduction in HIV-related morbidity and mortality has been observed. However,despite these advances, HIV-infected patients remain at highrisk for liver-related disease and mortality.94–97 The patho-physiology of liver fibrosis in patients with HIV is a multifacto-rial process. Control of HIV replication will likely mitigate theeffects of the virus in liver fibrosis, but mitochondrial toxicityand insulin resistance from the treatment may negativelyimpact the hepatic environment. In addition, ROS from meta-bolic disturbances such as alcohol consumption, NAFLD, HBVand HCV infection play a major role in the balance betweenimmune response and fibrosis. Key challenges facingresearchers today are to translate our understanding of HIV-mediated liver damage into the development of anti-fibrotictherapies and to identify biomarkers that could allow for indi-vidualized approaches to therapy.

Acknowledgments

Funded in part by the Virgo consortium, the Dutch govern-ment (project number FES0908 to AB), the American Collegeof Gastroenterology (CRA to JDD), and the United States’National Institutes of Health (grant AI096925 to PRB).


None


Interpreted studies, wrote majority of the manuscript (JDD),provided guidance in writing, revised the article for importantintellectual content (PRB), wrote part of the manuscript,edited content, interpreted studies (AB).

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[97] Sterling RK, Wegelin JA, Smith PG, Stravitz RT, Luketic VA, Fuchs M, et al.Similar progression of fibrosis between HIV/HCV-infected and HCV-infectedpatients: Analysis of paired liver biopsy samples. Clin Gastroenterol Hepatol2010;8:1070–1076. doi: 10.1016/j.cgh.2010.08.004.



Review Article

Efficacy and Safety of Daclatasvir in Hepatitis C: An Overview

Nesrine Gamal#, Stefano Gitto# and Pietro Andreone*

Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy

Abstract

Hepatitis C virus (HCV) infection is a growing public healthconcern, with 184 million people infected worldwide. Duringthe past decade, interferon has been the backbone of HCVtreatment, even though it remains far from ideal. The latestdevelopment of the new direct antivirals has drasticallychanged the treatment approach for chronic hepatitis C(CHC). Inhibitors of the HCV NS5A region have garneredremarkable interest among treating physicians, due to theirhigh potency and favourable safety profile. In particular,treatment with daclatasvir (DCV) has yielded high rates ofvriologic response in patients infected with genotype (Gt) 1and Gt 3, when used in combination with other antivirals of adifferent class, such as sofosbuvir. Although few data areavailable for DCV treatment of the other Gts, the results inpatients with Gt 2 and Gt 4 infection appear promising, as dothose for unique patient populations. NS5A-resistant viralvariants can pre-exist or emerge after treatment failure forthe HCV NS5A inhibitors. Nonetheless, DCV-resistant viralvariants continue to be sensitive to interferon and otherclasses of antivirals such as NS3/4A and NS5B inhibitors.Herein, we aimed to provide an overview of the currentknowledge about DCV in the treatment of CHC.© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

Hepatitis C virus (HCV) infection is an increasing public healthconcern, with an estimated 184 million people infected acrossthe globe.1 Moreover, approximately 700,000 deaths annuallyare reported as resulting fromHCV-related complications, mostof which involving cirrhosis and hepatocellular carcinoma.2

For the past decade, interferon (IFN) has been the back-bone of all anti-HCV treatment strategies. However, all of theregimens utilizing IFN yielded relatively unsatisfactory ratesof virologic response, along with poor safety profiles. Indeed,

research to define the molecular mechanisms underlying HCVreplication has facilitated the development of new antiviralmolecules that halt the process by targeting/blocking variousfactors at a variety of steps.3 The HCV viral genome encodes asingle open reading frame of; 3000 amino acids. It is cleavedby host and viral proteases into three structural proteins (thenucleocapsid and envelope glycoproteins: core, E1, and E2)and seven non-structural ones (protein p7, derived from E2cleavage and NS2, NS3, NS4A, NS4B, NS5A, and NS5B).3

A plethora of direct-acting antivirals (DAAs) is now avail-able. The first generation DAAs consist of the NS3/4A serineprotease inhibitors, such as (telaprevir and boceprevir. Thesecond-generation DAAs consist of the NS3/4A proteaseinhibitors, such as simeprevir, asunaprevir (ASN), paritapre-vir and grazoprevir, the NS5B non-nucleoside polymeraseinhibitors, such as dasabuvir, and nucleotide analogues, suchas sofosbuvir (SOF). Recently, a new class of DAAs has beendeveloped, and these include ledipasvir, daclatasvir (DCV),elbasvir, velpatasvir and ombitasvir. This new class is madeup exclusively of NS5A replication complex inhibitors, andeach has shown excellent antiviral efficacy and a low barrierto resistance.4

NS5A is a phosphorylated protein that has been studiedextensively.5,6 Its critical roles in viral replication, assembly andsecretion6 suggest it as a good target of antiviral molecules.

DCV/BMS-790052 (Bristol-Myers Squibb) is an NS5Ainhibitor. This class of DAAs has two distinct mechanisms ofaction: a) blocking of HCV-RNA replication to prevent for-mation of the so-called ‘membranous web’ in which functionalHCV-RNA replication takes place; and b) rapid inhibition ofintracellular virion assembly.7 The benefits of DCV include apicomolar potency, a pharmacokinetic profile that permitsonce-daily dosing, and broad genotypic coverage in vitro.8,9

In addition, DCV is highly selective for HCV, as evidenced byits lack of clinically important antiviral activity against otherviruses [e.g. human immunodeficiency virus (HIV)].10 InJune 2014, The European Medicines Agency’s Committee forMedicinal Products for Human Use (CHMP) granted marketingapproval for DCV in association with other medicines for thetherapy of chronic hepatitis C (CHC). Moreover, the combina-tion of DCV and ASN (an HCV NS3/4A protease inhibitor) wasapproved in Japan for the treatment of patients infected withGt 1 HCV.11

Pharmacokinetics

The pharmacokinetic patterns of DCV have been studied inboth healthy and chronically infected patients. In HCV-infectedsubjects, the peak plasma concentrations occurred within2 hours after multiple oral doses of DCV tablet administeredin doses ranging from 1 to 100 mg once daily.10 In regards to



