Download Original article The protease inhibitors ritonavir and saquinavir

Antiviral Therapy 2010 15:243–251 (doi: 10.3851/IMP1509)
Original article
The protease inhibitors ritonavir and saquinavir
influence lipid metabolism: a pig model for the rapid
evaluation of new drugs
Eskild Petersen1*, Huiling Mu 2,3, Trine Porsgaard 2, Lone S Bertelsen1
Department of Infectious Diseases, Aarhus University Hospital, Skejby, Aarhus, Denmark
BioCentrum-DTU, Technical University of Denmark, Kgs, Lyngby, Denmark
3
Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Copenhagen, Denmark
1
2
*Corresponding author e-mail: [email protected]
Background: Studies of the effects of antiretroviral drugs
on lipid metabolism are limited by the availability of suitable models. We have thus developed an animal model utilising Göttingen mini-pigs. The normal lipid metabolism
of mini-pigs closely reflects that of humans and they are
expected to have similar reactions to antiretroviral drugs.
Methods: The pigs were treated orally with high doses
of the protease inhibitors ritonavir and saquinavir for
4 weeks. The model allows repeated concomitant biopsies
from liver, muscle, adipose tissue and plasma samples.
Results: The study showed a general decrease in polyunsaturated fatty acids; changes in both saturated and
monounsaturated fatty acids were also apparent after
antiretroviral treatment. The changes were observed after
4 weeks of treatment. At 4 weeks post-treatment, the
levels of all fatty acids were lower compared with pretreatment levels, suggesting a prolonged effect of the
antiretroviral drug treatment lasting beyond the 4 week
post-treatment observation period.
Conclusions: The Göttingen mini-pig model is a promising
animal model for rapid screening of the metabolic effects
induced by antiretroviral drugs.
Introduction
Antiretroviral treatment is known to contribute to the
development of lipodystrophy and such treatment can
decrease levels of high-density lipoprotein (HDL) cholesterol, increase glucose levels and promote insulin resistance [1]. Both protease inhibitors and non-­nucleoside
reverse transcriptase inhibitors are associated with
an increase in levels of low-density lipoprotein (LDL)
cholesterol, insulin resistance and impaired glucose
tolerance, as well as an increased risk of cardiovascular disease [2,3]. Protease inhibitors inhibit triglyceride synthesis and glucose transport in adipocytes, but
increase the synthesis of triglycerides in hepatocytes [4].
HIV infection itself might also contribute to the development of chronic metabolic syndrome [5].
Antiretroviral drugs, therefore, influence the metabolism towards a more diabetogenic phenotype and
increase the risk of the patient developing chronic
metabolic syndrome [6–8]. During treatment with
antiretroviral drugs, glucose release from the liver
is increased, together with the secretion of very low©2010 International Medical Press 1359-6535 (print) 2040-2058 (online)
AVT-09-OA-1352_Peterson.indd 243
density lipoproteins and increased lipolysis in adipose
tissue has also been described [9]. The mechanisms
behind these changes are not known, but some studies indicate that antiretroviral drugs inhibit enzymes in
the host [10,11] and cause mitochondrial toxicity [12].
Also, genetic analysis has implicated mutations in the
gene coding for resistin as a predictor for the development of metabolic syndrome during highly active antiretroviral therapy (HAART) [13]. Treatment of HIV
is life-long and a slow development of metabolic side
effects can be difficult to predict before the drug has
been used for several years [14]. Once the symptoms of
lipodystrophy or lipoatrophy are established, they are
difficult to treat or reverse [15].
Information on the metabolic side effects of antiretroviral drugs often takes years to accumulate; therefore,
the prospect of screening new drugs for metabolic side
effects in an animal model is very attractive. The metabolism of fatty acids in humans and pigs is very similar
[16]; thus, the mini-pig represents a suitable model and
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E Petersen et al.
was used in the present study to examine responses to
antiretroviral drugs.
The aim of this study was to establish an animal model
to predict metabolic changes caused by antiretroviral
drugs by looking at changes in the lipid metabolism
using short-term high-dose antiretroviral treatment in
pigs before, during and after 4 weeks treatment.
Methods
Experimental animals
Two Göttingen mini-pigs were kept in the stables of
Aarhus University Hospital, Aarhus, Denmark. Each
pig had a body weight of 20 kg. The animals were fed
standard food and had access to water ad libitum.
