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Antimicrobial Agents and Chemotherapy, February 1998, p. 369-376, Vol. 42, No. 2
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Inhibitory Effect of
2'-Fluoro-5-Methyl-
-L-Arabinofuranosyl-Uracil on Duck
Hepatitis B Virus Replication
Stéphanie
Aguesse-Germon,1
Shwu-Huey
Liu,2
Michèle
Chevallier,3
Christian
Pichoud,1
Catherine
Jamard,1
Christelle
Borel,1
Chung K.
Chu,4
Christian
Trépo,1
Yung-Chi
Cheng,2 and
Fabien
Zoulim1,*
INSERM U271, 69003 Lyon,1 and
Department of Pathology,
Laboratoires Merieux, 69007 Lyon,3
France;
Department of Pharmacology, Yale University School
of Medicine, New Haven, Connecticut
065102; and
Department of Medicinal
Chemistry, University of Georgia, Athens, Georgia
306024
Received 27 May 1997/Returned for modification 16 September
1997/Accepted 5 November 1997
 |
ABSTRACT |
The antiviral activity of
2'-fluoro-5-methyl-
-L-arabinofuranosyluracil
(L-FMAU), a novel L-nucleoside analog of
thymidine known to be an inhibitor of hepatitis B virus (HBV)
replication in hepatoma cells (2.2.1.5 cell line), was evaluated in the
duck HBV (DHBV) model. Short-term oral administration (5 days) of
L-FMAU (40 mg/kg of body weight/day) to experimentally
infected ducklings induced a significant decrease in the level of
viremia. This antiviral effect was sustained in animals when therapy
was prolonged for 8 days. The histological study showed no evidence of
liver toxicity in the L-FMAU-treated group. By contrast,
microvesicular steatosis was found in the livers of
dideoxycytidine-treated animals. L-FMAU administration in
primary duck hepatocyte cultures infected with DHBV induced a
dose-dependent inhibition of both virion release in culture
supernatants and intracellular viral DNA synthesis, without clearance
of viral covalently closed circular DNA. By using a cell-free system
for the expression of an enzymatically active DHBV reverse
transcriptase, it was shown that L-FMAU triphosphate exhibits an inhibitory effect on the incorporation of dAMP in the viral
DNA primer. Thus, our data demonstrate that L-FMAU
inhibits DHBV replication in vitro and in vivo. Long-term
administration of L-FMAU for the eradication of viral
infection in animal models of HBV infection should be evaluated.
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INTRODUCTION |
The development of new antiviral
drugs for the therapy of chronic hepatitis B virus (HBV) infection
remains a major problem since alpha interferon therapy is moderately
active and its use is often limited because of dose-dependent side
effects (14, 40). Therefore, the efficacies of nucleoside
analogs, such as lamivudine and famciclovir, have been assessed in
chronically HBV-infected patients to improve the response rate to
antiviral therapy for chronic HBV infection. However, resistant viruses with mutations in the catalytic domain of the viral polymerase may be
selected in 10 to 25% of the patients after 12 months of treatment,
depending on the clinical setting (1, 21, 33). It is
therefore important to continue research to design new nucleoside analogs which could provide the basis for the development of new antiviral strategies for combating the emergence of resistant mutants.
Due to their high antiviral activities and very good selectivity
indices, compounds which belong to the -L-nucleoside analog family may represent potential candidates (2, 26). 2'-Fluoro-5-methyl--L-arabinofuranosyluracil
(L-FMAU) is a novel -L-nucleoside analog
derived from thymidine. It was found to be a potent inhibitor of HBV
replication in a stably transfected human hepatoma cell line (2.2.1.5)
and to have a level of low cytotoxicity in vitro (6). In
this cell line, it was further demonstrated that L-FMAU
inhibits HBV without affecting the host DNA synthetic machinery
(27). By contrast to D-FMAU and to
1-(2'-deoxy-2'-fluoro-1--D-arabinofuranosyl)-5-iodouracil (D-FIAU), L-FMAU did not decrease the
mitochondrial DNA content, did not affect mitochondrial function, and
was not incorporated into cellular DNA (27).
Considering its potent inhibitory activity against HBV DNA synthesis
and its minimal inhibitory effect on the cellular machinery, L-FMAU has been further explored for development as a
potential anti-HBV drug. Since 40 to 50 copies of viral covalently
closed circular (CCC) DNA are maintained in the nuclei of infected
cells and serve as templates for new viral DNA synthesis when antiviral therapy is withdrawn (13, 37), the ability of
L-FMAU therapy to clear viral CCC DNA should be evaluated.
Furthermore, because duck HBV (DHBV) reverse transcription is primed by
the synthesis of a short DNA primer (GTAA) covalently linked to a
conserved tyrosine residue of the amino-terminal domain of the viral
polymerase (35, 39), the potential antipriming activity of
L-FMAU should also be considered. Therefore, we have
evaluated in more detail its anti-HBV activity in the DHBV model
(23). This model provides relevant tools for the study of
the modes of action of new anti-HBV agents. A primary duck hepatocyte
culture system and studies with experimentally infected ducklings have
been used to investigate the inhibition of viral DNA synthesis in
hepatocytes, the clearance of CCC DNA from infected cells, and the
toxicities of new antiviral agents (10, 13, 20, 29, 34, 38).
