Download Original article Anti-hepatitis B virus activity in vitro of combinations

Antiviral Chemistry & Chemotherapy 19:165–176
Original article
Anti-hepatitis B virus activity in vitro of combinations
of tenofovir with nucleoside/nucleotide analogues
Yuao Zhu1*, Maria Curtis1, Xiaoping Qi1, Michael D Miller1 and Katyna Borroto-Esoda1
Gilead Sciences, Inc., Durham, NC, USA
1
*Corresponding author: E-mail: [email protected]
Background: Long-term management of some chronic hepatitis B patients might require combination therapy using
drugs with distinct resistance profiles to sustain viral suppression and to reduce the resistance-associated failure.
Tenofovir disoproxil fumarate (TDF), approved for hepatitis
B virus (HBV) and HIV-1 treatment, is active against wildtype HBV and HBV containing YMDD mutations, which
confer resistance to emtricitabine (FTC), lamivudine (3TC)
and telbivudine (LdT) and contribute to entecavir (ETV)
resistance. We therefore evaluated the in vitro anti-HBV
activity of tenofovir (TFV), the active parent drug of TDF,
combined with FTC, 3TC, ETV, LdT and adefovir (AFV).
Methods: The anti-HBV activities of the compounds were
tested using the AD38 cell line that expresses wild-type
HBV from a tetracycline-controllable promoter. Intracellular HBV DNA levels were quantified using real-time
PCR assay and cytotoxicities were assessed with XTT
assays. The antiviral data of the drug combinations were
evaluated using MacSynergy analyses on the basis of
the Bliss independence model as well as isobologram
analyses on the basis of the Loewe additivity theory.
Results: All drug combinations tested, FTC+TFV, 3TC+TFV,
ETV+TFV, LdT+TFV and AFV+TFV, showed additive antiviral interactions as analysed by MacSynergy. Isobologram analyses revealed that these combination pairs
were additive, with the exception of FTC+TFV, which
demonstrated slight synergistic activity. No cytotoxic
or antagonistic effects were observed with any of the
combinations tested.
Conclusions: The combination of TFV with FTC, 3TC, ETV,
LdT or AFV had additive to slightly synergistic anti-HBV
effects in vitro. These results support the use of TDF as
a component in combination regimens with currently
available anti-HBV nucleoside analogues.
Introduction
Chronic hepatitis B (CHB) infection is a significant
global public health problem affecting an estimated
350–400 million individuals and leading to 1 million
annual deaths worldwide from resultant illnesses, such
as cirrhosis and hepatocellular carcinoma [1,2]. Two
categories of drugs are used in CHB therapy: the interferons, including standard interferon-α or pegylated
interferon-α, and nucleoside/nucleotide hepatitis B virus
(HBV) reverse transcriptase (RT) inhibitors. Interferon-α
and the chemically modified pegylated interferon-α are
cytokines with immunomodulatory and antiviral activities. They are only effective in approximately one-third
of indicated patients and are associated with significant
side effects [3–5]. Monotherapy with an individual
nucleoside/nucleotide analogue is the current standard
of care for many patients [6]. There are five approved
nucleoside/nucleotide analogues, including lamivudine
(3TC), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT) and tenofovir disoproxil fumarate (TDF).
© 2009 International Medical Press 1359-6535
Zhu.indd 165
Several other analogues are in various stages of drug
development, including emtricitabine (FTC), clevudine
and pradefovir. Because of the persistent nature of CHB
infection, which is largely attributable to the stability
of HBV covalently closed circular DNA (cccDNA) [7],
these therapies rarely produce hepatitis B surface antigen seroconversion and therefore require prolonged
administration to control disease in most patients.
Long-term therapy, however, can be associated with the
emergence of resistant HBV strains, leading to loss of
therapeutic benefit and liver disease progression.
Resistance to 3TC results from the selection of HBV
RT rtM204V and rtM204I (YMDD) mutations and
occurs in approximately 20% of patients per year of
treatment [8]. Long-term use of other nucleoside/nucleotide analogues are also associated with resistance
development. FTC, an l-­nucleoside cytosine analogue
approved for the treatment of HIV-1 that is structurally
similar to 3TC, also selected the rtM204V and rtM204I
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Y Zhu et al.
mutations although at a frequency lower than 3TC [9].
LdT is an l-nucleoside analogue of thymidine and has
been shown to be more efficacious than 3TC at reducing serum HBV DNA in clinical trials [10]. However,
its long-term usage also resulted in virological breakthrough from rtM204V and rtM204I mutations [10].
ETV, a deoxyguanosine analogue, has partially reduced
activity against rtM204V and rtM204I mutants [11].
Long-term usage of ETV selects for a number of resistance mutations in HBV RT, including I169T, T184S/G,
S202I/G and M250V, which occur in addition to the
3TC rtM204V and rtM204I mutations [12,13]. By contrast, ADV maintains both in vitro and clinical efficacy
against 3TC resistance mutations [14,15], but its longterm administration selects for the resistance mutations
rtN236T and/or rtA181V/T [16,17].
TDF, an oral prodrug of tenofovir (TFV), showed
potent anti-HBV efficacy in vivo [18,19] and was
recently approved in the USA and EU for CHB treatment. TDF is also a potent inhibitor of HIV type-1
(HIV-1) and is a recommended component of antiHIV-1 therapies for HIV-1-infected patients as well as
for patients coinfected with HIV-1 and HBV [20]. TFV
is structurally similar to adefovir (AFV), and showed
similar in vitro activity against wild-type HBV [21–27].
It competitively inhibits HBV RT activity by its incorporation into virus DNA, resulting in chain termination
[23]. Like AFV, TFV is also active against 3TC-­resistant
HBV [11]. HBV resistance to TDF remains to be identified and confirmed. Previously, one report found
that two patients coinfected with HIV-1 and HBV
who were receiving antiviral treatment including TDF
were found to have HBV with the rtA194T mutation
in combination with the rtL180M+M204V mutations
[28]. In vitro phenotypical analyses showed a reduced
susceptibility of virus containing the rtA194T alone or
in combination with the rtL180M+M204V mutations
to TFV [28]. However, these results were not reproduced by a different group [23]. Therefore, whether
rtA194T is associated with resistance to TDF remains
to be resolved. The ongoing large scale clinical trials, as
well as the increased clinical usage of TDF, will help to
answer this question.
