Adv a nce s in Molecul a r Imaging, 2011, 1, 1-11
doi:10.4236/ami.2011.11001 Published O nlin e April 2011 ( mi)
Copyright © 2011 SciRes. AMI
Evaluation of 64Cu-DOTA- and 64Cu-CBTE2A-Galectin-3
Peptide as a PET Radiotracer for Breast Carcinoma
Se nthil R. Kumar1,2, S usan L. De uts ch er1,2
1Depa r tment of Biochemis tr y, University of Missou ri -Columbia School of Medicine, Columbia, MO 65211, USA
2Research Divi sion , Harry S. Truman Vetera n s Hospital, Columbia MO 65201, USA
E-mail: kumars@misso u ri. e d u, deutsc hers@ missouri.e d u
Received March 3, 2011; Re vised Apr il 3, 2011; Ac c ept ed April 30, 2011
A bstract
Galectin-3 (Gal-3) is a β-galactosidase binding protein that modulates various cellular processes including
cell adhesion, and metastasis. We evaluated the tumor targeting and imaging properties of a galectin-3 bind-
ing peptide originally selected from bacteriophage display, in a mouse model of human breast carcinoma
expressing galectin-3. A galectin-3 binding peptide, ANTPCGPYTHDCPVKR, was synthesized with a
Gly-Ser-Gly (GSG) spacer and 1,4,7,10, tetraazacyclododecane-N,N’,N’’,N’’’ -tetracetic acid (DOTA) or
4,11-bis(carboxymethyl)-1,4,8,11 tetrazabicyclo[6.6.2]hexadecane 4,11-diacetic acid (CB-TE2A), and radi-
olabeled with 64Cu. The synthesized peptides 64Cu-DO3A-(GSG)-ANTPCGPYTHDCPVKR (64Cu-DO3A-
pep) and 64Cu-CB-TE2A-(GSG)-AN TPCGPYT HDCPVKR( 64Cu-CB-TE2A-pep) demonstr at ed an IC50 va lue
of (97 ± 6.7) nM and (130 ± 10.2) nM, r esp ect i vel y, t o cultured MDA-MB-435 breast carcinoma cells in vi-
tro in a competitive displacement binding study. T he t u mor t is su e u pt a ke i n S C ID mice b ea r i ng M DA-MB-
435 tumors was (1. 2 ± 0. 18 ) %ID/g (64Cu-DO3A-pep) a nd (0.85 ± 0.0.9) %ID/g (64Cu-CB-TE2A-pep) a t 30
min, respectively. While liver retention was moderate with both radiolabeled peptides the kidney retention
was observed to be high. Radiation dose delivered to the tumor was estimated to be 42 mGy/mCi and 129
mGy/mCi with CB-TE2A and DO3A peptides, respectively. Imaging studies demonstrated tumor uptake
wit h both 64Cu-DO3A- and 64Cu-CB-TE2 A-(GSG)-ANTPCGPYTHDCPVKR after 2 h post injection. These
studies suggest that gal-3 binding peptide could be developed into a PET imaging agent for galec-
tin-3-expr essing breast tumors.
Keywords: Galectin-3, Breast Cancer, PET Imaging, Peptide, Radi otr acer
1. Introduction
Galectin-3 (Gal-3) is a 31 kDa protein, which has high
affinity for β-galactosides, and possesses a highly con-
served carbohydrate recognition domain (CRD) [1].
Gal-3 is found in the cytoplasm, but depending on cell
types and proliferative states, it can also be detected on
the cell surface [2], within the nucleus [3], and in the
extracellular compartment [4,5]. Through its interaction
with specific ligands, gal-3 is involved in multiple bio-
logical pr ocesses such a s adhesion, apoptosis, inflamma-
tion, and metastasis [1,6,7]. Gal-3 has been found to be
upregulated in a variety of human cancers including
breast carcinoma [8]. Metastatic breast carcinoma cells
express higher levels of gal-3 and have significantly in-
creased adhesion to monolayers of endothelial cells
compar ed with their n on-m etastatic counterparts [4].
