ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2006, p. 2471–2477
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 7
Intracellular Metabolism and In Vitro Activity of Tenofovir
against Hepatitis B Virus
William E. Delaney IV,* Adrian S. Ray, Huiling Yang, Xiaoping Qi, Shelly Xiong,†
Yuao Zhu, and Michael D. Miller
Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, California 94404
Received 2 February 2006/Returned for modification 15 March 2006/Accepted 25 April 2006
Tenofovir is an acyclic nucleotide analog with activity against human immunodeficiency virus (HIV) and
hepatitis B virus (HBV). Tenofovir disoproxil fumarate (tenofovir DF), a bis-alkoxyester prodrug of tenofovir,
is approved for the treatment of HIV and is currently being developed to treat chronic hepatitis B. In this
report, we further characterize the in vitro activity of tenofovir against HBV as well as its metabolism in hepatic
cells. We show that tenofovir is efficiently phosphorylated to tenofovir diphosphate (TFV-DP) in both HepG2
cells and primary human hepatocytes. TFV-DP has a long intracellular half-life (95 h) and is a potent and
competitive inhibitor of HBV polymerase (Ki? 0.18 ?M). Tenofovir has a 50% effective concentration of 1.1
?M against HBV in cell-based assays, and potency is improved >50-fold by the addition of bis-isoproxil
progroups. Tenofovir has previously demonstrated full activity against lamivudine-resistant HBV in vitro and
clinically. Here we show that the major adefovir resistance mutation, rtN236T, confers three- to fourfold-
reduced susceptibility to tenofovir in cell culture; the clinical significance of this susceptibility shift has not yet
been determined. The rtA194T HBV polymerase mutation recently identified in tenofovir DF-treated HIV/
HBV-coinfected patients did not confer in vitro resistance to tenofovir as a single mutation or in a lamivudine-
resistant viral background. Overall, the antiviral and metabolic profile of tenofovir supports its development
for the treatment of chronic hepatitis B.
Tenofovir belongs to a class of acyclic phosphonate nucleo-
tide analogs which have demonstrated clinical utility against a
broad spectrum of viral infections (reviewed by De Clercq and
Holy ´ ). Tenofovir has selective activity against retroviruses
and hepadnaviruses and is currently approved for the treat-
ment of human immunodeficiency virus (HIV) as the bis-
alkoxyester prodrug tenofovir disoproxil fumarate (tenofovir
DF). Tenofovir DF is orally bioavailable, and the promoieties
are cleaved during adsorption to release tenofovir into sys-
temic circulation (reviewed by Kearney et al. ). With re-
spect to hepadnaviruses, tenofovir was shown to be active
against duck and human hepatitis B viruses (HBV) in vitro (7)
and more recently against woodchuck hepatitis virus in vivo
(15). Clinically, 48 weeks of tenofovir DF treatment in HIV/
HBV-coinfected patients has resulted in serum HBV DNA
reductions of 4.7 to 5.5 log10copies/ml in patients with either
wild-type or lamivudine-resistant HBV infection (5, 28). The
clinical activity seen against lamivudine-resistant viruses is con-
sistent with in vitro studies which have demonstrated that
tenofovir is active against all major patterns of lamivudine
resistance mutations (35).
There are now three oral antiviral agents approved to treat
chronic hepatitis B: the nucleotide adefovir dipivoxil (ADV)
and the nucleosides lamivudine and entecavir. While each of
these agents produces multilog suppression of serum HBV
DNA, they induce only modest rates of HBV surface antigen
seroconversion and thus require long-term administration to
control disease in most patients. The need for long-term ther-
apy necessitates drug safety and the ability to delay or manage
the emergence of resistant HBV strains. While lamivudine is a
well-tolerated drug, resistance emerges in approximately 20%
of patients per year of monotherapy (13). In contrast, long-
term studies with ADV and more limited experience with en-
tecavir indicate that the selection of drug resistance is consid-
erably slower with these agents. ADV retains clinical efficacy
against lamivudine-resistant mutants (17, 18) but selects the
mutation rtN236T and, less frequently, rtA181V (14). Ente-
cavir shows reduced susceptibility to lamivudine-resistant mu-
tants in vitro and clinically and an accelerated development of
entecavir resistance mutations (rtI169T, rtT184S/G, rtS202I,
and rtM250V) in lamivudine-resistant patients (27, 35). Since
resistance leads to loss of virologic suppression and the re-
sumption of liver disease, new agents and treatment modalities
will be necessary to adequately treat patients as new mutations
On a molar basis, the in vitro antiviral activity of tenofovir is
similar to adefovir. However, tenofovir DF is used clinically at
higher doses (300 mg) without the reversible nephrotoxicity
observed using high doses of ADV (30 mg or greater) (8–10).
