High in Vivo Stability of 64Cu-Labeled Cross-Bridged Chelators Is a Crucial Factor in Improved Tumor Imaging of RGD Peptide Conjugates

Article abstract of DOI:10.1021/acs.jmedchem.7b01671

Although the importance of bifunctional chelators (BFCs) is well recognized, the chemophysical parameters of chelators that govern the biological behavior of the corresponding bioconjugates have not been clearly elucidated. Here, five BFCs closely related in structure were conjugated with a cyclic RGD peptide and radiolabeled with Cu-64 ions. Various biophysical and chemical properties of the Cu(II) complexes were analyzed with the aim of identifying correlations between individual factors and the biological behavior of the conjugates. Tumor uptake and body clearance of the ⁶⁴Cu-labeled bioconjugates were directly compared by animal PET imaging in animal models, which was further supported by biodistribution studies. Conjugates containing propylene cross-bridged chelators showed higher tumor uptake, while a closely related ethylene cross-bridged analogue exhibited rapid body clearance. High in vivo stability of the copper-chelator complex was strongly correlated with high tumor uptake, while the overall lipophilicity of the bioconjugate affected both tumor uptake and body clearance.

Full text of DOI:10.1021/acs.jmedchem.7b01671

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Article
High in vivo stability of 64Cu-labeled cross-bridged chelators is a
crucial factor in improved tumor imaging of RGD peptide conjugates
Swarbhanu Sarkar, Nikunj Bhatt, Yeong su Ha, Phuong Tu Huynh, Nisarg Soni, Woong Hee Lee, Yong Jin
Lee, Jung Young Kim, Darpan N. Pandya, Gwang Il An, Kyo Chul Lee, Yongmin Chang, and Jeongsoo Yoo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01671 • Publication Date (Web): 14 Dec 2017
Downloaded from http://pubs.acs.org on December 15, 2017
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High in vivo stability of Cu-labeled cross-bridged chelators is a crucial factor
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in improved tumor imaging of RGD peptide conjugates
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Swarbhanu Sarkar,a,† Nikunj Bhatt,a,† Yeong Su Ha,a,† Phuong Tu Huynh,a Nisarg Soni,a
Woonghee Lee,a Yong Jin Lee,b Jung Young Kim,b Darpan N. Pandya,a Gwang Il An,b Kyo Chul
Lee,b Yongmin Chang,a Jeongsoo Yoo a,*
aDepartment of Molecular Medicine, BK21 Plus KNU Biomedical Convergence Program,
Kyungpook National University, Daegu 41944, South Korea. Fax: (+82) 53ꢀ426ꢀ4944.
bMolecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul
139ꢀ706, South Korea.
†Contributed equally to this work
Corresponding Author
* Eꢀmail: yooj@knu.ac.kr. Phone: +82ꢀ53ꢀ420ꢀ4947. Fax: +82ꢀ53ꢀ426ꢀ4944
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Abstract:
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Although the importance of bifunctional chelators (BFCs) is well recognized, the chemophysical
parameters of chelators that govern the biological behavior of the corresponding bioconjugates
have not been clearly elucidated. Here, five BFCs closely related in structure were conjugated with
a cyclic RGD peptide and radiolabeled with Cuꢀ64 ions. Various biophysical and chemical
properties of the Cu(II) complexes were analyzed with the aim of identifying correlations between
individual factors and the biological behavior of the conjugates. Tumor uptake and body clearance
of the 64Cuꢀlabeled bioconjugates were directly compared by animal PET imaging in animal
models, which was further supported by biodistribution studies. Conjugates containing propylene
crossꢀbridged chelators showed higher tumor uptake, while a closely related ethylene crossꢀ
bridged analog exhibited rapid body clearance. High in vivo stability of the copperꢀchelator
complex was strongly correlated with high tumor uptake, while the overall lipophilicity of the
bioconjugate affected both tumor uptake and body clearance.
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Introduction
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Radioactive copper is one of most actively studied radionuclides. It comprises several
radioisotopes (60Cu, 61Cu, 62Cu, 64Cu or 67Cu) having different decay modes, which are utilized for
a wide range of applications from nuclear imaging to radiotherapy.1ꢀ2 Among these, Cuꢀ64 is most
commonly used for the preparation of radiopharmaceuticals because of its attractive physical
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properties (β+ 19%, β− 38%, t1/2 = 12.7 h) and good availability.3ꢀ4 Some Cuꢀlabeled imaging
probes are already used in humans and more are in clinical trials.5 Recently, the use of radioactive
copper for radiotherapy has gained more interest because of progress made in the production of the
therapeutic nuclide Cuꢀ67 (β− 100%, t1/2 = 61.8 h).1, 6
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Even though several small copper complexes such as CuꢀATSM and CuꢀPTSM have
shown great potential as PET (positron emission tomography) imaging probes in the clinic,7ꢀ8 most
applications are as biomolecule conjugates, in which peptides or antibodies are radiolabeled with
radioactive copper ions using bifunctional chelators (BFCs).9ꢀ10 Ideally, BFCs will easily form
robust copper complexes and should not lose copper ions under physiological conditions.11ꢀ12 They
also need to undergo straightforward conjugation with biomolecules, after which the intrinsic
targeting properties of these compounds should not deteriorate.13 For this, great efforts have been
made worldwide to develop better chelators for copper ions.10, 14ꢀ18 In particular, the observation
that the widely employed chelator DOTA (1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7,10ꢀtetraacetic
acid) loses copper ions in vivo accelerated the development of crossꢀbridged chelators that form
ultrastable copper complexes. Initially, Wong and Anderson developed the crossꢀbridged chelator
ECBꢀTE2A
(ethylene
crossꢀbridged
1,8ꢀN,N′ꢀbisꢀ(carboxymethyl)ꢀ1,4,8,11ꢀ
tetraazacyclotetradecane),19 and recently propylene crossꢀbridged TE2A (PCBꢀTE2A) was added
by our group.20
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However, traditional BFCs such as DOTA, TETA, and DTPA derivatives are still most
commonly chosen as chelators for the radiolabeling of biomolecules with Cuꢀ64.21ꢀ22 Although
several studies have directly compared the effects of different chelators on radiolabeled
bioconjugates, it remains unclear what properties of chelators are the most important determinants
of the biological properties of bioconjugates, especially regarding tumor uptake, because of the
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small number of chelators compared and the nonꢀsystematic structural variations between them.10,
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In this work, we carefully chose five BFCs with systematic variations in structure, focusing
particularly on crossꢀbridged chelators. Five chelators were conjugated with the same RGD peptide
and used for both in vitro and in vivo studies, including PET imaging. Various factors, including in
vivo stability, lipophilicity, mode of conjugation, overall charge, linker, and specific activity, were
directly compared and correlations between these parameters and tumor targeting efficacy were
closely examined.
