Download the evaluation of radiolabeled artesunate on tissue distribution in

Am. J. Trop. Med. Hyg., 75(5), 2006, pp. 817–826
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
THE EVALUATION OF RADIOLABELED ARTESUNATE ON TISSUE
DISTRIBUTION IN RATS AND PROTEIN BINDING IN HUMANS
QIGUI LI,* LISA H. XIE, ADAM HAEBERLE, JING ZHANG, AND PETER WEINA
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland
Abstract. The present study reports the tissue distribution, pharmacokinetics, mass balance, and elimination of [14C]
artesunate (AS) following single intravenous administration in rats. Protein binding was performed with rat and human
plasma. Radioactivity and drug levels in blood, plasma, tissues, urine, and feces up to 192 hours were collected and
measured. The mean terminal half-life of plasma (76 h) and blood (105 h) radioactivity was prolonged compared with
that of unchanged AS (0.43 h) and dihydroartemisinin (0.75 h), an active metabolite of AS. Drug was widely distributed
after 1 hour in select tissues. After 24 hours, the radioactivity rapidly declined in all tissues except spleen until 96 hours.
Only 1% of total radioactivity was detected in brain tissue. AS revealed a higher binding capacity with human and rat
plasma proteins (73–81%). The radioactivity in whole blood was higher (two to fourfold) than that in plasma throughout
the period of the treatment, suggesting that AS binding to RBCs may relate to its powerful antimalarial activity.
certain artemisinin derivatives requires knowledge of the penetration of the cerebrospinal fluid (CSF) by these drugs. It has
been shown that artemisinin derivatives cross the blood–brain
barrier in rats;24 however, concentrations of artemether, another artemisinin derivative, in the CSF of dogs were low and
there was no detectable penetration by its active metabolite,
DHA.20 Artesunate is converted stoichiometrically to DHA
with peak plasma concentrations occurring after 10 minutes.8,16 DHA is highly lipid soluble and has a low molecular
mass (284 Da), favoring penetration of CSF.25 DHA has a
relatively low solubility in water and would be able to cross
cell membranes. After AS treatments in human subjects no
AS was detected in CSF; DHA levels in CSF increased with
time while DHA levels in plasma fell, suggesting a continuing
influx but a slower efflux of DHA. DHA may accumulate in
CSF during frequent artemisinin dosing.25
There is little or no information on the distribution and
excretion of AS and DHA using radiolabeled drug and specific analytical techniques. For tissue distribution, a radioimmunoassay method was used to study distribution and excretion of AS in rats in China.26 This method detected the parent
compound and its metabolites in various tissues. Ten minutes
after IV administration of AS the greatest to least drug concentration in the organs and tissues were found to be in the:
intestines > brain > liver > kidney > testicle > muscle > fat >
heart > serum > eyeball > spleen > lung. After 60 minutes, the
concentration of drug and metabolites significantly decreased
in all tissues; data showed brain, fat, intestine, and serum
maintained relatively high concentrations. Drug and metabolites detected in other tissues was very low. Less than 1% of
the injected dose was found in the urine and feces collected
during the first 24 hours after administration. This method did
not discriminate between AS and DHA, but measured total
drug and metabolites.
A previously published paper has shown preliminary results of AS tissue distribution in rats, but did not reveal quantitative and kinetic profiles of AS in each organ.26 There have
been no published reports of pharmacokinetics, mass balance
and elimination data for AS or DHA, including no distribution data on AS in the brain and red blood cells. The present
study was undertaken to ascertain the tissue distribution,
pharmacokinetics, mass balance, and elimination of radiolabeled AS, allowing separation and measurement of AS and
DHA in rats.
INTRODUCTION
Artesunate (AS), an intravenous (IV) drug for the management of severe and complicated malaria, has been used for
decades in malaria endemic parts of the world. Clinical data
suggests the drug is both safe and highly effective.1,2 The
effectiveness of AS has been attributed to its rapid and extensive hydrolysis to dihydroartemisinin (DHA), which is
threefold to fivefold more active, and toxic, than the parent
compound.3–6 DHA rapidly and effectively fights all stages of
the parasite life cycle and completely inhibits parasite growth
within the shortest exposure time as compared with all other
artemisinin and conventional antimalarial drugs.7
The pharmacokinetic parameters of AS in humans are very
well known. Detailed pharmacokinetic data for AS and its
active metabolite DHA have been reported in adults8–10 and
children11 infected with malaria, and in healthy volunteers.12,13 Published work has shown AS to have an elimination half-life (t1/2) of 2–5 minutes and DHA to have a t1/2 on
the order of 40–90 minutes. In animals the pharmacokinetic
(PK) profile of both drugs has been clearly defined. The conversion of AS to DHA in human pharmacokinetic studies has
been shown to be much more efficient than in animal species.
The ratio of AUCDHA to AUCAS in humans had a range of
4.3–9.7, compared with rats and dogs with a ratio of only
0.5–1.1.9,14–18 The ratio in humans was 4.5–13.5 times higher
than that in animals.
Artemisinin derivatives are potent antimalarial drugs, but
concerns have been raised as to their neurotoxic potential.
Brain stem injury associated with the artemisinin derivatives,
arteether (AE) and artemether (AM), has been shown in
laboratory animals under experimental conditions, but despite their widespread use evidence of neurotoxicity at clinical
doses in humans is weak.19–22 In vitro testing has shown inhibition of neuronal development by artemisinin drugs at concentrations achieved in plasma during the management of
malaria.23
An understanding of in vivo neurotoxicity secondary to
* Address correspondence to Qigui Li, Division of Experimental
Therapeutics, Walter Reed Army Institute of Research, 503 Robert
Grant Avenue, Silver Spring, MD 20307. E-mail: [email protected]
.army.mil
817
818
LI AND OTHERS
MATERIALS AND METHODS
Chemicals. The test unlabeled compound used in this study
is artesunic acid (4-(10’dihydro-artemisinin-oxymethyl) succinate; AS), which was manufactured as a phosphate salt with
0.3 M PBS and obtained from the Walter Reed Chemical
Inventory System. Unlabeled AS (WR256283, BN: BQ38841,
Lot N# 2.03) was synthesized and manufactured by Knoll AG
and rebottled by BASF Pharmaceuticals. It was supplied
through the Division of Experimental Therapeutics at Walter
Reed Army Institute of Research (WRAIR, Silver Spring,
MD). One lot of [16-14C] artesunic acid (Lot No. 10839-113)
was used in this study and it was synthesized by Research
Triangle Institute (Research Triangle Park, NC). The stated
specific activity and radiochemical purity was 17.63 mCi/
mmol (45.86 ␮Ci/mg) and 97.0%, respectively. Unlabeled AS
and radiolabeled compounds were stored at approximately
−20°C.
