Competitive Displacement of Serum
Protein Binding of Radiopharmaceuticals
with Amino Acid Infusion Investigated with
N-Isopropyl-p-123I-Iodoamphetamine
Noriyuki Kuga1,2, Naoto Shikano3, Norito Takamura4, Ryuichi Nishii5, Keishi Yamasaki6, Masato Kobayashi7,
Shigeki Nagamachi1, Shozo Tamura1, and Keiichi Kawai2,71Radiological Division, University of Miyazaki Hospital, University of Miyazaki, Miyazaki, Japan; 2Division of Health Science,

Graduate School of Health Sciences, Kanazawa University, Ishikawa, Japan; 3Department of Radiological Sciences, Ibaraki
Prefectural University of Health Sciences, Ibaraki, Japan; 4School of Pharmaceutical Sciences, Kyusyu University of Health andWelfare, Miyazaki, Japan; 5Research Institute, Shiga Medical Center, Shiga, Japan; 6Faculty of Pharmaceutical Sciences, Sojo
University, Kumamoto, Japan; and 7Biomedical Imaging Research Center, University of Fukui, Fukui, Japan

When a therapeutic drug is competitively displaced at the binding sites of serum proteins, the free fraction of the drug will be increased, with an increase in the manifestation of pharmacologic
properties. In the case of molecular imaging probes, total clearance and tissue distribution are increased in such circumstances. The aim of this study was to observe the increase in
cerebral accumulation of N-isopropyl-p-123I-iodoamphetamine(123I-IMP) using the protein-binding displacement method withamino acid infusion. Methods: 123I-IMP binding to human serum
was investigated and identified. In addition, protein-binding sites
and the specific binding sites of human serum albumin (HSA) anda1-acid glycoprotein (AGP) were examined by ultrafiltration.
Then, serum-binding sites and the displacement effects of aminoacid infusion, including Proteamin 12X Injection and Kidomin,
were confirmed in vitro. Subsequently, displacement of 123IIMP serum protein binding with Proteamin amino acid infusion
was tested in monkeys. A scintigraphic study of 123I-IMP in monkeys loaded with or without Proteamin was performed, and time–
activity-curves of 123I-IMP brain accumulation in monkeys were
evaluated. Results: 123I-IMP was bound to HSA site II and
AGP to nearly equal extents. Compared with control conditions,loading with Proteamin and Kidomin markedly increased free
fractions of binding site markers for HSA site II (14C-diazepam:
0.95% 6 0.04% for control, 1.40% 6 0.06% for Proteamin,
1.62% 6 0.05% for Kidomin) and AGP (3H-propranolol:
10.60% 6 0.32% for control, 13.18% 6 0.14% for Proteamin,
13.82% 6 0.72% for Kidomin). Amino acid infusions were thus
suitable for use as displacers for binding site II and AGP. Withuse of Proteamin amino acid infusion to displace protein binding,the free fraction of 125I-IMP (14.95% 6 0.74%) was significantly
increased in serum (19.24% 6 0.87%). In a 123I-IMP scintigraphic
study of monkeys, average cerebral uptake in 2 monkeys increased by 1.34-fold with Proteamin. Our findings suggested
that Proteamin treatment increased the free fraction of 123IReceived Nov. 11, 2008; revision accepted Mar. 16, 2009.
For correspondence or reprints contact: Naoto Shikano, Department ofRadiological Sciences, Ibaraki Prefectural University of Health Sciences,4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japan.
E-mail: sikano@ipu.ac.jp
COPYRIGHT ª 2009 by the Society of Nuclear Medicine, Inc.

