Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, July 1998, p. 1805-1810, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Anti-Hepatitis B Virus Activity and Metabolism of
2',3'-Dideoxy-2',3'-Didehydro-
-L(
)-5-Fluorocytidine
Yong-Lian
Zhu,
Ginger E.
Dutschman,
Shwu-Huey
Liu,
Edward G.
Bridges, and
Yung-Chi
Cheng*
Department of Pharmacology, Yale University
School of Medicine, New Haven, Connecticut 06520
Received 14 November 1997/Returned for modification 7 March
1998/Accepted 9 April 1998
 |
ABSTRACT |
2',3'-Dideoxy-2',3'-didehydro-
-L(
)-5-fluorocytidine
[L()Fd4C] was found to be at least 10 times more potent
than -L-2',3'-dideoxy-3'-thiacytidine [L()SddC; also called 3TC, or
lamivudine]against hepatitis B virus (HBV) in culture. Its
cytotoxicity against HepG2 growth in culture was also greater than that
of L()SddC (3TC). There was no activity of this
compound against mitochondrial DNA synthesis in cells at
concentrations up to 10 µM. The dynamics of recovery of virus from
the medium of cells pretreated with equal drug concentrations were
slower with L()Fd4C than with L()SddC
(3TC). L()Fd4C could be metabolized to mono-, di-, and
triphosphate forms. The degree of L()Fd4C phosphorylation
to the 5'-triphosphate metabolite was higher than the degree of
L()SddC (3TC) phosphorylation when equal
extracellular concentrations of the two drugs were used. The apparent
Km of L()Fd4C phosphorylated
metabolites formed intracellularly was higher than that for
L()SddC (3TC). This may be due in part to a
difference in the behavior of L()Fd4C and
L()SddC (3TC) towards cytosolic deoxycytidine kinase.
Furthermore, L()Fd4C 5'-triphosphate was retained longer
within cells than L()SddC (3TC) 5'-triphosphate.
L()Fd4C 5'-triphosphate inhibited HBV DNA polymerase in
competition with dCTP with a Ki of 0.069 ± 0.015 µM. Given the antiviral potency and unique pharmacodynamic properties of L()Fd4C, this compound should be considered
for development as an expanded-spectrum anti-HBV drug.
| |
INTRODUCTION |
Hepatitis B virus (HBV) infection is
one of the most serious health issues in the world today (1,
3). -L()-2',3'-Dideoxy-3'-thiacytidine [L()SddC; also called 3TC, or lamivudine] (Fig.
1) is the first -L()
nucleoside analog identified by us and others to have potent activity
against HBV in culture (4, 8, 12, 17). This drug exerts its
action by inhibiting HBV DNA synthesis due to the preferential
interaction of the L()SddC (3TC) 5'-triphosphate metabolite with HBV DNA polymerase (4). Unlike
dideoxycytidine (ddC, or zalcitabine), a -D(+)
nucleoside analog with the natural nucleoside conformation in DNA and
RNA, L()SddC (3TC) does not have potent activity
against mitochondrial DNA (mtDNA) synthesis, which is important for
maintaining the function of cells (4). Clinical trials of
L()SddC (3TC) for the treatment of chronic HBV
infection are ongoing and look promising (2, 7, 13, 16, 18,
19). Its potential value for HBV-infected patients undergoing
liver transplantation is also being evaluated, since L()SddC (3TC) can suppress HBV DNA serum levels in
these patients. However, apparent L()SddC
(3TC)-resistant HBV emerged in some patients upon long-term treatment
(13, 16, 18). The HBV-resistant genotype appears to be
associated with the mutation of methionine to valine or isoleucine in
the YMDD motif of HBV DNA polymerase (13, 16, 18). This
mutation was previously demonstrated and could render duck HBV
resistant to L()SddC (3TC) (11). It is not
clear if this mutation alone can lead to resistance and, if so, to what
degree HBV resistance to L()SddC (3TC) develops.
One approach to overcoming clinical drug resistance is to use higher
dosages of L()SddC (3TC) given the therapeutic index of the compound in vitro. However, the potency of
L()SddC (3TC) against HBV in the clinic could be a
limiting factor given the dosage application. The antiviral
potency is determined not only by its antiviral activity but also by
the pharmacodynamic nature of its active metabolite,
L()SddC (3TC) 5'-triphosphate, in vivo. A more potent
compound with more favorable pharmacodynamic behavior of
intracellularly active metabolites would be worth exploring.
