Gadolinium Complex of 1,4,7,10-Tetraazacyclododecane-1,4,7-Trisacetic Acid (DO3A)-Ethoxybenzyl (EOB) Conjugate as a New Macrocyclic Hepatobiliary MRI Contrast Agent

Article abstract of DOI:10.1021/acs.jmedchem.7b00060

We report the synthesis of a macrocyclic Gd chelate based on a 1,4,7,10-Tetraazacyclododecane-1,4,7-Trisacetic acid (DO3A) coordinationn cage bearing an ethoxybenzyl (EOB) moiety and discuss its use as a T₁ hepatobiliary magnetic resonance imaging (MRI) contrast agent. The new macrocyclic liver agent shows high chelation stability and high r₁ relaxivity compared with linear-Type Gd chelates, which are the current clinically approved liver agents. Our macrocyclic, liver-specific Gd chelate was evaluated in vivo through biodistribution analysis and liver MRI, which demonstrated its high tumor detection sensitivity and suggested that the new Gd complex is a promising contrast agent for liver cancer imaging.

Full text of DOI:10.1021/acs.jmedchem.7b00060

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Article
A Gadolinium Complex of 1,4,7,10-Tetraazacyclododecane-1,4,7-
trisacetic acid (DO3A)-Ethoxybenzyl (EOB) Conjugate
as a New Macrocyclic Hepatobiliary MRI Contrast Agent
Ah Rum Baek, Hee-Kyung Kim, Subin Park, Gang Ho Lee, Hyo Jeung Kang, Jae
Chang Jung, Joon-Suk Park, Hun-Kyu Ryeom, Tae-Jeong Kim, and Yongmin Chang
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2017
Downloaded from http://pubs.acs.org on May 23, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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of their duties.

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A Gadolinium Complex of 1,4,7,10ꢀTetraazacyclododecaneꢀ
1,4,7ꢀtrisacetic acid (DO3A)ꢀEthoxybenzyl (EOB) Conjugate
as a New Macrocyclic Hepatobiliary MRI Contrast Agent
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Ah Rum Baek,1, Hee-Kyung Kim,2 Subin Park,3 Gang Ho Lee,4 Hyo Jeung
Kang,5 Jae-Chang Jung,3 Joon-Suk Park,6 Hun-Kyu Ryeom,7 Tae-Jeong Kim,8,*
and Yongmin Chang,1,2,7*
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1Department of Medical & Biological Engineering, Department of Molecular
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Medicine & BK21 Plus KNU Biomedical Convergence Program, Department
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of Biology, Department of Chemistry, Department of Pharmacy, Department
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of Radiology, and Institute of Biomedical Engineering Research, Kyungpook
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National University, 80 Daehakro, Bukgu, Daegu 702ꢀ701, Korea Laboratory
Animal Center, DaeguꢀGyeongbuk Medical Innovation Foundation 41061
Chumbokꢀro 80, Dongꢀgu, Daegu, Korea
Keywords: Gadolinium, Contrast agent, MRI, Liver, Chelate
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Abstract
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We report the synthesis of a macrocyclic Gd chelate based on a 1,4,7,10ꢀ
tetraazacyclododecaneꢀ1,4,7ꢀtrisacetic acid (DO3A) coordinationn cage bearing
an ethoxybenzyl (EOB) moiety and discuss its use as a T1 hepatobiliary
magnetic resonance imaging (MRI) contrast agent. The new macrocyclic liver
agent shows high chelation stability and high r1 relaxivity compared with linearꢀ
type Gd chelates, which are the current clinically approved liver agents. Our
macrocyclic, liverꢀspecific Gd chelate was evaluated in vivo through
biodistribution analysis and liver MRI, which demonstrated its high tumor
detection sensitivity and suggested that the new Gd complex is a promising
contrast agent for liver cancer imaging.