1378

IMP, yielding rapid and pronounced cerebral accumulation in
vivo. Conclusion: Amino acid infusion can improve brain accumulation by competitive displacement of serum protein bindingin vivo. Further similar studies are needed with other radiopharmaceuticals.
Key Words: 123I-IMP; cerebral accumulation; amino acid infusion; serum protein binding; displacement
J Nucl Med 2009; 50:1378–1383
DOI: 10.2967/jnumed.108.060095

I

t is generally accepted that only free drugs in serum
exhibit pharmacologic activity. Coadministration of drugs
with high protein-binding affinity may induce competitive
displacement at specific binding sites and thus result inincreased free drug concentration (1–3), with greater
biologic activity of each drug than if either were administered alone. However, the increase in free drug concentration
is often only transient, as a result of drug distribution andenhanced clearance.
Proteins bind a wide variety of drugs and thus significantly affect the pharmacokinetics and pharmacologic efficacy of such drugs. Contributions to drug binding inplasma include those of albumin, a1-acid glycoprotein(AGP), lipoproteins, and immunoglobulins (IgG). Several
drugs bind with high affinity to one of the human serum
albumin (HSA) sites and AGP (4,5). Sudlow et al. characterized 2 sites for drug binding, site I (also referred to as thewarfarin-binding site) and site II (the benzodiazepinebinding site) of HSA (6). Although interaction of many
drugs with AGP has been reported, the binding site of AGP
has not been determined. It has recently been shown that
AGP contains a wide drug-binding region for basic, acidic,
and neutral drugs and that the binding sites on AGP are not

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 50 • No. 8 • August 2009

completely separate and in fact partially overlap and influence one another (5,7).
Approximately 70% of N-isopropyl-p-123I-iodoamphetamine (123I-IMP, Fig. 1), a diagnostic agent for determination of regional cerebral blood flow in SPECT, is bound toserum proteins (8,9). If inhibitors with high protein-binding
affinity could displace 123I-IMP serum protein binding,
cerebral accumulation of this tracer would be increased. In
fact, Kawai et al. reported an increase in 123I-IMP cerebral
accumulation in monkeys using the protein-binding displacement method with 6MNA (a major metabolite ofnabumetone) (10). We noted that an amino acid infusion
could be administered more easily and safely than drugs.
Amino acid infusions are widely used for parenteral nutrition and consist of water, glucose, salts, amino acids,
vitamins, and sometimes emulsified fats. They are required
for medical management of a variety of patients in a
hypermetabolic state, as well as those who have neurologic
disease, gastrointestinal disease, cancer, or psychiatric illness (11).
The purpose of this study was to investigate the displacement effects of 123I-IMP binding to 2 major human serum
proteins, HSA and AGP. To identify the major binding
proteins, we also evaluated the binding sites and effects ofamino acid infusions on 123I-IMP serum protein binding toboth HSA and AGP. In addition, we investigated whether
cerebral accumulation of 123I-IMP was increased by aminoacid infusion, using the competitive displacement method.
MATERIALS AND METHODS
Materials
Reagent-grade chemicals were used in this study. HSA (essentially fatty acid–free albumin), AGP, and human IgG were acquired
from Sigma Chemical Co. Potassium warfarin, verapamil hydrochloride (Eisai), diazepam (Takeda Pharmaceutical Co., Ltd.), andpropranolol hydrochloride (Dainippon Sumitomo Pharma Co., Ltd.)
were also obtained. Amino acid infusions included Kidomin from
Otsuka Pharmaceutical Factory, Inc., and Proteamin 12X Injection
from Tanabe Seiyaku Co., Ltd. (Table 1). 14C-warfarin (2.1 MBq/mmol) was synthesized and purified by Amersham Biosciences, and14C-diazepam (2.0 MBq/mmol) and 3H-propranolol (902.8 GBq/mmol) were synthesized and purified by Perkin-Elmer Life Sciences 123I-IMP (82.8 GBq/mmol) and 125I-IMP (10.5 GBq/mmol)(Nihon Medi-Physics) were used as molecular imaging probes. The
radiochemical purities of the IMP and radiolabeled compounds
were greater than 95.0%. Human pooled serum was prepared fromblood samples obtained from 7 healthy male and female subjects.
All subjects discontinued any medication for at least 5 d before
blood sampling. The concentrations of albumin and AGP were
determined to be 767 and 13 mM, respectively, using a COBAS
Integra 400 Plus (Roche Diagnostics).

FIGURE 1. Chemical
structure of 123I-IMP.

