Intracellular pharmacokinetics of once versus twice daily zidovudine and lamivudine in adolescents.
ABSTRACT Zidovudine (ZDV) and lamivudine (3TC) metabolism to triphosphates (TP) is necessary for antiviral activity. The aims of this study were to compare ZDV-TP and 3TC-TP concentrations in adolescents receiving twice daily (BID) and once daily (QD) regimens and to determine the metabolite concentrations of ZDV and 3TC during chronic therapy on a QD regimen. Human immunodeficiency virus-infected patients (12 to 24 years) taking ZDV (600 mg/day) and 3TC (300 mg/day) as part of a highly active antiretroviral therapy regimen received QD and BID regimens of ZDV and 3TC for 7 to 14 days in a crossover design. Serial blood samples were obtained over 24 h on the QD regimen. Intracellular mono-, di-, and triphosphates for ZDV and 3TC were measured. The median ratio of BID/QD for ZDV-TP predose concentrations was 1.28 (95% confidence interval [CI] = 1.00 to 2.45) and for 3TC-TP was 1.12 (95% CI = 0.81 to 1.96). The typical population estimated half-lives (+/- the standard error of the mean) were 9.1 +/- 0.859 h for ZDV-TP and 17.7 +/- 2.8 h for 3TC-TP. Most patients had detectable levels of the TP of ZDV (24 of 27) and 3TC (24 of 25) 24 h after dosing, and half-lives on a QD regimen were similar to previously reported values when the drugs were given BID. Lower, but not significantly different, concentrations of ZDV-TP were demonstrated in the QD regimen compared to the BID regimen (P = 0.056). Although findings were similar between the BID and QD groups, the lower concentrations of ZDV and the number of patients below the level of detection after 24 h suggests that ZDV should continue to be administered BID.
- SourceAvailable from: Jean-Marc Tréluyer[Show abstract] [Hide abstract]
ABSTRACT: The population pharmacokinetic parameters of zidovudine (AZT), lamivudine (3TC), and their active intracellular metabolites in 75 naïve HIV-infected patients receiving an oral combination of AZT and 3TC twice daily as part of their multitherapy treatment in the COPHAR2-ANRS 111 trial are described. Four blood samples per patient were taken after 2 weeks of treatment to measure drug concentrations at steady state. Plasma AZT and 3TC concentrations were measured in 73 patients, and among those, 62 patients had measurable intracellular AZT-TP and 3TC-TP concentrations. For each drug, a joint population pharmacokinetic model was developed and we investigated the influence of different covariates. We then studied correlations between the mean plasma and intracellular concentrations of each drug. A one-compartment model with first-order absorption and elimination best described the plasma AZT concentration, with an additional compartment for intracellular AZT-TP. A similar model but with zero-order absorption was found to adequately described concentrations of 3TC and its metabolite 3TC-TP. The half-lives of AZT and 3TC were 0.81 h (94.8%) and 2.97 h (39.2%), respectively, whereas the intracellular half-lives of AZT-TP and 3TC-TP were 10.73 h (69%) and 21.16 h (44%), respectively. We found particularly a gender effect on the apparent bioavailability of AZT, as well as on the mean plasma and intracellular concentrations of AZT, which were significantly higher in females than in males. Relationships between mean plasma drug and intracellular metabolite concentrations were also highlighted both for AZT and for 3TC. Simulation with the model of plasma and intracellular concentrations for once- versus twice-daily regimens suggested that a daily dosing regimen with double doses could be appropriate.Antimicrobial Agents and Chemotherapy 07/2011; 55(7):3423-31. · 4.57 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: There is interest in evaluating the efficacy of lower doses of certain antiretrovirals for clinical care. We determined here the bioequivalence of plasma lamivudine (3TC) and intracellular 3TC-triphosphate (3TC-TP) concentrations after the administration of two different doses. ENCORE 2 was a randomized crossover study. Subjects received 3TC at 300 and 150 mg once daily for 10 days (arm 1; n = 13) or vice versa (arm 2; n = 11), separated by a 10-day washout. Pharmacokinetic (PK) profiles (0 to 24 h) were assessed on days 10 and 30. Plasma 3TC and 3TC-TP levels in peripheral blood mononuclear cells were quantified by high-performance liquid chromatography-tandem mass spectrometry. Within-subject changes in PK parameters (the area under the concentration-time curve from 0 to 24 h [AUC(0-24)], the trough concentration of drug in plasma at 24 h [C(24)], and the maximum concentration of drug in plasma [C(max)]) were evaluated by determining the geometric mean ratios (GMRs) adjusted for study arm, period, and intra-individual variation. Regimens were considered bioequivalent if the 90% confidence interval (90% CI) fell within the range of 0.8 to 1.25. A total of 24 subjects completed the study. The GM (90% CI) 3TC AUC(0-24)), expressed as ng·h/ml, for the 300- and 150-mg doses were 8,354 (7,609 to 9,172) and 4,773 (4,408 to 5,169), respectively. Bioequivalence in 3TC PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC(0-24), C(24), and C(max) were 0.57 (0.55 to 0.60), 0.63 (0.59 to 0.67), and 0.56 (0.53 to 0.60), respectively. The GM (90% CI) 3TC-TP AUC(0-24) values (pmol·h/10(6) cells) for the 300- and 150-mg doses were 59.5 (51.8 to 68.3) and 44.0 (38.0 to 51.0), respectively. Bioequivalence in 3TC-TP PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC(0-24), C(24), and C(max) were 0.73 (0.64 to 0.83), 0.82 (0.68 to 0.99), and 0.70 (0.61 to 0.82), respectively. We found that 3TC at 150 mg is not bioequivalent to the standard regimen of 300 mg, indicating that saturation of cytosine phosphorylation pathways is not achieved at a dose of 150 mg.Antimicrobial Agents and Chemotherapy 12/2011; 56(3):1427-33. · 4.57 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: To investigate the utility of metrics of CYP1A2 activity using caffeine as a probe, and saliva and plasma sampling with or without a 24-h caffeine abstinence. This was a cross-over pharmacokinetic