Clinical experience in HIV-1 treatment indicates that
combination antiretroviral therapy is superior to monotherapy in maintaining viral suppression and delaying
the emergence of drug-resistant virus [29]. Combination therapy might offer added benefits in treating
CHB because of the probable necessity for long-term
treatment with nucleoside/nucleotide analogues. One
aspect of such a benefit with combination therapy was
reduced resistance development and prolonged suppression of serum HBV DNA when ADV was added
on to 3TC treatment as opposed to switching from
3TC to ADV monotherapy [30,31]. Although in these
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Zhu.indd 166
studies combination therapy (ADV+3TC) was initiated
after 3TC monotherapy, de novo combination therapy
should be at least as beneficial. TDF could be used as an
important component of combination regimens for the
treatment of CHB, given its superior anti-HBV potency
as compared with AFV in vivo [18,19], its lack of crossresistance with the l-­nucleoside analogues and its effectiveness in the treatment of patients coinfected with
HIV-1 and HBV. We therefore investigated the in vitro
anti-HBV efficacy of TFV in combination with various
nucleoside analogues.
Methods
Cells and compounds
The AD38 cell line, which expresses HBV under the
control of an inducible tetracycline promoter, was used
[32]. TFV, AFV and FTC were synthesized by Gilead
Sciences (Foster City, CA, USA). ETV was extracted
from prescription tablets (Bristol–Myers Squibb, Farmington, CT, USA). 3TC and LdT were purchased from
a commercial source (Moraveck Biochemicals, Brea,
CA, USA).
Evaluation of 50% effective concentrations of
individual drugs
AD38 cells were seeded and cultured on 96-well plates as
described previously [32]. After incubation for 3 days,
cells were washed twice with phosphate-buffered saline
(PBS) and fed with drug-containing or plain media without tetracycline. After drug treatment for 3 days, culture
supernatants were replaced with fresh drug-containing
media and incubated for an additional day. Intracellular
HBV DNA was then extracted as previously described
[33] and quantified by real-time PCR. Briefly, 5 µl of the
above final cell lysate was added to a PCR reaction mixture that contained 0.9 µM of the forward primer, HBVF
(5′-CCGTCTGTGCCTTCTCATCTG-3′), 0.9 µM of
the reverse primer, HBVR (5′-AGTCCAAGAGTYCT
CTTATGYAAGACCTT-3′), 0.25 µM of HBV probe
(5′-6FAM-CCGTGTGCACTTCGCTTCACCTCTGCBHQ1-3′) and TaqMan® Universal PCR Master Mix
(Applied Biosystems, Foster City, CA, USA) in a final
volume of 50 µl. The PCR mixture was incubated at
95°C for 10 min followed by 40 cycles of incubation at
95°C for 15 s and 60°C for 1 min. Regression analyses
were performed using TableCurve 2D software (Systat
Software Inc., Richmond, CA, USA) to calculate the
50% effective concentration (EC50) values.
Drug combination assays
AD38 cells were seeded on 5 replicate 96-well plates
at 3×104 cells/well in culture media containing tetracycline as described above. After incubation at 37°C and
5% CO2 for 3 days, cells were washed twice with PBS
© 2009 International Medical Press
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Anti-HBV activity of combinations of tenofovir with other nucleoside/nucleotide analogues
Data analysis
The antiviral effects of TFV in combination with FTC,
3TC, ETV, LdT or AFV were assessed using the Bliss
independence [34] and Loewe additivity models [35].
The Bliss independence model is defined by the equation Exy=Ex+Ey-(ExEy), where Exy is the additive
effect of drugs x and y as predicted by their individual
effects (Ex and Ey). The MacSynergy II programme was
used to evaluate antiviral data according to the Bliss
independence model. MacSynergy II uses a non-parametric three-dimensional approach to quantify areas
where observed effects are significantly greater (synergy) or less (antagonism) than those predicted from
single-drug control data. Data sets were assessed at the
95% confidence level and interpreted as follows: volumes of synergy or antagonism <25 µΜ2 were considered insignificant, those >25–<50 µΜ2 were considered
minor but significant, those >50–<100 µΜ2 were considered moderate and potentially important in vivo and
those >100 µΜ2 were considered strong and likely to be
important in vivo [36].
The Loewe additivity model is defined by the equation dx/Dx+dy/Dy=1, where Dx and Dy are the doses
of individual drugs required to exert the same effect
as doses dx and dy used in combination. If the experimental product of this equation (termed the Loewe
combination index) is equal to 1, the data are considered additive; indices of <1 or >1 indicate synergy or
antagonism, respectively. Isobologram analyses were
used to evaluate antiviral activity according to the
Loewe additivity model. Dose–response curves were
generated for each drug alone and in combination and
used to determine EC50 values for each drug alone or
in the presence of the fixed concentration of the second drug. The x-­coordinate is the fractional inhibitory
concentration (FIC) and was calculated by dividing
the EC50 of drug A with a fixed overlay of drug B by
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Zhu.indd 167
With tetracycline
No drug
Figure 1. Two-drug combination matrix
Drug A
and fed with drug-containing or plain media without
tetracycline. The drug combinations were prepared in
a checker board fashion (concentrations of both drugs
increase from the lower left to the upper right corner)
as indicated in Figure 1. The highest concentrations
of each drug used in combination were 2 µM FTC,
2 µM 3TC, 0.04 mM ETV, 40 µM LdT, 20 µM AFV
and 40 µM TFV. After drug treatment for 3 days,
culture supernatants were replaced with fresh drug­containing media and incubated for an additional day.
After 4 days of drug treatment, intracellular HBV
DNA was extracted and quantified using a TaqMan®
PCR assay as described above. The average inhibition
of HBV DNA levels as a percentage of no drug control
was plotted using MacSynergy II software (University
of Michigan, Ann Arbor, MI, USA) to give a threedimensional presentation of the dose–response effect.