Keywords: Daclatasvir; NS5A inhibitor; Hepatitis C; Direct antivirals; Newantiviral therapy.Abbreviations: AE, adverse event; ASN, asunaprevir; DCV, daclatasvir; CTP,Child-Turcotte-Pugh; CHC, chronic hepatitis C; Gt, genotype; HCV, hepatitis Cvirus; Peg-IFN, pegylated-interferon; RBV, ribavirin; SAE, serious adverse event;SOF, sofosbuvir; SVR, sustained virologic response.Received: 06 September 2016; Revised: 10 October 2016; Accepted: 14 October2016qDOI: 10.14218/JCTH.2016.00038.#These authors contributed equally to the manuscript.*Correspondence to: Pietro Andreone, Department of Medical and SurgicalSciences, University of Bologna, Via Massarenti 9, Bologna 40138, Italy. Tel:+39-051-2143618, Fax: +39-051-345806, E-mail: [email protected]

effects of food intake, no reduction in DCV concentration wasobserved when the drug was administered after a light meal.Notably, DCV has been found to be primarily removed byhepatic metabolism and direct biliary excretion. As a conse-quence, renal impairment is not a contraindication for DCVadministration.10 Fundamentally, no dosage adjustment ofDCV is necessary for subjects with hepatic impairment [allclasses of Child-Turcotte-Pugh (CTP)].

Clinical Trials with Daclatasvir

DCV has been evaluated in more than 13,000 treatment-naïve and treatment-experienced CHC patients, harbouringinfections with all the various Gts. It has also been studied incombination with pegylated-interferon (Peg-IFN) or withother DAAs, with and without ribavirin (RBV). Based on Gt,we will subsequently describe all clinical trials that have beenconducted to assess both efficacy and safety of DCV. To thisend, we performed a standardized search of the MEDLINE,PubMed, Google Scholar and international conferenceabstract databases using the following search terms: ‘BMS-790052’, ‘daclatasvir’, ‘directly acting antivirals’ and ‘hep-atitis C.’ A manual exploration of references was alsoimplemented to recognize articles not found by the elec-tronic searches.

Interferon-based therapeutic regimens (Table 1)

Genotype 1

In the phase IIb study known as COMMAND-1, Hezode et al.12

assessed the effectiveness of Peg-IFN and RBV plus DCV intreatment-naïve Gt 1 and Gt 4 subjects. Three hundred andninety-five Gt 1 patients were randomized to receive standardtherapy plus DCV at 20 mg (arm A), DCV at 60 mg (arm B) or

placebo (arm C). The addition of DCV improved sustainedvirologic response (SVR) rates to 59%13 and 60% (B) com-pared to 38% in arm C. Notably, the DCV arms (A and B)were similar to the placebo arm (C) in rates of discontinuationand serious adverse events (SAEs) without safety eventsrelated to the experimental drug.

Lok et al.14 conducted a randomized, phase IIa, open-label,24-week treatment trial. Subjects with Gt 1 infection who pre-viously failed HCV antiviral treatment were randomized to thefollowing 5 different regimens, all including DCV once daily(60 mg): 1) DCV plus ASN (NS3 protease inhibitor, 200 mg)twice-daily (n=18); 2) DCV plus ASN once-daily (n=20);3) DCV plus ASN twice-daily plus Peg-IFN and RBV (n=20);4) DCV plus ASN once-daily plus Peg-IFN and RBV (n=21);5) DCV plus ASN twice-daily plus RBV (n=22). Indeed, ratesof SVR24 were highest in group 3 and group 4 (90% and 95%,respectively) and without virologic breakthrough. However, therate of such an event was rather high in group 5 (10/22, 46%,all Gt 1a). Headache, diarrhoea, and asthenia were the mostcommon adverse events (AEs). Grade 3 or 4 hematologic lab-oratory alterations occurred only in the groups treated withIFN-based therapy.

Genotype 2

Only one study reported data regarding DCV plus Peg-IFNand RBV in Gt 2 treatment-naïve subjects. Dore et al.15

reported data of a randomized, double-blind, phase IIbstudy that involved subjects infected with Gt 2 and Gt 3.Patients were assigned to receive 12 or 16 weeks of DCV(60 mg once daily) or 24 weeks of placebo, each combinedwith Peg-IFN and RBV. Among the patients with Gt 2 infec-tion, a similar SVR24 was achieved among those subjectstreated with DCV for 12 or 16 weeks (83%) versus thosewho received placebo (63%).

Table 1. Interferon-based trials utilizing daclatasvir

FirstAuthorRef Design

Genotype(n of subjects)

TreatmentStatus Cirrhosis Treatment Arms SVR (%)

Hezode12 Double-blindplacebocontrolled

1 (365) Naïve Yes A- DCV (20 mg)+Peg-IFN+RBVB- DCV (60 mg)+Peg-IFN+RBVC- Placebo+Peg-IFN/RBV

A- 59%B- 60%C- 38%

4 (30) Naïve Yes A- DCV (20 mg)+Peg-IFN+RBVB- DCV (60 mg)+Peg-IFN+RBVC- Placebo+Peg-IFN/RBV

A- 67%B- 100%C- 50%

Lok14 Open-label 1 (101) Null-responders

No 1- DCV+ASV twice daily2- DCV+ASV once daily3- DCV+ASV twice daily+Peg-IFN+RBV4- DCV+ASV once daily+Peg-IFN+RBV5- DCV+ASV twice daily+RBV

1- 83%2- 60%3- 90%4- 95%,5- 23%

Dore15 Double-blindplacebo control

2 (71) Naïve Yes A- DCV+Peg-IFN+RBV (12w)B- DCV+Peg-IFN+RBV (16w)C- Placebo+Peg-IFN/RBV

A- 83%B- 83%C- 63%

3 (80) Naïve Yes A- DCV+Peg-IFN+RBV (12w)B- DCV+Peg-IFN+RBV (16w)C- Placebo

A- 69%B- 67%C- 59%

Abbreviations: Peg-IFN, pegylated-Interferon; DCV, daclatasvir; SOF, sofosbuvir; RBV, ribavirin; ASV, asunaprevir; BEC, beclabuvir.


Gamal N. et al: Daclatasvir in CHC: an overview

Genotype 3

Only the above-reported study by Dore et al.15 assessed DCVplus standard therapy in Gt 3 treatment-naïve patients. Therates of SVR24 attained were 69%, 67%, and 59% of patientsin the 3 groups, respectively.

Genotype 4

In the above-cited COMMAND-1 study,12 30 Gt 4 treatment-naïve patients were randomized to receive DCV (20 mg or60 mg) or placebo plus Peg-IFN for 24 weeks. Notably, SVRrates for both subgroups (DCV 20 mg, 66.7% and 60 mg,100%) were high when compared with placebo (50%) andpatients infected with Gt 1.