study in 30 healthy male subjects who received a single oral 100mg caffeine dose after 24-h caffeine abstinence or after maintaining their regular caffeine intake (no caffeine abstinence). Serial blood and saliva samples were collected simultaneously over 24h. Caffeine and paraxanthine concentrations were measured using a validated HPLC assay. There was a strong correlation between the paraxanthine/caffeine AUC(0-24) ratio (reference metric) and the paraxanthine/caffeine concentration (C(t) ) ratio at 4h (C(4) ) in both saliva and plasma (r≥0.75). The paraxanthine/caffeine AUC(0-24) ratio in plasma and saliva did not differ between the 24-h caffeine abstinence and the no abstinence period (P>0.05). The optimal paraxanthine/caffeine C(t) that correlated with the plasma paraxanthine/caffeine AUC(0-24) ratio in the 24-h abstinence period was 2 and 4h (r=0.88) in plasma, and 4 and 6h in saliva (r=0.70), while it was the saliva 4h time-point in the no abstinence period (r=0.78). The saliva paraxanthine/caffeine concentration ratio at 4h was a suitable metric to assess CYP1A2 activity after oral administration of caffeine without the need for 24-h caffeine abstinence.The Journal of pharmacy and pharmacology. 09/2011; 63(9):1161-8.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2007, p. 3516–3522
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 51, No. 10
Intracellular Pharmacokinetics of Once versus Twice Daily Zidovudine
and Lamivudine in Adolescents?‡
Patricia M. Flynn,1,2*† John Rodman,3† Jane C. Lindsey,4Brian Robbins,1
Edmund Capparelli,5Katherine M. Knapp,1,2Jose F. Rodriguez,6,7
James McNamara,8Leslie Serchuck,9Barbara Heckman,10
Jaime Martinez,11and the PACTG P1012 Team
Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee1; Department of Pediatrics, University of
Tennessee Health Science Center, Memphis, Tennessee2; Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital,
Memphis, Tennessee3; Statistical & Data Analysis Center, Harvard School of Public Health, Boston, Massachusetts4;
Pediatric Pharmacology Research Unit, University of California, San Diego, California5; Department of Biochemistry,
University of Puerto Rico, San Juan, Puerto Rico6; Puerto Rico Forensic Sciences Institute, San Juan, Puerto Rico7;
National Institute of Allergy and Infectious Diseases8and National Institute of Child Health and Development,9
National Institutes of Health, Bethesda, Maryland; Frontier Science & Technology Research Foundation,
Amherst, New York10; and Department of Pediatrics, Stroger Hospital of
Cook County/CORE Center, Chicago, Illinois11
Received 29 December 2006/Returned for modification 15 February 2007/Accepted 19 July 2007
Zidovudine (ZDV) and lamivudine (3TC) metabolism to triphosphates (TP) is necessary for antiviral
activity. The aims of this study were to compare ZDV-TP and 3TC-TP concentrations in adolescents receiving
twice daily (BID) and once daily (QD) regimens and to determine the metabolite concentrations of ZDV and
3TC during chronic therapy on a QD regimen. Human immunodeficiency virus-infected patients (12 to 24
years) taking ZDV (600 mg/day) and 3TC (300 mg/day) as part of a highly active antiretroviral therapy regimen
received QD and BID regimens of ZDV and 3TC for 7 to 14 days in a crossover design. Serial blood samples
were obtained over 24 h on the QD regimen. Intracellular mono-, di-, and triphosphates for ZDV and 3TC were
measured. The median ratio of BID/QD for ZDV-TP predose concentrations was 1.28 (95% confidence interval
[CI] ? 1.00 to 2.45) and for 3TC-TP was 1.12 (95% CI ? 0.81 to 1.96). The typical population estimated
half-lives (? the standard error of the mean) were 9.1 ? 0.859 h for ZDV-TP and 17.7 ? 2.8 h for 3TC-TP. Most
patients had detectable levels of the TP of ZDV (24 of 27) and 3TC (24 of 25) 24 h after dosing, and half-lives
on a QD regimen were similar to previously reported values when the drugs were given BID. Lower, but not
significantly different, concentrations of ZDV-TP were demonstrated in the QD regimen compared to the BID
regimen (P ? 0.056). Although findings were similar between the BID and QD groups, the lower concentrations
of ZDV and the number of patients below the level of detection after 24 h suggests that ZDV should continue
to be administered BID.
Since its introduction, zidovudine (ZDV) treatment has un-
dergone many changes in dosage and frequency of administra-
tion. Initially, the adult recommended dosage was 250 mg
every 4 h for a total daily dose of 1,500 mg (16, 24). Both the
total daily dose and the frequency have decreased, and the
current recommended adult dosage is 600 mg daily, adminis-
tered as 300 mg twice daily (BID) (5, 23). Initial dosing regi-
mens were based on the short half-life, approximately 1 h, of
ZDV in plasma (4, 12, 20). Subsequent clinical trials have
demonstrated that lower total daily doses of ZDV are less toxic
than higher doses with no apparent decrease in efficacy (25–
27). The reason for this is that ZDV, like all of the nucleoside
reverse transcriptase inhibitors (NRTIs), undergoes a series of
three sequential phosphorylation reactions producing mono-
phosphates (MP), diphosphates (DP), and triphosphates (TP)
within the cell. The TP is the active metabolite of both ZDV
and lamivudine (3TC), another NRTI.
Recent studies of the intracellular metabolism of ZDV in
subjects have demonstrated that ZDV-TP is present within the
cell for an extended period of time, which suggests that anti-
viral activity will be present with less frequent dosing (1, 2, 8,
15, 19, 20, 21). Likewise, 3TC is given on a twice-daily schedule
based in part on a systemic half-life of 5 to 7 h in adults and
children and a longer half-life for 3TC-TP (11, 14, 21). The
adult and pediatric dosage schedules are supported by clinical
studies demonstrating efficacy and safety (7, 13). Similar to
changes in the ZDV dosing schedule, 3TC has been approved
for once daily (QD) administration. This change was based on
studies demonstrating that 300 mg once daily produced a sim-
ilar systemic area under the concentration-time curve (AUC)
to 150 mg twice daily (28).