Preliminary clinical data suggest that 300 mg of tenofovir DF
results in a significantly greater serum HBV DNA suppression
than the approved 10-mg dose of ADV (28). Based on long-
term safety data and initial efficacy data in patients with wild-
type or lamivudine-resistant HBV infection, tenofovir DF is
undergoing clinical development for the treatment of chronic
hepatitis B. We therefore profiled the in vitro properties of
tenofovir which are relevant for the treatment of HBV infec-
tion. Specifically, we sought to (i) confirm the antiviral mech-
anism of action of tenofovir and its prodrug against HBV, (ii)
* Corresponding author. Mailing address: Gilead Sciences Inc., 333
Lakeside Drive, Foster City, CA 94404. Phone: (650) 522-5598. Fax:
(650) 522-5890. E-mail: email@example.com.
† Present address: Covance Inc., Rm. 503, 457 Wu Lu Mu Qi (N) Rd.,
evaluate the activity of tenofovir against additional clinically
relevant drug resistance mutations, and (iii) investigate the
intracellular metabolism of tenofovir to its active diphosphate
species in hepatic cells.
MATERIALS AND METHODS
Cell culture. HepG2 cells were obtained from the American Type Culture
Collection (Manassas, VA) and maintained in humidified incubators at 37°C and
5% CO2. HepG2 cells were grown in minimal essential medium (American Type
Culture Collection) supplemented with 100 units/ml penicillin, 10 ?g/ml strep-
tomycin, and 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA). 2.2.15
cells were kindly provided by Brent Korba (Georgetown University, Washington,
D.C.) and maintained under conditions identical to HepG2 cells. Freshly plated
human primary hepatocytes were obtained from BD Biosciences (Bedford, MA)
and maintained in 12-well tissue culture plates with the supplier’s recommend
medium (Hepato-STIM; BD Biosciences).
Compounds. Tenofovir, tenofovir diphosphate (TFV-DP), tenofovir DF,
adefovir, adefovir diphosphate (AFV-DP), and ADV were synthesized by Gilead
Sciences (Foster City, CA). Lamivudine was obtained from Moravek Biochemi-
cals (Brea, CA).
HBV polymerase assays. The generation and purification of active HBV poly-
merase using recombinant baculovirus has been described previously (33, 34).
HBV DNA polymerase activity was measured by incorporation of ?-33P-labeled
dATP into acid-precipitatible products using activated calf thymus DNA as a
template as described previously (33, 34). Inhibition constants (Ki) were deter-
mined by fitting initial rates to Lineweaver-Burk plots based on the competitive
inhibition equation 1/V ? 1/Vmax? Km/Vmax(1 ? [I]/Ki) (1/S).
2.2.15 antiviral assays. 2.2.15 cells were seeded in 48-well plates and treated
with freshly prepared medium containing compound every 2 days for a 2-week
period. Seven doses of each compound were used at concentrations ranging from
0.001 ?M to 10 ?M in half-log increments. Following the treatment period, cells
were lysed by the addition of 200 ?l phosphate-buffered saline containing 0.5%
NP-40 (Sigma, St. Louis, MO) per well. Lysates were transferred to microcen-
trifuge tubes and centrifuged for 10 min at maximum speed to pellet nuclei.
Supernatants were transferred to clean tubes, and cytoplasmic DNA was ex-
tracted using QiaAmp DNA blood mini extraction kits (QIAGEN, Valencia,
CA). Purified DNA was transferred to nylon membranes using a slot blot man-
ifold as described previously (12). Viral DNA was detected by nucleic acid
hybridization using a33P-labeled HBV probe and quantified using a Storm 860
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Data were fit to the
logistic dose-response equation y ? a/[1?(x/b)c], and 50% effective concentra-
tion (EC50) values were calculated from the resulting equations as described
Generation and cloning of HBV encoding drug resistance mutations. The
plasmid pFBHBV-wt encodes a terminally redundant (1.3 unit-length), replica-
tion-competent HBV genome (genotype A; GenBank accession number
AF305422) in the pFastBacDual vector backbone (Invitrogen, Carlsbad, CA).