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INTRODUCTION
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Gdꢀbased contrast agents (CAs) are the most commonly used clinical
magnetic resonance imaging (MRI) CAs. By improving the visibility of specific
organs, blood vessels, or lesions, these CAs help physicians diagnose and treat a
wide variety of medical conditions.1 Until 2006, it was believed that most, if not
all, Gd was removed from the body rapidly after administration. However, in
the last ten years, several studies have reported that elevated levels of Gd can
remain in the body for a long period, which may cause nephrogenic systemic
fibrosis (NSF) in patients with severe kidney disease.2,3 NSF is a very serious
clinical condition involving fibrosis of the skin and internal organs.4ꢀ7 As
exposure to Gdꢀbased CAs has sharply increased owing to increased MRI
examinations worldwide, concerns about the risk of NSF have also increased.
More recently, researchers have found that, even in patients without severe
renal dysfunction, the use of Gdꢀbased CAs results in Gd accumulation in the
brain.8
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As safety concerns associated with the potential toxicity of Gd retained in the
human body have increased, emphasis has been placed on the development of
highly stable Gd complexes as CAs. In particular, the molecular structure of the
Gd complex may contribute to the degree of Gd retention in the body; there are
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two structurally distinct types of Gd complexes: linear and macrocyclic.
Macrocyclic Gd complexes consist of a closed, cageꢀlike ligand that surrounds
and binds tightly to the Gd ion, forming highly stable complexes less likely to
release free Gd into the body. Three macrocyclic Gd complexes are currently
approved by the U.S. Food and Drug Administration (FDA): 8 (GdꢀDOTA)9, 9
(GdꢀDO3Aꢀbutrol)10, and 10 (GdꢀHPꢀDO3A)11. Regarding biodistribution, as all
these complexes are nonprotein binding, they are nonspecific extracellular
agents.12
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However, the design of highly stable liverꢀspecific Gd complexes is
challenging. Recently, two linear Gd complexes have been approved by the
FDA as liverꢀspecific MRI CAs: 11 (GdꢀEOBꢀDTPA)13 and 12 (GdꢀBOPTAꢀ
DTPA)14. These liverꢀspecific linear Gd complexes have similar stabilities to
other nonspecific linear Gd complexes, including those associated with the
greatest number of NSF cases.15 There have been several attempts to synthesize
liverꢀspecific macrocyclic Gd complexes with high stability.16ꢀ21 In this study,
we designed and synthesized a new, highly stable macrocyclic Gd chelate based
on a 7 (DO3A) coordination cage bearing ethoxybenzyl (EOB) moieties. First,
we characterized the kinetic inertness of the complex with respect to
transmetalation, relaxivity, hydration state, and the water exchange kinetics of
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the macrocyclic Gd complex. In addition to forming a hydrophilic, stable
complex with Gd ions, our detailed pharmacokinetic and biodistribution studies
demonstrated that this new Gd chelate is rapidly taken up by hepatocytes.
Therefore, this complex is a new, small Gd chelate based on a macrocyclic
ligand that shows hepatobiliary specificity in vivo.
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RESULTS AND DISCUSSION
Synthesis and Characterization
Scheme 1 shows the synthetic procedures for the ligand 5 and the
corresponding Gd complex 6. Compound 1 and 2 were prepared according to a
literature method.22 Chlorination of 2 with thionyl chloride in diethyl ether
resulted in the formation of 3, which was used without further purification.
Ligand 5 was prepared by acid hydrolysis, with trifluoroacetic acid (TFA), of
the corresponding tert-butyl ester (tBu), which in turn was prepared from the
conjugate of tBu protected 7 with EOB moiety 3. Complexation of Gd(III) by 5
led to the formation of the corresponding complex 6 as a hygroscopic white
solid. Complex 6 was subsequently purified by preparative highꢀperformance
liquid chromatography (HPLC) (HPLC traces of 5 and 6 are shown in the
Supporting Information, Figure S1 and S2). The formation of 5 and 6 was
confirmed by microanalysis and spectroscopic techniques, such as 1H NMR and
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HRꢀFABꢀ or ESIꢀLC mass spectrometry (Figure 1 and Supporting Information,
Figure S3).
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Relaxivity
Table 1 shows the r1 and r2 relaxivities for 6, along with those of 11 and 12,
which are supplied for comparative purposes. The most significant feature
shown in Table 1 is that 6 exhibits higher r1 values than any of the clinically
used MRI CAs;23 the r1 value obtained for 6 is twice as high as that of 12. The
high relaxivity of 6 relative to those of the clinically used MRI CAs was
expected considering that ligand 5 forms heptadentate complexes with Gd(III)
ions.24,25 Consequently, 6 can have two innerꢀsphere water molecules (q = 2),
enabling more efficient relaxation.