An adolescent living with human immunodeficiency virus
(HIV) infection is a challenge to manage and responses to
highly active antiretroviral therapy (HAART) are not as suc-
cessful as in adult trials. In a trial of initial HAART in ado-
lescents, only 59% had achieved and maintained a viral load
* Corresponding author. Mailing address: Department of Infectious
Diseases, St. Jude Children’s Research Hospital, 332 N. Lauderdale
St., Memphis, TN 38105. Phone: (901) 495-2338. Fax: (901) 495-5068.
† P.M.F. and J.R. contributed equally to this study.
‡ This study is presented in honor of John Rodman, who passed
away in April 2006.
?Published ahead of print on 30 July 2007.
?400 copies/ml after 24 weeks of therapy (9). Adherence was
the only factor that correlated with virologic outcome. There
are significant problems with adherence to complex regimens
in this population. Frequent daily dosing and the numbers of
medications are listed as reasons for nonadherence among
adolescents enrolled in the REACH (for reaching for excel-
lence in adolescent care and health) project (A. Rogers, un-
published data). Reducing the dose frequency while preserving
viral suppression, is a major goal of adolescent caretakers.
Combination medications, including Combivir (GlaxoSmith-
Kline) that contains ZDV and 3TC, are popular agents among
adolescent caretakers. Less frequent drug administration could
potentially demonstrate improvements in adherence among
adolescents and thus improve the outcome of HAART ther-
Thus, we undertook the present study to compare the
steady-state concentrations of ZDV-TP and 3TC-TP in periph-
eral blood mononuclear cells when the same total daily dose of
Combivir is administered QD versus BID. We also sought to
describe the kinetics of phosphorylation of ZDV and 3TC in
peripheral blood mononuclear cells over 24 h at steady state
when Combivir is administered as a single daily dose and to
describe any differences in adherence that could be demon-
strated between a QD and a BID regimen. Our working hy-
pothesis was that a ?2-fold fluctuation in ZDV-TP and
3TC-TP between QD and BID administration would support
further investigation to determine whether this simpler dosing
regimen is appropriate for general clinical use.
MATERIALS AND METHODS
Study design. PACTG P1012 was a phase I, randomized, crossover study of two
schedules of combination NRTI therapy with ZDV and 3TC, administered as the
commercially available combination product Combivir. HIV-infected adolescents,
ages 12 to 24 years, who had received at least 4 weeks of ZDV and 3TC given
individually or as Combivir immediately prior to study entry were eligible for en-
rollment. Subjects also had to weigh more than 40 kg, have a hemoglobin level of ?9
g/dl, and have CD4?cell counts greater than 250 cells/mm3. Women of childbearing
age were required to be on adequate birth control. Subjects were excluded from
participation if they had underlying toxicities greater than or equal to grade 3
according to the Division of AIDS Pediatric Toxicity Table (http://rcc-tech-res.com
or had an active malignancy or opportunistic infection. Subjects received a
HAART regimen that contained a minimum of three drugs that included ZDV
and 3TC as two of the agents and either a protease inhibitor or a non-NRTI as
additional agents. NRTIs other than ZDV and 3TC were not allowed. The
Institutional Review Board at each participating center approved the study.
Study procedure. After enrollment, subjects completed a 7-day adherence
assessment. If adherence to Combivir was ?70% (defined as taking fewer than
10 of the prescribed 14 Combivir tablets during the 7 days) or if all scheduled
doses in the 24 h prior to the assessment were not taken, the subject was removed
from the study. Subjects that were able to achieve at least 70% adherence were
randomized to one of two groups: (i) initial therapy with zidovudine (600 mg)
plus lamivudine (300 mg) administered QD for the initial phase followed by
zidovudine (300 mg) and lamivudine (150 mg) administered BID (group A) or
(ii) BID dosing of these agents followed by QD dosing (group B). Subjects were
maintained on their QD regimen for 7 days and on the BID regimen for 7 to 14
days. After completion of the designated pharmacokinetic studies on the first
randomized regimen, the subjects were switched to the other regimen, and a
second pharmacokinetic assessment performed. The goal was to accrue 10 ad-
herent subjects in each group with evaluable pharmacokinetic data (20 subjects
Pharmacokinetic sampling for all subjects included a baseline trough sample
following the 7 days of BID Combivir used to assess adherence. After this
sample, the subjects began their first randomized regimen. For subjects receiving
the BID Combivir regimen, a predose sample was obtained after 7 to 14 days of
BID therapy prior to the morning dose. For subjects receiving the QD Combivir
regimen, a predose sample was taken in the morning at the time of the regularly
scheduled QD dosing, a single dose of two Combivir tablets (600 mg of zidovu-
dine and 300 mg of lamivudine) was administered, and samples for intracellular
pharmacokinetic analysis were obtained 2, 4, 6, 12, and 24 h afterward.
Adherence assessment. In addition to the initial 7-day adherence assessment
after enrollment, adherence to the study medication regimen was determined by
the self-reported number of missed doses in the 3 days prior to each study visit
using the standard PACTG modules (9).
Analytical methods: laboratory studies. Blood for intracellular pharmacoki-
netic studies was collected in cell preparation tubes (Becton Dickinson, Franklin
Lakes, NJ). A 48-ml portion of blood was obtained for predose pharmacokinetic
assessments when the subject was on a BID regimen. A 24-ml portion of blood
was obtained at each assessment for samples obtained when the subject was
taking the QD regimen. The cell preparation tubes were processed according to
the manufacturer’s instructions to recover mononuclear cells that were then
pelleted by centrifugation at 4°C. The cells were suspended in 1 ml of the
supernatant and recentrifuged. The supernatant was removed. Then the cells
were lysed by suspension in 70% methanol–30% MilliQ water (final concentra-
tion of 15 mM Tris at pH 7.0) with 200 ?l of methanol for every 10 million cells
for at least 15 min on ice. Cell counts were verified by using a hemacytometer.
The samples were centrifuged, and the supernatant was placed in a cryo-storage
vial and stored at ?70°C. The samples were transferred to the PACTG Core
Pharmacology laboratory at St. Jude Children’s Research Hospital for analysis.
