Evaluation of copper-64 labeled AmBaSar conjugated cyclic RGD peptide for improved MicroPET imaging of integrin αvβ3 expression

Article abstract of DOI:10.1021/bc900537f

Recently, we have developed a new cage-like bifunctional chelator 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) for copper-64 labeling and synthesized the positron emission tomography (PET) tracer ⁶⁴Cu-AmBaSar-RGD. In this study, we further evaluate the biological property of this new AmBaSar chelator by using ⁶⁴Cu-AmBaSar-RGD as the model compound. In vitro and in vivo stability, lipophilicity, cell binding and uptake, microPET imaging, receptor blocking experiments, and biodistribution studies of ⁶⁴Cu-AmBaSar-RGD were investigated, and the results were directly compared with the established radiotracer ⁶⁴Cu-DOTA-RGD. The ⁶⁴Cu-AmBaSar-RGD was obtained with high radiochemical yield (≥95%) and purity (≥99%) under mild conditions (pH 5.0-5.5 and 23-37 °C) in less than 30 min. For in vitro studies, the radiochemical purity of ⁶⁴Cu-AmBaSar-RGD was more than 97% in PBS or FBS and 95% in mouse serum after 24 h of incubation. The log P value of ⁶⁴Cu-AmBaSar-RGD was-2.44 ± 0.12. For in vivo studies, ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD have demonstrated comparable tumor uptake at selected time points on the basis of microPET imaging. The integrin α_vβ₃ receptor specificity was confirmed by blocking experiments for both tracers. Compared with ⁶⁴Cu-DOTA-RGD, ⁶⁴Cu-AmBaSar-RGD demonstrated much lower liver accumulation in both microPET imaging and biodistribution studies. Metabolic studies also directly supported the observation that ⁶⁴Cu-AmBaSar-RGD was more stable in vivo than ⁶⁴Cu-DOTA- RGD. In summary, the in vitro and in vivo evaluations of the ⁶⁴Cu-AmBaSar-RGD have demonstrated its improved Cu-chelation stability compared with that of the established tracer ⁶⁴Cu-DOTA-RGD. The AmBaSar chelator will also have general applications for ⁶⁴Cu labeling of various bioactive molecules in high radiochemical yield and high in vivo stability.

Full text of DOI:10.1021/bc900537f

Bioconjugate Chem. 2010, 21, 1417–1424
1417
Evaluation of Copper-64 Labeled AmBaSar Conjugated Cyclic RGD
Peptide for Improved MicroPET Imaging of Integrin rvꢀ3 Expression
Hancheng Cai, Zibo Li, Chiun-Wei Huang, Anthony H. Shahinian, Hui Wang, Ryan Park, and Peter S. Conti*
Molecular Imaging Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90033.
Received December 5, 2009; Revised Manuscript Received July 13, 2010
Recently, we have developed a new cage-like bifunctional chelator 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo
[6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) for copper-64 labeling and synthesized the positron
emission tomography (PET) tracer 64Cu-AmBaSar-RGD. In this study, we further evaluate the biological property
of this new AmBaSar chelator by using 64Cu-AmBaSar-RGD as the model compound. In Vitro and in ViVo stability,
lipophilicity, cell binding and uptake, microPET imaging, receptor blocking experiments, and biodistribution studies
of 64Cu-AmBaSar-RGD were investigated, and the results were directly compared with the established radiotracer
64Cu-DOTA-RGD. The 64Cu-AmBaSar-RGD was obtained with high radiochemical yield (g95%) and purity
(g99%) under mild conditions (pH 5.0-5.5 and 23-37 °C) in less than 30 min. For in Vitro studies, the
radiochemical purity of 64Cu-AmBaSar-RGD was more than 97% in PBS or FBS and 95% in mouse serum after
24 h of incubation. The log P value of 64Cu-AmBaSar-RGD was -2.44 ( 0.12. For in ViVo studies, 64Cu-
AmBaSar-RGD and 64Cu-DOTA-RGD have demonstrated comparable tumor uptake at selected time points on
the basis of microPET imaging. The integrin Rvꢀ3 receptor specificity was confirmed by blocking experiments
for both tracers. Compared with 64Cu-DOTA-RGD, 64Cu-AmBaSar-RGD demonstrated much lower liver
accumulation in both microPET imaging and biodistribution studies. Metabolic studies also directly supported
the observation that 64Cu-AmBaSar-RGD was more stable in ViVo than 64Cu-DOTA-RGD. In summary, the in
Vitro and in ViVo evaluations of the 64Cu-AmBaSar-RGD have demonstrated its improved Cu-chelation stability
compared with that of the established tracer 64Cu-DOTA-RGD. The AmBaSar chelator will also have general
applications for 64Cu labeling of various bioactive molecules in high radiochemical yield and high in ViVo stability.
their derivatives for 64Cu2+ labeling have limited stability in
ViVo due to the dissociation of 64Cu2+ from these BFCs, leading
to high retention in the liver. A class of cross-bridged TETA
derivatives has been developed that form stable complexes with
64Cu2+ and are less susceptible to in ViVo transchelation than
their nonbridged TETA analogues (9). However, harsh 64Cu2+
radiolabeling conditions (e.g., incubation at 85 °C under basic
conditions) for cross-bridged TETA derivatives may result in
the denaturation and hydrolysis of some biomolecules (10).