Results and discussion
Conjugation of BFCs with RGD peptides. Five BFCs were carefully selected for sideꢀbyꢀ
side comparison studies (Figure 1). Firstly, three crossꢀbridged chelators were selected. The
original crossꢀbridged chelator, CBꢀTE2A, was directly compared with its propylene analog PCBꢀ
TE2A. Another PCBꢀTE2A derivative, PCBꢀTE2AꢀBnꢀNCS, was also examined to assess the
effect of the additional functionality on the chelator backbone. The nonꢀcrossꢀbridged chelator
TE2AꢀBnꢀNCS was chosen for direct comparison with crossꢀbridged PCBꢀTE2AꢀBnꢀNCS. Finally,
a NOTA derivative (NOTAꢀBnꢀNCS), which is known to form a more stable copper complex than
DOTA in vivo,24 was compared with the others.
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These chelators can be also categorized into two groups depending on their conjugation
mode with biomolecules. Three chelators having an extra isothiocyanate group (ꢀNCS) conjugate
with cyclic RGDyK peptides (c(RGDyK)) via thiourea bonding, whereas ECBꢀTE2A and PCBꢀ
TE2A are linked to the peptide through amide bond formation using an acetate pendant arm
(Figure 2). Specifically, NOTAꢀBnꢀNCS and TE2AꢀBnꢀNCS, the chelators bearing NCS
functionality, were allowed to react with the free amine group on the lysine of the c(RGDyK)
peptide in the presence of diisopropylethylamine (DIEA), and then purified by semiꢀpreparative
HPLC. The formation of the both bioconjugates was confirmed by ESIꢀMS. We used standard
dicyclohexylcarbodiimide (DCC) coupling for the synthesis of PCBꢀTE2Aꢀc(RGDyK) (5). In this
protocol, the carboxylic group of PCBꢀTE2A was first activated by DCC/DIEA in dry DMSO, and
the subsequent addition of the peptide allowed the amino group of lysine to react with the activated
carboxylic acid group of PCBꢀTE2A; the bioconjugate was formed via an amide linkage. This
compound was then purified and confirmed by ESIꢀMS, as described above for the NCSꢀbearing
chelators. The other two bioconjugates, PCBꢀTE2AꢀBnꢀNCSꢀc(RGDyK) (3) and ECBꢀTE2Aꢀ
c(RGDyK) (4), were prepared according to the literature.20, 25ꢀ26 The yields of the isolated
conjugated products were 13%, 7%, and 9% for NOTA (1), TE2A (2), and PCBꢀTE2A (5),
respectively.
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Radiochemistry. Next, all bioconjugates were labeled with [64Cu]CuCl2 (37ꢀ370 MBq) at
a concentration of 1 µg/20 µL (Figure 2), which was followed by HPLC purification to separate
traces of free copper and minor impurities (Figure 3A–C, Supporting Information Figure S10).
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Although the radiochemical purity of all isolated Cuꢀbioconjugates was determined to be > 95%,
the radiolabeling conditions varied depending on the presence of crossꢀbridges on the chelators.
For the nonꢀcrossꢀbridged chelates, NOTA (1) and TE2A (2), quantitative radiolabeling was
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feasible at 50 °C within 15 min in simple water. However, the crossꢀbridged macrocycles, PCBꢀ
TE2AꢀBnꢀNCS (3), ECBꢀTE2A (4), and PCBꢀTE2A (5), were radiolabeled at 80 °C in buffer (pH
8.0) with an extended incubation time of 60 min (Figure. 2). The radiolabeling yields of all three
crossꢀbridged chelates were greater than 95%. Typical radiochemical yields were ~80% after
HPLC purification from the start of radiolabeling. Total radiolabeling time including HPLC
purification was less than 2 h.
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The measured specific activities of all Cuꢀbioconjugates was measured to be in the range
of 15ꢀ40 GBq/µmol (Figure 3D). The crossꢀbridged chelates showed slightly higher specific
activities than the nonꢀcrossꢀbridged chelates. Any meaningful correlation between specific
activities and tumor uptakes was not observed.
Effect of BFCs on overall lipophilicity of bioconjugates. The LogD values at pH 7.4 of
all radiolabeled bioconjugates are summarized in Figure 3D. As expected, the ECBꢀTE2A chelate
(64Cuꢀ4), which conjugates amides directly with peptide via an acetate arm, showed the highest
hydrophilicity (LogD ꢀ3.73). The PCBꢀTE2A chelate (64Cuꢀ5), which has an additional methylene
bridging carbon compared with ECBꢀTE2A, had the next highest hydrophilicity (LogD ꢀ3.49). The
overall charge of these two chelates after Cu(II) complexation is +1. The other three chelates,
which contain an additional lipophilic benzyl isothiocyanate group (BnꢀNCS), showed more
lipophilic characteristics. Among these three chelates, NOTAꢀBnꢀNCS (64Cuꢀ1), which has an
overall charge of ꢀ1 after complexation, showed higher hydrophilicity (LogD ꢀ3.19), while PCBꢀ
TE2AꢀBnꢀNCS (64Cuꢀ3), which contains a propylene crossꢀbridge and forms a neutral Cu(II)
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complex, was determined to be the most lipophilic (LogD ꢀ1.92). Another neutral complex, Cuꢀ
TE2AꢀBnꢀNCSꢀc(RGDyK) (64Cuꢀ2), showed much higher hydrophilicity than the PCBꢀTE2AꢀBnꢀ
NCS conjugate (64Cuꢀ3) (LogD ꢀ3.38). Several factors, such as the overall charge of the copper
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complex, the presence of an additional benzyl group, and the conjugation mode, thus seem to
contribute to the overall lipophilic behavior of the peptide conjugates.27
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Binding affinity assay. We then examined the αvβ3 integrin affinity of the five RGD
conjugates (Figure 3E). Surface receptor binding assays, using αvβ3 integrinꢀpositive U87MG cells
and 125Iꢀechistatin as a radioligand, indicated that all bioconjugates selectively bind to the cell
surface integrin receptor. The 50% inhibitory concentrations (IC50) of the bioconjugates were all
comparable and in the narrow range of 28–60 nM, which are similar to that of unꢀconjugated
c(RGDyK) peptide itself (22.6 ± 4.77 nM). It is interesting to note that the direct conjugation of
crossꢀbridged chelators with c(RGDyK) via an acetate group, resulted in minimal disturbance to its
affinity towards αvβ3 integrin. It has been reported previously that the conjugation mode of the
chelator has minimal impact on the integrin binding affinity of RGD conjugates.25, 28ꢀ29 The ECBꢀ
TE2A (4) and PCBꢀTE2A (5) conjugates showed even lower IC50 values than the other three
chelators with slightly long spacers between the chelator and the RGD moiety, although the
differences were not large (Figure 3D).
