LAMIVUDINE TREATMENT FOR CHRONIC HEPATITIS B P. Honkoop ISBN 90-9011356-8 All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system of any nature, or transmitted in any form by any means, electronic, mechanical, photocopying, recording or otherwise, included a complete or partial transcription, without the prior pennission of the author. W Print: Offse.drui<kerij Ridderprin. B.Y" Ridderkerk LAMIVUDINE TREATMENT FOR CHRONIC HEPATITIS B LAMIVUDINE BEHANDELING VAN CHRONISCHE HEPATITIS B PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof. dr P. W.C. Akkermans M.A. en volgens besluit van het college voor promoties. De openbare verdediging zal plaatsvinden op woensdag 22 april 1998 om llAS uur door Pieter Honkoop geboren te Kampen Promotie commissie Promotor: Prof. dr S.W. Schalm Promotie commissie: Prof. dr W.J. Mooi Prof. dr H.R. Scholte Prof. dr AD.M.E. Osterhaus Co-promotor: Dr R.A. de Man The clinical studies on lamivudine were supported by a grant from Glaxo-\Vellcome. This study was performed at the department of Hepatogastroenterology of the Erasmus University Hospital, Dijkzigt, Rotterdam, The Netherlands. Financial support for this thesis was kindly given by G1axo-Wellcome, Byk, Janssen-Cilag, Schering-Plough, Smith Kline Beecham, MSD, Boehringer Mannheim, Roche, Astra, Yamanouchi and Zambon. "Gee! OilS heden ons dagelijks brood" Mattheus 6: J J Aan Willlla en Pieler jr. Aan mijn moeder Tel' nagedachtenis aan mijn vader Lamivudine treatment for chronic hepatitis B Contents Page 1 Introduction 2 Short term effects of lamivudine monotherapy Initial experience Dynamics of hepatitis B virus infection in vivo 13 3 Six months lamivudine monotherapy Biochemical ejJects Lamivudine therapy for chronic hepatitis B: a six-month randomized dose-ranging study 25 Virological e!focts Quantitative HBV -DNA assessment by means of the limiting dilution polymerase chain reaction in chronic hepatitis B patients during a 24-weeks course of lamivudine 37 Histological e!focts Histological improvement in patients with chronic hepatitis B virus infection treated with lamivudine 47 4 Effects of lamivudine and a-Interferon combination therapy Virological and biochemical Combination alpha-interferon and lamivudine therapy for alpha­ interferon resistant chronic hepatitis B infection 55 Contents 5 Lamivudine therapy and mitochondrial toxicity Mechanism of mitochondrial toxicity in nucleoside analogue therapy Mitochondrial injury: lessons from the fialuridine trial 71 Mitochondrial mOlphology and function during lamivudine therapy Effect of lamivudine on morphology and function of mitochondrial in patients with chronic hepatitis B 83 6 Lamivudine withdrawal hepatitis Characteristic hepatitis B reactivation after lamivudine therapy Hepatitis B reactivation with hepatic decompensation 97 Incidence and management of severe acute exacerbation after lamivudine therapy Severe acute exacerbation of chronic heaptitis B virus infection after withdrawal of lamivudine 103 7 Lamivudine resistance and viral mutations Incidence and patterns Lamivudine resistance in inuuunocompetent chronic hepatitis B: Incidence and patterns 117 Virological characteristics Identification of more than one mutation in the hepatitis B virus polymerase gene arising during prolonged lamivudine treatment 125 Clinical impact Clinical impact of lamivudine resistance in chronic hepatitis B 135 8 Future clinical studies on lamivudine in chronic hepatitis B 141 9 Summary / Samenvatting / Dankwoord / Curriculum vitae / Abbrevations 153 Lamivudine treatment for chronic hepatitis B Introduction 1 INTRODUCTION P. Honkoop, R.A. de Man and S.W. Sehalm Department of Hepatogastroentero!ogy, University Hospital Rotterdam, Dijkzigt, The Netherlands. Lamivudine treatment for chronic hepatitis B 2 Introduction The hepatitis B virus The hepatitis B virus (HBV) is one of the smallest human viruses known and belongs to the family of Hepadnaviridae; it was the first human hepatitis virus that could be characterized. Before the discovery of the virus two types of transmission of infectious hepatitis were distinguished on the basis of epidemiological observations: the classical hepatitis (type A) was transmitted by the faecal-oral route, while type B was transmitted parentally.' In 1963, B8 Blumberg discovered a previously unknown antigen in the blood of an Australian aboriginal (Australia antigen) and within a few years this was found to be related to the parentally transmitted type B hepatitis.' In the early seventies the virus was seen by electron microscopy3 and the genome was found to be a small, circular DNA that was partially double-stranded (figure I). The nucleotide sequence of the virus contains only 3200 nucleotides (3.2 kb) and