Cellular Pharmacology of 9-( -D-1,3-dioxolan-4-yl) Guanine and its Lack of Drug Interactions with Zidovudine in Primary Human Lymphocytes

Article (PDF Available)inAntiviral chemistry & chemotherapy 18(6):343-6 · February 2007with21 Reads
DOI: 10.1177/095632020701800606 · Source: PubMed
Abstract
Amdoxovir, currently in Phase II clinical trials, is rapidly converted to 9-(beta-D-1,3-dioxolan-4-yl)guanine (DXG) by adenosine deaminase in vitro and in humans. The cellular pharmacology of DXG in primary human lymphocytes, including dose-response relationships, intracellular half-life of DXG triphosphate (DXG-TP), and combination studies were determined. DXG produced high levels of DXG-TP with a long half-life (16 h) in activated human peripheral blood mononuclear cells. Since zidovudine (ZDV) and DXG select for different resistance mutations, co-formulation of the these two drugs is an attractive proposition. A combination study between DXG and ZDV showed no reduction of DXG-TP or ZDV-TP. Taken together, these results suggest that an appropriately designed DXG prodrug could be given once a day and that co-formulation with ZDV might be a possibility.

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Available from: Raymond F Schinazi
Antiviral Chemistry & Chemotherapy 18:343–346
Short communication
Cellular pharmacology of 9-(β-
D-1,3-dioxolan-4-yl)
guanine and its lack of drug interactions with
zidovudine in primary human lymphocytes
Brenda I Hernandez-Santiago, Aleksandr Obikhod, Emilie Fromentin, Selwyn J Hurwitz and
Raymond F Schinazi*
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory
University School of Medicine, and Veterans Affairs Medical Center, Decatur, GA 30033, USA
*Corresponding author: Tel: 404 728 7711; Fax: 404 728 7726; E-mail: rschina@emory.edu
Amdoxovir, currently in Phase II clinical trials, is
rapidly converted to 9-(β-
D-1,3-dioxolan-4-
yl)guanine (DXG) by adenosine deaminase in vitro
and in humans. The cellular pharmacology of DXG
in primary human lymphocytes, including
dose–response relationships, intracellular half-life
of DXG triphosphate (DXG-TP), and combination
studies were determined. DXG produced high
levels of DXG-TP with a long half-life (16 h) in acti-
vated human peripheral blood mononuclear cells.
Since zidovudine (ZDV) and DXG select for
different resistance mutations, co-formulation of
the these two drugs is an attractive proposition. A
combination study between DXG and ZDV
showed no reduction of DXG-TP or ZDV-TP. Taken
together, these results suggest that an appropri-
ately designed DXG prodrug could be given once
a day and that co-formulation with ZDV might be
a possibility.
Keywords: cellular pharmacology, DXG, nucleoside
analogues, NRTI
343©2008 International Medical Press 0956-3202
The emergence of resistant HIV strains during therapy has
made it a major challenge to develop drugs that delay,
prevent or attenuate the onset of resistance. Therefore,
several nucleoside reverse transcriptase inhibitors (NRTIs)
are under development as second line therapies for individ-
uals infected by viruses with common mutations such as
M184V and thymidine analogue mutations (TAMs).
Although a number of NRTIs in development are pyrim-
idines, such as Racivir, Dexelvucitabine (2
,3-didehydro-
2
,3-dideoxy-5-fluorocytidine, reverset, D-D4FC, DFC,
RVT), AVX-754 (SPD-754, (-)dOTC, (-)-2
-deoxy-3-
oxa-4
-thiocytidine) and β-D-dioxolane-thymine (DOT),
only a few are purines. Amdoxovir (AMDX, (-)-β-
D-2,6-
diaminopurine dioxolane, DAPD) is a purine nucleoside in
Phase II clinical trials for the treatment of HIV-1 infections
(http://clinicaltrials.gov/show/NCT00432016).
DAPD was developed as a nucleoside analogue
prodrug that is deaminated by adenosine deaminase to
the 2
-deoxyguanosine analogue, 9-(β-D-1,3-dioxolan-4-
yl)guanine (DXG), to circumvent the limited aqueous
solubility and oral bioavailability of DXG. Activation of
DXG requires intracellular phosphorylation to the
triphosphate DXG-TP, which is a potent and selective
inhibitor of HIV-1, HIV-2 and hepatitis B virus (HBV)
in human cell lines. The antiretroviral spectrum of DXG-
TP includes potent activity against wild-type and drug-
resistant forms of HIV-1 reverse transcriptase (RT),
including RT enzymes containing M184V/I, TAMs
(specifically M41L, D67N, K70R, L210W, T215Y/F
and K219Q/E/N) and the 69SS double-insert mutations,
and against HBV in human cell lines in vitro (Chin et al.,
2001, Seigneres et al., 2002, Ying et al., 2000). Resistance
in HIV-1 develops slowly in vitro, and is associated with
mutations at K65R or L74V (Bazmi et al., 2000).
