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Antimicrobial Agents and Chemotherapy, August 1999, p. 2046-2050, Vol. 43, No. 8
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Hydroxyurea Enhances the Activities of Didanosine,
9-[2-(Phosphonylmethoxy)ethyl]adenine, and
9-[2-(Phosphonylmethoxy)propyl]adenine against Drug-Susceptible
and Drug-Resistant Human Immunodeficiency Virus Isolates
Sarah
Palmer,*
Robert
W.
Shafer, and
Thomas C.
Merigan
Center for AIDS Research at Stanford,
Stanford University Medical Center, Stanford, California 94305-5107
Received 30 October 1998/Returned for modification 8 March
1999/Accepted 13 May 1999
 |
ABSTRACT |
We assessed the effects of hydroxyurea (HU) at a concentration of
50 µM on the in vitro activities of 2',3'-dideoxyinosine (ddI),
9-[2-(phosphonylmethoxy)ethyl]adenine (PMEA), and
9-[2-(phosphonylmethoxy)propyl]adenine (PMPA) against a
wild-type human immunodeficiency virus (HIV) type 1 (HIV-1) laboratory
isolate and a panel of five well-characterized drug-resistant HIV
isolates. Fifty micromolar HU significantly increased the activities of
ddI, PMEA, and PMPA against both the wild-type and the drug-resistant
HIV-1 isolates. In fixed combinations, both ddI and PMEA were
synergistic with HU against wild-type and drug-resistant viruses.
| |
TEXT |
The experimental human
immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors
9-[2-(phosphonylmethoxy)ethyl]adenine (PMEA) and
9-[2-(phosphonylmethoxy)propyl]adenine (PMPA) are acyclic phosphonate analogs of AMP (3, 26, 32). Although they
already contain a single phosphate, PMEA and PMPA, like other
nucleoside analogs, rely on intracellular kinases for phosphorylation
to their active diphosphate forms (27, 28). The diphosphates of PMEA and PMPA and the triphosphate of 2',3'-dideoxyinosine (ddI)
(ddATP) compete with the cellular nucleotide dATP for the active
binding sites on the RT enzyme (1, 8). Therefore, the
antiretroviral activities of PMEA, PMPA, and ddI are dependent on two
factors: (i) the activities of intracellular phosphorylating enzymes
and (ii) the ratio of the amount of phosphorylated drug to the amount
of competing intracellular nucleoside triphosphate pools.
The anticancer agent hydroxyurea (HU) is used for the treatment of
myleoproliferative disorders (9, 34). HU is a potent inhibitor of the cellular enzyme ribonucleotide reductase, which catalyzes the reduction of ribonucleotides to deoxyribonucleotides (14). Cells exposed to HU show measurable reductions in
several deoxynucleotide pools, with the reduction of dATP pools being the most pronounced (4, 10-12, 24). These decreases in
deoxynucleotide pools effectively block cellular DNA synthesis
(4).
HU increases the anti-HIV activities of ddI and
2'-
-fluoro-2',3'-dideoxyadenosine, probably due to the favorable
shift in the ratio of adenosine drug triphosphates versus competing
cellular dATP pools which favors the binding of drug triphosphates to
RT (4, 10-13, 18, 24). Due to these promising in vitro
results, several clinical trials of ddI in combination with HU have
been initiated (5-7, 17, 35, 36).
In the present study, we investigated the effects of HU on the anti-HIV
activities of the three adenosine analogs PMEA, PMPA, and ddI. We
assessed the interaction of HU with these drugs against wild-type HIV
and versus a panel of drug-resistant HIV strains. We also analyzed the
cytotoxicity of HU alone and in combination with PMEA, PMPA, or ddI.
HIV-1 strains.