Interferon-free therapeutic regimens (Table 2)

Genotype 1

Everson et al.16 reported data from 66 Gt 1 treatment-naïve,non-cirrhotic subjects that had been randomly allocated toreceive DCV (60 mg, once daily), ASN (200 mg, twice daily),and the NS5B non-nucleoside polymerase inhibitor BMS-791325 (75 mg or 150 mg, twice daily) for 12 or 24 weeks.Sixty-one of the patients (92%) achieved SVR12. Notably, therates of SVR were similar among the 12- and 24-week groups.During the study, two patients experienced virologic break-through and one patient relapsed (3/66, 4.5%). There wereno deaths or discontinuations resulting from SAEs, or evenAEs, related to the study drugs.

Table 2. Interferon-free trials utilizing daclatasvir

FirstAuthorRef Design

Genotype(n ofsubjects) Status Cirrhosis Treatment Arms Svr (%)

Lok14* Open-label 1 (101) Null-responders No A- DCV+ASV twice dailyB- DCV+ASV once dailyC- DCV+ASV twice daily+Peg-IFN+RBVD- DCV+ASV once daily+Peg-IFN+RBVE- DCV+ASV twice daily+RBV

A- 83%B- 60%C- 90%D- 95%,E- 23%

Everson16 Open-label 1 (66) Naïve No DCV+ASV+BMS-791325

92%

Sulkowski17 Open-label 1 (167) Naïve andExperienced

No DCV+SOF6RBV Naïve 98%Experienced 98%

2 (26) Naïve andExperienced


3 (18) Naïve andExperienced


Manns18 Open-label 1b (747) Naïve,Experienced andIFN-ineligible

Yes DCV+ASV Naïve 90%Experienced 82%Ineligible 82%

Poordad19 Open-label 1 (415) Naïve andExperienced

No DCV+ASV+BEC Naïve 92%Experienced 89%

Muir21 Open-label 1 (202) Naïve andExperienced

Yes DCV+ASV+BEC6RBV Naïve 93%Experienced 87%

Nelson24 Open-label 3 (152) Naïve andExperienced

Yes DCV+SOF Naïve 90%Experienced 86%

Leroy25 Open -label 3 (50) Naïve andExperienced

Yes DCV/SOF+RBV Naïve 92%Experienced 89%

Welzel22 Open-label 3 (102) Naïve andexperienced

Yes DCV+SOF6RBV Naïve 94%Experienced 82%

Hezode26 Open-label 3 (282) Naïve andexperienced

Yes DCV+SOF6RBV With RBV: 87%Without RBV: 83%

Hassanein28 Open-label 4 (21) Naïve No A- DCV+BEC (75 mg)+ASVB- DCV+BEC (150 mg)+ASV

A- 91%B- 90%

Abbreviations: DCV, daclatasvir; SOF, sofosbuvir; RBV, ribavirin; ASV, asunaprevir; BEC, beclabuvir.*cited also in Table 1.



The antiviral combination of DCV and SOF, with or withoutRBV, was evaluated in an open-label study by Sulkowskiet al.17 in which 167 Gt 1 subjects (126 naïve, 41 experienced)and 44 Gt 2 or Gt 3 treatment-naïve patients were randomlyassigned to receive DCV (60 mg) plus SOF (400 mg), with orwithout RBV, for 12 or 24 weeks. One hundred and sixty-fourof the 167 Gt 1 patients (98%) attained SVR12. Notably, ratesof SVR12 were similar for patients infected with the variousviral subtypes (Gt 1a, 98%; Gt 1b, 100%), IL28B genotype(CC, 93%; non-CC, 98%), and RBV treatment (with, 94%;without, 98%). As expected, RBV recipients had amajor reduc-tion in haemoglobin level. Fatigue, headache, and nausea werethe most common AEs reported. Serious AEs occurred in10 patients, and in 2 of those cases (<1%) led to treatmentdiscontinuation.

HALLMARK-DUAL18 was a phase III multicohort studyconcerning Gt 1b patients who were treatment-naïve andprevious non-responders/ineligible for Peg-IFN/RBV, includingpatients with cirrhosis. The treatment-naïve patients (n=307)received DCV (60 mg, once daily) plus ASN (100 mg, twicedaily) or placebo for 12 weeks. The non-responders (n=205)and ineligible, intolerant patients (n=235) received the sametherapy for 24 weeks. This IFN-free regimen led to SVR12 in182 (90%) of the patients in the treatment-naive cohort, 168(82%) in the non-responder cohort, and 192 (82%) in the ineli-gible, intolerant cohort. Among the treatment-naïve patients,no differences were found in terms of SVR12 between patientswith or without cirrhosis (91% and 89%, respectively). Like-wise, nomeaningful differences were observed in terms of SVRamong the previously-treated or IFN-ineligible patients withcirrhosis. Overall, the rates of SAEs were low (5%–7%)across all treatment groups. Similarly, grade 3 or 4 laboratoryanomalies and AE-related discontinuations were uncommon.No deaths were recorded.

UNITY-1 was an open-label international study19 thatinvolved 312 treatment-naïve and 103 treatment-experiencedpatients with Gt 1 infection who had been enrolled and admin-istered DCV (30 mg), ASN (200 mg), and beclabuvir20 (anNS5B non-nucleoside inhibitor; 75 mg) for 12 weeks. SVR12was obtained by 379 of the 415 patients (91.3%; 95% CI:88.6%–94.0%). In particular, this finding involved 287 ofthe 312 treatment-naïve patients (92.0%) and 92 of the103 treatment-experienced patients (89.3%). Rates of virologicfailure were low (n=34/415, 8%). AEs leading to treatment dis-continuation occurred in less than 1%. Notably, one patient diedat post-treatment week 3 for a reason not related to the study.

The all-oral regimen of DCV plus ASN and BEC, with theaddition of RBV, was also evaluated for patients with Gt 1infection and compensated cirrhosis in the UNITY-2 study.21

One hundred and twelve treatment-naïve and 90 treatment-experienced patients were treated for 12 weeks. In thetreatment-naïve patients, SVR12 was achieved by 98% and93% with and without RBV, respectively. Comparable rates ofSVR12 were achieved among the treatment-experiencedpatients (93% and 87%, with and without RBV, respectively).In total, SVR12 was achieved by 88% of those receiving thefixed-dose combination alone and by 95% of those with RBVadded to the regimen. Based on further analysis of the sub-groups, the investigators suggested that inclusion of RBV withthe regimen may be considered for patients with Gt 1a infec-tion. The treatment was considered safe and well toleratedwith only three serious AEs (2.5%) reported that had beenconsidered as treatment-related, and in 4 cases (3.3%) anAE led to treatment discontinuation.