In the studies reported herein, we describe the anti-HBV
activity, metabolism, and pharmacodynamic properties of
2',3'-dideoxy-2',3'-didehydro--L()-5-fluorocytidine [L()Fd4C](Fig. 1) in comparison with those of
L()SddC (3TC), including its behavior toward
deoxycytidine kinase and the interaction of L()Fd4C
5'-triphosphate with virion-associated HBV DNA polymerase. Preliminary studies of the anti-HBV and anti-human
immunodeficiency virus (HIV) activities of L()Fd4C
were previously reported by us and others (10, 15).
| |
MATERIALS AND METHODS |
L()Fd4C and
2',3'-dideoxy-2',3'-didehydro--L()-cytidine
[L()d4C]were synthesized in the laboratory of the
late Tai-Shun Lin at Yale University (15).
[3H]L()Fd4C,
[3H]L()SddC
([3H]3TC), and [3H]deoxycytidine
were purchased from Moravek Biochemicals (Brea, Calif.), and the
-L() nucleoside analogs were further purified by using
a chiral column (Cyclobond I 2000, 500 by 10 cm; Advanced Separation
Technologies, Inc., Whippany, N.J.) with water as the mobile phase. All
other chemicals were of the highest grade available. L()Fd4C 5'-triphosphate was synthesized by scientists
from Vion Pharmaceuticals (New Haven, Conn.).
The procedures for assessing anti-HBV activity, cell growth, and
intracellular mtDNA content were described previously (8). Briefly, 6-day-old cultures of 2.2.15 cells (kindly provided by G. Acs), clonal derivatives of HepG2 that secrete hepatitis B virions,
were treated with various drug concentrations for 6 days; fresh medium
and drug were added on day 3, and the culture medium was processed for
analysis of HBV DNA on day 6. For studies of the reversal of drug
anti-HBV activity, 2.2.15 cultures were treated under various drug
conditions. After 6 days of drug treatment, fresh medium without drug
was added, and the cells were cultured an additional 12 days in the
absence of drug. After 6 days without drug and after 12 days without
drug, the culture medium was processed for analysis of HBV DNA. Virions
secreted into the culture medium were obtained by a polyethylene
glycol precipitation method. HBV DNA isolated from these virions was
analyzed by Southern blot analysis. Inhibition of viral DNA replication
was assessed by comparison of HBV DNA from drug-treated and untreated
cultures using hybridization of the blots to an HBV-specific probe
followed by autoradiography. Quantitative densitometry and computer
generation of images of autoradiographs were performed on a Molecular
Dynamics Personal Densitometer SI by using ImageQuaNT image analysis
software.
For metabolism studies, HepG2 cells were used 2 days after they reached
confluency. Cells were treated under the conditions indicated and were
washed twice with ice-cold phosphate-buffered saline at the time of
harvesting. Nucleotides were extracted from cells in 60% cold
methanol. The samples were centrifuged and supernatant was analyzed as
previously described (16) by ion-exchange high-performance liquid chromatography (HPLC) using a Whatman Partisil-SAX column.
Crude extract from aged HepG2 cells was prepared by suspending cells in
buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM ATP-MgCl2, and 2 mM dithiothreitol (5). After three
freeze-thaw cycles, the extract was centrifuged and the supernatant was
removed. Streptomycin sulfate was added to a concentration of 1%, and
following centrifugation ammonium sulfate was added to the supernatant
to bring the final concentration to 60%. The ammonium
sulfate-precipitated pellet was resuspended in the
ATP-MgCl2-containing buffer and dialyzed to remove ammonium
sulfate. The deoxycytidine kinase assay was performed as previously
described (5) by using 100 µM substrate {64 mCi of
[3H]deoxycytidine/mmol, 8 mCi of
[3H]L()Fd4Ci/mmol, or 27 mCi of
[3H]L()SddC
([3H]3TC)/mmol} in the presence or absence of
inhibitor (300 µM bromovinyldeoxyuridine [BVdU] or 200 µM
deoxycytidine). Kinetic determinations were performed by
Lineweaver-Burk plots using concentration ranges of 0.625 to 10 µM
[3H]deoxycytidine, 6.5 to 162 µM
[3H]L()Fd4C, and 1.25 to 20 µM
[3H]L()SddC
([3H]3TC).
Hepatitis B viral particles isolated by the polyethylene glycol
precipitation method were used to assess the inhibition of HBV DNA
polymerase by L()Fd4C 5'-triphosphate. The assay
mixture consisted of virion-associated HBV DNA polymerase in a solution containing 50 mM Tris-HCl (pH 7.5), 340 mM KCl, 22 mM
-mercaptoethanol, 0.4% Nonidet P-40, 70 µM each dATP, dTTP, and
dGTP, and 0.175 µM [
-32P]dCTP (11 Ci/mmol). The
details of the assay and gel electrophoresis of the DNA have been
described elsewhere (8).
| |
RESULTS |
Comparison of the anti-HBV activities of L()Fd4C,
L()d4C, and L()SddC
(3TC).