Drug B
The two columns to the right are the negative (with tetracycline) and positive
(no drug) controls. The left most column and bottom row each represents
twofold serial dilutions of drugs A and B alone. The two drugs were combined in
a checker board fashion across the drug gradients as represented by the circles.
the EC50 of drug A alone. The y-coordinate is the fixed
c­ oncentration of drug B divided by the EC50 of drug
B alone. These points were plotted on a graph to generate the isobologram. On this same graph, a line representing additivity was included (coordinates [1,0] to
[0,1]). Data points that were above the additivity line
represent antagonism between the compounds, whereas
data points below the additivity line represent synergy
between the compounds. Statistical evaluation of the
data was conducted on the basis of deviation from
additivity (D-value) and tested by a one-tailed Student’s
t-test [37]. D-­values between -0.2 and -0.1 with a statistically significant P-value (P<0.05) were representative of a slight synergistic effect, whereas a D-value of
-0.5 with P<0.05 could be interpreted as strong synergy [37]. The same interpretation could be applied to
positive D-values as indicative of antagonism.
Cytotoxicity assays
To assess cytostatic or cytotoxic effects of the drug
combinations, AD38 cells were seeded into 96-well
plates at a density of 3×104 cells/well and exposed to
compounds for 4 days with a treatment schedule identical to that described above for the antiviral assays.
Each drug was tested alone and in combination with
TFV at the highest doses used for antiviral combination assays. Specifically, TFV, FTC, 3TC, ETV, LdT
and AFV single drugs were tested at 40 µM, 2 µM,
2 µM, 0.04 mM, 40 µM and 20 µM each, respectively, whereas combinations of drugs were assayed
at 2 µM FTC+40 µM TFV, 2 µM 3TC+40 µM TFV,
0.04 mM ETV+40 µM TFV, 40 µM LdT+20 µM TFV
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Table 1. Anti-HBV activity of individual compounds
Compound
Mean EC50, µM (±sd)
TFV
FTC
3TC
ETV
LdT
AFV
5.46 (1.77)
0.22 (0.13)
0.25 (0.23)
0.01 (0.001)
9.49 (1.57)
5.24 (3.62)
AFV, adefovir; EC50, 50% effective concentration; ETV, entecavir; FTC, emtricitabine;
HBV, hepatitis B virus; LdT, telbivudine; TFV, tenofovir; 3TC, lamivudine.
and 20 µM AFV+20 µM TFV. Plain culture media
and a serial dilution of DMSO were used as negative and positive controls, respectively. Following the
drug treatment, cell viability was assessed by sodium
3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis
(4-methoxy-6-nitro) benzene sulfonic acid hydrate
(XTT) cleavage assay and optical density measured at
450 nm absorbance was read as previously described
[36]. The absorbance reading corresponded to the
metabolic activities of live cells.
Results
Anti-HBV activity of test compounds in the AD38
cell line
AD38 cells express high levels of wild-type HBV
(genotype D and subtype ayw) from an integrated
HBV genome under the transcriptional control of a
tetracycline-­controllable promoter (tetracycline-off and
HBV expressed after removal of tetracycline) [32]. To
test anti-HBV activity of various nucleoside/nucleotide
analogues, AD38 cells were incubated with the test
compounds for 4 days following release of tetracycline
suppression and intracellular HBV DNA were extracted
and quantified using a TaqMan® real-time PCR assay.
The HBV DNA levels from the positive control wells
(without tetracycline or drug) were consistently >200fold that from negative controls (with tetracycline), a
signal/noise ratio allowing differentiation of a range of
effects on HBV DNA levels (data not shown). The average EC50 values for each individual drug obtained with
the AD38 cell line are summarized in Table 1 and the
results are within the range of previously reported data
generated with other HBV expression systems using
real-time PCR assays as a detection method [24–27].
Anti-HBV activity of drug combinations
To test the anti-HBV activity of two-drug combinations
using AD38 cells, drug A or B was added in a checker
board fashion as depicted in Figure 1. Each combination assay experiment was carried out with 5 replicate
96-well plates because of the inherent variability of
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Zhu.indd 168
in vitro HBV expression. As a control experiment for
the assay system to evaluate drug interactions, the combination of FTC+FTC was assessed. Using MacSynergy
analyses on the basis of the Bliss independence model,
the results indicated that this combination was additive
with a synergy volume of 0 and an antagonism volume
of 16.48 µM2% at the 95% confidence interval (CI;
Figures 2A and 2B). A careful inspection indicated that
inhibition of HBV DNA at the highest test drug concentrations reached a plateau, resulting in the observed
small and statistically insignificant antagonism volume.
Excluding the data from the row and column corresponding to the highest drug concentrations resulted in
both synergy and antagonism volumes of 0. Analyses
of the same data using the isobologram method [37]
on the basis of the Loewe additivity model resulted in a
D-value of 0.063, which was not significantly different
from 0 (Student’s t-test P=0.38; Figure 2C).
Each TFV drug combination pair was independently
tested ≥3×, with the exception of the TFV+LdT and
TFV+AFV combinations. TFV+LdT was tested twice.
The TFV+AFV combination was tested 5×, as data
were noticeably more variable than with other combination pairs. One example of each of the anti-HBV
three-dimensional dose–response plots of TFV+FTC,
TFV+3TC, TFV+ETV, TFV+LdT and TFV+AFV combinations is shown in Figure 3. Each drug alone (x- or
y-axis, where the other drug is absent) showed a dose–
response and an EC50 similar to the values indicated
in Table 1. Also, a general trend of dose–response surface for each combination pair is visually indicated,
suggesting consistency of data.
Data analyses using MacSynergy demonstrated
that each of the TFV+FTC, TFV+3TC, TFV+ETV,
TFV+LdT and TFV+AFV combinations had mean
synergy and antagonism volumes at the 95% CI that
were within the range of -25–25 µM2% (Table 2). Volumes between -25–25 µM2% at the 95% CI are considered statistically insignificant, indicating additive
interactions [34,36]. Therefore by using MacSynergy
analyses, all the tested drug combinations were considered additive in anti-HBV activity with no evidence of
antagonism. A sample three-dimensional synergy plot
at the 95% CI from MacSynergy analyses for each of
the TFV combination pairs is shown in Figure 4.