An important real-world study by Welzel et al.22 evaluateda large cohort of 458 patients infected with Gt 1, 2, 3, 4 and5, among which 42% had decompensated cirrhosis. Therecommended therapeutic regimen was DCV plus SOF withor without RBV for 24 weeks. Patients were infected primar-ily with HCV GT 1b (36%) and 1a (33%). In the modifiedintention-to-treat analysis, SVR12 was achieved by 149/155 (96%) of GT 1a patients and 150/169 (89%) of GT 1bpatients. Notably, rates of virologic response were high irre-spective of the severity of hepatic disease.22

Genotype 2

In the above-mentioned trial by Sulkowski et al.,17 the DCVplus SOF combination (with or without RBV) was evaluatedin 26 treatment-naïve Gt 2 patients, including those withcirrhosis. After randomization, all subjects received the DCV(60 mg) plus SOF (400 mg) treatment for 24 weeks. Theoverall rate of SVR12 was 92%. Notably, the addition of RBVdid not provide any additional benefit in terms of SVR but didincrease side effects.17

A recently published small trial conducted by Mangiaet al.23 explored the IFN-free and RBV-free combination treat-ment with DCV plus SOF among patients with Gt 2 infectionand including treatment-experienced patients and those withCPT class A&B cirrhosis. A small cohort of 20 patients wereincluded in this study, and 19 received the DCV plus SOF com-bination for 12 or 24 weeks. Surprisingly, all patients attainedSVR, supporting the use of this regimen for 12 weeks in non-cirrhotics or 24 weeks in cirrhotic Gt 2 patients who are RBV-intolerant, including those with decompensated disease.23

Genotype 3

The combination of DCV plus SOF, with or without RBV, wasassessed in the same trial by Sulkowski et al.17 in 18 Gt 3infected, treatment-naïve patients, including patients withcirrhosis. In this subgroup, the SVR12 was 89%. As forpatients infected with Gt 1 and Gt 2, the inclusion of RBVproduced a negative impact on the safety profile, withoutadding any additional benefit in terms of SVR.

In the phase III ALLY-3 study, Nelson et al.24 evaluated a12-week therapeutic protocol with DCV (60 mg) plus SOF(400 mg) in patients infected with Gt 3 and who were eithertreatment-naïve (n=101) or treatment-experienced (n=51).Patients with cirrhosis (up to 50% in each cohort) were eligiblefor the study as well. During the course of treatment, no viro-logic breakthrough was registered. SVR12 rates were 90% (91/101) and 86% (44/51) in the treatment-naïve and treatment-experienced subjects, respectively. Among the non-respond-ers, 5 of 7 patients (71.4%) previously treated with a SOF-based regimen and 2 of 2 (100%) who previously failed treat-ment with alisporivir achieved SVR12. As expected, SVR rateswere lower in patients with severe fibrosis (63% vs. 96%). Theadministration of this DCV plus SOF treatment regimen led tono AEs that necessitated treatment withdrawal. Only one SAE(<1%), not related to study medications, was registered.

ALLY-3+ is a recently published phase III study thatevaluated DCV plus SOF and RBV in treatment-naïve andtreatment-experienced Gt 3 patients with advanced fibrosis orcirrhosis. The subjects had been randomized in a 1:1 mannerto receive either a 12- or 16-week treatment regimen.Overall, 50 patients were treated and 45 of those achievedSVR12 (90% overall; 92% among the treatment-naïve and



89% among the treatment-experienced). All patients withadvanced fibrosis attained SVR12 in both the 12- and 16-weekarms. Among the patients with cirrhosis, SVR12 was achievedin 83% and 89% of cases in the 12- and 16-week arms,respectively. This treatment regimen was considered safe andwell tolerated since no treatment interruptions related to AEswere observed.25

A similar study was the real-life experience of the Euro-pean DCV compassionate use program (CUP; AI444-237),in which the combination of DCV plus SOF, with or withoutRBV, for 24 weeks in Gt 3 patients with advanced liver diseasewas evaluated. Approximately 70% of the participants weretreatment-experienced. In total, 485 patients of differentgenotypes were included in this program and 460 wereincluded in the primary efficacy analysis. SVR12 was achievedby 82/93 (88%) of the patients infected with Gt 3, including88% among those treated with DCV plus SOF and 89%treated with DCV plus SOF and with RBV.22

Of note, improvements in liver function were observed,with few cases of discontinuation due to AEs, of treatment-correlated serious AEs, or of grade 3/4 laboratory anomalies.

The French ATU (Temporary Authorisation for Use)program for DCV provided early, pre-market authorizationaccess to DCV for HCV patients with advanced liver diseaseand without other antiviral treatment options. The primaryefficacy population (n=284) was composed of patientswithin the safety population (n=468) who had availableHCV-RNA data at 12 weeks after treatment discontinuation.Approximately 73% of the enrolled subjects were treatment-experienced, including patients with advanced fibrosis(15%) and cirrhosis (79%). All patients were randomizedto receive either a 12- or 24-week course of DCV plus SOF,with or without RBV at the discretion of the investigator. Theinterim analysis confirmed high rates of overall SVR (82%–100%). However, the secondary analysis of the subgroupsdemonstrated a rather unsatisfactory SVR12 rate among thecirrhotic patients who had undergone the 12-week treat-ment course (72%); thus, 24 weeks was suggested as theoptimal duration to treat such patients.26

Such data about the duration of treatment in cirrhoticpatients were confirmed by Eley et al.27 who concluded thatcirrhotic patients infected with Gt 3 required a longer durationof treatment (>12 weeks) with the DCV plus SOF combinationtherapy or with the addition of RBV, in case they should betreated for only 12 weeks.

Genotype 4

In the study by Hassanein et al.,28 21 Gt 4 treatment-naïveadults were randomized in a 1:1 manner to receive twice-daily oral regimen of BEC at 75 mg or at 150 mg, each withDCV (30 mg) and ASN (200 mg), for 12 weeks. In this explor-atory study, SVR12 was achieved by 90.9% (10/11) of thepatients who received BEC at 75 mg and by 90% (9/10) ofthe patients who received 150 mg. No serious AEs or discon-tinuations due to AEs were reported.