One of the key steps in the initiation of metastasis is
surface adhesion between tumor cells and endothelial
cells [9,10]. Previous studies from our laboratory dem-
onstrated that gal-3 is involved in heterotypic (carcino-
ma-endothelial cells) and homotypic (carcinoma-car-
cinoma) cellular adhesion via interactions with the tu-
mor-specific Thomsen-Friedenreich (TF) mucin-type
disaccharide, Gal 1-3GalNAc, expressed on most human
adenocarcinomas [11]. Further, TF acts as a ligand for
gal-3, and facilitates the mobilization of gal-3 to the sur-
face of endothelial cells [11,12]. Thus, gal-3-carbohydrate
int eraction s ar e crucial in can cer cell adhesion to the en-
dothelium. Carbohydrate-based inhibitors that block
gal-3-TF interactions [13,14], via binding to the CRD of
gal-3 have previously been reported. However, such in-
hibitors bound other galectin family members due to
their conserved carbohydrate binding residues which
Copyright © 2011 SciRes. AMI
undermine specificity [13]. We hypothesized that short
synthetic peptides which bind to unique regions of the
CRD of gal-3 with high specificity and affinity, may
represent an alternative approach for targeting gal-3 on
carcinoma cells.
Further, gal-3 molecules cluster at sites of cancer cell
in tera ction and could be a potential tar get for cancer im-
aging and/or therapy [15]. Previously, we reported the
selection of a gal-3 binding peptide ANTPCGPYTH-
DCPVKR (G3-C12) by combinatorial bacteriophage
(phage) display techniques [16]. We also demonstrated
successful imaging of human breast tumors expressing
gal-3 wi t h 111In-radiolabeled 1,4,7, 10-t etrazac yclododecane
-1,4,7,10-tetraacetic acid (DO3A)-(GSG)-G3-C12 [17] by
single photon emission computed tomography (SPECT)
in a mouse model. However, alternate imaging modali-
ties have emerged in clinical medicine to generate high
resolution images non-invasively. One such modality is
positron emission tomography (PET), which utilizes the
delivery of a pharmacologically significant molecule
containing a positron-emitting radionuclide (e.g. 64Cu
(half-life [t1/2] = 12.7 h) to a tissue or organ of interest
The bifunctional chelator DOTA has been used for
chelating 64Cu, however, its ability to chelate many +2
and +3 metal ions, an d its re lat ive instability comp ared to
other chelators such as CB-TE2A, have made it less fa-
vourable [19,20]. CB-TE2A has been reported to form
very stable complexes with 64Cu with less tranchelation
of the metal in vivo [21]. In the present study, we com-
pared both 64 Cu-DO3 A-GSG-G3 -C12(64Cu-DO3 A-pep-
tide) and 64Cu-CB-TE2A-GSG-G3-C12 (64Cu-CB-TE2A-
peptide) to evaluate the biodistrubtion and PET imaging
properties of the peptides in a mouse model of human
br east carcin oma .
2. Materials and Methods
2.1. Chemicals and Reagents
Amino acids and resin were purchased from Advanced
Chem Tech (Louisville, KY). The bifunctional chelator
CB-TE2A was synthesized as previousl y described [22].
DOTA was purchased from Macrocyclics (Richardson,
TX). Copper-64 was purchased from Nuclear Reactor
Laborator y, University of Wisconsin (Madison, WI). All
other reagents in this study were obtained from Fisher
Scientific Company (Pittsburgh, PA) unless otherwise
specifi ed.
2.2. C e ll Lines
Huma n br east carcin oma cel l l in e MDA-MB-4 35 and the
normal mammary epithelial cell line 184A-1 were ob-
tained from American Type Tissue Culture. The MDA-
MB-435 cells were maintained as monolayer cultures in
RPMI-1640 medium supplemented with 10% FBS, sodium
pyruvate, non-essential amino acids, and L-glutamine. The
184A-1 cells were grown in human mammary epithelial
cell media (HuMEC) media supplemented with bovine
pituitary extract. Cell cultures were maintained at 37˚C
in a 5% CO2 humidif ied incubator.
2. 3 . Peptid e Synthesis
Solid phase synthesis of DO3A- and CB-TE2A-G3-C12
peptide (ANTPCGPYTHDCPVKR) with a Gly-Ser-Gly
(GSG) amino acid spacer between the chelator and ami-
no terminus of the peptide was carried out using an Ad-
vanced ChemTech 396 multiple peptide synthesizer
(Advanced Chem Tech, Louisville, KY). The chelator
CB-TE2A was coupled to the amino terminus of the li-
near GSG-G3 -C12 through an intermediate preparation
of the activated mixed anhydride by use of DIC/DIEA/
DMF as described earlier [22]. Reverse pha se h igh pres-
sure liquid chromatography (RP-HPLC) analysis of syn-
thesized peptides was performed using a C18 column
(218TP54; Vydac, Hesparia, CA). The mobile phase
consisted of a linear gradient system, with solvent A
corresponding to 100% water with 0.1% trifluoroacetic
acid (TFA) and solvent B corresponding to 100% aceto-
nitrile with 0.1% TFA. Identities of the peptides were
confirmed by electrospray ionization mass spectrometry
(ESI -MS) (Mass Consortium Corporation, San Diego,
2.4. Radiol abeling of DO3A- and
CB-TE2A-(GSG)-G3-C12 Pept ide
Radiolabeling CB-TE2A-(GSG)-G3 -C12 peptide with
64Cu was performed as follows. CB-TE2A-(GSG)-G3-
C12 (30 μg) was radiolabeled with ~ 30.0 MBq (810 μCi)
64Cu in ammonium acetate (0.4 M) pH 7.0, at 80˚C for
60 min. The reaction buffer was purged extensively with
nitrogen prior to radiolabeling in order to minimize pep-
tide oxidation, and contained Tris-(2-carboxyet hyl) phos-
phine hydr ochloride (TCEP) in order to prevent disulfide
bond formation between cysteines. The resulting radi-
olabeled peptide conjugates were peak purified using a
Phenomenex (Torrence, CA) Jupiter 5u C18 300 Å 250 ×
4.6 mm columns and RP-HPLC (10% - 95% acetoni-
trile/0.1% TFA) for 30 min in order to separate the radi-
ol abel e d peptide from their nonr adi olabe led counterparts.