The HBV producer cell line HepG2 2.2.15 was used to
evaluate the anti-HBV activities of L()Fd4C,
L()d4C, and L()SddC (3TC). Our
studies showed that L()Fd4C and
L()d4C had potent anti-HBV activities. Antiviral effects
were measured by analysis of extracellular HBV DNA obtained from
secreted viral particles. Aged 2.2.15 cells were exposed to
L()Fd4C, L()d4C, or
L()SddC (3TC) for 6 days as indicated (Fig.
2). The amount of HBV DNA secreted from
these cells was analyzed as previously described (8). The
results show that each -L() nucleoside analog
decreased the amount of extracellular HBV DNA in a dose-dependent
manner. Secretion of HBV DNA from 2.2.15 cells was decreased by 50% at
a concentration of 1 nM by L()Fd4C, which was
approximately 7 and 15 times more potent than L()d4C and
L()SddC (3TC), respectively. There was no effect of
any of these analogs on cellular mtDNA content at concentrations up to
10 µM. The concentration required to inhibit 50% of HepG2 growth in
3-day cultures was estimated to be 20 µM for
L()Fd4C, 14 µM for L()d4C, and more
than 50 µM for L()SddC (3TC). None of these
compounds had any effect on HBV surface antigen secretion from 2.2.15 cells at concentrations up to 10 µM.
View larger version (66K):
[in this window]
[in a new window]
|
FIG. 2.
HBV inhibition by -L() nucleoside
analogs. (Top) Computer-generated autoradiograph of samples isolated
from HepG2 2.2.15 culture medium hybridized to a HBV DNA probe.
(Bottom) Graph of densitometric data from the autoradiograph: HBV DNA
secretion in drug-treated cultures. The DNA was processed and analyzed
as described in Materials and Methods.
|
|
To address the issue of the reversibility of the anti-HBV activity upon
removal of the drug from culture, 2.2.15 cells were
treated with drug
for 6 days and cultured an additional 12 days
in the absence of drug,
with medium changes every 3 days. The
levels of HBV DNA secreted into
the culture medium were assessed
on days 6 and 12 post-drug removal.
The results indicated that
the anti-HBV activities of all three analogs
are reversible and
that the time to reversal of the anti-HBV activity
is dependent
upon the initial drug concentration (Table
1). At equal concentrations,
the degree
of reversal at day 12 was much lower for
L(
)Fd4C than
for
L(
)d4C or
L(
)SddC (3TC). This
suggests that the continual
presence of the nucleoside analog is
required to maintain inhibitory
activity.
Intracellular formation of phosphorylated metabolites in HepG2
cells.
Because the 5'-triphosphate metabolite of
L()Fd4C is a potential substrate for viral DNA
polymerase, it is important to determine its phosphorylation profile in
cell culture. The experiment was done by incubating the human
hepatocellular carcinoma cell line HepG2 with 2 µM
[3H]L()Fd4C. At the indicated
times, cells were harvested, washed, and methanol extracted, and the
soluble fraction was analyzed by ion-exchange HPLC as described in
Materials and Methods. There was no detectable
[3H]L()Fd4C associated with the
methanol-insoluble fraction (less than 0.1 pmol/106 cells)
with up to 24 h of drug exposure. Radioactivity was associated with four peaks that were subsequently identified as
L()Fd4C, L()Fd4C
5'-monophosphate, L()Fd4C 5'-diphosphate, and
L()Fd4C 5'-triphosphate (Fig.
3A). The time-dependent formation of
intracellular L()Fd4C phosphorylated metabolites is
depicted in Fig. 3B. There was a linear increase in the amount of
L()Fd4C phosphorylated metabolites up to 24 h.
Although the proportions of the phosphorylated metabolites varied with
time, L()Fd4C 5'-diphosphate was the predominant
metabolite at 4 h, whereas the proportion of
L()Fd4C 5'-triphosphate increased with time. The
dose-dependent formation of phosphorylated metabolites of
L()Fd4C was compared with that of
L()SddC (3TC) at 24 h post-drug exposure. As
shown in Fig. 4, there were
concentration-dependent increases in the phosphorylated metabolites of
both compounds. The amount of L()Fd4C phosphorylated metabolites formed is greater than that of L()SddC
(3TC) phosphorylated metabolites. When the reciprocal of total
intracellular phosphorylated metabolite formation was plotted against
the reciprocal of the extracellular drug concentration, a linear
relationship was observed, with apparent Kms of
100 µM for L()Fd4C and 4.4 µM for
L()SddC; the relative maximum amount of formation was
33 for L()Fd4C and 1 for L()SddC
(3TC).
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 3.
