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Nuclear Medicine and Biology 36 (2009) 99 – 106
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Serum protein binding displacement: theoretical analysis using ahypothetical radiopharmaceutical and experimental analysis with
123
I-N-isopropyl-p-iodoamphetamine
Keiichi Kawaia,b , Ryuichi Nishiic , Naoto Shikanod,⁎,1 , Nobuo Makinoe , Noriyuki Kugaa,f ,Mitsuyoshi Yoshimotoa , Seishi Jinnouchig , Shigeki Nagamachig ,Shozo Tamurag , Norito TakamurahaDivision of Health Science, Graduate School of Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 9200-942, Japanb
Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japanc
Research Institute, Shiga Medical Center, Moriyama City, Shiga 524-8524, Japand
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Inashiki-gun, Ibaraki 300-0394, Japane
Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, Inashiki-gun, Ibaraki 300-0394, Japanf
Department of Radiology, University of Miyazaki Hospital, Kiyotake-cho, Miyazaki-gun, Miyazaki 889-1692, Japang
Department of Radiology, Faculty of Medicine, University of Miyazaki, Miyazaki-gun, Miyazaki 889-1692, JapanhSchool of Pharmaceutical Sciences, Kyusyu University of Health and Welfare, Nobeoka City, Miyazaki 882-8508, Japan
Received 21 August 2008; received in revised form 6 October 2008; accepted 9 October 2008

Abstract
Introduction: The binding of radiopharmaceutical to serum proteins is thought to be an important factor that restricts its excretion andaccumulation in tissue. We calculated the effect of inhibitors of serum protein binding using a hypothetical radiopharmaceutical. In vitro
experiments and protein binding inhibitor-loaded monkey scintigraphy were then conducted using 123I-N-isopropyl-p-iodoamphetamine(IMP) as the radiopharmaceutical.
Methods: Free fraction ratios of radiopharmaceutical were calculated with one radiopharmaceutical, two serum proteins and two specificinhibitors in the steady state at various serum protein concentrations. In vitro protein binding inhibition studies using human, rat and monkey
sera were performed with site-selective displacers of specific binding sites: 400 μM 6-methoxy-2-naphthylacetic acid (6MNA; a major
nabumeton metabolite) as a serum albumin Site II inhibitor and 400 μM erythromycin (ETC) as an α1-acid glycoprotein (AGP) site inhibitor.
Scintigraphy with or without 6MNA loading of monkeys was performed.
Results: The theoretical findings roughly corresponded to the experimental results. Approximately 75% of IMP bound to serum albumin Site
II and AGP in the species examined. The free fraction of IMP (25.0±0.6% for human, 22.8±0.4% for monkey, 23.7±0.3% for rat) increased
with loading of specific protein binding inhibitors (6MNA: 28.0±0.3% for human, 24.5±0.7% for monkey, 24.3±0.2% for rat; ETC: 26.3±
0.4% for human, 29.5±1.1% for monkey, 26.0±0.7% for rat) and was serum protein concentration dependant based on the results ofcalculations. Simultaneous administration of 6MNA and ETC produced a higher free fraction ratio of IMP (31.9±1.0% for human, 34.6±
0.4% for monkey, 27.0±0.3% for rat) than summation of the single administrations of 6MNA and ETC (domino effect) in human, rat andmonkey sera. Rapid cerebral accumulation was observed with 6MNA loading in monkey scintigraphy.
Conclusions: 6MNA appears to change the pharmacokinetics and brain accumulation of IMP in monkeys. Further studies in human are
required.
© 2009 Elsevier Inc. All rights reserved.
Keywords: N-Isopropyl-p-iodoamphetamine; Pharmacokinetics; Protein binding; Human serum albumin⁎ Corresponding author. Tel.: +81 298 40 2217; fax: +81 298 40 23171.
E-mail address: sikano@ipu.ac.jp (N. Shikano).
1
Current address: Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki
300-0394, Japan.
0969-8051/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2008.10.005