In this study, we also used an in vitro assay for the expression of an
enzymatically active viral reverse transcriptase which was first
described by Wang and Seeger (35) and used the assay to
study the mechanism of inhibition of DHBV reverse transcription by new
anti-HBV compounds (9, 30, 35, 38, 39). Our results show
that L-FMAU exhibits antiviral activity in vivo in
experimentally infected ducklings and primary duck hepatocytes and that
it has an inhibitory effect on the enzymatic activity of the DHBV
reverse transcriptase.
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MATERIALS AND METHODS |
Drugs.
L-FMAU was synthesized in the Department
of Medicinal Chemistry, University of Georgia, as described previously
by Chu et al. (6). Its triphosphate form
(L-FMAU-TP) and
2',3'-dideoxy--L-5-fluorocytidine (-L-F-ddC) were synthesized in the Department of
Pharmacology, Yale University. Dideoxythymidine-triphosphate (ddTTP)
and dideoxycytidine (ddC) were purchased from Boehringer Mannheim and
Sigma, respectively.
An in vitro assay for the expression of enzymatically active DHBV
reverse transcriptase.
The DHBV polymerase was expressed from
plasmid pHP, which contains the DHBV polymerase gene under the control
of the SP6 promoter and the sequence coding for the RNA template of
reverse transcription, as described previously (35, 39). The
polymerase gene was transcribed and translated in a coupled
transcription-translation rabbit reticulocyte lysate system (TNT
coupled reticulocyte system; Promega), and the reverse transcriptase
reaction was performed as described previously (38).
To study the inhibitory effect of L-FMAU-TP on viral
minus-strand DNA elongation as a result of TMP incorporation, the
translation mixture was incubated at 30°C for 30 min with an equal
volume of a reaction mixture containing 100 mM Tris-HCl (pH 7.5), 30 mM
NaCl, 20 mM MgCl2, dGTP, dATP, and dCTP (200 µM each) and
[
-32P]TTP (3,000 Ci/mmol, 0.60 µM). The inhibitors
L-FMAU-TP and ddTTP were added at the concentrations
indicated below.
To analyze the synthesis of the viral DNA primer, whose sequence is
5'-GTAA-3', the DHBV polymerase was incubated with dGTP (final
concentration, 100 µM), TTP or inhibitors (L-FMAU-TP or ddTTP) at increasing concentrations, and [-32P]dATP
(3,000 Ci/mmol; final concentration, 0.3 µM). The kinetics of
[-32P]dAMP incorporation into the viral DNA primer was
also analyzed at different times after incubation of the polymerase
mixture with dGTP, TTP (final concentration, 100 µM) or inhibitors
(L-FMAU-TP or ddTTP; final concentration, 50 µM) and
[-32P]dATP (3,000 Ci/mmol; final concentration, 0.3 µM).
Radiolabelled viral DNA covalently attached to polymerase was subjected
to electrophoresis through 0.1% sodium dodecyl sulfate
(SDS)-10%
polyacrylamide gels as described previously (
35,
39).
The
dried gels were exposed to X-ray film, and the viral DNA was
quantified
by laser densitometry as previously described in detail
(
9,
30,
38).
Primary hepatocyte cultures.
Primary hepatocyte cultures
were prepared from 4-week-old Pekin ducks chronically infected with
DHBV. The procedures of liver perfusion and hepatocyte isolation and
the culture conditions were described previously (34).
Hepatocytes were seeded at confluence onto 35-mm petri dishes, and the
serum-free growth medium was changed daily. The addition of the drugs
to the culture medium at the indicated concentrations was carried out
from day 3 to day 10 postseeding. Cellular toxicity was analyzed by
daily examination with a light microscope, cellular DNA gel
electrophoresis, and measurement of the lactic acid level in cell
supernatants (Lactate PAP; Biomerieux, Marcy l'Etoile, France).
Experimental inoculation of ducklings.
Five-day-old
ducklings were inoculated intravenously with a DHBV-positive serum
specimen known to be infectious, and each duckling received 1.5 × 107 viral genome equivalents by following a protocol
described by Lambert et al. (16). Ducklings were
administered nucleoside analogs orally 3 days postinoculation according
to the protocols described in Results. Animal weight and lactic acid
levels were monitored daily during the study period.
Analysis of viral DNA.
DHBV DNA from experimentally infected
ducklings and from hepatocyte culture supernatants was detected by a
DNA spot hybridization assay at different time points, as indicated
below, by using a Hybridot Manifold apparatus (Gibco BRL). Fifty
microliters of serum or 800 µl of culture supernatant were spotted
directly onto nitrocellulose filters (Schleicher & Schuell). After
denaturation and neutralization, the filters were hybridized with a
full-length DHBV genomic DNA probe labelled with 32P. The
filters were autoradiographed, and the spots were counted in a
scintillation counter (38).