TABLE 1. Content of Amino Acid Infusions
Amino acid infusion
Content
L-arginine
L-histidine
L-isoleucine
L-leucineL-lysine
L-methionine
L-phenylalanine
L-threonine
L-tryptophan
L-valine
GlycineL-proline
L-serine
L-aspartic acidL-glutamic acidL-tyrosine
L-cystine
L-cysteine
L-alanine
Total free amino acids

Proteamin
35.1
17.1
22.8
43.4
26.9
14.5
29.5
21.2
4.6
29.4
104.4
46.2
22.2
7.6
3.5
1.6
0.5
—
46.1
476.6

Kidomin
12.9
11.3
34.3
53.4
17.3
10.1
15.1
14.7
6.1
42.7
—
13.0
14.3
3.8
3.4
0.1
—
4.1
14.0
270.6

Data are mmol/L. Infusions also contain electrolytes andcarbohydrates.

Identification of Protein Binding of 123I-IMP
Serum protein binding of 123I-IMP (111 MBq/mL) was measured by ultrafiltration (Ultracent-10; Tosoh). Centrifugal filter
devices consisted of 2 compartments separated by a cellulose
membrane, which had a molecular weight cutoff of 10,000 Da.
123I-IMP binding to human serum protein and to purified serum
proteins such as HSA, AGP, and immunoglobulin (IgG) was
examined. Mixtures (900 mL) of 123I-IMP at 220 kBq in 20 mL ofsaline and serum (or HSA, AGP, or IgG solution) were centrifuged
at 3,000 rpm for 10 min at room temperature (model 5900;
Kubota). The radioactivities (counts/20 mL) of the initial sample[I] and filtrate [F] were measured by a well-type scintillation
counter (ARC-360; Aloka), and the free fraction and proteinbinding rate were then determined using the following equations:Free fraction ð%Þ 5 ½F=½I · 100
Protein-binding rate ð%Þ 5 100 2 free fraction

Eq. 1
Eq. 2

Human pooled sera were adjusted to an albumin concentration of
500 mM with phosphate buffer (pH 7.4). For use with purified
human serum protein, solutions of HSA (740 mM, 4.9 g/100 mL ofphosphate buffer), AGP (17 mM, 75.0 mg/100 mL of phosphatebuffer), and IgG (62 mM, 1.0 g/100 mL of phosphate buffer) wereprepared at a normal human serum concentration.
Examination of Specific Sites of Protein Binding by
123I-IMP on HSA and AGP
Effects of specific sites of protein binding by 123I-IMP on HSA
and AGP were also examined in human pooled serum (albumin
concentration, 500 mM) by ultrafiltration. Warfarin as a marker of
HSA binding site I, diazepam as a marker of HSA binding site II,

SERUM PROTEIN BINDING OF IMP • Kuga et al.