The rtA194T, rtL180M, and rtM204V mutations were introduced by site-di-
rected mutagenesis using the following primers: rtA194T, 5?-TCA GTG GTT
CGT AGG ACT TTC CCC CAC TGT TTG-3?; rtL180M, 5?-AGT CCG TTT
CTC ATG GCT CAG TTT ACT-3?; rtM204V, 5?-GCT TTC AGC TAT GTG
GAT GAT GTG GTA-3? (bolded sequence indicates the mutated codon). The
reverse transcriptase domains of all constructs were sequenced to confirm that no
additional mutations had been introduced. Patient baseline and rtN236T-con-
taining viruses were cloned by full-length PCR amplification as described by
Gunther et al. (6), followed by SapI digestion and ligation into the HBV expres-
sion vector pHY106 as previously described (36).
Transient antiviral assays. HepG2 cells were seeded into six-well culture
dishes at a density of 1 ? 106cells/dish. Sixteen hours postseeding, cells were
transfected with 5 ?g of plasmid DNA encoding the HBV genome of interest
using the Fugene 6 transfection reagent (Roche, Indianapolis, IN). The following
day, cells were fed fresh medium containing compound. Cells were treated for 1
week, after which intracellular HBV replicative intermediates were isolated as
previously described (4). Viral DNA was fractionated on 1% agarose gels and
transferred to nylon membranes (Roche) using standard Southern blotting pro-
cedures (25). Viral DNA was quantified, and EC50values were calculated as
Analysis of intracellular tenofovir metabolites. HepG2 cells were seeded in
12-well tissue culture plates and grown to confluence (average of 8.8 ? 105
cells/well). Fresh primary hepatocytes were purchased in 12-well tissue culture
plates. Uptake and phosphorylation experiments were initiated by the addition of
10 ?M tenofovir to cell culture medium. At time points of 2, 6, and 24 h,
drug-containing medium was aspirated and cells were washed two times with 2.5
ml ice-cold phosphate-buffered saline. Cells were then scraped into 500 ?l 70%
methanol and stored at ?20°C to facilitate the extraction of metabolites. Cellular
debris was removed by centrifugation for 10 min at 15,000 rpm in a microcen-
trifuge, and supernatants were dried by speed vacuum. Samples were resus-
pended in 10 ?l tetrabutyl ammonium acetate containing 2 picomole of
diphosphate (as an internal standard) per 200,000 cells. Transient ion-pairing
high-performance liquid chromatography coupled to positive ion electrospray
tandem mass spectrometery (LC/MS/MS) was used to analyze tenofovir and
its phosphorylated metabolites. Comparative experiments performed with
adefovir were conducted as described above using 10 ?M adefovir as the
concentration for cell incubation. Details of the LC/MS/MS conditions have
recently been published (22).
Tenofovir is a competitive inhibitor of HBV polymerase.
Tenofovir is a nucleotide analog, suggesting that it may inhibit
viral replication by competing with natural nucleotides for
binding to the active site of HBV polymerase. To test this
hypothesis, we used a previously established quantitative HBV
DNA polymerase assay (33, 34). The catalytic activity of HBV
polymerase was measured in the absence or presence of three
different concentrations of TFV-DP. HBV polymerase activity
was inhibited in a dose-dependent manner without a change in
Vmax, confirming that TFV-DP acts by competitive inhibition
with respect to the natural substrate, dATP (Fig. 1). The Kiof
TFV-DP was determined to be 0.18 ?M, which is 2.1-fold
lower than the Kmof dATP (0.38 ?M) (33).