Hezode et al.26 explored the efficacy of DCV plus SOF, withor without RBV, in the French ATU programme; patients wereinfected with HCV Gt 4 (n=2015), 5 (n=26) and 6 (n=5). Thisstudy protocol included adult patients with the following con-ditions: 1) METAVIR fibrosis score of F3 or above; and 2) withany fibrosis score: extrahepatic manifestations, post-livertransplant HCV recurrence or indication for liver or kidneytransplant. Patients were treated for 12 or 24 weeks. In this

real-life study, the rates of SVR12 were generally high, 91%,100% and 100% among the Gt 4, Gt 5 and Gt 6 patients,respectively. Treatment terminations due to AEs were uncom-mon (1%) and none were considered drug-related.

Unique patient populations

Liver transplant recipients

HCV recurrence after liver transplantation is associated withlower rates of patient survival. Indeed, SVR is associated withincreased life expectancy in such cases.30,31

Herzer et al.32 were the first to describe the first use ofDCV, SMV and RBV as an all-oral triple regimen administeredto 6 liver transplant recipients with recurrent CHC; this cohortincluded one patient that was infected with Gt 1a and5 patients infected with Gt 1b. All subjects were treated for24 weeks. Notably, although none of the 6 patients respondedto previous treatment using the standard IFN-based therapy,4 exhibited SVR24. Furthermore, clinical measures of liverfunction improved substantially for all patients and AEs werefew and limited to RBV-induced moderate anaemia. Impor-tantly, adjustments to the immunosuppressant dosage werenot required.

In the phase 3 ALLY-1 trial, Poordad and colleagues33 used a12-week course of SOF plus DCV with RBV to treat 53 patientswith HCV recurrence after liver transplant and 60 patientswith advanced cirrhosis; infections with Gt 1 (76%), Gt 2,Gt 3, Gt 4, and Gt 6 were represented. Patients were bothtreatment-naïve and treatment-experienced. Enrolled patientshad CPTclass A, B, or C cirrhosis and Model for End-Stage LiverDisease (MELD) scores ranging from 8 to 27. Interestingly, theoverall SVR12 rate was 82% for the Gt 1 patients withadvanced cirrhosis and 95% and 91% for the patients withGt 1 and Gt 3, respectively, in the post-transplant arm. Nodeaths occurred during the study. SAEs were observed in17% of the advanced cirrhosis patients and 9% of the post-transplant subjects. However, no drug-related events wereconsidered to have been related to the study drugs.

A larger scale study conducted by Fontana et al.34 exploredthe DCV-based regimens in liver transplant recipients whoalso had severe recurrent CHC. The study included 97 livertransplant recipients, of which 93% had Gt 1 HCV infectionand 31% had biopsy-proven cirrhosis. The mean MELD scorewas 13±6, and 12% of the patients were CTP class C. Anti-viral regimens comprised DCV plus SOF (n=77), DCV plusSMV (n=18), and DCV plus SMV and SOF (n=2); RBV wasadministered to 35% of the patients. Overall, 84/97 (87%)patients attained SVR12. Eight deaths occurred during thestudy and none were attributed to therapy. Notably, hepaticfunction, as expressed by both CTP and MELD scores, consid-erably improved with the beginning of antiviral therapy.

HCV/HIV co-infected patients

The DCV plus SOF combination has shown efficacy in patientswith HCV and HIV type 1 (HIV-1) co-infection. ALLY-235 wasan open-label clinical trial that included 151 treatment-naïveand 52 treatment-experienced CHC patients, all infected withHIV-1. The treatment-naïve subjects were randomly assignedin a 2:1 manner to receive either 12 weeks or 8 weeks of DCV(60 mg, once daily, with dose modification for concomitantantiretroviral therapy) plus SOF (400 mg, once daily). Thetreatment-experienced patients were assigned to undergo



12 weeks of therapy at the same doses. The patients wereinfected with HCV Gt 1 (83%), Gt 2, Gt 3 and Gt 4, and14% had compensated cirrhosis. Rates of SVR12 across allgenotypes were 97% after 12 weeks of treatment, irrespec-tive of their anti-HCV treatment history or a concomitant anti-retroviral regimen, without disruption in their HIV-1 virologiccontrol. Nonetheless, HCV relapse was more common (24%)after 8 weeks of treatment than after 12 weeks of treatment,suggesting that the 12-week course might be the preferredduration of treatment for most patients with HIV/HCV co-infection. Notably, there were no study drug discontinuationsbecause of AEs, and SAEs were rarely reported.

An interesting Austrian real-life study36 investigated bothsafety and efficacy of DCV and SOF combination in 31 HIV/HCVco-infected patients with advanced liver disease. Approxi-mately half of the patients in the study were treatment-experienced, and the various treatment durations were:12 weeks in the Gt 1 and Gt 4 patients without cirrhosis;24 weeks in the Gt 1 and Gt 4 patients with cirrhosis; and24 weeks in the Gt 3 patients. If HCV-RNA was detectable at4 weeks before the end of therapy, the duration was extendedby 4 weeks at a time. Surprisingly, 100% of the enrolledpatients attained SVR12. Notably, HCV eradication determineda remarkable improvement in liver stiffness (median change,-3.6 kPa) and in liver enzymes.

Chronic haemodialysis patients with CHC

The DCV plus ASN combination therapy was assessed inJapanese haemodialysis subjects with Gt 1 infection, includingpatients with compensated cirrhosis.37 Overall, 21 patientscompleted a 24-week treatment course of DCV (60 mg, oncedaily) and ASN (100mg, twice daily). Rates of SVR12 were high(20/21, 95%). Furthermore, this combination had an excellentsafety profile and even significantly improved levels of serumalanine aminotransferase and albumin. Recently, Toyodaet al.38 investigated the safety and viral responses of thesame combination (DCV plus ASN for 24 weeks) in 28 patientswith Gt 1 infection who were receiving hemodialysis, and 56who had no renal dysfunction. The two groups included cirrhoticsubjects (39%). The rates of SVR were comparable betweenthe two groups; the rate of SVR12 was 100% in patients receiv-ing haemodialysis and 94.6% in subjects without renal dys-function. In addition, no important AEs were reported.

