0022-3565/00/2933-0996$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 293:996–1001, 2000 /2188/823036
Vol. 293, No. 3
Printed in U.S.A.
Preclinical and Clinical Evidence for Disappearance of LongCirculating Characteristics of Polyethylene Glycol Liposomes at
Low Lipid Dose1
PETER LAVERMAN, ADRIENNE H. BROUWERS, ELS TH. M. DAMS, WIM J. G. OYEN, GERT STORM,
NICO VAN ROOIJEN, FRANS H. M. CORSTENS, and OTTO C. BOERMAN
Department of Nuclear Medicine, University Medical Center Nijmegen, Nijmegen, the Netherlands (P.L., A.H.B., E.Th.M.D., W.J.G., F.H.M.C.,
O.C.B.); Utrecht Institute for Pharmaceutical Science, Utrecht University, Utrecht, the Netherlands (G.S.); Department of Cell Biology and
Immunology, Faculty of Medicine, Free University, Amsterdam, the Netherlands (N.v.R.)
Received for publication February 7, 2000
This paper is available online at http://www.jpet.org
Soon after the discovery of liposomes, it was recognized
that liposomes labeled with ␥-radiation-emitting radionuclides could be used as radiopharmaceuticals to visualize
pathological processes, such as infections and tumors, in
vivo. However, liposomes in their classical composition were
less suitable because of their short circulatory half-life and
high uptake in liver and spleen. The circulatory half-life of
these so-called “conventional” liposomes could be enhanced
by reducing their size, giving them a positive surface charge,
and including phospholipids with saturated acyl chains (Gregoriadis et al., 1974; Juliano and Stamp, 1975). In addition, it
was shown that the blood residence time of conventional
liposomes increases with the lipid dose given. Beaumier et al.
(1983) showed in mice that small liposomes (⬃80 nm) at 6 mg
of liposomal lipid/kg b.wt. had relatively high liver uptake
[60%ID (percentage of the injected dose), 23 h postinjection
(p.i.)], whereas at doses exceeding 120 mg/kg b.wt., liver
Received for publication October 11, 1999
1
This study was supported by Grant NGN 55.3665 from the Technology
Foundation (Technologiestichting STW), the Netherlands.
same effect was observed in rabbits where enhanced clearance
was observed at a dose level of 0.02 ␮mol/kg. The circulatory
half-life decreased from 10.4 to 3.5 h (at 1.0 and 0.02 ␮mol/kg,
respectively). At the lowest dose level, liposomes were mainly
taken up by the liver and to a lesser extent by the spleen.
Injection of a low dose of PEG liposomes in macrophagedepleted rabbits resulted in normal pharmacokinetics, suggesting involvement of macrophages in the effectuation of the rapid
elimination of the liposomes from the circulation. Most importantly, the rapid clearance of low-dose PEG liposomes was also
observed in humans when relatively low lipid doses were administered. This study showed that at very low lipid doses the
biodistribution of PEG liposomes is dramatically altered.
uptake was much lower (20%ID, 23 h p.i.). The blood clearance pattern in these experiments virtually mirrored the
liver uptake. These experiments demonstrated that the liver
uptake mainly resulted from saturable phagocytosis by
Kupffer cells.
The development of liposomes coated with the hydrophilic
polymer polyethylene glycol (PEG) greatly broadened the
application of liposomes (Woodle and Lasic, 1992). PEGcoated liposomes (also known as stealth liposomes) are considered to have long-circulating properties irrespective of
lipid composition, surface charge, and/or lipid dose. In various preclinical studies, as well as in a clinical study, we have
shown that PEG liposomes labeled with 99mTc-hexamethylpropylene-amine-oxime performed well in visualizing various
types of infection and inflammation (Boerman et al., 1997;
Dams et al., 1998, 1999, 2000). The development of an improved labeling method, based on the bifunctional chelator
hydrazinonicotinamide (HYNIC), enabled us to reduce the
administered lipid dose to 20% of the initially used dose
(Laverman et al., 1999). Recently, however, Utkhede and
ABBREVIATIONS: %ID, percentage of injected dose; PEG, polyethylene glycol; p.i., post injection; MPS, mononuclear phagocyte system; HYNIC,
hydrazinonicotinamide; DSPE, distearoylphosphatidyl-ethanolamine; PHEPC, partially hydrogenated egg phosphatidyl choline.
