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Antimicrobial Agents and Chemotherapy, September 2000, p. 2572-2574, Vol. 44, No. 9
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Use of Saliva Specimens for Monitoring Indinavir
Therapy in Human Immunodeficiency Virus-Infected Patients
Uwe
Wintergerst,1,*
M.
Kurowski,2
B.
Rolinski,3
M.
Müller,2
E.
Wolf,4
H.
Jaeger,4 and
B.
H.
Belohradsky1
Department of Infectious Diseases and
Immunology1 and Department of Clinical
Chemistry,3 Children's Hospital of the
Ludwig-Maximilians University, 80337 Munich, KIS-Curatorium for
Immunodeficiencies, 80336 Munich,4 and
HIV-Lab, c/o Auguste-Viktoria Hospital, 12157 Berlin,2 Federal Republic of Germany
Received 12 November 1999/Returned for modification 13 February
2000/Accepted 6 June 2000
 |
ABSTRACT |
Indinavir concentrations were determined in plasma and saliva over
a random period of 4 h. On average, levels in saliva were 70% ± 38% of the corresponding levels in plasma. These findings suggest that
saliva might serve as an appropriate specimen for monitoring of plasma
indinavir levels in patients treated with indinavir.
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TEXT |
The protease inhibitor indinavir
(IDV) has been shown to be an important component of triple-drug
regimens for the treatment of human immunodeficiency virus (HIV)
infection and to have excellent efficacy (6, 7). However,
its use may be associated with severe side effects such as the
development of lipodystrophy (4) or renal calculi
(8).
Pharmacokinetic studies of IDV have revealed considerable
interindividual differences in such parameters as peak concentration or
area under the concentration-time curve (AUC) (9, 12). To
date, there is preliminary evidence that trough levels of indinavir in
plasma correlate with a reduction in the viral load (1, 9,
12). However, an efficient and safe therapeutic range or
desirable target levels are still under debate.
To establish such target levels, large studies with frequent blood
sampling are required. These are cumbersome to perform and costly and
necessitate the attendance of medical staff. In contrast, monitoring of
IDV concentrations in saliva would have obvious advantages: sample
collection is easy and inexpensive and can be carried out by the
patient, even at home. The use of saliva samples would facilitate
large-scale studies on the relation of drug levels to efficacy.
Moreover, the risk of HIV transmission to the medical staff and
discomfort for the patients would be minimized. The goal of our study
was thus to evaluate the feasibility of using saliva for determination
of IDV concentrations.
Patients and methods.
Ten asymptomatic HIV-infected male
outpatients currently treated with antiretroviral combination therapies
that included IDV were asked to participate in the study. None of the
patients suffered from acute parotitis. The mean CD4 cell count was
337 ± 257/µl, and the patients' disease classifications
according to the Centers for Disease Control and Prevention were 1xB3,
1xC1, and 8xC3. The mean age of the patients was 42 ± 9 years,
and the mean weight was 71 ± 3.7 kg. Antiretroviral treatment was
as follows: 7 patients received 800 mg of IDV three times a day
(t.i.d.) in combination either with stavudine and lamivudine or with
zidovudine and lamivudine; one patient received 1,200 mg of IDV t.i.d.
with nevirapine and lamivudine; one patient received 300 mg of IDV two
times a day (b.i.d.) with ritonavir, stavudine, didanosine, efavirenz,
and hydroxyurea; and one patient received 400 mg of IDV b.i.d. with ritonavir, zidovudine, lamivudine, and efavirenz.
Blood and simultaneous saliva samples were collected every 60 min over
a period of 4 h during a routine visit to the outpatient department. To avoid interruption of the therapeutic schedule, patients
were advised to adhere to their routine drug regimen and to record the
time of the last drug intake. Thus, the time lag between the last dose
and the first sampling ranged from 0 to 6 h, and drug intake was
not under observation for most patients. For all patients except those
also taking ritonavir, IDV was administered while the patients were in
the fasted state (1 h before or 2 h after a meal).
Blood samples (2 ml placed in tubes containing EDTA) were drawn with a
peripheral indwelling catheter. Saliva was collected
by having the
patient spit into a collection tube. Blood and saliva
samples were
centrifuged within 2 h to remove cellular elements
or mucous, and
supernatants were stored at
80°C until IDV concentrations
were
measured.
The concentrations of IDV were determined by liquid
chromatography-tandem mass spectrometry. Plasma and saliva samples were
diluted 1:3 with ammonia phosphate buffer (0.1 M).
N-Ethyl-diazepam
was added as an internal standard, and
online extraction was performed
on an activated diol silica column
(ADS-6; Merck, Darmstadt, Germany).
