Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, February 2001, p. 577-582, Vol. 45, No. 2
Department of Pharmacology and
Therapeutics, University of Liverpool, Liverpool, United Kingdom L69
3GE1; Department of Medical
Microbiology, University of Liverpool, Liverpool, United Kingdom L69
3GA2; and Department of Infectious
Diseases and Tropical Medicine, North Manchester General Hospital,
Crumpsall, Manchester4; and Department
of Genitourinary Medicine, Royal Liverpool University Hospital,
Liverpool,3 United Kingdom
Received 11 May 2000/Returned for modification 6 September
2000/Accepted 25 October 2000
Intracellular phosphorylation of stavudine (d4T) and
zidovudine (ZDV) was investigated in peripheral blood mononuclear cells (PBMCs) isolated from ZDV-naive and ZDV-experienced human
immunodeficiency virus (HIV)-positive patients. An in vivo study
measured the amount of d4T triphosphate (d4TTP), while an ex vivo study
assessed the capacity of cells to phosphorylate added d4T. Endogenous
dTTP was also measured. d4TTP and dTTP were determined in vivo using a
reverse transcriptase chain termination assay. In ex vivo studies, d4T
(1 µM) was incubated in resting and phytohemagglutinin-stimulated (10 µg ml The sequence in which human
immunodeficiency virus (HIV) nucleoside analogues such as zidovudine
(ZDV) and stavudine (d4T) should be used has been the subject of much
debate in recent years. Preliminary observations from in vitro studies
and some small clinical studies have suggested that the intracellular
phosphorylation of these compounds may vary with duration of therapy or
prior use of another nucleoside analogue [1, 4; J. P. Sommadossi, X. Zhou, J. Moore, D. R. Havlir, G. Friedland, C. Tierney, L. Smeaton, L. Fox, D. Richman, and R. Pollard, 5th Conf.
Retrovir. Opportunistic Infect., 1998).
The intracellular phosphorylation of these drugs to their active
triphosphates is essential for anti-HIV activity. The ratio of drug
triphosphates to endogenous deoxynucleoside triphosphates (dNTPs) most
accurately reflects the direct competition between drug triphosphate
and dNTP for HIV reverse transcriptase (RT) (6, 7, 21).
Treatment failure is multifactorial. In addition to viral resistance
and lack of adherence, it has been postulated that decreased
intracellular phosphorylation may contribute to the loss of therapeutic
benefits of drugs seen over time (4, 10). A number of
factors have been implicated, including previous drug experience and
the disease stage. HIV infection may alter the capacity of peripheral
blood mononuclear cells (PBMCs) to phosphorylate drugs by reducing
thymidine kinase activity (12, 13). Prolonged use of one
nucleoside or prior exposure to other nucleoside analogues may modulate
the activity of phosphorylating enzymes. Thymidine kinase has a
600-fold-lower affinity for d4T than for thymidine and ZDV and
represents the rate-limiting step for d4T phosphorylation
(11). Thus, changes in d4T phosphorylation in
ZDV-experienced patients would be the most sensitive indicator of
ZDV-induced down-regulation of thymidine kinase, if this mechanism was
important. The ALTIPHAR study reported that patients exposed to ZDV had
decreased formation of d4T and lamivudine (3TC) triphosphates compared
to those who were ZDV naive (Sommadossi, et al., 5th Conf. Retrovir.
Opportunistic Infect.).
We investigated the intracellular phosphorylation of d4T in PBMCs from
ZDV-naive and ZDV-experienced patients. An in vivo study measured the
amount of d4T triphosphate (d4TTP) and dTTP formed, and an ex vivo
study examined the capacity of PBMCs to phosphorylate added d4T.
Materials.
Lymphoprep was purchased from Nycomed Pharma AS,
Oslo, Norway. d4T and [methyl-3H]d4T were
gifts from Bristol-Myers Squibb, Wallingford, Conn. ZDV was donated by
the MRC AIDS Research Project. [methyl-3H]ZDV,
[3H]dTTP (58 mCi mmol Subjects.