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ABSTRACT
This study describes the effect of the lipid dose of 99mTcpolyethylene glycol (PEG) liposomes in the low-dose range
(0.02–1.0 ␮mol/kg) on the pharmacokinetics and biodistribution
in rats, rabbits, and humans. The biodistribution and pharmacokinetics of 99mTc-PEG liposomes at various dose levels were
studied in rats and rabbits with a focal Escherichia coli infection. Scintigraphic images were recorded on a gamma camera.
In addition, the role of macrophages in the biodistribution of a
low-dose PEG liposome injection was studied. Finally, the
pharmacokinetics of 99mTc-PEG liposomes at two lipid dose
levels was studied in four patients. At a dose level of 0.03
␮mol/kg, the blood level in rats at 4 h postinjection was significantly lower than at the highest dose level (1.1 ␮mol/kg). The
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Dose Effect of PEG Liposomes
Tilcock (1998) showed that the circulation time of PEG liposomes at very low lipid dose levels was strongly reduced,
whereas uptake in the liver and the spleen was clearly enhanced. To further elucidate this phenomenon, we investigated in detail the effect of lipid dose on the pharmacokinetics and biodistribution of PEG liposomes in two animal
species as well as in humans.
In both rats and rabbits with an artificially induced focal
infection, we determined the pharmacokinetics and the biodistribution of radiolabeled PEG liposomes at various lipid
dose levels. Furthermore, we investigated the effect of two
successive injections of PEG liposomes at low lipid dose. In
rabbits we also studied the effect of macrophage depletion on
the blood clearance of liposomes at low lipid dose. Finally, in
four patients the blood clearance and biodistribution of PEG
liposomes at two lipid dose levels was assessed.
Materials and Methods
Partially hydrogenated egg-phosphatidylcholine (PHEPC) with an
iodine value of 35 was a gift from Lipoı̈d GmbH (Ludwigshafen,
Germany). Distearoylphosphatidylethanolamine (DSPE) and the
PEG-2000 derivative of DSPE were purchased from Avanti Polar
Lipids (Alabaster, AL). N-Hydroxysuccinimidyl-hydrazinonicotinate
hydrochloride (S-HYNIC) was synthesized as described by Abrams
et al. (1991) with minor modifications (Claessens et al., 1996). The
hydrazinonicotinamide derivative of DSPE (HYNIC-DSPE) was prepared as described previously (Laverman et al., 1999). Clodronate
(Cl2MBP) was a gift from Boehringer Mannheim GmbH (Mannheim,
Germany).
Liposome Preparation
PEG liposomes were prepared as described previously (Laverman
et al., 1999). Briefly, a lipid mixture in methanol/chloroform (10:1)
was prepared with a molar ratio of 1.85:0.15:0.07:1 (PHEPC/PEGDSPE/HYNIC-DSPE/cholesterol) unless stated otherwise. After
evaporation of the organic solvents, the resulting lipid film was
dispersed in HEPES buffer [10 mM HEPES (ICN Biomedicals, Inc.,
Costa Mesa, CA), 135 mM NaCl at pH 7.4] at room temperature.
Liposomes were sized by multiple extrusion through two stacked
polycarbonate membranes with a medium pressure extruder (Lipex
Biomembranes, Inc., Vancouver, BC). After sizing, the suspension
was dialyzed against HEPES buffer overnight at 4°C with four buffer
changes to remove unconjugated S-HYNIC. Liposomes were stored
in HEPES buffer at 4°C.