Transmetalation Kinetics
In the human body, various endogenous metal ions are present, such as Zn(II),
Cu(II), and Ca(II). These metal ions can compete with Gd(III) for the ligands,
resulting in the loss of Gd ions,26 which causes various diseases, such as NSF,
or the deposition of Gd ions in the brain. In particular, the concentration of
Zn(II) in human blood is higher (55–125 ꢁM) than those of other metal ions,
such as Cu(II) (1–10 ꢁM). Moreover, the association constants toward DTPA
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and DOTA are similar to those of Gd(III). Thus, it is important to assess the
stability of Gd complexes in the presence of Zn(II) ions.
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The kinetic inertness of 6 can be evaluated against those of clinically used
MRI CAs in phosphate buffer (PBS). In the case of transmetalation by
diamagnetic Zn(II) ions, insoluble Gd2(PO4)3 is formed in PBS solution, and the
paramagnetic relaxation rate of the solution decreases. Consequently, the
p
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normalized paramagnetic relaxation rate at a given time (R1 (t)/R1 (0)) can be
used to measure the degree of transmetalation. Figure 2 shows that the cyclic
octadentate 8 has high kinetic inertness, with more than 90% of the longitudinal
relaxation rate maintained 72 h after the initial measurement. Complex 6, which
has a cyclic heptadentate structure, shows a relatively high kinetic stability
compared with that of the octadentate group. The difference in the kinetic
stability arises from differences in the coordination numbers of each complex.
In contrast to acyclic complexes, the longitudinal relaxation rate maintained by
6 was considerably higher. Complex 6 maintained more than 75% of the initial
longitudinal relaxation rate, whereas acyclic Gd complexes 11 and 12
maintained just 30–50% during the same measurement period.
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Cell Viability Assay
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Figure 3 shows the results of cell viability tests with 6. The cells were
incubated with the Gd complex for 24 h. The viability percentage of the cell line
was almost 100% at all concentrations, indicating that 6 has negligible cell
toxicity in the typical clinical concentration range. In addition, in normal
hepatocyte cell toxicity tests, the proliferation percentage of 6 was similar to
those of 8 and 11 over 72 h, as shown in the Supporting Information (Figure
S4).
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Biodistribution
The in vivo biodistribution of 6 was quantitatively measured by inductively
coupled plasma atomic emission spectrometry (ICPꢀAES) (Figure 4). The
clinical MR CAs, 8 and 11, are used for comparative purpose. The ICPꢀAES
data shows the high accumulation of 6 in the liver, at up to 50% of the
administrated Gd dose. In case of 11, it had faster liver accumulation rate than 6
within 30 min, reaching almost 50%. Whereas 8 presents typical ECF agent
property without tissueꢀspecific property. After 6 h, 6 was rapidly excreted from
the liver. In addition to the liver, the kidneys and bladder show relatively high
Gd accumulation, resulting from glomerular excretion via the renal pathway.
Therefore, the in vivo biodistribution data strongly suggest that 6 is excreted by
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both renal and hepatobiliary routes. This dual excretion pattern of 6 is very
similar to the excretion pattern of the liverꢀspecific linear Gd agent 11.27
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In Vivo MR Images of Normal Animals
In vivo MRI experiments were performed with 6 and liverꢀspecific MRI CAs,
such as 11 and 12. Figure 5a–c shows the coronal T1ꢀweighted images, and
Figure 5d–f shows the axial T1ꢀweighted images of 6ꢀweekꢀold male Institute of
Cancer Research (ICR) mice treated with 6 and currently used liver MRI CAs.28
The most characteristic features of 6 as a CA are that the signal enhancement in
the liver and gallbladder is comparable with those of liverꢀspecific CAs, such as
11 and 12, and the contrastꢀtoꢀnoiseꢀratio (CNR) curves for 6, 11, and 12 show
similar trends (Supporting Information, Figure S5). The CNR of 6 in the liver
and gallbladder is slightly lower than those of 11 and 12. However, this results
from the higher initial renal excretion ratio of 6 at 20 min, as shown in Figure
S5. All three liverꢀspecific CAs show biliary excretion via the bile duct,
confirming hepatobiliary uptake, as seen in Figure 5.29,30 The clearance of 6 is
confirmed by the T1ꢀweighted wholeꢀbody coronal images 24 h after
intravascular injection (Supporting Information, Figure S6).