For concentrating, the peak-purified peptides were per-
colated through Empore high efficiency (C18) extraction
disk cartridges (St. Paul, MN), and eluted with 400 μl of
an 8:2 ethanol/sterile saline solution. The ethanol was
evap ora t ed un der a str eam of ni tr ogen and wa s diluted to
Copyright © 2011 SciRes. AMI
the appropriate volume with sterile saline. Radiochemi-
cal yields for 64 Cu-DO3 A-peptide and 64Cu-CB-TE2A-
peptide averaged 45% and 40%, respectively. Radio-
chemical purity of both 64 Cu-la beled peptides was found
t o be ≥ 98% pure.
For non-radioactive Cu-labeling, 0.5 mg samples of
DO3A- or CB-TE2A-peptide were dissolved in 500 μl of
ammonium acetate buffer (as described previously for
DO3A and CB-TE2A)/0.8 mM copper(II) sulfate-pen-
tahydrate solution. The solutions were heated at identical
conditions as mentioned above for radiolabeling, and
allowed to cool to room temperature. Native copper
(natCu)-peptides were then peak purified by RP-HPLC
and determined to be ≥ 97% pure. ESI-MS was per-
formed to check the integri t y of the peptides.
2.5. C e ll Binding Assay
Approximately 1.0 × 106 of MDA-MB-435 or 184A-1
cells/tube were incubated at 37˚C for different time in-
tervals (10, 30, 45, 60, 90 and 120 min) with 1 × 105 cpm
64Cu-DO3A- or 64Cu-CB-TE2A-peptide in 0.3 mL of cell
binding media (RPMI 1640 with 25 mM HEPES, pH 7.4,
0. 2% BSA, 3 mM 1,10-phenathroline). At different time
points, the cells were pelleted by centrifugation and
washed twice with ice-cold 0.01 M PBS, pH 7.4, 0.2%
BSA. After removing the supernatant by centrifugation,
the radioactivity associated with the cells was quantitated
in a Wallac γ counter (PerkinElmer Life and Analytical
Sciences Inc., Waltham, MA). Cell binding ability was
reported as total radioactivity (cpm) that was bound to
the cell s.
2.6. In Vitr o C ompe tit ive Ce ll Binding Affinit y
and Serum Stability Studies
A competitive displacement binding assay was used to
det er min e th e fifty-percent inhibitory concentration (IC50)
for radiolabeled G3-C12 peptide using natCu-DO3A- or
natCu-CB-TE2A-peptide as the displacement ligand. Ap-
proximately, 2.0 × 106 MDA-MB-435 cells/tube were
suspended in cell binding media along with 2.5 × 104
cpm of each 64Cu-DO3A or 64Cu-CB-TE2 A-peptide and
a range of concentrations (10-5 - 10-12 M) of respective
non-radiolabeled counterparts and incubated at 37˚C for
60 min . The supernatant was removed after pell etin g the
cells by centrifugation. The cell pellet was further
washed twice with 0.5 mL of ice-cold binding buffer.
Cell-ass ociated rad ioactivity w as meas ure d in a γ counter
and the binding affinity was determined using Grafit
software (Er itha cu s Software Limited, Sur rey, UK) .