Drugs
Ritonavir was kindly donated by Abbott Laboratories
(Abbott Park, IL, USA) and saquinavir by Roche Pharmaceuticals (Basel, Switzerland). Drug concentration
measurements were performed by HPLC at the Laboratory of Clinical Pharmacology, Southern University,
Odense, Denmark. The drugs were emulsified in water
and blackcurrent taste was added; the emulsion was
then mixed with the normal pig feed. Pig A received
6,900 mg ritonavir and pig B received 21,500 mg
saquinavir plus 1,500 mg ritonavir daily. After 3 days,
the pigs accepted the feed and nothing was left of the
feed–drug mixture.
The amount of drugs available for the study were
limited, which explains why only two animals were
treated and the choice of doses.
Samples
Before the study, after 4 weeks of treatment and 4 weeks
post-treatment, the pigs where anaesthetized and plasma
samples obtained from the ear vein. At the same time,
tissue biopsies were taken from the liver, muscle and subcutaneous adipose tissue. Samples were transported in
liquid nitrogen and kept at -80°C until analysis.
Plasma levels of cholesterol and triglycerides
Plasma levels of HDL cholesterol, total cholesterol and
triglycerides were analysed using the Vitros 5.1 system
for routine clinical biochemical analysis (Ortho Clinical
Diagnostics, Rochester, NY, USA). LDL cholesterol was
calculated as total cholesterol - HDL cholesterol - 0.45×
triglycerides.
Lipid analysis of plasma, fat and liver tissue
Tissue samples were homogenized, total lipids together
with internal standards were extracted with chloroform/
methanol and separated on a silica thin-layer chromatography plate (Art 5721; Merck, Darmstadt, Germany),
which was developed in a closed chamber with hexane/
244 AVT-09-OA-1352_Peterson.indd 244
diethyl ether/acetate acid (80/20/1, v/v/v). Following
visualization by spraying with 2,7-dichlorofluorescein
(0.2% in ethanol), triglycerides and phospholipids were
scraped off and methylated by boron trifluoride (BF3)catalysed methylation. The fatty acid methyl esters were
analysed on a gas chromatograph with a 60 mm fused
silica capillary column (SP-2380) and the injector and
detector temperature were at 270°C. The carrier gas
was helium. The initial oven temperature was 70°C for
0.5 min and the temperature programming was as follows: 15°C min-1 to 160°C, 1.5°C min-1 to 200°C (maintained for 15 min) then 30°C min-1 to 225°C (maintained for 5 min). Peak areas were calculated using a
Hewlett–­Packard computing integrator. The fatty acids
were identified by comparing the retention time with
standards of known fatty acid composition (NU-ChekPrep, Elysian, MN, USA). The concentration of fatty
acids was calculated using internal standards.
Animal ethics approval
The study was approved by the Committee for Animal
Experiments, Ministry of Justice, Denmark, approval
number 2004/561-932.
Results
Pig A received 6,900 mg ritonavir daily and pig B
received 21,500 mg saquinavir plus 1,500 mg ritonavir daily. The ritonavir dose for humans is 1,200 mg
daily administered as two doses; the saquinavir dose for
humans is 2 g daily administered as two doses together
with 100 mg ritonavir twice daily to boost concentration. The total doses given per animal were thus 5.75×
the daily dose for ritonavir and 10× the daily dose for
saquinavir boosted with 7.5× the usual boosting dose
of ritonavir. The pigs had a body weight of 20 kg.
Thus, the weight-adjusted dose, assuming a standard
body weight of adult humans of 70 kg, was 20× the
standard ritonavir dose for humans and 35× the standard saquinavir dose for humans boosted with 26× the
standard ritonavir boosting dose.
Plasma concentrations of the drugs after 4 weeks
treatment (morning concentrations before feeding)
were 170 ng/ml for ritonavir and 1,542 ng/ml for
saquinavir (Table 1). Minimum plasma concentration
(Cmin) in humans receiving ritonavir 500 mg twice daily
vary considerably and the Cmin has been found to be
between 50 and 700 ng/ml [17,18]. The mean Cmin concentration after saquinavir administration in humans
has been found to be between 928 ng/ml and 1,444 ng/
ml after 48 and 4 weeks administration [17,19]. Thus,
we achieved concentrations of saquinavir close to the
median of what can be seen in humans after 4 weeks
administration; plasma concentrations of ritonavir were
in the middle of the range seen in humans (Table 1).