(A) Resolution by ion-exchange HPLC of intracellular
metabolites detected in HepG2 cells exposed to 2 µM
[3H]L()Fd4C (11 mCi/mmol). The
metabolites were identified as L()Fd4C (I),
L()Fd4C 5'-monophosphate (II),
L()Fd4C 5'-diphosphate (III), and
L()Fd4C 5'-triphosphate (IV). (B) Time course of
intracellular metabolites detected in HepG2 cells after incubation with
2 µM [3H]L()Fd4C (11 mCi/mmol).
|
|
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 4.
Intracellular concentrations of metabolites detected in
HepG2 cells after 24 h of incubation with either
[3H]L()Fd4C (11 mCi/mmol) (A) or
[3H]L()SddC
([3H]3TC) (40 mCi/mmol) (B) at the indicated
concentrations. The metabolites were identified as the 5'-monophosphate
(II), the 5'-diphosphate (III), the 5'-triphosphate (IV), and an
unidentified metabolite that eluted between the nucleoside and the
monophosphate (V).
|
|
Retention of L()Fd4C phosphorylated metabolites
in comparison with that of L()SddC (3TC)
phosphorylated metabolites in HepG2 cells.
Retention of
L()Fd4C phosphorylated metabolites in HepG2 cells was
compared with that of L()SddC (3TC) phosphorylated
metabolites by incubating HepG2 cells with 2 µM
[3H]L()Fd4C or
[3H]L()SddC
([3H]3TC). After 24 h of exposure to
radiolabeled drug, the cells were washed, and fresh culture medium was
added. At various times following drug removal, cells were harvested
and analyzed for phosphorylated metabolites. Fresh medium without drug
was replaced at 8 h to prevent the reuptake of drug secreted from
cells. Figure 5 shows the intracellular
content of phosphorylated metabolites at various times following drug
removal. The apparent intracellular half-life of the
-L() nucleoside analog phosphorylated metabolite was
approximately 20 h for L()Fd4C and 4 h for
L()SddC (3TC). At 24 h after drug removal, there
was still approximately 10 µM L()Fd4C
5'-triphosphate in cells.
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 5.
Intracellular concentration of metabolites detected in
HepG2 cells after removal of drug from the culture medium. HepG2 cells
were exposed to either 2 µM
[3H]L()Fd4C (11 mCi/mmol) (A) or 2 µM [3H]L()SddC
([3H]3TC) (40 mCi/mmol) (B) for 24 h. The
metabolites were identified as explained for Fig. 3 and 4.
|
|
Phosphorylation of L()Fd4C and
L()SddC (3TC) by HepG2 cellular extracts.
Extracts of HepG2 cells were prepared in order to examine the
phosphorylation of L()Fd4C and
L()SddC (3TC) as described in Materials and Methods.
BVdU, a potent inhibitor of mitochondrial deoxypyrimidine nucleoside
kinase, could not inhibit the phosphorylation of
L()Fd4C and L()SddC (3TC), whereas
deoxycytidine inhibited their phosphorylation. This suggests that
neither of these -L() nucleoside analogs serves as a
substrate for mitochondrial deoxypyrimidine nucleoside kinase in these
cell extracts (9). The concentration-dependent phosphorylation of L()Fd4C and
L()SddC (3TC) was examined in the cellular extracts.
The apparent Kms of L()Fd4C
and L()SddC (3TC) were determined to be 100 and 11 µM, respectively, in crude HepG2 cellular extracts and were not
dissimilar to the values obtained from purified deoxycytidine kinase
for L()Fd4C and L()SddC (3TC)
(9). The difference in the relative
Vmax values for L()Fd4C and
L()SddC (3TC) was about 36-fold (Table
2).
Inhibition of virion-associated HBV DNA polymerase by
L()Fd4C 5'-triphosphate.
The effect of
L()Fd4C 5'-triphosphate on the activity of
virion-associated HBV DNA polymerase in vitro was examined. There is a
dose-dependent inhibition of [-32P]dCTP
incorporation into viral DNA, as shown in Fig.
6. The Ki was
calculated to be 69 ± 15 nM by using the competitive inhibition equation as previously described (6).
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 6.
Inhibition of endogenous HBV DNA polymerase by
L()Fd4C 5'-triphosphate. (A) Computer-generated
autoradiograph of HBV DNA analyzed by agarose gel electrophoresis. (B)
Quantitation of HBV DNA by densitometry. The assay was performed as
described in Materials and Methods.
|
|
| |
DISCUSSION |
L()Fd4C is among the most potent anti-HBV
compounds discovered. The introduction of a fluorine atom at the 5 position of cytosine enhances the antiviral activity against both HBV
and HIV with minimal effects on cytotoxicity. The cytotoxicity of L()Fd4C is dependent on the cell line used. The
concentration required to inhibit 50% of the growth of HepG2 cells is
about 2 to 3 times greater than that for CEM cells. Based on the
concentration required to inhibit HBV or HIV replication and that
required to inhibit cell growth in culture, L()Fd4C
is at least as impressive as L()SddC (3TC) as an
antiviral compound. The anti-HBV activity of L()Fd4C
is reversible, like that of L()SddC (3TC). However, at equal concentrations, it takes longer for drug-treated 2.2.15 cells
to resynthesize viral DNA after removal of L()Fd4C
than after removal of L()SddC (3TC). This could be
due to differences in the rates of removal of these compounds from the
termini of HBV DNA, a possibility which is currently under
investigation, and/or to differences in the retention of intracellular
active metabolites.