Recently, a new class of bifunctional chelators has been
synthesized on the basis of the sarcophagine (3,6,10,13,16,19-
hexaazabicyclo [6.6.6] icosane, also called Sar) for 64Cu2+
radiolabeling, resulting in extraordinarily stable 64Cu-Sar based
complexes in Vitro and in ViVo under physiological conditions
(11, 12). The carboxylate and amino derivatives of the diamSar
have also been developed by our group and Smith’s group,
respectively (13-16). These derivatives allow them to be readily
cross-linked to amine or carboxyl residues on peptides and
antibody molecules through amide bonds, yielding PET probes
with improved pharmacokinetics, and dynamics due to the
increased stability (1, 15).
The integrin Rvꢀ3 receptor has been the attractive target of
intensive research given its major role in several distinct
processes, such as tumor angiogenesis and metastasis, and
osteoclast mediated bone resorption (17, 18). The molecular
imaging of integrin Rvꢀ3 expression will allow the detection of
cancer and other angiogenesis related diseases, patient stratifica-
tion, and treatment monitoring of antiangiogenesis based
therapy (19, 20). Although we and others have successfully
developed various DOTA conjugated RGD peptides for mul-
timodality imaging of integrin Rvꢀ3 expression (19-23), the
loss of 64Cu from the chelator has led to unfavorable high
INTRODUCTION
The advancement of molecular imaging based on positron
emission tomography (PET) is mainly driven by the discovery
of imaging biomarkers and targeted molecules labeled with
positron emitting radioisotopes (1-3). PET tracers have been
widely applied in molecular imaging as these agents exhibit
high binding affinity toward various promising biological targets,
such as receptors or enzymes expressed during different phases
of tumor growth or in other uncontrolled diseases (3, 4).
Targeted copper-64 labeled molecules have shown promise for
diagnostic PET imaging, as well as for radiotherapy, due to the
favorable nuclear characteristics of the isotope (t1/2 ) 12.7 h,
ꢀ+ 17.4%, Emax ) 0.656 MeV, ꢀ- 39%, Emax ) 0.573 MeV)
and its availability with high specific activity (5). The half-life
of 64Cu is more suitable for developing biomolecule-based PET
tracers, which usually require longer circulation times to achieve
optimal targeting and uptake. Furthermore, 64Cu-based PET
tracers may be used to provide in ViVo pharmacokinetic profiles
of radiopharmaceuticals which contain the therapeutic radio-
nuclide copper-67 (t1/2 ) 61.5 h, ꢀ- 100%, Emax )0.121 MeV)
(6). In ViVo stable attachment of 64Cu2+ to targeted biomolecules
requires the use of a bifunctional chelator (BFC) (7). More and
more ligands have been investigated as BFCs to complex 64Cu2+
due to the development of copper coordination chemistry. Three
of the most common chelators for 64Cu2+ labeling are the
macrocyclic ligands 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-
tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclotetradecane-
N,N′,N′′,N′′′-tetraacetic acid (TETA), and cross-bridged TETA
ligands (2, 5, 7, 8). It is well known that DOTA, TETA, and
* To whom correspondence should be addressed. E-mail: pconti@
usc.edu.
10.1021/bc900537f  2010 American Chemical Society
Published on Web 07/28/2010

1418 Bioconjugate Chem., Vol. 21, No. 8, 2010
Cai et al.
Figure 1. Chemical structures of 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD.
retention in the liver, resulting in high background. Therefore,
the choice of a more stable BFC is preferred. As we mentioned
above, we have developed a carboxylate derivative of diamSar,
4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-
ylamino) methyl) benzoic acid (AmBaSar) (15, 16), which could
be conjugated to the primary amines in biomolecules through
amide bonds. We also reported the synthesis of a new PET tracer
64Cu-AmBaSar-RGD (Figure 1a). In this study, we further
evaluated the conjugation and labeling conditions of 64Cu-
AmBaSar-RGD. Moreover, the in Vitro and in ViVo stability,
lipophilicity, cell uptake, microPET imaging, and biodistribution
studies of 64Cu-AmBaSar-RGD and the established 64Cu-DOTA-
RGD (Figure 1b) were investigated to obtain a direct comparison
of the two agents.
analytical HPLC was operated at the same conditions with a
Rainin Microsorb-MV C18 column (5 µm, 250 × 4.6 mm) and
the mobile phase starting from 98% solvent A and 2% solvent
B (0-2 min) to 50% solvent A and 50% solvent B at 25 min.
Radio-TLC was performed on MKC18 silica gel 60 Å plates
(Whatman, NJ) with MeOH/20% NaOAc ) 3:1 as the eluent.
The plates were read with a Bioscan AR2000 imaging scanner
(Washington, DC) and Winscan 2.2 software. Mass spectrometry
using LC-MS was operated by the Proteomics Core Facility of
the USC School of Pharmacy.