Animals. Male Sprague-Dawley rats obtained from Charles
River Laboratories were used in this study. On arrival, the
animals were acclimated for 7 days (quarantine). The animals
were housed individually and maintained in a room with a
temperature range of 64–79°F, 34–68% relative humidity, and
12-h light:dark cycles. Food and water were supplied ad libitum during quarantine and throughout the study. The animals
were fed a standard rodent maintenance diet. The animal
protocol was approved by IACUC, WRAIR, and the research
was conducted in compliance with the Animal Welfare Act
and other federal statutes. Regulations relating to animals
and experiments involving animals adhered to principles
stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition.
Pharmacokinetic (PK) study. Traditional PK studies involve intermittent blood sampling and subsequent determination of drug concentrations in plasma. The Culex automated blood sampler provides a way for blood withdrawal at
preprogrammed intervals, which is more accurate and less
labor intensive.27 Six rats in the PK group were implanted
with a jugular vein catheter under an isoflurane anesthesia.
After the surgery, rats were allowed to recover for 96 hours
with free access to food and water. The rats were dosed single
IV at 5 mg/ml/kg with [14C] AS (20 ␮Ci/kg) through the tail
vein. A 100–200 ␮L blood sample was withdrawn from the
jugular vein catheter into a cooled vial, containing powdered
heparin, at 5, 10, 30 minutes and 1, 1.5, 3, 6, 12, 24, 48, 72, 96,
and 192 hours post-dosing according to a preset schedule.
Tissue distribution of [14C] artesunate study. Rats treated
with a single [14C] AS dose of 5 mg/20 ␮Ci/kg intravenously (5
rats per group) were anesthetized by isoflurane and euthanized via cardiac puncture at 1, 6, 24, 48, 72, 96, and 192 hours,
post-dosing. Blood, liver, brain, eyes, adrenals, muscle, lungs,
heart, liver, kidneys, spleen, stomach, small intestine, large
intestine, perirenal fat, and bone marrow samples were removed from each animal by gross dissection. The gastrointestinal tract (GI) was separated into the stomach (esophagus
and stomach), small intestine, and large intestine. Contents
were procured from each GI segment. After flushing with
saline, these and other tissues were stored. Tissues were
rinsed gently, but thoroughly with water to remove remaining
traces of blood before storage. Dissecting instruments were
also washed between tissue procurements to avoid crosscontamination.
For most of the tissues 20% (w/w) aqueous homogenates of
the tissue were prepared. Aqueous homogenates of the fecal
and GI content samples were also prepared. Metabolism
cages were washed with a solution of 2% Count-off威 and the
volume measured. Radioactivity in the plasma samples and in
aliquots (approx. 0.02–1.0 mL) of urine and cage washings
was determined. The blood cells (primarily erythrocytes),
perirenal fat (approx. 0.2 g), bone marrow samples (approx.
0.1 mL), and approximately 300-␮L aliquots of the homogenates of feces, liver, kidneys, heart, brain, stomach, and carcass were solubilized with SolvableTM tissue solubilizer (Perkin-Elmer Life and Analytical Sciences, Boston, MA). The
homogenates of feces, stomach content, and intestinal contents were extracted with 50% methanol for 24 hours. Aliquots of blood and pooled urine samples from selected intervals were also analyzed for AS content directly.
Total radioactivity in the collected tissue samples were
quantified in duplicate. Weighed tissue aliquots (∼0.2 g) were
digested overnight in 2 mL of Solvable威 tissue solubilizer
(Perkin-Elmer Life and Analytical Sciences, Boston, MA) at
50°C. Colored samples were decolorized by adding a maximum of 0.2 mL of 30% H2O2 for 4 hours. Scintillation cocktail (Hyonic Fluor; Perkin-Elmer Boston, MA) was then
added and radioactivity determined in a Perkin-Elmer TriCarb 3100 TR scintillation spectrophotometer (Packard Instrument Co., Downers Grove, IL). Feces and intestinal contents were thoroughly homogenized before taking aliquots.
Plasma was also analyzed using the HPLC-ECD system to
measure the concentration of unchanged AS, as described by
Li et al.,17,18 to assay AS and its main and active metabolite,
dihydroartemisinin (DHA).
Mass balance and elimination of [ 14 C] artesunate
study. Samples of urine and feces were collected for a period
of 192 hours after a single IV injection at 1, 8, 24, 48, 72, 96,
120, 144, 168, and 192 hours from individual stainless steel
Nalgene metabolic cages (Nalgene Company, Rochester,
New York) designed for separate collection of urine and feces. Metabolic cages were washed with a solution of 2%
Count-off威 (Perkin-Elmer Life and Analytical Sciences, Boston, MA) and the volume measured. The volume of each
urine and cage rinse was measured. Duplicate portions (0.1
mL urine; 1 mL cage rinse) of each sample were radioassayed
after the addition of scintillation cocktail. The weights of individual fecal samples were obtained and the samples were
subsequently homogenized in 4 volumes of deionized water.
Quadruplicate portions (1.0 mL) of each homogenate were
decolorized with 30% hydrogen peroxide, and digested with
Solvable® tissue solubilizer. After acidification with glacial
acetic acid and addition of scintillation cocktail, samples were
assayed for radioactivity. In addition, urine samples collected
from each time point after dosing were assayed by HPLCECD to obtain a metabolite profile of the radioactivity in
each sample. The samples were kept in at −20°C for 2 weeks
prior to analysis.
Metabolic profile of [14C] artesunate study. Metabolic profile evaluation of [14C] AS in plasma and whole blood, as well
as unchanged AS in plasma was conducted in individual rats.