The antiviral activities of the drugs and
drug combinations were assessed against six different HIV type 1 (HIV-1) strains: a wild-type laboratory isolate (HIVNL4-3),
three recombinant isolates containing ddI resistance mutations
(HIVK65R, HIVL74V, and
HIVL74V, M184V), one molecularly constructed
multinucleoside-resistant strain
(HIVV75I, F77L,F116Y, Q151M) (15), and a
recently reported multidrug-resistant clinical isolate containing
six major RT mutations
(HIVM41L, D67N, M184V, L210W, T215Y, K219N) (30).
Sequence analysis of HIV-1 strains.
A 1.3-kb fragment of cDNA
encompassing HIV-1 protease and the first 300 codons of RT was
sequenced from each cultured supernatant as described previously
(38). Briefly, purified viral RNA (Qiagen Viral RNA
Extraction Kits Qiagen, Chatsworth, Calif.) was reverse transcribed and
amplified by PCR with the Superscript-One-Step-RT-PCR Reagent (Life
Technologies, Gaithersburg, Md.) and two primers, MAW-26 and RT21
(23). A 5-µl aliquot of the first PCR product was used for
a second-round nested PCR with primers PRO-1 (29) and RT20
(23). Approximately 70 ng of the 1.3-kb product was sequenced by dye-labelled dideoxyterminator cycle sequencing (Applied Biosystems, Foster City, Calif.). Isolate sequences were compared to
both patient plasma sequences and the consensus B sequence from the Los
Alamos HIV Sequence Database (21).
Drug susceptibility assays.
In vitro drug susceptibility
assays were performed by a modified AIDS Clinical Trials Group-U.S.
Department of Defense consensus method (virology manual for ACTG HIV
laboratories, 1997). Peripheral blood mononuclear cells (PBMCs) were
preinfected with titrated viral stocks for 4 h at 37°C in a
humidified atmosphere of 5% CO2. Each microtiter plate
well contained 100,000 preinfected PBMCs and eight serial drug
dilutions in cell media of ddI, PMEA, PMPA, 3'-azido-3'-deoxythymidine
(AZT), 2'-deoxy-3'-thiacytidine (3TC), or indinavir (IDV) in the
presence or absence of 50 µM HU. A 50 µM concentration of HU was
used since it is in the range of the average steady-state HU
concentration in serum during HIV treatment (35 to 56 µM)
(37). An 8:1 series of combinations of ddI and HU or PMEA
and HU was also analyzed. The drug dilutions were chosen to span the
50% inhibitory concentration (IC50) of each single drug
(2, 3, 25, 26, 32). The drugs were combined in fixed
clinically achievable ratios, based on the relative potencies of the
drugs, by the median-effect method of analyzing drug interactions.
Control wells containing cells and virus were coincubated on each plate.
To enable assay standardization and comparison, the 50% tissue culture
infective dose of each isolate was maintained at between 30 and 100. After a 7-day incubation at 37°C and in a humidified atmosphere of
5% CO2, viral growth was determined by a p24 antigen assay
with supernatants (Dupont Pharmaceuticals, Wilmington, Del.). The
percent inhibition of viral growth compared to the viral growth in the
control wells without drugs was calculated. Results were expressed as
the mean IC50 of four to six values obtained in two to
three different experiments per isolate.
The results for the two-drug combinations were calculated by using a
computer program that follows the median-effect principle.
The computer
constructs a median-effect plot of log fraction affected/fraction
unaffected against the log of the dose of the two separate drugs
and
the dose of the combination. A combination index (CI), which
compares
the amount of drug which gives a 50% effect when used
in combination
with that which gives a 50% effect when the drug
is used alone, is
calculated. A CI of <1 indicates synergy, and
a CI of >1 indicates
antagonism. However, in accordance with variation
in raw data, CIs of
between 0.8 and 1.2 were considered to represent
additivism.
Cytotoxicity assays.
Thymidine uptake analyses were used to
assess the effects of the drugs on cellular DNA synthesis.