Syntheses of AmBaSar-RGD and DOTA-RGD. AmBaSar
could be activated and conjugated to the c(RGDyK) peptide in
a water-soluble procedure as reported earlier (15). The conjuga-
tion can also be performed in organic phase according to
literature procedures (24, 25). Briefly, the solution of AmBaSar
(4.5 mg, 0.01 mmol), HATU (3.8 mg, 0.01 mmol), HOAt (1.4
mg, 0.01 mmol), and dimethyl sulfoxide (DMSO, 0.5 mL) were
stirred at room temperature for 10 min. Seven equivalents of
DIPEA (9.1 mg, 0.07 mmol) and c(RGDyK) (1.2 mg, 0.002
mmol) in 300 µL of DMSO were then added to the mixture at
0 °C. The mixture was stirred for 3 h at room temperature, and
the solvent removed in Vacuo. The residue was dissolved in
acetonitrile/water (1:3) containing 0.1% TFA and purified by
semipreparative HPLC. AmBaSar-RGD was obtained as a white
solid material after lyophilization. DOTA was activated and
conjugated to c(RGDyK) as reported earlier (23). DOTA-RGD
was also purified by semipreparative HPLC and confirmed by
mass spectrometry. AmBaSar-RGD and DOTA-RGD conjugates
were dissolved in 0.1 N ammonium acetate buffer solution (pH
5-5.5) and stored at -20 °C for future use in radiolabeling
reactions.
Copper-64 Labeling and Formulation. The 64Cu-AmBaSar-
RGD was prepared using a method developed in our laboratory
(15) with minor modifications as follows: [64Cu]Acetate
(64Cu(OAc)2) was prepared by adding 37-111 MBq of 64CuCl2
in 0.1 N HCl into 300 µL of 0.1 N ammonium acetate buffer
(pH 5.0-5.5), followed by mixing and incubating for 15 min
at room temperature. The AmBaSar-RGD (about 2-5 µg in
100 µL 0.1 N ammonium acetate buffer) was added to the above
64Cu(OAc)2 solution. The resulting mixture was incubated at a
temperature of 23-37 °C for 30 min. The 64Cu-AmBaSar-RGD
was determined and purified by semipreparative HPLC. The
radioactive peak containing 64Cu-AmBaSar-RGD was collected
and concentrated by rotary evaporation to remove organic
solvent, and the radioactivity was reconstituted in 500-800 µL
of phosphate buffered saline (PBS) and passed through a 0.22
µm Millipore filter into a sterile dose vial for use in the
experiments below.
EXPERIMENTAL PROCEDURES
General. All reagents and solvents were purchased from
Sigma-Aldrich Chemicals (St. Louis, MO, USA) and used
without further purification unless otherwise stated. N,N-
Diisopropylethylamine (DIPEA), 1-hydroxy-7-azabenzotriazole
(HOAt), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU), and Chelex 100 resin (50-100
mesh) were obtained from the Sigma-Aldrich Chemical Co. The
c(RGDyK) peptide was purchased from Peptides International,
Inc. (Louisville, KY, USA). DOTA was purchased from
Macrocyclics (Dallas, TX, USA). The chelator AmBaSar was
prepared following a method developed in our laboratory (16).
64CuCl2 solution was ordered from University of Wisconsin
(Madison, WI) or Washington University School of Medicine
(St. Louis, MO), which both were produced through the
64Ni(p,n)64Cu nuclear reaction. Water was purified using a
Milli-Q ultrapure water system from Millipore (Milford, MA,
USA), followed by passing through a Chelex 100 resin before
bioconjugation and radiolabeling.
Analytic and semipreparative reversed phase high perfor-
mance liquid chromatography (HPLC) were accomplished on
two Waters 515 HPLC pumps, a Waters 2487 absorbance UV
detector, and a Ludlum Model 2200 radioactivity detector, which
were operated by Waters Empower 2 software. The purification
of AmBaSar, DOTA conjugated c(RGDyK) peptides, and 64Cu-
labeled peptides were performed on a Phenomenex Luna C18
reversed phase column (5 µm, 250 × 10 mm). The flow rate
was 3 mL/min for semipreparative HPLC, with the mobile phase
starting from 98% solvent A (0.1% TFA in water) and 2%
solvent B (0.1% TFA in acetonitrile) (0-2 min) to 60% solvent
A and 40% solvent B at 40 min. The separation was monitored
using a radiodetector and UV at 218 and 254 nm. The analytic
HPLC was performed with the same mobile phase gradient
system and detection, except that a Phenomenex Luna C18
reversed phase analytic column (5 µm, 250 × 4.6 mm) was
used at a flow rate of 1 mL/min. For the in ViVo stability study,
Details of the 64Cu-labeling DOTA-RGD procedure were
reported earlier (23). Briefly, 37-111 MBq of 64CuCl2 in 0.1
N HCl was diluted in 300 µL of 0.1 N ammonium acetate buffer

Improved MicroPET Imaging
Bioconjugate Chem., Vol. 21, No. 8, 2010 1419
(pH 5.5) and added to the DOTA-RGD solution (about 2-5
µg in 100 µL of 0.1 N ammonium acetate buffer). The reaction
mixture was incubated at 45 °C for 45 min. The radiochemical
yield was determined by radio-TLC and HPLC. 64Cu-DOTA-
RGD was then purified by semipreparative HPLC, and the
radioactive peak containing the desired product was collected.
