ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2005, p. 1898–1906
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 5
Selective Intracellular Activation of a Novel Prodrug of the Human
Immunodeficiency Virus Reverse Transcriptase Inhibitor Tenofovir
Leads to Preferential Distribution and Accumulation in
William A. Lee,1* Gong-Xin He,1Eugene Eisenberg,1Tomas Cihlar,1Swami Swaminathan,1
Andrew Mulato,1and Kenneth C. Cundy2
Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404,1and XenoPort, Inc.,
3410 Central Expressway, Santa Clara, California 950512
Received 15 July 2004/Returned for modification 7 October 2004/Accepted 31 December 2004
An isopropylalaninyl monoamidate phenyl monoester prodrug of tenofovir (GS 7340) was prepared, and its
in vitro antiviral activity, metabolism, and pharmacokinetics in dogs were determined. The 50% effective
concentration (EC50) of GS 7340 against human immunodeficiency virus type 1 in MT-2 cells was 0.005 ?M
compared to an EC50of 5 ?M for the parent drug, tenofovir. The (L)-alaninyl analog (GS 7340) was
>1,000-fold more active than the (D)-alaninyl analog. GS 7340 has a half-life of 90 min in human plasma at
37°C and a half-life of 28.3 min in an MT-2 cell extract at 37°C. The antiviral activity (>10? the EC50) and
the metabolic stability in MT-2 cell extracts (>35?) and plasma (>2.5?) were also sensitive to the stereo-
chemistry at the phosphorus. After a single oral dose of GS 7340 (10 mg-eq/kg tenofovir) to male beagle dogs,
the plasma bioavailability of tenofovir compared to an intravenous dose of tenofovir was 17%. The total
intracellular concentration of all tenofovir species in isolated peripheral blood mononuclear cells at 24 h was
63 ?g-eq/ml compared to 0.2 ?g-eq/ml in plasma. A radiolabeled distribution study with dogs resulted in an
increased distribution of tenofovir to tissues of lymphatic origin compared to the commercially available
prodrug tenofovir DF (Viread).
Highly active antiretroviral therapy (HAART) for the treat-
ment of human immunodeficiency virus is effective in reducing
plasma viral loads below current assay detection limits and is
responsible for significant reductions in AIDS-related mortal-
ity in the United States (13). Combinations of protease and
reverse transcriptase inhibitors are extremely potent at block-
ing de novo infection; however, they have no effect on latently
infected cells. The half-lives of these latent cellular reservoirs
were originally estimated to be ?3 years, leading to the con-
clusion that it may not be possible to eradicate human immu-
nodeficiency virus (HIV) from an infected individual by using
current HAART (2). It has subsequently been shown that even
in patients who have undetectable plasma viremia (?50 copies/
ml), low-level replication is ongoing (11, 15, 36), resulting in
repopulation of latent reservoirs and thus accounting for the
long apparent half-lives observed (12, 22, 23, 35). The failure
of HAART to completely shut down virus replication in vivo is
a function of both the intrinsic potency of the drug regimen
and its distribution to the cellular sites of virus replication. The
lymphatic tissues and the peripheral blood mononuclear cells
(PBMCs) are the primary sites of virus replication and poten-
tial virus latency (9, 19). A drug targeting strategy that selec-
tively enhances active drug concentrations in these tissues
without excessive systemic exposure is conceptually attractive
and would potentially lead to a more effective HAART with
fewer potential side effects.
(PMPA) (Fig. 1) is a nucleotide analog that inhibits HIV re-
verse transcriptase and shows potent in vitro and in vivo activ-
ity against HIV (3, 7) but has low oral bioavailability in pre-
clinical models (6). An oral prodrug of tenofovir, tenofovir
disoproxil fumarate (tenofovir DF; Viread) (Fig. 1), is indi-
cated in combination with other antiretrovirals for the treat-
ment of HIV infection. The long intracellular half-life (?50 h)
of the active diphosphate metabolite of tenofovir in resting
PBMCs (26) allows this drug to be administered once daily.
The prodrug tenofovir DF was designed to undergo rapid
metabolism to the parent drug, tenofovir, in the systemic cir-
culation after oral administration. Interestingly, in preclinical
studies with dogs, the intracellular levels of tenofovir in
PBMCs were fivefold higher after oral administration of teno-
fovir DF than after an equivalent subcutaneous exposure of
tenofovir. Correspondingly, in human clinical trials, the change
in HIV virus load was threefold higher after oral administra-
tion of tenofovir DF than after an equivalent exposure of
intravenously (i.v.) administered tenofovir (5). The “en-
hanced” anti-HIV activity observed in patients with the oral
prodrug relative to the intravenously administered parent drug
may be attributable to an increase in the intracellular concen-
tration of tenofovir, which is likely the result of better intra-
cellular distribution of the oral prodrug.
These results led us to explore a new class of orally admin-
istered tenofovir prodrugs designed to circulate systemically as
the prodrug and to undergo selective conversion to tenofovir
inside cells. In this report, we describe the in vitro and in vivo
characterization of GS 7340, an isopropylalaninyl monoami-
* Coresponding author. Mailing address: Gilead Sciences, Inc., 333
Lakeside Dr., Foster City, CA 94404. Phone: (650) 522-5716. Fax:
(650) 522-5899. E-mail: Bill_Lee@Gilead.com.
date phenyl monoester prodrug of tenofovir (Fig. 1). This
molecule demonstrates extremely potent in vitro activity and
selective targeting to lymphoreticular tissues and PBMCs in
vitro and in vivo.