This study was carried out between May and
December 1998. Healthy volunteers and HIV-positive patients (aged 20 to
60 years; CD4 counts, 12 × 106 to 640 × 106/liter) were recruited. All patients were receiving
antiretroviral therapy that included d4T; patients were excluded if
their current regimens included either ZDV or hydroxyurea. Approval for
the study was obtained (from the ethics committees of the Royal
Liverpool University Hospital and North Manchester Health Authority),
and each participant provided written informed consent.
Blood sampling.
A single blood sample (25 ml) was obtained
by venipuncture at different times after dosing (range, 0 to 12 h).
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.2.577-582.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Influence of Prior Exposure to Zidovudine on
Stavudine Phosphorylation In Vivo and Ex Vivo
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1; 72 h) PBMCs for 24 h. After washing
and methanol extraction, radiolabeled anabolites were detected by
high-performance liquid chromatography. d4TTP reached its highest level
2 to 4 h after dosing (0.21 ± 0.14 pmol/106
cells; n = 27 [mean ± standard deviation]).
Comparison of ZDV-naive and ZDV-experienced individuals showed no
significant difference in levels of d4TTP (ZDV naive, 0.23 ± 0.17 pmol/106 cells [n = 7] versus ZDV
experienced, 0.20 ± 0.14 pmol/106 cells
[n = 20]; P = 0.473) or the
d4TTP/dTTP ratio (0.14 ± 0.12 [n = 7] and
0.10 ± 0.08 [n = 20], respectively;
p = 0.391). Ex vivo data demonstrated no significant
difference in the formation of d4TTP or total d4T phosphates in naive
and experienced patients (0.086 ± 0.055 pmol/106
cells in ZDV-naive patients [n = 17] versus
0.081 ± 0.038 pmol/106 cells in ZDV-experienced
patients [n = 22]; P = 0.767). The
ability of HIV-infected patients to phosphorylate d4T in vivo and ex
vivo was unchanged with increasing exposure to ZDV.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1), and
[3H]dATP (18 mCi mmol1) were purchased from
Moravek Biochemicals, Brea, Calif. d4TTP was synthesized by Sierra
Biotech) Poly(rA) · oligo(dTP)12-18 was purchased from
Pharmacia Biotech. Sequenase version 2.0 DNA polymerase and
oligonucleotides (19) were purchased from United States
Biochemical (Cleveland, Ohio). HIV RT was obtained from Amersham,
Little Chalfont, United Kingdom. All other drugs and chemicals were
purchased from Sigma Chemical Company Ltd. 3H-nucleoside
analogues were of 
97% purity.
dTTP assay. Intracellular dTTP pools were measured by an enzymatic assay adapted from that of Sherman and Fyfe (19) as previously described (20).
Determination of the d4TTP standard inhibition curve. In vivo d4TTP concentrations were determined by using an RT assay similar to that previously described for the quantification of ZDV triphosphate (ZDVTP) (16, 17). A total volume of 50 µl of the reaction mixture contained the following final concentrations: 50 mM Tris HCl (pH 7.8 to 8.5), 1 mM dithiothreitol, 6 mM MgCl2, 30 mM KCl, 0.1% Triton X-100, 2 mM CuSO4, 0.025% bovine serum albumin; 0.5 mM EDTA, 4 pmol (0.3 µCi) of [3H]dTTP, 0.002 µg of poly(rA) · oligo(dT)12-18, 5 µl of cell extract (containing a known amount of dTTP), and either 0, 0.025, 0.05, 0.075, or 0.1 pmol of d4TTP. d4TTP in PBMCs from HIV patients was determined from 10-µl extracts.
Initially, d4TTP, [3H]dTTP, cell extract, Tris-HCl, and KCl were incubated with CuSO4 at room temperature for 20 min to inactivate any RNase present in the extract. Bovine serum albumin and EDTA were then added, and the mixture was incubated for a further 15 min to remove excess CuSO4. The template primer was included prior to initiation of the reaction by the addition of 0.4 U of HIV RT, which was then incubated in a water bath (37°C) for 60 min. The reaction mixtures (20 µl) were spotted onto filter paper (presoaked with 5% trichloroacetic acid plus 1% sodium pyrophosphate solution). Following heat drying, the filter papers were washed three times with the same cold solution (10 ml; 5 min) and finished by washing them twice with 95% ethanol (vol/vol; 3 ml). After being heat dried, the filter papers were transferred to scintillation vials and the radioactivity was counted.Incubation of PBMCs with 3H-nucleoside analogues ex
vivo.