The extract was split with 80 ?l (4 million cells) used for 3TC analysis and the
remainder used for ZDV metabolite analysis. Both samples were analyzed by
cartridge separation of metabolites, followed by dephosphorylation and by ra-
dioimmunoassay (RIA) analysis according to procedures similar to those previ-
ously described (17–19), but with the following modifications. Samples used in
the analysis of 3TC were eluted from QMA cartridges (Waters Accell Plus
QMA; Millipore, Milford, MA) at the following volumes and concentrations of
KCl. The uncharged/parent compound was eluted with 3.0 ml of 5 mM KCl, 3TC
ethanolamine-choline was eluted with 6.0 ml of 25 mM KCl, 3TC-MP was eluted
with 7.0 ml of 50 mM KCl, 3TC-DP was eluted with 15 ml of 75 mM KCl, and
3TC-TP was eluted with 5.0 ml of 700 mM KCl. The fractions were dephosphor-
ylated with 1 U of acid phosphatase (sweet potato type XA, P1435; Sigma, St.
Louis)/ml, dried overnight with a SpeedVac, and reconstituted with water. The
samples were cleaned, and the salt was removed with SepPak C-18 cartridges
(Waters Division, Millipore Corp.). The ZDV samples were separated by using
a QMA cartridge with the following elution procedure. ZDV/uncharged were
eluted with 3 ml of 5 mM KCl, ZDV-MP was eluted with 12.0 ml of 50 mM KCl,
ZDV-DP was eluted with 15 ml of 75 mM KCl, and the ZDV-TP was eluted with
4.0 ml of 700 mM KCl. These samples were then dephosphorylated with acid
phosphatase, dried, and reconstituted with 3.0 ml of water and cleaned with
SepPak C-18 cartridges. The resultant ZDV and 3TC were measured with their
The limit of quantitation for both the ZDV and 3TC RIA assays was 0.15
ng/ml. Because of the expected concentration differential between intracellular
metabolites of ZDV, the final fractions containing the ZDV and ZDV-MP were
diluted more than ZDV-DP and ZDV-TP so that 0.15-ng/ml equaled concen-
trations of 28 fmol of ZDV-MP, 7.5 fmol of ZDV-DP, and 7.5 fmol of cells
ZDV-TP/106cells (17, 18). The 0.15 ng of LOQ/ml for the 3TC RIA equaled 0.16
pmol/106cells for all 3TC metabolites because they were all diluted to the same
volume. Triplicate control concentrations of 0.25, 2.5, and 15 ng/ml were run for
the ZDV assay and of 0.15, 0.5, 1, and 10 ng/ml were run for the 3TC assay.
The AUC of the intracellular metabolite concentrations were calculated by
using the trapezoidal method, and the half-life determined by a mixed-effects
modeling of the terminal portion of the log concentration profile for ZDV-TP
and 3TC-TP (4-6 through 24-h samples) using the program NONMEM (FOCE
subroutine with interaction). Several subjects had ZDV-TP concentrations ap-
proaching or falling below the limit of quantitation near the end of the dose
interval. To prevent potential bias through exclusion of these patients with rapid
elimination of the ZDV-TP, ZDV-TP samples with concentrations below the
limit of quantitation were included with reduced weight compared to other
samples. The ZDV-TP concentrations that were below the limit of quantitation
but above the limit of detection were set at their estimated concentration, while
those below the limit of detection were set at half of the limit of detection. An
additional additive random error, fixed to half of the limit of quantitation, was
applied to these samples. Individual subject parameter estimates were generated
by empirical Bayesian post-hoc subroutine.
Analytical methods: statistical. We calculated noncompartmental pharmaco-
kinetic parameters for all metabolites on the QD regimen, substituting half the
lower limit of quantification for any samples where the metabolite concentrations
were undetectable. Since there are no established therapeutic ranges for intra-
VOL. 51, 2007 ONCE VERSUS TWICE DAILY ZDV AND 3TC IN ADOLESCENTS3517
cellular amounts of either ZDV-TP or 3TC-TP, we were unable to directly
compare measured values to established ranges. We compared the within-subject
predose and 24-h measurements on the QD regimen by using a one-sample
signed-rank test and compared the predose values for all metabolites on the QD
and BID regimens using nonparametric Wilcoxon rank-sum tests to test for
treatment (dosing frequency) by order interactions, order effects, and treatment
We also calculated the ratios of predose levels of all metabolites to assess our
hypothesis that ?2-fold differences in the ratios of predose ZDV-TP and
3TC-TP between BID and QD schedules would support additional clinical in-
vestigation. Ratios are sensitive to extreme values, so medians and 95% confi-
dence intervals (CIs) for the median were used as summary statistics. CIs were
calculated by using the nonparametric CIPCTLDF option in SAS, version 9 (10).
A signed rank test was used to test whether the ratio of metabolite levels were
significantly different from 1 and from 2 – the ‘acceptable’ difference. Correla-
tions based on ranks rather than actual values were used as they are less sensitive
to extreme values.
Subject characteristics. Thirty-six subjects were accrued to
the present study between June 2001 and July 2002. One sub-
ject did not achieve the targeted 70% adherence during the
first week and was removed from the study. Of the 35 remain-
ing subjects, 21 were randomized to group A (QD followed by
BID) and 14 to group B (BID followed by QD). Because of an
imbalance in the number of evaluable subjects by group, the
randomization for order was shut down in June 2002 after the
enrollment of 26 subjects. At that time, there were 4 of 11
evaluable in group A and 11 of 15 evaluable in group B. At the
conclusion of the study, evaluable pharmacokinetic data on
adherent subjects was available for analysis on 14 of the 21
subjects in group A and in 13 of the 14 subjects in group B.
Subject characteristics are shown in Table 1. There were no
statistically significant differences between the evaluable and
nonevaluable patients nor those randomized or assigned to
group A or group B. Reasons for nonevaluability of the phar-
macokinetic data included four subjects with samples insuffi-
cient to assay and four who did not come for the visit or who
had been given the incorrect dose or regimen of the study
Safety. Both study regimens were well tolerated. During the
initial week of observation for adherence, two subjects re-
ported adverse effects. One subject reported a grade 2 diarrhea
that resolved prior to the beginning the first randomized reg-
imen. Another reported back pain that persisted for the re-
mainder of the study. While receiving the QD regimen, three
subjects developed a new adverse event greater than or equal
to grade 2. These included low back pain (grade 2), increased
bilirubin (grade 2), and decreased serum glucose (grade 2). On
the BID regimen, two subjects had new adverse events of
increased bilirubin: one grade 2 and one grade 3.