©2010 International Medical Press
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A pig model for the rapid evaluation of antiretroviral drugs
Table 1. Plasma drug concentrations
Treatment
Before start of treatment Drug concentration, ng/ml
After 4 weeks treatment
4 Weeks after treatment ended
Ritonavir (animal A)
Saquinavir (animal B)
Ritonavir boost (animal B)
0
0
0
169.8
1,541.5
53.1
0
0
0
Table 2. Triglyceride and cholesterol levels in plasmaa
Parameter
Normal level, mmol/l
Before start of treatment
After 4 weeks treatment
Ritonavir (animal A)
Triglyceride, mmol/l
HDL cholesterol, mmol/l LDL cholesterol, mmol/l
Total cholesterol, mmol/l
Saquinavir (animal B)
Triglyceride, mmol/l
HDL cholesterol, mmol/l LDL cholesterol, mmol/l
Total cholesterol, mmol/l
<2.5 0.23
0.25
>0.9 0.9
0.9
<4.5 1.4
1.3
<6 2.4
2.3
<2.5 0.28
0.51
>0.9
0.9
1.1
<4.5 1.1
1.0
<6 2.1
2.3
4 Weeks after treatment ended
0.15
0.8
1.0
1.9
0.14
0.8
0.6
1.5
Methods for measuring the human compounds were used and the normal levels are normal values in humans. HDL, high-density lipoprotein; LDL, low-density lipoprotein.
a
Both ritonavir and saquinavir did not influence total
triglyceride or cholesterol levels during the 4 weeks
treatment, but triglyceride levels decreased below
pretreatment levels after treatment had been stopped
(Table 2). Saquinavir treatment almost doubled triglyceride levels, but once the treatment had ended these
decreased to levels lower than those measured before
the start of treatment. LDL cholesterol levels decreased
from 1.0 mmol/l to 0.6 mmol/l 4 weeks after treatment
stopped (Table 2).
Ritonavir treatment almost tripled total liver triglyceride levels from 2.82 µg/mg to 8.16 µg/mg after the end of
treatment, but at the same time levels decreased in muscle (30.21 µg/mg to 4.39 µg/mg), adipose tissue (681 µg/
mg to 487 µg/mg) and plasma (186.6 µg/ml to 78.0 µg/
ml; Figure 1). The changes were seen in both the mono­unsaturated fatty acids 18.1n-9 and polyunsaturated
fatty acids C18:2n-6 and C20:4n-6, and the amount of
triglycerides increased above pretreatment levels after the
drug administration were stopped (Figure 1).
Saquinavir treatment more than doubled the triglyceride levels in muscle, but not in the liver and adipose
tissue where levels increased 4 weeks after the treatment
was stopped (Figure 2). There was a 50% increase in
total plasma triglycerides, but this normalized after
treatment was stopped (Figure 2). The changes were
seen in both saturated and unsaturated fatty acids in
muscle and adipose tissue, but were more pronounced
in the unsaturated C18:1n-9 and C18:2n-6 fatty acids in
plasma (Figure 2). The levels of unsaturated C18:1n-9
Antiviral Therapy 15.2
AVT-09-OA-1352_Peterson.indd 245
and C18:2n-6 fatty acids in plasma and muscle increased
during treatment and fell after the washout period. This
observation was in contrast to liver and muscle tissues, where the unsaturated C18:1n-9 and C18:2n-6
fatty acids in plasma decreased during treatment and
increased after the washout period (Figure 2).
Ritonavir treatment resulted in a 25% increase in
total phospholipids in the liver, muscle and plasma
(adipose tissue was not analysed; Figure 3). The total
amount of phospholipids in the liver increased during
treatment, but decreased 4 weeks after washout; in the
muscle; there was an increase after 4 weeks of treatment, but the levels were back to the starting level 4
weeks after treatment ended (Figure 3). Phospholipids
in plasma increased after 4 weeks of treatment and
decreased 4 weeks after treatment ended. The changes
were mostly caused by changes in the unsaturated fatty
acids C18:1n-9, C18:2n-6 and C20:4n-6, although
changes were observed in both saturated and unsaturated fatty acids (Figure 3). In both liver and muscles the
levels of unsaturated fatty acids decreased to below pretreatment levels after the treatment ended (Figure 3).