Freshly isolated PBMCs were stimulated with
phytohemagglutinin (PHA) (10 µg ml1) and cultured for
72 h in RPMI medium supplemented with 10% fetal calf serum (4 ml)
and L-glutamine (2 mM) at 37°C in an incubator with
humidified 5% CO2. Freshly isolated nondividing resting
PBMCs and PHA-stimulated PBMCs were then incubated with d4T (1 µM; 5 µCi) for 24 h at 37°C in an incubator with humidified 5%
CO2. Experiments were performed in triplicate.
20°C until they
were analyzed by high-performance liquid chromatography (HPLC).
The coefficient of variation for repeated analysis of ZDV and d4T
phosphates extracted from the same sample was less than 10%. The
extent of and variability in the phosphorylation of ZDV and d4T were
similar to those in previous reports (2, 3, 18). In
resting cells, total phosphates of ZDV, d4T, and thymidine were always
detectable following a 24-h incubation. The limit of quantification of
individual phosphate anabolites for d4T and thymidine was 0.003 pmol/106 cells, and for ZDV the limit was 0.0003 pmol/106 cells, corresponding to a peak height of three
times the baseline.
HPLC analysis. Cell extracts were reconstituted in double-distilled H2O and separated by HPLC on an anion-exchange column (Partisil 10-SAX; 25 cm by 4.6 mm) as previously described (2, 3). Samples were eluted with increasing buffer concentrations, and phosphate metabolites were identified by cochromatography with authentic standards or occasionally by their elution order as previously established (2, 3). The eluent was collected in insert vials at 30-s collection times. Scintillant (4 ml) was added to the collected fractions, which were then counted in a liquid scintillation counter.
Statistical analysis. Data from resting and PHA-stimulated PBMCs were standardized to picomoles per million cells. In resting cells, the results were calculated using cell counts taken at the conclusion of the drug incubations. However, since PHA causes clumping, which prevents hemocytometer cell counting at the end of the incubations, cell counts prior to mitogen stimulation were used in these incubations. d4TTP data obtained in vivo were analyzed by an unpaired t test. d4TTP data obtained ex vivo were analyzed by the Mann-Whitney U test.
Multiple linear regression followed by analysis of variance was performed to assess the dependence of d4TTP on time on and time since ZDV therapy both in vivo and ex vivo. This analysis was also used to investigate the dependence of total d4T phosphates on time on and time since ZDV therapy ex vivo. Simple linear regression analysis was used (see Fig. 2 and 3).| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Validation of d4T assay. The recovery of known amounts of d4TTP in the presence of cell extracts varied between 97 and 109%. Consistent inhibitory curves with high reproducibility were obtained with correlation coefficients (r2) greater than 0.98. The lower limit of detection was 0.025 pmol. The coefficient of variation for repeated analysis of d4TTP from the same extract was less than 10%. The observed values are similar to those previously reported using LCMS (Sommadossi et al., 5th Conf. Retrovir. Opportunistic Infect.). Intracellular levels of d4TTP, like the triphosphates of the other nucleoside analogues, show high inter- and intraindividual variation (18, 20; D. J. Back, P. G. Hoggard, S. E. Gibbons, J. Lloyd, M. G. Barry, S. H. Khoo, and C. Loveday, 12th World AIDS Conf., abstr. 42260, 1998; Sommadossi et al., 5th Conf. Retrovir. Opportunistic Infect.).
d4TTP in PBMCs from HIV-infected individuals.
d4TTP
concentrations were highest 2 to 4 h after dosing (0.21 ± 0.14 pmol/106 cells; n = 27) (Fig.
