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
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1906LEE ET AL.ANTIMICROB. AGENTS CHEMOTHER.