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In vitro and in vivo stability studies. First, we measured the serum stability of the Cuꢀ
bioconjugates; no significant demetallation was observed for any chelator. For the least stable
conjugate, the NOTA conjugate (64Cuꢀ1), 95% of the complex remained intact after 24 h
incubation with fetal bovine serum at 37 °C, while the three crossꢀbridged chelators showed less
than 1% decomplexation (Supporting Information Figure S1).
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The plasma protein binding of CuꢀBFCsꢀc(RGDyK) was measured to be 17.5%, 12.9%,
12.5%, 15.8%, and 9.6% for NOTA (64Cuꢀ1), TE2A (64Cuꢀ2), PCBꢀTE2AꢀBnꢀNCS (64Cuꢀ3),
ECBꢀTE2A (64Cuꢀ4), and PCBꢀTE2A (64Cuꢀ5), respectively (Supporting Information Figure S2).
No correlation between protein binding and stability or lipophilicity was observed.30
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Next, we directly analyzed the in vivo stability of the Cuꢀlabeled bioconjugates in blood,
liver, and kidney 1 h postꢀinjection into SpragueꢀDawley (SD) rats. In contrast to the in vitro
serum stability data, there were large differences in in vivo stability between the different chelators
(Fig. 4). In general, the three crossꢀbridged chelators showed much higher robustness compared
with the two nonꢀcrossꢀbridged chelators. In particular, PCBꢀTE2AꢀBnꢀNCS (64Cuꢀ3) showed
exceptionally high stability in all three tissues. The blood demetallation of PCBꢀTE2AꢀBnꢀNCS
(64Cuꢀ3) was only 5.8%, whereas the blood demetallation of NOTA (64Cuꢀ1) and TE2A (64Cuꢀ2)
was five times higher (30% and 40%, respectively), as determined by quantification of the
demetalated free 64Cu ion peak (Supporting Information Figure S3). The other crossꢀbridged
chelators, which use one acetate group for peptide conjugation, showed intermediate inertness in
blood (24% and 20% for ECBꢀTE2A (64Cuꢀ4) and PCBꢀTE2A (64Cuꢀ5), respectively). In liver, the
stability differences between the crossꢀbridged and nonꢀcrossꢀbridged chelators were even more
pronounced. All three crossꢀbridged chelators showed approximately 10% demetallation, which
was only one quarter of that observed for the nonꢀcrossꢀbridged chelators (40% and 45% for
NOTA (64Cuꢀ1) and TE2A (64Cuꢀ2), respectively). A very similar pattern was observed in the
kidneys; demetallation was in the range of 8–12% for the crossꢀbridged chelators, whereas NOTA
and TE2A showed approximately fourꢀfold higher demetallation (34% and 45%, respectively). The
ultrahigh robustness of PCBꢀTE2AꢀBnꢀNCS can be explained by its tight clam shellꢀlike holding
of the copper ion with two acetate arms;20 in contrast, the other two crossꢀbridged chelators use
only one acetate arm for coordination to Cu(II) ions.
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Animal PET imaging studies. A full comparative study to evaluate the in vivo behavior
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and tumor targeting efficacy of all Cuꢀlabeled bioconjugates was performed using animal PET
imaging studies in U87MG tumorꢀbearing nude mice (n = 3 per bioconjugate). Representative
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maximum intensity projection (MIP) PET images collected 1 h, 4 h, and 24 h postꢀinjection are
displayed in Figure 5. At first glance, most activity was found in clearance organs such as the liver,
kidneys, intestine and bladder at 1 and 4 h postꢀinjection (Figure 5A ꢀ J). After 24 h, most of the
abdominal activity had been cleared from the body, and tumors became more easily
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distinguishable from the background for all five Cuꢀbioconjugates (Figure 5K ꢀ O). However,
upon closer inspection, all five conjugates showed distinct clearance and tumor uptake patterns.
Tumors could be clearly identified from the background as early as 1 h postꢀinjection in the case of
the three crossꢀbridged chelators (Figure 5C–E), while the tumors could not be clearly identified
until 24 h postꢀinjection using the two nonꢀcrossꢀbridged chelators (Figure 5K & L). With respect
to absolute tumor uptake, the two PCBꢀTE2A conjugates exhibited distinctively higher tumor
uptake compared with the other three chelators (Figure 5C, H, M & 5E, J, O). The clearance
patterns of the five conjugates were also different. The ECBꢀTE2A conjugate (64Cuꢀ4) exhibited
the fastest body clearance via the urinary route,31 while the two propylene crossꢀbridged chelators
showed higher liver and kidney uptake at all time points compared with the other chelators. In
particular, the PCBꢀTE2AꢀBnꢀNCS bioconjugate (64Cuꢀ3) showed significant activity in the liver
and intestine until 24 h postꢀinjection (Figure 5M).