These results were also analysed using the isobologram
method [34,37]. One example isobologram for each combination is shown in Figure 5. D-values calculated from
combinations of TFV+3TC, TFV+ETV, TFV+LdT and
TFV+AFV varied between -0.17–0.24, but each time that
the values were outside of the -0.1–0.1 range the P-values
were not statistically significant (>0.05). These small or
statistically insignificant D-values were indicative of additive interactions between each of the tested combination
pairs (Table 3). Data from TFV+AFV were more variable,
© 2009 International Medical Press
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Anti-HBV activity of combinations of tenofovir with other nucleoside/nucleotide analogues
Figure 2. FTC+FTC combination
B
00
0.
0.
FTC, µM
25
0.
06
25
0.
0
0. . 5
12
0. 0 3 5
00 12
78 5
12
5
0.
5
FTC, µM
0
5
12
03
00
FTC, µM
0.
25
0.
25
06
0.
25
56
25
01
06
39
0.
0.
0
0.
-25
06
0
25
25
39
50
100
75
50
25
0
-25
-50
-75
-100
25
75
Synergy, %
Inhibition, %
100
01
56
A
FTC, µM
C
2.5
FIC (FTC)
2
1.5
1
0.5
0
0
0.5
1
1.5
2
FIC (FTC)
(A) Three-dimensional inhibition plot. (B) Synergy plot at the 95% confidence interval from MacSynergy analysis. (C) Isobologram analysis plot showing means ±se. The
line between coordinates (0,1) and (1,0) indicates additivity. FIC, fractional inhibitory concentration. FTC, emtricitabine.
and results from only 1 of the 5 experiments could be
analysed with the isobologram method. It is unclear why
this combination pair had more variability in anti-HBV
activity. Interestingly, TFV+FTC combinations consistently resulted in negative D-values ranging from -0.25
to -0.12 (D-values between -0.2 and -0.1 indicate weak
synergy [37]), although statistical significance was not
consistently shown (P-values ranged from 0.0007 to
0.22). Therefore, isobologram analyses revealed that all
the tested combinations were additive with the exception
of the TFV+FTC combination, which showed a weak
synergistic effect.
Effects of drug combinations on cytotoxicity and
cytostasis
To exclude the possibility that the observed anti-HBV
activities of the tested drug combinations were a result
of cytotoxicity and/or cytostatic effects of the drug
Antiviral Chemistry & Chemotherapy 19.4
Zhu.indd 169
treatments, experiments were conducted at the highest
drug concentrations of each drug alone and in combination with TFV (as tested for anti-HBV activity) to
evaluate toxicity. The XTT-based cytotoxicity assays
were conducted ≥3× for each drug or drug combination. No significant differences (Student’s t-test,
P>0.05) were observed between the untreated control
and any of the drug-treated cultures. Treatment with
5% DMSO resulted in a >50% reduction in the assay
signals (Figure 6).
Discussion
Several new drugs have been developed in recent years for
the treatment of CHB. However, the available anti-HBV
agents only prevent virus replication and have no direct
or permanent effect on eliminating the existing cccDNA,
the viral form that does not undergo semi-conservative
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Y Zhu et al.
Figure 3. Three-dimensional dose–response plots of drug combinations
A
B
100
100
Inhibition, %
Inhibition, %
75
50
25
0
75
50
25
-25
0
1
1
20
20
25
25
06
0.
25
06
0.
25
0.
0
TFV, µM
25
31
C
3TC, µM
0.
TFV, µM
25
1.
0.
5
5
25
31
0
25
1.
0.
FTC, µM
D
100
75
Inhibition, %
50
25
0
-25
75
50
25
0
02
1.
25
5
5
00
00
12
5
0.
0
5
62
LdT, µM
0.
TFV, µM
6
15
0.
TFV, µM
25
0.
5
2.
0.
10
20
5
25
31
0
25
1.
0.
ETV, µM
20
Inhibition, %
100
e
Inhibition, %
75
50
25
0
-25
10
10
2.
5
5
2.
0.
5
62
62
5
0.
6
15
0.
AFV, µM
25
TFV, µM
0
One example of the three-dimensional graph for each tested drug combination is shown. (A) tenofovir (TFV)+emtricitabine (FTC). (B) TFV+lamivudine (3TC). (C) TFV+entecavir
(ETV). (D) TFV+telbivudine (LdT). (E) TFV+adefovir (AFV).
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Zhu.indd 170
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Anti-HBV activity of combinations of tenofovir with other nucleoside/nucleotide analogues
Table 2. MacSynergy analyses of anti-HBV drug combination results
Drug
combination
Mean synergya
volume, µM2% (±sd)
Mean antagonisma
volume, µM2% (±sd) Net effect
TFV+FTC
TFV+3TC
TFV+ETV
TFV+LdT
TFV+AFV
5.88 (6.67)
7.16 (12.41)
0.4 (0.69)
3.5 (4.95)
2.33 (5.20)
-1.88 (3.26)
-11.35 (4.64)
-9.53 (4.95)
-8.81 (11.22)
-13.57 (6.46)
Additive
Additive
Additive
Additive
Additive
Mean (±sd) synergy and antagonism volumes at the 95% confidence interval. AFV, adefovir; ETV, entecavir; FTC, emtricitabine; HBV, hepatitis B virus; LdT, telbivudine;
TFV, tenofovir; 3TC, lamivudine.
a
Figure 4. MacSynergy analyses of antiviral combination data
B
0
3TC, µM
0.
31
25
1.
25
20
5
Synergy, %
0
31
25
0.
1.
25
D
0
25
62
5
0.
0.
15
6
0
0.
2.
5
Synergy, %
5
31
25
25
TFV, µM
25
1.
00
1.
25
5
02
0.
20
0.