MATERIALS AND METHODS
Chemicals. The synthesis of tenofovir and tenofovir DF was described previ-
ously (1, 31). The monoamidate prodrugs of tenofovir were prepared by using a
modified procedure from the literature (32). Detailed procedures and identifi-
cation will be published elsewhere. The radiolabeled analogs [14C]tenofovir DF
(specific activity, 42 mCi/mmol) and [14C]GS 7340 (specific activity, 53 mCi/
mmol) were obtained from Moravek Biochemicals (Brea, Calif.). The radio-
chemicals were verified by high-performance liquid chromatography (HPLC)
before use and were estimated to be ?98% pure. All other chemicals and
solvents were obtained from commercial sources.
In vitro antiviral activity and cytotoxicity. Triplicate serial dilutions of the test
compounds were incubated in 96-well plates with MT-2 cells (20,000 cells/well)
infected with HIV-1 IIIb at a multiplicity of infection of 0.01. After 5 days at
37°C, the virus-induced cytopathic effect was determined by using a colorimetric
cell viability assay based on the metabolic conversion of 2,3,-bis(methoxy-4-nitro-
5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) as previously described in
the literature (34). The concentration of each compound that inhibited the
virus-induced cytopathic effect by 50% (EC50) was estimated from the inhibition
plots. To determine the compound cytotoxicity, uninfected MT-2 cells in 96-well
plates (20,000 cells/well) were incubated with appropriate serial dilutions of
tested compounds for 5 days, followed by the XTT-based cell viability assay. Cell
growth was expressed as a percentage of the signal relative to untreated control.
The concentration of each drug that reduced the cell growth by 50% was esti-
mated from the inhibition plots.
In vitro metabolism studies. MT-2 cell extract was prepared from MT-2 cells
according to a previously published procedure (21). Extract (80 ?l) was trans-
ferred into a screw-cap centrifuge tube and incubated at 37°C for 5 min. Test
compounds were dissolved in HEPES buffer (0.2 mg/ml) containing 0.010 M
HEPES, 0.05 M potassium chloride, 0.005 M magnesium chloride, and 0.005 M
DL-dithiothreitol, and 20 ?l was added to the MT-2 cell extract. Aliquots (each,
20 ?l) were taken at specified times and mixed with 60 ?l of methanol containing
0.015 mg/ml of 2-hydroxymethylnaphthalene (internal standard). The mixture
was centrifuged at 15,000 ? g for 5 min, and the supernatant was analyzed by
HPLC. The same procedure was employed for the human plasma (pooled from
George King Biomedical Systems, Inc.), except that test compounds were dis-
solved in Tris-buffered saline containing 0.05 M Tris, 0.0027 M KCl, and 0.138 M
NaCl (pH 7.5).
The reverse-phase gradient HPLC method used to analyze samples from the
MT-2 cell extract and plasma metabolism studies employed a 4.6- by 250-mm,
5-?m particle size Zorbax Rx-C8column (MAC-MOD Analytical, Inc.; Chadds
Ford, Pa.) with UV detection at 260 nM. The mobile phase was varied from 50
mM potassium phosphate (pH 6.0)/CH3CN (95:5) to 50 mM potassium phos-
phate (pH 6.0)/CH3CN (50:50) over 30 min at a flow rate of 1.0 ml/min.
Human whole blood was incubated for 1 h at 37°C separately with
radiolabeled GS 7340, tenofovir DF, and tenofovir at a concentration of 5 ?g-eq
tenofovir per ml (17.4 ?M). The blood was subjected to treatment with the
Ficoll-Paque sodium diatriozate solution (described below). The treatment re-
sulted in the formation of multiple layers containing different cell types. The
bottom layer contained mostly erythrocytes (RBCs) aggregated by Ficoll-Paque.
The PBMC layer was washed and extracted with 70% methanol. Aliquots of the
plasma and RBC layers (0.5 ml) were also extracted. Radioactivity in all layers
was measured by oxidation/scintillation counting and by a comparison with ra-
dioactivity from the standard solutions. All extracts were reconstituted in water
and analyzed by HPLC with radiometric flow detection (8). The experiment was
repeated, incubating with [14C]GS 7340 at 0.7, 2.3, 6.9, and 20.8 ?M.
MT-2 cells (107) were incubated in a standard cell culture medium with 10 ?M
of [14C]GS 7340 at 37°C for 24 h. At specified time points, an aliquot of the cell
suspension was taken, and cells were counted, washed three times with ice-cold
phosphate-buffered saline (PBS), and extracted with 70% methanol. The super-
natants were analyzed using HPLC with radiometric flow detection (8).