The detailed pharmacokinetic behavior of the 64Cuꢀlabeled conjugates was analyzed by
PET imageꢀbased quantification. Regions of interest (ROIs) were drawn on liver, kidney, and
muscle, as well as tumor tissue. The resulting quantitative data along with the tumorꢀtoꢀorgan
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ratios are summarized in Table 1. One of the strongest distinctions among the five Cuꢀlabeled
peptides was the higher concentration of activity in tumors for two propylene crossꢀbridged
chelators. The PCBꢀTE2AꢀBnꢀNCS conjugate (64Cuꢀ3) exhibited the highest tumor uptakes at all
time points (3.76 ± 0.21, 3.89 ± 0.07, 3.49 ± 0.10 %ID/g at 1, 4, 24 h, respectively), which was
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followed by the PCBꢀTE2A conjugate (64Cuꢀ5) (3.45 ± 0.16, 3.10 ± 0.10, 2.34 ± 0.32 %ID/g). The
ethylene crossꢀbridged chelator (64Cuꢀ4) showed the third highest tumor uptake until 4 h (2.23 ±
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0.11, 1.99 ± 0.21 %ID/g at 1 and 4 h, respectively). In contrast to the nonꢀcrossꢀbridged chelators,
which showed maximum uptake of less than 2 %ID/g in tumors at all time points, tumors could be
visualized clearly using peptide conjugates of the three crossꢀbridged chelators. Even though the
absolute tumor uptake of the ECBꢀTE2A conjugate (64Cuꢀ4) was lower than that of the PCBꢀTE2A
conjugates (64Cuꢀ5), the tumors were more unambiguously visualized using the ECBꢀTE2Aꢀ
conjugated peptide at 1 and 4 h because uptake of this conjugate in the neighboring liver and
intestine was considerably lower than uptake of the other conjugates. Liver uptake of the ECBꢀ
TE2A conjugate (64Cuꢀ4) was less than half that of the PCBꢀTE2AꢀBnꢀNCS conjugate (64Cuꢀ3)
(1.97 ± 0.38 vs 3.91 ± 0.16 at 1 h, 1.66 ± 0.25 vs 3.15 ± 0.57 at 4 h, 0.79 ± 0.13 vs 2.73 ± 0.11
%ID/g at 24 h, respectively), resulting in the highest tumorꢀtoꢀliver ratios at all time points (1.13,
1.20, 1.41 at 1, 4, and 24 h, respectively). In contrast to all other chelators, the ECBꢀTE2A
conjugate (64Cuꢀ4) was rapidly excreted, mostly via the urinary route rather than the hepatobiliary
route, which has also been observed by other groups.25, 32 However, the PCBꢀTE2A conjugate
(64Cuꢀ5), which has only one extra methylene carbon in the crossꢀbridge compared with the ECBꢀ
TE2A conjugate, resembled the PCBꢀTE2AꢀBnꢀNCS conjugate (64Cuꢀ3) more closely than the
ECBꢀTE2A conjugate in tumor uptake and clearance patterns. The PCBꢀTE2A conjugate (64Cuꢀ5)
showed preferential excretion through the liver and intestine rather than the kidneys (3.91 ± 0.22
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vs 2.97 ± 0.45 at 1 h, 3.13 ± 0.10 vs 2.25 ± 0.46 at 4 h, 1.83 ± 0.10 vs 1.60 ± 0.38 %ID/g at 24 h
for liver and kidney, respectively). Considering that the chelators differ in their linker (the
presence of the additional benzyl NCS group), the overall charge after Cu(II) complexation
(neutral vs +1), and conjugation mode (thiourea vs amide), the closer resemblance of the PCBꢀ
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TE2A conjugate (64Cuꢀ5) to the PCBꢀTE2AꢀBnꢀNCS conjugate (64Cuꢀ3) than the ECBꢀTE2A
conjugate (64Cuꢀ4) is striking, and could be considered a good example of the importance of the
bifunctional chelator in 64Cuꢀlabeled peptide conjugates.25, 28 The only plausible explanation of this
observation is that the high hydrophilicity of the ECBꢀTE2A conjugate (logD ꢀ3.73) facilitates
urinary excretion.33
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The improved tumor targeting efficacy—in terms of absolute tumor uptake and high tumorꢀ
toꢀbackground ratios—associated with the three crossꢀbridged chelators can be mostly attributed to
the high in vivo stability of the corresponding Cu(II)ꢀchelator complexes. As seen in Figure 4, the
copper complexes of the three crossꢀbridged chelators showed much higher in vivo stability than
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the complexes of the nonꢀcrossꢀbridged chelators. Among the three crossꢀbridged chelators, Cuꢀ
PCBꢀTE2AꢀBnꢀNCSꢀc(RGDyK) (64Cuꢀ3), which exhibited the highest tumor uptake and
prolonged tumor retention (Table 1), showed approximately fourꢀfold lower demetallation in blood
compared with the other two chelators. Ultrastable 64Cuꢀlabeled chelators could be delivered to the
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tumor region, minimizing the release of free Cu(II) ions, which increases background activity
and results in lower tumorꢀtoꢀorgan ratios.25, 34 The lipophilicity of chelators also seems to have
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some effect on tumor uptake of Cuꢀlabeled bioconjugates.35 Even though the in vivo stability of
64CuꢀPCBꢀTE2Aꢀc(RGDyK) (64Cuꢀ5) was comparable with that of the ECBꢀTE2A analog (64Cuꢀ4),
the PCBꢀTE2A conjugate, which had higher lipophilicity than the ECBꢀTE2A conjugate (LogD ꢀ
3.49 vs ꢀ3.73), exhibited >1.5ꢀfold higher tumor uptake at all three time points.
Biodistribution studies. Biodistribution studies of 64CuꢀTE2AꢀBnꢀNCSꢀc(RGDyK) (64Cuꢀ
2) and 64CuꢀPCBꢀTE2Aꢀc(RGDyK) (64Cuꢀ5) were performed for the first time in the current study
(Figure 6); biodistribution data for the other radiotracers have been reported previously.20, 24, 32
Both of the 64Cuꢀbioconjugates showed αvβ3 integrinꢀmediated specific tumor uptake. In
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agreement with the PET imaging data, tumor uptake of CuꢀPCBꢀTE2Aꢀc(RGDyK) (64Cuꢀ5) was
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twoꢀfold higher than that of the TE2AꢀBnꢀNCS conjugate (64Cuꢀ2) 1 h and 24 h postꢀinjection
(4.70 ± 0.35 vs 2.36 ± 0.41 at 1 h, 3.82 ± 0.23 vs 1.78 ± 0.04 %ID/g at 24 h). The PCBꢀTE2A
conjugate (64Cuꢀ5) exhibited prolonged activity in tumors, and more than 80% of the activity
observed 1 h postꢀinjection remained after 24 h (Figure 6B). The activity in the other organs
cleared gradually over time, resulting in high tumorꢀtoꢀorgan ratios at 24 h (22.6, 6.5, and 4.9 for
blood, muscle, and liver, respectively). The relatively low tumorꢀtoꢀkidney ratio of the PCBꢀTE2A
conjugate (1.8) can be attributed to its preference for renal excretion, presumably because of its
high hydrophilicity25 (LogD −3.49) and the net positive charge of the tracer.36ꢀ37 In contrast to the
PCBꢀTE2A analog (64Cuꢀ5), the more lipophilic neutral 64CuꢀTE2AꢀBnꢀNCSꢀc(RGDyK)
conjugate (64Cuꢀ2) was excreted via both the liver and kidneys (Figure 6A), resulting in a lower
tumorꢀtoꢀliver ratio at all time points (0.71 vs. 2.1, 0.75 vs. 1.8, and 1.8 vs. 4.9, respectively).
