http://jac.oxfordjournals.org/content/55/5/608.full.pdf

JAC
Journal of Antimicrobial Chemotherapy (2005) 55, 608–611
doi:10.1093/jac/dki095
Advance Access publication 6 April 2005
Combination of nucleoside analogues in the treatment of chronic
hepatitis B virus infection: lesson from experimental models
Fabien Zoulim*
INSERM U271, 151 Cours Albert Thomas, 69003 Lyon, France
Owing to the persistence of hepatitis B virus (HBV) and the selection of drug-resistant mutants, a new
concept of antiviral therapy for chronic hepatitis B relies on the combination of nucleoside analogues.
In experimental models of HBV infection, several key points concerning these combinations were
addressed. (i) Is it possible to achieve a synergic antiviral effect with polymerase inhibitors? (ii) Is it
possible to impact on intracellular viral covalently closed circular DNA? (iii) What is the impact of the
cross-resistance patterns of the different nucleoside analogues? (iv) What is the effect of viral load
suppression on the restoration of specific antiviral cellular responses? The clinical impact of these key
issues is discussed in the perspective of new clinical trials.
Keywords: HBV, antiviral, therapy
Introduction
Hepatitis B virus (HBV) is the leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide.1 Antiviral therapy of chronic HBV infection remains a clinical
challenge. Indeed, owing to its unique mechanism of replication,
HBV is able to persist in the infected host despite efficient antiviral therapy, and then to escape antiviral drugs.2,3 HBV has a
DNA genome that is replicated via a reverse transcription step.
The template for viral mRNA transcription, viral covalently
closed circular DNA (cccDNA), is located in the nucleus of
infected cells and its half-life is believed to be long and to be
mainly related to the fate of infected cells. As HBV replication
does not usually lead to a cytopathic effect, clearance of HBV
cccDNA and HBV infection is closely related to the death of
infected cells induced by the host immune response. However,
in most chronic carriers, the anti-HBV immune response is
defective. Altogether, this leads to the development of chronic
HBV infection. In the presence of nucleoside analogues that
potently inhibit the viral polymerase, viral production is
decreased but the pool of intranuclear cccDNA persists owing to
its long half-life. Long-term antiviral therapy is therefore
required in most patients with chronic hepatitis B who do not
mount a vigorous immune response, to avoid relapse of viral
replication after treatment withdrawal. However, the major
drawback of prolonged therapy is the selection of drug-resistant
mutants generated by the spontaneous error rate of the viral
polymerase.2 Therefore, one of the major research areas is the
evaluation of combination therapy to delay or prevent the occurrence of drug-resistant mutants.
The major questions are whether a combination of nucleoside
analogues can achieve antiviral synergy, thereby reducing the
risk of resistance development? Can it prevent the formation
of cccDNA in newly infected cells? Can it decrease the pool of
cccDNA in already chronically infected cells? Has the use of
drugs that do not share the same cross-resistance profile the
capacity to delay the development of multidrug-resistant
mutants? And can all these effects on HBV replication result in
restoration of immune response to achieve a sustained control of
viral replication?
Mechanism of action of nucleoside analogues
Although all orally available drugs target the viral polymerase,
nucleoside analogues have different mechanisms of action on the
viral genome replication machinery.2,4 Several drugs such as adefovir, entecavir and amdoxovir inhibit the priming of reverse
transcription, a unique enzymic reaction that results in the synthesis of a short DNA primer covalently attached to a conserved
tyrosine residue of the viral polymerase. Other compounds inhibit the elongation of viral minus-strand DNA, such as lamivudine, emtricitabine and elvucitabine. The mechanism of action
of clevudine involves a weak effect on the priming reaction but
a stronger inhibitory activity on plus-strand DNA synthesis. The
mechanism of action of telbivudine has not been described so
far. Based on these distinct modes of action, one may speculate
that the combination of drugs targeting different steps of viral
genome replication may lead to an additive or synergic effect.
Therefore, the evaluation of the combination of drugs such as
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Leading article
emtricitabine and clevudine,5 which are under clinical investigation,6,7 is relevant for the future development of combination
therapy.