Warnings and precautions

In clinical trials, approximately 2400 subjects with CHC havebeen treated with the recommended dose of DCV in combi-nation with another anti-HCV drug in clinical studies. Overall,this drug was found to be safe and well tolerated in all testedcombinations. In particular, fatigue (14%), headache (14%),nausea (8%), and diarrhoea (5%) were among the mostcommonly reported AEs among patients treated with DCV/SOF combination with and without RBV.10 However, in 2015,the FDA advised against the concomitant use of amiodaronewith DCV in combination with SOF, as this can cause serioussymptomatic bradycardia.39

Drug-drug interactions

DCV is a CYP3A4 substrate, as well as a substrate andinhibitor of P-gp. Additionally, it inhibits organic anion trans-porting polypeptide (OATP) 1B1, organic cation transporter

(OCT) 1 and breast cancer resistance protein (BCRP). There-fore, attention must be paid during concomitant administra-tion of substrates of P-gp, OATP 1B1, OCT1 or BCRP. In fact,they may prolong their therapeutic effect and adverse reac-tions. Notably, DCV dose should be reduced to 30 mg oncedaily when co-administered with strong inhibitors of CYP3A4(e.g. telaprevir, boceprevir, atazanavir/ritonavir, cobicistat,clarithromycin, telithromycin, and ketoconazole). Similarly,DCV dose should be increased to 90 mg/day when it isadministered along with moderate CYP3A inducers (e.g.bosentan, dexamethasone, and nafcillin). Its co-administrationwith strong inducers of CYP3A4 is contraindicated. Drug-druginteractions and dose recommendations of DCV with com-monly used medications are summarized in Table 3.

Onset of resistance: A real clinical problem?

So far, HCV NS5A-targeting molecules are amongst the mostpotent antivirals with broad activity against HCV genotypesand subtypes.40 However, the first generation NS5A-inhibitorsshowed a moderately low genetic barrier, leading to emer-gence of drug-resistant mutants.41 Such mutations have beendescribed in the N-terminal region of HCV NS5A.41 The in vitroresistance profile of DCV is well known, thanks to the develop-ment of HCV replicon system, cell culture-adaptive viruscomplex and human hepatocyte chimeric mice. Importantly,the resistance seems to give cross-resistance to other NS5A-inhibitors, while DCV-resistant variants remained completelysensitive to IFN-alpha and to other classes of DAAs, such asHCV NS3/4A and HCV NS5B-inhibitors.

Regarding Gt 1, the genetic barrier to resistance is signifi-cantly lower for subtype 1a in comparison with 1b. In Gt 1a,Q30E and Y93N determined the highest levels of resistance.41

In Gt 2a, HCV-NS5A F28S, L31M, C92R, and Y93H are themajor resistance mutations described.42 Zhou et al.43 eval-uated NS5A polymorphisms and their impact on responserates in patients with Gt 2 infection who were treated withDCV-based regimens. In particular, the authors performed426 Gt 2 NS5A sequencing and concluded that high SVRrates were obtained in treated patients regardless of viralsubtype or baseline NS5A polymorphisms. In Gt 3a, the posi-tion residues 31 and 93 have been identified as locations forDCV-resistance, through which DCV has sub-nanomolarpotency, with EC50 ranging from 120 to 870 pmol/L.44

A typical example of DCV-resistance can be found in theCOMMAND-1 study.12 In this trial, significantly lower rates ofSVR and higher percentages of on-treatment virologic failurewere observed in Gt 1a infection. Of note, in a study involvingGt 1b subjects treated with DCV in combination with the NS3protease inhibitor ASN, NS3 resistance variants quickly dis-appeared. Conversely, NS5A resistance variants were detect-able throughout the 48 weeks of observation.45

The impact of pre-existing drug-resistant substitutions onvirologic response to DCV and ASN combination therapy wasrecently evaluated in 31 patients with Gt 1b who were treatedfor 24 weeks.46 Three subjects experienced virologic break-through, and two patients relapsed. Virologic failure wasassociated with the onset of both NS5A-L31/Y93 and NS3-D168 variants. NS5A-L31/Y93 variants continued to occur athigh frequency, through post-treatment week 103 and up to170, while NS3-D168 variants were replaced by wild-typein all considered subjects. Recently, Kinugasa et al.47 con-ducted a study that indicated the possible implications oflow frequency RAVs in Gt 1b patients who had been treated



for 24 weeks with ASV and DCV. Viral sequences in regions3 and 5A before treatment were examined with direct sequenc-ing, next-generation sequencing and the PCR-invader method.The authors concluded that the presence of RAVs at a low fre-quencymight not alter the outcomes of antiviral therapy. More-over, while direct sequencingmay not detect RAVs for ASV plusDCV therapy that occur at a low frequency (<12%), deepsequencing and PCR-invader methods can reveal such RAVs.

DCV resistance-associated variants were recently describedin Gt 4 subjects. Bartolini et al.48 assessed the NS5A variability

in 5 Gt 4 treatment-naïve patients to analyse the resistance-associated variants in virologic failure; the patients weretreated with standard therapy plus DCV. Among the patientswho experienced virologic breakthrough, multiple substitutionsassociated with DCV-resistance were observed at the NS5Aamino acid positions 28, 31 and 93.

HCV NS5A-resistant variants exist naturally and appearfrequently after virologic response failure following subopti-mal treatment, including with HCV NS5A inhibitors. DCV-resistant variants tend to persist after the drug termination,

Table 3. Precautions and interactions of daclatasvir with commonly used medications

No Dose Adjustment Administer with Caution Contraindicated

Antivirals, HCV� Sofosbuvir

� Simeprevir

� Peg-IFN and ribavirin

Antibacterials� Erythromycin

Anticonvulsants� Carbamazepine

� Oxcarbazepine

� Phenobarbital

� Phenytoin

Antivirals, HIV or HBV� Tenofovir disoproxil fumarate

� Lamivudine

� Zidovudine

� Emtricitabine� Abacavir

� Didanosine

� Stavudine

Anticoagulants� Dabigatran etexilate (not recommendedin specific renal impairment groups)

Antimycobacterial� Rifampicin

Acid reducing agents� Famotidine

Proton pump Inhibitors� Omeprazole

Cardiovascular agents� Digoxin (initiate treatment using thelowest appropriate digoxin dosage)

� Nifedipine

� Amlodipine

Corticosteroids� Systemic dexamethasone

Antibacterials� Azithromycin

� Ciprofloxacin

Lipid lowering agents (monitorfor HMG-CoA reductase inhibitorassociated side effects, i.e. myopathy)� Rosuvastatin

� Atorvastatin

� Fluvastatin

� Simvastatin

� Pitavastatin

� Pravastatin

Herbal supplements� St. John’s wort(Hypericum perforatum)

Anticoagulants� Warfarin

Antidepressants� Escitalopram

Antifungal� Fluconazole

Hormonal contraceptives� Ethinyl estradiol/norgestimate

Immunosuppressants� Cyclosporine

� Tacrolimus

� Sirolimus

Narcotic analgesics� Buprenorphine/naloxone

� Methadone



and cross-resistance has been observed to all HCV NS5Ainhibitors.39 Nevertheless, DCV-resistant variants remainedfully sensitive to IFN-alpha and other classes of DAAs, suchas NS3/4A and NS5B inhibitors. Notably, clinical costs of pre-viously selected resistant variants are still to be defined.