Prolonged retention in tumor and fast blood clearance also resulted in an increase in the tumorꢀtoꢀ
blood ratio for 64CuꢀPCBꢀTE2Aꢀc(RGDyK) (64Cuꢀ5) compared with 64CuꢀTE2AꢀBnꢀNCSꢀ
c(RGDyK) (64Cuꢀ2) at all time points (6.8 vs. 4.4, 30.9 vs. 7.7, and 22.6 vs. 15.7 at 1, 4 and 24 h,
respectively). In blocking studies, tumor uptake of TE2AꢀBnꢀNCS (64Cuꢀ2) and PCBꢀTE2A (64Cuꢀ
5) conjugates was reduced by 54% (1.09 ± 0.16 %ID/g) and 77% (1.08 ± 0.15 %ID/g),
respectively, by coꢀinjection of c(RGDyK) peptide, which clearly indicates that tumor uptake was
mediated by specific binding of radiotracers to the αvβ3 integrin receptor (Figure 6A & B).
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Conclusions
In the current study, the effects of bifunctional chelators on the pharmacokinetic behavior
of 64Cuꢀradiolabeled peptides were evaluated. Five closely related chelators, including three crossꢀ
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bridged macrocycles, were conjugated with cyclic RGD peptides and radiolabeled with Cuꢀ64 ions
in high yield. Various properties, such as in vitro and in vivo stability, LogD, specific activity,
selective binding affinity, and protein binding, were measured. For biological evaluation, direct
comparison of tumor uptake and body clearance among the five bioconjugates was achieved by
animal PET imaging studies. Biodistribution studies further supported the imaging data.
Significant differences in tissue uptake and clearance patterns were observed depending on
the chelator utilized in the peptide conjugate. The three crossꢀbridged chelators resulted in higher
tumor visibility compared with the nonꢀcrossꢀbridged chelators. In particular, propylene crossꢀ
bridged chelators, which exhibited ultrahigh in vivo stability, showed higher tumor uptake, while
the analogous ethylene crossꢀbridged chelator, which had the highest hydrophilicity, showed rapid
body clearance. In our study, the in vivo stability of copper complexes was strongly correlated
with tumor uptake of bioconjugates. Tumor targeting and body clearance, and therefore tumorꢀtoꢀ
background ratios, also showed strong correlations with the overall lipophilicity of the 64Cuꢀ
bioconjugates. No significant correlation between other factors (i.e., overall charge of the Cu(II)
complex, linker, or conjugation mode of the chelator) and biological behavior was observed,
although these factors might together contribute to the overall lipophilicity of the 64Cuꢀ
bioconjugates. The effect of the chelator on the specific activity, selective binding affinity, and
protein binding of the peptide conjugates was also minimal.
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In conclusion, propylene crossꢀbridged TE2A derivatives show potential as promising
bifunctional chelators for the radiolabeling of short peptides with radioactive copper ions, at least
for RGD peptides. Further studies using PCBꢀTE2A chelators for radiolabeling of other diseaseꢀ
specific peptides for radioactive copperꢀbased imaging and therapeutic applications are highly
anticipated.
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Experimental Section
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Materials and methods. All chemicals or solvents used in the study were purchased from Sigmaꢀ
Aldrich (St. Louis, USA) unless otherwise stated. c(RGDyK) was purchased from FutureChem
(Seoul, Korea). Cuꢀ64 was produced at KIRAMS (Seoul, Korea) by the 64Ni(p,n)64Cu nuclear
reaction using an MC50 Cyclotron (Scanditronix, Sweden). All radioꢀTLC measurements were
performed using a Bioscan 2000 imaging scanner (Bioscan, Washington, D.C., USA).
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Conjugation of BFCs to c(RGDyK). The BFCs used in the study had two different functional
groups (ꢀNCS or ꢀCOOH), and two different coupling reactions were employed to conjugate the
chelators to c(RGDyK) peptide depending on the functionality. For the synthesis of TE2AꢀBnꢀ
NCSꢀc(RGDyK) (2), N,Nꢀdiisopropylethylamine (20 mmol) was added to a solution containing
TE2AꢀBnꢀNCS (10 ꢁmol, 4.8 mg) and c(RGDyK) (10 ꢁmol, 6.2 mg) in dimethylformamide
(DMF; 1 mL), and the resulting mixture was stirred at room temperature for 16 h. A similar
method was followed for the synthesis of NOTAꢀBnꢀNCSꢀc(RGDyK) (1). Here NOTAꢀBnꢀNCS
(11 ꢁmol, 5 mg) and c(RGDyK) (32 ꢁmol, 10 mg) were used and the reaction was continued with
N,Nꢀdiisopropylethylamine (40 mmol, 5 mg). Subsequently, the bioconjugates were purified by
semiꢀpreparative HPLC. HPLC conditions for TE2AꢀBnꢀNCSꢀc(RGDyK) (2) and NOTAꢀBnꢀ
NCSꢀc(RGDyK) (1) were as follows. For TE2AꢀBnꢀNCSꢀc(RGDyK) (2), an Agilent preparative
C18 column (5 ꢁm, 21.2 × 100 mm) with the mobile phase (flow rate 3 mL/min) starting from
95% solvent A [0.1% TFA in water] and 5% solvent B [0.1% TFA in acetonitrile] [0–2 min] to
35% solvent A and 65% solvent B at 32 min was used. The peak containing the TE2AꢀBnꢀNCSꢀ
c(RGDyK) (2) conjugate was collected at a retention time of 21.7 min, and it showed a retention
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time of 12.8 min through analytical HPLC (Vydac TP C18, 3 ꢁm, 4.6 × 100 mm; flow rate 1
mL/min, the mobile phase consisting of 0.1% TFA/H2O (solvent A) and 0.1% TFA/acetonitrile
(solvent B) with a gradient consisting of 1% B to 70% B in 20 min). For NOTAꢀBnꢀNCSꢀ
c(RGDyK) (1), a Waters ꢁbondapak C18 column (10 ꢁm, 7.8 × 300 mm) with the mobile phase
(flow rate 3 mL/min) starting from 95% solvent A (0.1% TFA in water) and 5% solvent B (0.1%
TFA in acetonitrile) (0ꢀ2 min) to 35% solvent A and 65% solvent B at 32 min was used. The peak
containing NOTAꢀBnꢀNCSꢀc(RGDyK) conjugate (1) was collected at a retention time of 17.5 min.
All fractions containing the product were combined and lyophilized to provide the final product as
a white powder (isolated yield 7.3% and 13% for TE2AꢀBnꢀNCSꢀc(RGDyK) (2) and NOTAꢀBnꢀ
NCSꢀc(RGDyK) (1), respectively).