1) and were still detectable at 12 h
postdose in the one patient analyzed. Comparison of d4TTP
concentrations (2 to 4 h postdose) in ZDV-naive and
ZDV-experienced individuals showed no significant difference (ZDV
naive, 0.23 ± 0.17 pmol/106 cells [n = 7], versus ZDV experienced, 0.20 ± 0.14 pmol/106 cells [n = 20]; P = 0.473).
Similarly, there was no significant difference in dTTP levels (ZDV
naive, 1.91 ± 0.90 pmol/106 cells [n = 7] versus ZDV experienced, 2.73 ± 1.81 pmol/106
cells [n = 20]; P = 0.761) or the d4TTP/dTTP
ratio in ZDV-naive and ZDV-experienced individuals (0.14 ± 0.12 [n = 7] and 0.10 ± 0.08 [n = 20], respectively; P = 0.391).
|
Incubation of PBMCs with 3H-nucleoside analogues.
Total intracellular phosphate formation in resting and 72-h
PHA-stimulated PBMCs is shown in Table 1.
Following cell activation, significant increases were seen in the total
phosphorylation of d4T (1 µM), ZDV (0.1 µM), and thymidine (1 µM)
in PBMCs isolated from both healthy volunteers and HIV patients.
However, there was marked intersubject variation. The extent of total
phosphorylation of ZDV and d4T was lower in resting PBMCs from healthy
volunteers than in those from HIV patients. Formation of d4TTP was also
lower in resting PBMCs from healthy volunteers (Table 1). Following PHA
activation, the total phosphorylation of thymidine and ZDV was
significantly greater in healthy volunteers than in HIV patients (Table
1).
|
d4T phosphorylation in resting HIV-infected PBMCs. d4T phosphorylation in resting PBMCs from HIV patients was further investigated to assess whether prior experience of ZDV reduced anabolite formation. However, no significant difference was seen in total intracellular d4T phosphates in cells isolated from ZDV-naive and ZDV-experienced HIV patients (0.086 ± 0.055 pmol/106 cells in ZDV-naive patients [n = 17] versus 0.081 ± 0.038 pmol/106 cells in ZDV-experienced patients [n = 22]; P = 0.767). Similarly, there was no significant difference between the d4TTP concentrations in these two groups (0.008 ± 0.005 pmol/106 cells in ZDV-naive patients [n = 17] versus 0.008 ± 0.005 pmol/106 cells in ZDV-experienced patients [n = 22]; P = 0.597).
Effect of ZDV experience on d4T phosphorylation in vivo and ex
vivo.
The effect on d4T phosphorylation of the length of time that
ZDV-experienced patients had been on a combination regimen including ZDV is illustrated for the in vivo (Fig.
2a) and ex vivo (Fig. 2b) studies. In
both instances, there was no significant change in d4TTP with either
time on ZDV therapy or time since ZDV therapy (in vivo multiple
correlation coefficient (r2) = 0.0016, P = 0.997; ex vivo r2 = 0.0126, P = 0.875). Figures 2 and 3 illustrate
the relationships between d4TTP and these parameters. The ex vivo data
also demonstrated that there was no correlation between total d4T
phosphates and either time on ZDV or time since ZDV therapy was last
administered (multiple correlation coefficient
r2 = 0.0255; P = 0.703).
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Since there has been considerable concern surrounding the correct sequence for the use of nucleoside analogues, we designed this study to look in the clinical setting for differences in intracellular phosphorylation of d4T in ZDV-naive patients as well as those who had previously received ZDV for various amounts of time. In vivo assessment of PBMCs from patients receiving d4T revealed no differences in formation of d4TTP between ZDV-naive and ZDV-experienced patients. In addition, no difference was observed in the amount of endogenous dTTP or the ratio of intracellular d4TTP to dTTP. Furthermore, there was no relationship between the amount of d4TTP or dTTP formed and either the prior duration of ZDV treatment or the time elapsed since ZDV was discontinued. As patients switched from drug therapy combinations including ZDV to d4T, as shown in Fig. 3, the relationship between the time since ZDV therapy and the d4TTP level demonstrates that the effect of time on d4T also does not affect d4TTP formation.