Blood samples (7–8 mL) were collected from animals at 5, 10,
30 minutes and at 1, 1.5, 3, 6, 12, 24, 48, 72, 96, and 192 hours
post-dosing when rats were euthanized. About 2 mL of
plasma and urine, and 1–2 g of feces were used. Conjugates
were hydrolyzed by glucuronidase (1000 FU/ml) at pH 4.6,
819
TISSUE DISTRIBUTION OF [14C] ARTESUNATE IN RATS
38°C for 24 hours. Active enzyme was checked by phenophthaleinglucuronide at the end of incubation. Urine and fecal samples were incubated for 48 hours and fresh enzyme
was added after 24 hours. Total radioactivity in free, conjugated, and the remaining aqueous fractions were measured
with liquid scintillation counters. Protein precipitates were
dissolved in Solvable® (200 mg in 1 mL) and counted for
radioactivity after addition of scintillation cocktail.
Protein binding of [14C] artesunate study. Human blood
samples (5 men and 5 women) were freshly obtained from
subject volunteers at the Department of Clinical Trials,
WRAIR. Rat samples were freshly collected from 5 male and
5 female rats. Equilibrium dialysis was performed using Teflon dialysis chambers and Diachema standard membranes as
described by Li et al.28 The GD-4/90 Equilibrium Dialyzer
(MM Developments, Ottawa) contained 20 micro-dialysis
cells (2.0 mL/chamber). The molecular weight cutoff for dialysis membranes was 5000 D. Equilibrium was reached after
4 hours when cells were rotated at 16 rpm and 37°C. Drugs
were dissolved in plasma samples with various concentrations
(0.2–78125 ng/ml) and all experiments were carried out for 5
hours.
HPLC-ECD method. HPLC with reductive electrochemical detection (HPLC-ECD) was performed utilizing a model
BAS 200B liquid chromatography system (Bioanalytical Systems, West Lafayette, IN). The system had 3 mobile phase
reservoirs, solenoid proportioning valves, a dual piston pump,
a pulse dampener, a column and detector oven, dual thinlayer electrodes with Ag/AgCI reference electrode, and a
Rheodyne injector for manual injection that was modified for
reductive work. The system was also equipped for mobile
phase heating and sparging. Stainless steel connectors and
tubing were used throughout the system. For simultaneous
determination of AS and its major metabolite DHA (artemisinin as an internal standard), the Waters ␮Bondapack
(Waters Associates, Milford, MA), CN column (4.6 mm × 30
cm), and a mobile phase consisting of 30% acetonitrile: 70%
0.1 M acetic acid/NaOH buffer (pH 4) was used. Compounds
were detected via reductive electrochemical detection. Data
was acquired and analyzed using a Waters model 820chromatography data system, Maxima program (Waters Associates, Milford, MA). Reproducibility was good with a
lower limit of quantitation of 2.5 ng/ml for AS and 4 ng/ml for
DHA. The inter- and intra-day coefficient of variation for
accuracy and precision was ± 10%.
Detector response linearity studies were performed by preparing 6 duplicate calibrations covering a range of 2.5–1000
ng/ml for AS and DHA. Linear regression lines were obtained by plotting the peak area ratios (target peak areas
divided by internal standard peak areas). To evaluate the
reproducibility (within and between-days precision) of the
method, duplicate analyses (N ⳱ 6) of plasma spiked with AS
were carried out. The absolute recoveries of AS were determined by comparing the peak areas of spiked plasma samples
and reference samples. The reference samples were injected
directly into the HPLC for both detections. The limit of detection for AS and DHA was determined as the lowest concentration giving a response with 1.0–2.5 ng/ml plasma in the
spiked serum sample for AS and DHA (signal-to-noise ratio
of 3). The limit of quantitation (lowest concentration of the
calibration curve) was 1.8 ng/ml in spiked plasma for AS.
Data evaluation. For PK, the concentration-time data of
[14C] AS in plasma and whole blood collected during the
192-hours treatment period was fitted to a 3-compartment
open model using a nonlinear, extended least-square fitting
procedure (WinNonlin 4.1, Scientific Consulting, Inc. Apex,
NC) using weighted (1/concentration) nonlinear regression.
The area under the curve (AUC) was determined by the linear trapezoidal rule with extrapolation to infinity based on
the concentration of the last time point divided by the terminal rate constant. Extrapolations to time zero were done using
zero concentration for intravenous dosing and using C0 values
determined from the 3-compartment model equation at time
zero by IV route. Mean clearance rate (CL) was determined
by dividing the dose by the AUCinf for IV injection. Mean
residence time (MRT) was determined by dividing the area
under the first moment curve (AUMC) by the AUC. The
volume of distribution at steady state (Vss) was calculated as
the product of CL and MRT. The concentration-time data of
unchanged AS was fitted to a 2-compartment open model.
The ratio of DHA to AS was calculated by AUCDHA/
AUCAS. Statistical analysis was conducted with Microsoft Excel using a Student’s t test for dependent samples to compare
means of paired and unpaired samples between the two
groups.
RESULTS
Dose formulation analysis. The results of the analysis of the
IV formulations of [14C] AS are designated as 5 mg/20 ␮Ci/ml
in this study. Chemical concentration and radioactivity analysis of samples taken from each dosing solution indicated the
formulation was homogeneous. The mean concentration of
AS in the IV formulation was 4.77–5.20 mg/ml. The radioactivity content of the IV formulation was detected between
19.87 and 21.68 ␮Ci/ml. The radiochemical purity of [14C] AS
was determined to be greater than 97% while cold AS was
greater than 99%.
Pharmacokinetics of [14C] artesunate, unchanged artesunate, and dihydroartemisinin. Pharmacokinetic parameters
calculated from mean levels of total radioactivity, unchanged
AS, and unchanged DHA in either whole blood or plasma
after single IV administration of [14C] AS are shown in Table
1. The concentration-time data of [14C] AS in plasma and
blood (Figure 1) collected during the 192 hours of the treatment period were fitted to a 3-compartment open model.