0
TFV, µM
ETV, µM
100
75
50
25
0
-25
-50
-75
-100
20
100
75
50
25
0
-25
-50
-75
-100
10
C
Synergy, %
TFV, µM
0
FTC, µM
5
12
0.
5
1
5
12
0.
0
TFV, µM
100
75
50
25
0
-25
-50
-75
-100
1
100
75
50
25
0
-25
-50
-75
-100
20
Synergy, %
A
LdT, µM
0
25
0.
15
6
0.
62
5
25
1.
AFV, µM
0
2.
5
10
100
75
50
25
0
-25
-50
-75
-100
10
Synergy, %
e
TFV, µM
Effects of each drug combination pair are represented as synergy plots at the 95% confidence interval by MacSynergy analyses (MacSynergy II; University of Michigan,
Ann Arbor, MI, USA). A representative graph for each drug combination is shown. (A) tenofovir (TFV)+emtricitabine (FTC). (B) TFV+lamivudine (3TC). (C) TFV+entecavir
(ETV). (D) TFV+telbivudine (LdT). (E) TFV+adefovir (AFV).
Antiviral Chemistry & Chemotherapy 19.4
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Y Zhu et al.
In addition, no clinical study has demonstrated that any
currently approved drugs completely inhibited HBV
DNA replication as a monotherapy, which could allow
for the rise of drug resistance mutations from the residual virus replication in some patients. Aside from the
issue of whether drug resistance mutations pre-existed
DNA replication but plays a central role in maintaining
an infected state [7]. Because cccDNA has a very long
half-life [38,39] and is probably distributed into daughter
cells during cell division [39,40], it will likely take a very
long period of treatment, even with a very effective drug,
to significantly clear the virus from infected hepatocytes.
Figure 5. Isobolograms of anti-HBV activities of the drug combinations
A
B
1.4
1
1.2
0.8
1
FIC (TFV)
FIC (TFV)
1.2
0.6
0.4
0.8
0.6
0.4
0.2
0.2
0
0
0
0.2
0.4
0.6
0.8
1
0
1.2
0.5
1.5
2
FIC (3TC)
FIC (FTC)
C
1
D
2.5
1.6
1.4
2
FIC (TFV)
FIC (TFV)
1.2
1.5
1
1
0.8
0.6
0.4
0.5
0.2
0
0
0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
FIC (LdT)
FIC (ETV)
FIC (AFV)
e
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1.0
1.2
FIC (TFV)
A representative graph for each drug combination is shown. Data are means ±se. (A) tenofovir (TFV)+emtricitabine (FTC). (B) TFV+lamivudine (3TC). (C) TFV+entecavir (ETV).
(D) TFV+telbivudine (LdT). (E) TFV+adefovir (AFV). FIC, fractional inhibitory concentration; HBV, hepatitis B virus.
172
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© 2009 International Medical Press
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Anti-HBV activity of combinations of tenofovir with other nucleoside/nucleotide analogues
or evolved because of the low level of ongoing virus
­replication, long-term antiviral treatments (3TC, ADV,
LdT and ETV) against a single target (RT) are associated with the emergence of drug resistance mutations,
or predicted to be so with the ensuing loss of therapeutic
benefits [41]. Combination therapy using drugs with at
least additive interactions and without cross-resistance
might provide the added efficacy necessary to reduce the
risk of antiviral drug resistance.
Data on nucleoside/nucleotide drug combinations
against HBV, either from preclinical or clinical studies,
are still limited. One of the earlier preclinical studies by
Korba [42] showed that combinations of 3TC and penciclovir had synergistic activity against HBV in HepG2
2.2.15 cells. Using the woodchuck hepatitis virus model,
the combination of 3TC and famciclovir was shown to
have additive to synergistic antiviral effects in chronically infected woodchucks [43]. In duck hepatitis B virus
(DHBV)-­infected duck primary hepatocytes, AFV, 3TC
and penciclovir showed additive or synergistic antiviral
effects when used in combination [44]. Also using the
DHBV model, Seigneres et al. [45] reported enzymatic,
cell culture and in vivo results demonstrating that
­combinations of FTC, amdoxovir and clevudine were
more efficacious than any of the drugs alone in antiviral
activity. More recently, Delaney et al. [46] showed that
combinations of AFV and 3TC, FTC, ETV, LdT or TFV
had additive to synergistic effects in anti-HBV activity in
a stably transfected cell line that constitutively expresses
a wild-type genotype A HBV. Clinically, de novo
3TC+ADV combination therapy was compared with
3TC monotherapy in treatment-naive hepatitis B e antigen (HBeAg)-positive patients [47]. Initially, both treatments resulted in an equally effective antiviral response
by week 16, with a 4–5 log10 copies/ml serum HBV DNA
reduction. However, by week 52, the ADV+3TC group
maintained viral suppression with a median 5.22 log10
copies/ml reduction in HBV DNA as compared with a
3.41 log10 copies/ml reduction in the 3TC monotherapy
group, largely owing to the development of 3TC resistance in the 3TC monotherapy group. In another study,
30 treatment-naive HBeAg-positive patients were treated
with ADV+FTC combination therapy or ADV monotherapy [48]. At week 48, the combination group showed
Table 3. Isobologram analyses of anti-HBV drug combination results
Drug combination
D-value range
P-value range
Net effect
TFV+FTC
TFV+3TC
TFV+ETV
TFV+LdT
TFV+AFVa
-0.25–-0.12
-0.17–0.12
0.036–0.24
0.066–0.047
-0.12
0.0007–0.22
0.02–0.44
0.1–0.36
0.25–0.32
0.14
Synergistic
Additive
Additive
Additive
Additive
a
For this combination n=1. AFV, adefovir; ETV, entecavir; FTC, emtricitabine; HBV, hepatitis B virus; LdT, telbivudine; TFV, tenofovir; 3TC, lamivudine.