After removal of the solvent by rotary evaporation, the residue
was reconstituted in 500-800 µL of PBS and passed through
a 0.22 µm Millipore filter into a sterile multidose vial for use
in the following experiments.
Determination of log P Value. The partition coefficient value
was expressed as log P. log P of 64Cu-AmBaSar-RGD was
determined by measuring the distribution of radioactivity in
1-octanol and PBS. A 5 µL sample of 64Cu-AmBaSar-RGD in
PBS was added to a vial containing 1 mL each of 1-octanol
and PBS. After vigorously vortexing for 10 min, the vial was
centrifuged for 5 min to ensure the complete separation of layers.
Then, 3 × 10 µL of each layer was pipetted into other test tubes,
and radioactivity was measured using a gamma counter (Perkin-
Elmer Packard Cobra). The measurement was repeated three
times, and log P values were calculated according to a known
formula.
In Vitro Stability Assay. The in Vitro stability of 64Cu-
AmBaSar-RGD and 64Cu-DOTA-RGD was studied at different
time points. Briefly, 3.7 MBq of 64Cu-AmBaSar-RGD and 64Cu-
DOTA-RGD was pipetted into 1 mL of PBS, fetal bovine serum
(FBS), and mouse serum, respectively. After incubation at 37
°C for 3, 18, and 24 h, an aliquot of the mixture was removed
from the PBS solution and the radiochemical purity was
determined with HPLC. For the solution of FBS and mouse
serum, the aliquots were added to 100 µL of PBS with 50%
TFA. After centrifugation, the upper solution was taken and
filtered for HPLC analysis.
Integrin Receptor Binding Assay and Cell Uptake
Study. U87MG human glioblastoma cell line (integrin Rνꢀ3-
positive) was obtained from the American Type Culture
Collection (ATCC, Manassas, VA) and maintained under
standard conditions according to ATCC as follows: the U87MG
glioma cells were grown in Gibco’s Dulbecco’s medium
supplemented with 10% fetal bovine serum (FBS), 100 IU/mL
penicillin, and 100 µg/mL streptomycin (Invitrogen Co, Carls-
bad, CA), at 37 °C in humidified atmosphere containing 5%
CO2. The U87MG glioma cells were grown in culture until
sufficient cells were available.
The in Vitro integrin-binding affinity and specificity of
AmBaSar-RGD and RGD were assessed via competitive cell
binding assays using 125I-echistatin as the integrin Rνꢀ3-specific
radioligand by a minor modification of a method previously
described (26, 27). In detail, U87MG cells were harvested,
washed three times with PBS, and resuspended (2 × 106 cells/
mL) in the binding buffer. Filter multiscreen DV plates (96-
well, pore size, 0.65 µm, Millipore) were seeded with 105 cells
and incubated with 125I-echistatin (0.74 kBq/well) in the presence
of increasing concentrations of different RGD peptide analogues
(0-1,000 nmol/L). The total incubation volume was adjusted
to 200 µL. After the cells were incubated for 2 h at room
temperature, the plates were filtered through a multiscreen
vacuum manifold and washed three times with binding buffer.
The hydrophilic polyvinylidenedifluoride (PVDF) filters were
collected, and the radioactivity was determined using a gamma
counter. The best-fit 50% inhibitory concentration (IC50) values
for U87MG cells were calculated by fitting the data with
nonlinear regression using GraphPad Prism (GraphPad Software,
Inc.). Experiments were performed on triplicate samples.
60 min, and 120 min. The blocking experiment was performed
by incubating U87MG cells with 64Cu-AmBaSar-RGD (37 kBq/
well) in the presence of 2 µg of c(RGDyK). The tumor cells
were then washed three times with chilled PBS and harvested
by 0.1 N NaOH solution containing 0.5% sodium dodecyl
sulfate (SDS). The cell suspensions were collected and measured
in a gamma counter.
Animal Model. Athymic nude mice (about 10-20 weeks
old, with a body weight of 25-35 g) were obtained from Harlan
(Charles River, MA). All animal experiments were performed
according to a protocol approved by the University of Southern
California Institutional Animal Care and Use Committee. The
U87MG human glioma xenograft model was generated by a
subcutaneous injection of 5 × 106 U87MG human glioma cells
into the front flank of athymic nude mice. The tumors were
allowed to grow 3-5 weeks until 200-500 mm3 in volume.
Tumor growth was followed by caliper measurements of the
perpendicular dimensions.