Like L()SddC (3TC), L()Fd4C could
be phosphorylated to mono-, di-, and triphosphate metabolites. The
level of formation of phosphorylated metabolites in cells is higher for
L()Fd4C than for L()SddC (3TC).
Also, the apparent Km and the relative amount of
phosphorylated metabolites in whole cells are similar to those with
HepG2 extracts. Deoxycytidine inhibits the formation of
L()Fd4C and L()SddC (3TC)
metabolites in HepG2 cell extracts, suggesting that
L()Fd4C, L()SddC (3TC), and
deoxycytidine share a common kinase for their phosphorylation to
monophosphate metabolites. The inhibition of deoxycytidine
phosphorylation by BVdU suggests the presence of mitochondrial
deoxypryrimidine nucleoside kinase in the HepG2 cell extracts. However,
the absence of an effect of BVdU on L()Fd4C and
L()SddC (3TC) phosphorylation implies that
mitochondrial deoxypyrimidine nucleoside kinase does not phosphorylate
these two -L() nucleoside analogs. Indeed, the enzyme
responsible for the phosphorylation of L()Fd4C has
been shown to be cytosolic deoxycytidine kinase (9). That
study also shows that mitochondrial deoxypyrimidine nucleoside kinase does not phosphorylate L()Fd4C. The major metabolite
of L()Fd4C and L()SddC (3TC) is the
diphosphate nucleotide as early as 4 h after drug exposure. This
suggests that, for both -L() nucleoside analogs, the
step of conversion of the diphosphate metabolite to the triphosphate
metabolite is not efficient. However, this step appears to be more
efficient for L()Fd4C than for
L()SddC (3TC) when equal extracellular drug
concentrations are compared. This is supported by the observation that
longer drug exposure times decrease the
diphosphate-to-triphosphate ratio of L()Fd4C faster than that of L()SddC (3TC). Whether this is
due to a difference between L()SddC (3TC)
5'-diphosphate and L()Fd4C 5'-diphosphate in behavior
towards the enzyme for phosphorylation or a difference in the
intracellular levels of these two diphosphate metabolites is not clear.
L()Fd4C metabolites have a much longer retention time
in HepG2 cells than L()SddC (3TC) metabolites. This
could partly explain why the time to reappearance of HBV DNA after drug removal is greater for L()Fd4C-treated cells than for
L()SddC (3TC)-treated cells.
L()Fd4C 5'-triphosphate is inhibitory to the
incorporation of dCTP into viral DNA by HBV DNA polymerase. The
Ki is estimated to be much lower than the
intracellular content of L()Fd4C 5'-triphosphate at a
2 µM extracellular concentration of L()Fd4C. It is
likely that L()Fd4C 5'-triphosphate is an alternative
substrate for the viral enzyme and can be incorporated into viral DNA
as a DNA chain terminator. This is currently under investigation,
including the interaction of L()Fd4C 5'-triphosphate with human DNA polymerase , , 
, and 
(14).
In summary, L()Fd4C is much more potent than
L()SddC (3TC) against HBV in cell culture. It is
likely that the dosage required to suppress HBV replication in patients
will be lower for L()Fd4C than for
L()SddC (3TC). This could be important in view of the current dosage of L()SddC (3TC), which is 100 mg per
day for the treatment of patients with chronic HBV infection.
Escalation of the dosage, if required to overcome viral resistance to
L()SddC (3TC), could be achieved with much less
L()Fd4C than L()SddC (3TC),
assuming that there is the same degree of viral resistance to
L()Fd4C. The higher potency against HBV of
L()Fd4C compared to L()SddC (3TC)
is partly due to the higher levels of phosphorylated metabolites of
L()Fd4C compared to those of
L()SddC (3TC), which may reflect differences in the
behavior of these two analogs towards cytosolic deoxycytidine kinase.
L()Fd4C 5'-triphosphate is most likely the active
metabolite that can inhibit HBV DNA replication. The intracellular
retention time of phosphorylated L()Fd4C metabolites is about 5 times longer than that of phosphorylated
L()SddC (3TC) metabolites. This suggests that
less-frequent doses of L()Fd4C than of
L()SddC (3TC) are required, assuming that there is no major difference in pharmacokinetic behavior between these two analogs.