Figure 6. Cytotoxicity assay
Alone
Plus TFV
0.8
Absorbance, nm
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Untreated
5%
DMSO
TFV
No drug
3TC
ETV
LdT
AFV
Drug treatment
The absorbance readings were averages of three replicate wells. AFV, adefovir; ETV, entecavir; LdT, telbivudine; TFV, tenofovir; 3TC, lamivudine
Antiviral Chemistry & Chemotherapy 19.4
Zhu.indd 173
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Y Zhu et al.
a median HBV DNA reduction of 3.95 log10 copies/ml as
compared with a 2.44 log10 copies/ml reduction in the
ADV monotherapy group.
In this study, we used TFV as a common component
of in vitro combinations with 3TC, FTC, ETV, LdT
or AFV, and assessed their anti-HBV activities in the
AD38 cell line. Two types of analyses were performed
to evaluate the degree of synergy between these combinations. MacSynergy and isobologram analyses, on the
basis of the Bliss independence or the Loewe additivity
models, respectively, demonstrated that with the exception of TFV+FTC, these combinations were additive
with respect to inhibition of HBV DNA replication. The
combination of TFV+FTC had a slight synergistic effect
according to isobologram analyses. None of the drug
combinations had any significant antagonistic effects.
Also, none of the drugs alone or in combination with
TFV showed any cytotoxic effects at the highest tested
combination doses. One combination pair, TFV+AFV,
was previously tested in a different cell line [46]. Data
analyses using the MacSynergy method in both studies
indicated that the two drugs were additive with regards
to anti-HBV activity, albeit with some degree of variability in this study. Data analyses using the isobologram
method could only be carried out on one set of data
in this study, which yielded a slightly negative D-value
(-0.12). However, this did not achieve statistical significance and visual inspection of the isobologram curve
(Figure 5E) also suggested additive anti-HBV activity
for this combination.
It is not unexpected that analyses of the same data
using MacSynergy and isobologram methods, which
are each based on a different theory, would lead to
slightly different results [34,46,49], as was shown for
the TFV+FTC combination in this study. The basis
for such a disagreement is mostly attributable to the
shape of the individual dose–response curves of each
drug. Both models are in agreement when two drugs
have identical exponential dose–response curves [50].
It is unclear what mechanism contributed to the slight
synergistic interaction between TFV and FTC observed
in the isobologram analyses of this study. One possible explanation could be deduced from published drug
metabolism studies in CEM cells [36]. In those studies,
when 10 µM each of TFV and FTC were included in
culture media, the intracellular concentrations of TFV
diphosphate and FTC triphosphate each were significantly increased compared with when individual drugs
were added alone [36]. This observation correlated
with an additive to synergistic anti-HIV-1 effect of the
TFV+FTC combination in peripheral blood mononuclear cells, and strong synergy in MT-2 cells [36].
Whether higher levels of the active metabolites of TFV
and FTC are formed in the AD38 cell line used in this
study, is unknown.
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Zhu.indd 174
Because TFV remained active against mutations
known to be associated with resistance to 3TC, FTC,
LdT or ETV [11,26,27], the finding in this study that
TFV is at least additive with each of these nucleoside analogues supports a clinical evaluation of therapy using any of these combinations. Among the
combinations, TDF+FTC (Truvada®) is widely used
for the treatment of HIV-1 AIDS. Because TDF and
FTC each showed potent antiviral activity in treating
CHB [9,18,19], it was assumed that their combination would be more potent against HBV, particularly
because the two drugs do not share cross-resistance.
Therefore, TDF+FTC combination therapy is already
used in disease management in patients with prolonged
viraemia following TDF monotherapy [18,19], and
has been used successfully in treating patients with
ADV-­resistant HBV mutations [51]. Double-blind
randomized clinical trials to evaluate the efficacy of
TDF+FTC are currently ongoing. As with TDF+FTC,
TDF+3TC combination therapy might also offer added
activity while reducing the risk of resistance [52–55].
Other combinations, such as TDF+LdT and TDF+ETV
could offer added benefits for long-term patient treatment because of the additive interaction as well as lack
of cross-resistance [11,26,27]. However, unlike the
combinations of TDF+FTC or TDF+3TC, there are little data on the safety profile of TDF+LdT or TDF+ETV;
therefore, the overall clinical benefit of these combinations is unknown. By contrast, TDF+ADV combination therapy, although additive in antiviral activity
as demonstrated in this study, should not be used in
the treatment of CHB. TFV and AFV are structurally
similar analogues, with similar activities against wildtype and various drug resistance mutations [11,33]. In
particular, previous in vitro studies have demonstrated
low-level cross-resistance to TFV of the rtA181V and
rtN236T ADV-associated mutations [33]. Although
the clinical significance of this observed in vitro crossresistance has not been demonstrated, the use of combination therapy consisting of TDF and ADV would
not be recommended at this time despite the lack of
antagonism demonstrated in vitro.
In summary, the combinations of TFV plus FTC,
3TC, ETV, LdT or AFV each showed additive to
slightly synergistic anti-HBV activity in vitro. There
was no evidence of in vitro cytotoxicity with any of the
drug combinations at the tested drug concentrations.
These results provide a rationale for and support the
use of TDF as an important component in combination
therapy for the treatment of CHB.
Acknowledgements
We thank our colleagues, William Delaney and Shelly
Xiong, for their scientific discussions.
© 2009 International Medical Press
2/3/09 17:20:41
Anti-HBV activity of combinations of tenofovir with other nucleoside/nucleotide analogues
Disclosure statement
YZ, MC, XQ, MDM and KBE are all employees of
Gilead Sciences, Inc. (Durham, NC, USA).
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Lavanchy D. Hepatitis B virus epidemiology, disease
burden, treatment, and current and emerging prevention
and control measures. J Viral Hepat 2004; 11:97–107.
Lee WM. Hepatitis B virus infection. N Engl J Med 1997;
337:1733–1745.
Wong DKH, Cheung AM, O’Rourke K, Naylor CD,
Detsky AS, Heathcote J. Effect of alpha-interferon treatment
in patients with hepatitis B e antigen-positive chronic hepatitis
B. A meta-analysis. Ann Intern Med 1993; 119:312–323.
Lau GKK, Piratvisuth T, Luo KX, et al. Peginterferon alfa-2a,
lamivudine, and the combination for HBeAg-positive chronic
hepatitis B. N Engl J Med 2005; 352:2682–2695.