DNA sequence analysis of the catalytic site of the reverse
transcriptase domain of the DHBV polymerase gene was performed
with
circulating virions at the end of the 8-day administration
of
L-FMAU. One hundred microliters of serum was submitted to
digestion
with proteinase K and SDS (final concentrations, 1 mg/ml and
1%,
respectively), and DNAs were extracted with phenol-chloroform.
Viral DNA encompassing the catalytic site of the polymerase gene
was
amplified by PCR with primers p-pol-1 (nucleotide positions
171 [5']
to 185 [3']) and p-pol-2 (nucleotide positions 1812 [3']
to 1833 [5']). The PCR products were then directly sequenced with
the
Sequenase PCR product sequencing kit (United States Biochemicals)
according to the manufacturer's recommendations.
Intrahepatic viral DNA from experimentally inoculated ducklings was
extracted by a procedure described in detail by Jilbert
et al.
(
15). Liver samples were snap frozen in liquid nitrogen
and
were stored at
80°C and then analyzed for viral DNAs. One
hundred
milligrams of liver was homogenized in 0.01 M Tris-HCl
(pH 7.5)-0.01 M
EDTA, and the homogenate was divided into two
parts, one for the
isolation of total viral DNA and one for the
isolation of
non-protein-bound, CCC viral DNA. Five micrograms
of the total DNA or
the CCC DNA preparation was subjected to electrophoresis
on 1.5%
agarose gels. Southern blot analysis was carried out as
described
previously (
15), and viral DNAs were detected by
hybridization
with a
32P-labelled probe representing the
complete viral genome (
15).
To analyze the viral DNA in primary hepatocytes, cells were rinsed with
phosphate-buffered saline (PBS) and stored at
80°C
for DNA
isolation. Intracellular viral CCC DNA (non-protein-bound
DNA) and
replicative intermediates (protein-bound DNA) were isolated
as
described by Summers et al. (
31). Viral DNA (corresponding
to 0.5 µg of cellular DNA) was analyzed by electrophoresis through
1.5% agarose gels, transferred by blotting onto nylon membranes
(Hybond N+; Amersham), and hybridized with a full-length DHBV
genomic
DNA probe labelled with
32P.
Anti-pre-S antibody detection in the serum of DHBV-infected
ducklings.
Anti-pre-S antibody detection in the serum of infected
ducklings was performed by an enzyme-linked immunosorbent assay (ELISA) with a recombinant pre-S protein as described previously
(3).
Western blot analysis of intrahepatic viral proteins.
Fifty
milligrams of duck liver was pulverized in liquid nitrogen and was
resuspended in 10 volumes of lysis buffer (20 mM Tris [pH 7], 150 mM
NaCl, 1 mM EDTA, 1 mM MgCl2, 5 mM KCl, 1% Nonidet P-40, 1 µg of leupeptin per ml, 1 µg of pepstatin A per ml, 2 mM
phenylmethylsulfonyl fluoride) (8). The cell extracts were
clarified by centrifugation (12,000 × g, 20 min,
4°C). One hundred fifty micrograms of proteins was subjected to
electrophoresis through an SDS-15% polyacrylamide gel and was
transferred to a polyvinylidene difluoride-Immobilon P transfer
membrane (Immobilon P; Millipore). The membranes were soaked in 5%
nonfat dry milk and 0.1% Tween 20 in PBS for 30 min and incubated for
1 h with anti-DHBV core rabbit polyclonal antibodies (final
dilution, 1:5,000). Goat anti-rabbit immunoglobulin G antibodies
conjugated to horseradish peroxidase (Dako) were then added for 30 min
at room temperature. The membranes were washed (5% nonfat dry milk and
0.1% Tween 20 in PBS), and a chemiluminescence reaction was performed
according to the manufacturer's instructions (ECL kit, Amersham,
France).
Analysis of liver histology in experimentally infected
ducklings.
Coded, formalin-fixed liver tissue sections embedded in
paraffin were sectioned at a thickness of 3 µM, stained with
hematoxylin, eosin, and safran, and examined under a light microscope.
The levels of hepatocyte necrosis (acidophilic bodies), portal tract inflammation, intralobular inflammation, steatosis, and ductular proliferation were assessed as described previously (5).
| |
RESULTS |
In vivo inhibition of DHBV replication in experimentally infected
ducklings after oral administration of L-FMAU.
Ducklings were inoculated at 5 days of age by following a protocol
which has already been described in detail (16). DHBV viremia was reproducibly detectable at day 4 postinoculation and reached a peak at day 6. This experimental model allowed us to study
the inhibitory effect of L-FMAU therapy on DHBV replication in vivo and assess its toxicity and compare these with the effect and
toxicity of ddC. In preliminary experiments, it was determined that
oral administration of L-FMAU for 4 days at a dosage of 40 mg/kg of body weight once a day or 20 mg/kg twice a day gave similar antiviral effects but was followed by a rebound of viremia after drug
withdrawal (data not shown). The results of Southern blot analysis of
intrahepatic viral DNA at the end of therapy indicated that viral DNA
synthesis was significantly decreased in L-FMAU-treated animals compared to that in control and ddC-treated animals (Fig. 1A).