Radioactivity derived from [14C]AS was eliminated from
plasma in 3 phases with half-lives of 0.12, 13.2, and 76.2 hours,
and from blood in 3 phases with half-lives of 0.1, 26.5, and
104.7 hours. Unchanged AS was eliminated from plasma in 2
apparent phases with half-lives of 0.14 and 0.43 hours. Unchanged DHA, the active metabolite of AS, was eliminated
from the plasma in 2 apparent phases with half-lives of 0.21
and 0.75 hours (Table 1).
Peak concentrations (Cmax) of [14C] AS and unchanged AS
in plasma at 0.1 hour were 4,271 ng equivalents/ml and 1,627
ng/ml, respectively. The corresponding AUCs of [14C] AS and
unchanged AS were 21,702 ng equivalents⭈h/ml and 392 ng⭈h/
ml, suggesting AS is extensively metabolized in rats. Similar
to the AUC, the volume of distribution at steady state (Vss)
was 16,081 mL/kg for radiolabeled drug and 3,982 mL/kg for
unchanged AS. Calculated clearance values for radioactivity
and unchanged AS in plasma were 237 and 12,719 mL/hr/kg,
respectively (Table 1).
820
LI AND OTHERS
TABLE 1
Comparison of main pharmacokinetic parameters of [14C] artesunate in plasma and blood, as well as unchanged artesunate (AS) and dihydroartemisinin (DHA) in plasma following a single intravenous dose at 5 mg/20 ␮Ci/kg in rats*
Parameters
Dose
14
C-Artesunate
5 mg/20 ␮Ci/kg
Unchanged AS
5 mg/kg
Unchanged DHA (metabolite of AS)
Sample
PK fitting
Plasma
3-compartment
Blood
3-compartment
Plasma
2-compartment
Plasma
2-compartment
Cmax (ng/ml
Tmax (hr)
AUCinf. (ng⭈g.h/ml)
t1⁄2-alpha (hr)
t1⁄2-beta (hr)
t1⁄2-gamma (hr)
CL (ml/hr/kg)
Vss (ml/kg)
MRT (hr)
Ratio of DHA/AS
Cmax at 1st peak (ng/ml)
Tmax at 1st peak (hr)
Cmax at 2nd peak (ng/ml)
Tmax at 2nd peak (hr)
4271 ± 1482
0.0
21702 ± 2949
0.12 ± 0.05
13.24 ± 6.14
76.17 ± 16.68
237.2 ± 35.2
16081 ± 4191
66.98 ± 8.60
4527 ± 1016
0.0
66213 ± 5918
0.10 ± 0.01
26.54 ± 2.95
104.7 ± 24.48
81.46 ± 7.93
6183 ± 1732
79.01 ± 24.17
1627 ± 415
0.0
392.2 ± 63.3
0.14 ± 0.02
0.43 ± 0.04
–
12719.3 ± 1751.0
3982 ± 1022
0.31 ± 0.05
468 ± 92
0.27 ± 0.04
376.1 ± 53.8
0.21 ± 0.03
0.75 ± 0.20
–
2631 ± 692
0.083
744.4 ± 200.2
3.25 ± 1.47
2999 ± 789
0.083
1316 ± 178
3.75 ± 1.84
1026 ± 183
0.083
–
–
1.00 ± 0.31
0.98 ± 0.22
468 ± 92
0.27 ± 0.04
–
–
* DHA ⳱ dihydroartemisinin, a main and more active metabolite of artesunate; PK ⳱ pharmacokinetics; MRT ⳱ mean residence time; MAT ⳱ mean arrival time; – ⳱ no data is available.
Mean ± SD (n ⳱ 6).
Following the IV administration of [14C] AS two peaks of
radioactivity were detected (Figure 1, re-plot). The first peak
concentration of radioactivity, 2,631 and 2,999 ng equivalents/
g, was present in plasma and whole blood, respectively, at 5
minutes (earliest measured time point) after dosing. The second peak formed at 3.3 hours with plasma at 744 ng equivalents/ml and at 3.8 hours for whole blood with 1316 ng equivalents/g. The peak concentration of unchanged AS was 1026
ng/ml, but a second peak was not detected in the unchanged
AS. Levels of radioactivity in blood decreased at a slower rate
than in plasma, indicating a majority of the radioactivity in
blood was associated with red blood cells, thus reducing drug
clearance and distribution (Figure 1).
The individual and mean plasma concentration-time curves
following the single IV injections of unchanged AS and its
FIGURE 1. Total radioactivity (ng equivalents per ml) of [14C]
artesunate in whole blood (dashed line with triangular markers) and
in plasma (solid line with circular markers), and plasma concentration
of unchanged artesunate (AS) (dotted line with dot makers in replot) and unchanged dihydroartemisinin (DHA, dashed line empty
circle in re-plot) following a single 5 mg/20 ␮Ci/kg intravenous injection of [14C] AS in rats (N ⳱ 6). This figure appears in color at
www.ajtmh.org.
active metabolite, DHA, are shown in Figure 1. The conversion of AS to DHA is presented in Table 1. DHA had a peak
plasma drug concentration (Cmax) of 468 ng/ml at 0.27 hours
after dosing. The AUC ratio for unchanged DHA (376 ng⭈h/
ml) to unchanged AS (392 ng⭈h/ml) was 0.98 following the
single dose.
Tissue distribution of [14C] artesunate. In tissues collected
at 1 hour, approximately 68% of radioactivity in the total
measured tissues was amassed in the small intestine and its
content. Lesser amounts of radioactivity were distributed in
other tissues (Table 2). The total amount of radioactivity in all
measured tissues per gram was 90,502 ng equivalents (Table
2). Six hours after the injection, high levels of radioactivity
were still present in the intestine, colon, and in their contents
with slow loss; however, drug distribution was significantly
increased in other tissues such as kidney, spleen, liver, heart,
adrenals, blood, muscle, colon, and colon content (Figure 2).
The total amount of radioactivity in all measured tissues per
gram at 6 hours increased to 107,210 ng equivalents. The
remarkable increases of radioactivity in other tissues at 6
hours may relate to re-distribution from tissues.