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In Vivo MR Images of Liver Cancer Model
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The induction of liver cancer in the mice was confirmed by bioluminescence
imaging (Figure 6c). Using MRI, we made the liver cancer diagnosis using 6.
Figure 6a and b shows the axial T1ꢀweighted MR images of hepatocellular
carcinoma (HCC) model nude mice; the images were obtained by tail vein
injection with 6 (Figure 6a), as well as with 8 (Figure 6b), which was used for
comparison. Unlike ECF agents, such as 8, the new hepatobiliary MRI CA 6 is
taken up by normal hepatocytes.31,32 It is wellꢀknown that the human normal
hepatocyte has hydrophobic drugꢀtransporting proteins called organic anionꢀ
transporting polypeptides (OATPs) in its membrane, which play a significant
role in drug absorption. Lipophilic xenobiotics can pass through the OATP drug
transporters in normal liver tissue. However, OATPs are considerably decreased
in most HCC.33,34 Therefore, lipophilic organic drugs cannot pass through HCC
cells, resulting in low MR intensity. These agents are thought to pass via
OATP1 to the basolateral membrane of the hepatocyte. Thereafter, the agents
are excreted via multidrug resistance protein 2 (MRP2).
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The differences in uptake of 6 between normal hepatocytes and tumor cells
demonstrate a significant difference in MR signal enhancement. Thus, the
inflow of paramagnetic agents to the liver is greatly enhanced in normal
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hepatocytes, as shown in Figure 6a. In contrast, liver cancer cells usually have
abnormal OATP1 expression, resulting in inhibition of the uptake of 6 and the
demonstration darker MR contrast compared with normal liver tissue. In the
case of the nonspecific extracellular agent 8, the MR images show
indistinguishable contrast enhancement between normal liver tissue and tumor
regions (Figure 6b). Therefore, 6 has significant clinical potential for detecting
liver tumors and differentiating between normal liver cells and HCC.
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CONCLUSION
The stability of Gdꢀbased MRI CAs is of serious concern because free Gd
(Gd3+) is toxic and is associated with the development of NSF. Therefore, the
ability of a ligand to bind tightly to Gd ions is an important safety consideration.
In the current study, we designed and synthesized a new liverꢀspecific
macrocyclic Gd complex 6 for liver MRI, which has high chelation stability.
Complex 6 showed much higher kinetic stability than liverꢀspecific linear Gd
complexes 11 and 12. Furthermore, the r1 relaxivity of 6 (8.07 mM−1 s−1) was
much higher than those of 11 and 12 (r1 = 4.70 and 4.00 mM−1 s−1,
respectively). The biodistribution and in vivo MR images showed biliary
excretion via the bile duct, confirming hepatobiliary uptake. To demonstrate the
possible clinical potential for liver imaging, contrastꢀenhanced in vivo MR
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images were obtained using 6 for the liver cancer animal model. MR images
using 6 as a CA clearly demonstrated a significant difference in signal
enhancement between normal liver and tumor tissues, whereas for the
nonspecific extracellular agent 8, no distinguishable contrast enhancement
between the two tissue types was observed. Therefore, we conclude that, in
every respect, macrocyclic complex 6 represents a new class of practical, liverꢀ
specific MRI CAs with significant benefits over its linear counterparts.
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Experimental Methods
General Remarks
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All commercial reagents were purchased from Aldrich or TCI and used as
received unless otherwise stated. Deionized water was used for all experiments.