However, L-FMAU administration could not clear viral CCC DNA from the liver (Fig. 1B).
Furthermore, a pulse therapy with three doses of 20 mg of
L-FMAU per kg given orally every 12 h before
intravenous inoculation of infectious serum resulted in a delay in the
onset of viremia by 1 day in all five treated animals compared to the
time of onset in five control animals (data not shown).
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FIG. 1.
Oral administration of L-FMAU decreases
viral DNA synthesis in the livers of experimentally infected ducklings
but does not clear viral CCC DNA. Protein-bound (A) and protein-free
(B) viral DNAs were extracted from the livers of experimentally
infected birds and were subjected to Southern blot analysis at the
cessation of therapy. Liver samples from four control animals, four
animals that received L-FMAU at 20 mg/kg twice a day, per
os, for 4 days, and four animals that were treated with ddC at 50 mg/kg
twice a day, per os, for 4 days were available for analysis. The
positions of relaxed circular (RC), linear (L), CCC, and
single-stranded (SS) DNAs are indicated, as are the therapeutic
protocols.
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In the next experiment, we analyzed the antiviral efficacy of a more
prolonged administration of
L-FMAU in experimentally
infected ducklings. Four animals received an oral dosage of 40
mg of
L-FMAU per kg once a day for 5 days, four ducklings were
given the same dosage for 8 days, and four birds served as controls.
Figure
2 shows that administration of
L-FMAU for 5 days was associated
with a 55% inhibition of
the peak of DHBV viremia, followed by
a weak and transient rebound of
viremia 5 days after drug withdrawal.
Administration of
L-FMAU for 8 days induced a 72% inhibition of
the peak of
DHBV viremia which was not followed by a rebound of
viremia during the
2-week posttreatment follow-up (Fig.
2). In
this experiment, the mean
area under the curve for viremia, which
reflects total virus
production, was significantly lower for the
group of
L-FMAU-treated animals than for the control group
(
P = 0.0482; Wilcoxon-Mann Whitney test, Monte Carlo
modification
for small number; Stat-Xact-3 software). DNA sequence
analysis
of the DHBV polymerase gene was performed after amplification
of circulating viral DNA by PCR at the end of the 8 days of
administration
of
L-FMAU to all four treated animals. The
results showed the
absence of amino acid sequence variation in the
catalytic site
of the viral polymerase (data not shown). Determination
of lactic
acid levels in the plasma of the animals which received
L-FMAU
therapy for 8 days showed no significant increase
compared with
the levels in the control animals (Fig.
2). Then, after
drug withdrawal
we determined whether viral infection had been cleared
from the
liver. Analysis of viral DNA replicative intermediates by gel
electrophoresis and Southern blot hybridization of the livers
of
infected ducklings 16 days after
L-FMAU withdrawal showed
the
persistence of DHBV single-stranded and relaxed circular DNAs,
as
well as viral CCC DNA, indicating that short-term administration
of
L-FMAU is not able to clear viral infection from the liver,
despite the dramatic drop in the level of viremia (Fig.
3A). Analysis
of intrahepatic viral
proteins by Western blot analysis of the
same liver samples showed a
similar expression of DHBV core proteins
in
L-FMAU-treated
animals and in the control birds (Fig.
3B).
Since in both groups of
L-FMAU-treated animals and the control
animals, the level
of viremia dropped and DHBV DNA was detectable
only by PCR assay, we
asked whether an anti-DHBV antibody response
could also be responsible
for the clearance of circulating virus.
However, detection of
anti-pre-S antibody in the serum of these
animals by an ELISA did not
show any appearance of antibody (data
not shown).
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FIG. 2.
Oral administration of L-FMAU inhibits DHBV
viremia in experimentally infected ducklings. Ducklings at 5 days of
age were inoculated with a DHBV-positive serum specimen.
L-FMAU was given orally at day 3 postinoculation by two
different protocols. A group of four ducklings received
L-FMAU at a dosage of 40 mg/kg once a day from day 3 to day
7 postinoculation (5 days of treatment). A second group of four
ducklings was given L-FMAU at a dosage of 40 mg/kg once a
day from day 3 postinoculation for 8 days. A third group of four
ducklings served as controls. (A) Analysis of viremia levels. Viral DNA
in serum was analyzed by a dot blot assay. The level of mean viral DNA
in serum in each group of animals was plotted. (B) Analysis of lactic
acid levels. The lactic acid levels in experimentally infected birds
were determined before inoculation and at days 7, 10, 12, and 14 postinoculation. The mean serum lactic acid concentration in each group
of animals was plotted.
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FIG. 3.
