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Antimicrobial Agents and Chemotherapy, November 2000, p. 3097-3100, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Simultaneous Quantitation of Intracellular
Zidovudine and Lamivudine Triphosphates in Human Immunodeficiency
Virus-Infected Individuals
Jose F.
Rodriguez,1,2,*
Jorge L.
Rodriguez,1
Jorge
Santana,3
Hermes
García,4 and
Osvaldo
Rosario2
Department of
Biochemistry1 and Department of
Medicine,3 School of Medicine, Medical Sciences
Campus, University of Puerto Rico, and Puerto Rico Health
Department (CLETS),4 San Juan, and
Department of Chemistry, Rio Piedras Campus, University of
Puerto Rico, Rio Piedras,2 Puerto Rico
Received 11 February 2000/Returned for modification 28 May
2000/Accepted 21 August 2000
 |
ABSTRACT |
Highly active antiretroviral therapy (HAART) is the standard
treatment for infection with human immunodeficiency virus (HIV). The
most common HAART regimen consists of the combination of at least one
protease inhibitor (PI) with two nucleoside reverse transcriptase
inhibitors (NRTIs). Contrary to PIs, NRTIs require intracellular
activation from the parent compound of their triphosphate moiety to
suppress HIV replication. Simultaneous intracellular determination of
two NRTI triphosphates is difficult to accomplish due to their
relatively small concentrations in peripheral blood mononuclear cells
(PBMCs), requiring large amounts of blood from HIV-positive patients.
Recently, we described a method to determine intracellular zidovudine
triphosphate (ZDV-TP) concentrations in HIV-infected patients by using
solid-phase extraction and tandem mass spectrometry. The limit of
quantitation (LOQ) for ZDV-TP was 0.10 pmol, and the method was
successfully used for the determination of ZDV-TP in HIV-positive
patients. In this study, we enhanced the aforementioned method by the
simultaneous quantitation of ZDV-TP and lamivudine triphosphate
(3TC-TP) in PBMCs from HIV-infected patients. The LOQ for 3TC-TP was
4.0 pmol, with an interassay coefficient of variation and an accuracy
of 7 and 12%, respectively. This method was successfully applied to
the simultaneous in vivo determination of the ZDV-TP and 3TC-TP
pharmacokinetic profiles from HIV-infected patients receiving HAART.
| |
INTRODUCTION |
Highly active antiretroviral therapy
(HAART) has been used successfully for treatment of human
immunodeficiency virus (HIV) since the discovery of protease inhibitors
(PIs) (3, 4, 20). HAART treatment includes a broad category
of antiretroviral drug combinations with the goals of decreasing plasma
HIV-1 RNA levels below the limit of detection, limiting disease
progression, and delaying the appearance of resistant mutants
(12). The most common HAART regimen consists of the
combination of one PI with two nucleoside reverse transcriptase
inhibitors (NRTIs). This triple drug combination has shown dramatic
improvements in viral suppression over the combination of the two
nucleosides zidovudine and lamivudine (ZDV and 3TC, respectively)
(8-10).
Contrary to PIs, NRTIs require intracellular activation from the parent
compound of their triphosphate (TP) moiety to suppress HIV replication.
ZDV and 3TC are not active against HIV; they need to be metabolized to
5'-ZDV-TP (ZDV-TP) and 5'-3TC-TP (3TC-TP) to act as competitive
inhibitors of HIV reverse transcriptase or be incorporated into the
viral genome (2, 7, 11, 23). Studies conducted with
HIV-infected populations have not established any relationship between
ZDV or 3TC concentrations in plasma and the efficacy of these agents
(19). On the other hand, a recent study showed a linear
relationship between ZDV-TP intracellular concentrations and an
increase in the percent change in CD4+ cells from baseline
in HIV-infected adults (5). Furthermore, several studies
have shown that intracellular concentrations of NRTI-TPs correlated
better with virologic responses than the parent plasma NRTI levels
(J. P. Sommadossi, M. A. Valentin, X. J. Zhou, M. Y. Xie, J. Moore, V. Calvez, M. Desa, and C. Kotlama, Program Abstr.