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1
The H NMR experiments were carried out using a Bruker Advance 500
spectrometer at the Center for Instrumental Analysis, Kyungpook National
University (KNU). NMR chemical shifts are given as δ values with reference to
tetramethylsilane (TMS) as an internal standard and coupling constants are in
Hz. Highꢀresolution FABꢀmass spectra were obtained using a JMSꢀ700 model
(Jeol, Japan) mass spectrophotometer at the Korean Basic Science Institute
(KBSI). A HPLC system (LCꢀforte/R, Kyoto, Japan) equipped with a Luna 10
ꢁm C18 column (250 × 21.2 mm, Pheonomenex, Inc., Torrance, CA, USA) and
a UVꢀVis detector (220 nm) was used for purifications and characterization.
Elution conditions were as follows: (Method A) a mixture of an aqueous
solution of TFA (0.1%, v/v) and an acetonitrile solution of TFA (0.1%, v/v),
10–100% acetonitrile solution over 25 min, flow rate = 12 mL/min. (Method B)
an aqueous ammonium acetate solution (10 mM) and an acetonitrile solution of
aq. ammonium acetate buffer (10 mM buffer, 10%, v/v), 20–100% acetonitrile
solution over 35 min. Purities of 5 and 6 of >95% were obtained by preparative
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reversedꢀphase HPLC with UVꢀvis detection at 220 nm (Supporting
Information, Figure S1 and S2).
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Synthesis and Characterization
4ꢀEthoxybenzaldehyde (1)
To a mixture of pꢀhydroxybenzaldehyde (4 g, 32.75 mmol) and K2CO3 (13.58
g, 98.26 mmol) in DMF (150 mL), ethyl bromide (2.69 mL, 36.03 mmol) was
added dropwise. After stirring for 18 h at RT, K2CO3 residue was filtered and
the filtrate was diluted with water (150 mL). Then, the solution was washed
with diethyl ether (3 × 150 mL). The organic layers were dehydrated with
anhydrous Na2SO4, and the solvent was evaporated under reduced pressure.
1
Yield: 4.49 g (91.30%), H NMR (CDCl3); δ = 9.88 (s, 1H; CHO), 7.83 (d, J =
58.4 Hz, 2H; ArH), 6.99 (d, J = 58.4 Hz, 2H; ArH), 4.12 (q, J = 56.9 Hz, 2H;
CH2), 1.46 (t, J = 56.9 Hz, 3H; CH3).
(4ꢀEthoxyphenyl)methanol (2)
NaBH4 (0.83 g, 21.98 mmol) was gradually added to 1 (3 g, 19.98 mmol) in
methanol (50 mL) at 0 °C. Then, the reaction mixture was stirred for 1 h at RT.
When the reaction was completed, the solvent was evaporated under reduced
pressure and water was added (80 mL). The mixture was extracted with ethyl
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acetate (3 × 50 mL). The organic layer was dried over anhydrous MgSO4,
filtered, and evaporated to give a crude product, which was further purified by
1
chromatography on silica (hexane/ethyl acetate, 7:3). Yield: 2.36 g (77.6%), H
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NMR (DMSOꢀd6); δ = 7.19 (d, 2H, J = 8.8 Hz), 6.82 (d, 2H, J = 8.4 Hz), 4.95
(s, 1H), 4.41 (d, 2H, J = 5.6 Hz), 3.96–4.02 (m, 2H), 1.32 (t, 3H, J = 7.2 Hz).
1ꢀ(Chloromethyl)ꢀ4ꢀethoxybenzene (3)
To compound 2 (3 g, 19.71 mmol) in diethyl ether (30 mL), thionyl chloride
(2.86 mL, 39.42 mmol) was carefully added at 0 °C and stirred for 5 h at RT.
The reaction mixture was extracted with saturated NaHCO3 solution (3 × 30
mL). Subsequently, the diethyl ether layer was dried over anhydrous Na2SO4,
filtered, and evaporated to obtain a pale yellowish oil. No further purification
was carried out. Yield: 3.34 g (99.8%).
2, 2′, 2′′ꢀ(10ꢀ(4ꢀEthoxybenzyl)ꢀ1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀ
triyl)triacetic acid (5)
To a mixture of Bu protected 7 (6.69 g, 20.28 mmol) and K2CO3 (5.60 g,
t
40.56 mmol) in acetonitrile (50 mL), a solution of 3 in acetonitrile (30 mL) was
added dropwise at RT, and the mixture was stirred overnight. Any inorganic
residues were removed by filtration, and then, acetonitrile was evaporated under
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reduced pressure. The obtained crude product was dissolved in
dichloromethane/TFA (1:1, 40 mL) at RT for deprotection of tBu. After 5 h, the
solvent was removed under reduced pressure. The residue was precipitated in
cold diethyl ether and then dried under vacuum. Further purification was
accomplished using preparative HPLC, as follows: Rt = 10.45 min (Method A).