From 24–192 hours, measured radioactivity rapidly declined in all tissues except for the spleen (Figure 2). The total
amount of radioactivity in all measured tissues per gram was
40,278, 30,406, 22,581, 16,704, and 8,117 ng equivalent at 24,
48, 72, 96, and 192 hours, respectively (Table 2). At 192 hours
after dosing, residual activity (close to 2.57% of total amount
collected) was still detected in some tissues and the highest
levels of radioactivity were measured in spleen, kidney,
adrenals, and heart; trace amounts were found in other tissues. Radioactivity in the blood and plasma was measured in
quantifiable levels at 192 hours after the single IV injection
(Figure 1). Radioactivity in whole blood was always higher
(2.1–4.2 fold) than in plasma throughout the period measured
(Figure 1). Unchanged AS was eliminated within 2 hours
(Table 1 and Figure 1), indicating long-lasting radioactivity to
be potentially resultant of metabolites.
During the treatment period, measured levels of radioactivity were more than twofold higher in the brain than in
821
TISSUE DISTRIBUTION OF [14C] ARTESUNATE IN RATS
TABLE 2
Distribution (ng equivalents/tissue gram), half-lives, and area under the curve (AUC of [14C]-AS in main organs and tissues of the rats following
single intravenous dose at 5 mg/20 ␮Ci/kg and animals were euthanized at 1, 6, 24, 48, 72, 96, and 192 hr after dosing (n = 5)
Organs
1 hr
6 hr
24 hr
48 hr
72 hr
96 hr
192 hr
(ng equivalents per gram of organ and tissue)
Blood
Plasma
Brain
Eyes
Adrenals
Fat
Muscle
Lungs
Heart
Spleen
Kidneys
Liver
Stomach
Stomach content
Large intestine (LI)
LI content
Small intestine (SI)
SI content
Bone marrow
Total amount
% of total recovery
687.93
499.05
691.86
1373.05
2688.05
473.38
404.29
612.84
811.25
3707.27
2401.76
3638.56
756.99
327.62
1429.32
1186.58
11463.6
55906.5
1441.56
90501.51
28.66
990.04
740.54
591.37
560.03
3122.01
501.38
815.55
1568.24
1967.07
6448.14
7234.39
5417.10
1753.69
2171.11
6006.64
9421.07
7453.89
50448.0
107210.28
33.95
868.38
317.41
735.46
461.82
1874.82
96.45
242.76
2579.44
4313.07
5282.39
4975.95
2088.16
782.51
213.63
2548.93
6601.61
1484.20
4192.94
617.65
40277.56
12.75
291.10
133.25
365.69
208.62
1602.52
122.27
214.15
458.43
626.10
6132.07
6643.95
1866.09
483.68
187.00
1194.68
3831.90
967.61
5077.14
298.86
114.83
257.87
248.95
1372.77
132.87
252.51
452.96
696.07
6096.56
3209.11
1787.88
495.14
116.70
1463.99
1378.43
733.54
3471.98
30406.25
9.63
22581.03
7.15
145.28
59.03
168.75
44.97
1049.10
70.81
129.71
335.15
581.53
6539.26
2341.82
1261.59
356.16
28.86
443.89
842.04
417.34
1734.62
154.16
16704.08
5.29
51.17
22.97
123.52
60.00
754.01
57.48
100.12
191.87
338.66
3524.37
1372.32
544.39
209.30
56.83
193.98
255.77
202.40
57.47
8116.63
2.57
Half-lives
Tissue AUC
% of Total
(hr)
(ng⭈hr/g)
AUC
98.8 ± 25.7
63.7 ± 10.7
94.2 ± 11.4
62.1 ± 12.9
109.6 ± 11.7
115.5 ± 10.4
105.8 ± 6.3
123.8 ± 6.7
108.6 ± 6.6
154.3 ± 15.7
94.2 ± 13.5
78.9 ± 3.3
93.5 ± 5.4
77.9 ± 4.1
69.5 ± 10.6
65.6 ± 11.9
74.9 ± 11.6
29.4 ± 3.8
40.0 ± 9.5
75.37 ± 5.64
104303 ± 81099
29425 ± 2455
70723 ± 12828
42505 ± 6012
374210 ± 29758
32176 ± 4533
54555 ± 9693
160034 ± 15147
251987 ± 10129
1860779 ± 315462
832224 ± 182922
363288 ± 36099
122124 ± 6683
48694 ± 14480
246379 ± 63649
462496 ± 147705
249033 ± 10233
1310061 ± 819635
52186 ± 8680.79
6635878.91
1.57
0.44
1.07
0.64
5.64
0.48
0.82
2.41
3.80
28.04
12.54
5.47
1.84
0.73
3.71
6.97
3.75
19.74
0.81
100.00
Mean ± SD (n ⳱ 5).
plasma; the AUC of radioactivity from 0–192 hours was
70,723 ng⭈h/g in the brain and 29,425 ng⭈h/ml in the plasma.
The highest levels of the radioactivity were detected in the
spleen. The measured amount was 3,707 ng equivalents/g at 1
hour and rapidly increased to 6,448 ng equivalents/g at 6
hours after dosing. In the post 6-hour period the amount
remained between 5,283 to 6,539 ng/g until 96 hours (Table
2). At 192 hours the radioactive amount was measured at
3,524 ng equivalent/g. During the 192-hours treatment period
the radioactivity in spleen was the highest with an AUC of
1,860,779 ng⭈h/g and the highest percentage of total radioactivity (28.0%) when compared with other tissues (Table 1). In
other measured tissues the highest to lowest AUCs were
found as follows: small intestine content (1,310,061 ng⭈h/g),
kidneys (832,224 ng⭈h/g), large intestine content (462,496
ng⭈h/g), adrenals (374,210 ng⭈h/g), liver (363,288 ng⭈h/g),
heart (251,987 ng⭈h/g), small intestine (249,033 ng⭈h/g), large
intestine (246,379 ng⭈h/g), and lung (160,034 ng⭈h/g).
The longest half-life of radioactivity was measured in the
spleen at 154.3 hours, followed by the lungs (123.7 hours), fat
FIGURE 2. Mean distribution profile of total radioactivity (ng equivalents per gram tissue) of radiolabeled artesunate (AS) in various tissues
(column) at 1, 6, 24, 48, 72, 96, and 192 hours following a single 5 mg/20 ␮Ci/kg intravenous injection of [14C]AS in rats (n ⳱ 5). This figure appears
in color at www.ajtmh.org.