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K. Kawai et al. / Nuclear Medicine and Biology 36 (2009) 99–106

1. Introduction
The binding of drugs to serum proteins is an importantfactor that restricts both drug excretion and accumulation intissue [1]. The free drug hypothesis assumes that only thefree fraction of the drug in plasma can penetrate the tissue
and that drug transport in tissue is governed by the free drugconcentration gradient between plasma and tissue, until
equilibrium is reached [1,2].
Several drugs bind with high affinity to one of the serum
albumin sites and to α1-acid glycoprotein (AGP), and there
are individual variations in free/bound drug ratios [3]. The
specific binding sites in human serum albumin have been
well characterized. In fact, three sites for drug binding havebeen reported: Site I (also known as the warfarin bindingsite), Site II (the benzodiazepine binding site) and Site III(the digitoxin binding site). Of these, Sites I and II are known
to be the main binding sites of various therapeutic drugs [4].
However, few studies have examined competitive displacement by radiopharmaceuticals of specific serum protein
binding sites.
In this study, free fraction ratios of hypotheticalradiopharmaceutical or radiolabeled ligand (L) in model

sera were calculated in an attempt to clarify the influence ofthe drug–radiopharmaceutical interaction with protein
binding on molecular imaging. Free fraction ratios ofradioactive ligand were calculated with one radiopharmaceutical (L), two serum proteins (P1 and P2) and two
specific inhibitors (I1 and I2) (referred to as the LP2I2model) at the steady state. Because the concentration ofserum protein changes depending on the disease condition,
free fraction ratios with various P1 and P2 concentrations
were also calculated.
Although 123I-N-isopropyl-p-iodoamphetamine (IMP) is
widely used for cerebral blood flow assessment [5,6], theeffects of serum protein binding displacement by coadministered drugs are unknown. It is noteworthy that the obtained
values determined with SPECT may or may not be
influenced by serum protein binding as well as bymetabolism. Furthermore, it is important to establish themagnitude of the influence of plasma protein binding ofradiopharmaceuticals. We compared the theoretical resultswith experimental results of serum protein binding displacement by IMP and the specific inhibitor for Site II of serum
albumin, 6-methoxy-2-naphthylacetic acid (6MNA; a nabumeton metabolite), and the specific inhibitor for the AGP

Fig. 1. LP2I2 model serum with one hypothetical radioactive ligand (L), two serum proteins (P1 and P2) and two specific inhibitors (I1 and I2) at steady state. L canbind to free P1 and P2. I1 or I2 competitively inhibits binding of L to P1 or P2, respectively. K1, K2, K1′ and K2′ are binding constants (A). Chemical structures ofradioactive ligand and specific serum protein binding inhibitor for the in vitro study (B).

K. Kawai et al. / Nuclear Medicine and Biology 36 (2009) 99–106

binding site, erythromycin (ETC), in human, monkey and rat
sera (Fig. 1). As an in vivo study, we used scintigraphy toevaluate the effects of 6MNA on the biodistribution of IMP
in monkeys intravenously injected with 6MNA.
2. Materials and methods
2.1. Materials
Reagent-grade chemicals were used in this experiment.
ETC and 6MNA were purchased from Abbott Japan (Tokyo,
Japan) and SmithKline Beecham Co., Ltd. (West Sussex,UK), respectively. IMP (Nihon Medi-Physics Co., Ltd.,Tokyo, Japan) was used as the radiopharmaceutical. The
radiochemical purity of the IMP was greater than 98.0%.
2.2. Model serum protein binding calculations
In this study, free fraction ratios of hypothetical radiopharmaceutical or radiolabeled ligand (L) that assume IMPin the model sera were calculated in an attempt to clarify the
influence of drug–radiopharmaceutical interactions withprotein binding on imaging. Radiolabeled ligand binds
serum protein-1 (P1), which assumes serum albumin, andalso serum protein-2 (P2), which assumes AGP (Fig. 1). I1