5th Conf. Retroviruses Opportunistic Infect., abstr. 262, p. 146;
J. P. Sommadossi, X. J. Zhou, J. Moore, D. V. Havlir, G. Friedland, C. Tierny, L. Smeaton, L. Fox, D. Richmann, and R. Pollard,
Program Abstr. 5th Conf. Retroviruses Opportunistic Infect., abstr. 3, p. 79).
Several approaches have been reported for the individual determination
of ZDV-TP and 3TC-TP (6, 13, 15-18, 21, 22, 24). A recent
approach was developed in which strong anion-exchange-solid-phase extraction separated ZDV anabolites (ZDV-MP, ZDV-DP, and ZDV-TP), followed by enzyme digestion and quantification by radioimmunoassay (18). A similar approach was employed by the same group to
determine intracellular levels of 3TC-TP (17). The
combination of both methods was used to individually measure ZDV-TP and
3TC-TP concentrations in HIV-infected subjects. Limitations of the
aforementioned method include the lack of an internal standard in the
quantitation process and the use of parent compounds (ZDV and 3TC) to
produce the calibration curve instead of ZDV-TP and 3TC-TP.
Another approach has been proposed to measure intracellular 3TC
metabolites by a combination of solid-phase extraction and high-performance liquid chromatography (HPLC) with UV detection (22). The use of UV detection is possible with 3TC
metabolites (3TC-MP, 3TC-DP, and 3TC-TP) because of the large amounts
(picomoles per 106 cells instead of femtomoles per
106 cells) formed in vivo. However, as well as in the
aforementioned methods, no internal standard was used with this
methodology. In addition, this method can only be used for 3TC, since
ZDV does not produce the large amounts of intracellular metabolites
made by 3TC.
In this study, we report the simultaneous determination of
intracellular ZDV-TP and 3TC-TP concentrations in human peripheral blood mononuclear cells (PBMCs) with azidodeoxyuridine (AZdU) as the
internal standard. With this methodology, the limits of quantitation
(LOQ) for 3TC-TP and ZDV-TP are 4.0 and 0.10 pmol, respectively. This
method was successfully used to determine the in vivo pharmacokinetic
profile of ZDV-TP and 3TC-TP from HIV-infected patients receiving HAART.
| |
MATERIALS AND METHODS |
Chemicals.
ZDV, AZdU, sodium acetate, and acid phosphatase
(type XA) were obtained from Sigma Chemical Co. (St. Louis, Mo.).
ZDV-TP, 3TC, and 3TC-TP were purchased from Moravek Biochemicals (Brea, Calif.). Potassium chloride, acetonitrile, methanol, and glacial acetic
acid (American Chemical Society certified) were obtained from Fisher
Scientific (Fairlawn, N.J.). Strong anion-exchange Sep-Pak plus
(SAX-QMA) cartridges were purchased from Waters Co. (Milford, Mass.).
XAD resin was obtained from Serva (Heidelberg, N.Y.). RPMI 1640, glutamine, nonessential amino acids, penicillin-streptomycin, and fetal
calf serum were obtained from BioWhittaker (Baltimore, Md.).
Preparation of standard solutions.
ZDV-TP and 3TC-TP
standard solutions were prepared by serial dilution starting with a
stock concentration of 500 µM for ZDV-TP and 1,000 µM for 3TC-TP
spiked with PBMCs from HIV-negative individuals.
Sample collection from HIV-infected patients.