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For the conjugation of PCBꢀTE2A to c(RGDyK), a typical amide coupling reaction was
applied. N,N'ꢀdicyclohexylcarbodiimides (10 mmol) and N,Nꢀdiisopropylethylamine (20 mmol)
were added to a solution of PCBꢀTE2A (20 ꢁmol, 7.2 mg) and c(RGDyK) (10 ꢁmol, 6.2 mg) in
DMSO (1 mL), and the reaction was allowed to continue at room temperature for 16 h. Finally, the
pure PCBꢀTE2Aꢀc(RGDyK) (5) was isolated as a white solid by HPLC purification [Zorbax
Agilent PrepꢀC18; 21.2 × 100 mm; mobile phase starting from 95% solvent A (0.1% TFA in
water) and 5% solvent B (0.1% TFA in acetonitrile) (0–2 min) to 35% solvent A and 65% solvent
B at 32 min; flow rate 3 ml/min], which was followed by lyophilization (9.4% yield). Analytical
HPLC identified the retention time of PCBꢀTE2Aꢀc(RGDyK) (5) to be 12.2 min (Vydac TP C18, 3
ꢁm, 4.6 x 100 mm; flow rate 1 mL/min, with the mobile phase consisting of 0.1% TFA/H2O
(solvent A) and 0.1% TFA/acetonitrile (solvent B) with a gradient consisting of 1% B to 70% B in
20 min). The other bioconjugates, ECBꢀTE2Aꢀc(RGDyK) (4) and PCBꢀTE2AꢀBnꢀNCSꢀ
c(RGDyK) (3) were prepared according to the published literature.20, 26
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The formation of all bioconjugates was confirmed by ESIꢀMS. For TE2AꢀBnꢀNCSꢀ
c(RGDyK), the calculated and identified m/z values for C50H76N14O12S [M+H]+ were 1097.56 and
1097.54 respectively (Supporting Information Figure S4). For NOTAꢀBnꢀNCSꢀc(RGDyK) (1), the
calculated and identified m/z values for C47H67N13O14S [M+H]+ were 1070.47 and 1070.5,
respectively (Supporting Information Figure S5). For PCBꢀTE2Aꢀc(RGDyK) (5), the calculated
and identified m/z values for C44H71N13O11 [M+H]+ were 958.5 and 958.2, respectively
(Supporting Information Figure S6).
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The chemical purities of the five isolated BFCꢀc(RGDyK) peptides were characterized by
employing the second HPLC conditions. More than 95% chemical purity were obtained in all five
peptide conjugates (Supporting Information Figure S9).
Radiolabeling with 64Cu. Two different strategies were used for the radiolabeling of BFCsꢀ
c(RGDyK). For nonꢀcross bridged macrocycles NOTAꢀBnꢀNCSꢀc(RGDyK) (1) and TE2AꢀBnꢀ
NCSꢀc(RGDyK) (2), 64CuCl2 (18.5–148 MBq) in 100 ꢁL MilliQ water (with no carrier added) was
first mixed with 10 ꢁg of BFCsꢀc(RGDyK) in 100 ꢁL MilliQ water followed by incubation at 50
°C for 15 min using a ThermoMixer (800 rpm, Eppendorf). The radiolabeling progress was
monitored by radioꢀTLC using Whatman MKC18F TLC plates developed with 10%
NH4OAc/methanol (3:7). Under this condition, 64CuꢀNOTAꢀBnꢀNCSꢀc(RGDyK) (64Cuꢀ1) and
64CuꢀTE2AꢀBnꢀNCSꢀc(RGDyK) (64Cuꢀ2) gave Rf values in the range of 0.9–1.0. For the cross
bridged macrocycles, PCBꢀTE2AꢀBnꢀNCSꢀc(RGDyK) (3), ECBꢀTE2Aꢀc(RGDyK) (4), and PCBꢀ
TE2Aꢀc(RGDyK) (5), the radiolabeling was performed in 0.1 M NH4OAc buffer (pH 8.0) with an
elongated incubation time of 60 min at 80 °C. Crossꢀbridged macrocycles gave Rf values of 0.8ꢀ0.9
under the same TLC condition. The 64Cuꢀlabeled peptide was then purified by reverseꢀphase
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HPLC (RPꢀHPLC) using a Waters Grace smart RP C18 column (4.6 × 250 mm, 5 µm), with the
mobile phase consisting of 0.1% TFA/H2O or pure H2O (solvent A, 0.1% TFA/H2O and H2O was
used for crossꢀbridged and nonꢀcrossꢀbridged macrocycles, respectively) and 0.1%
TFA/acetonitrile or acetonitrile (solvent B, 0.1% TFA/acetonitrile and acetonitrile was used for
crossꢀbridged and nonꢀcrossꢀbridged macrocycles, respectively) and a gradient consisting of 1% B
to 70% B in 20 min with a 1ꢀmL/min flow rate. The radiotracers were collected in a fraction
collector in 1–2 mL of HPLC eluent. The solvents were then evaporated at 37 °C and the activity
was reconstituted in saline or 10% DMSO/saline, followed by passage through a 0.22ꢀꢁm
Millipore filter into a sterile dose vial for use in animal experiments. The radiochemical purities of
64Cuꢀlabeled NOTAꢀBnꢀNCS (64Cuꢀ1), TE2AꢀBnꢀNCS (64Cuꢀ2), PCBꢀTE2AꢀBnꢀNCS (64Cuꢀ3),
ECBꢀTE2A (64Cuꢀ4), and PCBꢀTE2A (64Cuꢀ5) bioconjugates after HPLC purification were 99%,
96%, 99%, 99%, and 95%, respectively (Figure 3A–C, Supporting Information Figure S7). The
chemical purities for the same samples based on UVꢀHPLC chromatograms were 98.3%, 96.5%,
96.9%, 98.1%, and 97.7%, respectively.
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Cold Cu(II)ꢀBFCsꢀc(RGDyK) complexes were prepared by the reaction of BFCsꢀ
c(RGDyK) conjugates with 5 equivalent CuCl2 in 0.1 M NH4OAc (pH 8.0) buffer at 80 °C for 1 h,
and used as internal standard for HPLC characterization without further purification. HRMS
(FAB) for CuꢀECBꢀTE2Aꢀc(RGDyK) (Cuꢀ4). calcd. for C43H68CuN13O11 [M]+ 1005.4457; found
1005.4460. HRMS (FAB) for CuꢀPCBꢀTE2Aꢀc(RGDyK) (Cuꢀ5). calcd. for C44H70CuN13O11 [M]+
1019.4614; found 1019.4618.
In vitro serum stability. In vitro serum stability of all five radiotracers was performed by adding
the 64Cuꢀbioconjugate (50 ꢁL, 7.4ꢀ13 MBq) to fetal bovine serum (500 ꢁL). The mixture was then
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incubated in a ThermoMixer (800 rpm) at 37 °C, and demetallation was analyzed by radioꢀTLC
(10% NH4OAc:MeOH = 3:7) at 0 h, 4 h, and 24 h.