1379and propranolol and verapamil as markers of AGP were added toeach serum sample at respective therapeutic concentrations (200mM) before addition of the radioactive sample. The molar ratio of
HSA to each drug was 46.9. Free fraction rates were obtainedusing Equation 1. The control experiment used saline as a substitute for site marker. Adsorption of drugs onto the membrane andapparatus was negligible (1,2,12).
Examinations of Specific Sites of Protein Binding by
Amino Acid Infusions in SerumThe effects of amino acid infusion as a displacer were also
examined in human serum (albumin concentration, 700 mM) byultrafiltration. Proteamin and Kidomin were used because of theircomposition and content of amino acids. For confirmation ofserum protein-binding sites of amino acid infusions, binding site
markers such as 14C-warfarin (1.23 MBq/mL) and 14C-diazepam(3.7 MBq/mL) for binding sites I and II, respectively, and 3Hpropranolol (37 MBq/mL) for AGP were used. Mixtures (200 mL)of binding site marker (5 mL at 7.4 kBq/mL), amino acid infusion(2 mL), and serum were centrifuged at 3,000 rpm for 10 min at
25C (model 8800; Kubota). The molar ratios of HSA/L-tryptophan
in Proteamin and HSA/L-tryptophan in Kidomin were 15.2 and
11.4, respectively. Radioactivities (counts/10 mL) of the initialsample [I] and filtrate [F] were measured with a liquid scintillation
counter (LSC-5100; Aloka) after the addition of liquid scintillator(Clear-sol II; Nacalai Tesque) to scintillation vials. Free fraction
rates were then determined using Equation 1.
Effects of Amino Acid Infusions on Binding of 123I-IMP
The effects of amino acid infusions as displacers on serum
protein binding of 123I-IMP were examined. In this in vitro study,
125I-IMP (37 MBq/mL) was used instead of 123I-IMP. The free
fraction of 125I-IMP was determined by ultrafiltration as described above, and free fractions were then calculated using
Equation 1, with relative free fraction rates calculated as follows:
Relative free fraction rate (fold) 5 free fraction of sample/freefraction of control. HSA concentration in human serum wasadjusted to 700 mM with phosphate buffer (pH 7.4). The AGP
concentration in human serum was also adjusted to 20 mM byadding AGP-purified serum protein, and 2-mL aliquots of aminoacid infusion were added to 200 mL of a solution of 125I-IMP (1kBq in 10 mL) and human serum. Normal saline solution was used
as a control.
Displacement of 123I-IMP serum protein binding was attemptedin monkeys with amino acid infusion. A scintigraphic studyof 123I-IMP in 2 monkeys with or without loaded Proteaminwas performed. Fasting Japanese monkeys (4.0 kg) were anesthetized with pentobarbital (12.5 mg/kg of body weight, intraperitoneally) after Ketalar (5.0 mg/kg of body weight,
intramuscularly) and then received 123I-IMP (37 MBq in a 1.0mL bolus via the left cephalic vein, intravenously). To avoid
circulatory disorder, saline was infused via the right cephalic vein.
Supplemental heat to maintain body temperature was provided bya warm blanket. The depth of anesthesia was monitored byobserving the pupils and using pulse oximetry. The animals
recovered from anesthesia 5 h after the imaging study and werethen maintained in the normal fashion in our facility. Dynamic
whole-body planar data were acquired every 1 min for 60 min
with a 2-head scintillation camera (Prism 2000; Picker International). Subsequently, brain SPECT was performed over 30 min(30 s/frame) using a 3-head scintillation camera (Prism 3000;

1380

FIGURE 2. Protein binding of 123I-IMP to serum,
HSA, AGP, and IgG at concentrations of 740, 17, and62 mM, respectively. Eachcolumn represents mean 6
SD of 3 independent experiments.

Picker International). For the displacement study, 5.0 mL ofProteamin (142.0 mg/kg of body weight, intravenously) was
loaded just before the 123I-IMP injection. After 123I-IMP injection,
Proteamin was also administered at 0.5 mL/min for 30 min (14.2
mg/kg of body weight/min, intravenously). All animal experiments were approved by the Ethics Committee of the Universityof Miyazaki.
Statistical Analysis
Data are expressed as mean 6 SD. Groups were comparedusing the Student t test.

RESULTS
Determination of Protein Binding of 123I-IMP

Binding by 123I-IMP of human serum protein and purified serum protein was examined using ultrafiltration. In thein vitro binding assay shown in Figure 2, the percentage
binding of 123I-IMP to human protein was 78.34% 6
0.86%. In addition, 123I-IMP bound moderately and almost
equally to both HSA and AGP.
Examinations of Specific Protein-Binding Sites for
123I-IMP on HSA and AGP

Warfarin and diazepam are drugs that bind specifically tosites I and II, respectively, of HSA (7,13), whereas propranolol and verapamil bind specifically to AGP (14,15).
Free fractions of 123I-IMP were significantly increased,compared with control, by the HSA site II marker diazepam
and the AGP site marker propranolol (increases of 1.11and 1.16-fold, respectively). There were no effects on

FIGURE 3. Effects ofbinding site markers on
123I-IMP protein binding.
Normal saline solution wasused as control. Concentration of HSA in humanserum was adjusted to 500
mM. Each site marker was
loaded at 200 mM final concentration. Each column
represents mean 6 SD of
3 independent experiments. *P , 0.05.