101

and I2, which assume 6MNA and ETC, respectively, were
the specific inhibitors of P1 and P2, respectively. Therefore,
this is a one-ligand, two-serum-protein and two-specificinhibitor model (denoted the LP2I2 model). Free fraction
ratios of L were calculated repeatedly using step-by-step
approximation until values reached the steady state. The
normal range of human serum albumin and AGP is 3.8–
5.0 g/dl and 70.0–80.0 mg/dl, respectively [7]. Because theconcentration of serum protein changes depending on the
disease condition, free fraction ratios with various P1 and P2
concentrations were also calculated. The scheme of calculation of the concentration of L, [L], in the steady state is
described in the Appendix A. Parameters determined fromthe normal range of clinical laboratory tests and therapeutic
concentrations of inhibitors were used in this calculation.
2.3. Measurement of IMP free fraction ratios ofprotein binding
We determined the IMP free fraction ratios of protein
binding using the method published by Nishio et al. [8]. The
human and monkey blood samples were obtained from thecephalic vein, while the rat blood samples were obtained byheart puncture. In brief, the serum protein binding of IMP
was evaluated by ultrafiltration (Ultracent-10; Tosoh, Tokyo,

Fig. 2. Results of calculations. The following parameters were used: K1=8000 M−1, K2=11,000 M−1, K1′=5.0×106 M−1, K2′=3.5×104 M−1. For Panels A and B,
P1: 2–7 g/dl, P2: 75 mg/dl, L: 1.25×10−9 M, I1: 400 μM, I2: 400 μM. For Panels C and D, P1: 4 g/dl, P2: 10–100 mg/dl, L: 1.25×10−9 M, I1: 400 μM, I2: 400 μM.

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K. Kawai et al. / Nuclear Medicine and Biology 36 (2009) 99–106

Fig. 3. Calculated in vitro displacement of IMP protein binding in rat serum (A and B). Proposed mechanisms by which the free fractions of L are determinedwith administration conditions I1 (C), I2 (D) and I1+I2 (E) are illustrated. The free fraction of L is increased by competitive inhibition with I1 (C). The free fractionof L does not increase as much by competitive inhibition with I2 because of P2 to P1 transfer of L (D). The combination of both binding inhibitors appears to exert
a synergistic effect on the displacement of L serum binding, the domino effect (E).

Japan). Inhibitors of albumin binding were examined inhuman, Japanese monkey and rat sera. Mixtures (0.90 ml) ofa radioactive tracer (IMP at 220 kBq in 20.0 μl saline;
1.25×10−9 M) and serum were centrifuged at 3000 rpm for
10 min at room temperature (Model 5900; Kubota, Tokyo,
Japan). The123 I free iodide in the radiotracer and “sticking”
of radioactivity to the Ultracent-10 were not observed. The
radioactivity (count/20.0 μl) of the initial sample [I] andfiltrate [F] was measured using a well-type scintillation
counter (ARC-360; Aloka, Japan), and the protein binding
rate and free fraction rate were then determined using thefollowing equations: Free fraction (%)=[F]/[I]×100, andProtein binding rate (%)=100−free fraction. The sera were
adjusted to albumin concentrations of 375 μM withphosphate buffer (0.067 M, pH 7.4) as described elsewhere[9–11]. 6MNA was tested as a displacer of serum albuminbinding site (Site II), while ETC was tested as a displacer ofthe AGP binding site. Test compounds were added at thetherapeutic concentration of 400 μM before addition of theradioactive sample [7].