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Plasma protein binding assay. Plasma was prepared by centrifuging (10,000 rpm) blood (SD rat,
male, 6 weeks) at 4 °C for 10 min just before the experiment. PDꢀ10 columns (GE healthcare)
were first preconditioned with 1 mL of 1% bovine serum albumin in 0.1 M DTPA, which was
followed by repeated washing with saline (50 mL). 64CuꢀBFCꢀc(RGDyK) (0.55–0.75 MBq, 50 ꢁL)
was then incubated with plasma (1 mL) and PBS (1 mL) separately for 30 min at 37 °C. After the
incubation, each sample was loaded on the preconditioned PDꢀ10 column and eluted with saline;
30 fractions (500 µL each) were collected per sample in Eppendorf tubes. Radioactivity of each
fraction was measured using a γ counter and a graph of radioactivity (CPM) vs saline volume (mL)
was plotted.
Specific activity measurement. The specific activities of all the 64Cuꢀbioconjugates were
measured following previous reports.20 Briefly, a calibration curve was first prepared by injecting
various amounts (0–2 µg) of CuꢀBFCsꢀc(RGDyK) into HPLC (λmax = 220 nm). Nonꢀradioactive
Cuꢀcomplexes were prepared by incubating the BFCsꢀc(RGDyK) conjugates with excess CuCl2
(10 mole equivalent) at 90 °C for 1 h. The pH of the reaction medium was maintained at 8 by
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adding NaOH solution (0.1 M). Subsequently, a known amount of activity of the purified Cuꢀ
BFCsꢀc(RGDyK) (7.4–11.1 MBq) was injected into HPLC and the peak area obtained in the UV
chromatogram was used to calculate specific activity (GBq/µmol).
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Determination of partition coefficient. LogD values of all Cuꢀbioconjugates were determined
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by the octanol/PBS method. Briefly, 5 ꢁL of the tracer (1.85 MBq) was added to a mixture of 1ꢀ
octanol and phosphate buffered saline (pH 7.4) (1:1 v/v, 1 mL) and the resulting mixture was
vortexed vigorously for 5 min at room temperature and centrifuged (10,000 rpm, 5 min) thereafter
to ensure complete separation of both layers. Next, a 100ꢀꢁL aliquot was taken from each phase
and counted separately using a γꢀcounter. The partition coefficient was calculated as the ratio of
counts in 1ꢀoctanol to that in PBS. The LogD7.4 values were reported as an average of six
measurements.
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Integrin receptor binding assay. The cellꢀbinding assay of the bioconjugates was performed
using 125Iꢀechistatin on U87MG cells (2 × 106/100ꢀL, resuspended in a binding buffer (20 mM tris,
pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MnCl2, and 0.1% bovine serum albumin) and IC50
values were compared to that of c(RGDyK). For this assay, equal volumes of all nonradioactive
ligands, NOTAꢀBnꢀNCSꢀc(RGDyK) (1), TE2AꢀBnꢀNCSꢀc(RGDyK) (2), PCBꢀTE2AꢀBnꢀNCSꢀ
c(RGDyK) (3), ECBꢀTE2Aꢀc(RGDyK) (4), and PCBꢀTE2Aꢀc(RGDyK) (5), and radioactive 125Iꢀ
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echistatin (3.7 kBq, PerkinꢀElmer, Branford, CT) were used. Various concentrations (10− to 10−
M) of the ligands were incubated at room temperature for 60 min which was followed by the
removal of the reaction medium. The cells were then washed with PBS (three times), harvested,
and the bound 125Iꢀechistatin was counted in a γꢀcounter (1480 WIZARD, Wallac). Data were
analyzed with GraphPad Prism 5 (GraphPad software, Inc., San Diego, CA) to calculate
corresponding IC50 values. Each experiment was conducted in triplicate.
In vivo stability studies. In a typical experiment, approximately 37 MBq of radiolabeled
conjugates was injected intravenously in SD rats (n = 2, male, 210 ꢀ 220 g). At 1 h postꢀinjection,
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the animals were sacrificed and blood and organs of interest (liver and kidney) were collected. To
determine the stability of the bioconjugates in blood, the blood sample was immediately
centrifuged (13,000 rpm for 10 min). Then, to the supernatant (200 µL), a solution mixture of
acetonitrile/water/trifluoroacetic acid (50:45:5, 100 µL) was added. The mixture was then kept at 4
°C for 10 min and centrifuged again for 10 min at 13,000 rpm. The supernatant was then filtered
through a 0.22ꢀµm filter and injected into HPLC (Grace smart RP C18 column, 5 ꢁm, 4.6 × 250
mm). The eluent [0.1% TFA/H2O (solvent A) and 0.1% TFA/acetonitrile (solvent B), a gradient of
1% B to 70% B in 20 min at a flow rate of 1 mL/min] was collected with a fraction collector (1.0
min/fraction), and the radioactivity of each fraction was measured using a γꢀcounter. To determine
the stability of 64Cuꢀbioconjugates in the liver and kidney, the collected organs were first
homogenized, which was followed by the addition of PBS (5 mL) and vigorous vortexing for 5
min. The mixture was then centrifuged for 10 min (13,000 rpm). To the supernatant (200 µL), 100
µL of a mixture of acetonitrile:water:trifluoroacetic acid (50:45:5) was added, which was followed
by mixing and incubation at 4 °C for 10 min; samples were then centrifuged for 10 min at 13,000
rpm. The supernatant was then filtered through a 0.22ꢀµm filter, and the filtrate was injected to
reverseꢀphase HPLC; the activities of collected fractions were measured with a γꢀcounter. The in
vivo stability of 64CuꢀNOTAꢀNCSꢀRGD (64Cuꢀ1), 64CuꢀTE2AꢀNCSꢀRGD (64Cuꢀ2), and 64Cuꢀ
PCBꢀTE2AꢀRGD (64Cuꢀ5) were measured for the first time in the current manuscript, and the data
were compared to that of 64CuꢀPCBꢀTE2AꢀBnꢀNCSꢀRGD (64Cuꢀ3) and 64CuꢀECBꢀTE2AꢀRGD
(64Cuꢀ4), which were reported by us previously.20
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The activities found in the precipitate from blood, liver and kidneys, were less than 10%
and more than 90% of total tissue activities were used for the analysis.
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Animal models. All the animal experiments were conducted in compliance with the guidelines set
by the Institutional Animal Care and Use Committee requirements of Kyungpook National
University. The procedures were reviewed and approved by the animal ethics committee of
Kyungpook National University (Approval Number: 2016ꢀ0024). Specifically, 6ꢀweek old
BALB/c female nude mice were used to prepare xenograft tumor models using U87MG cell lines.
