Nuclear Medicine and Biology 40 (2013) 366–370

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Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio

Pharmacokinetic Alteration of 99mTc-MAG3 using Serum Protein Binding
Displacement Method☆
Kodai Nishi a, Masato Kobayashi a, Ryuichi Nishii b, Naoto Shikano c, Norito Takamura d, Noriyuki Kuga a, b,
Keishi Yamasaki e, Shigeki Nagamachi b, Shozo Tamura b, Masaki Otagiri e, Keiichi Kawai a, f,⁎aSchool of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Ishikawa 920-0942, Japan
Department of Radiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japanc
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ami, JapandSchool of Pharmaceutical Sciences, Kyusyu University of Health and Welfare, Miyazaki, Japane
Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japanf
Biomedical Imaging Research Center, University of Fukui, Fukui, Japanba r t i c l e

i n f oArticle history:
Received 20 October 2012
Received in revised form 26 November 2012
Accepted 1 December 2012
Keywords:
99m
Tc-MAG3
Pharmacokinetics
Serum protein binding
Competitive displacement
Renograma b s t r a c t
Introduction: When a radiopharmaceutical is simultaneously administered with a medicine that has highafﬁnity for the same plasma protein, the radiopharmaceutical is released at higher concentrations in blood,leading to enhanced transfer into target tissues. This is known as the serum protein binding displacement
method. In this study, we investigated the pharmacokinetic alteration of technetium-99m-labeled
mercaptoacetylglycylglycylglycine (99mTc-MAG3) using the serum protein binding displacement method.
Methods: Rat and human serum protein binding rates of 99mTc-MAG3 were measured by ultraﬁltration with orwithout displacers of human serum albumin (HSA) binding sites I and II (200 μM and 400 μM loading). Male
Wistar rats were injected with 99mTc-MAG3 (740 kBq/0.3 mL saline) via the tail vein, and biodistribution wasassessed at 2, 5, 10 and 15 min. Dynamic whole-body images were obtained for 99mTc-MAG3 (11.1 MBq/
0.3 mL saline)-injected rats, with or without HSA displacers.
Results: 99mTc-MAG3 strongly bound to HSA (87.37%±2.13%). Using HSA site I displacers, the free fraction of99m
Tc-MAG3 increased signiﬁcantly (1.20 to 1.47 times) when compared with controls. For biodistribution
and imaging, rapid blood clearance was observed with bucolome (BCL) loading, which is an HSA site I
displacer. With BCL loading, peak times for rat renograms were respectively shifted from 240 s to 110 s, andfrom 170 s to 120 s.
Conclusions: We found that 99mTc-MAG3 bound to the HSA binding site I. It was conﬁrmed thatpharmacokinetic distribution of 99mTc-MAG3 is altered by presence of BCL, which leads to increases in thefree fraction of 99mTc-MAG3, and BCL produced rapid blood clearance and fast peak times on rat renograms.
The serum protein binding displacement method using 99mTc-MAG3 and BCL, a safe displacer for humans,
may be applicable to clinical study and lead to better diagnostic images with shorter waiting times and lower
radiation doses for patients.
© 2013 Elsevier Inc. All rights reserved.

1. Introduction
Most radiopharmaceuticals have a weak or moderate afﬁnity foralbumin and acid glycoproteins, often depending on their lipophilicity[1]. When the afﬁnity of the radiopharmaceuticals for blood
components increases, the radiopharmaceuticals do not readilytransfer into target tissue. Changes in the pharmacokinetics and☆ Disclosure/Conﬂict of interest: The authors declare no conﬂicts of interest.
⁎ Corresponding author. School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Ishikawa 920-0942,
Japan. Tel.: +81 76 265 2527; fax: +81 76 234 4366.
E-mail address: kei@mhs.mp.kanazawa-u.ac.jp (K. Kawai).
0969-8051/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nucmedbio.2012.12.002

distribution of radiopharmaceuticals would thus be expected toenhance the transfer into target tissues [2].
In the distribution of medicines for medical diagnosis ortreatment, medicines move along with systemic blood circulation,
and are transferred to the intravascular, interstitial and intracellularspaces by diffusion and transportation of unbound medicine (freefraction). The free fraction of medicines arriving at target tissues is
considered to provide pharmacological activity [3–5]. Thus, theconcentration of the pharmacological agent is typically determinedby the level of protein binding.
When one medicine is simultaneously administered with a second
medicine that has high afﬁnity for the same plasma protein, the ﬁrst
medicine is released at higher concentrations and produces a stronger
pharmacological action. This method is known as the serum protein