The ex vivo study examined the capacity of PBMCs to phosphorylate d4T. In agreement with the in vivo study, there was no difference in the capacities of resting and PHA-stimulated cells from ZDV-naive or ZDV-experienced patients to phosphorylate d4T. Similarly, no relationship was observed between d4T phosphorylation and the length of time on ZDV or the interval since ZDV was discontinued.
In the ALTIS trial (14), treatment-naive patients receiving a combination of d4T plus 3TC experienced a 1.66-log-unit drop in virus load at 24 weeks compared to a 0.55-log-unit drop in ZDV-experienced patients. Since neither population had previously received d4T or 3TC and sequencing did not reveal significant differences in virological resistance patterns, it was suggested that prior exposure to ZDV reduced the potency of the new combination of nucleoside analogues. ALTIPHAR (the pharmacological arm of ALTIS [Sommadossi et al., 5th Conf. Retrovir. Opportunistic Infect.]) reported significantly decreased formation of d4T and 3TC triphosphates in a subgroup of patients who were both ZDV experienced and nonresponders (<1-log-unit drop in virus load; n = 6) compared with another subgroup of treatment-naive patients who were responders (>1-log-unit drop in virus load; n = 10). This gave rise to the speculation that prior ZDV treatment may impair subsequent d4T and 3TC phosphorylation. There are alternative explanations for this finding, e.g., the large inter- and intrapatient variability seen in d4T phosphorylation (18, 20; Back et al., 12th World AIDS Conf., Sommadossi et al., 5th Conf. Retrovir. Opportunistic Infect.). Also, concentrating on a group of nonresponders who had prior ZDV therapy may have selected a group of individuals who are poor metabolizers of nucleoside analogues and for whom treatment is thus at increased risk of failing. Preliminary data suggest a correlation between ZDV and 3TC phosphorylation (C. V. Fletcher, S. P. Kawle, D. Weller, T. N. Kakuda, P. L. Anderson, L. Bushman, R. C. Brundage, and R. P. Remmel, Abstr. 7th Conf. Retrovir. Opportunistic Infect., abstr. 94, 2000) and that the amount of ZDV or 3TC triphosphate is related to the rise in CD4 cells and decline in HIV RNA following treatment (C. Fletcher, Program Abstr. First Int. Workshop Clin. Pharmacol HIV Ther., abstr. 6.2, 2000).
In agreement with other studies (8, 9, 15), the formation of ZDV, d4T, and thymidine triphosphates was enhanced with PHA stimulation. This effect was more marked in healthy volunteers than in HIV-positive patients. However, we found that in resting, unstimulated cells, healthy volunteers had lower levels of endogenous nucleotides compared to HIV-positive patients. Several factors may account for these findings. For example, PBMCs from HIV-positive patients may already have high levels of basal activation or may demonstrate a degree of anergy to mitogen stimulation (5). The relative proportions of different cell populations that make up PBMCs also vary with HIV disease, and these subpopulations of cells may respond differently to PHA stimulation. Some of these factors may contribute to the apparently lower levels of thymidine kinase activity seen in PHA-stimulated PBMCs from HIV-infected patients.
Our data highlight the pitfalls of using PHA stimulation when assessing intracellular drug phosphorylation in HIV-positive patients. Moreover, in vitro data from cell lines chronically infected with HIV which suggest reduced phosphorylation with increasing drug exposure (1) are not confirmed. We would caution against extrapolating the findings of such studies to HIV-positive patients and stress the importance of using resting, unstimulated PBMCs in future clinical studies assessing drug phosphorylation.