822
LI AND OTHERS
(115.4 hours), adrenals (109.6 hours), heart (108.6 hours),
muscle (105.8 hours), and blood (98.7 hours). In contrast,
radioactivity found in the small intestine, large intestine, and
their contents had short half-lives of 29.4–74.8 hours.
Mass balance determination. The total recovery of radioactivity in excreta (urine and feces) and cage rinses collected
from animals through 192 hours after single IV administration
is shown in Table 3. Through 192 hours after injection, approximately 56.1%, 38.5%, and 1.6% of the dose was eliminated in urine, feces, and cage rinses, respectively. The urinary excretion was the major route of elimination, accounting
for 48–66% of the administered dose. Feces excretion was the
secondary route of elimination, accounting for 37–44% of the
administered dose. The recovery of [14C] AS from urine, feces
and cage washing solution was 96% of total dose during the 9
days of collection.
Urinary and fecal elimination. The elimination parameter
estimates after intravenous dosing of [14C] AS at 5 mg/kg are
presented in Table 3. Results showed that greater than 73%
of the total dose was excreted within first 24 hours. The maximum [14C] radioactivity concentration in urine was observed
at 0.48 days with 381 ␮g equivalents per rat (Figure 3). The
elimination of [14C] label from urine occurred in 2 phases, fast
and slow, with half-lives of 0.37 and 2.06 days (Table 3). Similar to urine, fast and slow elimination phases were also exhibited in feces. A peak concentration of 301 ␮g equivalents
per rat was seen in fecal matter at 0.53 days after dosing
(Figure 3). The half-lives of [14C] radioactivity were 0.57 and
2.54 days for fast and slow phases in feces, respectively. The
elimination of radioactivity in feces was delayed with a lag
time of 0.29 days (Table 3), while the lag time for urine was
0.02 days (Figure 3).
Metabolite profiles of [14C] artesunate. Unchanged AS was
rapidly and completely eliminated from rats within 2–3 hours
following single IV administration. The total radioactivity
TABLE 3
Percentages of renal and fecal excretion of total radiolabel following
single intravenous administration of [14C] artesunate with phosphate buffer (pH 8.1) formulation at 5 mg/20 ␮Ci/kg to rats
(n ⳱ 5)*
Duration (days)
Urine (%)
Feces (%)
0–0.04
1.61 ± 1.07
0.04–0.33
19.19 ± 2.73
1.10 ± 0.58
0.33–1
26.76 ± 6.94
24.63 ± 5.45
1–2
5.07 ± 0.96
7.50 ± 1.98
2–3
1.50 ± 0.33
2.60 ± 0.60
3–4
0.83 ± 0.13
1.25 ± 0.34
4–5
0.47 ± 0.17
0.56 ± 0.11
5–6
0.33 ± 0.09
0.36 ± 0.11
6–7
0.20 ± 0.11
0.27 ± 0.08
7–8
0.13 ± 0.06
0.18 ± 0.09
Total
56.06 ± 7.70
38.46 ± 6.26
Ratio of excretion
(urine/feces)
1.48 ± 0.26
Balance (% of dose)
96.07 ± 11.03
Elimination parameters
Peak height (␮g/rat) 381.06 ± 46.72 300.96 ± 89.58
Peak time (days)
0.48 ± 0.13
0.53 ± 0.04
Fast elimination
phase (days)
0.37 ± 0.03
0.57 ± 0.07
Slow elimination
phase (days)
2.06 ± 0.46
2.54 ± 0.46
* All values expressed as % of dose, mean ± SD, n ⳱ 5.
Cage wash (%)
1.55 ± 0.29
FIGURE 3. Individual elimination profiles of total radioactivity
(␮g equivalents per rat) of radiolabeled artesunate (circular markers)
in urine (top) and feces (bottom), as well as computer fitted curves
(solid line) by pharmacokinetic parameters following a single 5 mg/20
␮Ci/kg intravenous injection of [14C]AS in rats during 8 days collection (N ⳱ 5).
(metabolites) lasted longer than 192 hours in plasma, urine,
and feces. Conjugation studies showed drug conjugation (up
to 90% of the dose in plasma and urine) is a major metabolic
pathway of [14C] AS, and that conjugation is time-dependent
in rats (Table 4). Metabolites of AS were quantified before
and after hydrolysis with beta-glucuronidase to give free and
conjugated fractions of plasma, urine, and feces. Metabolites
arising from AS in the free fraction were 12.3 ± 4.4% and 10.4
± 2.5% of the total administered dose in plasma and urine;
those arising from the total conjugated fractions (glucuronide
and other conjugations) were 88.6% and 89.6% of administered dose in plasma and urine, respectively. Glucuronide
conjugation was 8% in plasma and 18% in the urine (Table 4).
Within 30 minutes after dosing the drug conjugated fraction
was less than the free fraction; the ratio of conjugated to free
ranged from 0.57–0.97 in plasma. From 0.5 hours post dosing,
the ratio increased to 1.97 at 1 hour and then rapidly increased to 19.36 at 48 hours. After 48 hours the ratio increased moderately to 25.11 at 192 hours.