Patients
signed an informed consent form approved by the Medical Sciences Campus
Institutional Review Board at the University of Puerto Rico. Blood
samples (16 ml) were drawn at predose (time zero) and 1, 2, 4, 8, 12, 16, and 24 h postdose from five HIV-infected patients treated with
the standard ZDV (300 mg twice a day [BID]) and 3TC therapy (150 mg
BID). Measurements were performed at steady state, since patients had
been receiving ZDV or 3TC therapy for at least 24 weeks. PBMCs (usually
16 million cells) were separated from erythrocytes by centrifugation at
1,500 × g for 20 min at room temperature. PBMCs were
recovered and counted in a Coulter Z2 series system (Hialeah, Fla.),
followed by an extraction with 70% methanol, and stored at 
80°C
until analysis.
Intracellular ZDV-TP and 3TC-TP isolation.
SAX-QMA was used
for the separation of ZDV and 3TC nucleotides as previously described
(22). Briefly, the cartridges were preconditioned with 500 µl of deionized water. The cell extract sample was loaded onto the
cartridge and eluted under reduced pressure. The parent drugs were
eluted with a serial wash of 150 and 500 µl of water. ZDV-MP and
3TC-MP were obtained in the fraction rinsed with 400 µl of 100 mM
KCl, and ZDV-DP and 3TC-DP were eluted with 400 µl of 120 mM KCl. The
fraction containing ZDV-TP and 3TC-TP was eluted with 500 µl of 400 mM KCl. Recovery experiments were performed comparing SAX-QMA
cartridges with SAX-HPLC by using [3H]ZDV-TP and
[3H]3TC-TP formed from PBMC incubation experiments.
Recoveries were found to be >95% for both nucleotides. Cleavage of
phosphate groups was accomplished by the addition of 2 U of acid
phosphatase per ml for 30 min at 37°C, adjusting the fraction to pH
4.0 with sodium acetate. After enzyme digestion, AZdU (internal
standard, 100 ng/ml) was added to the extract and recovered
simultaneously with ZDV and 3TC by using an XAD column. The XAD column
was preconditioned with water before sample loading. The sample was
desalted with 5 ml of water. ZDV, 3TC, and AZdU were eluted with 2.0 ml
of methanol. Desalting and recovery of ZDV, 3TC, and AZdU by using XAD
columns were >98%. Samples were dried in a Labconco CentriVap console (Kansas City, Miss.) and reconstituted with 100 µl of mobile phase, prior to HPLC-mass spectrometry (MS)/MS analysis.
Liquid chromatography-tandem MS.
HPLC analysis was performed
on a Hewlett-Packard 1100 system (San Fernando, Calif.) using a
Hypersil C18 reversed-phase column (100 by 2.1 mm, 3 µm).
The mobile phase consisted of a methanol and acetonitrile mixture
(30:10 [vol/vol]) with 0.25% acetic acid at a flow rate of 200 µl/min. An injection of 20 µl was sufficient for the detection of
ZDV, 3TC, and AZdU. A Micromass Quattro II triple quadrupole mass
spectrometer (Manchester, United Kingdom) was used for the analysis in
the selective reaction monitoring (SRM) mode. Sample introduction was
through ESI in the positive ion mode. The cone voltage was set between
15 and 20 V, and the source temperature was set at 120°C. Ions were
collisionally activated at a collision energy of between 3 and 7 eV
with a cell pressure of approximately 7 × 10
4 mbar
of argon. SRM data were acquired and analyzed with MassLynx software
(v. 3.0)
Data analysis.
Concentrations of analytes were determined by
using ZDV/AZdU and 3TC/AZdU peak area ratios. Calibration curves from
ZDV-TP and 3TC-TP standard solutions were prepared every time a series of samples were analyzed. Linear regression analyses were performed with five ZDV-TP and 3TC-TP standard concentrations. Regression coefficients (r) were better than 0.998 for all calibration curves.
| |
RESULTS AND DISCUSSION |
Ion chromatograms for ZDV-TP and 3TC-TP.