Mean particle size of the liposomal preparations was determined
by a dynamic light scattering system (Malvern 4700; Malvern Instruments Ltd., Malvern, UK). The mean size of the liposomes was
90 nm, with a polydispersity index of 0.1. This index ranges from 0.0
for an entirely monodisperse dispersion to 1.0 for a completely polydisperse dispersion. Phospholipid concentration in the liposome dispersions was determined using the colorimetric method described by
Rouser et al. (1970).
Radiolabeling
Labeling of the liposomes was performed as described earlier
(Laverman et al., 1999) with minor modifications. Briefly, various
volumes of liposomes (85 ␮mol of phospholipid/ml) were adjusted to
a final volume of 100 ␮l with HEPES buffer and added to a mixture
of 10 mg of Tricine, 10 ␮g of stannous sulfate, and 99mTcO4⫺ in saline.
The mixture was incubated for 20 min at room temperature. Radiochemical purity was determined using instant thin-layer chromatography on silica gel strips (Gelman Sciences, Inc., Ann Arbor, MI) with
0.15 M sodium citrate buffer, pH 5.5, as the mobile phase. When
radiochemical purity was less than 95%, liposomes were further
purified by gel filtration on a PD-10 column with HEPES buffer as
the eluent.
Animal Experiments
Rat Model of Infection. An abscess was induced in the left calf
muscle of young, male, randomly bred Wistar rats (220 –240 g) with
approximately 1 ⫻ 109 colony-forming units of Escherichia coli in 0.1
ml of a 1:1 suspension of autologous blood and normal saline. During
the procedure, animals were anesthetized with ether. After 24 h,
when swelling of the muscle was apparent, the radiolabeled liposomes were injected i.v. via the tail vein.
Rabbit Model of Infection. An abscess was induced in the left
thigh muscle of female New Zealand White rabbits (2.2–2.8 kg) with
approximately 1.5 ⫻ 1010 colony-forming units of E. coli in 0.5 ml of
normal saline. During the procedure, animals were anesthetized
with a 0.6-ml s.c. injection of a mixture of fentanyl 0.315 mg/ml and
fluanisone 10 mg/ml (Hypnorm; Janssen Pharmaceuticals, Buckinghamshire, UK). After 24 h, when swelling of the muscle was apparent, the radiolabeled liposomes were injected i.v. via the lateral ear
vein.
Gamma Camera Imaging and Biodistribution Studies.
Twenty-four hours after E. coli inoculation, groups of five rats received 10 MBq 99mTc-PEG liposomes via the tail vein. Rabbits (five
per group) were injected with 15 MBq 99mTc-PEG liposomes via the
lateral ear vein. From each group, at least three animals were placed
prone on a gamma camera (Orbiter; Siemens Medical Systems, Inc.,
Hoffman Estates, IL) equipped with a parallel-hole low-energy collimator, and images were recorded at 5, 30, 60, 120, and 240 min p.i.
After acquisition of 100,000 counts, the images were stored digitally
in a 256 ⫻ 256 matrix.
Blood samples from the rabbits were collected at 1, 30, 60, 120,
and 240 min after injection of the radiopharmaceutical to determine
the blood clearance of the radiolabeled liposomes. Data were analyzed by nonlinear least-squares fit using a biexponential model.
After completion of the final image, animals were sacrificed by a
lethal dose of sodium phenobarbital. Samples of blood, infected thigh
muscle, noninfected contralateral thigh muscle, lung, spleen, liver,
kidney, and intestine were collected. The dissected tissues were
weighed and counted in a shielded well-type gamma counter (Wizard; Pharmacia-LKB, Uppsala, Sweden). To correct for physical decay, injection standards were counted simultaneously. The measured
activity in the dissected tissues was expressed as %ID per gram of
tissue (%ID/g).