In vitro serum stability of the radiolabeled peptide
conjugates was tested by incubating 30.0 MBq (810 μCi)
of 64Cu-DO3A- or 64Cu-CB-TE2A-peptide in 0.3 mL of
mouse serum at 37˚C for 0.5, 1, 2, 4 and 24 h, respec-
tively. At various time points, 30 μL were removed and
the proteins precipitated with 30 μL acetonitrile. The
samples were centrifuged at 12,500 rpm for 5 min and
the cleared lysate (~ 25 μl aliquots) was analyzed by
RP-HPLC with a 0% - 95% gradient acetonitrile in 30
min to assess the integrity of the radioconjugates.
2. 7 . Phar macokineti c S t ud ies i n Mic e Beari ng
Human Br ea st Tumors
All animal studies were conducted in accordance with
hi gh est stan dard s of car e accor ding to the National Insti-
tute of Health guide for care and use of laboratory ani-
mal s an d th e policy an d procedur e for animal r esearch at
the Harry S. Truman Veterans Hospital. Female 4 - 6
week old SCID (ICR-SCID) mice were obtained from
Taconic (Hudson, NY). The mice were implanted sub-
cutaneously in the right shoulder with 1 × 107 MDA-
MB-435 human breast carcinoma cells. Fully grown tu-
mors ranged from 0.28 - 0.85 g (3 - 4 wk after inocula-
tion). Each mouse (three mice per time point) was in-
jected in the tail vein with 0.185 MBq (5 μCi) of
64Cu-DO3A- or 64Cu-CB-TE2A-peptide in 100 μl sal ine.
The mice were sacrificed by cervical dislocation and
their tissues and organs excised at different time points
(30 min, 1, 2, 4, and 24 h) post injection (p.i.). The ex-
cised tissues were weighed, and the tissue activity was
mea sur ed in a γ counter. Th e per cent injected dose (%ID)
and percent injected dose per gram (%ID/g) were deter-
mined for each tissue. Whole blood %ID and %ID/g
wer e determin ed assum ing th e blood accoun ted for 6.5 %
of the body w e igh t of the mouse. Tumor blocking studies
was performed by administering natCu-DO3A- or natCu-
CB-TE2A-peptide (120 μg) in MDA-MB-435 tumor
mice (n = 3) 20 min before administering respective ra-
diolabeled count erparts. After 2 h, a biodistribution study
were performed as describe above and %ID/g was de-
termined for ea ch tissu e.
Inhibition experiments with bovine serum albumin
fragments (BSA-f) were performed to reduce the kidney
retention of the radiolabeled peptides during biodistribu-
tion studies [23]. Briefly, 3.0 g of BSA in 50 mM am-
monium car bona t e (15 mL) was subjected to trypsin (335
mg) digestion at 37˚C for 24 h. The trypsin-digested
sample was filtered using 50 kDa Centripep (YM-50)
centrifugal filters (Millipore, Billerica, MA). The filtrate
containing albumin fragments < 50 kDa, was further
fractionated using an YM-3 filter (3 kDa cutoff) which
yielded albumin fragments < 3 kDa (filtrate), and frag-
ments with molecular weight range of 3 - 50 kDa (resi-
due). The 3 - 50 kDa albumi n frag me nt(s) (200 g) sample
in normal saline (100 μL) was pre-injected into the
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MDA-MB-435 tumor bearing mice (n = 3) via the tail
vein. Aft er 5 min, 0. 185 MBq (5 μCi ) of 64Cu-DO3A- or
64Cu-CB-TE2A-peptide was administered into the mice.
The mice were sacr ificed after 2 h, an d th e k idney uptake
of t he radiolabeled p epti d es was measured in a γ-counter.
Control experiments (n = 3) were run in parallel with
pre-injection of normal saline instead of albumin frag-
2.8. Do s i me try S tudies
The biodistribution data was processed with a dosimetry
model to determine doses to all organs and to th e tumor
in the mouse model. The dosimetry model being used is
a derivation on that developed by Hui et al. [24] to pr o-
vide realistic beta dose estimates for organs in mice that
received therapeutic radiopharmaceuticals for high ener-
gy betas. This model is required because the beta range
of candidate radioisotopes such as 64Cu is large relative
t o th e si z e of m an y of th e or g an s. Th i s m ode l ha s al r ea d y
been u sed to ca lcu lat e the n ecessa r y dosi m etr y va lu es for
several isotopes incl uding 64Cu [25].