Saquinavir had no influence on the levels of total
phospholipids in muscle, but total phospholipids in the
plasma decreased during and after treatment, whereas
levels increased in the liver (Figure 4). The decrease in
plasma levels was mainly caused by a decrease in C18:0,
whereas unsaturated C18:1n-9 and C20:4n-6 remained
unchanged and C18:n2-6 almost doubled during treatment (Figure 4).
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E Petersen et al.
Figure 1. Triglyceride levels in a mini-pig model before, during and 4 weeks after treatment with ritonavir
Before
4 Weeks treatment
A
4 Weeks washout
35
30
25
20
15
10
5
Fatty acid
s
al
To
t
er
6
th
n:4
C
20
18
C
O
n-
9
9
:2
:1
n-
:0
18
C
18
C
:1
16
C
C
16
:0
Triglyceride level, µg/mg
al
To
t
s
er
6
th
O
C
20
:4
n-
n-
6
9
:2
18
C
C
18
:1
n-
:0
n7
18
C
:1
16
C
Fatty acid
200
180
160
140
120
100
80
60
40
20
0
n7
D
16
C
l
Fatty acid
800
700
600
500
400
300
200
100
0
:0
Triglyceride level, µg/mg
C
To
ta
C
C
16
:1
n7
C
18
:0
C
18
:1
n9
C
18
:2
n6
C
20
:4
n6
O
th
er
s
0
16
:0
Triglyceride level, µg/mg
To
ta
l
C
18
:1
n9
C
18
:2
n6
C
20
:4
n6
O
th
er
s
C
18
:0
C
16
:0
C
16
:1
n7
Triglyceride level, µg/mg
B
9
8
7
6
5
4
3
2
1
0
Fatty acid
Levels of triglycerides in (A) liver, (B) muscle, (C) fatty tissue and (D) plasma before, during and after (washout) treatment with ritonavir.
Discussion
Both ritonavir and saquinavir belong to the protease
inhibitor group of drugs. Despite the very high doses
of ritonavir and saquinavir used in this study, plasma
levels much above levels found in humans were not
obtained. This could be due to a very rapid drug
metabolism – both drugs are metabolised through the
cytochrome P450 system – or it could be a result of
inadequate administration through mixing the drugs
with the feed; however, the feeds were always finished.
The lower drug concentration could also be a result of
continuous feeding over the day, as compared with the
twice-daily dosing in humans. Nevertheless, the results
on lipid metabolism should still be comparable between
humans and the pig model with respect to alterations
in fatty acid composition in plasma and tissue. The
246 AVT-09-OA-1352_Peterson.indd 246
very high doses needed indicate that the drug turnover
by the cytochrome P450 system is very rapid, and it
could be argued that the high doses were responsible
for the observed changes in lipid composition; however, plasma levels reflect tissue concentrations and we
believe that plasma concentrations are more important
than the doses.
Increased plasma levels of triglycerides and LDL,
and reduced levels of HDL are found in patients receiving antiretroviral therapy [20]. Moreover, ritonavir is
known to increase triglyceride levels in a dose-dependent
manner [21,22]. Saquinavir but not ritonavir increased
triglyceride levels, which then reverted to baseline levels
or below after the washout period.