Pharmacokinetic data. Because patients receiving the QD
dosing regimens had predose and 24-h measurements that
should be similar for all metabolites tested, we compared the
two values using a one-sample signed-rank test of the within-
patient differences. There were no statistically significant dif-
ferences between the two measurements (P ? 0.300 for all), so
we used the predose values for all subsequent analyses.
Using values from the frequent sampling of patients receiv-
ing the QD regimen, noncompartmental pharmacokinetic pa-
rameters were calculated and are shown in Table 2. The pop-
ulation estimate for half-life of ZDV-TP was 9.1 ? 0.9 h
TABLE 1. Subject characteristics for the 36 patients enrolled and the 27 patients with evaluable pharmacokinetic sampling
All accrued subjects (n ? 36)
Subjects with adequate pharmacokinetic
assessments (n ? 27)
Group A (n ? 21)Group B (n ? 15)Group A (n ? 14)Group B (n ? 13)
Gender (no. of subjects)
Race or ethnicity (no. of subjects)
No. of subjects at age (yr):
15 to ?18
19.7 (17.0, 21.4)
20.0 (17.2, 22.7)
19.8 (17.8, 21.4) 20.0 (18.0, 22.3)
Baseline CD4?count (cells/mm3)
550 (425, 640)531 (421, 830) 550 (378, 620)670 (421, 935)
Both PI and non-NRTI
a*, The 25th and 75th percentiles are given in parentheses. PI, protease inhibitor.
3518FLYNN ET AL.ANTIMICROB. AGENTS CHEMOTHER.
(standard error of the mean) and 17.7 ? 2.8 h for 3TC-TP.
Post-hoc estimates of individual subjects ZDV-TP and 3TC-TP
half-lives were similar to the population estimate model (me-
dians of 8.4 and 18.2 h for ZDV-TP and 3TC-TP, respectively).
There was no statistically significant correlation between
ZDV-TP AUC and 3TC-TP AUC (Pearson correlation coef-
ficient ? ? 0.12, P ? 0.558). Figure 1 shows the concentration
curves for intracellular ZDV and 3TC MP, DP, and TP.
We assessed (i) treatment by order, (ii) order, and (iii)
treatment effects in all subjects with complete data on both the
QD and BID regimen (n ? 26 for all outcomes except 3TC-TP
with n ? 25) using Wilcoxon rank-sum tests. A statistically
significant interaction (P ? 0.031) for 3TC-DP was found, but
there were no other significant interactions or order effects.
Since median 3TC-DP levels were lower on the QD regimen in
both order groups and since the one interaction was only
marginally significant, we show summaries for all metabolites
collapsed over order and include all subjects with available
data on either the QD or BID regimens in Table 3. There was
considerable intersubject variability in the concentrations of
metabolites. On the QD regimen, 24 of 27 (89%) of the sub-
jects had predose measurable concentrations of ZDV-TP, and
24 of 25 (96%) had measurable concentrations of 3TC-TP. On
the BID regimen, 23 of 26 (88%) of the subjects had predose
measurable concentrations of ZDV-TP, and 26 of 27 (96%)
had a measurable concentration of 3TC-TP. A marginally sig-
nificant difference in ZDV-TP between the QD and the BID
regimens (P ? 0.056) was demonstrated with higher ZDV-TP
values on the BID regimen. The median predose ZDV-TP
concentration on the BID regimen was 15.4 fmol/106cells
compared to 9.3 fmol/106cells on the QD regimen.
The ratio of BID concentrations relative to the QD concen-
trations was calculated for 26 of the 27 evaluable subjects for
all metabolites except 3TC-TP (n ? 25). All 95% CI values for
the median ratios of the metabolites covered the value of 1.
The ZDV-TP ratio (median BID/QD ratio ? 1.28 [95% CI ?
1.00 to 2.45; interquartile range ? 0.86 to 2.80]) was statisti-
cally significantly different from the value of 1 (signed-rank test
P ? 0.005) but not significantly different from a value of 2 (P ?
0.405). Therefore, predose ZDV-TP values were not outside
our targeted acceptable difference of twofold for the two reg-
imens. The median ratio for the BID/QD 3TC-TP was 1.12
(95% CI ? 0.81 to 1.96; interquartile range ? 0.65 to 2.04). We
saw no evidence that background antiviral medications (pro-
tease inhibitors or non-NRTIs) influenced TP values (data not
Finally, we demonstrated that ZDV-TP measurements on
the standard BID regimen were correlated with values on the
TABLE 2. Noncompartmental pharmacokinetic parameters for ZDV and 3TC metabolites in subjects receiving 600 mg of ZDV and 300 mg
of 3TC QD (n ? 27)
ParameterZDV-MP ZDV-DPZDV-TP3TC-MP 3TC-DP3TC-TP
Median AUC tra
Mean half-life (h) ? SEM
9.1 ? 0.85
17.7 ? 2.8
aZDV metabolite AUCs were calculated in femtomoles, and 3TC metabolite AUCs were calculated in picomoles.
FIG. 1. Time-concentration curves for ZDV and 3TC TP (A), DP (B), and MP (C) after a single dose of 600 mg of ZDV and 300 mg of 3TC
showing median and 95% CI values for each metabolite.
VOL. 51, 2007 ONCE VERSUS TWICE DAILY ZDV AND 3TC IN ADOLESCENTS3519
QD regimen (Spearman correlation coefficient ? ? 0.60, P ?