Combination of drugs and antiviral synergy
Although anti-HBV drugs mainly target the viral polymerase
activity and not other steps of viral replication, it was shown
that antiviral synergy could be obtained by combining adefovir,
lamivudine and penciclovir in duck HBV (DHBV)-infected primary hepatocyte cultures.8,9 In a hepatoma cell line expressing
wild-type HBV, an additive effect was also observed with the
combination of adefovir and thymidine analogues (lamivudine,
emtricitabine and telbivudine).10 Antiviral synergy was shown
against HBV in the 2215 cell line, and in vivo in the woodchuck
model.11,12 However, the few combination trials performed in
patients with chronic hepatitis B have not shown a benefit of the
combination on the early virological response. Differences may
be observed in the long term in terms of the selection of drugresistant mutants, which may be delayed by using agents that do
not share the same cross-resistance profile.
In a recent study, we showed in the DHBV model an additive
effect of the combination of drugs acting on the priming of
reverse transcription (amdoxovir), elongation of minus-strand
DNA (emtricitabine), elongation of plus-strand DNA (clevudine)
on the viral polymerase activity, intracellular viral DNA synthesis in primary duck hepatocyte cultures and on virus production in vivo in experimentally infected animals.13 Antiviral
synergy per se was not observed, which may suggest that the
combination strategies should use drugs with clearly distinct
mechanisms of action, such as polymerase inhibitors and interferon-a (as demonstrated in recent clinical trials of lamivudine
plus pegylated interferon14), or the use of new compounds inhibiting viral assembly or morphogenesis or viral entry, etc. In the
woodchuck model, we found that the combination of clevudine
and emtricitabine resulted in a sharp decrease in viraemia
levels,5 which seemed to be more pronounced than in historical
controls receiving single drugs alone. Interestingly, as also
observed previously in woodchucks treated with clevudine
alone, a slow rebound of viraemia was observed after treatment
cessation in animals who received the combination therapy. This
unusual evolution of viraemia following drug withdrawal compared with all the other nucleoside analogues tested so far may
be attributable to an unknown effect of clevudine.5,15,16 Furthermore, in Phase II clinical trials this slow rebound of viraemia
was also observed in patients.7 This unique antiviral activity profile may lead to a new concept of antiviral therapy for chronic
hepatitis B.
Effect of combination therapy on viral cccDNA
One of the major problems of antiviral therapy of chronic HBV
infection is the effect of anti-HBV agents on de novo cccDNA
formation after viral entry.17 – 19 So far, none of the nucleoside
analogues that are available was able to inhibit this critical step
of viral infection, implying that the residual viraemia during
antiviral therapy may still result in the infection of new cells,
thus delaying the clearance of viral infection. Even the combination of amdoxovir, emtricitabine and clevudine could not
prevent the formation of cccDNA in experimentally infected primary hepatocytes.13
Another issue is to determine whether antiviral therapy may
have an effect on already formed cccDNA in chronically
infected cells, thus resulting in cell curing. As polymerase
inhibitors do not have a direct effect on cccDNA formation, this
may be due to the inhibition of intracellular recycling of viral
nucleocapsid to the nucleus secondary to the inhibition of viral
DNA synthesis. This has been observed with single drug administration in the woodchuck model with clevudine and entecavir,16,20 and in clinical trials with entecavir and adefovir
dipivoxil.21 Interestingly, it was shown that a combination of
nucleoside analogues may have an additive effect on the intracellular cccDNA levels in already infected cells, which suggests
that the additive effect observed on viral DNA synthesis may
also result in an additive effect on cccDNA levels.13 Therefore,
this implies an important role of combination therapy to decrease
intrahepatic viral load during long-term therapy.
The decrease in cccDNA levels observed in woodchucks treated with clevudine alone or in combination with emtricitabine
may explain the slow relapse of viral replication post-treatment.