We have clearly demonstrated that prior use of ZDV does not adversely affect the phosphorylation of d4T, both as a measured amount of drug triphosphate within cells and in regard to the capacity of these cells to phosphorylate added d4T. The choice of whether to use d4T or ZDV when initiating therapy should be based upon the likelihood of toxicity in the patient and the risk of primary drug resistance or subsequently generating cross-resistance to other nucleoside analogues rather than on drug phosphorylation.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Pharmacology and Therapeutics, University of Liverpool, New Medical Building, Ashton St., Liverpool, United Kingdom L69 3GE. Phone: 0151 794-5565. Fax: 0151 794-5540. E-mail: patrick{at}liv.ac.uk.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Avramis, I. A., R. Kwock, M. M. Solorzano, and E. Gomperts. 1993. Evidence of in vitro development of drug resistance to azidothymidine in T-lymphocytic leukemia cell lines (Jurkat E6-1/ZAT 100) and in pediatric patients with HIV infection. J. Acquir. Immune Defic. Syndr. 6:1283-1287. |
| 2. | Barry, M., M. Wild, G. Veal, D. Back, A. Breckenridge, N. Fox, N. Beeching, F. Nye, P. Carey, and D. Timmins. 1994. Zidovudine phosphorylation in HIV-infected patients and seronegative volunteers. AIDS 8:F1-F5[Medline]. |
| 3. | Barry, M., S. Khoo, G. Veal, P. Hoggard, S. Gibbons, E. Wilkins, O. Williams, A. Breckenridge, and D. J. Back. 1996. The effect of zidovudine dose on the formation of intracellular phosphorylated metabolites. AIDS 10:1361-1367[Medline]. |
| 4. | Cinatl, J. R., Jr., J. Cinatl, H. Rabenau, H. W. Doerr, and B. Weber. 1994. Failure of antiretroviral therapy: role of viral and cellular factors. Intervirology 37:307-314[Medline]. |
| 5. | Clerici, M., N. I. Stocks, R. A. Zajac, N. Boswell, D. R. Lucey, C. S. Via, and G. Shearer. 1989. Detection of three distinct patterns of T helper cell dysfunction in asymptomatic, human immunodeficiency virus-seropositive patients. J. Clin. Investig. 84:1892-1899. |
| 6. | De Clercq, E. 1992. HIV inhibitors targeted at the reverse transcriptase. AIDS Res. Hum. Retrovir. 8:119-134[Medline]. |
| 7. |
Furman, P. A.,
J. A. Fyfe,
M. H. St. Clair,
K. Weinhold,
J. L. Rideout,
G. A. Freeman,
S. Nusinoff Lehrman,
D. Bolognesi,
S. Broder,
H. Mitsuya, and D. W. Barry.
1986.
Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase.
Proc. Natl. Acad. Sci. USA
83:8333-8337 |
| 8. |
Gao, W.-Y.,
R. Agbaria,
J. S. Driscoll, and H. Mitsuya.
1994.
Divergent anti-human immunodeficiency virus activity and anabolic phosphorylation of 2',3'-dideoxynucleoside analogs in resting and activated human cells.
J. Biol. Chem.
269:12633-12638 |
| 9. | Gao, W.-Y., T. Shirasaka, D. G. Johns, S. Broder, and H. Mitsuya. 1993. Differential phosphorylation of azidothymidine, dideoxycytidine and dideoxyinosine in resting and activated peripheral blood mononuclear cells. J. Clin. Investig. 91:2326-2333. |
| 10. | Groschel, B., J. Cinatl, and J. Cinatl, Jr. 1994. Viral and cellular factors for resistance against antiretroviral agents. Intervirology 40:400-407. |
| 11. |
Ho, H.-T., and M. J. M. Hitchcock.
1989.
Cellular pharmacology of 2',3'-dideoxy-2',3'-didehydrothymidine, a nucleoside analog active against human immunodeficiency virus.
Antimicrob. Agents Chemother.
33:844-849 |
| 12. | Jacobsson, B., S. Britton, Q. He, A. Karlsson, and S. Eriksson. 1995. Decreased thymidine kinase levels in peripheral blood cells from HIV-seropositive individuals: implications for zidovudine metabolism. AIDS Res. Hum. Retrovir. 11:805-811[Medline]. |
| 13. | Jacobsson, B., S. Britton, Y. Tornevik, and S. Eriksson. 1998. Decrease in thymidylate kinase activity in peripheral blood mononuclear cells from HIV-infected individuals. Biochem. Pharmacol. 56:389-395[CrossRef][Medline]. |
| 14. |
Katlama, C.,
M. A. Valantin,
S. Matheron,
A. Coutelier,
V. Calvez,
D. Descamps,
C. Longuet,
M. Bonmarchand,
R. Tubiana,
T. De Sa,
R. Lancar,
H. Agut,
F. Brun-Vezinet, and D. Costagliola.
1998.