A total of 5 × 106 cells was inoculated subcutaneously into the right flank of the mice, and tumors
of the appropriate size required approximately 15 days to develop.
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Animal PET imaging. Small animal PET images were obtained using an Inveon PETꢀcomputed
tomography (PET/CT) scanner (Siemens). All radiolabeled tracers (18.5–22.2 MBq, 200 µL 10%
DMSO/saline) were injected via the tail vein into female nude mice bearing xenograft U87MG
tumors on their right flank. At 1, 4, and 24 h postꢀinjection, the mice were anesthetized with
isoflurane (1–2 % in oxygen), positioned prone, and imaged using a PET scanner. CT images were
acquired successively immediately after PET imaging without adding any additional contrast
agent. All images were reconstructed using the 2ꢀdimensional orderedꢀsubsets expectation
maximum (OSEM) algorithm. Regions of interest (ROIs) were manually drawn around the
perimeters of the organs over 5ꢀ7 consecutive slices on transverse PET images entirely covering
the most intense areas of 64Cuꢀlabelled BFCꢀc(RGDyK) uptake in the tumor, liver, kidney and
muscle, while avoiding nearby tissues. Activity counts in the ROIs were calculated as the
percentage of the injected dose per gram (%ID/g), and no corrections for attenuation were
necessary. Since the location of ROIs on the PET images could not be accurately defined, we used
the corresponding transverse CT images as a reference. The PET and CT volumes were manually
coꢀregistered for each of the scans (Inveon Research Workplace 4.0).
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Biodistribution study. Under anesthesia using 1–2% isoflurane in O2, tumorꢀbearing female
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BALB/c nude mice (n = 3 per group) were subjected to intravenous injection of the purified Cuꢀ
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bioconjugates (0.74 MBq, 200 µL saline) via tail vein. The animals were sacrificed at 1, 4, and 24
h postꢀinjection and tissues of interest were explanted, weighted, and counted using a γꢀcounter
(Wallac). Uptake in each tissue was expressed as percent of injected dose per gram (%ID/g) and
was calculated by comparing the samples with a standard solution derived from the injected
64
solution. For the blocking study, nonꢀradioactive c(RGDyK) was coꢀinjected (5 mg/kg) with Cuꢀ
labelled tracers in tumor models and biodistribution was conducted 1 h postꢀinjection.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI:
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Serum stability studies of CuꢀBFCsꢀc(RGDyK), plasma protein binding studies of Cuꢀ
BFCsꢀc(RGDyK), in vivo stability studies of 64CuꢀBFCsꢀc(RGDyK), mass spectra of BFCsꢀ
c(RGDyK) conjugates, UV HPLC profiles of BFCsꢀc(RGDyK) conjugates, UV/radioꢀHPLC
chromatogram of 64CuꢀPCBꢀTE2AꢀBnꢀNCSꢀc(RGDyK) and 64CuꢀECBꢀTE2Aꢀc(RGDyK), mass
spectra of CuꢀECBꢀTE2Aꢀc(RGDyK) and CuꢀPCBꢀTE2Aꢀc(RGDyK).
AUTHOR INFORMATION
Corresponding Author
*J.Y.: phone, +82ꢀ53ꢀ420ꢀ4947; eꢀmail, yooj@knu.ac.kr.
ORCID
Jeongsoo Yoo: 0000ꢀ0002ꢀ0752ꢀ5745
Author Contributions
S.S, N.B and Y.S.H contributed equally to this work.
ACKNOWLEDGMENTS
This work was supported by an R&D program through the National Research Foundation
of Korea funded by the Ministry of Science, ICT & Future Planning (No. 2016R1A2B4011546,
2013R1A4A1069507,
2017M2C2A1014006,
2017M2A2A6A02018506,
and
2016H1D3A1907667) and supported by a grant of the Korea Health Technology R&D Project
through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of
Health & Welfare, Republic of Korea (grant number: HI17C0221).
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ABBREVIATIONS USED
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BFCs, bifunctional chelators; RGD, arginylglycylaspartic acid; PET, positronꢀemission
tomography; ATSM, diacetylꢀbis(N4ꢀmethylthiosemicarbazone); PTSM, pyruvaldehyde bis(N4ꢀ
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methylthiosemicarbazone);
ECBꢀTE2A, ethylene
DOTA, 1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7,10ꢀtetraacetic acid;
crossꢀbridged 1,8ꢀN,N′ꢀbisꢀ(carboxymethyl)ꢀ1,4,8,11ꢀ
tetraazacyclotetradecane; PCBꢀTE2A, propylene crossꢀbridged 1,8ꢀN,N′ꢀbisꢀ(carboxymethyl)ꢀ
1,4,8,11ꢀtetraazacyclotetradecane; TETA, 1,4,8,11ꢀtetraazacyclotetradecaneꢀN,N',N'',N'''ꢀtetraacetic
acid;
bis(carboxymethyl)ꢀ16ꢀ(4ꢀisothiocyanatobenzyl)ꢀ1,4,8,11ꢀtetraazabicyclo[6.6.3]heptadecane;
TE2AꢀBnꢀNCS, 1,8ꢀN,N′ꢀbisꢀ(carboxymethyl)ꢀ4ꢀN″ꢀ(4′ꢀisothiocyanatophenethyl)ꢀ1,4,8,11ꢀ
DTPA,
diethylenetriaminepentaacetic
acid;
PCBꢀTE2AꢀBnꢀNCS,
4,11ꢀN,N′ꢀ
tetraazacyclotetradecane; NOTA, 1,4,7ꢀtriazacyclododecaneꢀN,N′,N″ꢀtriacetic acid); NOTAꢀBnꢀ
NCS, 2ꢀSꢀ(4ꢀisothiocyanatobenzyl)ꢀ1,4,7ꢀtriazacyclononaneꢀ1,4,7ꢀtriacetic acid; HPLC, high
performance liquid chromatography; DIEA, N,Nꢀdiisopropylethylamine; ESIꢀMS, electrospray
ionization mass spectrometry; HRMS, high resolution mass spectrometry; FAB, fast atom
bombardment; DCC, dicyclohexylcarbodiimides; DMSO, dimethylsulfoxide; IC, inhibitory
concentrations; SD rat, SpragueꢀDawley rat; MIP, maximum intensity projection; TLC, thin layer
chromatography; rpm, revolutions per minute; DMF, dimethylformamide; TFA, trifluoroacetic
acid ; %ID/g, percent injected dose per gram; CPM, counts per minute; PBS, phosphate buffered
saline; ROI, region of interest; CT, computed tomography.
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