Adherence. Adherence at all study visits was excellent with
no more than one patient reporting a missed Combivir dose
over the 3 days prior to the study visit at which their pharma-
cokinetic data were collected for both the QD and the BID
regimens. Because subjects could not proceed through the
steps of the study without very good adherence, the study
population was probably not representative of the general ad-
olescent population, so these results need to be interpreted
This study demonstrates that the intracellular active metab-
olites, ZDV-TP and 3TC-TP, persist within the cell for long
periods of time at detectable levels that are likely to inhibit
viral replication. Although the ZDV-MP concentration fluctu-
ated greatly over the 24-h dose interval, the other metabolites,
including ZDV-TP and 3TC-TP, only showed modest fluctua-
tion over this interval. Pharmacokinetic modeling yielded a
half-life for ZDV-TP of 9.1 h, while that for 3TC-TP was
17.7 h. These half-lives are similar to previously reported val-
ues from adult populations: 7 to 11 h for ZDV-TP (1, 21) and
16 to 32 h for 3TC-TP (1, 14, 21, 28).
We witnessed considerable intersubject variability in the
rates of phosphorylation, with the ZDV-TP and 3TC-TP pre-
dose concentrations spanning an ?40-fold range (coefficients
of variation of 74% for ZDV-TP QD, 97% for ZDV-TP BID,
90% for 3TC-TP QD, and 97% for 3TC-TP BID). This broad
variability is a concern when trying to extend the dose interval
since a substantial number of subjects would have very low
concentrations with the QD dosing.
A priori, we had chosen a ratio of BID/QD predose concen-
trations of ZDV-TP and 3TC-TP of ?2 to support additional
studies of QD ZDV and 3TC. When the same total daily doses
were administered either QD or BID, the ratios of predose
levels between all of the 3TC metabolites all differed by less
than a factor of 2. The same was true for ZDV, ZDV-MP, and
ZDV-DP. The ZDV-TP ratio was significantly different from 1
but not different from 2. Despite the ratios of predose concen-
trations falling below our threshold value of 2 for all of the
metabolites, the lower levels of the active metabolite ZDV-TP,
the substantial intersubject variability, the relatively short
ZDV-TP half-life, and findings from preclinical and relatively
recent adult clinical studies require reassessment of these cri-
teria. Preclinical studies using ZDV as a continuous infusion or
as a QD dose in a murine model of AIDS encephalopathy
demonstrated improved antiviral effect of continuous infusion
ZDV compared to QD dosing (3). Similarly, Drusano et al.
reported findings that did not support a QD dosing of ZDV in
an in vitro hollow-fiber model system that has been predictive
for other nucleosides (6).
In addition, clinical studies of pharmacokinetics and phar-
macodynamics comparing ZDV at 300 mg BID versus 600 mg
QD have been performed in adults. The COD10001 study
demonstrated that mean half-lives were similar in the two
dosing regimens (6.3 h for the QD regimen versus 5.48 h for
TABLE 3. Comparison of predose (and 24 h for QD) values for ZDV and 3TC metabolites on QD and BID regimens collapsed over order
P (dose effect)d
aThat is, the number of measurements where one-half the lower limit of quantification (LLOQ) was imputed.
bMedian and 95% CI reported in fmol/106cells for ZDV and in pmol/106cells for 3TC.
cCV, coefficient of variation.
dSignificance value as determined by the Wilcoxon rank-sum test for differences in median values on the QD versus the BID regimen.
3520 FLYNN ET AL.ANTIMICROB. AGENTS CHEMOTHER.
the BID regimen), and both groups had similar declines in viral
loads over 14 days of monotherapy (22). In COD20002, 32
antiretroviral naive subjects were randomized, in a 1:1 ratio, to
receive either ZDV 600 mg QD or 300 mg BID as mono-
therapy. Viral loads over the first 14 days demonstrated a trend
suggesting that the subjects on the BID dosing regimen
achieved lower viral loads (P ? 0.056) and more rapid declines
in viral load (P ? 0.065) compared to the QD group (22).
These studies differ from the present study in several signifi-
cant ways. In the present study, the QD and BID regimen were
given in addition to either a protease inhibitor or a non-NRTI
or both as part of a HAART regimen. In addition, we opted to
study subjects who had been receiving their current HAART
regimen for at least 4 weeks and were at steady state. Although
our ZDV-TP half-life was somewhat longer at 9.1 h in adoles-
cents, it was still short enough to result in lower predose
ZDV-TP concentrations with QD dosing. In addition, we did
not investigate viral dynamics since many of our subjects had
undetectable viral loads at the start of the study. Thus,
despite a potential gain in adherence, the lower levels of
ZDV-TP taken in consideration with findings from other
studies have dissuaded us from pursuing further study of a
QD ZDV regimen.
In our study, we were able to demonstrate that ZDV-TP
measurements on the standard BID dosing regimen were cor-
related with ZDV-TP concentrations on a QD dose. This find-
ing may be important if future technologies allow easy mea-
surement of ZDV-TP. If monitoring of ZDV-TP becomes
practical, one may be able to individualize patient dosing reg-
imens in patients with high ZDV-TP levels on a BID regimen
and to assess a QD regimen. In adolescent patients, removing
barriers to adherence by simplifying dosing regimens is criti-
cally important and requires additional research efforts. How-
ever, because the measurement of ZDV-TP is a complex re-
search laboratory procedure, extending the ZDV dose interval
in patients with high predose ZDV-TP is not practical or
Another possible strategy to make daily ZDV more effective
would be to use increased doses of ZDV to achieve higher
ZDV-TP levels. Although we did not study doses other than
600 mg QD and 300 mg BID in our study, Fletcher and co-
workers demonstrated systemic exposure to ZDV-TP in-
creased using a 700-mg daily dose compared to a 500-mg daily
dose (1, 8). In addition, Anderson et al. noted a 125% increase
in ZDV-TP concentrations when the ZDV dose was increased
by 130% (1). These data indicate that, despite substantial sub-
ject variability, measurements of ZDV-TP do change as a func-
tion of dose. However, higher doses may be associated with
greater toxicity and cost and would need to be studied.
This is the first evaluation of intracellular NRTI metabolites
in adolescent HIV-infected subjects. Our findings are in accord
with previous studies in older populations. The estimates of the
ZDV and 3TC metabolites were in the range of those previ-
ously reported, and peak ZDV-TP concentrations were in the
range previously reported (1, 19, 28; Rogers, unpublished).