K. Nishi et al. / Nuclear Medicine and Biology 40 (2013) 366–370

binding displacement method. Our study group has already reported
the use of the serum protein binding displacement method with 123IN-isopropyl-p-iodoamphetamine ( 123I-IMP). We found that thepharmacokinetics of 123I-IMP were markedly changed, and that thebrain accumulation of 123I-IMP was improved in a Japanese monkey
study [6,7].
Technetium-99m-labeled mercaptoacetylglycylglycylglycine( 99mTc-MAG3) is widely used in renal scintigraphy and has very
high afﬁnity (90% binding) for plasma protein at typical clinical doses[8]. However, the binding site of 99mTc-MAG3 has not been identiﬁed.
In this study, we determined the binding site of the 99mTc-MAG3 andapplied the protein binding displacement method in order to alter thepharmacokinetics of 99mTc-MAG3. In an in vitro study, the serum
protein binding site of 99mTc-MAG3 was identiﬁed using ultraﬁltration analysis with rat and human serum. Subsequently, the protein
binding displacement method was applied to a rat study in order toexplore the possibility of clinical application.
2. Materials and methods
2.1. Materials
Reagent-grade chemicals, HSA (essentially fatty acid-free albumin), AGP and human immunoglobulin G (IgG) were acquired from
Sigma Chemical (St. Louis, MO). Valproic acid and oleic acid sodium
salts were obtained from Tokyo Chemical Industry (Tokyo, Japan), andoctanoic acid sodium salt was purchased from Katayama Chemical(Osaka, Japan). Bucolome (BCL), Paramidin (Grelan Pharmaceutical,
Tokyo, Japan), potassium warfarin (Eisai, Tokyo, Japan), cefazolin
sodium (Fujisawa Pharmaceutical, Osaka, Japan) and ibuprofen(Kaken Pharmaceutical Co., Ltd., Tokyo, Japan) were kind gifts.
99m
Tc-MAG3 (Daiichi Radioisotope Laboratories, Tokyo, Japan) wasused as a radiopharmaceutical.
2.2. Measurement of protein binding rates of 99mTc-MAG3
Serum protein binding of 99mTc-MAG3 was evaluated by ultraﬁltration (Ultracent-10; Tosoh, Tokyo, Japan) [9–11]. Mixtures (0.9 mL)of 99mTc-MAG3 (740 kBq in 20 μL saline) and serum (or HSA, AGP or
IgG solution) were centrifuged at 3000 rpm for 10 min at room
temperature (Model 5900; Kubota, Tokyo, Japan). Radioactivities(counts/20 μL) of the initial solutions and ﬁltrates were measured
using a well-type scintillation counter (ARC-360; Aloka, Tokyo,
Japan), and their protein binding and free fraction rates were
determined. With regard to bufferized solvent, 0.067 M phosphatebuffer (adjusted to pH 7.4) was used. Human and rat sera wererespectively adjusted at an albumin concentration of 500 μM and375 μM with phosphate buffer. For puriﬁed human serum protein,
HSA (740 μM, 4.9 g in 100 mL phosphate buffer), AGP (17 μM, 75 mg
in 100 mL phosphate buffer) and IgG (62 μM, 1.0 g in 100 mL
phosphate buffer) solutions were prepared at normal human serum
concentrations. Effects of displacers of albumin binding were also
examined in human and rat sera.

367

Table 1
Displacement effects of inhibitors on 99mTc-MAG3 binding to plasma protein.

Site-speciﬁc
inhibitor
Concentration
Site I

Site II

Control
BCL
Valproic acid
WarfarinCefazolin
Ibuprofen
Octanoic acid
Oleic acid

HSA

RSA

99m

Tc-MAG3 free
fraction (%)

99m
Tc-MAG3 free
fraction (%)

200 μM

200 μM

12.23
11.98
11.50
11.13
10.18
9.60
8.74

400 μM

10.20
13.74
13.02
13.57
14.76
10.53
9.86
9.44

32.76
28.48
33.57
28.58
28.48
N/A
N/A

400 μM

24.75
43.85
29.30
43.28
33.52
33.04
N/A
N/A

N/A: not analyzed.