Indications and current approval

On the 24th of July 2015, the US-FDA approved DCV (60 mg)for use with SOF to treat CHC Gt 3 infection in adultpatients.1,49,50 This approval has been recently expanded toinclude: Gt 1-infected patients, HCV/HIV-co-infected patients,and patients with advanced cirrhosis or post-transplant recur-rence of HCV.49

Conclusions and future perspectives

DCV/BMS-790052 is the first-in-class HCV NS5A replicationcomplex with potent antiviral activity. It is a new, oral, DAAwith a pan-genotypic action against HCV. It determines notonly a rapid and strong HCV-RNA decline but also a favourablesafety profile. Like all NS5A inhibitors, DCV should be admin-istered in association with another potent DAA of a differentclass in order to minimize the risk of resistance. According tothe recently published HCV Recommendations of the Euro-pean Association for the Study of Liver (EASL),50 DCV at themaximum dosage (60 mg) plus SOF represents a good IFN-free strategy for Gt 1-, Gt 2-, Gt 3- and Gt 4-infected patients.Although the above-cited EASL recommendations suggestthat RBV inclusion is still questionable, even for treatment-experienced patients, the aforementioned trial by Poordad19

concludes that RBV does not seem to improve the rates ofSVR; rather, it worsens the safety profile.

Regarding Gt 1, DCV has provided quite high rates of SVR(>90%) in all available trials and in different combinations(with SOF or ASN alone, ASN with BEC, and with or withoutRBV).51 In Gt 2 and Gt 3 treatment-naïve patients, the SVRof DCV plus SOF is again very good (92% and 90–92%,respectively). Likewise, excellent results were reported forthe historically difficult-to-treat Gt 3 patients who failed aprevious anti-HCV treatment, with rates reaching up to100%,26 including in patients with advanced liver disease.Indeed, the recently published data suggest that a 24-weektreatment course is the optimal duration of treatment withDCV in patients with cirrhosis.26,27 Pertaining to Gt 4 patients,the combination of DCV and ASN plus BEC and DCV plus SOFwith or without RBV provided relatively high SVR rates (90%and 91%, respectively). Still, no data are available regardingGt 2 and Gt 4 treatment-experienced patients. Notably, allDCV-based therapeutic regimens have provided very highrates of SVR in unique patient populations, such as: HIV/HCV co-infected, liver transplant recipients, and patientswith CHC who are on chronic haemodialysis, includingpatients with advanced liver disease.33,35–37 In regards toreal-world experience, Welzel et al. has recently evaluated485 Gt 1 and Gt 3 CHC patients, of whom 42% had cirrhosisand CPT B/C. Interestingly, SVR12 (modified intention-to-treat) was achieved by 91% of the patients.

Regarding the safety profile, SAEs and AEs leading totreatment discontinuation were rather rare in all of the DCVIFN-free trials. Obviously, the inclusion of RBV in such IFN-free regimens would only worsen the safety profile withoutadding real benefit in terms of SVR.19

To conclude, DCV is a potent HCV NS5A protein inhibitorwith high pan-genotypic virologic efficacy and a favourablesafety profile, with low rates of discontinuations. Indeed, thepromising results concerning the new DCV-containing regi-mens will lead to relevant changes to the treatment algorithmfor CHC. Future large-scale trials are warranted to assess allDCV-containing regimens in patients with Gt 2 and Gt 4infection and in patients with advanced hepatic disease.


None


Manuscript writing (NG, SG), data collection (SG, NG, PA),discussion and approval of the manuscript (PA, NG, SG), andcritical revision (PA, NG, SG).

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Case Report

Massive Hemolysis Causing Renal Failure in AcuteHepatitis E Infection

Pragya Karki1, Sarthak Malik2, Bipadabhanjan Mallick2, Vishal Sharma*2 and Surinder S Rana2

1Department of Internal Medicine, PGIMER, Chandigarh, India; 2Department of Gastroenterology, PGIMER, Chandigarh, India

Abstract

Acute viral hepatitis is usually a self-limiting illness. However,it can lead to complications that can be life-threatening, suchas acute liver failure. Glucose 6 phosphate dehydrogenase(G6PD) deficiency in the setting of acute viral hepatitis canlead to a massive hemolysis, manifesting as acute kidneyinjury and markedly raised bilirubin levels; although cases arerare. Here, we report such a case. The patient had a viralhepatitis E infection and presented with kidney injury requiringdialysis. Examination showed very high mixed hyperbilirubi-nemia due to massive intravascular hemolysis. The patientexperienced a long, protracted course of illness, requiringrenal replacement therapy with other supportive manage-ment, which led to improvement over a period of four weeks.This case highlights the importance of recognizing associatedhemolysis in a patient with viral hepatitis who presents withvery high bilirubin levels or associated kidney injury. Suchpatients will require aggressive supportive care with promptfluid and electrolyte management.© 2016 The Second Affiliated Hospital of Chongqing MedicalUniversity. Published by XIA & HE Publishing Inc. All rightsreserved.

Introduction

Hepatitis E virus (HEV) is an important cause of acute viralhepatitis in the developing world. Glucose-6 phosphate dehy-drogenase (G6PD) deficiency reportedly affects 2.2–14% ofthe general population in North India.1 Though HEV usuallypresents as a mild self-limiting illness, its association withG6PD deficiency may lead to exaggerated intravascularhemolysis, severe hyperbilirubinemia and acute renal failure.

Case report

A 48-year-old man presented to emergency services withcomplaint of a two-week period of yellowish discoloration ofthe eyes and urine. For four days prior to presentation, these

symptoms had been accompanied by reduced urine output.One day prior to presentation, the patient developed slurringof speech with altered sleep-wake pattern. The patient hadhistory of fever, which subsided with onset of jaundice; thefever was reportedly intermittent and not associated withchills or rigors, and which subsided by antipyretic. There wasno history of any alcohol or drug intake, or of any previousneurological symptoms or family history of Wilson’s disease.