Efficacy and tolerability of stavudine plus lamivudine in treatment-naive and treatment-experienced patients with HIV-1 infection.
Ann. Intern. Med.
129:525-531 |
| 15. | Münch-Petersen, B., G. Tyrsted, and B. Dupont. 1973. The deoxyribonucleoside-5'-triphosphate (dATP and dTTP) pool in phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Exp. Cell Res. 79:249-256[CrossRef][Medline]. |
| 16. | Phiboonbanakit, D. A., M. G. Barry, and D. J. Back. 1998. Quantification of intracellular zidovudine triphosphate by enzymatic assay. Br. J. Clin. Pharmacol. 46:294P. |
| 17. |
Robbins, B. L.,
J. Rodman,
C. McDonald,
R. V. Srinivas,
P. M. Flynn, and A. Fridland.
1994.
Enzymatic assay for measurement of zidovudine triphosphate in peripheral blood mononuclear cells.
Antimicrob. Agents Chemother.
38:115-121 |
| 18. | Rodman, J. H., B. Robbins, P. M. Flynn, and A. Fridland. 1996. A systematic and cellular model for zidovudine plasma concentrations and intracellular phosphorylation in patients. J. Infect. Dis. 174:490-499[Medline]. |
| 19. | Sherman, P. A., and J. A. Fyfe. 1989. Enzymatic assay for deoxynucleoside triphosphates using synthetic oligonucleotides as template primers. Anal. Biochem. 180:222-226[CrossRef][Medline]. |
| 20. | Sim, S. M., P. G. Hoggard, S. D. Sales, D. Phiboonbanakit, C. A. Hart, and D. J. Back. 1998. Effect of ribavirin on zidovudine efficacy and toxicity in vitro: a concentration dependent interaction. AIDS Res. Hum. Retrovir. 14:1661-1666[Medline]. |
| 21. |
St. Clair, M. H.,
C. A. Richards,
T. Spector,
K. J. Weinhold,
M. H. Miller,
A. J. Langlois, and P. A. Furman.
1987.
3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase.
Antimicrob. Agents Chemother.
31:1972-1977 |
Antimicrobial Agents and Chemotherapy, February 2001, p. 577-582, Vol. 45, No. 2
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.2.577-582.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Durand-Gasselin, L., Pruvost, A., Dehee, A., Vaudre, G., Tabone, M.-D., Grassi, J., Leverger, G., Garbarg-Chenon, A., Benech, H., Dollfus, C.
(2008). High Levels of Zidovudine (AZT) and Its Intracellular Phosphate Metabolites in AZT- and AZT-Lamivudine-Treated Newborns of Human Immunodeficiency Virus-Infected Mothers. Antimicrob. Agents Chemother.
52: 2555-2563
[Abstract] [Full Text] -
Rodriguez-Torres, M., Torriani, F. J., Soriano, V., Borucki, M. J., Lissen, E., Sulkowski, M., Dieterich, D., Wang, K., Gries, J.-M., Hoggard, P. G., Back, D., for the APRICOT Study Group,
(2005). Effect of Ribavirin on Intracellular and Plasma Pharmacokinetics of Nucleoside Reverse Transcriptase Inhibitors in Patients with Human Immunodeficiency Virus-Hepatitis C Virus Coinfection: Results of a Randomized Clinical Study. Antimicrob. Agents Chemother.
49: 3997-4008
[Abstract] [Full Text] -
Joly, V., Flandre, P., Meiffredy, V., Brun-Vezinet, F., Gastaut, J.-A., Goujard, C., Remy, G., Descamps, D., Ruffault, A., Certain, A., Aboulker, J.-P., Yeni, P.
(2002). Efficacy of Zidovudine Compared to Stavudine, Both in Combination with Lamivudine and Indinavir, in Human Immunodeficiency Virus-Infected Nucleoside-Experienced Patients with No Prior Exposure to Lamivudine, Stavudine, or Protease Inhibitors (Novavir Trial). Antimicrob. Agents Chemother.
46: 1906-1913
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»