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 50 • No. 8 • August 2009

FIGURE 4. Free fraction rates of radiolabeled chemicals (14C-warfarin [A],14C-diazepam [B], and 3H-propranolol[C]) loaded with Proteamin (PTA) andKidomin (KDM) in normal human serum.
Concentration of HSA in human serum
was adjusted to 700 mM, and 2.0-mL
aliquots of amino acid infusion wereadded to 200.0 mL of radiolabeledchemical–human serum mixture. *P ,0.02 vs. control. **P , 0.003 vs. control.

binding rate of the HSA site I marker warfarin (Fig. 3). 123IIMP thus appeared to bind site II on HSA and AGP.
Examinations of Specific Sites of Protein Binding by
Amino Acid Infusions in SerumAs shown in Figure 4, Proteamin and Kidomin markedly
increased not only the free fraction of 14C-diazepam (0.95% 6
0.04% for control, 1.40% 6 0.06% for Proteamin, and1.62% 6 0.05% for Kidomin) but also that of 3H-propranolol(10.60% 6 0.32% for control, 13.18% 6 0.14% forProteamin, and 13.82% 6 0.72% for Kidomin). 14C-diazepam
concentrations in human pooled serum were, with Proteaminand Kidomin, increased by 1.47- and 1.71-fold, respectively,
over the control level. The free fraction of 3H-propranolol
was also increased (by 1.24- to 1.30-fold) over the control
level. The free fraction of 14C-warfarin was reduced. Thesefindings indicated that the 2 amino acid infusions we tested
are suitable displacers for binding site II on HSA and AGP.
Effects of Amino Acid Infusion on Binding of 123I-IMP

We evaluated the relative free fraction rates of 125I-IMP
with amino acid infusions as displacers of serum protein
binding in vitro. The free fractions of 125I-IMP in humanpooled serum with Proteamin and Kidomin were significantly increased to 1.29 and 1.12 times the control level,
respectively (Table 2). 123I-IMP scintigraphy was therefore
performed on 2 monkeys with or without Proteamin.

TABLE 2. Free Fraction of 125I-IMP and Relative Free
Fraction Rate with Proteamin and Kidomin
Amino acidinfusionControl (saline)
Proteamin
KidominFree
fraction (%)
14.95 6 0.74
19.24 6 0.87
16.79 6 0.87

Relative free
fraction rate
(fold)—
1.29 6 0.06
1.12 6 0.06

*By paired t test. Control vs. amino acid infusion.

P*
—
,0.02
,0.02

Dynamic whole-body images and time–activity curves of
123I-IMP for each organ in monkeys are shown in Figures
5A and 6, respectively. Rapid cerebral accumulation wasobserved with Proteamin loading in both monkeys. The
average cerebral uptake of 2 monkeys increased by 1.34fold with Proteamin. Particularly, the plateaus of the time–
activity curves were slightly shifted within 15 min withProteamin loading in monkey 2. The plateaus of radioactivity in the brain of monkey 1 were significantly increased(1.44-fold), compared with the control condition, whereas
relative changes in regional cerebral accumulation on brain
SPECT (Fig. 5B) were not observed. These findings suggested that Proteamin treatment increased the free fractionof 123I-IMP and thus yielded rapid and pronounced cerebral
accumulation in vivo.
DISCUSSION

Systemically administered drugs bind reversibly to serum proteins, and only free drugs in serum exhibit pharmacologic effects. The binding of drugs to proteins is an
important factor inhibiting tissue distribution and excretion.
The usefulness of protein-binding displacement has beenreported. Takamura et al. suggested that coadministrationof bucolome as a displacer of binding site I on HSA iseffective in treating nephritic syndrome (16). 123I-IMP
exhibits high protein binding. We therefore attempted toincrease cerebral accumulation of 123I-IMP using the competitive displacement method with amino acid infusion,
which is applied clinically for parenteral nutrition andeasily administered to maintain high blood concentrationsof parenteral formula.
In our in vitro binding assay, approximately 78% of 123IIMP was bound to human serum protein, which has a highaffinity to HSA site II (one of the specific binding sites of
HSA) and AGP (Figs. 2 and 3). Yamasaki et al. havesuggested that L-tryptophan in amino acid fluids specifically inhibits binding site II of HSA (12). Furthermore, we
found that displacement by amino acid infusions might

SERUM PROTEIN BINDING OF IMP • Kuga et al.