Likewise, 3TC-TP concentrations were similar to those re-
ported (1, 14, 28; Rogers, unpublished). The proportion of
3TC metabolites as the 3TC-TP moiety was higher than seen
by Moore et al. (14). In that study 3TC accounted for only 15
to 20% of the intracellular phosphates compared to the ca.
45% observed in the present study. Unlike Anderson et al. (1),
we were unable to find a correlation between concentrations of
ZDV-TP and 3TC-TP. Given variability in measurements and
different methodologies among studies, these findings suggest
similar metabolic handling of ZDV and 3TC in this young
Our study had several limitations. Because of imbalance in
the evaluability of pharmacokinetic data during the early
stages of accrual, randomization to the two groups was
stopped, compromising the crossover design and limiting the
interpretability of the tests for interaction and order effects.
Given the high variability in metabolite concentrations, our
study was too small to assess for potential age or gender effects
as reported by Anderson et al., and no analyses by gender were
performed (1). In addition, the BID predose sample was ob-
tained prior to the scheduled morning dose and did not follow
an observed dose.
In conclusion, we have demonstrated that most patients
have detectable levels of the TP of ZDV and 3TC 24 h after
dosing and that half-lives on a QD regimen were similar to
previously reported values when the drugs were given BID.
There was considerable variability, and some patients had non-
measurable concentrations of ZDV-TP after 24 h. The predose
intracellular MP and DP metabolite concentrations of ZDV
and 3TC on QD were all within 70% of those seen with BID
therapy. However, ZDV-TP and 3TC-TP concentrations with
QD therapy were lower as a percentage of the BID values, with
the ZDV-TP changes reaching marginal significance due to its
shorter half-life. Despite these similarities between the QD
and BID ZDV dosing regimens, the lower concentrations of
ZDV-TP and the lack of a targeted nadir linked with virologic
efficacy, previously published literature, and the increasing
number of QD antiviral agents do not support further study of
a QD dose of 600 mg of ZDV.
Thus study was supported in part by National Institutes of Health
grants P41-EB001978 and UO1 AI41089, by the Pediatric AIDS Clin-
ical Trials Group of the National Institute of Allergy and Infectious
Diseases, and by the American Lebanese-Syrian Associated Charities.
Staff at participating PACTG 1012 sites included J. Martinez and K.
Bojan, The CORE Center for the Prevention, Care, and Research of
Infectious Disease, Chicago, IL; S. Nesheim and R. Dennis, Emory
University Hospital, Atlanta, GA; A. Kovacs and J. Homans, Los
Angeles County Medical Center (USC), Los Angeles, CA; K. Knapp
and S. DiScenza, St. Jude Children’s Research Hospital, Memphis,
TN; R. B. Van Dyke and M. Sillio, Tulane University Charity Hospital,
New Orleans, LA; P. Palumbo, University of Medicine and Dentistry
of New Jersey, Newark, NJ; and J. F. Rodriguez, University of Puerto
Rico, San Juan, Puerto Rico.
1. Anderson, P. L., T. N. Kakuda, S. Kawle, and C. V. Fletcher. 2003. Antiviral
dynamics and sex differences of zidovudine and lamivudine triphosphate
concentrations in HIV-infected individuals. AIDS 17:2159–2168.
2. Barry, M. G., S. H. Khoo, G. J. Veal, P. G. Hoggard, S. E. Gibbons, E. G.
Wilkins, O. Williams, A. M. Breckenridge, and D. J. Back. 1996. The effect
of zidovudine dose on the formation of intracellular phosphorylated metab-
olites. AIDS 10:1361–1367.
3. Bilello, J. A., J. L. Eiseman, H. C. Standiford, and G. L. Drusano. 1994.
Impact of dosing schedule upon suppression of a retrovirus in a murine
model of AIDS encephalopathy. Antimicrob. Agents Chemother. 38:628–
4. Blum, M. R., S. H. Liao, S. S. Good, and P. de Miranda. 1988. Pharmaco-
kinetics and bioavailability of zidovudine in humans. Am. J. Med. 85:89–94.
5. Collier, A. C., S. Bozzette, R. W. Coombs, D. M. Causey, D. A. Schoenfeld,
VOL. 51, 2007 ONCE VERSUS TWICE DAILY ZDV AND 3TC IN ADOLESCENTS3521
S. A. Spector, C. B. Pettinelli, G. Davies, D. D. Richman, and J. M. Leedom.
1990. A pilot study of low-dose zidovudine in human immunodeficiency virus
infection. N. Engl. J. Med. 323:1015–1021.
6. Drusano, G. L., P. A. Bilello, W. T. Symonds, D. S. Stein, J. McDoweell, A.
Bye, and J. A. Bilello. 2002. Pharmacodynamics of abacavir in an in vitro
hollow-fiber model system. Antimicrob. Agents Chemother. 46:464–470.
7. Eron, J. J., S. L. Benoit, J. Jemsek, R. D. MacArthur, J. Santana, J. B.
Quinn, D. R. Kuritzkes, M. A. Fallon, and M. Rubin. 1995. Treatment with
lamivudine, zidovudine or both in HIV-positive subjects. N. Engl. J. Med.
8. Fletcher, C. V., E. P. Acosta, K. Henry, L. M. Page, C. R. Gross, S. P. Kawle,
R. P. Remmel, A. Erice, and H. H. Balfour, Jr. 1998. Concentration-con-
trolled zidovudine therapy. Clin. Pharm. Ther. 64:331–338.
9. Flynn, P. M., B. J. Rudy, S. D. Douglas, J. Lathey, S. A. Spector, J. Martinez,
M. Silio, M. Belzer, L. Friedman, L. D’Angelo, J. McNamara, J. Hodge,
M. D. Hughes, J. C. Lindsey, et al. 2004. Virologic and immunologic out-
comes after 24 weeks in HIV-1-infected adolescents on highly active anti-
retroviral therapy. J. Infect. Dis. 190:271–279.
10. Hahn, G. J., and W. Q. Meeker. 1991. Statistical intervals: a guide for
practitioners. John Wiley & Sons, Inc., New York, NY.