It has been proposed that HAART results in excess
circulating lipids. These lipids become deposited in
the muscles and liver, because HAART reduces lipid
©2010 International Medical Press
23/3/10 12:14:57
A pig model for the rapid evaluation of antiretroviral drugs
Figure 2. Triglyceride levels in a mini-pig model before, during and 4 weeks after treatment with saquinavir
Before
4 Weeks treatment
B
1
C
C
To
ta
l
C
18
:0
C
18
:1
n9
C
18
:2
n6
C
20
:4
n6
O
th
er
s
0
Fatty acid
Fatty acid
D
200
150
100
50
Fatty acid
s
al
To
t
er
6
9
n-
th
O
:4
n:2
20
C
9
n:1
C
18
:0
18
C
18
C
C
:1
:0
16
n7
0
C
s
250
al
To
t
er
6
th
n:4
O
6
n:2
20
C
9
18
C
18
:1
n-
:0
C
18
C
n7
:1
16
C
C
16
:0
Triglyceride level, µg/mg
1000
900
800
700
600
500
400
300
200
100
0
16
C
Triglyceride level, µg/mg
l
2
To
ta
3
16
:1
n7
C
18
:0
C
18
:1
n9
C
18
:2
n6
C
20
:4
n6
O
th
er
s
5
4
16
14
12
10
8
6
4
2
0
16
:0
Triglyceride level, µg/mg
6
C
16
:0
C
16
:1
n7
Triglyceride level, µg/mg
A
4 Weeks washout
Fatty acid
Levels of triglycerides in (A) liver, (B) muscle, (C) fatty tissue and (D) plasma before, during and after (washout) treatment with saquinavir.
storage in adipocytes and results in insulin resistance
[8]. Indeed, smaller adipocytes in adipose tissue have
been observed in HIV-infected patients during HAART
compared with controls [23]. Although we found an
increase in total plasma triglycerides after 4 weeks
of saquinavir treatment, a decrease was seen during
ritonavir treatment. This observation suggests that the
drugs might induce changes in the lipid metabolism
by different mechanisms. For both drugs, the levels of
phospholipids and triglycerides decreased below pretreatment levels 4 weeks post-treatment.
Lipoatropic tissue from patients on HAART has
reduced levels of adipogenic transcription factors [24]
and reduced messenger RNA expression of adiponectin and leptin [25], all of which indicate that apoptotic mechanisms are increased in the fat tissue during
HAART [26]. Indeed, increased adipocyte apoptosis
Antiviral Therapy 15.2
AVT-09-OA-1352_Peterson.indd 247
has been reported in biopsies of subcutaneous tissue
from patients on HAART [27]. Another study found
a direct inhibition by saquinavir on lipoprotein lipase
and at the same time increased lipolysis, but no effect
was seen on protein synthesis [28].
It can be argued that a 4-week trial time is too short,
but changes in insulin resistance have been described
in humans even after a single dose [29]. Measurements of blood glucose and insulin levels would be an
interesting addition to the measurements of triglycerides and phospholipids, as would examination of the
underlying mechanisms for lipid dysregulation – all of
which would be possible in the mini-pig model. HIV
infection, especially in patients with measurable viral
load, promotes a higher risk of developing chronic
metabolic syndrome. The Göttingen mini-pig model
can be used as a model of non-HIV-infected humans;
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E Petersen et al.
however, the HIV infection adds an additional risk
factor and we believe that the changes found here
would be augmented if we had a porcine retroviral
model available.
In the present study, treatment resulted in changes
in both saturated, monounsaturated and polyunsaturated fatty acids. These data indicate an effect mediated through elongases and desaturases, which are key
enzymes in the synthesis of fatty acids and might be
influenced directly by inhibition with the antiretroviral
drugs. Alternatively, the drugs might interact with regulatory factors like the sterol regulatory element binding
proteins, which are regulatory factors of stearoyl Coenzyme A desaturase 1 and 2 [30,31], and peroxisome
proliferator activated receptor α [32]. The prolonged
effect of the treatment seen even 4 weeks after the end
of treatment could indicate that we observe an indirect
effect of the drugs and not a direct inhibitory effect.
A recent study of genome-wide changes in the human
proteome during HAART did not report changes in
elongases or desaturases [33]. A study of elongases and
desaturases in Japanese women found that an increased
level of ∆9 and ∆6 desaturase activity and decreased
levels of ∆5 desaturase and elongase activity were associated with high body mass index and high levels of
HDL [34]. Indeed, inhibition of the elongase Elovl6 in
mice resulted in obesity and insulin resistance [35].