An Eurospher RP-18 (30 mm) column
was used as a filter between
the LC-system (Merck-Hitachi, Darmstadt,
Germany) and the mass
spectrometry interface (API 365; PE-SCIEX,
Langen, Germany). The
lower limit of detection for both plasma and
saliva was 0.5 ng/ml,
the intra-assay variability was 2.8%, and the
interassay variability
was 6.5% (at 100 and 5,000 ng/ml). The method
was linear between
20 and 20,000 ng/ml (M. Kurowski, M. Mueller, K. Arasteh, and
C. Moecklinghoff, Abstr. 39th Intersci. Conf. Antimicrob.
Agents
Chemother., abstr. 320, p. 13,
1999).
The maximum concentration of drug (
Cmax) was
determined by visual inspection of the time-concentration curve. The
AUC was
determined by the log-trapezoidal method. Values were
interpreted
by linear regression analysis and with the plots of Bland
and
Altman (
3). IDV and reference material were kind gifts
of MSD,
Munich, Germany. The internal standard was purchased from
Bio-Rad
(Hercules, Calif.).
Results.
Forty-eight plasma IDV concentrations for 10 patients
were related to the corresponding concentrations in saliva. The
mean ± standard deviation (SD) Cmaxs in
plasma and saliva were 5,074 ± 5,168 and 3,340 ± 3,558 ng/ml, respectively. The mean AUCs for plasma and saliva were
9,196 ± 10,452 and 5,432 ± 5,872 ng · h/ml (62% ± 26% of that for plasma), respectively. On average, the IDV
concentrations in saliva were 70% ± 38% of the corresponding levels
in plasma. Linear regression analysis (Fig.
1) yielded correlation coefficients of
0.80 (P < 0.0001) for single values and 0.82 (P = 0.0035) for the respective AUCs. However, analysis of data for different times after the last intake of the drug revealed
that the best correlation between concentrations in plasma and saliva
was for samples collected more than 5 h after dosing (Table
1; Fig. 1). To better evaluate the
suitability of saliva as a substitute for determination of the IDV
concentration in plasma, data were plotted as described by Bland and
Altman (3) (Fig. 2A and B).
Descriptive analysis of the plots shows that the differences in the
concentrations in saliva and plasma tend to increase with increases in
the concentrations in plasma, indicating a closer relation between the
concentrations in saliva and plasma at lower concentrations, which
occurs at the end of the dosing interval.
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FIG. 1.
Scattergrams of IDV concentrations in plasma versus
those in saliva. (A) Whole sampling time (n = 48). (B)
Sampling time from >5 to 10 h after intake (n = 15).
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TABLE 1.
Evaluation of the ratio of the concentration in
saliva/concentration in plasma and linear regression analysis for the
different time periods after intake of IDV
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FIG. 2.
Bland-Altman (3) plots of the difference in
concentration in saliva and concentration in plasma versus the mean
concentrations in saliva and plasma. (A) Whole sampling time
(n = 48). (B) Sampling time from >5 to 10 h after
intake (n = 15).
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Discussion.
The feasibility of using saliva to monitor drug
concentrations has been clearly demonstrated for a variety of drugs,
e.g., phenytoin, zidovudine, and theophylline (2, 5, 10,
11).
The present study demonstrates that IDV enters the salivary
compartment. On average, IDV concentrations in saliva were lower
than
those in plasma but higher than the assumed unbound fraction
of the
drug in plasma (approximately 40%) (
13). Whether protein
binding in saliva or blood-saliva barrier-related mechanisms contribute
to this finding cannot be concluded from our
data.
In general, there was a good agreement between the IDV levels in plasma
and saliva. This was particularly true at the end
of the sampling
period suggesting a slow equilibration between
the blood and the
salivary gland compartment. Prediction of the
levels in plasma shortly
after drug intake seems to be less reliable.
However, there is evidence
in the literature that the AUC or trough
levels of IDV are associated
with antiretroviral efficacy (
1,
12). Determination of the
AUC, however, necessitates repeated
blood sampling over prolonged
periods of time, which is difficult
to perform in an outpatient
setting. Therefore, determination
of the trough concentration is more
convenient. As it is considered
important to keep trough levels above
approximately 200 ng/ml
(
1,
12), the small difference
between the concentration in
saliva and the concentrations in plasma
(Bland-Altman plot [
3])
at concentrations below 500 ng/ml (a difference of about 100 ng/ml)
for samples gathered 5 to
10 h after IDV administration indicate
that saliva might be a
substitute with which monitoring of trough
concentrations in plasma is
easy to perform. Furthermore, determination
of IDV concentrations in
saliva could provide an easy method for
determination of patient
adherence to the treatment
regimen.
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ACKNOWLEDGMENTS |
We are indebted to the patients who participated in this study. We
thank C. Moser-Juenemann (practice of E. Jaegel-Guedes and H. Jaeger)
for excellent cooperation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunology, University Children's Hospital, Lindwurmstraße 4, 80337 Munich 2, Federal Republic of Germany. Phone: 49-89 5160 5318. Fax:
49-89 5160 3964. E-mail:
uwe.wintergerst{at}KK-i.med.uni-muenchen.de.
| |
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Antimicrobial Agents and Chemotherapy, September 2000, p. 2572-2574, Vol. 44, No. 9
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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