DHBV is not cleared from the livers of experimentally
infected ducklings after L-FMAU withdrawal. (A) Analysis of
intrahepatic viral DNA. A preparation of enriched protein-bound viral
DNAs, extracted from the livers of experimentally infected birds, was
subjected to Southern blot analysis 16 days after the cessation of
therapy. Liver samples from three control animals, four animals that
received L-FMAU for 8 days, and four animals that were
treated for 5 days were available for analysis. The positions of
relaxed circular (RC), linear (L), CCC, and single-stranded (SS) DNAs
are indicated, as are the therapeutic protocols (controls, oral
administration of L-FMAU for 5 or 8 days). (B) Analysis of
intrahepatic viral proteins. Viral core proteins were analyzed by
Western blotting of the same liver samples, as described in Material
and Methods. MW, molecular mass.
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L-FMAU therapy of DHBV-infected ducklings is not toxic
during short-term administration.
To analyze whether short-term
L-FMAU administration may be toxic in vivo in ducklings, we
examined the effect of L-FMAU treatment (20 mg/kg twice a
day for 4 days, 40 mg/kg once a day for 4 days, or 80 mg/kg every other
day for 4 days) on survival and liver histology and compared the effect
with the effect of another oral dosage of ddC (50 mg/kg twice a day)
given to another group of four animals from day 3 to day 6 postinoculation. This dosage of ddC was chosen since previous
experiments had shown that this therapeutic regimen is associated with
significant clinical toxicity and a negligible antiviral effect in
ducklings (38). Analysis of intrahepatic viral DNA did not
show any significant decrease in viral DNA synthesis in ddC-treated
animals (Fig. 1). All four animals treated with ddC started to lose
weight at the end of therapy and eventually died on days 4 (one
animal), 6 (two animals), and 7 (one animal) posttreatment. By
contrast, in comparison with the clinical condition of the control
animals, none of the 12 animals treated with L-FMAU showed
any clinical signs of toxicity. Liver histology was analyzed under code
without knowing the treatment regimen that the animals received, and
the results are summarized in Table 1 and
Fig. 4. Microscopic examination of liver sections from the control
animals showed a commonly observed pattern of mild viral hepatitis with
ballooning degeneration (swollen hepatocytes), inflammatory
infiltration of the portal tracts, and rare aspects of hepatocyte
necrosis (Fig. 4A). For
L-FMAU-treated animals, the histological aspect was similar
to the one observed in the control group, regardless of the treatment
protocol (Fig. 4B). The inflammation in the portal tracts and the
lobules was somewhat weaker in L-FMAU-treated animals. In
three of four ddC-treated animals, typical signs of liver injury
characterized by microvesicular steatosis (accumulation of lipid
droplets in the cytoplasms of hepatocytes) and acidophilic necrosis of
hepatocytes were observed (Fig. 4C and D).
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FIG. 4.
Analysis of liver histology revealing microvesicular
steatosis in ddC-treated animals and no histological sign of toxicity
during short-term administration of L-FMAU. (A) Liver
histology for one control animal. Signs of viral hepatitis are
observed: ballooning hepatocytes, portal tract infiltration, and
lobular inflammation (obj., 25). (B) Liver histology for one
L-FMAU (40 mg/kg every day)-treated animal. A pattern
similar to that in the control animals is observed. (C) Liver histology
for one ddC (50 mg/kg twice a day)-treated duckling. Typical signs of
microvesicular steatosis (accumulation of lipid droplets in the
cytoplasms of hepatocytes) and acidophilic necrosis of hepatocytes are
shown. (D) Liver histology for the same ddC (50 mg/kg twice a
day)-treated duckling but at a higher magnification (obj., 40), which
shows intracytoplasmic lipid droplets.
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L-FMAU inhibits DHBV DNA synthesis in primary duck
hepatocyte cultures.
The inhibitory effect of L-FMAU
was further investigated with cultures of primary hepatocytes from
chronically infected ducklings. Different concentrations of
L-FMAU and -L-F-ddC (0.01 to 10 µM) were
studied in parallel. Three days after plating, hepatocytes were
incubated with nucleoside analogs which were renewed on a daily basis
for 7 consecutive days. Quantification of the virion DNA released in
the culture supernatant showed a reproducible and significant
inhibitory effect of L-FMAU which was concentration dependent (Fig. 5A). The 50% inhibitory
concentration (IC50) of L-FMAU for virion DNA
release was 0.1 µM. At concentrations of 1 and 10 µM, the
inhibitory effect reached a plateau (80 to 90% inhibition). We also
confirmed our previous data showing that -L-F-ddC
inhibits DHBV replication in primary hepatocytes.
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FIG. 5.
L-FMAU inhibits DHBV DNA synthesis in DHBV
DNA-infected primary duck hepatocytes. L-FMAU or
-L-F-ddC was added at the indicated concentrations 3 days after plating for 6 days. Cells were harvested at the end of
therapy (day 10 [D10]) and 3 days after drug release (day 13 [D13]). The viral DNA released in the supernatant of primary
hepatocyte culture was analyzed by a dot blot assay (A). Intracellular
protein-bound (B) and protein-free (C) viral DNAs were extracted and
subjected to Southern blot analysis. The positions of relaxed circular
(RC), linear (L), CCC, and single-stranded (SS) DNAs are indicated. The
results of a typical experiment are shown.