BCL, warfarin, valproic acid and cefazolin were used as displacersof HSA binding site I, while ibuprofen, octanoate and oleate were usedas displacers of HSA binding site II. These were added to serum
samples at the concentrations required for sufﬁcient competitive
inhibition (200 μM) and at double doses (400 μM) in order to conﬁrm
concentration dependence. Warfarin and octanoate were added toserum samples immediately prior to injection of radiotracer in order
to avoid deactivation in serum samples. Binding rate to protein was
calculated using the following formula:100−ð100  ½F=½SÞ½%

where, [S] is total radioactivity of the mixture consisting of plasma
protein and radiopharmaceutical, and [F] is radioactivity of the ﬁltrateobtained by centrifuging the mixture.
2.3. Biodistribution studies of 99mTc-MAG3 in rats
Animal studies were approved by the Animal Care Committee atthe University of Miyazaki, and were conducted in accordance with
the international standards for animal welfare and institutionalguidelines. To assess the competitive displacement of protein binding
with BCL in vivo, ﬁve male Wistar rats (age, 6 weeks) wereadministered 99mTc-MAG3 (740 kBq) in 0.1 mL of saline by intravenous bolus injection via the tail vein. Rats were sacriﬁced at 2, 5, 10
and 15 min after injection of radiotracer by cervical dislocation underdeep ether anesthesia, and blood and organs of interest were excised.
After measuring organ weights, radioactivities were determinedusing a scintillation counter. Accumulation ratios (% dose/organ and% dose/g of tissue) were determined after decay correction. In vivo
displacement studies on serum protein binding of radiopharmaceuticals were performed with BCL pretreatment (20 mg/kg body weight,
i.v., 10 min before injection of radiotracer).
2.4. Scintigraphic studies of 99mTc-MAG3 in rats

Fig. 1. Binding rates of 99mTc-MAG3 with human serum protein. 99mTc-MAG3 boundstrongly to HSA.

Wistar rats weighing 400 g were administered 99mTc-MAG3(11.1 MBq) in 0.3 mL of saline via the tail vein. Dynamic wholebody images (10 s/frame for 20 min) were obtained with a scintillation camera (Prism 3000; Picker International, Cleveland, OH).
Time–activity curves were obtained from the main tissue region using
a supercomputer (Odyssey; Picker International), and regions ofinterest (ROIs) in the kidneys were examined on renograms. In thesame rats, scintigraphic studies with displacer treatment were also
carried out. BCL (20 mg/kg body weight) was loaded by intravenous
infusion for 10 min using a syringe pump (A-99; Razel Scientiﬁc
Instruments, Stamford, CT) at 5–15 min before 99mTc-MAG3 injection.

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K. Nishi et al. / Nuclear Medicine and Biology 40 (2013) 366–370

Table 2
Biodistribution of 99mTc-MAG3 in rats at 10 min after administration with or without
BCL (20 mg/kg) loading.
Organ

Control group(% dose/g tissue)

BCL-loading group(% dose/g tissue)

Blood
Brain
SpleenPancreas
StomachLiver
KidneyHeart
Lung

0.317±0.073
0.010±0.001
0.052±0.008
0.046±0.001
0.024±0.024
0.151±0.001
6.191±0.187
0.101±0.016
0.195±0.030

0.047±0.044⁎
0.001±0.001⁎
0.009±0.008⁎
0.006±0.007⁎
0.040±0.036⁎
0.033±0.026⁎
0.651±0.324⁎
0.014±0.010⁎
0.041±0.037⁎⁎ : Pb.01 vs. controls.