revealed 4 overlapping genes for the production of seven viral proteins. Pre·52 1- Sill/ace antigen related peptides 2- Core antigen related peptides 3- HBxAg 4- HBV-DNA polymerase Pre-core 2 Figure 1. The genollle of the hepatitis B virus consisting of 3200 nue/eotides representing 4 open readingfi'ames drawn as arrows. (Smface, Core, X and Polymerase) The first open reading frame encodes a family of hepatitis B surface antigen (HBsAg)­ related polypeptides that make up the outer envelop of the virus. The major HBsAg polypeptide is encoded by the 8 gene. The pre-82 sequences are cotranslated with 8 sequences to yield pre-821S or middle-sized polypeptides. Cotranslation of the upstream 3 Lamivudine treatment for chronic hepatitis B pre-Sl sequences, yield pre-SlIpre-S2/S or large sized polypeptides. The second HBV open reading frame, the C gene, encodes the major hepatitis B core antigen (HBcAg) polypeptide and hepatitis B e antigen (HBeAg), these are the major components of the virus inner nucleocapsid. These particles are important targets for the immune response against the virus. The soluble HBeAg is secreted into serum and associated with virus replication and high infectivity. The third HBV open reading frame encodes the hepatitis B x antigen (HBxAg), which function is not clear. It is probably one of the co-factors in the pathogenesis of hepatocellular carcinoma.' The fourth and largest HBV open reading frame encodes the HBV -DNA polymerase, which is responsible for the endogenous DNA­ polymerase activity in virus particles. HBV-DNA polymerase has been shown to be the reverse transcriptase (RT) polypeptide of the virus. In this context, any mutation that affects the polymerase activity will not only affect the amount of virus produced, but also the number of templates available to encode virus proteins, and elicit appropriate and timely immune responses. Chronic hepatitis B Inoculation with the hepatitis B virus causes hepatocellular necrosis and inflammation, which severity ranges from asymptomatic to acute liver failure. Acute infection wiH resolve spontaneously in 90% of the adults; the reason why in certain patients the acute infection will not resolve but progresses to chronic hepatitis B is not yet clear. Both viral factors and host factors can probably influence the outcome of the acute infection. Chronic infection with the hepatitis B virus is a serious liver disorder which can result in chronic active hepatitis, cirrhosis and primary hepatocellular carcinoma. It is estimated that 5% of the world population is chronic HBsAg carrier,' the majority of these will even not show any disease progression. In our population about half of the HBsAg carriers did not show any signs of liver inflammation (normal serum transaminases) or viral replication (HBV-DNA negativity by PCR). Currently patients with detectable HBV-DNA by PCR in combination with the lack of any signs of liver inflammation do rarely show progression of their liver disease. Longitudinal studies on the natural history of chronic liver disease due to HBV have shown that ongoing replication (HBeAg either HBV-DNA positivity by hybridization technique) 01' liver inflammation are features which predict unfavourable evolution of the disease.'·7 .• Permanent termination of HBV replication is found to be the 9 IO 1I major event that will influence outcome. • . Spontaneous remission of disease activity may occur in approximately 10% of HBeAg positive carriers per year. Antiviral therapy The ultimate goal of antiviral therapy in chronic hepatitis B is to prevent progression of liver disease to cirrhosis, hepatocellular carcinoma or liver failure and subsequently 4 Introduction improve life expectancy and quality of life (table I). Ihis can be obtained by elimination of the virus from the body and the induction of protective antibodies against the virus. However, improvement of prognosis can be achieved by suppression of HBV replication or induction of HBeAg ser.oconversion despite the continued presence of HB~Ag. This situation of viral latency is associated with normalization of serum transaminase levels and decrease in necro-illflammatory activity on liver biopsies. Therefore, in most clinical studies, the therapeutic outcome (response) is defined as clearance of HBeAg and HBV­ DNA in serum, and subsequently improvement of liver disease. Table 1. Goals of antiviral therapy for chronic hepatitis B • Loss of markers of viral replication (HBV-DNA I HBeAg) II Nonnalization of serum transaminase levels (AL T) II Disappearance of liver cell inflammation • Improvement of symptoms (quality of life) • Decrease of progression of liver disease II Decrease in incidence of hepatocellular carcinoma .. Improvement