Protein binding of [14C] artesunate with rat and human
plasma. [14C] AS revealed a higher binding percentage with
human and rat plasma proteins (73–81%) when incubated
TISSUE DISTRIBUTION OF [14C] ARTESUNATE IN RATS
823
TABLE 4
Percentage distribution, concentration, and half-lives of [14C] artesunate in total, free, glucuronide and other conjugation fractions in plasma or
urine following a single intravenous dose at 5 mg/20 ␮Ci/kg in rats*
Time
Plasma (n ⳱ 3)
0
1
6
24
48
72
96
168
192
AUC (ng⭈h/ml)
t1/2 (h)
% of total
Urine (n ⳱ 5)
1
6
24
48
72
96
120
144
168
192
AUC (ng⭈h/ml)
t1/2 (h)
% of total
Total
Free fraction
Glucuronide conjugation fraction
Other conjugation fraction
3030.1 ± 77.6
540.9 ± 27.6
1070.1 ± 219.2
255.7 ± 21.9
135.5 ± 19.6
92.4 ± 12.8
63.4 ± 8.6
20.6 ± 3.3
14.2 ± 2.5
28101 ± 3286
44.47 ± 3.68
99.22 ± 11.60
245.5 ± 16.4
91.3 ± 13.1
187.4 ± 29.2
24.8 ± 9.6
8.8 ± 6.3
6.5 ± 5.0
4.9 ± 3.9
2.1 ± 1.9
1.6 ± 1.5
3491.1 ± 1255.6
56.61 ± 7.45
12.33 ± 4.43
144.2 ± 21.3
43.1 ± 5.7
113.9 ± 14.9
15.8 ± 1.9
6.9 ± 0.6
5.1 ± 0.4
3.8 ± 0.2
1.6 ± 0.1
1.2 ± 0.1
2252.2 ± 193.5
58.09 ± 4.86
7.95 ± 0.68
658.4 ± 106.1
204.5 ± 68.3
585.8 ± 148.5
205.7 ± 27.1
103.6 ± 3.2
79.1 ± 2.0
61.8 ± 2.0
29.5 ± 1.7
23.1 ± 1.5
22577.8 ± 2615.2
67.58 ± 2.53
79.72 ± 9.23
82995 ± 25899
49996 ± 8819
19118 ± 2097
5265 ± 1809
1793 ± 763
774 ± 295
413 ± 135
256 ± 89
172 ± 72
120 ± 61
1671670 ± 212023
48.51 ± 13.24
100.56 ± 12.75
7969 ± 1872
4820 ± 1119
1836 ± 428
512 ± 132
193 ± 62
97 ± 38
57 ± 26
36 ± 19
24 ± 13
16 ± 10
173616 ± 41760
38.16 ± 7.51
10.44 ± 2.51
17041 ± 4213
9758 ± 2578
3235 ± 965
654 ± 228
154 ± 63
48 ± 24
21 ± 12
11 ± 7
7±5
4±3
298884 ± 87123
35.27 ± 10.97
17.98 ± 5.24
62694 ± 10648
38967 ± 5507
15300 ± 2476
4169 ± 1101
1399 ± 443
620 ± 190
349 ± 101
226 ± 64
156 ± 45
110 ± 33
1189805 ± 194459
50.59 ± 8.82
71.58 ± 5.24
* AUC ⳱ area under curve; t1/2 ⳱ half-life.
with undiluted plasma samples at concentrations of 0.2–78125
ng/ml at 37°C for 5 hours. The binding percent of [14C] AS
was shown to be concentration-dependent. At higher concentrations (> 125 ng/ml) the binding percentage declined from
75–81% to 62–66%, indicating that the maximum binding
percentage occurred in the concentration range of 0.2–125
ng/ml (Figure 4). The binding capacity of AS was not significantly different between male and female in human plasma (P
⳱ 0.15), but was significantly different between with male
and female in rat plasma (P < 0.05). When compared with
human plasma, the bound portion (75%) of radiolabeled AS
was about 7% less than that (82%) in rat plasma (Figure 4).
DISCUSSION
Previous studies have established the role of biliary excretion in the elimination of artemisinin metabolites from rats16
and mice.29 As early reports on tissue distribution and metabolism indicate, AS was transformed rapidly into DHA
when it was intravenously injected in rats. The order of distribution in tissues was: heart, muscle, lung, spleen, kidney,
brain, blood, and liver. After 2 hours, no DHA was detected
in the organs.26,29
In the present studies, the tissue distribution and metabolic
profiles of AS revealed similar data to the published literature. The outcomes of this study indicate very little AS is
eliminated by direct excretion from kidneys (< 1% of dose);
rather most (99%) occurs via metabolism (hydrolysis and oxidation). Data presented in this study allows a comprehensive
characterization of AS with tissue distribution and its metabolic profiles.
Artesunate underwent rapid hydrolysis, glucuronylation,
biliary excretion, and likely enterohepatic circulation. The results of the tissue distribution studies show 65–82% of total
radioactivity to be found in the content of small intestine
within the first hour after IV dosing. At 1 hour after dosing
most of the [14C] was present in the liver, where it was excreted into the intestines via bile ductules; this suggests the
higher hepatic biotransformation of AS is similar to metabolic
profiles of other artemisinin compounds.16,30 The biliary metabolites of AS in rats were predominantly from the hydrolysis pathway (first to DHA), followed by glucuronylation
and other conjugated products from multiple oxidative pathways for both compounds (AS and DHA). DHA is the major
metabolite of AS. Figure 5A shows DHA presented almost
immediately in the free fraction of plasma at 1 minute postdosing. AS and DHA were rapid and extensively metabolized
into various polar metabolites; the unchanged AS disappeared from the plasma within minutes. DHA is the metabolite found in the greatest quantities (Figure 5B). In the glucuronide conjugated fraction of the plasma, unchanged AS,
unchanged DHA, a few less polar and a greater number of
more polar metabolites were detected. The unchanged AS
and DHA were the major components of the conjugated
plasma fraction (Figure 5C), indicating conjugation is also a
major metabolic pathway of AS and DHA. In addition, the
two peaks of radioactivity were detected in blood and plasma
and the most of radioactivity was excreted into the intestines,
showing a potential enterohepatic circulation of [14C] AS in
rats.
Urinary and fecal excretion data obtained after IV administration of [14C] AS demonstrated that approximately 73%
of total radioactivity was eliminated in the first 24 hours.
824
LI AND OTHERS
FIGURE 4. Individual protein binding of [14C]AS in male humans
(top) and male rats (bottom) plasma (triangle markers) and mean
value in the humans and rats plasma (solid line) after incubation with
variable concentrations of AS (0.2, 1, 5, 25, 125, 625, 3125, 15625, and
78125 ng/ml) at 37°C for 5 hours (N ⳱ 3).
Fifty-six percent of the dose was eliminated in urine and 40%
in feces within 192 hours after dosing; radioactivity recovered
in the cage rinses (approximately 2%) may have been derived
from urine due to reduced fecal contamination in the new
metabolic cage system. The finding that fecal elimination of
radioactivity after an IV dose was less than that observed in
urine suggests a majority of [14C] AS may be first excreted in
bile and re-absorbed from intestines.