MicroPET Imaging and Blocking Experiment. MicroPET
scans and imaging analysis were performed using a rodent
scanner (microPET R4; Siemens Medical Solutions) as previ-
ously reported (26). About 11.1 MBq of 64Cu-AmBaSar-RGD
or 64Cu-DOTA-RGD was intravenously injected into each mouse
under isoflurane anesthesia. Ten minute static scans were
acquired at 1, 2, 4, and 20 h post-injection. The images were
reconstructed by a 2-dimensional ordered-subsets expectation
maximum (OSEM) algorithm. For each microPET scan, regions
of interest were drawn over the tumor, normal tissue, and major
organs on the decay-corrected whole-body coronal images. The
radioactivity concentration (accumulation) within the tumor,
muscle, liver, and kidneys were obtained from the mean value
within the multiple regions of interest and then converted to %
ID/g. For the receptor blocking experiment, mice bearing
U87MG tumors were scanned (10 min static) at the 2 h time
point after the coinjection of 11.1 MBq of 64Cu-AmBaSar-RGD
or 64Cu-DOTA-RGD with 10 mg/kg c(RGDyK) per mouse.
Biodistribution Studies. The U87MG tumor bearing nude
mice (n ) 3) were injected with 0.37 MBq of 64Cu-AmBaSar-
RGD or 64Cu-DOTA-RGD to evaluate the biodistribution of
these tracers. All mice were sacrificed and dissected at 20 h
after the injection of the tracers. Blood, U87MG tumor, major
organs, and tissues were collected and weighed wet. The
radioactivity in the tissues was measured using a gamma counter.
The results were presented as the percentage injected dose per
gram of tissue (% ID/g). For each mouse, the radioactivity of
the tissue samples was calibrated against a known aliquot of
the injected activity. The mean uptake (% ID/g) for each group
of animals was calculated with standard deviations.
Metabolic Stability. The metabolic stabilities of 64Cu-
AmBaSar-RGD and 64Cu-DOTA-RGD were evaluated in an
athymic nude mouse bearing a U87MG tumor. Sixty minutes
after the intravenous injection of 11.1 MBq of 64Cu-AmBaSar-
RGD or 64Cu-DOTA-RGD, the mouse was sacrificed, and the
relevant organs were harvested. The blood was collected
immediately and centrifuged for 5 min at 14,000 rpm. Then,
50% TFA in 100 µL of PBS was added to the upper serum
solution, followed by mixing and centrifugation for 5 min. The
upper solution was then taken and injected for HPLC analysis.
The liver, kidneys, and tumor were homogenized using a
homogenizer, suspended in 1 mL of PBS buffer, and then
centrifuged for 5 min at 14,000 rpm. For each sample, after the
removal of the supernatant, 50% TFA in 100 µL PBS was added
to the solution, followed by mixing and centrifugation for 5
min. The upper solution was then taken and injected for HPLC
analysis. The eluent was collected with a fraction collector (1.5
min/fraction), and the radioactivity of each fraction was
measured with a gamma counter.
The cell uptake study was performed as described with some
modifications (25). Cells were incubated with 64Cu-AmBaSar-
RGD (37 kBq/well) or 64Cu-DOTA-RGD at 37 °C for 30 min,

1420 Bioconjugate Chem., Vol. 21, No. 8, 2010
Cai et al.
than 97% of 64Cu-AmBaSar-RGD remained intact in the PBS
and FBS, and more than 95% of 64Cu-AmBaSar-RGD remained
intact in mouse serum. We also found that 64Cu-DOTA-RGD
demonstrated similar stability results in Vitro under the above
experimental conditions (data not shown).
In Vitro Cell Binding Affinity and Uptake. We compared
the receptor-binding affinity of AmBaSar-RGD and c(RGDyK)
using a competitive cell-binding assay (Figure 3A). All peptides
inhibited the binding of 125I-echistatin to Rνꢀ3 integrin-positive
U87MG cells in a dose-dependent manner. The IC50 value for
AmBaSar-RGD (53.26 ( 0.51 nmol/L) was comparable to that
of c(RGDyK) (36.55 ( 0.65 nmol/L). These results had shown
that chelator (such as AmBaSar or DOTA) conjugation had a
minimal effect on the receptor-binding avidity (27).
Figure 2. In Vitro stability of 64Cu-AmBaSar-RGD in PBS (pH 7.4),
FBS, and mouse serum for incubation under 37 °C for 3, 18, and 24 h.
The cell uptake study of 64Cu-AmBaSar-RGD or 64Cu-DOTA-
RGD was performed on U87MG tumor cells, and the cell uptake
was expressed as radioactivity (cpm) per 106 cells after decay
correction as shown in Figure 3B. The cell uptake study revealed
that both 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD could
bind to U87MG tumor cells, but relatively low amounts of
activity (only about 0.1-0.4% was internalized). However, this
binding could be efficiently blocked by an excess amount of
cold c(RGDyK) peptide, which demonstrated the binding
specificity of the radiolabeled ligands.
Statistical Analysis. Quantitative data were expressed as the
mean ( SD. Means were compared using one-way ANOVA
and Student’S t test. P values <0.05 were considered statistically
significant.