Effect of Macrophage Depletion. To study the involvement of
macrophages on the in vivo behavior of PEG liposomes, liver and
splenic macrophages in rabbits were depleted by injection of multilamellar liposomes containing dichloromethylene-bisphosphonate
(Cl2MBP or clodronate). Clodronate liposomes and PBS-containing
liposomes (serving as control) were prepared as described previously
by Van Rooijen and Sanders (1994). Two female New Zealand White
rabbits (2.0 –2.5 kg) were injected i.v. with 4 ml of clodronate liposomes (containing approximately 20 mg of clodronate), and two rabbits were injected with 4 ml of PBS liposomes. After 48 h, when
depletion of the macrophages in liver and spleen is most optimal
(Van Rooijen and Sanders, 1994), all rabbits were injected i.v. with
10 MBq 99mTc-PEG liposomes (0.02 ␮mol/kg). Gamma camera imaging was acquired at 5 min, 2, and 4 h p.i. After the final images were
recorded, rabbits were sacrificed, tissues were dissected and the
biodistribution of the radiolabel was determined as described above.
Parts of liver and spleen were snap-frozen in dry ice-cooled isopentane and stored at ⫺80°C. Cryostat sections were stained for acid
phosphatase to confirm macrophage depletion (Zysk et al., 1997).
Clinical Studies
Four patients (two males, two females, ages 45, 56, 57, and 64
years), referred to the Department of Nuclear Medicine for imaging
of infection and inflammation, were included in our study. Informed
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Reagents
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Laverman et al.
Vol. 293
consent was obtained from each patient. The study protocol had been
approved by the ethical review board of the University Hospital
Nijmegen. Three patients were suspected of infection or inflammation of the musculoskeletal system, and one patient had possible
abdominal pathology (Table 1).
All four patients received 740 MBq 99mTc-PEG liposomes (0.5 or
0.1 ␮mol/kg b.wt.) and were imaged at 4 and 24 h p.i. of the radiopharmaceutical. Scintigraphic images were obtained with a MultiSpect-2 gamma camera connected to an ICON computer system
(Siemens Inc., Hoffman Estates, IL). All images were collected in
digital format in a 256 ⫻ 256 matrix. Whole body scans were recorded at 4 and 24 h p.i., scan speed 15 and 6 cm/min, respectively.
The scintigraphic results were analyzed by drawing regions of interest over the liver and the spleen. An injection standard was recorded
simultaneously to permit the calculation of the %ID in both organs.
Blood samples were collected at 0, 5, 15, 30 min, and 1, 2, 4, and
24 h after injection of the liposomes. The samples were weighed, and
radioactivity was counted and expressed as %ID. Data were analyzed by nonlinear least-squares fit using a biexponential model.
TABLE 2
Effect of lipid dose on the biodistribution of PEG liposomes in rats at
various lipid dose levels (%ID/g ⫾ S.D.) at 4 h p.i.
Phospholipid Dose, ␮mol/kg
Organ
Blood
Muscle
Abscess
Lung
Spleen
Kidney
Liver
Intestine
0.03
0.4
1.1
3.59 ⫾ 0.31
0.09 ⫾ 0.08
0.46 ⫾ 0.13
0.96 ⫾ 0.13
5.32 ⫾ 1.09
1.47 ⫾ 0.24
1.67 ⫾ 0.31
0.50 ⫾ 0.08
4.07 ⫾ 0.23
0.07 ⫾ 0.01
0.98 ⫾ 0.16
1.18 ⫾ 0.31
5.48 ⫾ 1.12
1.48 ⫾ 0.10
1.47 ⫾ 0.43
0.72 ⫾ 0.15
4.86 ⫾ 0.48
0.08 ⫾ 0.02
1.16 ⫾ 0.28
1.51 ⫾ 0.22
6.71 ⫾ 1.12
1.69 ⫾ 0.14
1.54 ⫾ 0.34
0.73 ⫾ 0.09
All mean values are expressed as mean ⫾ S.D. Statistical analysis
was performed using the one-way ANOVA and corrected for multiple
comparisons by the Bonferroni procedure. The level of significance
was set at P ⬍ .05.