2.4. In vivo monkey study
Female Japanese monkeys (4 kg) were anesthetized withpentobarbital (50 mg/kg body weight, ip) after ketalar(5 mg/kg body weight, im) and were then administered
1.0 ml saline containing IMP (111 MBq, iv via the left
cephalic vein, bolus). Saline was infused via the rightcephalic vein to avoid a circulation failure. A warm patch
was used to maintain body temperature. The depth ofanesthesia was monitored based on the condition of thepupils and using pulse oximetry. Dynamic planar data for
the whole body was collected (1 frame/min for 30 min)using a two-headed scintillation camera (Prism 2000; Picker
International, Cleveland, OH, USA). In the displacement
study, 6MNA (20 mg/kg body weight, iv) was administered
to the same monkey immediately prior to IMP injection.
Time–activity curves were obtained for the main tissue
regions using a supercomputer (Odyssey; Picker International). The monkey studies were conducted three times in
two different monkeys to confirm reproducibility. All

K. Kawai et al. / Nuclear Medicine and Biology 36 (2009) 99–106

animal experiments were approved by the Ethics Committeeof the University of Miyazaki.
2.5. Statistical analysis
The values obtained in each experiment are expressed as
the mean±S.D. Statistical comparisons between groups were
performed using Student's t test.
3. Results
3.1. Calculations
Fig. 2 shows the calculated free fraction ratio of
hypothetical radiopharmaceutical L versus P1 or P2 concentration. As shown in Fig. 2A and C, the free fraction
ratios of L increased upon addition of the inhibitors,
compared to the control. The changes in the free fraction
ratio of L depended on the P1 and P2 concentration.
Interestingly, as shown in Fig. 2B and D, simultaneousadministration of I1 and I2 produced a higher free fraction
ratio of L than the single administration of I1 or I2 (domino
effect). This effect increased as the P1 concentration
decreased. In contrast, the effect increased as the P2
concentration increased.

103

Fig. 3A shows the free fraction ratio of L when the P1
concentration was 4 g/dl under various I1 and I2 administration conditions. Coadministration of I1 and I2 produced ahigher free fraction ratio of L than single administration of I1
or I2 (Fig. 3B).
3.2. Measurement of IMP free fraction ratios in serum
The percentage of IMP bound to human serum protein
was found to be 69.1±1.9%, and thus, we next evaluated theeffects of the site-specific inhibitors (Fig. 4) on serum protein
binding of IMP. IMP bound to human serum albumin and
AGP almost equally. In human serum, the free fraction of
IMP with 6MNA loading increased significantly, by 1.1fold, when compared to the controls (free fraction of IMP
without loading) in the presence of the human serum
albumin Site II inhibitor, 6MNA. The free fraction of IMP
increased significantly, by 1.1-fold, when compared to thefree fraction of IMP without loading (controls), in thepresence of the AGP inhibitor, ETC. Simultaneous administration of 6MNA and ETC produced a 1.3-fold-higher free
fraction ratio of IMP than summation of the data from thesingle administrations of 6MNA and ETC. Similar effects
were observed in the Japanese monkey and rat sera studies(Fig. 4A and B).

Fig. 4. In vitro displacement of IMP protein binding in rat serum. Inhibitor was loaded at a final concentration of 400 μM. ⁎Pb.01 versus control, n=6 (A).
Observed domino effect in human, monkey and rat sera (B).

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K. Kawai et al. / Nuclear Medicine and Biology 36 (2009) 99–106

Fig. 5. Representative IMP whole-body scintigraphy of monkey. The arrowheads indicate the brain. 6MNA (20 mg/kg body weight) was simultaneously injectedwith IMP. Dynamic planar data for the whole body were collected (1 frame/min for 60 min) by scintillation camera (A). The time–activity curve of Panel A (B).

3.3. In vivo monkey study
Representative IMP whole-body planar scintigraphy of aJapanese monkey and the resultant time–activity curves areshown in Fig. 5A and B, respectively. Rapid cerebral
accumulation was observed with 6MNA loading. The
plateau level of brain radioactivity was 1.3-fold higher
than in the controls. In vitro monkey serum analysis revealed
that the free fraction of IMP with 6MNA loading increased
significantly (1.3-fold), from 27.9% without loading to
36.9% with 6MNA loading.