To date, close to 200 subjects have safely received
DAPD in seven Phase I and II studies
(www.rfspharma.com). DAPD has an excellent safety
profile in subjects receiving treatment for up to 96 weeks
and is very effective at decreasing viral load in HIV-
infected individuals, including those with extensive NRTI
mutations. A Phase I/II pharmacokinetic/pharmacody-
namic study in HIV-infected individuals to identify a
potential co-formulation of DAPD with 3
-azido-3-
deoxythymidine (zidovudine; ZDV) has been completed
and suggests that ZDV enhances the activity of DAPD
(Murphy R, Zala C, Ochoa C, Tharnish P, Mathew J,
Schinazi 11/2/08 14:00 Page 343
Fromentin E, Asif G, Hurwitz SJ, Kivel NM &
Schinazi RF [2008] Pharmacokinetics and potent anti-
HIV-1 activity of amdoxovir plus zidovudine in a random-
ized double-blind placebo-controlled study. 15th Conference
on Retroviruses and Opportunistic Infections. Boston, MA,
USA, 3–6 February 2008. Abstract J126).
Although extensive pharmacology studies have been
conducted in humans, in vitro cellular pharmacology
including assessments of dose–response relationships, accu-
rate determination of the intracellular half-life of DXG-TP,
and combination studies have yet to be published.
To achieve a better understanding of DXG phos-
phorylation, phytohaemagglutinin-stimulated primary
human peripheral blood mononuclear cells (PBMCs)
(1×10
6
cells/per time point) were incubated in the pres-
ence of different concentrations of [8-
3
H]-DXG (1, 3, 5,
7, 10, 20 and 30 μM) for 4 h. To determine the cellular
half-life of DXG-TP, [
3
H]-DXG (30 μM) was incubated
in PBMCs for 4 h at 37°C in a 5% CO
2
atmosphere. The
cells were then washed three times with drug-free
medium to remove extracellular DXG and re-incubated
in drug-free cell culture medium for specific time periods
(0, 1, 2, 4, 8, 12, 24 and 48 h). Combination studies with
ZDV were also performed in which radiolabelled-DXG
(10 μM) was co-incubated with ZDV (0.1, 1 and 10 μM)
or [
14
C]-ZDV (10 μM) was co-incubated with DXG (1,
10 and 100 μM) for 2 h in PBMCs. All studies were
conducted in triplicate.
Cells were then processed to remove extracellular
DXG: at selected times cells were centrifuged for 10 min
at 350×g at 4˚C; the pellets were resuspended and washed
two to three times with cold phosphate-buffered saline
(PBS); viable cells were counted using Vi-cell XR
counter (Beckman Coulter, Fullerton, CA, USA; viability
>98%). The intracellular metabolites of DXG were then
extracted by incubation for 2 h at -20˚C with 60%
methanol/water (1 ml), and the extracts collected and
centrifuged at 14,000 rpm (Eppendorf Centrifuge Model
5415C, Hamburg, Germany) for 5 min, before being dried
under a gentle filtered air flow and stored at -20˚C until
they were assayed. The residues were resuspended in 100
μl
of water and aliquots were injected into a high-pressure
liquid chromatography (HPLC) system.
Separation of DXG metabolites was performed by ion-
pairing reverse phase HPLC on a Columbus 5 μm C
18
column (250×4.6 mm; Phenomenex, Torrance, CA, USA)
using a Varian Pro Star HPLC model 210 with manual injec-
tion (Walnut Creek, CA, USA). The mobile phase consisted
of buffer A (25 mM ammonium acetate with 5 mM tetra-
butylammonium phosphate [TBAP]; pH 7.0) and buffer B
(methanol). Elution was performed using a multistage linear
gradient of buffer B from 10% to 50%. The limit of detection
was ~0.01 pmol/10
6
cells. Radioactivity was quantified using
a 2500 TR liquid scintillation analyzer (PerkinElmer, Life
and Analytical Sciences, Wellesley, MA, USA). DXG-TP
was identified based on an authentic standard.
BI Hernandez-Santiago et al.
344 ©2008 International Medical Press
1
3
5 7 10 20 30
0
5
10
15
20
25
30
35
40
[
3
H]-DXG, µM
[
3
H]-DXG-TP intracellular
concentration, pmol/10
6
cells
Figure 1. DXG-TP levels in PHA-stimulated PBMCs
after incubation with different concentrations of
DXG for 4 h
[
3
H]-DXG (250–1,000 dpm/pmol) phosphorylation in phytohaemagglu-
tinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs)
for 4 h. DXG, 9-(β-
D-1,3-dioxolan-4-yl)guanine; TP, triphosphate.