Activities of tenofovir and tenofovir DF against HBV in
2.2.15 cells. We used the HepG2 2.2.15 cell line, which stably
expresses wild-type HBV, to assay the anti-HBV activities of
tenofovir and its bis-alkoxyester prodrug, tenofovir DF, in cell
culture. Adefovir, ADV (bis-alkoxyester prodrug of adefovir),
and lamivudine were also tested in parallel. 2.2.15 cells were
treated with drug for 2 weeks, and replicating cytoplasmic
DNA was then extracted, quantified by Southern blotting, and
used to calculate EC50values (Table 1). Tenofovir and adefo-
vir had similar antiviral activities in 2.2.15 cells (EC50values
were 1.1 ?M and 0.8 ?M, respectively). The potency of teno-
fovir increased approximately 50-fold to 0.02 ?M by the addi-
FIG. 1. Competitive inhibition of HBV polymerase by TFV-DP.
HBV polymerase was incubated with template (activated calf thymus
DNA) deoxynucleoside triphosphates and ?-33P-labeled dATP. Inhi-
bition of labeled dATP incorporation was measured in the presence of
the indicated concentrations of TFV-DP. Data are presented as a
2472 DELANEY ET AL.ANTIMICROB. AGENTS CHEMOTHER.
tion of the bis-isoproxil promoieties. The activity of adefovir
increased about 10-fold with the addition of bis-pivoxylmethyl
Activity of tenofovir against adefovir-resistant HBV. The
major resistance mutation to adefovir dipivoxil is an aspara-
gine-to-threonine change at position 236 of HBV polymerase
(rtN236T). To investigate potential cross-resistance, we tested
baseline and postbreakthrough HBV isolates from two patients
who developed rtN236T while on adefovir dipivoxil therapy. In
transient–cell-based antiviral assays, rtN236T conferred 3- to
4.2-fold resistance to tenofovir (Table 2). The same clinical
isolates had 7.3- to 13.8-fold susceptibility shifts to adefovir
and 2.3- to 3.5-fold susceptibility shifts to lamivudine.
The rtA194T mutation does not confer resistance to tenofo-
vir in vitro. A recent study identified rtA194T as an emerging
mutation in two HBV/HIV-coinfected patients receiving long-
term tenofovir DF therapy; in vitro analysis indicated this HBV
mutation resulted in a 7.6-fold reduction in tenofovir suscep-
tibility (26). To study this mutation, we engineered rtA194T
into wild-type and lamivudine-resistant (rtL180M?rtM204V)
laboratory strains of HBV and assayed susceptibility to teno-
fovir and lamivudine using transient-replication assays. Our
data indicated that the rtA194T mutation resulted in a 1.5-fold
increase in the EC50of tenofovir (Table 3). The lamivudine-
resistant mutant rtL180M?rtM204V HBV displayed a 2.1-fold
increase in the EC50for tenofovir. The addition of rtA194T to
the lamivudine-resistant background did not significantly
change the susceptibility of the virus to tenofovir compared to
the rtL180M?rtM204V mutant (Table 3).
Tenofovir is efficiently phosphorylated in hepatic cells. To
investigate the phosphorylation of tenofovir in hepatic cells, we
incubated HepG2 cells and primary human hepatocytes with
10 ?M tenofovir for 2, 6, or 24 h and then quantified intracel-
lular metabolites by LC/MS/MS (Fig. 2A and B). In both cell
types, there were time-dependent increases in the amount of
intracellular tenofovir, with the highest levels being reached at
24 h; this is consistent with the low inherent permeability of
charged nucleotides. Time-dependent increases were observed
for both tenofovir monophosphate (TFV-MP) and TFV-DP.
Notably, TFV-DP, the species active against HBV polymerase,
was efficiently formed and achieved higher intracellular con-
centrations than tenofovir. TFV-DP reached levels of 4.7 ?M
and 6 ?M in primary hepatocytes and HepG2 cells, respec-
tively. TFV-MP was the least abundant metabolite detected
(?1.5 ?M), suggesting that once formed it was efficiently con-
verted into diphosphate.
We also performed parallel experiments with adefovir in
both cell types (Fig. 2C and D). Adefovir achieved slightly
lower diphosphate levels in each cell type (3.9 ?M and 2.4 ?M
in primary human hepatocytes and HepG2 cells, respectively).