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Southern blot analysis of intracellular viral DNA showed a decrease in
the intensity of replicative intermediates at the end
of the treatment
schedule for
L-FMAU-treated cultures (Fig.
5B).
At a
concentration of 10 µM,
L-FMAU induced a dramatic
inhibition
of viral DNA synthesis, as shown by the profound decrease in
viral
single-stranded DNA. However, viral CCC DNA was still detected
at
the end of therapy (Fig.
5C). These results were similar to
those
obtained with
-
L-F-ddC.
Rebound of viral replication after drug release was analyzed in
hepatocyte cultures. After
L-FMAU and
-
L-F-ddC were withdrawn,
we observed a rebound in viral
replication with an increase in
the level of replicative intermediates,
whose intensities remained
weaker compared to those in the control
cultures (Fig.
5B). This
particular pattern was observed when
L-FMAU or
-
L-F-ddC was administered
at
concentrations of 10 µM.
No significant signs of cytotoxicity were observed during the daily
microscopic examination of cultured cells. Testing of
the culture
supernatant for lactic acid levels showed no increase
in lactic acid
levels during cell culture, regardless of the therapeutic
protocol
(control cultures or
-
L-F-ddC or
L-FMAU
administration).
Inhibitory effect of L-FMAU-TP on viral minus-strand
DNA synthesis.
The inhibitory effect of the triphosphate form of
L-FMAU on the synthesis of viral minus-strand DNA was
analyzed by an in vitro assay for the expression of an enzymatically
active DHBV reverse transcriptase. Viral DNA synthesis was studied by
the incorporation of deoxynucleoside triphosphates (dGTP, dATP, dCTP) and radiolabelled [-32P]TTP. Nascent viral DNA
covalently linked to the DHBV polymerase was analyzed through 0.1%
SDS-10% polyacrylamide gels. The incorporation of
[-32P]TMP in the presence of increasing concentrations
of ddTTP (IC50 = 3 µM) was reproducibly inhibited. The
level of inhibition of [-32P]TMP incorporation in
elongating viral minus-strand DNA by L-FMAU-TP was weaker
since concentrations of 10 µM induced a 40% inhibition. These
results suggest that, in vitro, L-FMAU-TP is a rather weak inhibitor of [-32P]TMP incorporation in viral
minus-strand DNA (data not shown).
Then we asked whether
L-FMAU-TP or ddTTP can terminate the
synthesis of the short oligonucleotide primer for reverse transcription
(POL-GTAA). The DHBV polymerase was therefore incubated with dGTP,
TTP,
or inhibitors (
L-FMAU-TP or ddTTP) at increasing
concentrations
and [
-
32P]dATP for 30 min. The amount
of [
-
32P]dAMP incorporated into the DNA primer was
measured in the presence
of the different thymidine analog
triphosphates. The results obtained
at 30 min showed an increasing
level of incorporation of [
-
32P]dAMP in the primer in
the presence of increasing concentrations
of TTP, reaching a plateau at
a TTP concentration of 100 µM. In
the presence of increasing
concentrations of ddTTP or
L-FMAU-TP,
the incorporation of
[
-
32P]dAMP decreased to the background level (no TTP
analog added).
The results indicate that a complete inhibition of viral
primer
synthesis could not be obtained under our in vitro conditions.
Figure
6A presents typical results
obtained with 50 µM TTP analog.
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FIG. 6.
Inhibitory effect of L-FMAU-TP on the
synthesis of the viral DNA primer for DHBV reverse transcription. The
sequence of the short DNA primer covalently linked to the DHBV
polymerase is 5'-GTAA-3'. (A) The DHBV polymerase was incubated with
different concentrations of thymidine-TP analogs (TTP or ddTTP or
L-FMAU-TP) together with dGTP and
[-32P]dATP (3,000 Ci/mmol; final concentration, 0.3 µM) for 30 min. The incorporation of [-32P]dAMP was
also analyzed in control reactions without TTP, without dGTP and TTP,
or without DHBV polymerase. Reactions were analyzed with a 0.1%
SDS-10% polyacrylamide gel and autoradiographed. The figure shows the
results obtained with the TTP analog at a concentration of 50 µM. (B)
Kinetics of [-32P]dAMP incorporation into primer DNA
by the DHBV polymerase. The DHBV polymerase was incubated with
thymidine-TP analogs at a concentration of 50 µM ( , TTP;  ,
ddTTP;  , L-FMAU-TP) together with dGTP (100 µM) and
[-32P]dATP (3,000 Ci/mmol; final concentration, 0.3 µM). The incorporation of [-32P]dAMP in the viral
primer was measured at different time points, as indicated. The
activity at 30 min in the presence of TTP was arbitrarily defined as
100%.
|
|
The kinetics of [
-
32P]dAMP incorporation into the
viral DNA primer was also analyzed at different times after incubation
of
the polymerase mixture with cold dGTP, TTP (final concentration,
100 µM) or inhibitors (
L-FMAU-TP or ddTTP; final
concentration,
50 µM), and [
-
32P]dATP (Fig.