0
1
10
100
10 20 30 40 50 60
Time, h
Intracellular DXG, pmol/10
6
cells
Figure 2. Decay study of DXG-TP in PHA-stimulated
PBMCs
The half-life (t
1/2
) of DXG-TP in phytohaemagglutinin (PHA)-
stimulated peripheral blood mononuclear cells (PBMC) after 4 h
incubation with [
3
H]-DXG (30 μM, 250 dpm/pmol). The average half-
life derived from three experiments was 16.04 ±0.61 h. DXG, 9-(β-
D-
1,3-dioxolan-4-yl)guanine; TP, triphosphate.
Schinazi 11/2/08 14:00 Page 344
Cellular pharmacology of DXG
The quantification of ZDV-TP was performed using a
sensitive and specific liquid chromatography tandem mass
spectrometry (LC-MS/MS) method. Dried extracts were
reconstituted in ultra-pure water (100 μl) containing
lamivudine-triphosphate (3TC-TP; 100 nM) as internal
standard (IS) and filtered (0.22 μm nylon centrifuge tube)
at 16,000g for 5 min to remove insoluble particulates;
45 μl were injected on the column. The separation was
accomplished using a Dionex Packing Ultimate 3000
modular LC system (Dionex, Sunnyvale, CA, USA)
consisting of a quaternary pump, vacuum degasser, ther-
mostated autosampler and column compartment. A weak
anion exchange chromatography was performed on a
Biobasic AX, 1×100 mm, 5 μm column; 20% acetonitrile
was maintained during the entire run (25 min). The initial
mobile phase consisted of 20 mM ammonium acetate. A
pH gradient was accomplished in 4 min using 2 mM
ammonium phosphate adjusted to pH 11 with ammo-
nium hydroxide. The flow rate, 100 μl/min, was increased
to 200 μl/min at the end of the run to remove any late-
eluting impurities. The autosampler temperature was
maintained at 4°C and the column temperature was main-
tained at 20°C. The retention times were 6.41 min for
ZDV-TP and 3TC-TP (IS).
A TSQ Quantum Ultra triple quadrupole mass
spectrometer (Thermo Electron Corp., Waltham, MA,
USA) was used for detection. The mass spectrometer was
operated with a spray voltage of 3.0 kV, sheath gas at 50
(arbitrary units), ion sweep gas at 0.3 (arbitrary units),
auxiliary gas at 0 (arbitrary units), and a capillary temper-
ature of 350°C. The collision cell pressure was maintained
at 1.9 mTorr. Two positive ion selected reactions were
monitored for ZDV-TP (m/z 508
m/z 81; collision
energy 20 V) and for 3TC-TP (m/z 470
m/z 112; colli-
sion energy 27 V). Thermo Xcalibur software was used to
control both the HPLC and the mass spectrometer and to
perform data analysis. Calibration curves were generated
using ZDV-TP standard serially diluted in blank PBMCs
suspensions ranging from 100 fmol/10
6
cells to
10,000 fmol/10
6
cells. The limit of quantification was
100 fmol/10
6
cells; r
2
values were 0.999.
DXG was phosphorylated in primary human PBMCs
and the major metabolite was DXG-TP. DXG-TP reached
concentrations up to 14 pmol/10
6
cells after incubation with
10 μM DXG, and the ratio of DXG to DXG-TP was ~1:3
(Figure 1). The cellular accumulation of DXG-TP was linear
and was not saturated at concentrations =30 μM (Figure 1).
The intracellular half-life (t
1/2
) of DXG-TP was
measured in PBMCs after 4 h incubation with 30 μM
DXG (Figure 2). DXG-TP reached a concentration of 29.8
±5.7 pmol/10
6
cells and declined with a half-life of 16.0
±0.6 h. This relatively long half-life suggests that prodrugs
delivering DXG are candidates for once daily dosing.
ZDV is an attractive drug for co-formulation with
DXG, as these drugs are activated by different phosphory-
lation pathways and select for different resistance muta-
tions. Furthermore, previous reports suggest that the
mutation K65R in HIV-1 reverse transcriptase, which
confers cross-resistance to DXG and DAPD, can revert
ZDV-resistant virus to ZDV sensitivity (Gu et al., 1999,
Mewshaw et al., 2002). Previous in vitro studies have also
suggested that DXG could act synergistically with ZDV,
Antiviral Chemistry & Chemotherapy 18.6
345
30
25
20
15
10
5
0
DXG-TP
DXG (10 µM) + ZDV (10 µM)
DXG (10 µM)
DXG (10 µM) + ZDV (0.1 µM)
DXG (10 µM) + ZDV (1 µM)
Intracellular concentration,
pmol/10
6
cells
1
1.2
1.4
1.6
0.8
0.4
0.6
0.2
0
ZDV-TP
Intracellular concentration,
pmol/10
6
cells
ZDV (1 µM)
ZDV (1 µM) + DXG (1 µM)
ZDV (1 µM) + DXG (10 µM)
ZDV (1 µM) + DXG (100 µM)
Figure 3. A competition study between DXG and
ZDV to measure potential drug–drug interactions at
the phosphorylation level
Nucleoside triphosphate concentrations in the competition study
between: (A) [8-
3
H]-DXG (10 μM, 500 dpm/pmol) with different
concentrations of ZDV (0.1, 1 and 10 μM) and (B) ZDV (1 μM) with
different concentrations of DXG (1, 10 and 100 μM) in human periph-
eral blood mononuclear cells at 2 h. DXG, 9-(β-
D-1,3-dioxolan-4-yl)
guanine; TP, triphosphate, ZDV, zidovudine.