4. Discussion
As predicted from the calculation using the LP2I2 modelin Fig. 1, the free fraction of IMP was affected by the change
in human serum albumin and AGP. The change in serum
albumin was more effective than that in AGP, as shown in
Fig. 2. The free fraction of IMP would increase in a patientwith low serum protein concentration and decrease in apatient with high serum protein concentration. Possible
causes of a decrease in human serum albumin include renal

damage, hepatic damage, diabetes, inflammatory disorders(rheumatism, cancer and infection), nephrosis and hemorrhage (due to surgery, blood in urine and hematochezia).
Human serum albumin increases in hemodialysis due to theconcentrated blood. On the other hand, AGP increases inrenal damage, hemodialysis and inflammatory disorders(rheumatism, cancer and infection), while AGP decreases inhepatic damage, nephrosis and hemorrhage (due to surgery,blood in urine and hematochezia) [12].
Figs. 3 and 4 show that the theoretical and experimental
results roughly corresponded, although species differenceswere observed in the experimental data. Furthermore, thefree fraction data differed between the theoretical andexperimental data, possibly caused by naturally occurring
compounds in the sera. Although the principle wasprovisionally proven by the calculation, the appropriate
binding constants K1, K2, K1′ and K2′ should be determined
experimentally in future research.
We predicted the domino effect using the LP2I2 model.
Significant inhibition of 6MNA and ECT showed that IMP
was bound to Site II on human serum albumin and to thebasic binding site on AGP (Fig. 4A). The combination ofboth binding inhibitors appears to exert a synergistic effectK. Kawai et al. / Nuclear Medicine and Biology 36 (2009) 99–106

on the displacement of IMP serum binding (domino effect,
Fig. 3E). The domino effect was observed in all sera studied
in this experiment (Fig. 4B).
We used 6MNA for loading in the monkey study based on
the results of Fig. 2. As shown in Fig. 5, 6MNA loading
resulted in high cerebral accumulation (1.30 times control
level) and rapid clearance of IMP. The free fraction of IMP inin vitro monkey serum increased with 6MNA loading (1.32
times control level). Acceleration of cerebral accumulation
with 6MNA loading was considered the result of IMP
protein binding displacement on only the serum albuminbinding site. This condition corresponds to Fig. 3C. The useof binding site inhibitors may result in more rapid productionof better diagnostic images using lower radiation doses.
There are several clinical implications of these findings,
and the following points should be noted when a protein
binding inhibitor is used. The distribution of drugs depends
not only on their interaction with proteins but also on anumber of other factors, such as metabolism and excretion[1]. For example, an increase in IMP brain uptake by loading
imipramine has been demonstrated in an animal study, andthe imipramine bound plasma proteins and erythrocytes [2].
Moretti et al. [13] found that post-loading of imipramine
resulted in an increase in the brain activity of IMP in rabbits,
which was thought to occur because IMP competed withimipramine in a metabolic reaction related to the mixed
function oxidase system in the lung. On the other hand, Riant
et al. [2] reported that in vivo studies demonstrated that theprotein-bound drug as well as imipramine or hormone, as
measured in vitro, is partly available for transport into tissue,
including brain, depending on the nature of the drug and of
the binding protein.
The safety aspects of administering this kind of protein
binding inhibitor to patients require analysis and discussion.
Because the protein displacer affects not only the binding ofIMP but also the binding of other drugs or small molecules,
the use of a protein displacer needs to be investigatedcarefully not only in patients taking other medications but
also in patients with low plasma proteins, dialysis patients,and so forth. Nabumeton, with a major metabolite of 6MNA,
is easily administered perorally [14], and its pharmacokinetics with continuous and single oral administration havebeen well studied in humans [15]. Although the risks of
NSAIDs including nabumeton are generally low, clinicallysafe inhibitors without pharmacological activity need to be
identified.
In conclusion, we studied serum protein binding displacement by theoretically using a hypothetical radiopharmaceutical and experimentally with IMP. The theoretical resultswith the LP2I2 model roughly corresponded to the experimental findings in human, rat and monkey sera withadministration of IMP, 6MNA and ECT. In the in vivo
monkey scintigraphy study, rapid cerebral accumulation wasobserved with 6MNA loading. The search for strategies toimprove imaging may facilitate further research into the
combined use of radiopharmaceuticals with drugs.