6B). In
the presence of TTP, the results showed an
increasing level of
incorporation of [
-
32P]dAMP with time. A
time-dependent inhibition of incorporation
by ddTTP and
L-FMAU-TP was observed, suggesting that ddTTP and
L-FMAU-TP may terminate the synthesis of the short primer.
Other
experiments showed that
L-FMAU-TP and ddTTP do not
inhibit the
incorporation of [
-
32P]dGMP in viral
minus-strand DNA (data not shown).
| |
DISCUSSION |
In this report, we present data on the mode of action of
L-FMAU in the inhibition of hepadnavirus replication and on
its antiviral activity in vivo in the DHBV model. L-FMAU
was shown to inhibit HBV replication in the human hepatoma cell line
2.2.1.5, which permanently replicates the HBV genome. It was also shown
to have a very good selectivity index (>2,000) in this cell line
(6) and not to be incorporated into cellular DNA
(27).
In this study, the in vivo administration of L-FMAU (40 mg/kg/day) by the oral route to experimentally infected ducklings showed a potent inhibition of viral replication which may support the
therapeutic utility of this drug if its absence of in vivo toxicity is
confirmed during long-term administration. The study of intrahepatic
viral DNA at the end of 4 days of therapy showed that
L-FMAU administration is associated with decreased viral DNA synthesis (Fig. 1). Administration for 5 days could inhibit viral
replication significantly but was followed by a transient rebound of
viremia 5 days after drug withdrawal (Fig. 2), as has also been
observed with other drugs (2, 10, 13, 38). Interestingly, a
more prolonged protocol with the administration of L-FMAU
to ducklings for 8 days could prevent the rebound of viremia after drug
withdrawal and was not associated with increased serum lactic acid
levels (Fig. 2). Statistical analysis showed that, although the number
of animals was small, there was a significant trend for decreased viral
production in L-FMAU-treated animals compared with that in
the controls (P < 0.05). Southern blot analysis of
intrahepatic viral DNA 2 weeks after drug withdrawal showed the
persistence of viral CCC DNA and replicative intermediates, as was also
observed in tissue culture (Fig. 3 and 5), accompanied by the
persistence of viral core protein expression, as determined by Western
blot analysis (Fig. 3). This emphasizes the need for a prolonged
therapeutic protocol in order to cure infected hepatocytes (10,
20, 22, 38). Although DHBV pol gene mutants were not
selected during short-term administration of L-FMAU, it
remains to be determined whether this phenomenon could be observed
during long-term therapy. After drug withdrawal, the persistence of
viral DNA and proteins was associated with a low level of viremia that was below the limit of detection of our dot blot assay. This may suggest either a decrease in viral particle secretion from infected hepatocytes or an enhanced clearance of viral particles from the serum.
However, our attempts to detect antibodies against envelope proteins in
the serum of DHBV-infected ducklings during the course of experimental
infection did not show any antibody response. Analysis of the liver
histology of infected ducklings showed the absence of significant signs
of liver toxicity with a short-term treatment with L-FMAU
(Fig. 4). With regard to toxicity, it is noteworthy that the drug was
administered when the birds were rapidly growing and cell division,
including hepatocytes, was occurring at a significant rate. In control
animals as well as in L-FMAU-treated animals, a typical
pattern of mild acute hepatitis characterized by portal tract
inflammation and rare hepatocyte necrosis was observed. Since most
studies have shown the absence of major liver damage during the natural
course of experimental infection in ducklings (24), our
observation may be related to the dose of the inoculum, as was recently
shown by Jilbert's group (14a). This further confirms the
absence of toxicity of L-FMAU administration to mice for 30 days at a dosage of 25 mg/kg/day (4a). However, it will be
necessary to confirm the absence of the in vivo toxicity of
L-FMAU during long-term administration in ducks and
woodchucks. Indeed, the administration of D-FMAU to
woodchucks was associated with severe toxicity (11), which can now be explained by the inhibitory effect of D-FMAU on
mitochondrial function (7, 19, 28). In our experiment, a
typical pattern of liver toxicity as a result of ddC administration was
observed: microvesicular steatosis and acidophilic necrosis, which are
typical histological signs of mitochondrial toxicity (Fig. 4 and Table 1). These histological signs have been reported with the use of several
nucleoside analogs such as fialuridine (7, 18, 25). Because
it has been indicated that ddC interferes with mitochondrial DNA
synthesis (4), the liver injury observed in these animals
may be related to the inhibitory effect of ddC on mitochondrial DNA
polymerase. This liver toxicity may have been responsible, at least in
part, for the deaths of all ddC-treated birds.