RESULTS
Chemistry and Radiolabeling. The identity of AmBaSar-
RGD and DOTA-RGD conjugates was confirmed by HPLC and
mass spectrometry as previously reported (15, 23). The Am-
BaSar-RGD conjugate was obtained in about 80% yield after
HPLC purification for both aqueous and organic phase proce-
dures. The 64Cu-labeling (n ) 7) was achieved in more than
95% decay-corrected yield for 64Cu-AmBaSar-RGD with a
radiochemical purity of >99% and 80% radiochemical yield for
64Cu-DOTA-RGD with a radiochemical purity >98%. 64Cu-
AmBaSar-RGD and 64Cu-DOTA-RGD were analyzed and
purified by HPLC. The HPLC retention times of 64Cu-AmBaSar-
RGD and 64Cu-DOTA-RGD were 26.5 and 21.5 min, respec-
tively, under the analytical conditions. For radio-TLC analysis,
the free 64Cu2+ remained at the origin of the TLC plate, while
the Rf values of 64Cu-DOTA-RGD and 64Cu-AmBaSar-RGD
were about 0.8-1.0. The specific activity of 64Cu-DOTA-RGD
and 64Cu-AmBaSar-RGD was estimated to be about 10.1-22.2
GBq/µmol. Both tracers were used immediately after the
formulation.
log P Value and in Vitro Stability. The octanol/water
partition coefficients (log P) for 64Cu-AmBaSar-RGD and 64Cu-
DOTA-RGD were -2.44 ( 0.12 and -2.80 ( 0.04, respectively
(23), which demonstrated that both tracers are rather hydrophilic.
The in Vitro stability of 64Cu-AmBaSar-RGD was also studied
in PBS (pH 7.4), FBS, and mouse serum for different time
intervals (3, 18, and 24 h) at physiological temperature of 37
°C. The stability was presented as the percentage of intact 64Cu-
AmBaSar-RGD on the basis of the HPLC analysis, and the
results are shown in Figure 2. After 24 h of incubation, more
MicroPET Imaging. The tumor-targeting efficacy and bio-
distribution patterns of 64Cu-AmBaSar-RGD and 64Cu-DOTA-
RGD were evaluated in nude mice bearing U87MG human
glioma xenograft tumors (n ) 3) at multiple time points (1, 2,
4, and 20 h) with static microPET scans. Representative decay-
corrected coronal images at different time points were shown
in Figure 4A. The U87MG tumors were clearly visualized with
high tumor-to-background contrast for both tracers. The uptake
of 64Cu-AmBaSar-RGD in U87MG tumors was 2.04 ( 0.14,
1.85 ( 0.16, 1.87 ( 0.11, and 0.97 ( 0.05% ID/g at 1, 2, 4,
and 20 h p.i., respectively. 64Cu-DOTA-RGD demonstrated
similar tumor uptake with the value of 2.03 ( 0.10, 1.97 (
0.05, 1.88 ( 0.29, and 0.88 ( 0.07% ID/g at the above time
points, respectively (Figure 4B). However, the biodistribution
patterns of 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD were
significantly different, especially in the kidneys and liver. The
renal uptake of 64Cu-AmBaSar-RGD were 2.83 ( 0.77, 2.68
( 0.55, 2.49 ( 0.50, and 1.43 ( 0.35% ID/g at 1, 2, 4, and
20 h p.i., respectively, while the corresponding uptake for 64Cu-
DOTA-RGD was 1.52 ( 0.46, 1.33 ( 0.23, 1.55 ( 0.61, and
1.58 ( 0.13% ID/g, respectively. This was lower than that of
64Cu-AmBaSar-RGD from 1 to 4 h, and reached comparable
levels at the 20 h time point. The liver uptake for 64Cu-
AmBaSar-RGD was 0.89 ( 0.13, 0.76 ( 0.13, 0.75 ( 0.14,
and 0.64 ( 0.06% ID/g at 1, 2, 4, and 20 h p.i.. The
corresponding uptake value for 64Cu-DOTA-RGD was 2.28 (
Figure 3. In Vitro cell integrin receptor binding assay and cell uptake study. (A) In Vitro inhibition of 125I-echistatin binding to integrin on U87MG
cells by c(RGDyK), AmBaSar-RGD, and DOTA-RGD; (B) U87MG cell uptake of 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD at 0.5, 1, and 2 h
in the absence/presence of an excess amount of c(RGDyK) peptide.

Improved MicroPET Imaging
Bioconjugate Chem., Vol. 21, No. 8, 2010 1421
Figure 4. MicroPET study of U87MG tumor-bearing mice. (A) Coronal images of nude mice bearing the U87MG tumor at 1, 2, 4, and 20 h p.i.
of 64Cu-AmbaSar-RGD and 64Cu-DOTA-RGD; (B) time activity curves of 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD in the U87MG tumor, liver,
and kidneys (n ) 3). Tumors are indicated by arrows.
1.08, 2.31 ( 1.34, 2.15 ( 0.90, and 2.52 ( 1.43% ID/g,
respectively, which were significantly higher than those of 64Cu-
AmBaSar-RGD at all time points.
organs, which is shown in Figure 6B. For 64Cu-AmBaSar-RGD,
the ratio of tumor uptake to muscle, heart, lung, liver, and kidney
uptake was 14.20 ( 3.84, 7.33 ( 1.55, 3.34 ( 0.70, 1.18 (
0.05, and 0.43 ( 0.06, respectively, while the corresponding
values for 64Cu-DOTA-RGD were 5.97 ( 2.25, 1.78 ( 0.37,
1.27 ( 0.37, 0.33 ( 0.09, and 0.66 ( 0.04, respectively.