A
B
Schinazi 11/2/08 14:00 Page 345
lamivudine and nevirapine (Gu et al., 1999). On the basis
of these studies, we conducted a combination study with
DXG and ZDV to measure potential drug–drug interac-
tions at the phosphorylation level. When DXG (10 μM)
was combined with different ZDV concentrations (up to
10 μM), no significant effect was noted on DXG-TP
levels (Figure 3). Similar results were observed when DXG
(concentrations up to 100 μM) were co-incubated with
ZDV (1 μM). No reduction in ZDV-TP was observed in
primary human PBMCs (Figure 3). These results were
expected given that these nucleoside analogues do not
share kinases in their phosphorylation pathways.
In summary, human PBMCs accumulated high levels of
DXG-TP, which declined. The combination study between
DXG and ZDV demonstrated no reduction in DXG-TP or
ZDV-TP formation. On the basis of these preclinical
studies, the results of a recently completed Phase I/II study
on the usefulness of DAPD with ZDV (http://clinical-
trials.gov/show/NCT00432016; Murphy R, Zala C, Ochoa
C, Tharnish P, Mathew J, Fromentin E, Asif G, Hurwitz SJ,
Kivel NM & SchinazI RF [2008] Pharmacokinetics and
potent anti-HIV-1 activity of amdoxovir plus zidovudine in
a randomized double-blind placebo-controlled study. 15th
Conference on Retroviruses and Opportunistic Infections.
Boston, MA, USA, 3–6 February 2008. Abstract J126), a
multinational Phase IIb study involving at least 60 HIV-1-
infected individuals is planned using a fixed-dose combina-
tion of DAPD with ZDV, as add-on therapy in
treatment-experienced HIV-1-infected persons.
Acknowledgements
This work was supported in part by National Institutes of
Health grants 5R37-AI-41980, 4R37-AI-025899,
1RO1-AI-071846 and 5P30-AI-50409, and by the
Department of Veterans Affairs. RFS is the inventor of
Amdoxovir and may receive future royalties from the sales
of this drug.
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346 ©2008 International Medical Press
Received 16 November 2007, accepted 15 January 2008
Schinazi 11/2/08 14:00 Page 346
  • [Show abstract] [Hide abstract] ABSTRACT: In vitro selection studies and data from large genotype databases from clinical studies have demonstrated that tenofovir disoproxil fumarate and abacavir sulfate select for the K65R mutation in the human immunodeficiency virus type 1 polymerase region. Furthermore, other novel non-thymine nucleoside reverse transcriptase (RT) inhibitors also select for this mutation in vitro. Studies performed in vitro and in humans suggest that viruses containing the K65R mutation remained susceptible to zidovudine (ZDV) and other thymine nucleoside antiretroviral agents. Therefore, ZDV could be coformulated with these agents as a "resistance repellent" agent for the K65R mutation. The approved ZDV oral dose is 300 mg twice a day (b.i.d.) and is commonly associated with bone marrow toxicity thought to be secondary to ZDV-5'-monophosphate (ZDV-MP) accumulation. A simulation study was performed in silico to optimize the ZDV dose for b.i.d. administration with K65R-selecting antiretroviral agents in virtual subjects using the population pharmacokinetic and cellular enzyme kinetic parameters of ZDV. These simulations predicted that a reduction in the ZDV dose from 300 to 200 mg b.i.d. should produce similar amounts of ZDV-5'-triphosphate (ZDV-TP) associated with antiviral efficacy (>97% overlap) and reduced plasma ZDV and cellular amounts of ZDV-MP associated with toxicity. The simulations also predicted reduced peak and trough amounts of cellular ZDV-TP after treatment with 600 mg ZDV once a day (q.d.) rather than 300 or 200 mg ZDV b.i.d., indicating that q.d. dosing with ZDV should be avoided. These in silico predictions suggest that 200 mg ZDV b.i.d. is an efficacious and safe dose that could delay the emergence of the K65R mutation.
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