In some animals this was accompanied by a decline in the number of infected cells to undetectable levels as assessed by immunostaining of liver sections for viral antigens.5 It is also
noteworthy that in one study, the combination of clevudine followed by vaccination against the woodchuck hepatitis virus
(WHV) envelope proteins led to the clearance of circulating
viral antigens.22
Influence of the cross-resistance profile of nucleoside
analogues
It is important to note that most of the nucleoside analogues
administered in monotherapy may select for drug-resistant
mutants, as this was shown with HIV.23 The profile of crossresistance of these mutants has been studied in vitro after transfection of the mutants in hepatoma cell lines and the study of
their susceptibility to other drugs.2 It was shown that lamivudine
and emtricitabine share the same cross-resistance profile with the
selection of the M204V or M204I polymerase mutants that are
susceptible to adefovir. To date, telbivudine is associated with
the emergence of the M204I mutant, which is resistant to lamivudine and emtricitabine. Adefovir selects for the A181V and
N236T mutants, which are susceptible to lamivudine. Entecavir
is less active against lamivudine-resistant strains and selects for
specific resistance mutations on a genetic background of lamivudine resistance mutations. Clevudine is not active against the
lamivudine-resistant strains and was shown to select for the
same resistant mutants in woodchucks chronically infected with
WHV.24 It also exhibits some antiviral activity against the adefovir-resistant strains in vitro. Elvucitabine is not active against
the lamivudine-resistant mutants but is active against the adefovir-resistant strains. Tenofovir shows a good antiviral efficacy
against the lamivudine-resistant strains and slightly decreased
activity against the adefovir-resistant mutants. A tenofovir-resistant mutant was also recently described. Given the cross-resistance profile of these drugs, the rationale is to combine the drugs
that would inhibit the emergence of drug-resistant strains to one
or the other drug. This may lead to an improved management
of antiviral therapy of chronic HBV infection in the long term.
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Leading article
Restoration of specific anti-HBV immune response
Several studies have shown that the decrease in viral load
induced by lamivudine therapy is associated with the subsequent
restoration of the CD4 and then the CD8 cellular immune
response against HBV.25,26 Another recent study showed that the
activation of the specific cellular immune response depends on a
relatively low level of viral load.27 However, the recent results
of clinical trials with entecavir and telbivudine, which showed a
stronger inhibition of viral load compared with lamivudine, did
not show a statistical difference in the rate of hepatitis B e antigen (HBeAg) seroconversion.
In our study in the woodchuck model, the intrahepatic delivery of recombinant adenovirus vectors encoding the woodchuck
interferon-g did not lead to an added benefit to the combination
of emtricitabine and clevudine, suggesting that chronically
infected woodchuck hepatocytes may be refractory to the antiWHV activity of interferon-g.5 This has been confirmed by other
teams, one study showing that interferon-a had more antiviral
efficacy than interferon-g in the woodchuck model.28,29
Perspectives for the treatment of chronic hepatitis B
The current understanding of chronic HBV infection and its
treatment suggest that the patients who are more likely to seroconvert anti-HBeAg antibodies should first receive a course of
pegylated interferon-a.30 In this setting, pegylated interferon
therapy may be the best option because of the possibility of
short-term therapy and the absence of selection of resistant
mutants. On the other hand, the majority of patients who are
non-seroconverters or are infected with a pre-core mutant will
require long-term maintenance therapy to control viral replication and liver disease. In this view, the development of clevudine
and emtricitabine opens new avenues in the management of
these patients. Clevudine, with its unusual antiviral activity profile, may be the first nucleoside analogue to be used as a relative
short-term treatment and to achieve sustained control of viral
replication even after treatment withdrawal. On the other hand,
emtricitabine, as well as other drugs in development, offers a
new option for combination of nucleoside analogues that do not
share the same cross-resistance profile. For instance, it may be
used in combination with adefovir or tenofovir. The evaluation
of such combination strategies will need to rely on accurate endpoints and timing for such analysis.
Acknowledgements
This work was supported in part by fundings from the European
Community (HepBvar project, contract QLRT2001-00977; and
viRgil network contract LSHM-CT-2004-503359).
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