The product was obtained by lyophilization as a hygroscopic white solid. Yield:
2.99 g (45.93%), HRꢀFABMS (m/z): calcd for C23H37O7N4: 481.2662 [MH]+;
found, 481.2662.
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[Gd(DO3AꢀEOB)(H2O)2] (6)
Ligand 5 (0.36 g, 0.749 mmol) was dissolved in deionized water (10 mL), to
which was added gadolinium(III) chloride hexahydrate (0.28 g, 0.749 mmol).
The pH of reaction mixture was carefully adjusted to 7–7.5 with aq. NaOH (1
M). After 18 h, the solvent was evaporated to obtain the crude product, which
was further purified by HPLC as follows: Rt = 15.92 min (Method B). Finally,
the product was lyophilized to obtain a hygroscopic white solid. Yield: 0.24 g
(50.20%), HRꢀFABMS (m/z): calcd for C23H33GdN4O7Na, 658.1494, [MNa]+;
found, 658.1494.
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Relaxivity
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T1 measurements were carried out using an inversion recovery method with a
variable inversion time (TI) at 1.5 T (64 MHz, GE Healthcare, Milwaukee, WI,
USA). The MR images were acquired at 35 different TI values ranging from 50
to 1750 ms. T1 relaxation times were obtained from the nonlinear least square fit
of the signal intensity measured at each TI value. For T2 measurements, the
Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence was adapted for multiple
spinꢀecho (SE) measurements. The MR images were acquired at 34 different
echo times (TEs) ranging from 10 to 1900 ms. T2 relaxation times were obtained
from the nonlinear least squares fit of the mean pixel values for multiple SE
measurements at each TE. Subsequently, R1 and R2 relaxivities were calculated
from the linear fit of each relaxation time with concentration (1, 0.5, 0.25,
0.125, and 0.0625 mM).
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Transmetalation Kinetics
The kinetic inertness study was performed according to a literature
method.35,36 This method is based on measuring the evolution of the water
p
p
proton longitudinal relaxation rate, R1 (t)/R1 (0), of a buffer solution (PBS, pH
7.4) containing equimolar Gd complex and zinc chloride over time. The
samples were prepared using a 2.5 mM solution of 6 solution of various
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commercial MRI CAs, 8, 11, 12 and 13 (GdꢀDTPAꢀBMA) as controls. Then, 10
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ꢁL of a 250 mM zinc chloride solution was added to 1 mL of a buffered
p
solution of the Gd complex, and the mixture was vigorously stirred. The R1
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relaxation rate was obtained after subtraction of the diamagnetic contribution of
the water proton relaxation from the observed relaxation rate R1. The
measurements were carried out using a 3 T wholeꢀbody system (Magnetom Tim
Trio, Simens, Korea Institute of Radiological & Medical Science) at RT.
Cell Viability Assay
In this study, human embryonic kidney cells (HEKꢀ293), which are widely
used for in vitro cell cytotoxicity evaluation, were chosen. Cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco)
supplemented with heatꢀinactivated fetal bovine serum (FBS) (10%), penicillin
(100 IU/mL), and streptomycin (100 mg/mL), which were all purchased from
Gibco. The medium was replaced every 2 days, and the cells were split into a
96ꢀwell plate (1 × 104 cells/well/200 ꢁL). Aqueous solutions of 6 (50–500 ꢁM)
were added into the serumꢀfree media and incubated for 24 h. Then, cell
counting kitꢀ8 (CCKꢀ8) (10 ꢁL) was added to each well. The solution was
removed after 4 h at 37 °C. The optical density at 450 nm was determined using
a microplate reader (Bioꢀrad550 Reader, Molecular Devices, Sunnyvale, CA,
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USA) to evaluate the cell cytotoxicity.37 In addition, normal hepatocytes were
used to test Gd toxicity in the liver environment over 72 h. The methodology for
the normal cell proliferation test was the same as that used for the above HEKꢀ
293 cell viability test.