Figure
1 shows the ion chromatograms obtained
from an HIV-infected patient treated with ZDV or 3TC. The upper panel
shows the ion chromatogram for ZDV (after removal of the phosphate
groups from ZDV-TP) with a retention time of 3.08 min. The middle panel shows the ion chromatogram of 3TC (after removal of the phosphate groups from 3TC-TP) with a retention time of 3.41 min, which provides a
higher signal than ZDV. This result is not unexpected, since in vitro
and in vivo studies have demonstrated that the intracellular production
of 3TC-TP is higher than that of ZDV-TP (1, 2, 17, 22). In
the lower panel, the chromatogram of AZdU (internal standard) shows a
strong signal with a retention time of 2.74 min. We determined that
AZdU did not interfere with the signal of ZDV or 3TC, despite the
proximity in their retention times (data not shown). Similarly, 3TC did
not interfere with the signal of ZDV or AZdU. In addition, endogenous
nucleotides or other compounds from cell samples do not interfere with
the quantitation process of ZDV-TP or 3TC-TP. The short retention times
for the three compounds increase the throughput of samples (20 samples/h), providing the opportunity to quantify ZDV-TP or 3TC-TP
concentrations in clinical trials.
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FIG. 1.
Chromatograms for ZDV (top) and 3TC (middle) obtained
from an HIV-infected individual. The solution was processed throughout
the complete procedure, and AZdU was used as the internal standard
(bottom). Retention times for ZDV, 3TC, and AZdU were 3.08, 3.41, and
2.72 min, respectively. Chromatographic conditions are described in
Materials and Methods. The y axis is the ion intensity
obtained for each compound.
|
|
Standard curve and statistics.
Figure
2 shows a typical calibration curve
constructed from the ratio of the areas from 3TC and AZdU chromatograms
plotted against 3TC-TP concentration. This calibration curve was
obtained by using different 3TC-TP standard concentrations spiked with PBMCs and passed through the entire methodology (SAX-QMA, enzyme digestion, and XAD column). The LOQ obtained with this methodology is
4.0 pmol (coefficient of variation [CV], <8%, n = 5; accuracy, <15%, n = 5). The regression
coefficients for similar calibration curves were better than 0.998. We
have previously reported calibration curves for ZDV-TP with regression
coefficients better than 0.999 and an LOQ of 0.10 pmol (6).
The quantitation process for ZDV-TP was not affected by the
simultaneous quantitation of 3TC-TP, as shown in Table
1.
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FIG. 2.
Calibration standard curve for 3TC-TP constructed from
standard solutions (4, 21, 43, 85, and 128 pmol) and passed through the
complete methodology. The data plotted represent the average of two
determinations for each concentration. Error bars reside within the
points if not shown. The equation describing the complete range is
y = 0.121x 0.018, with a regression
coefficient (r) of 0.998.
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|
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TABLE 1.
Performance evaluation of the method for simultaneous
determination of 3TC-TP and
ZDV-TP concentrationsa
|
|
In the validation process for ZDV-TP, we pointed out the necessity of
the internal standard to obtain precise and accurate
results
(
6). For 3TC-TP, we found a similar situation. With
AZdU
(internal standard), we improved the 3TC-TP regression coefficient
for
the calibration curve from 0.937 to 0.998. Furthermore, the
intra-assay
variability improved with AZdU for a 3TC-TP concentration
of 43.0 pmol
from a CV of 6% to 1%. The accuracy (percent error)
of the method
with AZdU improved from 42% to 3% for 4.0 pmol (
n = 5) and from 37% to 8% for 43.0 pmol (
n = 5).
Thus, the use of
AZdU as the internal standard improved dramatically
the accuracy
of the assay and provided a precise method to measure low
concentrations
of 3TC-TP.
Table
1 shows the results for the interassay variability (10.0, 50.0, and 150.0 pmol) used in the validation process. These
results confirm
the excellent accuracy and precision of this new
methodology. For all
concentrations studied, recoveries were
93%
and the CV was <10%.