Phytohemagglutinin-stimulated PBMCs were plated at 100,000 cells per
well and exposed to 3.1, 12.5, and 50 µM concentrations of ddI, PMEA,
or PMPA in the presence or absence of HU at concentrations of 25 to 500 µM. Control cells, without drugs, were coincubated on each plate and
were used for comparison when measuring inhibition of cellular DNA
synthesis by the drugs. The plates were incubated at 37°C for 7 days.
Sixteen hours prior to cell harvest, 50 µCi of
[3H]thymidine was introduced into all wells. The cells
were harvested onto preprinted filter paper with rinsings of water and
95% ethanol. After drying at 37°C, scintillant was added and the
counts on the filters were determined with a Wallac beta counter (LKB
Wallac, Turku, Finland).
The presence of 50 µM HU decreased the IC
50s of ddI in
vitro, which enhanced the anti-HIV activity of ddI against all viral
strains analyzed (Table
1). The
IC
50 of ddI for many of the ddI-resistant
viral
strains was reduced to less than the range for the wild
type in
the presence of HU. These observations are consistent
with previous
studies showing that HU at concentrations of 50
to 100 µM increased
the activity of ddI (
11,
12). Moreover,
recent
clinical studies have shown that patients who respond to
ddI and HU
therapy may harbor ddI-resistant viral strains (
7).
Further
in vitro analysis found that these resistant strains are
phenotypically
sensitive to inhibition by ddI and HU (
19). Similar
to ddI,
the IC
50s of the acyclic adenosine derivatives PMEA
and
PMPA were decreased by the presence of 50 µM HU for all
viruses
analyzed, including five drug-resistant strains:
HIV
K65R, HIV
L74V,
HIV
L74V, M184V;
HIV
V75I, F77L, F116Y, Q151M;
and
HIV
M41L, D67N, M184V, L210W, T215Y, K219N.
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TABLE 1.
Effect of HU on antiviral activities of ddI, PMEA, and
PMPA against a wild-type laboratory isolate and drug-resistant
HIV-1 strains
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|
The HU-induced reductions in IC
50s of ddI, PMEA, and PMPA
for the wild-type isolate were from 8- to >26-fold (Table
1), whereas
in parallel experiments the reduction in IC
50s of AZT, 3TC,
and
IDV for the wild type were between 2- and 5-fold (data not shown).
The HU-induced fold decrease in IC
50s of these drugs for
the wild-type
strain could be ranked as PMPA
ddI > PMEA > AZT > 3TC > IDV.
The differences of these drug
IC
50s may be attributed to the effects
of HU, a known
inhibitor of ribonucleotide reductase, upon intracellular
nucleotide
pools (
4,
10-12,
24). Cells exposed to HU experience
a
severe loss in dATP pools. After 5 days of continuous exposure
to HU,
the levels of dATP pools remain lower than those in control
cells not
exposed to HU (
11). In contrast, studies show that
natural
dTTP and dCTP pools and the thymidine and deoxycytidine
phosphorylating
enzymes are elevated in cells exposed to HU (
4,
10-12,
24).
Consequently, in HU-treated cells, the ratio of phosphorylated
adenosine analogs (ddATP, PMEA diphosphosphate, or PMPA diphosphate)
to
natural dATP may be substantially higher than the ratios of
AZT-triphosphate/dTTP or 3TC-triphosphate/dCTP. The shift of the
phosphorylated adenosine analogs/dATP ratio favors the binding
of the
analog to RT and is the probable cause of the more pronounced
effect of
HU on the anti-HIV activities of the adenosine analogs
(ddI, PMEA, and
PMPA) versus AZT and 3TC. The anti-HIV activity
of IDV is independent
of intracellular nucleotide levels, which
may explain the limited
effect of HU upon the activity of this
protease inhibitor. Furthermore,
HU at a concentration of 50 µM
was found to inhibit viral growth by
approximately 30%, or 0.3-fold;
therefore, the fold decreases in drug
IC
50s were not overly influenced
by the inherent anti-HIV
activity of
HU.