Marcellin P, Lau GK, Bonino F, et al. Peginterferon alfa-2a
alone, lamivudine alone, and the two in combination in
patients with HBeAg-negative chronic hepatitis B. N Engl J
Med 2004; 351:1206–1217.
Zoulim F, Perrillo R. Hepatitis B: reflections on the
current approach to antiviral therapy. J Hepatol 2008; 48
Suppl 1:S2–S19.
Seeger C, Mason WS. Hepatitis B virus biology. Microbiol
Mol Biol Rev 2000; 64:51–68.
Lai C-L, Dienstag J, Schiff E, et al. Prevalence and clinical
correlates of YMDD variants during lamivudine therapy
for patients with chronic hepatitis B. Clin Infect Dis 2003;
36:687–696.
Gish RG, Trinh H, Leung NW, et al. Safety and antiviral
activity of emtricitabine (FTC) for the treatment of chronic
hepatitis B infection. A two year study. J Hepatol 2005;
43:60–66.
Lai CL, Gane E, Liaw YF, et al. Telbivudine versus
lamivudine in patients with chronic hepatitis B. N Engl J
Med 2007; 357:2576–2588.
Yang H, Qi X, Sabogal A, Miller M, Xiong S,
Delaney WE, IV. Cross-resistance testing of next-generation
nucleoside and nucleotide analogues against lamivudineresistant HBV. Antivir Ther 2005; 10:625–633.
Tenney DJ, Levine SM, Rose RE, et al. Clinical emergence
of entecavir-resistant hepatitis B virus requires additional
substitutions in virus already resistant to lamivudine.
Antimicrob Agents Chemother 2004; 48:3498–3507.
Colonno RJ, Rose R, Baldick CJ, et al. Entecavir resistance
is rare in nucleoside naive patients with hepatitis B.
Hepatology 2006; 44:1656–1665.
Perrillo R, Hann HW, Mutimer D, et al. Adefovir dipivoxil
added to ongoing lamivudine in chronic hepatitis B with
YMDD mutant hepatitis B virus. Gastroenterology 2004;
126:81–90.
Peters MG, Hann HW, Martin P, et al. Adefovir dipivoxil
alone or in combination with lamivudine in patients with
lamivudine-resistant chronic hepatitis B. Gastroenterology
2004; 126:91–101.
Angus P, Vaughan R, Xiong S, et al. Resistance to adefovir
dipivoxil therapy associated with the selection of a novel
mutation in the HBV polymerase. Gastroenterology 2003;
125:292–297.
Locarnini S, Qi X, Arterburn S, et al. Incidence and
predictors of emergence of HBV mutations associated
with ADV resistance during 4 years of ADV therapy for
patients with chronic hepatitis B. 40th Annual Meeting of
the European Association for the Study of the Liver. 13–17
April 2005, Paris, France. Abstract 36.
Heathcote EJ, Gane E, DeMan R, et al. Randomized,
double-blind comparison of tenofovir DF (TDF) versus
adefovir dipivoxil (ADV) for the treatment of HBeAgpositive chronic hepatitis B (CHB): Study GS-US-174-0103.
Hepatology 2007; 46:861A.
Antiviral Chemistry & Chemotherapy 19.4
Zhu.indd 175
19. Marcellin P, Buti M, Kraslev Z, et al. Randomized, doubleblind comparison of tenofovir DF (TDF) versus adefovir
dipivoxil (ADV) for the treatment of HBeAg-negative
chronic hepatitis B (CHB): Study GS-US-174-0102.
Hepatology 2007; 46:290A–291A.
20. Panel on Antiretroviral Guidelines for Adults and
Adolescents. Guidelines for the use of antiretroviral agents
in HIV-1-infected adults and adolescents. US Department of
Health and Human Services. (Updated 3 November 2008.
Accessed 20 February 2009.) Available from http://www.
aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf.
21. Heijtink RA, Kruining J, de Wilde GA, Balzarini J,
De Clercq E, Schalm SW. Inhibitory effects of acyclic
nucleoside phosphonates on human hepatitis B virus
and duck hepatitis B virus infections in tissue culture.
Antimicrob Agents Chemother 1994; 38:2180–2182.
22. Ying C, De Clercq E, Nicholson W, Furman P, Neyts J.
Inhibition of the replication of the DNA polymerase
M550V mutation variant of human hepatitis B virus by
adefovir, tenofovir, L-FMAU, DAPD, penciclovir and
lobucavir. J Viral Hepat 2000; 7:161–165.
23. Delaney WE, IV, Ray AS, Yang H, et al. Intracellular
metabolism and in vitro activity of tenofovir against
hepatitis B virus. Antimicrob Agents Chemother 2006;
50:2471–2477.
24. Villet S, Pichoud C, Villeneuve JP, Trepo C, Zoulim F.
Selection of a multiple drug-resistant hepatitis B virus strain
in a liver-transplanted patient. Gastroenterology 2006;
131:1253–1261.
25. Lacombe K, Ollivet A, Gozlan J, et al. A novel hepatitis
B virus mutation with resistance to adefovir but not to
tenofovir in an HIV-hepatitis B virus-co-infected patient.
AIDS 2006; 20:2229–2231.
26. Brunelle MN, Lucifora J, Neyts J, et al. In vitro Activity of
2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]-pyrimidine
against multidrug-resistant hepatitis B virus mutants.
Antimicrob Agents Chemother 2007; 51:2240–2243.
27. Villet S, Ollivet A, Pichoud C, et al. Stepwise process for the
development of entecavir resistance in a chronic hepatitis B
virus infected patient. J Hepatol 2007; 46:531–538.
28. Sheldon J, Camino N, Rodes B, et al. Selection of hepatitis
B virus polymerase mutations in HIV-coinfected patients
treated with tenofovir. Antivir Ther 2005; 10:727–734.
29. Clavel F, Hance AJ. HIV drug resistance. N Engl J Med
2004; 350:1023–1035.