Similar results were obtained for the ZDV-TP
quality control standards
(0.10, 0.50, and 5.00 pmol). Thus, the
methodology is suitable to
measure intracellular 3TC-TP and ZDV-TP
from HIV-infected patients
treated with ZDV or
3TC.
Patient samples.
Figure 3 shows
the concentration-time curves for 3TC-TP and ZDV-TP from five
HIV-infected patients treated with ZDV or 3TC. Patients were taking the
standard dose for these antiretroviral agents: ZDV, 300 mg BID; and
3TC, 150 mg BID. For the pharmacokinetic study, measurements were
performed at steady state, and patients did not take the dose at
12 h in order to obtain the complete profile for 24 h.
Despite the large variability in intracellular 3TC-TP and ZDV-TP
concentrations between patients, differences between the
pharmacokinetic profiles of the two metabolites are evident.
Intracellular 3TC-TP concentrations are 20-fold higher than those of
ZDV-TP, due to the greater efficiency of the 3TC phosphorylation
process (2). The elimination process appears to be different
for both nucleotides, since the estimated median 3TC-TP half-life for
these five patients is approximately 32 h (range, 23 to 49 h), while for ZDV-TP, it is approximately 11 h (range, 5 to
13 h). In all five patients, a robust signal was observed for
3TC-TP at 24 h, while only one out of five had measurable concentrations of ZDV-TP. In a recent study, Moore et al. measured the
intracellular concentration of 3TC-TP in 10 HIV-infected patients (14). The median intracellular half-life for 3TC-TP was
reported to be 15 h (range, 6 to 32 h). The discrepancy
between both studies can be attributed to the large variability in the
measurements made by Moore et al. and to the small number of patients
used in both studies. They used HPLC to separate their metabolites, and
quantitation was performed by radioimmunoassay. They did not use an
internal standard in their methodology, which may account for their
large variability (CV, >50%). The lack of an internal standard also
diminishes the certainty of the measurements, since the method cannot
account for losses during the experimental procedure for each sample.
This could explain the higher 3TC-TP intracellular concentrations
observed in our study.
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FIG. 3.
Intracellular pharmacokinetic profile for 3TC-TP (top)
and ZDV-TP (bottom) from five HIV-infected patients. Patients stayed
overnight at the University of Puerto Rico-Clinical Research Center
facilities, and samples were collected at predose (time zero) and 1, 2, 4, 8, 12, 16, and 24 h postdose. The line represents the median
value for each time point.
|
|
The values observed for 3TC-TP and ZDV-TP are similar to those reported
previously by other methodologies. Interestingly,
the ZDV-TP
concentrations between 2 and 4 h are similar to those
obtained by
Fletcher et al. for patients that had an increase
in the percent change
of CD4
+ cells from baseline (
5). 3TC-TP
concentrations are in the
same range observed by other laboratories
(
17,
22). To the
best of our knowledge, this is the first
time that intensive intracellular
pharmacokinetic profiles have been
obtained for ZDV-TP and 3TC-TP,
providing the opportunity to understand
better the in vivo intracellular
interactions between these
antiretroviral agents. These interactions
will provide information that
could be essential for the clinical
management of HIV-infected
patients. We now have the capacity
to establish a correlation between
intracellular pharmacological
parameters and drug efficacy or toxicity.
These studies are currently
in progress at our
institution.
| |
ACKNOWLEDGMENTS |
This work was supported in part by Public Health Service grants
2U01AI32906, 1P20RR11126, G12-RR03051, AI34858, and R01AI39191 (J.F.R.).
We acknowledge the technical assistance of Marianela Pérez and
Raúl Blanco.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, P.O. Box 365067, School of Medicine, Medical Sciences
Campus, University of Puerto Rico, San Juan Puerto Rico 00936-5067. Phone and Fax: (787) 754-4929. E-mail:
j_rodriguez{at}rcmaxp.upr.clu.edu.
| |
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Antimicrobial Agents and Chemotherapy, November 2000, p. 3097-3100, Vol. 44, No. 11
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