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Acknowledgments
The authors wish to thank Nihon Medi-Physics Co., Ltd.,for generously supplying IMP and Grelan PharmaceuticalCo., Ltd.; Eisai Co., Ltd.; Fujisawa Pharmaceutical Co., Ltd.;
Kaken Pharmaceutical Co., Ltd.; and SmithKline BeechamCo., Ltd., for kindly providing the chemical regents.

Appendix A. Scheme
P1: serum protein 1
P2: serum protein 2
L: radioactive ligand
I1: specific inhibitor of P1
I2: specific inhibitor of P2
P1 + LfP1  L;

K1 = ½P1  L=½P1 ½L

ð1ÞP2 + LfP2  L;

K2 = ½P2  L=½P2 ½Lð2ÞP1 + I1 fP1  I1 ;K V1 = ½P1  I1 =½P1 ½I1 

ð3Þ

P2 + I2 fP2  I2 ;K V2 = ½P2  I2 =½P2 ½I2 ð4Þ

L can bind to free P1 and P2. I1 or I2 competitively inhibits
binding L with P1 or P2, respectively.
K1, K2, K1′ and K2′ are binding constants. The unit of thesebinding constants is M−1.
From Eqs. (1–4),½P1  L = K1 ½P1 ½L

ð5Þ

½P2  L = K2 ½P2 ½L

ð6Þ

½P1  I1  = K V1 ½P1 ½I1 

ð7Þ

½P2  I2  = K V2 ½P2 ½I2 

ð8ÞInitial concentration of P1, [P1]0, can be described asfollows:½P1 0 = ½P1  + ½P1  L + ½P1  I1 

ð9ÞFrom Eqs. (5), (7) and (9),
½P1 0 = ½P1 ð1 + K1 ½L + K V1 ½I1 Þ
Concentration of free P1, [P1], can be described as follows:½P1  = ½P1 0 =ð1 + K1 ½L + K V1 ½I1 Þð10ÞIn a similar way, the next expression consists of:½P2  = ½P2 0 =ð1 + K2 ½L + K V2 ½I2 Þ

ð11Þ

Initial concentration of L, [L]0, can be described asfollows:½L0 = ½L + ½P1  L + ½P2  L

ð12Þ

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K. Kawai et al. / Nuclear Medicine and Biology 36 (2009) 99–106

From Eqs. (5), (6) and (12),½L0 = ½Lð1 + K1 ½P1  + K2 ½P2 Þ
The above equation is rewritten as follows:½L = ½L0 =ð1 + K1 ½P1  + K2 ½P2 Þð13Þ

[L]/[L]0 is experimentally measured in this study.
In a similar way, the next expression consists of:½I1  = ½I1 0 =ð 1 + K V1 ½P1 Þ

ð14Þ

½I2  = ½I2 0 =ð 1 + K V2 ½P2 Þ

ð15Þ

Calculation of [L] in the steady state
Step 1. At first, free P1 concentrations [P1]1 and [P2]1 arecalculated from the initial concentrations of [P1]0, [P2]0,[P2]0, [L]0, [I1]0 and [I2]0 using Eqs. (10) and (11). At Step 1,[P1]1 and [P2]1 are not true values but approximate values,
because instead of initial concentrations, the free concentration should be used with this calculation.
Step 2. Then, from the free plasma protein concentrations[P1]1 and [P2]1 obtained above, the concentrations of free
radioactive ligand and specific inhibitors [L]1, [I1]1 and [I2]1
are calculated using Eqs. (13–15)). In addition, we tried to
calculate [P1]2 and [P2]2 using the values of [L]1, [I1]1 and [I2]1.
Step 3 and further steps. After Step 2, the accuracy of theapproximation is improved by repeating Step 2.
The values are obtained with the accuracy of six digits
within Step 10 or less.
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