This is particularly important in view of the potent activity of
L()Fd4C against HIV. Currently, two doses per day are
required for L()SddC (3TC), while one could be
required for L()Fd4C, making patient compliance
easier. If the longer retention time of phosphorylated
L()Fd4C metabolites in cells compared with that of
phosphorylated L()SddC (3TC) metabolites also holds for HIV-susceptible cells in patients as for CEM cells (9), then L()Fd4C could be more advantageous than
L()SddC (3TC) for the treatment of patients with HIV
infection. Based on the antiviral potency and unique pharmacodynamic
properties of L()Fd4C, this compound should be
explored further for its clinical potential.
| |
ACKNOWLEDGMENTS |
This work was supported by grants AI-38204 and CA-63477 from the
National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pharmacology, Yale University School of Medicine, 333 Cedar St., P.O. Box 802066, New Haven, CT 06520-8066. Phone: (203) 785-7118. Fax: (203)
785-7129. E-mail: cheng.lab{at}yale.edu.
Present address: Department of Pharmacology, Zhejiang Medical
University, Hangzhou, Zhejiang 310006, People's Republic of China.
| |
REFERENCES |
| 1.
|
Beasley, R. P.,
L. Y. Hwang,
C. C. Lin, and S. Chien.
1981.
Hepatocellular carcinoma and hepatitis B virus.
Lancet
ii:1129-1133.
|
| 2.
|
Benhamou, Y.,
E. Dohin,
F. Lunel-Fabiani,
T. Poynard,
J. M. Huraus,
C. Katlama,
P. Opolon, and M. Gentilini.
1994.
Efficacy of lamivudine on replication of hepatitis B virus in HIV-infected patients.
Lancet
345:396-397.
|
| 3.
|
Centers for Disease Control and Prevention.
1995.
Cases of selected notifiable diseases.
Morbid. Mortal. Weekly Rep.
1995:43-963.
|
| 4.
|
Chang, C.-N.,
S.-L. Doong,
J. H. Zhou,
J. W. Beach,
L. S. Jeong,
C. K. Chu,
C. H. Tsai,
D. C. Liotta,
R. F. Schinazi, and Y.-C. Cheng.
1992.
Deoxycytidine deaminase-resistant stereoisomer is the active form of (±)-2',3'-dideoxy-3'-thiacytidine in the inhibition of hepatitis B virus replication.
J. Biol. Chem.
267:13938-13942[Abstract/Free Full Text].
|
| 5.
|
Cheng, Y.-C.,
B. Domin, and L.-S. Lee.
1977.
Human deoxycytidine kinase. Purification and characterization of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia patients.
Biochim. Biophys. Acta
481:481-492[Medline].
|
| 6.
|
Cheng, Y.-C., and W. H. Prusoff.
1973.
Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (ID50) of an enzymatic reaction.
Biochem. Pharmacol.
22:3099-3108[Medline].
|
| 7.
|
Dienstag, J. L.,
R. P. Perrillo,
E. R. Schiff,
M. Bartholomew,
C. Vicary, and M. Rubin.
1995.
A preliminary trial of lamivudine for chronic hepatitis B infection.
N. Engl. J. Med.
333:1657-1661[Abstract/Free Full Text].
|
| 8.
|
Doong, S. I.,
C. H. Tsai,
R. F. Schinazi,
D. C. Liotta, and Y. C. Cheng.
1991.
Inhibition of the replication of hepatitis B virus in vitro by 2',3'-dideoxy-3'-thiapyrimidine and related analogues.
Proc. Natl. Acad. Sci. USA
88:8495-8499[Abstract/Free Full Text].
|
| 9.
|
Dutschman, G. E.,
E. G. Bridges,
S.-H. Liu,
E. Gullen,
X. Guo,
M. Kukhanova, and Y.-C. Cheng.
1998.
Metabolism of 2',3'-dideoxy-2',3'-dehydro--L()-5-fluorocytidine and its activity in combination with clinically approved anti-human immunodeficiency virus -D(+) nucleoside analogs in vitro.
Antimicrob. Agents Chemother.
42:1799-1804[Abstract/Free Full Text].
|
| 10.
|
Faraj, A.,
R. F. Schinazi,
A. Joudawlkis,
Z. Lesnikowski,
A. McMillan,
C. D. Morrow, and J.-P. Sommadossi.
1997.
Effects of -D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine 5'-triphosphate (-D-D4FC-TP) and its -L-enantiomer 5'-triphosphate (-L-D4FC-TP) on viral DNA polymerases.
Antivir. Res.
34:A66.
|
| 11.
|
Fischer, K. P., and D. L. J. Tyrrell.
1996.
Generation of duck hepatitis B virus polymerase mutants through site-directed mutagenesis which demonstrate resistance to lamivudine [()--L-2',3'-dideoxy-3'-thiacytidine] in vitro.