Unlike tenofovir, AFV-DP levels were equal to (in primary
human hepatocytes) or lower than (in HepG2 cells) adefovir
levels, suggesting a lower overall phosphorylation efficiency
than tenofovir. Like tenofovir, AFV-MP was the least abun-
dant species, suggesting it was efficiently converted to diphos-
TFV-DP has a long half-life in hepatic cells. To determine
the half-life of TFV-DP in hepatic cells, we incubated primary
human hepatocytes in 10 ?M tenofovir for 24 h and then
measured TFV-DP concentrations over a 4-day period follow-
ing drug removal (Fig. 3A). Consistent with the previous ex-
periment, approximately 7 ?M TFV-DP was formed after a
24-h incubation with tenofovir. TFV-DP remained detectable
throughout the 4-day period after drug withdrawal, and a half-
life of 95 ? 6 h was calculated using a single exponential decay
model. We also determined the half-life of AFV-DP in parallel
(Fig. 3B). Following a 24-h incubation with adefovir, 3.2 ?M
AFV-DP was formed and the diphosphate had an intracellular
half-life of 75 ? 1 h.
The anti-HIV properties of tenofovir and tenofovir DF have
been well characterized both in vitro and clinically. While in
vitro and preliminary clinical data have demonstrated that
TABLE 1. Anti-HBV activities of tenofovir and related compounds
in HepG2 2.2.15 cells
1.1 ? 0.3
0.02 ? 0.01
0.8 ? 0.2
0.1 ? 0.01
0.06 ? 0.01
aMean ? standard deviation from the indicated number of independent
TABLE 2. In vitro activities of tenofovir, adefovir, and lamivudine
against HBV, isolated from two patients, encoding the adefovir
resistance mutation rtN236T
0.33 ? 0.11
0.26 ? 0.17
0.04 ? 0.01
1.01 ? 0.84
3.64 ? 2.78
0.12 ? 0.06
0.13 ? 0.03
0.21 ? 0.01
0.03 ? 0.02
0.55 ? 0.07
1.55 ? 0.91
0.07 ? 0.2
aPretherapy isolates were confirmed not to contain rtN236T.
bMean ? standard deviation from the indicated number of independent
cPreviously reported (1).
TABLE 3. In vitro susceptibilities of wild-type, rtA194T, rtL180M?
rtM204V, and rtA194T?rtL180M?rtM204V HBV to tenofovir
in a transient–cell-based antiviral assay
0.13 ? 0.043
0.19 ? 0.059
0.27 ? 0.16
0.31 ? 0.041
aMean ? standard deviation from the indicated number of experiments (n).
bThe fold resistance is the ratio of the mutant EC50and the wild-type EC50.
The reported value is the mean of three or more independent experiments.
VOL. 50, 2006IN VITRO METABOLISM AND ANTI-HBV ACTIVITY OF TENOFOVIR 2473
tenofovir is a potent anti-HBV agent, detailed studies on its
mechanism of action, resistance profile against newer muta-
tions, and metabolism in hepatic cells have not been reported.
Furthermore, while tenofovir is structurally similar to adefovir
(differing only by addition of a single methyl group in the
acyclic linker region), it cannot be assumed that the two mol-
ecules will behave similarly with respect to antiviral activity,
resistance profile, or metabolism. The aim of our studies was to
provide a specific in vitro evaluation of the properties of teno-
fovir relevant to its development as an anti-HBV agent.
Our enzymatic studies confirmed tenofovir inhibits HBV
replication through competitive inhibition of the viral polymer-
ase. The Kiof TFV-DP for HBV polymerase (0.18 ?M) is
similar to that of AFV-DP (0.10 ?M) and slightly lower than
the Kmof the natural substrate, dATP (0.38 ?M) (33). This is
analogous to what was observed for HIV reverse transcriptase,
FIG. 2. Phosphorylation kinetics of tenofovir to mono- and diphosphate species in HepG2 cells and primary human hepatocytes. Primary
human hepatocytes (A) and HepG2 cells (B) were treated with 10 ?M tenofovir. At the indicated time points, cells were washed and lysed and
the amounts of intracellular tenofovir, TFV-MP, and TFV-DP were quantified by LC/MS/MS. Parallel experiments were performed with 10 ?M
adefovir in primary hepatocytes (C) and HepG2 cells (D) to determine levels of adefovir, AFV-MP, and AFV-DP.