The HU-induced decreases in drug IC
50s were greatest for
the HIV
K65R, HIV
L74V, and
HIV
L74V, M184V recombinant isolates
(Table
1). Recent
studies have shown that the specific activity
is diminished for mutant
RT enzymes containing ddI-resistant mutations
including enzymes with
K65R or L74V mutations (
20,
31). The
combination of reduced
specific activity and HU-induced reduction
in cellular nucleotide pools
may cause the increased susceptibilities
of these recombinants to
inhibition by antiretroviral drugs in
the presence of
HU.
The IC
50s of HU remained relatively constant (approximately
83 µM) for wild-type and resistant viral strains. These observations
suggest that the RT gene mutations of the viral strains in this
study
have little effect on the inherent anti-HIV activity of
HU (Table
2).
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TABLE 2.
Antiviral susceptibilities of a laboratory HIV-1 isolate
and drug-resistant isolates to two-drug combinations
|
|
At clinically achievable concentrations, the combinations ddI-HU (1:8)
and PMEA-HU (1:8) synergistically inhibited the two
drug-resistant
viral strains tested, whereas the combination of
ddI-HU synergistically
inhibited the wild-type isolate at high
concentrations of drug (Fig.
1). The mechanism for the synergistic
interactions is unknown and may reflect the different modes of
action
of HU (decreasing cellular nucleotides) and the nucleoside
and
nucleotide analogs (RT inhibition) (
16,
33).
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FIG. 1.
Inhibition of HIV-1 isolates, a wild-type strain
(HIVNL4-3 [ ]) and two drug-resistant
strains (HIVV75I, F77L, F116Y, Q151M [ ] and
HIVM41L, D67N, M184V, L210W, T215Y, K219N
[ ]), by the combinations ddI-HU (A) and PMEA-HU (B). The
variations in the raw data were <20%.
|
|
Analysis of thymidine uptake assay results revealed a reduction in
cellular DNA synthesis in the presence of HU (Table
3).
Although the measurements of
thymidine uptake may be affected
by the HU-induced increase in
intracellular dTTP pools and the
extended half-life of these pools in
the presence of HU, this
observation suggests that HU has a cytostatic
effect on cells
(
4,
22).
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TABLE 3.
Effects of ddI, PMEA, and PMPA on the thymidine uptake of
PHA-stimulated PMBCs in the presence or absence HU at
designated concentrations
|
|
In conclusion, the presence of HU at low, clinically tolerated
concentrations enhances the anti-HIV activities of ddI, PMEA,
and PMPA.
This HU-induced increase in the activities of ddI, PMEA,
and PMPA
against HIV is also observed in clinical isolates that
are
resistant to one or more of these compounds. The two-drug
combinations ddI-HU and PMEA-HU synergistically inhibited
drug-resistant
viral strains. This study provides evidence that
supports the
need for clinical trials with HU (i) in combination with
ddI against
ddI-resistant patient isolates and (ii) in combination with
two
recently developed adenosine analogs, PMEA and PMPA. Moreover,
the
strategy of combining highly specific HIV inhibitors with
relatively
nonspecific inhibitors is an approach to drug resistance
that should be
tested
clinically.
| |
ACKNOWLEDGMENTS |
We thank Gilead Sciences, Foster City, Calif., for the kind gifts
of PMEA and PMPA. We especially thank Darcy Levee and Kristi Cooley for
excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Present address: Southern
Research Institute/Serquest, Department of Infectious Disease Research,
431 Aviation Way, Frederick, MD 21701-4756. Phone: (301) 694-3232. Fax:
(301) 694-7223. E-mail: palmer{at}SRI.org.
| |
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Antimicrobial Agents and Chemotherapy, August 1999, p. 2046-2050, Vol. 43, No. 8
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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