Blocking Experiment. The integrin Rνꢀ3 receptor specificity
of 64Cu-AmBaSar-RGD was confirmed by a blocking experi-
ment where the tracers were coinjected with the c(RGDyK)
peptide (10 mg/kg). As can be seen from Figure 5, the U87MG
tumor uptake in the presence of nonradiolabeled c(RGDyK)
peptide (0.09 ( 0.03% ID/g) is significantly lower than that
without c(RGDyK) peptide blocking (1.85 ( 0.16% ID/g) (P
< 0.05) at 2 h p.i. The uptake of 64Cu-AmBaSar-RGD in other
organs (heart, intestine, kidneys, lungs, liver, and spleen) was
also significantly decreased, which correlates well with previous
references (23). Similarly, the integrin Rνꢀ3 specificity of64Cu-
DOTA-RGD was confirmed by blocking experiments (Figure
5).
Biodistribution Studies. To validate the accuracy of small
animal PET quantification, we also performed a biodistribution
experiment by using the direct tissue sampling technique. The
data shown as the percentage administered activity (injected
dose) per gram of tissue (% ID/g) in Figure 6A. Both 64Cu-
AmBaSar-RGD and 64Cu-DOTA-RGD mainly accumulated in
the kidneys, liver, stomach, intestine, and tumor. After 20 h
p.i., the kidneys and liver uptake reached 1.51 ( 0.27 and 0.55
( 0.06% ID/g for 64Cu-AmBaSar-RGD and 0.98 ( 0.40 and
2.16 ( 0.85% ID/g for 64Cu-DOTA-RGD, respectively. This
difference was consistent with the excretion pattern from the
microPET imaging results. On the basis of the biodistribution
results, we also calculated the contrast of the tumor to main
Metabolic Stability of 64Cu-AmBaSar-RGD and 64Cu-
DOTA-RGD. The metabolic stability of 64Cu-AmBaSar-RGD
and 64Cu-DOTA-RGD were determined in mouse blood and in
liver, kidney, and tumor homogenates at 1 h after the intravenous
injection of the radiotracer into a U87MG tumor-bearing mouse
according to the literature procedures (28, 29). The radioactivity
of each sample was analyzed by HPLC, and the representative
radio-HPLC profiles were shown in Figure 7. There was only
one major radiolabeled metabolite with a retention time between
3.0 to 6.0 min. It has been well demonstrated that the cylic-
RGDyk peptide is stable in ViVo. Therefore, it is highly possible
that this solvent front peak corresponds to free Cu. Nonetheless,
its identity needs to be confirmed in our future studies. The
amount of intact tracer in the blood, tumor, liver, and kidneys
were approximately 88%, 95%, 98%, and 98% for 64Cu-
AmBaSar-RGD and 38%, 87%, 34%, and 74% for 64Cu-DOTA-
RGD at 1 h post-injection, respectively.
DISCUSSION
The established BFC DOTA is the most widely used Cu
chelation ligand for molecular imaging and radiotherapy (4, 7, 30).
DOTA has been conjugated to various macromolecules includ-

1422 Bioconjugate Chem., Vol. 21, No. 8, 2010
Cai et al.
Figure 5. MicroPET imaging of 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD on the U87MG xenograft mouse model at 2 h p.i. with/without a
blocking dose of c(RGDyK) peptide: (A) microPET coronal images. (B) Quantitative analyses of microPET imaging of the U87MG tumor, muscle,
liver, and kidneys (n ) 3). Arrows indicate the tumor positions.
Figure 6. (A) Biodistribution data for 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD in mice bearing U87MG glioma xenografts (mean ( SD, n )
3) at 20 h p.i.; (B) the ratios of tumor to main organ uptake of 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD.
ing proteins (31), antibodies (32), peptides (30), as well as
nanoparticles to track their distributions in ViVo (33, 34).
However, under physiological conditions, Cu may be removed
from the Cu-DOTA complex and transferred to copper-binding
proteins. The significant loss of Cu from the conjugate could
lead to high uptake in the liver and fail to reflect the real
distribution of the macromolecules. Therefore, the limited
stability of the copper chelate in ViVo has hindered its further
applications (5). For example, although we have demonstrated
that 64Cu-DOTA-RGD could be a suitable tracer for PET
imaging of integrin Rνꢀ3 expression in breast cancer (23),
unfavorable hepatobiliary excretion was also observed due to
64Cu2+ dissociation from the DOTA-RGD conjugate in ViVo.