FIG. 3. Intracellular half-life of TFV-DP in primary human hepatocytes. Primary human hepatocytes were incubated with 10 ?M tenofovir
(A) or 10 ?M adefovir (B) for 24 h to accumulate diphosphate. Cells were then washed, fed fresh medium without drug, and then lysed at the
indicated times for quantification of intracellular diphosphate levels by LC/MS/MS. Using a single exponential decay model, intracellular
diphosphate half-lives of 95 ? 6 and 75 ? 1 h were calculated for tenofovir and adefovir, respectively.
2474 DELANEY ET AL.ANTIMICROB. AGENTS CHEMOTHER.
where TFV-DP had a Kiof 0.16 ?M, AFV-DP had a Kiof 0.07
?M, and the Kmof dATP was 0.33 ?M (16, 32). Overall,
tenofovir appears to inhibit HIV and HBV by the same mech-
anism (inhibition of viral DNA polymerization). However, it
should be noted that assays to study inhibition in greater detail
(e.g., individual contributions of nucleotide incorporation and
excision) are not currently available for HBV (31).
In cell culture, tenofovir had an EC50of 1 ?M against
wild-type HBV using the stable cell line HepG2.2.15; this is
similar to previous reports which have used either 2.2.15 cells
or other stable cell lines (7, 35). Addition of the two soproxil
progroups to tenofovir lowers the EC50to 0.02 ?M, a 50-fold
increase in activity. This improvement in potency is likely due
to significantly improved cellular permeability as the two hy-
droxyl groups of the phosphonic acid are covered with un-
charged lipophilic promoieties. Indeed, analysis of permeabil-
ity using a Caco-2 assay indicated that tenofovir DF had
moderate cell permeability while tenofovir had low cell per-
meability (data not shown).
Clinical experience with famciclovir, lamivudine, ADV, and
entecavir has indicated that resistance can be selected to all these
compounds. Although resistant mutants emerge relatively slowly
during ADV and entecavir therapy, it can be expected that resis-
tance will become an increasing problem, since most patients
require long-term therapy to adequately control disease. The
cross-resistance profiles of all new agents should be carefully
evaluated to guide the development of rational treatment regi-
mens. Like adefovir, tenofovir retains activity against all of the
major patterns of lamivudine resistance mutations in vitro and
clinically (17, 18, 35). Our results indicated that tenofovir showed
a small but reproducible decrease in susceptibility (3- to 4.2-fold)
to clinical HBV isolates bearing rtN236T, the most common
adefovir-associated resistance mutation. These findings are in
agreement with a recent report by Brunelle et al., who observed
a 4.5-fold shift in the tenofovir EC50after introduction of
rtN236T into a laboratory strain of HBV (2).
It is important to note that the in vitro rtN236T susceptibility
shift observed for tenofovir is smaller than that of adefovir
(7.3- to 13.8-fold) and that tenofovir DF is administered clin-
ically at a dose 30 times higher than adefovir dipivoxil (300 mg
versus 10 mg, respectively). The combination of a smaller sus-
ceptibility shift and a significantly higher dose may enable
tenofovir DF to effectively suppress serum HBV DNA in pa-
tients with the rtN236T mutant. Indeed, two recent reports
indicated that two patients with rtN236T had serum HBV
DNA reductions of ?4 log10copies/ml when switched from
ADV to tenofovir DF therapy (20, 30). Similarly, patients with
rtN236T also respond to therapy with 100 mg of lamivudine
despite a 2.1- to 3.5-fold in vitro decrease in lamivudine sus-
ceptibility (19). Future studies are needed to determine the
best treatment options for patients with rtN236T; however,
early clinical data indicate that tenofovir DF and lamivudine
should each be explored.
We also studied the impact of the rtA194T mutation, which
was recently reported to emerge in two HIV/HBV-coinfected
patients receiving tenofovir DF plus lamivudine as part of their
antiretroviral treatment regimen (26). Our phenotypic analysis
indicated that rtA194T did not cause a significant change in
tenofovir susceptibility either alone or when expressed in com-
bination with lamivudine resistance mutations (EC50values
changed 1.5- to 2.5-fold). These results do not agree with those
reported by Sheldon et al., who observed a 7.6-fold change with
the single rtA194T mutation and a ?10-fold increase in the
EC50when rtA194T was expressed with lamivudine resistance
mutations (26). The discrepancy might be explained by differ-
ences between the EC50assays used in the two labs. Our results
were obtained using standard Southern blotting procedures to
quantify intracellular HBV replication, whereas Sheldon et al.