1381

FIGURE 5. 123I-IMP whole-body scintigraphy (A) and brain SPECT (B) with orwithout Proteamin loading in same monkey (monkey 1). Proteamin (PTA; 5.0 mL)
was loaded just before 123I-IMP injection.
After 123I-IMP injection, Proteamin was
also administered at 0.5 mL/min for 30
min.

affect AGP and binding site II of HSA (Fig. 4). The aminoacid infusions we tested are thus suitable for clinical use as
displacers of binding site II of HSA and AGP.
These amino acid infusions were also found to havedisplacement effects on 125I-IMP in human pooled serum.
The free fraction of 125I-IMP loaded with amino acidinfusion, especially Proteamin, was significantly increased(1.29 times), compared with control (Table 2). Species
differences were observed in displacement effects (10). A
monkey study was then performed to confirm the displacement effects of Proteamin found in human serum studies,
since a species similarity was expected between monkeys
and humans. In the monkey scintigraphic study, rapid
cerebral accumulation and a slight shift of time–activity

curve plateaus were observed with Proteamin loading (Figs.
5 and 6). The radioactivity plateaus in brain were 1.24- and1.44-fold higher in Proteamin-loading monkeys than thosein control monkey 1 and control monkey 2, respectively.
Kawai et al. reported that the combination of both binding
inhibitors (of HSA site II and AGP) appears to exert asynergistic effect in displacement of IMP serum binding(10). It thus appeared that Proteamin treatment increased
the free fraction of 123I-IMP and displaced 123I-IMP serum
binding via a domino effect, yielding rapid and pronounced
cerebral accumulation in vivo. The initial gradients of thebrain accumulation curve with Proteamin loading were 1.3and 1.4-fold higher in Proteamin-loading monkeys than incontrol monkey 1 and control monkey 2, respectively. We

FIGURE 6. Time–activity curves of
123I-IMP monkey 1 (A) and monkey 2(B) on dynamic scintigraphy in samemonkey. Findings with or without Proteamin (PTA) loading are shown for 123IIMP accumulation in monkey.

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THE JOURNAL OF NUCLEAR MEDICINE • Vol. 50 • No. 8 • August 2009

observed that the IMP uptake rate in the brain wasincreased just after administration. Furthermore, we observed different initial gradients in lung accumulation
curves between Proteamin loading and control (Fig. 6).
The free drug hypothesis assumes that only the free fractionof the drug in plasma can penetrate tissue and that drugtransport in tissue is governed by the free drug concentration gradient between plasma and tissue, until equilibrium
is reached (17,18). Thus, it appeared that Proteamin treatment increased the free 123I-IMP concentration gradient,with a tendency toward shift and a short time to peak value.
However, the mechanism of IMP uptake into brain capillaries requires further study. These findings suggested thepossibility of controlling the pharmacokinetics of 123I-IMP
and imaging in clinical applications. Such a possibility
could improve not only cerebral accumulation but also thesignal-to-noise ratio of scintigraphic images. On the other
hand, the possibility exists that reduced administration of
123I-IMP can yield image quality equal to that currently
obtainable, with control of diagnostic costs. The displacement method could easily be applied to human studies
under the conditions of the present examination. Displacement of radiopharmaceutical binding to serum proteins, we
believe, provides a higher target organ accumulation andshortened imaging times, as well as higher signal-to-noise
ratios in imaging.
CONCLUSION

This study showed that application of amino acidinfusions as displacers of serum protein binding significantly increased the free fraction of IMP in vitro in serum
and improved cerebral accumulation in monkey brain onwhole-body dynamic scintigraphy. Amino acid infusions
could thus be clinically useful for competitive displacement and regulation of the tissue distribution and kinetics
of molecular imaging probes with high protein binding.
Further similar studies are needed with other radiopharmaceuticals.
ACKNOWLEDGMENTS

We thank Hideo Arita, Yasuyoshi Kuroiwa, Dr. Seigo
Fujita, Dr. Takashi Jinnouchi, and the rest of the staff of the
Department of Radiology, University of Miyazaki Hospital,

as well as the members of the Kawai Laboratory ofKanazawa University, for technical assistance.
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