Pharmacokinetic parameters were calculated from the
blood and plasma levels of radioactivity after IV injection of
[14C] AS. The studies indicated the terminal half-life of radioactivity was approximately 1.4 times longer in whole blood
(t1/2 ⳱ 104.7 hours) than in plasma (t1/2 ⳱ 76.2 hours). The
calculated steady state volume of distribution (Vss) of radioactivity in whole blood and in plasma was 6,183 and 16,081
mL/kg, respectively; this is an indication of wide-extravascular distribution of the radiolabel or tissue binding. The
clearance of radioactivity from whole blood was estimated to
be 81 mL/hr/kg, which was lower than the rate of hepatic
blood flow.31
Unchanged AS was eliminated from plasma with a terminal
half-life of 0.43 hours after IV administration. The Vss of
unchanged AS was estimated to be 3,982 mL/kg, indicating
AS has a relatively small volume of distribution. The calcu-
lated plasma clearance of AS was 12,719 mL/hr/kg, which is
higher than hepatic blood flow (4,800 mL/hr/kg). This is probably related to uptake and binding of AS in red blood cells.
When corrected for an AS blood/plasma ratio of 2.44, the
hepatic blood clearance is 5,213 mL/hr/kg, which is reasonably close to hepatic blood flow. The results indicate even
though the distribution of unchanged AS is apparently limited
to total body water, the radioactivity (metabolites of AS)
derived from [14C]AS was extensively distributed throughout
rats and lasted for an extended period (192 hours).
The average ratio of blood to plasma was 2.44 during the
192-hour period. When calculated with rat hematocrit, the
ratios of the concentrations in red blood cells (RBC) to
plasma (ml/ml) were 1.2–3.1 during the 0–1.5 hours, and from
3.4–6.4 during the 3–192 hour period after dosing. The median ratio of RBC to plasma was 4.03 during the treatment
time of 192 hours. High concentrations of [14C] AS present in
RBC is very important for the antimalarial agent. The drug
concentration in RBC is about 4 times higher than that in
plasma, indicating that the powerful antimalarial potency of
AS in animal species and humans may relate to the high drug
levels in the RBC.
An understanding of artemisinin-associated neurotoxicity
in vivo requires knowledge of the penetration of the cerebrospinal fluid (CSF) by these drugs. Such data is sparse. Artemisinin derivatives cross the blood–brain barrier in rats.24
Artesunate is converted stoichiometrically to DHA, which is
highly lipid soluble and has a low molecular mass (284 Da),
favoring penetration of CSF.25 Since DHA has relatively low
solubility in water, it should be able to cross cell membranes
and be conjugated. After AS treatments in patients, no AS
was detected in CSF but DHA levels in CSF increased with
time while DHA levels in plasma fell, suggesting continued
influx, with a slower efflux of DHA. DHA may accumulate in
CSF during frequent AS dosing.25 In our study, the radioactivity in brain tissues (AUC: 70,723 ng⭈h/g) was more than 2
times higher than that in plasma (AUC: 29,425 ng⭈h/g). The
half-life of [14C] AS in brain tissue was 94.2 hours, which was
also longer than that in plasma, 63.7 hours. The results indicated that the resident time of [14C] AS is longer in the brain
than in plasma. Also, there was 1% of total dose [14C] AS
detected in rat brain tissue. This may reflect a sink effect of
DHA and/or other metabolites and an uptake transfer by
lipid-rich brain structures.25 In this time, the other metabolites present in the brain tissue are unknown. In addition,
whether the metabolites in the brain are associated with the
neurotoxicity is unknown.
The present study showed AS to be completely hydrolyzed
to DHA within 2–3 hours following the intravenous administration in rats. As a result of approximately 73% of total
dose was eliminated in the first 24 hours, and 90% of the dose
appears as glucuronide and other conjugations in blood,
urine, and feces. However, the majority of elimination of
[14C] AS is through urinary excretion (56% dose) due to a
possible drug re-absorption in the intestines and enterohepatic circulation as suggested by multiple concentration peaks
in blood, plasma, and tissues. The long-lasting metabolites of
AS and DHA (> 192 hours) may be also related to enterohepatic circulation. The binding capacities of [14C] AS with
human and rat plasma at higher percentages, 73–81%, are
seen in a concentration-dependent manner. High tissue distribution was found in rat spleen, followed by bone marrow,
TISSUE DISTRIBUTION OF [14C] ARTESUNATE IN RATS
825
FIGURE 5. Metabolic patterns (solid line) of radiolabel distribution with standard references (dotted line) of alpha-dihydroartemisinin
(␣-DHA), ␤-dihydroartemisinin (␤-DHA), and artesunate (AS) in plasma free fraction at 1 min (A), 5 min (B), plasma glucuronidate conjugation
fraction at 5 min (C), and urine glucuronidate conjugation fraction at 8 hr (D) after single intravenous injection at dose of 5 mg/20␮Ci/kg of
[14C]AS in rats displayed together in radiochromatography.
kidney, adrenals, and heart during the 192-hour period. In rat
brain, the total concentration of [14C] was 2.4-fold higher than
that in plasma, indicating the radioactivity could easily penetrate the brain–blood barrier. Whether the metabolites in
the brain are associated with the neurotoxicity is unknown.
Total radioactivity distributed in RBC was about twofold to
fourfold higher than that in plasma, suggesting that the powerful antimalarial potency of AS in treatment of blood stage
malaria may relate to the high RBC binding.
Received March 16, 2006. Accepted for publication July 14, 2006.
Disclaimer: The opinions or assertions contained herein are the private views of the author and are not to be construed as official, or as
reflecting true views of the Department of the Army or the Department of Defense.
Financial support: This study was supported by the United States
Army Research and Materiel Command.
Authors’ addresses: Qigui Li, Lisa H. Xie, Adam Haeberle, Jing
Zhang, and Peter Weina, Division of Experimental Therapeutics,
Walter Reed Army Institute of Research, 503 Robert Grant Avenue,
Silver Spring, MD 20910-7500, Tel: (301) 319-9351, Fax: (301) 3197360, E-mails: [email protected]; [email protected]
.mil; [email protected]; [email protected]
.army.mil; [email protected].
Reprint requests: Dr. Qigui Li, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant
Avenue, Silver Spring, MD 20910-7500. Tel: (301) 319-9351, Fax:
(301) 319-7360, E-mail: [email protected].
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