Results
The lipid dose dependence of the biodistribution of 99mTcPEG liposomes was studied in rats with an E. coli infection in
the left calf muscle. Three groups of five rats received 99mTcPEG liposomes either at 1.1, 0.4, or 0.03 ␮mol of phospholipid/kg b.wt. As shown in Table 2, at the lowest lipid dose (0.03
␮mol/kg), the blood level of the 99mTc-PEG liposomes was
significantly lower as compared with the blood level after
injection of the highest lipid dose of 1.1 ␮mol/kg (P ⬍ .01).
Abscess uptake was significantly reduced (P ⬍ .01), probably
as a result of the accelerated blood clearance. To investigate
whether this lipid dose effect is species-specific, the biodistribution of PEG liposomes was also studied at various lipid
dose levels in rabbits. Four groups of five rabbits each received 99mTc-PEG liposomes at either 1.0, 0.5, 0.1, or 0.02
␮mol of phospholipid/kg b.wt. The biodistribution as determined ex vivo (i.e., counting the radioactivity in the tissues
after dissection) was studied at 4 h p.i. Results are shown in
Fig. 1. Compared with the data obtained at the highest dose
level of 1.0 ␮mol/kg, significant differences were observed at
the lowest dose level of 0.02 ␮mol/kg. The blood level at 4 h p.i
was reduced by 50% (P ⬍ .01). As observed in the studies in
rats, abscess uptake was reduced at the two lowest dose
levels. At the 0.02 ␮mol/kg dose level, radioactivity in the
liver and spleen was strongly elevated (P ⬍ .05), whereas
uptake in all other organs was markedly decreased.
Fig. 1. Biodistribution at 4 h p.i. of 99mTc-PEG liposomes at four phospholipid dose levels in rabbits with an E. coli infection in the left thigh
muscle. Values are expressed as %ID/g of tissue. The error bars represent
S.D. (n ⫽ 5/group). 䡺, 0.02 ␮mol/kg; s, 0.1 ␮mol/kg; ^, 0.5 ␮mol/kg; o, 1.0
␮mol/kg.
In addition, the blood clearance of the radiolabeled liposomes was studied in rabbits by drawing blood samples at
several time points p.i. As depicted in Fig. 2, removal of the
PEG liposomes from the blood was relatively slow at the
highest lipid dose levels (1.0 and 0.5 ␮mol/kg; t1/2␣ ⫽ 10.4
and 9.3 h, respectively), which is concordant with our observations in previous studies. At the 0.1 ␮mol/kg dose level, the
initial clearance was faster (t1/2␣ ⫽ 6.7 h), whereas at the
lowest dose level of 0.02 ␮mol/kg, the circulatory half-life
appeared even more reduced (t1/2␣ ⫽ 3.5 h). The main differences in the clearance profiles were observed in the distribution phase (t1/2␣), whereas the clearance pattern during the
elimination-phase (t1/2␤) remained similar at all lipid dose
levels.
Representative scintigraphic images of the biodistribution
TABLE 1
Clinical characteristics, administered phospholipid dose, verification procedures, and blood clearance in patients
Patient
Sex
Age
Suspected Focus
Phospholipid
Dose
Follow-up
t1/2␣
Scintigraphy
Procedure
Result
Ig, Bo
Ba
Ig
Ig
Bursitis
M. Crohn
No infection
No infection
␮mol/kg
1
2
3
4
Ig,
M
M
F
F
64
56
57
45
Hip
Abdomen
Hip
Spine
0.5
0.5
0.1
0.1
⫹
⫹
⫺
⫺
In-IgG scan; Bo, Bone scan (99mTc-MDP); Ba, Barium enema; ⫹, positive scan; ⫺, negative scan.