Isolation of CD4?T cells and monocytes from whole blood. Whole human
blood was incubated for 1 h at 37°C with 17.4 ?M [14C]GS 7340. PBMCs were
obtained by density gradient centrifugation over Ficoll-Paque. CD4?T helper
(Th) cells or monocytes were isolated from PBMCs by depletion of non-Th cells
and nonmonocytes, respectively. The non-Th cells were indirectly magnetically
labeled using a cocktail of hapten-conjugated CD8, CD11B, CD16, CD19, CD36,
and CD56 antibodies and paramagnetic beads coupled to an anti-hapten mono-
clonal antibody (Miltenyi Biotec, Inc., Auburn, CA). For depletion of nonmono-
cytes, the T cells, NK cells, B cells, dendritic cells, and basophils from PBMCs
were indirectly magnetically labeled using a cocktail of hapten-conjugated CD3,
CD7, CD19, CD45RA, CD56 and anti-immunoglobulin antibodies and paramag-
netic beads coupled to an anti-hapten monoclonal antibody. The magnetically
labeled cells were depleted by retention on an extraction column in the magnetic
field. The eluted respective cell types (CD4 or monocytes) were lysed and
analyzed for tenofovir metabolites by radiochromatography (8).
In vivo administration and sample collection. The in-life phase was conducted
in accordance with the recommendations of the Guide for the Care and Use of
Laboratory Animals (National Institutes of Health publication 86-23) and was
approved by the Institutional Animal Care and Use Committee at Stanford
Research Institute (Menlo Park, CA). Male beagle dogs (four to six/group; body
weight, 10 ? 2 kg) were used for the studies. Prodrugs were formulated as
solutions in 50 mM citric acid and administered as a single dose by oral gavage.
For PBMCs, blood samples were collected at 0 (predose), 2, 8, and 24 h postdose.
For plasma, blood samples were collected at 0 (predose), 5, 15, and 30 min and
1, 2, 3, 4, 6, 8, 12, and 24 h postdose. Blood (1.0 ml) was processed immediately
for plasma by centrifugation at 2,000 rpm for 10 min. Plasma samples were
frozen and maintained at ?70°C until analyzed.
PBMC preparation. Whole blood (8 ml) drawn at specified time points was
mixed in equal proportion with PBS, layered onto 4 ml of Ficoll-Paque solution
(Pharmacia Biotech), and centrifuged at 400 ? g for 40 min. The PBMC layer
was removed and washed once with PBS. The formed PMBC pellet was recon-
stituted in 0.5 ml of PBS, and cells were resuspended and counted with a
hemocytometer. The number of cells multiplied by the mean single-cell volume
was used to calculate intracellular concentrations. A reported value of 200
femtoliters was used as the resting PBMC volume (28).
Determination of tenofovir and GS 7340 and GS 7339 in plasma and PBMCs.
The concentration of tenofovir in dog plasma samples was determined by deriv-
atizing tenofovir with chloroacetaldehyde to yield a highly fluorescent N1,N6-
ethenoadenine derivative (18). Plasma (100 ?l) was mixed with 200 ?l of 0.1%
trifluoroacetic acid in acetonitrile to precipitate proteins. Samples were then
evaporated to dryness under reduced pressure at room temperature. Dried
samples were reconstituted in 200 ?l of derivatization cocktail (0.34% chloro-
acetaldehyde in 100 mM sodium acetate, pH 4.5), vortexed, and centrifuged. The
supernatant was then transferred to a clean screw-cap tube and incubated at 95°C
for 40 min. Derivatized samples were then evaporated to dryness and reconsti-
tuted in 100 ?l of water for HPLC analysis. Conversion of intact prodrug to
tenofovir during the analysis procedures was determined to be ?10% with
Ribonucleotides present in the PBMC extracts were removed by selective
oxidation using a modified procedure of Tanaka et al. (33). PBMC extracts were
mixed 1:2 with methanol and evaporated to dryness under reduced pressure. The
dried samples were derivatized with chloroacetaldehyde as described above for
the plasma assay, mixed with 20 ?l of 1 M rhamnose and 30 ?l of 0.1 M sodium
periodate, and incubated at 37°C for 5 min. Following incubation, 40 ?l of 4 M
methylamine and 20 ?l of 0.5 M inosine were added, and samples were further
incubated at 37°C for 30 min. Samples were then evaporated to dryness under
reduced pressure and reconstituted in water for HPLC analysis. Independently,
it was demonstrated that the chloroacetaldehyde derivatization and periodate
oxidation resulted in ?6% conversion of the mono- and diphosphate metabolites
of tenofovir to the N1,N6-ethenoadenine derivative of tenofovir.
The HPLC system comprised a P4000 solvent delivery system with AS3000
autoinjector and F2000 fluorescence detector (Thermo Separation, San Jose,
CA). The column was an Inertsil ODS-2 column (4.6 by 150 mm). The mobile
phases were as follows: A, 5% acetonitrile in 25 mM potassium phosphate buffer
with 5 mM tetrabutyl ammonium bromide, pH 6.0; B, 60% acetonitrile in 25 mM
potassium phosphate buffer with 5 mM tetrabutyl ammonium bromide, pH 6.0.
FIG. 1. Structure.
VOL. 49, 2005INTRACELLULAR ACTIVATION OF NOVEL TENOFOVIR PRODRUG1899
The flow rate was 2 ml/min, and the column temperature was maintained at 35°C
by a column oven. The gradient profile was 90% A/10% B for 10 min for
tenofovir, followed by 65% A/35% B for 10 min for GS 7340. Detection was by
fluorescence with excitation at 236 nm and emission at 420 nm, and the injection
volume was 10 ?l. Data was acquired and stored by an Artemis data acquisition
system (Beckman, Palo Alto, CA).