2.5. Statistical analysis
All results for in vitro assays and rat studies represent the averageof least three experiments and are expressed as means±SD except
protein binding rate. Data were analyzed by ANOVA t-test, and Pb .05
was considered to indicate signiﬁcance.
3. Results
As shown in Fig. 1, strong binding of 99mTc-MAG3 with HSA wasobserved. Binding levels of 99mTc-MAG3 with HSA were almost equal
to those of serum. 99mTc-MAG3 scarcely binds with other proteins,
such as AGP and IgG. The effects of several displacers on serum protein
binding of 99mTc-MAG3 were then examined (Table 1). In thepresence of HSA site I displacers, such as BCL, warfarin, valproic acidand cefazolin, the free fractions of 99mTc-MAG3 were signiﬁcantly
elevated (1.20 to 1.47 times), as compared to controls. Displacement
rates increased markedly with concentration of HSA and RSA. On theother hand, there were no signiﬁcant differences in the presence of
ibuprofen, octanoate or oleate. Therefore, 99mTc-MAG3 is considered
to combine speciﬁcally with binding site I on HSA.
In the rat serum study, BCL and warfarin signiﬁcantly interactedwith 99mTc-MAG3 binding to serum. BCL was found to be a better
displacer than warfarin for clinical application, thereby allowing its
blood concentrations to be maintained at high levels and maximizing
its protein binding displacement effect. Table 2 shows the effects of
BCL treatment (20 mg/kg body weight) on 99mTc-MAG3 distribution
at 10 min after tracer injection. Accumulation with BCL loading was
signiﬁcantly lower in all organs. In particular, accumulation in the
blood was about 15%, and that in the kidney was 11% of control levels.
Thus, the presence of BCL, which is a displacer of binding site I canalter the pharmacokinetics of 99mTc-MAG3. 99mTc-MAG3 distribution
at various time intervals with and without BCL is shown in Fig. 2. The
dose of BCL was 20 mg/kg body weight, and this is a therapeutic dosein humans. Without BCL loading, moderate blood clearance wasobserved, and peak time of accumulation in kidney was seen at 5 minafter 99mTc-MAG3 injection. On the other hand, with BCL loading,
rapid blood clearance was observed. Peak time of accumulation in
kidney shifted to within 2 min after tracer injection. Subsequently,
radioactivity cleared rapidly from kidneys. Based on these results, BCL
treatment increased free 99mTc-MAG3 in vivo, and rates of kidney
accumulation and blood clearance increased as a result. Indeed, thefree fraction of 99mTc-MAG3 was substantially higher than in thecontrol group on ultraﬁltration analysis of rat serum in the BCLtreated group after 99mTc-MAG3 injection.
On 99mTc-MAG3 scintigraphy, time–activity curves peaked at an
early time point after 99mTc-MAG3 injection, and then decreased
gradually (Fig. 3). Table 3 shows the peak time and up-slope on therenograms of 99mTc-MAG3 in rats. With BCL loading, the renogram
pattern changed; in BCL-loaded rats, peak times shifted to 110 s and
120 s from 240 s and 170 s in controls, respectively.

4. Discussion
In the present study, we applied the serum protein binding
displacement method reported in our previous study to 99mTc-MAG3
scintigraphy. 99mTc-MAG3 is a radiopharmaceutical for in vivo
imaging and is widely used for the purpose of diagnosing renal
diseases [12–17], as it accumulates in the kidneys. We believed that it
would be very useful to regulate the pharmacokinetics and biodistribution of this radiopharmaceutical.
Generally speaking, competitive inhibition between the radiopharmaceutical and displacer for plasma protein binding requires adisplacer concentration of 1/3 that of protein concentration to obtaina sufﬁcient effect. For this reason, we set displacer concentration at
200 μM and 400 μM in order to conﬁrm concentration dependence. In
the in vitro study, the free fraction of 99mTc-MAG3 was signiﬁcantly
higher than in the control sample after administration of a site Ispeciﬁc agent (e.g., BCL, valproic acid, warfarin or cefazolin). On theother hand, site-II speciﬁc agents (e.g., ibuprofen, octanoic acid or
sodium oleate) did not increase the free fraction of 99mTc-MAG3.
Similarly, in rat serum, the free fraction of 99mTc-MAG3 increased withadministration of site I-speciﬁc agents. With the rise in displacement
rates with increasing concentrations of HSA and RSA, a clear
concentration dependence of displacement rates was conﬁrmed. For
this reason, the binding site of 99mTc-MAG3 is considered to be site I on
HSA, and we selected BCL as a site I-speciﬁc agent. BCL is a nonsteroidal anti-inﬂammatory medicine that can be safely administeredin large doses (600–1200 mg/day). It has been reported that BCL does
not affect urinary enzyme activities and that nephrotoxicity by thisagent is very low [18–21]. In addition, warfarin–BCL combination
therapy has been used to treat patients with nephritic syndrome [22].