Antimicrob. Agents Chemother.
40:1957-1960[Abstract].
|
| 12.
|
Furman, P. A.,
M. Davis,
D. C. Liotta,
M. Paff,
L. W. Frick,
D. J. Nelson,
R. E. Dornsife,
J. A. Wurster,
L. J. Wilson,
J. A. Fyfe,
J. V. Tuttle,
W. H. Miller,
L. Condreay,
D. R. Averett,
R. F. Schinazi, and G. R. Painter.
1992.
The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the () and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.
Antimicrob. Agents Chemother.
36:2686-2692[Abstract/Free Full Text].
|
| 13.
|
Honkoop, P.,
H. G. M. Niesters,
R. A. M. Deman,
A. D. M. E. Osterhaus, and S. W. Schalm.
1997.
Lamivudine resistance in immunocompetent chronic hepatitis B. Incidence and patterns.
J. Hepatol.
26:1393-1395[Medline].
|
| 14.
| Kukhanova, M., and Y.-C. Cheng. 1997. Unpublished data.
|
| 15.
|
Lin, T.-S.,
M.-Z. Luo,
M.-C. Liu,
Y.-L. Zhu,
E. Gullen,
G. E. Dutschman, and Y.-C. Cheng.
1996.
Design and synthesis of 2',3'-dideoxy-2',3'-didehydro--L-cytidine (-L-d4C) and 2',3'-dideoxy-2',3'-didehydro--L-5-fluorocytidine (-L-Fd4C), two exceptionally potent inhibitors of human hepatitis B virus (HBV) and potent inhibitors of human immunodeficiency virus (HIV) in vitro.
J. Med. Chem.
39:1757-1759[Medline].
|
| 16.
|
Ling, R.,
D. Mutimer,
M. Ahmed,
E. H. Boxall,
E. Elias,
G. M. Dusheiko, and T. J. Harrison.
1996.
Selection of mutations in the hepatitis B virus polymerase during therapy of transplant recipients with lamivudine.
Hepatology
24:711-713[Medline].
|
| 17.
|
Severini, A.,
X.-Y. Liu,
J. S. Wilson, and D. L. J. Tyrrell.
1995.
Mechanism of inhibition of duck hepatitis B virus polymerase by ()--L-2',3'-dideoxy-3'-thiacytidine.
Antimicrob. Agents Chemother.
39:1430-1435[Abstract].
|
| 18.
|
Tipples, G. A.,
M. M. Ma,
K. P. Fischer,
V. G. Bain,
N. M. Kneteman, and D. L. Tyrrell.
1996.
Mutation in HBV RNA-dependent polymerase confers resistance to lamivudine in vivo.
Hepatology
24:714-717[Medline].
|
| 19.
|
Tyrrell, D. L. J.,
M. C. Mitchell,
R. DeMon,
S. W. Schalm,
J. Main,
H. C. Thomas,
J. Fevery,
F. Nevens,
P. Beranck, and C. Vicary.
1993.
Phase II trial of lamivudine for chronic hepatitis B.
Hepatology
18:112A.
|
Antimicrobial Agents and Chemotherapy, July 1998, p. 1805-1810, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Mazzucco, C. E., Hamatake, R. K., Colonno, R. J., Tenney, D. J.
(2008). Entecavir for Treatment of Hepatitis B Virus Displays No In Vitro Mitochondrial Toxicity or DNA Polymerase Gamma Inhibition. Antimicrob. Agents Chemother.
52: 598-605
[Abstract]
[Full Text]
-
Paintsil, E., Dutschman, G. E., Hu, R., Grill, S. P., Lam, W., Baba, M., Tanaka, H., Cheng, Y.-C.
(2007). Intracellular Metabolism and Persistence of the Anti-Human Immunodeficiency Virus Activity of 2',3'-Didehydro-3'-Deoxy-4'-Ethynylthymidine, a Novel Thymidine Analog. Antimicrob. Agents Chemother.
51: 3870-3879
[Abstract]
[Full Text]
-
Hernandez-Santiago, B. I., Mathew, J. S., Rapp, K. L., Grier, J. P., Schinazi, R. F.
(2007). Antiviral and Cellular Metabolism Interactions between Dexelvucitabine and Lamivudine. Antimicrob. Agents Chemother.
51: 2130-2135
[Abstract]
[Full Text]
-
Lam, W., Li, Y., Liou, J.-Y., Dutschman, G. E., Cheng, Y.-c.
(2004). Reverse Transcriptase Activity of Hepatitis B Virus (HBV) DNA Polymerase within Core Capsid: Interaction with Deoxynucleoside Triphosphates and Anti-HBV L-Deoxynucleoside Analog Triphosphates. Mol. Pharmacol.