Pharmacokinetic calculations. Cmaxand Tmaxwere observed values. Tenofovir
exposures were expressed as areas under tenofovir concentration curves from
zero to 24 h (AUC0-24). The AUC values were calculated using the trapezoidal
rule. All other pharmacokinetic parameters, including the elimination half-life,
were calculated by noncompartmental methods using WinNonlin (Pharsight
Corp., Mountain View, CA).
Tissue distribution study. Two male beagle dogs (each, 10 to 12 kg) were
dosed orally (10 mg-eq tenofovir/kg; 30 to 35 ?Ci/kg) by gavage with [14C]GS
7340 or [14C]tenofovir DF. Dosing solutions of GS 7340 and tenofovir DF were
prepared 1 h prior to dosing by dissolving necessary quantities of GS 7340
(fumarate salt) or tenofovir DF and dried [14C]GS 7340 or [14C]tenofovir diso-
proxil (radiolabeled free base) in 50 mM citric acid (pH 2.2). Urine, as well as
cage rinse and feces, was collected at 24 h after administration. Plasma samples
were obtained at 0, 0.0833, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, and 24 h postdose.
Additionally, blood samples were collected at 0, 2, 8, and 24 h postdose and
processed for PBMCs as described above. At 24 h after the dose, the animals
were sacrificed, and tissues were removed for further analysis. Plasma, peripheral
blood mononuclear cells, whole blood, bile, cerebrospinal fluid, and urine were
analyzed for total radioactivity by oxidation. Feces were homogenized in water
(10% [wt/wt]), and aliquots of homogenate were processed by oxidation for
counting. The brain, liver, spleen, heart, lungs, kidneys, uterus, stomach, small
intestines, stomach contents, small intestine contents, and large intestine con-
tents were homogenized separately in water (20% [wt/wt]), and aliquots of each
homogenate were oxidized and counted.
In vitro anti-HIV activity. GS 7340 was synthesized from
(R)-PMPA and (L)-isopropyl alanine ester in a nonstereospe-
cific synthesis, resulting in the formation of equal amounts of
two stereoisomers at phosphorus. These two diastereomers,
GS 7339 and GS 7340, were subsequently separated by chro-
matography. To assess the ability of these prodrugs to cross the
cellular membrane and undergo intracellular metabolism to
tenofovir, their in vitro activities were measured against HIV-1
in MT-2 cells. The antiviral activities for the diastereomeric
mixture (GS 7171), the individual diastereomers (GS 7339 and
GS 7340), the diastereomic mixture of D-alaninyl analog (GS
7485), and the alaninyl monoamidate metabolite (GS 7160) are
shown in Table 1. Compared to tenofovir, the individual iso-
mers, GS 7339 and GS 7340, were 83- and 1,000-fold more
active, respectively, whereas the D-isopropyl alaninyl analog
(GS 7485) and the metabolite (GS 7160) showed activity sim-
ilar to tenofovir. The enhanced activities of the L-alaninyl pro-
drugs compared to those of tenofovir are a result of greater
cellular permeability and rapid conversion to tenofovir inside
the MT-2 cells. The dramatically reduced activity (1,000 fold)
of the D-alaninyl analog (GS 7485), relative to the L-alaninyl
analog (GS 7171) demonstrates a strong metabolic preference
inside the MT-2 cells for the L-amino acid (see below). The
12-fold-greater activity of GS 7340 compared to that of GS
7339 further suggests that intracellular metabolism is also sen-
sitive to stereochemistry at the phosphorus. The greater selec-
tivity index (?10?) for GS 7340 than tenofovir DF may reflect
the kinetics of cell loading; GS 7340 results in a higher initial
intracellular concentration of tenofovir, which may differen-
tially affect antiviral potency and cytotoxicity.
In vitro metabolism and accumulation in PBMCs. The an-
tiviral activities in tissue culture were determined in buffer
containing 5% heat-inactivated fetal calf serum, a medium in
which the prodrugs in this study were stable. To enhance the in
vivo accumulation of tenofovir inside cells, a prodrug must
exhibit greater stability in plasma relative to the intracellular
environment. Table 1 lists the in vitro half-lives of the mono-
amidate prodrugs and tenofovir DF in MT-2 cell extract and
human plasma. GS 7340 was metabolized 3-fold faster in MT-2
cell extract than in plasma, whereas tenofovir DF, a prodrug
designed to release tenofovir into systemic circulation, was
metabolized 170-fold faster in plasma. As inferred from the
antiviral activity, GS 7339 and the D-amino acid analogs (GS
7485) are more stable in MT-2 cell extract than plasma.
The putative first step in the conversion of the GS 7340 to
tenofovir is the hydrolysis of the amino acid ester, which is
sensitive to stereochemistry at both the amino acid and phos-
phorus. The metabolites observed from the degradation of GS
7340 were identified as the alaninyl amidate (GS 7160) in
MT-2 cell extract and the isopropyl alaninyl amidate (GS 7161)
in plasma (Fig. 2) by coinjection of authentic samples prepared
independently. In the MT-2 cell extract, the alaninyl amidate
metabolite was slowly hydrolyzed to tenofovir. No evidence of
further conversion to the mono- or diphosphate metabolites
was observed in the MT-2 cell extracts.