11. Johnson, M. A., K. H. P. Moore, G. J. Yuen, A. Bye, and G. E. Pakes. 1999.
Clinical pharmacokinetics of lamivudine. Clin. Pharmacokinet. 36:41–66.
12. Laskin, O. L., P. de Miranda, and M. R. Blum. 1989. Azidothymidine
steady-state pharmacokinetics in subjects with AIDS and AIDS-related com-
plex. J. Infect. Dis. 159:745–747.
13. McKinney, R. E., Jr., G. M. Johnson, K. Stanley, F. H. Yong, A. Keller, K. J.
O’Donnell, P. Brouwers, W. G. Mitchell, R. Yogev, D. W. Wara, A. Wiznia,
L. Mofenson, J. McNamara, and S. A. Spector. 1998. A randomized study of
combined zidovudine-lamivudine versus didanosine monotherapy in chil-
dren. J. Pediatr. 133:500–506.
14. Moore, K. H., J. E. Barrett, S. Shaw, G. E. Pakes, R. Churchus, A. Kapoor,
J. Lloyd, M. G. Barry, and D. Back. 1999. The pharmacokinetics of lamivu-
dine triphosphorylation in peripheral blood mononuclear cells from patients
infected with HIV-1. AIDS 13:2239–2250.
15. Peter, K., and J. G. Gambertoglio. 1998. Intracellular phosphorylation of
zidovudine and other nucleoside reverse transcriptase inhibitors for HIV
infection. Pharm. Res. 15:819–825.
16. Richman, D. 1991. Antiviral therapy of HIV infection. Annu. Rev. Med.
17. Robbins, B. L., T. T. Tran, F. H. Pinkerton, Jr., F. Akeb, R. Guedj, J. Grassi,
D. Lancaster, and A. Fridland. 1998. Development of a new cartridge ra-
dioimmunoassay for determination of intracellular levels of lamivudine
triphosphate in the peripheral blood mononuclear cells of human immuno-
deficiency virus-infected patients. Antimicrob. Agents Chemother. 42:2656–
18. Robbins, B. L., B. H. Waibel, and A. Fridland. 1996. Quantitation of intra-
cellular zidovudine phosphates by use of combined cartridge-radioimmuno-
assay methodology. Antimicrob. Agents Chemother. 40:2651–2654.
19. Rodman, J. H., P. M. Flynn, B. Robbins, E. Jimenez, A. D. Bardeguez, J. F.
Rodriguez, S. Blanchard, and A. Fridland. 1999. Systemic pharmacokinetics
and cellular pharmacology of in human immunodeficiency virus type-1-in-
fected women and newborn infants. J. Infect. Dis. 180:1844–1850.
20. Rodman, J. H., B. Robbins, P. M. Flynn, and A. Fridland. 1996. A systemic
and cellular model for zidovudine plasma concentrations and intracellular
phosphorylation in subjects. J. Infect. Dis. 74:490–499.
21. Rodriguez, J. F., J. L. Rodriguez, J. Santana, H. Garcia, and O. Rosario.
2000. Simultaneous quantitations of intracellular zidovudine and lamivudine
triphosphates in human immunodeficiency virus-infected individuals. Anti-
microb. Agent Chemother. 44:3097–3100.
22. Ruane, P. J., G. J. Richmond, E. DeJesus, C. E. Hill-Zabala, S. C.
Danehower, Q. Liao, J. Johnson, M. S. Shaefer, et al. 2004. Pharmacody-
namic effects of zidovudine 600 mg once/day versus 300 mg twice/day in
therapy-naive patients infected with human immunodeficiency virus. Phar-
23. Shepp, D. H., C. Ramirez-Ronda, L. Dall, R. B. Pollard, R. L. Murphy, H.
Kessler, R. Sherer, G. Mertz, G. Perez, D. J. Gocke, S. B. Greenberg, E.
Petersen, I. Frank, M. D. Moore, R. McKinnis, and J. F. Rooney. 1997. A
comparative trial of zidovudine administered every four versus every twelve
hours for the treatment of advanced HIV disease. J. Acquir. Immune Defic.
Syndr. Hum. Retrovirol. 15:283–288.
24. Smiley, L. 1989. Zidovudine treatment of subjects with acquired immune
deficiency syndrome and acquired immune deficiency syndrome-related
complex: long-term experience. J. Infect. 18(Suppl. 1):77.
25. Volberding, P. A., S. W. Lagakos, J. M. Grimes, D. S. Stein, H. H. Balfour,
Jr., R. C. Reichman, J. A. Bartlett, M. S. Hirsch, J. P. Phair, R. T. Mitsu-
yasu, et al. 1994. The duration of zidovudine benefit in persons with asymp-
tomatic HIV infection. JAMA 272:437–442.
26. Volberding, P. A., S. W. Lagakos, J. M. Grimes, D. S. Stein, J. Rooney, T. C.
Meng, M. A. Fischl, A. C. Collier, J. P. Phair, M. S. Hirsch, et al. 1995. A
comparison of immediate with deferred zidovudine therapy for asymptom-
atic HIV-infected adults with CD4 cell counts of 500 or more per cubic
millimeter. N. Engl. J. Med. 333:401–407.
27. Volberding, P. A., S. W. Lagakos, M. A. Koch, C. Pettinelli, M. W. Myers,
D. K. Booth, H. H. Balfour, Jr., R. C. Reichman, J. A. Bartlett, M. S. Hirsch,
et al. 1990. Zidovudine in asymptomatic human immunodeficiency virus
infection: a controlled trial in persons with fewer than 500 CD4-positive cells
per cubic millimeter. N. Engl. J. Med. 322:941–949.
28. Yuen, G. J., Y. Lou, N. F. Bumgarner, J. P. Bishop, G. A. Smith, V. R. Otto,
and D. D. Hoelscher. 2004. Equivalent steady-state pharmacokinetics of
lamivudine in plasma and lamivudine triphosphate within cells following
administration of lamivudine at 300 milligrams once daily and 150 milligrams
twice daily. Antimicrob. Agents Chemother. 48:176–182.
3522FLYNN ET AL.ANTIMICROB. AGENTS CHEMOTHER.