65: 400-406
[Abstract]
[Full Text]
-
Liou, J.-Y., Krishnan, P., Hsieh, C.-C., Dutschman, G. E., Cheng, Y.-c.
(2003). Assessment of the Effect of Phosphorylated Metabolites of Anti-Human Immunodeficiency Virus and Anti-Hepatitis B Virus Pyrimidine Analogs on the Behavior of Human Deoxycytidylate Deaminase. Mol. Pharmacol.
63: 105-110
[Abstract]
[Full Text]
-
Kamiya, N., Kubota, A., Iwase, Y., Sekiya, K., Ubasawa, M., Yuasa, S.
(2002). Antiviral Activities of MCC-478, a Novel and Specific Inhibitor of Hepatitis B Virus. Antimicrob. Agents Chemother.
46: 2872-2877
[Abstract]
[Full Text]
-
Krishnan, P., Liou, J.-Y., Cheng, Y.-C.
(2002). Phosphorylation of Pyrimidine L-Deoxynucleoside Analog Diphosphates. KINETICS OF PHOSPHORYLATION AND DEPHOSPHORYLATION OF NUCLEOSIDE ANALOG DIPHOSPHATES AND TRIPHOSPHATES BY 3-PHOSPHOGLYCERATE KINASE. J. Biol. Chem.
277: 31593-31600
[Abstract]
[Full Text]
-
Liou, J.-Y., Dutschman, G. E., Lam, W., Jiang, Z., Cheng, Y.-C.
(2002). Characterization of Human UMP/CMP Kinase and Its Phosphorylation of D- and L-Form Deoxycytidine Analogue Monophosphates. Cancer Res.
62: 1624-1631
[Abstract]
[Full Text]
-
Krishnan, P., Fu, Q., Lam, W., Liou, J.-Y., Dutschman, G., Cheng, Y.-C.
(2002). Phosphorylation of Pyrimidine Deoxynucleoside Analog Diphosphates. SELECTIVE PHOSPHORYLATION OF L-NUCLEOSIDE ANALOG DIPHOSPHATES BY 3-PHOSPHOGLYCERATE KINASE. J. Biol. Chem.
277: 5453-5459
[Abstract]
[Full Text]
-
Le Guerhier, F., Pichoud, C., Jamard, C., Guerret, S., Chevallier, M., Peyrol, S., Hantz, O., King, I., Trépo, C., Cheng, Y.-C., Zoulim, F.
(2001). Antiviral Activity of {beta}-L-2',3'-Dideoxy-2',3'-Didehydro-5-Fluorocytidine in Woodchucks Chronically Infected with Woodchuck Hepatitis Virus. Antimicrob. Agents Chemother.
45: 1065-1077
[Abstract]
[Full Text]
-
Kira, T., Grill, S. P., Dutschman, G. E., Lin, J.-S., Qu, F., Choi, Y., Chu, C. K., Cheng, Y.-C.
(2000). Anti-Epstein-Barr Virus (EBV) Activity of beta -L-5-Iododioxolane Uracil Is Dependent on EBV Thymidine Kinase. Antimicrob. Agents Chemother.
44: 3278-3284
[Abstract]
[Full Text]
-
Pelicano, H., Kukhanova, M., Cheng, Y.-C.
(2000). Excision of beta -D- and beta -L-Nucleotide Analogs from DNA by the Human Cytosolic 3'-to-5' Exonuclease. Mol. Pharmacol.
57: 1051-1055
[Abstract]
[Full Text]
-
Le Guerhier, F., Pichoud, C., Guerret, S., Chevallier, M., Jamard, C., Hantz, O., Li, X.-Y., Chen, S.-H., King, I., Trépo, C., Cheng, Y.-C., Zoulim, F.
(2000). Characterization of the Antiviral Effect of 2',3'-Dideoxy-2', 3'-Didehydro-beta -L-5-Fluorocytidine in the Duck Hepatitis B Virus Infection Model. Antimicrob. Agents Chemother.
44: 111-122
[Abstract]
[Full Text]
-
Sarafianos, S. G., Das, K., Clark, A. D. Jr., Ding, J., Boyer, P. L., Hughes, S. H., Arnold, E.
(1999). Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta -branched amino acids. Proc. Natl. Acad. Sci. USA
96: 10027-10032
[Abstract]
[Full Text]
-
Dutschman, G. E., Bridges, E. G., Liu, S.-H., Gullen, E., Guo, X., Kukhanova, M., Cheng, Y.-C.
(1998). Metabolism of 2',3'-Dideoxy-2',3'-Didehydro-beta -L(-)-5-Fluorocytidine and Its Activity in Combination with Clinically Approved Anti-Human Immunodeficiency Virus beta -D(+) Nucleoside Analogs In Vitro. Antimicrob. Agents Chemother.
42: 1799-1804
[Abstract]
[Full Text]
Top
Abstract
Introduction