111
t1/2␤
min
h
8
28
20
43
55
28
34
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Statistical Analysis
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of the radiolabel at various PEG liposome doses in rabbits are
shown in Fig. 3. High blood levels, represented by a clearly
visible heart region and large vessels, were observed on the
scintigrapic images of infected rabbits that received lipid
doses of 1.0, 0.5, and 0.1 ␮mol/kg. At these doses, liver,
spleen, and kidneys were visualized. Abscess uptake was
well visualized at the 1.0 and 0.5 ␮mol/kg dose level. At the
0.1 ␮mol/kg dose level, abscess uptake was less pronounced;
at 0.02 ␮mol/kg, the lowest dose level, the abscess was not
visible, coinciding with rapid blood clearance. At 4 h p.i., the
blood pool activity was very low. Activity mainly shifted to
the liver and, to a lesser extent, the spleen. With decreasing
lipid dose, increased renal elimination of the radiolabel was
observed, as shown by the well visualized bladder. In addition, bone marrow uptake was markedly higher at the lowest
lipid dose. Elevated uptake in the liver and bone marrow was
visible at 30 min and afterward.
To investigate whether saturation of the mononuclear
phagocyte system (MPS) or any other liposome consumptive
system in the body played a role in this phenomenon of rapid
clearance, two groups of rabbits received a first injection with
999
either 0.5 ml of PBS (control) or 0.5 ml of 0.1 ␮mol/kg PEG
liposomes. One hour after the first injection, rabbits received
0.02 ␮mol/kg 99mTc-PEG liposomes; images were recorded
1 h after this injection (data not shown). The rabbits of the
control group (injected with PBS) showed enhanced removal
of the radiolabeled liposomes from the blood pool, in line with
the results obtained at the 0.02 ␮mol/kg dose level discussed
above. After injection of the second dose (0.02 ␮mol/kg), the
rabbits of the other group (injected twice with liposomes)
showed a distribution similar to that observed at higher
doses, indicating saturation of the MPS or any other system
involved in the removal of liposomes from the blood during
the first PEG liposome injection.
To study the role of macrophages on the biodistribution of
PEG liposomes at a low dose level (0.02 ␮mol/kg), macrophages in rabbits were depleted by a single injection of large
multilamellar liposomes containing clodronate. Control rabbits received liposomes of the same lipid composition containing PBS instead of clodronate. Acid phosphatase staining
of sections of liver and spleen from rabbits treated with
clodronate liposomes confirmed the almost complete elimination of macrophages from these tissues. The macrophagedepleted rabbits showed a normal biodistribution, comparable with rabbits injected with higher doses of PEG liposomes.
Both of these groups showed high blood levels (0.50 ⫾
0.06%ID/g) and significantly lower liver uptake (0.19 ⫾
0.07%ID/g). Control rabbits (not depleted from macrophages)
showed a biodistribution pattern similar to the biodistribution observed after a single injection of low-dose PEG liposomes and elevated liver uptake. Accelerated blood clearance
was observed (0.35 ⫾ 0.05%ID/g, 4 h p.i.), as well as elevated
uptake in the liver (0.49 ⫾ 0.04%ID/g, 4 h p.i.). These data
strongly indicate that macrophages are involved in the rapid
clearance of low lipid dose PEG liposomes from the circulation.
To investigate whether the dose effect as observed in rats
and rabbits could also be observed in humans, the blood
clearance and biodistribution of radiolabeled PEG liposomes
at two lipid dose levels was studied in patients. As part of an
ongoing clinical trial, we investigated two patients at a lipid
dose level of 0.5 ␮mol/kg (the standard dose used in that
study) and two patients at a dose level of 0.1 ␮mol/kg. The
clinical characteristics of the patients, the injected phospholipid dose, the results of the 99mTc-PEG liposome scan, and
Fig. 3. Scintigraphic images of rabbits with an E. coli abscess in the left thigh muscle 4 h after injection of 99mTc-PEG liposomes at four phospholipid
dose levels.
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Fig. 2. Blood clearance of 99mTc-PEG liposomes at four phospholipid dose
levels determined in rabbits with an E. coli infection in the left thigh
muscle. Values are expressed as %ID/g of blood. The error bars represent
S.D. (n ⫽ 5/group). F, 0.02 ␮mol/kg; E, 0.1 ␮mol/kg; f, 0.5 ␮mol/kg; 䡺, 1.0
␮mol/kg.