To gain insight into the mechanism of action of L-FMAU, we
have studied its antiviral activity in primary duck hepatocyte cultures
chronically infected with DHBV. Our results demonstrated that
L-FMAU is a strong inhibitor of viral DNA synthesis and
virion DNA release (Fig. 5). The IC50 of L-FMAU
on virion DNA release in primary duck hepatocytes (0.1 µM) was found
to be similar to the one reported in human hepatoma cells permanently
transfected with HBV (6, 27). Viral single-stranded DNA
synthesis was significantly decreased, suggesting that
L-FMAU inhibits the reverse transcriptase step of DHBV
replication (Fig. 5). Furthermore, short-term therapy with
L-FMAU could not clear viral CCC DNA from infected cells,
as was also observed with other nucleoside analogs including
-L-F-ddC (38). Daily microscopic examination
and determination of lactic acid levels in primary hepatocyte culture supernatants treated with L-FMAU did not show any
significant sign of cellular toxicity, as has already been observed
with human hepatoma cells (6, 27). This is in contrast to
the observation made with the administration of D-FMAU, the
dextrorotatory analog of FMAU, and D-FIAU (fialuridine),
which proved to be toxic for mitochondrial functions and/or to be
incorporated into cellular DNA (7, 25, 27). The antiviral
efficacy of D-FMAU in the duck model was also evaluated by
earlier studies with a dosage of 2 mg/kg/day given intraperitoneally
for 5 days to adult ducks, but its toxic effect was not studied
(12). It was further shown that D-FIAU is a more
efficient substrate for mitochondrial thymidine kinase 2 than for
cytosolic thymidine kinase 1 (36) and that D-FIAU-TP, as well as D-FMAU-TP, inhibits
mitochondrial function through its incorporation into mitochondrial DNA
by DNA polymerase-
, leading to ultrastructural defects in the
mitochondria and the accumulation of intracytoplasmic lipid droplets
(19, 28). Although it was shown in previous studies and in
the present work that L-FMAU does not significantly inhibit
cellular functions at concentrations that inhibit HBV replication, it
remains to be shown that its administration in hepatocyte culture does
not induce any ultrastructural modifications of mitochondria. Moreover, results of recent studies have shown that administration of
L-FMAU at a dosage of 10 mg/kg/day for 12 weeks is not
toxic in woodchuck HBV-infected woodchucks (32).
Experiments performed in vitro with the DHBV polymerase expressed in a
reticulocyte lysate system showed an inhibitory effect of
L-FMAU-TP on the incorporation of radiolabelled TMP during viral minus-strand DNA synthesis, suggesting that L-FMAU-TP
is indeed an inhibitor of DHBV reverse transcription. The study of L-FMAU metabolism in human hepatocytes showed that the
L-FMAU-TP concentration may peak at 20 µM
(27), which would already account for a 40% inhibition of
viral reverse transcriptase. Under our in vitro conditions, the
intracellular metabolism of the nucleoside analog is not taken into
account. Depending on the half-life of the nucleoside analog
triphosphate form, a greater inhibitory effect may be obtained in
tissue culture. This hypothesis may be relevant to the case of
L-FMAU, since its triphosphate metabolite was shown to have
a long half-life in human hepatocytes (27). L-FMAU-TP was also shown to have a very potent activity
against the DNA-dependent DNA polymerase activity of HBV polymerase
(27). It may be hypothesized that L-FMAU-TP has
a combined inhibitory effect on both the reverse transcriptase and the
DNA polymerase activities of the hepadnavirus polymerase, which may
result in a strong inhibition of viral replication in hepatocytes. By
comparison with the results obtained by Staschke and Colacino
(30), L-FMAU-TP appears to be a weaker inhibitor
of TMP incorporation in viral minus-strand DNA than fialuridine
triphosphate, suggesting that these drugs have a different mode of
action. Interestingly, we could also show that L-FMAU-TP
inhibits the synthesis of the DNA primer for reverse transcription in a
time-dependent manner, suggesting that L-FMAU-TP may
terminate the synthesis of the short primer (Fig. 6). The lack of
complete inhibition of dAMP incorporation in the viral primer may be
due to the fact that some of the viral polymerase polypeptides prime
reverse transcription directly with dAMP in the first position, as was
previously demonstrated by biochemical and genetic approaches
(9, 17, 30). It has yet to be established that
L-FMAU-TP is indeed incorporated in viral minus-strand DNA.
In conclusion, our results suggest that L-FMAU displays a
strong inhibitory effect on hepadnavirus replication in primary hepatocytes and in vivo in ducklings. Long-term administration of
L-FMAU, alone or in combination with other
L-nucleosides, should be evaluated in the woodchuck model
to study its toxicity and its ability to prevent the appearance of
viral resistance and to eradicate viral infection.
| |
ACKNOWLEDGMENTS |
We thank L. Cova (INSERM Unit 271, Lyon, France) for critical
review of the manuscript, P. Chossegros (INSERM Unit 271, Lyon, France)
for statistical analysis, and Michael Nassal (University of Heidelberg,
Heidelberg, Germany) for providing the anti-DHBV-core rabbit polyclonal
antibody.
This work was supported in part by fundings from the Ligue Nationale
Française Contre le Cancer and grants AI 33655 and AI 38204 from
the National Institutes of Health. Stéphanie Aguesse-Germon was a
recipient of a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche, France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: INSERM U271, 151 cours Albert Thomas, 69003 Lyon, France. Phone: (33) 4 72 68 19 70. Fax: (33) 4 72 68 19 71. E-mail:
zoulim{at}lyon151.inserm.fr.
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Abstract
Introduction