The metabolism of GS 7340 was further explored by incu-
bating [14C]GS 7340 in fresh whole human blood. After a 1-h
incubation at 37°C, plasma, PBMCs, and RBCs were separated
and total radioactivity was analyzed by HPLC with radiometric
detection. The results are shown in Table 2, along with those of
tenofovir and tenofovir DF. The total recovered radioactivity
in PBMCs after a 1-h incubation with GS 7340 was 9.3-fold and
38-fold higher than with tenofovir DF or tenofovir, respec-
TABLE 1. In vitro anti-HIV-1 activity (EC50), cytotoxicity (CC50), and in vitro metabolic stabilities of tenofovir and tenofovir prodrugs
MT-2 cell extract Human plasma
GS 7485 (D-Ala)
5.0 ? 2.6
0.05 ? 0.03
0.01 ? 0.005
0.06 ? 0.04
0.005 ? 0.002
10 ? 4
10 ? 2.5
6,000 ? 2,700
50 ? 28
95 ? 37
40 ? 29
70.7 ? 10.1
28.3 ? 7.4
0.41 ? 0.20
231 ? 72
90 ? 12
aThe data represent means ? SD from two to four independent determinations.
bNR, not reactive; ND, not determined.
1900LEE ET AL.ANTIMICROB. AGENTS CHEMOTHER.
tively. The greater radioactivity in RBCs after incubation with
GS 7340 or tenofovir DF than with tenofovir can be accounted
for by the decreased permeability of the latter across RBC
membrane. The HPLC radiochromatograms depicting the
product distribution in PBMCs, plasma, and RBCs after incu-
bation of GS 7340 in whole blood are shown in Fig. 3. In
plasma, the major species present was intact GS 7340 (84%),
with minor amounts of GS 7161 (13%), GS 7160 (2%), and
tenofovir (1%). In RBCs, the major species observed was in-
tact GS 7340 (57%). In PBMCs, there was no intact GS 7340
detected, and the major species present were tenofovir (45%),
(21%), and GS 7160 (18%). The phosphorylation of tenofovir
in PMBCs to the mono- and diphosphate species after 1-h
incubation in whole blood was not saturable over a 30-fold
concentration range (Fig. 4). Additionally, in MT-2 cells incu-
bated with 10 ?M GS 7340, the formation of the diphosphate
metabolite was linear out to 24 h (Fig. 5.) The concentration of
the diphosphate species inside the MT-2 cells at 24 h exceeded
the initial extracellular concentration of GS 7340 by 250 fold.
These results are consistent with the data generated in MT-2
extract and isolated plasma; however, unlike the MT-2 extract,
conversion to the active diphosphate metabolite was readily
observed with intact MT-2 cells and PBMCs.
The whole-blood experiments were extended to determine
the distribution of tenofovir into CD4?T lymphocyte and
monocyte fractions isolated from the PBMCs, following incu-
bation with radiolabeled GS 7340. The results in Table 3 show
that the concentration of total GS 7340 metabolites in CD4?
cells was approximately 70% of that in the entire PBMC frac-
tion. The concentration of total GS 7340 metabolites in mono-
cytes was approximately half of that in CD4?cells. There are
no major differences in the relative concentration of phosphor-
ylated tenofovir species in CD4?cells or monocytes.
In vivo pharmacokinetics in samples from dogs. The diaste-
reomeric mixture, GS 7171, was administered as an oral solu-
tion in 50 mM citric acid (pH 2.3) at a dose of 10 mg-eq/kg (of
tenofovir) to five male beagle dogs. Figure 6 shows the result-
ing plasma levels of tenofovir and the individual diastereomers
(GS 7339 and GS 7340) from 0 to 24 h after administration.
Elimination of both diastereomers (GS 7340 and GS 7339) was
rapid relative to tenofovir in plasma. Consistent with the faster
metabolism observed in vitro (Table 1), GS 7340 was cleared
more quickly from plasma than GS 7339. To measure the
intracellular levels of tenofovir after oral administration of GS
7171, PBMCs were isolated from dog blood samples at 0, 2, 8,
and 24 h postadministration. After cells were counted, the
concentration of tenofovir in the PBMCs was analyzed by a
FIG. 2. Routes of metabolism.
FIG. 3. Radiochromatograms labeled with14C from plasma (A),
aggregated red blood cells (B), and PBMCs (C), obtained after incu-
bation of 17.4 ?M GS 7340 with whole human blood for 1 h at 37°C.
FIG. 4. Metabolism of [14C]GS 7340 in PMBCs after 1-h incuba-
tion in whole human blood at 37°C (mean ? SD; n ? 3 samples of
TABLE 2. Distribution of all tenofovir-related species after
incubation of14C-labeled drug in human whole blooda
Total amt recoveredb(?g-eq)
43.0 ? 7.8
48.10 ? 8.7
55.70 ? 4.5
1.25 ? 0.22
0.133 ? 0.029
0.033 ? 0.014
12.6 ? 3.02
10.5 ? 5.17
3.72 ? 1.86
aA total of 10 ?g-eq/ml of14C-labeled compound was incubated for 1 h at
37°C in human blood, followed by Ficoll-Paque separation of the blood fractions.
bMean ? SD, n ? 3.