Dose Effect of PEG Liposomes
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Laverman et al.
Discussion
In this study we have shown that the lipid dose of PEG
liposomes greatly affects the circulation time. At the low
dose, the PEG liposomes were cleared rapidly from the circulation. The liposomes were mainly taken up by the MPS
tissues (liver, spleen, and bone marrow). Several studies
(Allen and Chonn, 1987; Gabizon and Papahadjopoulos,
1988; Woodle and Lasic, 1992) have reported that liposomes
coated with PEG have long-circulating times independent of
Fig. 4. Anterior whole body scintigram of patient 2 (right) and patient 4
(left), 4 h p.i. of the 99mTc-PEG liposomes.
the administered lipid dose. Most of these studies focused on
the suitability of PEG liposomes for drug delivery in therapeutic applications. In general, in these applications relatively high lipid doses were used. In contrast, in nuclear
medicine applications it is common to administer tracer
amounts of radiopharmaceuticals. Studies on the blood clearance of PEG liposomes have been carried out using doses
ranging from 4 to 400 ␮mol of lipid/kg b.wt. (Allen and
Hansen, 1991), whereas PEG liposomes used for infection
imaging were injected at lipid dose levels of approximately
0.5 ␮mol/kg (Boerman et al., 1997; Dams et al. 1998, 1999,
2000). Recently, Utkhede and Tilcock (1998) were the first to
report on accelerated blood clearance of PEG liposomes after
administration of very low lipid doses in rabbits. They described strongly decreased circulatory half-lives at the 0.16
␮mol/kg dose level. In agreement with their studies, we
found enhanced clearance at the 0.02 ␮mol/kg dose level but
normal circulatory half-lives at a lipid dose of 0.1 ␮mol/kg.
This discordance may—at least partially— be related to differences in PEG liposome formulations. Utkhede and Tilcock
(1998) coated liposomes with PEG with a molecular weight of
5000, whereas we used PEG-2000. Maruyama et al. (1992)
have shown that PEG-5000 liposomes have significantly
shorter circulation times and higher uptake in liver and
spleen compared with PEG-2000 liposomes. This would point
to more intensive opsonization of PEG-5000 liposomes and
possibly a higher “threshold” dose above which the long circulation kinetics is lipid dose-independent. In line with our
results, Utkhede and Tilcock (1998) also report on increased
renal elimination of the radiolabel with decreasing lipid dose,
indicating enhanced processing and degradation of the PEG
liposomes within the phagocytic cells in the liver and the
spleen.
More importantly, in this study the lipid dose phenomenon
was also observed in humans. In humans, rapid elimination
of the radiolabeled PEG liposomes from the circulation was
observed at a phospholipid dose of approximately 0.1 ␮mol/
kg. The biodistribution pattern was similar to that observed
in the rats and rabbits, with high uptake in liver, spleen, and
bone marrow. Most likely, the liposomes are taken up by cells
of the MPS present in liver, spleen, and bone marrow. In
addition, bowel uptake was noted in one patient, indicating
processing of the liposomes trapped in the liver and subsequent hepatobiliary excretion.
At present, there is no consensus about the mechanism of
elimination of PEG liposomes from the circulation by the
MPS. Several reports suggested the presence of proteins that
may act as opsonins for these liposomes. From our experiments, as well as from literature (Oja et al., 1996), it appears
that this opsonin pool of proteins is limited in the case of PEG
liposomes. Oja et al. (1996) hypothesized that the amount of
protein bound to the liposomes dictates the liposome clearance properties. In this hypothesis, high doses of liposomes
correspond with relatively low protein amounts per particle
and therefore extend circulation times. This suggests that
“saturation” is in fact the result of depletion of blood opsonins, thus not resulting from MPS saturation. In our experiments, with two subsequent injections of liposomes, the second (lower lipid dose) injection showed a normal
biodistribution, suggesting that the pool of blood opsonins
was depleted by the first (higher lipid dose) injection. Although the clearance mechanism is still not fully understood,
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the pharmacokinetic data are summarized in Table 1. Whole
body scintigrams of patient 2 (high dose) and patient 4 (low
dose) are shown in Fig. 4. Patients 1 and 2, injected with
liposomes at the highest dose level, both showed a normal
biodistribution with activity in the heart region and the large
vessels and uptake in the liver and the spleen. In patient 2,
suffering from inflammatory bowel disease, focal uptake in
an affected bowel segment was visible. This disease entity
was confirmed by barium enema. By drawing regions of interest over the liver and the spleen, %ID in both organs was
calculated at 4 h p.i. In patients 1 and 2, liver uptake was 9
and 14%ID, respectively, whereas splenic uptake was 1 and
3%ID, respectively.