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Biodistribution
Complex 6 was administrated intravenously as a bolus in a tail vein of a 6ꢀ
weekꢀold male mouse (ICR, 25–27 g). The mice were anesthetized and killed by
means of exsanguinations from the vena cava at each time point (0.5, 1, 6, and
24 h after injection time). The Gd concentration was measured in tissues (liver,
spleen, intestine, heart, lung, kidneys, bladder, bile duct, and blood). In addition,
the Gd concentration was determined by digesting the tissues with nitric acid
(70%) at 180 °C for 120 min and measuring the concentration in the clear
diluted solution by ICPꢀAES (Optima, 7300DV, PerkinElmer, Waltham, MA,
USA). The detection limit of this method is 0.01 ppm.38
Orthotopic Xenograft Mouse Model
The orthotopic xenograft mouse model was approved by the Institutional
Animal Care and Use of Committee of DaeguꢀGyeongbuk Medical Innovation
Foundation (DGMIF) and performed in accordance with protocols approved by
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the Institutional Animal Care and Use of Committee. Fiveꢀweekꢀold male
athymic nude (BALB/c nu/nu) mice were purchased from Orient Bio
(Seongnam, Korea) and housed in a specific pathogenꢀfree facility at the
Laboratory Animal Center of DGMIF before use. The mice were inoculated
with luciferaseꢀexpressing human liver cancer cells (HepG2ꢀluc2) (1 × 106 cells
in 50 ꢁL HBSS), which were purchased from Perkin Elmer Inc. and were grown
in Eagle’s minimum essential medium (EMEM) (American Type Culture
Collection [ATCC]) containing 10% FBS) in the subcapsular parenchymal of
the left liver lobe. Four weeks after inoculation, the mice were imaged using an
in vivo imaging system (IVIS).
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Bioluminescence Imaging
Mice were intraperitoneally administered firefly Dꢀluciferin potassium salt at
a dose of 150 mg/kg body weight in Dulbecco’s PBS. Bioluminescence images
were acquired with the IVIS Lumina system (PerkinElmer). Image analysis was
performed using Living Image software by measuring the photon flux
(photons/[s·cm2·steradian]). The signal intensity was measured for
approximately 1 h, until it decayed considerably. Serial images were obtained
from all animals, and the mean photon flux relative to the peak signal was
determined.
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In Vivo MRI Experiments
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The in vivo MRI experiments were performed in accordance with the rules of
the animal research committee of Kyungpook National University (KNU). In
these studies, sixꢀweek male ICR mice with weights of 25–27 g were used. The
mice were anesthetized by 1.5% isoflurane in oxygen. MR images were
acquired before and after tail vein injection of each MRI CA (6, 11 and 12). The
CA injection dose was 0.1 mmol Gd/kg for MR images. After each
measurement, the mice were revived from anesthesia and placed in the cage
with free access to food and water. During these measurements, the animals
were maintained at approximately 37 °C using a warm water blanket.
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MR images were acquired with a 1.5 T system (GE Healthcare, Milwaukee,
WI, USA) equipped with a homemade birdcageꢀshaped RF coil for small
animals. The coil was of the receiver type with an inner diameter of 50 mm. The
image parameters of the coronal images for SE measurements were as follows:
repetition time (TR) = 300 ms; echo time (TE) = 13 ms; field of view (FOV) =
11 mm; 192 × 128 matrix size; 1.2 mm slice thickness; number of acquisition
(NEX) = 8; and scan time of each image = 2 min 37 s. Those of the axial images
were as follows: TR = 300 ms; TE = 13 ms; FOV = 6 mm; 192 × 128 matrix
size; 1.5 mm slice thickness; NEX = 4; spacing = 0.4; and scan time of each
image: 2 min 41 s. MR images were obtained 24 h after injection.