VOL. 49, 2005 INTRACELLULAR ACTIVATION OF NOVEL TENOFOVIR PRODRUG1901
precolumn fluorescent derivatization reverse-phase HPLC
method (18). Tenofovir concentration in PBMCs increased
rapidly within 2 h to levels that greatly exceeded plasma levels
(Fig. 6). The ratio of the total tenofovir AUC from 0 to 24 h in
PBMCs to that in plasma was ?90 after a single dose of GS
7171. GS 7339 and GS 7340 were also administered separately
to two sets of five dogs (10 mg-eq/kg of tenofovir). A bar graph
comparing the plasma and PBMC AUC0-24values for tenofo-
vir after administration of GS 7340, GS 7339, GS 7171, and
tenofovir DF is shown in Fig. 7. Oral administration of GS
7340, the more rapidly cleared isomer, resulted in a ?34-fold
increase in the AUC0-24in PBMCs relative to tenofovir DF
and a 6-fold increase relative to GS 7339. In the case of teno-
fovir DF, the AUC0-24ratio of tenofovir in PBMCs to plasma
was 4.7. Based on these data and in vitro metabolism studies,
the more rapidly cleared isomer GS 7340 was chosen as a lead
prodrug for further preclinical development.
The concentration of tenofovir in PBMCs determined by the
fluorescent derivatization method does not include the teno-
fovir mono- or diphosphate species. As part of the radiolabeled
distribution study (see below), PBMCs were collected at 2, 8,
and 24 h, and total radioactivity was determined. The concen-
tration of total tenofovir metabolites at 24 h was 63 ?g-eq/ml.
The calculated t1/2value in PBMCs was ?25 h, which is con-
sistent with the intracellular half-life observed in vitro with
resting lymphocytes (26).
The absolute bioavailabilities of tenofovir after oral admin-
istration of GS 7171, GS 7339, and GS 7340 were calculated by
comparing the resulting plasma tenofovir AUC0-24to that ob-
served after the i.v. administration of tenofovir itself. Data are
shown in Table 4. The bioavailabilities were 20, 13, and 17%,
respectively. The oral bioavailability of the prodrug (as pro-
drug) was ?70% at the 20-mg/kg dose and was calculated by
comparing the plasma levels of GS 7340 after oral administra-
tion to those after an i.v. bolus administration of GS 7340 (data
Tissue distribution of GS 7340 after oral administration in
dogs. The results of a tissue distribution study in dogs following
oral administration of [14C]GS 7340 or [14C]tenofovir DF are
shown in Table 5. Two dogs per group received a single oral
dose (10 mg-eq/kg; 35 ?Ci/kg), and tissues were harvested at
24 h postadministration. Except for the kidney and liver, ra-
dioactivity was generally higher in all tissues after administra-
tion of GS 7340 than with tenofovir DF. In the lymph nodes,
concentrations of radioactivity were 5- to 15-fold higher after
administration of GS 7340. In lung, ileum, spleen, bone mar-
row, and muscle, concentrations were consistently higher than
with tenofovir DF. The increased concentrations of radioactiv-
ity in the bile for GS 7340 suggest that there is a hepatic
FIG. 5. Formation of tenofovir and metabolites in MT-2 cells dur-
ing a 24-h incubation with 10 ?M [14C]GS 7340 (mean ? SD; n ? 3
samples of MT-2 cells).
05 10 152025
Tenofovir in Plasma
GS 7340 in Plasma
GS 7339 in Plasma
Total Tenofovir in PBMC
FIG. 6. Mean concentration versus time profiles for tenofovir, GS
7340, and GS 7339 in plasma and tenofovir in PBMCs after oral
administration of GS 7171 (10 mg-eq/kg tenofovir) to dogs (mean ?
SD for samples from four dogs).
GS 7339GS 7340 GS 7171
AUV (0-24) Tenofovir ( µg.h/mL)
FIG. 7. Tenofovir in PBMCs and plasma (AUC0-24 h) after subcu-
taneous administration of tenofovir and after oral administration of
tenofovir DF, GS 7339, GS 7340, and GS 7171 (10 mg-eq/kg) to dogs
(mean ? SD, n ? 5 dogs).
TABLE 3. Concentration of metabolites in CD4?lymphocytes and
monocytes isolated from whole human blood after incubation with
17 ?M14C-labeled GS 7340 for 1 h
Tenofovir Tenofovir-P Tenofovir-PP GS 7340
Monocytesb138 ? 55
218 ? 31 122 ? 8
89 ? 8
157 ? 15
73 ? 15
703 ? 36
364 ? 24
921 ? 64NDd
aValues are means ? SD (n ? 2 samples of whole blood).
bCalculated based on cell volumes of 0.2 pl/cell for T lymphocytes and 0.4
pl/cell for monocytes (4).
cBased on average volume of 0.2 pl/cell.
dND, not determined.
1902 LEE ET AL.ANTIMICROB. AGENTS CHEMOTHER.
clearance mechanism for either the intact prodrug or the me-
Although there is still much debate regarding the ultimate
reservoir for HIV, the evidence for ongoing HIV replication
during HAART in patients who have undetectable viremia is
compelling. Using an ultrasensitive viral RNA assay, Ramrat-
nam et al. have shown that low-level viral bursts can be de-
tected in samples from stable, long-term patients on HAART
(22). The number of bursts observed over a 6-month period
was directly correlated to the terminal viral elimination half-
life from plasma. These data suggest that the long apparent
half-lives (?44 mo) reported for “latent cells” (10) may be an
overestimation of the true latency half-life, since new latent
cells are being formed as a result of low-level virus replication.