Patients 3 and 4, injected with the lower lipid dose (0.1
␮mol/kg), showed strongly increased activity in liver, spleen,
and bone marrow and decreased activity in the heart region,
indicating enhanced clearance of the 99mTc-PEG liposomes
from the circulation. Uptake in the liver 4 h p.i. for patients
3 and 4 was 30 and 23%ID, respectively, which is approximately 2.5 times higher than at the 0.5 ␮mol/kg lipid dose
level. Splenic uptake in both patients was 7 and 17%ID,
respectively, which is nearly 5-fold higher than observed at
the 0.5 ␮mol/kg dose level. At 24 h p.i., uptake of the radiolabel in the intestines was noted in both patients, indicating
hepatobiliary excretion of the radiolabel after processing of
the liposomes in the liver.
Vol. 293
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Dose Effect of PEG Liposomes
Conclusion
Our studies demonstrated that the biodistribution of PEG
liposomes is lipid dose-dependent and dramatically altered at
very low lipid doses. Therefore, the clinical use of 99mTc-PEG
liposomes for imaging infection and inflammation requires
administration of lipid doses of at least 0.5 ␮mol/kg b.wt.
Acknowledgments
We thank Gerrie Grutters and Bianca Lemmers-de Weem (Central Animal Laboratory, University of Nijmegen, the Netherlands)
for skilled assistance in the animal experiments and Louis van
Bloois (Department of Pharmaceutics, Utrecht University, the Netherlands) for assistance in the phospholipid determinations.
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Send reprint requests to: Peter Laverman, University Medical Center Nijmegen, Department of Nuclear Medicine, P.O. Box 9101, NL-6500 HB Nijmegen, the Netherlands. E-mail: [email protected]
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it is hypothesized that PEG mediates a long circulation time
by opposing opsonization of liposomes (Liu et al., 1995). However, it is clear from our study that this mechanism is not
compatible with the rapid blood clearance occurring at the
low lipid dose. Perhaps minimal amounts of opsonins capable
of opsonizing the PEG liposomes are present in the circulation. From our biodistribution data (Fig. 1), this amount can
be estimated. Assuming that at higher lipid doses there is
mainly “passive uptake” of PEG liposomes in the liver
[11%ID; based on a liver of 82 g (Kozma et al., 1974)], on top
of this liver uptake there is an amount of liposomes that is
“actively” taken up by the Kupffer cells. At the lowest lipid
dose, this amount is 20%ID (31 ⫺ 11%). Thus, at this phospholipid dose, as well as at the 0.1 ␮mol/kg lipid dose level,
the actively taken up fraction amounts to 10 nmol of PEG
liposomes.
In a subsequent experiment, we showed that macrophages
are involved in the rapid elimination of the PEG liposomes
from the circulation. Injection of a low dose of PEG liposomes
in macrophage-depleted rabbits did not result in fast clearance of these liposomes from the circulation, thus suggesting
that macrophages are necessary to effectuate the clearance of
PEG liposomes from the circulation. Although PEG liposomes are considered to be “invisible” for the MPS (also
known as the “stealth” principle), we clearly illustrated that
this concept does not hold after injection of PEG liposomes at
low lipid dose. This might have great implications for the
application of PEG liposomes in nuclear medicine and drug
targeting.
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