Genetic polymorphisms in genes encoding enzymes
in the metabolism of polyunsaturated fatty acids influence plasma concentrations of fatty acids [36]. Other
explanations could be the low levels of adiponectin
found in patients treated with HAART [37]. Adiponectin functions as an insulin sensitizer in liver, muscle
and adipose tissue [37] and a low level of adiponectin
could be a compensatory response to the loss of peripheral fat [38]. One study found that protease inhibitor
Figure 3. Phospholipid levels in a mini-pig model before, during and 4 weeks after treatment with ritonavir
Before
4 Weeks treatment
B
al
To
t
er
s
th
6
O
n-
C
20
:4
:2
n
-6
-9
Fatty acid
C
18
:0
C
16
C
16
al
To
t
er
s
6
th
n:4
O
6
n-
20
:2
C
18
C
C
18
:1
n9
:0
18
7
C
:1
n
C
16
16
C
:0
5
0
:1
n
10
18
15
C
18
20
7
25
9
8
7
6
5
4
3
2
1
0
C
30
:1
n
Phospholipid level, µg/mg
35
:0
Phospholipid level, µg/mg
A
4 Weeks washout
Fatty acid
al
To
t
18
:1
n9
C
18
:2
n6
C
20
:4
n6
O
th
er
s
C
18
:0
C
C
C
16
:1
n7
1000
900
800
700
600
500
400
300
200
100
0
16
:0
Phospholipid level, µg/mg
C
Fatty acid
Phospholipid levels in (A) liver, (B) muscle tissue and (C) plasma before, during and after (washout) treatment with ritonavir.
248 AVT-09-OA-1352_Peterson.indd 248
©2010 International Medical Press
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A pig model for the rapid evaluation of antiretroviral drugs
as a rapid screening model for the effects of new antiretroviral drugs on lipid metabolism.
The study can easily be expanded to measure
­transcription levels in different tissues and levels of hormones or other signalling factors, as well as being an
illustrative model for studying the underlying mechanisms of lipid dysregulation. Moreover, the model can
be used to test other classes of antiretroviral drugs. The
study provides a model that can be used to screen newly
developed drugs for metabolic side effects.
treatment inhibits proteasomal degradation of nascent
apolipoprotein B, the principal protein component of
triglyceride- and cholesterol-rich plasma lipoproteins.
Protease inhibitors were also found to inhibit the
secretion of apolipoprotein B [39].
Reduced growth hormone response to insulin and
reduced insulin growth factor 1 (IGF1) levels have been
found during HAART [40], but a recent study of treatment with recombinant human leptin of patients with
HAART-induced lipoatrophy and metabolic syndrome
found that the effect was not mediated by IGF1 or
IGF1-binding proteins [41].
The present study was limited by the amount of drugs
available, which resulted in only one animal being treated
with each drug and the use of only two drugs. However,
we have shown that the model can be used for studying
changes in lipid composition in several tissues simultaneously over just 4 weeks; the model therefore has promise
Acknowledgements
Ritonavir was a kind gift from Abbott Laboratories
and saquinavir was a kind gift from Roche Pharmaceuticals. The plasma cholesterol and triglyceride
measurements were performed by the Department
of Clinical Chemistry, Aarhus University Hospital,
Figure 4. Phospholipid levels in a mini-pig model before, during and 4 weeks after treatment with saquinavir
Before
4 Weeks treatment
B
6
Phospholipid level, µg/mg
al
To
t
er
s
6
th
n:4
O
6
n:2
20
C
:1
16
9
C
C
C
C
16
n7
l
:0
0
To
ta
er
s
th
-6
O
20
:4
n
-6
9
:2
n
n-
18
:1
18
C
C
7
18
C
:1
n
16
16
C
C
:0
0
1
n-
5
2
18
10
3
:1
15
C
20
5
4
:0
25
18
30
C
18
35
:0
Phospholipid level, µg/mg
A
4 Weeks washout
Fatty acid
Fatty acid
l
To
ta
:4
n6
O
th
er
s
n6
C
20
n9
18
:2
C
18
:1
:0
C
18
C
C
16
:1
16
C
n7
900
800
700
600
500
400
300
200
100
0
:0
Phospholipid level, µg/mg
C
Fatty acid
Phospholipid levels in (A) liver, (B) muscle tissue and (C) plasma before, during and after (washout) treatment with saquinavir.
Antiviral Therapy 15.2
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E Petersen et al.
Skejby, Denmark and the drug concentrations were
performed by Ulrik Stenz Justesen, Institute of Public
Health, Clinical Pharmacology, University of Southern Denmark. The study was supported by the Clinical
Institute, Aarhus University.
Disclosure statement
The authors declare no competing interests.
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Accepted for publication 22 October 2009
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