Fig. 2. Biodistribution of 99mTc-MAG3 (740 kBq) with/without BCL in rats. BCL (20 mg/kg) was loaded 10 min before injection of 99mTc-MAG3. With BCL loading, blood levels werelower (A), and peak time for kidney accumulation was more rapid (B) than in the control group. *; not detected.

K. Nishi et al. / Nuclear Medicine and Biology 40 (2013) 366–370

369

Fig. 3. Renograms of 99mTc-MAG3 scintigraphy in control rat (A), and in BCL-treated rat (B). BCL (20 mg/kg) was loaded at 10 min before injection of 99mTc-MAG3. Peak time of therenogram shifted to 110 s (B) from 240 s (A).

From the results of the biodistribution study in BCL-treated rats,
blood clearance was clearly accelerated in the BCL-treated group, andthe free fraction of 99mTc-MAG3 in vivo was markedly higher in thetest group. In the BCL-treated group, accumulation of 99mTc-MAG3 in
the kidney increased rapidly to a peak value after administration, andthen decreased and was eliminated quickly, in contrast to the control
group without BCL treatment. 99mTc-MAG3 cleared rapidly from thekidney, which is the target organ of this radiotracer after BCL
treatment. Therefore, BCL induced the rapid elimination of radioactivity, and enhanced the clearance from the blood and organs.
On in vivo whole body dynamic imaging of rats, we conﬁrmed thatthe radioactivity increased gradually at the initial stages afteradministration until the peak time of 240 s in the control group;
however, in the BCL-treated group, radioactivity rose quickly and thepeak time was shortened to 120 s. Renal function is typically analyzed
by determining the peak time in renograms, and the slope of thestraight line in linear regression. By inhibiting the binding ability of99m
Tc-MAG3 to site I on HSA, renograms were ideally approximatedto simple curves by inhibiting the binding ability of 99mTc-MAG3 tosite I on HSA using BCL. These results suggest that the pharmacokinetics of 99mTc-MAG3 and subsequent imaging can be altered inclinical settings. The free fraction rate was equal to the in vitro human

Table 3
Peak times and up-slopes of renograms in rats.

Rat 1
Control
BCL-treatedRat 2
Control
BCL-treatedPeak time (s)Up-slope (counts/s)

240
110

1.166
2.208

170
120

0.941
2.000

serum data. Therefore, in the rat study, rapid accumulation in thekidney and accelerated clearance were achieved due to the increase infree 99mTc-MAG3 by BCL loading in vivo.
As described above, the protein binding displacement method
could be readily applied to human studies under similar conditions
using BCL. Displacement of radiopharmaceutical binding to serum
proteins may lead to better diagnostic images with shorter waitingtimes and lower radiation doses for patients.
5. Conclusion
We conﬁrmed that 99mTc-MAG3 binds to HSA binding site I, andapplied the serum protein binding displacement method using coadministration of BCL with binding afﬁnity for HSA site I in a rat study.
Therefore, the pharmacokinetics of 99mTc-MAG3 were altered by BCL,
and BCL produced rapid blood clearance and peak times on rat
renograms. There is a good possibility that the serum protein binding
displacement method of 99mTc-MAG3 and BCL, a safe displacer forhumans, can be applied to clinical study.
Acknowledgments
The authors are grateful to Nihon Medi-physics Co., Ltd., forgenerously providing radiopharmaceuticals, and would like toacknowledge Grelan Pharmaceutical Co., Ltd., Eisai Co., Ltd., Fujisawa
Pharmaceutical Co., Ltd., Kaken Pharmaceutical Co., Ltd., and SmithKline Beecham Co., Ltd., for kindly providing chemical reagents. This
work was supported by Grants-in-Aid for Scientiﬁc Research(#10770451, #11470193, #14770498, #13557075, #15659283,#16659322, #17390336 and #22591372) from the Ministry of
Education, Science, Sports and Culture of Japan, and the Japan Society
for the Promotion of Science. Financial support was also provided by
the Ibaraki Prefectural University Research Project (9808, 0118 and370

K. Nishi et al. / Nuclear Medicine and Biology 40 (2013) 366–3700220), Ibaraki Prefectural University Grants-in-Aid of the Encouragement for Young Scientists 2001, 2002, 2004, 2005, 2006, 2008, 2009
and 2010, and the Japan Atherosclerosis Research Foundation Grants
2008 and 2010.

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