Recently, we have developed a new cage-like BFC AmBaSar
for 64Cu2+ labeling and demonstrated that this 64Cu-complexing
moiety is a superior ligand for imaging application and targeted
radiotherapy due to its improved Cu-chelation stability compared
with that of DOTA (16). We also reported the synthesis of a
new PET tracer 64Cu-AmBaSar-RGD that could be possibly used
for imaging integrin Rνꢀ3 expression (15). The objective of this
study is to evaluate the 64Cu2+ labeling and imaging ability of
AmBaSar-RGD conjugates and compare the results with the
established chelator DOTA using the DOTA-RGD peptide as
the standard compound. These studies have demonstrated that
64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD have comparable
in Vitro stability, lipophilicity, and tumor uptake. However, 64Cu-
AmBaSar-RGD did demonstrate improved in ViVo stability and
biodistribution pattern compared with those of 64Cu-DOTA-
RGD.
From the chemistry point of view, there were no obvious
differences between the AmBaSar and DOTA conjugation to
the c(RGDyK) peptide. Both chelators used a carboxylate group
to directly conjugate with the lysine amine in the c(RGDyK)
peptide in high yield. However, for radiochemistry, the 64Cu2+
labeling condition of AmBaSar-RGD was more favorable, and
the 64Cu-AmBaSar-RGD could be obtained with higher radio-
chemical yield (g95%) and purity (g99%) under mild condi-
tions (pH 5.0-5.5, 23-37 °C) in less than 30 min, compared

Improved MicroPET Imaging
Bioconjugate Chem., Vol. 21, No. 8, 2010 1423
Figure 7. Metabolic stability of 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD in the mouse blood sample and in the liver, kidney, and U87MG
tumor homogenates at 1 h post-injection. The HPLC profiles of pure 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD (standard) are also shown.
with 90% radiochemical yield for 64Cu-DOTA-RGD after
incubation at 45 °C for 45 min.
a more stable Cu complex in ViVo than the established chelator
DOTA through direct comparison of their metabolic stability.
We would also like to point out that the rapid renal clearance
of 64Cu-AmBaSar-RGD, as opposed to the mixed renal and
hepatic clearance of 64Cu-DOTA-RGD is preferred as it will
provide better somatic contrast from the imaging perspective
and potentially reduce radiation exposure.
Under in Vitro conditions, both 64Cu-AmBaSar-RGD and
64Cu-DOTA-RGD were very stable in PBS, FBS, and mouse
serum solutions at physiological temperature. The log P value
is a very useful parameter that can be used to understand the
behavior of drug molecules and predict the distribution of a
drug compound in a biological system in combination with other
parameters (35). The log P values of 64Cu-AmBaSar-RGD and
64Cu-DOTA-RGD indicated that both tracers are rather hydro-
philic and that they should show rapid blood clearance and
preferential renal excretion if the Cu-chelation is stable in ViVo.
However, compared with 64Cu-AmBaSar-RGD, 64Cu-DOTA-
RGD had significantly higher liver uptake (∼2-3 times) on
the basis of the microPET imaging and biodistrbution studies,
which indicated that 64Cu-DOTA-RGD is less stable than 64Cu-
AmBaSar-RGD in ViVo. In microPET studies, the U87MG
tumor uptake of 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD
were comparable at selected time points, and the binding
specificity was proven by the blocking experiment. The bio-
distribution study was consistent with the microPET studies.
Similar tumor uptake of both tracers may be attributed to their
comparable hydrophilicity and the minimal impact of chelators
on the integrin binding affinity of c(RGDyK) peptides (36).
64Cu-AmBaSar-RGD cleared rapidly from the blood and
predominantly through the kidneys, and 64Cu-DOTA-RGD
mainly cleared through both kidneys and liver. This difference
might be because the Cu-chelation in 64Cu-AmBaSar-RGD is
more stable in ViVo compared with that in 64Cu-DOTA-RGD,
which would result in reduced nonspecific binding. To further
confirm this statement, we also studied the metabolic stability
of 64Cu-AmBaSar-RGD and 64Cu-DOTA-RGD in blood, liver,
kidneys, and tumors in nude mice bearing U87MG glioma
xenografts after 1 h p.i. Our study clearly demonstrated that
the amount of intact 64Cu-AmBaSar-RGD was much higher than
that of 64Cu-DOTA-RGD in blood, tumor, liver, and kidneys.
To the best of our knowledge, this is the first study that directly
demonstrates that a Sar-type chelator, such as AmBaSar, forms
CONCLUSIONS
64Cu-AmBaSar-RGD was obtained in high yield under mild
conditions for PET imaging of integrin Rνꢀ3 expression. This
tracer exhibited good tumor-targeting efficacy, excellent meta-
bolic stability, as well as favorable in ViVo pharmacokinetics.
In Vitro and in ViVo evaluation of the 64Cu-AmBaSar-RGD has
demonstrated its improved in ViVo Cu-chelation stability com-
pared with that of the established tracer 64Cu-DOTA-RGD. The
AmBaSar chelator will also have a general application for 64Cu
labeling of various bioactive molecules in high radiochemical
yield and high in ViVo Cu-chelation stability for future PET
applications.
ACKNOWLEDGMENT
This work was supported partially by research grant DE-
SC0002353 from the Department of Energy, the USC Depart-
ment of Radiology, and the Provost’s Biomedical Imaging
Science Initiative.
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