On examination, the patient was conscious but disoriented,and in appearance was pale and deeply icteric. Abdominalexamination revealed hepatomegaly at 3 cm below the rightcostal margin, with no splenomegaly. Shifting dullness waspresent. Neurological examination revealed asterixis. Therewas no focal deficit. Laboratory investigations revealed hemo-globin of 5.8 g/dL, total leucocyte count of 5700/mL, plateletcount of 320000/mL, total bilirubin of 53.39mg/dL (conjugatedfraction of 20.25 mg/dL), aspartate transaminase of 4553 U/L,alanine aminotransferase of 3907 U/L, lactate dehydrogenase(LDH) of 6230 U/L, serum creatinine of 3.4, prothrombin timeof 44 s (control: 12 s) and international normalized ratio of3.03. The workup for jaundice showed positivity for IgM anti-HEV (EIAgen HEV IgM kit; Adaltis, Spain) and negativity forhepatitis A antibody IgM (IgM anti-HAV kit; Adaltis), hepatitis Bsurface antigen (HBsAg) (Monalisa HBsAg Plus kit; Bio-Rad,USA) and hepatitis C virus antibody (Innotest HCV ab IV kit;Innogenetics, Belgium). Serum ceruloplasmin levels werenormal and autoimmune markers (antinuclear antibody,smooth muscle antibody, liver-kidney microsomal antibody)were negative. There was no Kayser-Fleischer (KF) ring.

In view of the markedly increased bilirubin and low hemo-globin, the possibility of intravascular hemolysis was kept inmind. On evaluation, the patient was found to be deficientfor G6PD, with raised plasma hemoglobin levels. Direct andindirect Coombs tests were negative. Urine examinationrevealed hemoglobinuria. The patient’s renal functions wors-ened, and creatinine gradually increased to 8.1 mg/dL. Con-sidering the coagulopathy, encephalopathy and intravascularhemolysis, a diagnosis of HEV-related acute liver failure withhemolysis with acute kidney injury was made. The patient wasmanaged with hemodialysis, transfusion of packed red cells,intravenous antibiotics and other supportive measures, and agradual improvement in the patient’s status occurred over thenext four weeks. Hemoglobin levels increased to 7.9 g/dL andliver and renal functions normalized (Fig. 1). The patient wasdischarged in stable condition and remains well one monthafter the discharge. At the three-month follow-up, a repeatanti-HCV antibody test was negative.



Keywords: Hepatitis E virus; Hemolysis; Anemia; Hemoglobinuria; G6PDdeficiency.Abbreviations: HEV, hepatitis E virus; G6PD, glucose 6 phosphate dehydrogen-ase; LDH, lactate dehydrogenase; KF, Kayser-Fleischer.Received: 12 September 2016; Revised: 16 October 2016; Accepted: 08 November2016qDOI: 10.14218/JCTH.2016.00042.*Correspondence to: Vishal Sharma, Department of Gastroenterology, PGIMER,Chandigarh 160012, India. Tel: +91-708-7008099, Fax: +91-172-2744401,E-mail: [email protected]

Discussion

HEV is one of the most common causes of acute viral hepatitisin northern India and is usually a self-limiting illness.2

However, in rare cases, it has been associated with severelife-threatening complications such as acute hemolytic crisisleading to acute renal failure on the background of G6PD defi-ciency. G6PD deficiency reportedly affects 2.2–14% of thenorth Indian population, and co-existence of both illnesses,though rare, has been documented.2,3 Acute viral hepatitisand HEV per se can lead to mild hemolysis; however, casesof severe hemolysis with markedly raised bilirubin with acutekidney injury, as in our patient, are very rare.3,4 Our patienthad severe intravascular hemolysis, as evidenced by the rapidfall in hemoglobin, marked elevation in mixed hyperbilirubi-nemia, hemoglobinuria, and markedly raised plasma LDH.Abid et al.3 were the first to describe this rare life-threateningassociation in a series of five cases, four of which experiencedrenal failure leading to a long, protracted course of illness.Occasional reports have also suggested that HEV can causemassive hemolysis and renal injury in patients with G6PDdeficiency.4,5

The mechanism of hemolysis in G6PD deficient patients isoxidative stress, which may be precipitated by drugs orinfection. Acute viral hepatitis leads to hepatic dysfunction,further leading to accumulation of harmful oxidants thatresult in reduced glutathione levels. Glutathione levels arealready low in red blood cells of G6PD deficient subjects,however, and the further reduction due to viral hepatitisprecipitates severe hemolysis.6,7 A recent review of all suchcases reported to date suggested that renal failure occurs inthe setting of high bilirubin levels, but the exact causeremained undetermined.8 It has been proposed that acutetubular necrosis and mechanical tubular obstruction due to

hematin and bilirubin may contribute.7 Wilson’s disease canalso present as an acute hemolytic crisis, however, it wasexcluded in our case according to the ceruloplasmin levelsbeing normal and the KF ring being absent. Our patient devel-oped acute renal failure, requiring three sessions of hemodial-ysis. During subsequent outpatient visits, testing of the viralmarkers (HBs antigen, IgM anti-HBc, HCV RNA and anti-HCV)was repeated and negative results obtained. We againrepeated testing for ceruloplasmin levels and 24-hour urinecopper level, and both were found to be normal. The possibilityof Wilson’s disease was very low, as Wilsonian acute liverfailure doesn’t improve short of liver transplant.

In light of the case presented herein, we suggest thatphysicians consider the possibility of hemolysis for patientswith acute viral hepatitis and very high bilirubin levels. Testingof G6PD levels, even when results are normal, should (ideally)be repeated at 6–8 weeks, as levels can be falsely normal dueto the presence of fresh red blood cells in the circulationfollowing a hemolytic episode. A patient with hemolysis whodevelops renal failure should be managed carefully, withoptimal correction of fluid and electrolyte imbalance, andavoidance of nephrotoxic and hemolysis-producing drugs.


None


Wrote the initial draft (PK), edited the article (VS, SSR),helped in literature search (SM, BM), participated in care ofthe patient (PK, SM, BM, VS, SSR).

Fig. 1. Bilirubin and creatinine changes in the index patient.


Karki P. et al: HEV-related massive hemolysis

References

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Karki P. et al: HEV-related massive hemolysis

Reviewer Acknowledgement


Editorial Office of Journal of Clinical and Translational Hepatology

We thank the following reviewers for their contribution and support in 2016.

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