30. Lampertico P, Vigano M, Manenti E, Lavarone M,
Lunghi G, Colombo M. Adefovir rapidly suppresses
hepatitis B in HBeAg-negative patients developing genotypic
resistance to lamivudine. Hepatology 2005; 42:1414–1419.
31. Lampertico P, Vigano M, Manenti E, Lavarone M, Sablon E,
Colombo M. Low resistance to adefovir combined with
lamivudine: a 3-year study of 145 lamivudine-resistant
hepatitis B patients. Gastroenterology 2007; 133:1445–1451.
32. Ladner SK, Otto MJ, Barker CS, et al. Inducible expression
of human hepatitis B virus (HBV) in stably transfected
hepatoblastoma cells: a novel system for screening
potential inhibitors of HBV replication. Antimicrob Agents
Chemother 1997; 41:1715–1720.
33. Qi X, Xiong S, Yang H, Miller M, Delaney WE, IV. In vitro
susceptibility of adefovir-associated hepatitis B virus
polymerase mutations to other antiviral agents. Antivir Ther
2007; 12:355–362.
34. Prichard MN, Shipman C, Jr. Analysis of combinations of
antiviral drugs and design of effective multidrug therapies.
Antivir Ther 1996; 1:9–20.
35. Elion GB, Singer S, Hitchings GH. Antagonists of nucleic
acid derivatives. VIII. Synergism in combinations of
biochemically related antimetabolites. J Biol Chem 1954;
208:477–488.
36. Borroto-Esdoa K, Vela JE, Myrick F, Ray AS, Miller MD.
In vitro evaluation of the anti-HIV activity and metabolic
interactions of tenofovir and emtricitabine. Antivir Ther
2006; 11:377–384.
175
2/3/09 17:20:41
Y Zhu et al.
37. Selleseth DW, Talarico CL, Miller T, Lutz MW, Biron KK,
Harvey RJ. Interactions of 1263W94 with other antiviral
agents in inhibition of human cytomegalovirus replication.
Antimicrob Agents Chemother 2003; 47:1468–1471.
38. Moraleda G, Saputelli J, Aldrich CE, Averett D, Condreay L,
Mason WS. Lack of effect of antiviral therapy in
nondividing hepatocyte cultures on the closed circular DNA
of woodchuck hepatitis virus. J Virol 1997; 71:9392–9399.
39. Zhu Y, Yamamoto T, Cullen J, et al. Kinetics of
hepadnavirus loss from the liver during inhibition of viral
DNA synthesis. J Virol 2001; 75:311–322.
40. Summers J, Mason WS. Residual integrated viral DNA after
hepadnavirus clearance by nucleoside analogue therapy.
Proc Natl Acad Sci U S A 2004; 101:638–640.
41. Locarnini S, Mason WS. Cellular and virological
mechanisms of HBV drug resistance. J Hepatol 2006;
44:422–431.
42. Korba BE. In vitro evaluation of combination therapies
against hepatitis B virus replication. Antiviral Res 1996;
29:49–51.
43. Korba BE, Cote P, Hornbuckle W, Schinazi R, Gerin JL,
Tennant BC. Enhanced antiviral benefit of combination
therapy with lamivudine and famciclovir against WHV
replication in chronic WHV carrier woodchucks. Antiviral
Res 2000; 45:19–32.
44. Colledge D, Civitico G, Locarnini S, Shaw T. In vitro
antihepadnaviral activities of combinations of penciclovir,
lamivudine, and adefovir. Antimicrob Agents Chaemother
2000; 44:551–560.
45. Seigneres B, Martin P, Werle B, et al. Effects of pyrimidine
and purine analogue combinations in the duck hepatitis
B virus infection model. Antimicrob Agents Chaemother
2003; 47:1842–1852.
46. Delaney WE, IV, Yang H, Miller MD, Gibbs CS, Xiong S.
Combinations of adefovir with nucleoside analogs produce
additive antiviral effects against hepatitis B virus in vitro.
Antimicrob Agents Chaemother 2004; 48:3702–3710.
47. Sung JJY, Lai JY, Zeuzem S, et al. A randomized doubleblind phase II study of lamivudine (LAM) compared to
lamivudine plus adefovir dipivoxil (ADV) for treatment
naive patients with chronic hepatitis B (CHB): week 52
analysis. J Hepatol 2003; 38:25–26. Abstract 69.
48. Lau GK, Cooksley H, Ribeiro RM, et al. Impact of early
viral kinetics on T-cell reactivity during antiviral therapy in
chronic hepatitis B. Antivir Ther 2007; 12:705–718.
49. Prichard MN, Prichard LE, Shipman C, Jr. Strategic
design and three-dimensional analysis of antiviral drug
combinations. Antimicrob Agents Chaemother 1993;
37:540–545.
50. Berenbaum MC. What is synergy? Pharmacol Rev 1989;
41:93–141.
51. Tan J, Degertekin B, Wong SN, Husain M, Oberhelman K,
Lok AS. Tenofovir monotherapy is effective in hepatitis B
patients with antiviral treatment failure to adefovir in the
absence of adefovir-resistant mutations. J Hepatol 2008;
48:391–398.
52. Benhamou Y, Fleury H, Trimoulet P, et al. Anti-hepatitis B
virus efficacy of tenofovir disoproxil fumarate in HIVinfected patients. Hepatology 2006; 43:548–555.
53. Peters MG, Andersen J, Lynch P, et al. Randomized
controlled study of tenofovir and adefovir in chronic
hepatitis B virus and HIV infection: ACTG A5127.
Hepatology 2006; 44:1110–1116.
54. Schmutz G, Nelson M, Lutz T, et al. Combination of
tenofovir and lamivudine versus tenofovir after lamivudine
failure for therapy of hepatitis B in HIV-coinfection. AIDS
2006; 20:1951–1954.
55. Bani-Sadr F, Palmer P, Scieux C, Molina JM. Ninety-sixweek efficacy of combination therapy with lamivudine and
tenofovir in patients coinfected with HIV-1 and wild-type
hepatitis B virus. Clin Infect Dis 2004; 39:1062–1064.
Received 30 October 2008, accepted 2 December 2008
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Zhu.indd 176
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