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The anatomical locations with enhanced contrast were identified for liver, bile
duct, heart, and kidney in postꢀcontrast MR images. For quantitative
measurements, the signal intensities in specific regions of interest (ROI) were
measured using Advantage Window software (GE Medical, USA). The CNR
was calculated using eq. (1), where SNR is the signalꢀtoꢀnoise ratio.39
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CNR = SNRpost ꢀ SNRpre
(1)
Liver Cancer In Vivo MR Images
In this in vivo MR study, 6 and 8 were tailꢀvein injected into liver cancer
model mice at a dosage of 0.1 mmol Gd/kg. As a liver cancer cell line, HepG2
cells were introduced into the livers of 5ꢀweekꢀold male nude mice (20–21 g).
The MR images were collected using a 1.5 T system equipped with a
homemade birdcageꢀtype coil for small animals. A fast SE sequence was used
to verify the liver cancer region using a T2ꢀweighted image and the following
image parameters: TR = 2000 ms; TE = 40 ms; FOV = 8 mm; 256 × 192 matrix
size; 2.0 mm slice thickness; NEX = 8; scan time of each image = 2 min 44 s;
and flip angle (FA) = 10°. In sequence, the T1ꢀweighted image was obtained to
estimate the liver cancer diagnosis capability of 6. The parameters for the T1ꢀ
weighted coronal images are as follows (those for the axial images are given in
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parentheses): TR = 300 ms; TE = 13 ms; FOV = 11 mm (6 mm); 192 × 128
matrix size; 1.2 mm slice thickness (1.5 mm); NEX = 8.00 (4.00); scan time = 2
min 37 s (2 min 41 s).
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
Molecular formula strings (CSV)
CNR profiles and coronal T1ꢀweighted whole body MR images
Chemical compound characterization (Highꢀresolution FAB mass, HPLC
spectra)
Cell proliferation data
AUTHOR INFORMATION
Corresponding Authors
* For Y.C.: Phone, (+)82ꢀ53ꢀ420ꢀ5471; Eꢀmail, ychang@knu.ac.kr
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* For T.J.K.: Phone, (+)82ꢀ53ꢀ950ꢀ4127; Eꢀmail, tjkim@knu.ac.kr
Authors to whom correspondence should be addressed
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Author Contributions
The manuscript was written through contributions of all authors. All authors
have given approval to the final version of the manuscript.
ACKNOWLEDGMENTS
This work was supported by the Basic Research Laboratory (BRL) Program
(2013R1A4A1069507) and by the Basic Science Research Program (Grant No.
2017R1A2B3003214) through the National Research Foundation funded by the
Ministry of Science, ICT and Future Planning.
ABBREVIATIONS
DO3A, 1, 4, 7, 10ꢀtetraazacyclododecaneꢀ1, 4, 7ꢀtrisacetic acid; DTPA,
diethylenetriaminepentaacetic acid; DOTA, tetraazacyclododecaneꢀ1, 4, 7, 10ꢀ
tetraacetic acid; EOB, Ethoxybenzyl; BOPTA, benzyloxypropionoicꢀ
tetraacetate; GdꢀDOTA, gadoterate meglumine; GdꢀHPꢀDO3A, gadoteridol;
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GdꢀDO3Aꢀbutrol, gadobutrol; GdꢀEOBꢀDTPA, gadoxetic acid; GdꢀBOPTAꢀ
DTPA, gadobenate dimeglumine; HPLC, high performance liquid
chromatography; MRI, magnetic resonance imaging; NSF, nephrogenic
systemic fibrosis; GB, gallbladder; OATP, organic anion transporter
polypeptide; MRP, multidrug resistance protein; ECF, extracellular fluid; TI,
inversion time; CPMG, Carr−Purcell−Meiboon−Gill pulse sequence; TE, echo
time; TR, repetition time; SE, spin echo; FOV, field of view; NEX, number of
acquisition; ROI, regions of interest; CNR, contrast to noise ratio; CAs, contrast
agents; HCC, hepatocellular carcinoma; CCKꢀ8, cell counting kitꢀ8; PBS,
phosphate buffered saline; DMEM, Dulbecco’s modified eagle’s medium; ICP,
inductively coupled plasma; EMEM, eagle’s minimum essential medium; FBS,
fetal bovine serum; IVIS, in vivo imaging system; HepG2ꢀluc2, luciferase
expressing human liver cancer cell line.
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