More-potent HAART therapies or therapies that better target
the sites of ongoing HIV replication are required to completely
halt virus replication. Currently, the intrinsic potencies of po-
tential drugs are optimized against HIV in tissue culture sys-
tems with PBMCs or immortalized cell lines to replicate the
virus. These assays are very useful for optimizing chemical
structure during the initial phases of drug design; however,
they do not provide any guidance into the pharmacokinetics,
intracellular half-life, or distribution of a drug in vivo. Intra-
cellular half-life and distribution are particularly important
because a significant fraction of memory CD4?T cells exists
outside of lymphoid tissue (24). Additionally, suboptimal drug
concentrations in replication competent compartments will se-
lect and amplify resistant virus (14, 25).
Tenofovir belongs to a class of nucleotide analogs that have
prolonged intracellular half-lives (20). The long intracellular
half-life of tenofovir is a result of rapid metabolism within the
cell to the nucleotide diphosphate and its limited efflux from
cells. The phosphonate moiety of tenofovir is recognized by
cellular kinases as a monophosphate, thereby bypassing the
initial phosphorylation step, which can be rate limiting for
nucleoside analogs (25). Because tenofovir is a dianion at phys-
iological pH, it has low cellular permeability, which is reflected
in its in vitro HIV-activity (EC50? 5 ?M). However, in vivo,
the long intracellular half-life of tenofovir results in a potent
and durable anti-HIV effect in both preclinical models and
clinical studies (30). The amidate prodrug GS 7340 was de-
signed to overcome the permeability limitations of tenofovir by
masking the dianion with a neutral promoiety and increasing
the plasma stability of the prodrug relative to its intracellular
stability. The in vitro anti-HIV potency of GS 7340 (EC50?
0.005 ?M) is comparable to the most potent protease inhibi-
tors and reflects the greater cell permeation of GS 7340 com-
pared to tenofovir. In addition to favorable potency and a long
intracellular half-life, the in vivo administration of GS 7340
results in an enhanced distribution to lymphatic tissue com-
pared to tenofovir DF. The concentration of tenofovir inside
PBMCs at 24 h was ?100 fold the concentration of tenofovir in
plasma at the same time point, and the ratio of AUC0-24for
tenofovir in PBMCs versus plasma was ?150 (Fig. 7). With
tenofovir DF, the PBMC/plasma ratio for AUC0-24 hwas 5.
Since the HPLC assay only measured tenofovir inside PBMCs
and not the mono- or diphosphate species, these differences
should be considered the minimum ratios. Using total reactiv-
ity at 24 h to compare the PBMC-to-plasma concentration, the
ratio for GS 7340 was 316, suggesting that the majority of the
tenofovir species inside cells exists as mono- or diphosphate.
In our drug optimization studies, we used PBMCs as a sur-
rogate marker for distribution to lymphatic tissues, since the
exchange of lymphocytes between blood and lymph is rapid
and only a small percentage of the total lymphocytes reside in
the blood compartment. The increased concentration of radio-
activity in the lymph nodes, ileum, lung, bone marrow, and
thymus after administration of GS 7340 relative to tenofovir
DF suggests that the amidate is preferentially metabolized to
tenofovir and accumulates in tissues that contain high concen-
trations of lymphatic cells. Although we do not have detailed
cellular distribution data for these tissues, the fact that teno-
fovir is present in tissues that have all been implicated as
possible HIV reservoirs is encouraging. It must be noted, how-
ever, that the expanded distribution and the higher intracellu-
lar levels of tenofovir after administration of GS 7340 open the
possibility of safety issues not observed with tenofovir DF.
Other in vitro studies have been published using an amidate
prodrug approach to mask the monophosphates of zidovudine
(AZT) and stavudine (d4T). The monoamidate prodrugs of
the monophosphate of AZT exhibit minimal or no enhance-
ment in in vitro anti-HIV activity (16, 17). The modest in vitro
effects seen with these AZT prodrugs have been explained in
part by the lability of the AZT monophosphate bond, which
can readily hydrolyze back to AZT. Selected amidate prodrugs
of d4T monophosphate do show significant increases in in vitro
activity relative to that of d4T itself (up to 100 fold), suggesting
that the subsequent phosphorylation is rapid compared to the
hydrolysis of the nucleoside phosphate bond (29). The princi-
pal advantage of this approach with nucleosides is the ability to
deliver the nucleoside monophosphate into the cell, thereby
removing a potential rate-limiting step in the formation of the
nucleoside triphosphate. The half-life of the nucleoside in cells
TABLE 4. Pharmacokinetics of tenofovir in plasma after oral administration of GS 7340, GS 7339, and GS 7171 in fasted dogsa
Drug (route)AUC0–24(?g · h/ml)
GS 7340 (p.o.)b
GS 7339 (p.o.)b
GS 7171 (p.o.)c
aData are means ? SD for groups of five dogs. p.o., oral.
bDose as tenofovir (mg-eq/kg) ? 10.85.
cDose as tenofovir (mg-eq/kg) ? 9.64.
d%F calculated from AUC0–?? 4.30 ?g-h/ml for a 1-mg/kg dose of intravenous tenofovir.