used a PCR assay to quantify extracellular HBV DNA follow-
ing transient transfection. Examining the clinical data does not
provide a clear association of rtA194T with viral load rebound:
one patient had a transient viral load increase of 1.5 logs after
the mutation emerged, while the second patient had continu-
ous viral load decline after the emergence of rtA194T and a ?9
log10decline in serum HBV DNA after the initiation of teno-
fovir DF plus lamivudine therapy. We are not aware of any
additional patients who have developed this mutation under
tenofovir DF therapy. Nevertheless, this residue should be
monitored closely during the clinical development of tenofovir
DF for chronic hepatitis B.
Studies of the anabolism of tenofovir to its active diphos-
phate form were previously restricted to lymphoid cells to
support the HIV indication of tenofovir DF (21, 24). Here we
have shown that tenofovir phosphorylation occurs efficiently in
a human hepatoblast cell line (HepG2) and in primary human
hepatocytes. In the CEM (lymphoid) cell line, TFV-DP achieved
levels of 5.2 ?M, which is similar to the levels we observed in
HepG2 cells (6.0 ?M). However, TFV-DP formation in pri-
mary human hepatocytes was significantly greater (4.7 ?M)
than in primary lymphocytes (1.0 ?M levels reached in periph-
eral blood mononuclear cells) (21). In parallel experiments,
the efficiency of TFV-DP formation was greater than that of
adefovir in both cell types that we tested; however, the differ-
ence in primary human hepatocytes (?2-fold) was smaller than in
HepG2 cells (2.5-fold).
Tenofovir diphosphate increased in a linear manner over the
24-hour incubation period. Linear increases over the same
time period were also observed for adefovir diphosphate when
run in parallel during these experiments as well as in previous
studies (22). The linear accumulation of adefovir and tenofovir
diphosphates over 24 hours is in contrast to most nucleoside
analogs, which usually reach a maximal intracellular concen-
tration after 8 to 12 h. However, this result is consistent with
the reduced permeability of adefovir and tenofovir due to the
presence of the two negative charges on the phosphonate moi-
ety. Similar studies conducted on T cells in our laboratory
indicate that tenofovir diphosphate levels begin to reach max-
imal concentrations after 48 hours of incubation (data not
shown). The diphosphates of both tenofovir and adefovir had
very long intracellular half-lives, which is consistent with ear-
lier studies demonstrating prolonged in vitro antiviral effects
with tenofovir and adefovir after drug removal (38). Overall,
the efficient phosphorylation and long half-life we observed for
tenofovir agree with the clinical results indicating that a single
daily dose of tenofovir DF will be phosphorylated to levels
sufficient to exert a potent antiviral effect in the liver.
Due to its favorable safety profile and efficacy, tenofovir DF
is a recommended first-line treatment option for HIV infection
(37). Multiple small investigator studies have demonstrated
that the 300-mg dose of tenofovir DF approved for HIV also
VOL. 50, 2006 IN VITRO METABOLISM AND ANTI-HBV ACTIVITY OF TENOFOVIR2475
results in a potent antiviral suppression of serum HBV DNA
(?4 log10reduction in serum HBV copies/ml) in coinfected
patients (5, 23, 28, 29). The in vitro data presented here have
confirmed that tenofovir inhibits HIV and HBV by similar
mechanisms (competitive inhibition of DNA polymerization)
and that tenofovir DF has potent cell-based anti-HBV activity
(EC50, 0.02 ?M). Tenofovir also has a favorable metabolic
profile in hepatic cells, the target compartment for antiviral
activity in vivo. Accordingly, enrollment has recently begun for
phase III studies of tenofovir DF in HBV e antigen-negative
and e antigen-positive chronic hepatitis B patients. We have
also shown that tenofovir is more efficacious than adefovir
against the rtN236T adefovir resistance mutation in vitro.
Since significantly higher doses of tenofovir DF are being used
clinically, these data suggest tenofovir should also be explored
for the treatment of adefovir-resistant HBV.
We thank Jennifer Vela for her technical assistance during metab-
olism studies. We also thank Katyna Borroto-Esoda, Steven Chuck,
Mick Hitchcock, Herve Mommeja-Marin, and Joe Quinn for critical
reading of the manuscript.
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