VOL. 49, 2005 INTRACELLULAR ACTIVATION OF NOVEL TENOFOVIR PRODRUG1903
The detailed mechanism for the selective intracellular tar-
geting of the amidate prodrugs is not fully understood. How-
ever, we have shown that the monoamidate prodrugs of teno-
fovir are more rapidly cleaved in the intracellular environment
than in plasma and that the tenofovir formed within the cell is
rapidly phosphorylated to give a long-lived metabolite. The
selectivity exhibited between plasma and intracellular extracts
is the basis for the preferential cellular loading observed in
vivo. The limited structure activity relationship presented in
this study demonstrates that the intracellular degradation of
the amidate prodrugs is sensitive to the stereochemistry at both
the amino acid and the phosphorus, suggesting that the process
is enzyme mediated. It has yet not been possible under buffer
or enzymatic conditions to isolate the monophenyl monoami-
date species, leading to the conclusion that this reaction is
spontaneous. Work by other investigators with the monoami-
TABLE 5. Tissue distribution of14C-labeled tenofovir DF and14C-labeled GS 7340 in dogs following an oral
dose of tenofovir of 10 mg-eq/kga
Tissue or fluid
Tenofovir DF GS 7340
Tissue concn ratio of GS 7340
to tenofovir DF
% Dose Concn (?g-eq/g)b
% Dose Concn (?g-eq/g)b
38.3 ? 14.3
87.9 ? 38.4
0.53 ? 0.06
52.9 ? 4.7
80.2 ? 3.4
4.34 ? 0.15
Iliac lymph nodes
Axillary lymph nodes
Inguinal lymph nodes
Mesenteric lymph nodes
0.51 ? 0.14
0.37 ? 0.25
0.28 ? 0.28
1.20 ? 0.64
5.42 ? 0.86
5.54 ? 0.42
4.12 ? 0.44
6.88 ? 0.78
Salivary gland (left ? right)
0.30 ? 0.20
0.23 ? 0.07
0.45 ? 0.1
1.9 ? 0.79
4.78 ? 1.31
1.80 ? 0.13
5.54 ? 0.10
3.47 ? 0.13
0.63 ? 0.11
0.57 ? 0.62
8.13 ? 0.19
3.51 ? 0.57
Testes (left ? right)
0.24 ? 0.03
1.95 ? 0.84
0.11 ? 0.00
0.46 ? 0.17
2.14 ? 0.38
1.99 ? 0.74
1.12 ? 0.17
1.97 ? 0.03
0.08 ? 0.03
0.2 ? 0.08
0.28 ? 0.05
2.05 ? 0.92
Eye (left ? right)
0.13 ? 0.06
0.16 ? 0.01
0.06 ? 0.03
0.95 ? 0.17
0.90 ? 0.07
0.24 ? 0.00
0.04 ? 0.00
1.93 ? 1.03
3.01 ? 1.37
4.96 ? 0.54
0.50 ? 0.19
2.57 ? 0.33
3.58 ? 1.99
9.63 ? 9.42
2.68 ? 0.27
4.16 ? 0.73
8.77 ? 1.13
4.61 ? 1.91
47.2 ? 42.2
25.0 ? 4.7
40.5 ? 4.9
Total GI tract contents
NA91.9 ? 34.0
Plasma at 24 h
PBMCs at 24 hc
Whole blood at 24 h
0.20 ? 0.09
0.85 ? 0.20
0.20 ? 0.02
63.2 ? 15.5
0.20 ? 0.00
aNA, not applicable; ND, not determined; ?LOD, below the limit of detection (0.02 ?g-eq/g).
bConcentrations are means ? SD for groups of two dogs.
cCalculated using typical recovery of 15 ? 106cells total and mean PBMC volume of 0.2 picoliters/cell.
1904LEE ET AL.ANTIMICROB. AGENTS CHEMOTHER.
dates of nucleosides has identified a cytosolic fraction from
hepatocytes thought to be responsible for cleavage of the P-N
bond (27). However, in the case of GS 7160, degradation at low
pH is extremely fast (t1/2? 1 min); therefore, the spontaneous
hydrolysis of the metabolite in a cellular compartment with a
low pH cannot be ruled out. Investigations to characterize the
specific enzymes and mechanism responsible for the conver-
sion of the prodrug to tenofovir are in progress. The prefer-
ential distribution into PBMCs and other lymphatic tissues is
likely due to the increased metabolic activity of these tissues
and the long intracellular half-lives of tenofovir and its mono-
and diphosphate metabolites.
In conclusion, the high concentrations of tenofovir observed
in lymphatic tissues after oral administration of GS 7340 are
expected to result in increased clinical potency relative to te-
nofovir DF and could have a profound effect on the low-level
virus replication that occurs in tissues with suboptimal drug
exposure during HAART. The parent drug, tenofovir, is
unique in this potential, due to its long half-life as the active
diphosphate in uninfected cells and the lack of significant in
vivo resistance development. With GS 7340, it should be pos-
sible to reduce the total dose of tenofovir, thereby minimizing
systemic exposure, while at the same time increasing antiviral
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