2.9. MicroPET / MicroCT Stud ies
In vivo microPET/CT of MDA-MB-435 tumor-bearing
mice with 64Cu-DO3A- or 64Cu-CB-peptides were per-
formed. Tissue data imaging analysis was performed on
a MDA-MB-435 tumor bearing mice in a microPET
scanner 2 h after intravenous injection of 12.0 MBq (324
μCi) of the radiolabeled peptides in a small animal PET
scanner (MOSAIC small animal PET unit (Philips,
USA)). This PET scanner has a transverse field of view
(FOV) of 12.8 cm and a gantry diameter of 21 cm, and
oper a tes in a 3-dimensional mode (3D). The mou se t o b e
imaged was laser aligned at the center of the scanner
FOV for imaging. The microPET image reconstruction
was performed with a 3D row-action maximum-likely-
hood algorithm (RAMLA). The microCT was performed
for the purpose of anatomic/molecular data fusion, and
concurrent image reconstruction was performed with a
Fanbeam (Feldkamp) filtered-backprojection algorithm.
Reconstructed DICOM (Digital Imaging and Communi-
cation in Medicine) PET images were imported into
AMIRA 3.1 software (Visage Imaging, Inc. San Diego,
CA) for subsequent image fusion with microCT and 3D
2.10 . Statis tic al Analy sis
Data are expressed as mean ± SD. Mean values were
compared using the Student’s t test. Differences were
considered statistically significant for P ≤ 0.05.
3. R esults a n d Discussi o n
The DO3A- and CB-TE2A-peptides was prepared by
solid-phase peptide synthesis using standard F-moc pro-
cedures [26]. In order to enhance the conformational
freedom, a GSG spacer was intr oduced bet ween th e che-
lators and the amino-termin us of the peptides [24]. natCu-
labeled peptides was prepared by heating aqueous solu-
tions of the peptides with CuSO4.5H2O at 85˚C for 60
min. natCu-labeled peptides were purified and analyzed
by mass spectrometry before cell binding assays. The
calculated and observed molecular weights of the natCu-
peptides were 2405.0 Da and 2405.3 Da (DO3A-peptide)
and 2342.0 and 2342.5 Da (CB-TE2A-peptide), respec-
tively. For radiolabeling, the peptides were labeled with
64Cu in ammonium acetate buffer (pH 7. 0) a t 85˚C for 1
h, and further purified by RP-HPLC in order to separate
the radiolabeled peptides from their non-radiolabeled
counterparts. 64Cu-DO3A- and 64Cu-CB-TE2A -peptide
eluted with a retention time (tr) of 13. 2 min and 13.8 min,
resp ectively, a minute a ft er the el ut ion of their res p ective
non-radiola beled coun ter parts. Th e rad iolabel ed pept ides
were obtained at a purity of ≥ 98%.
Specific binding of radiolabeled peptides was demon-
stra ted with MDA-MB-435 cells. Minimal or no binding
was o bs er ve d wit h 184A-1 con t r ol ce l ls . The time course
experiments (Figure 1) revealed the association of radi-
olabeled peptide with the MDA-MB-435 cells which
increased gradually and equilibrium was reached at 1 h,
beyond which no further increase in cell associated ra-
dioa ct i vi t y wa s o bserv ed . T ot al cel l bi n din g c apacity was
~1% compared to the initial total radioactivity added to
breast carcinoma cells. MDA-MB-435 human breast
carcinoma cells were originally derived from a pleural
effusion of a woman with metastatic ductal breast carci-
n oma [2 7]. However, there has been speculation that the
original MDA-MB-435 cancer cells were lost early after
their establishment and found to be identical with the
M-14 mel anoma cell lin e [28]. On the contrary, a recent
report revealed that both MDA-MB-435 and M-14 are of
MDA-MB-435 breast cancer origin [29]. Despite these
specu lat ion s, th e MDA-MB-435 cell lin e has been wide-
ly used as a breast carcinoma model and remains one of
the mo s t re liable in vivo m odel s of human breast cancer.
Competitive binding displacement assays in MDA-
MB-435 cells were performed using various concentra-
tions (10-5 to 10-12 M) of natCu-DO3A- or natCu-CB-
TE2A-pepti de a s the disp lacem en t r adi oli gan d. Decrease
in bound radioactivity to the cultured MDA-MB-435
cells was observed with increasing concentration of re-
spective non-radiolabeled peptide (Figure 2). Data anal-
ysis indicated that the IC50 value for the radiolabeled
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Figure 1. The gal-3 binding properties of the radiolabeled
64Cu-DO3A-peptide and 64 Cu -CB-TE2A-peptide. Approxi-
mately, 1.0 × 106 c ells/tube were incubated at 37˚C for dif-
ferent time intervals with 1.0 × 105 cpm radioligand. While
the radiolabeled peptide demonstrated binding to human
MDA-MB-435 breast carcinoma cells, minimal binding was
observed with normal mammary epithelial (184A1) cells.
peptides was (97 ± 6.7) nM (DO3A-peptide) and (130 ±
10.0) nM (CB-TE2A-peptide), respectively.
This affinity was found to be slightly higher than what
has been reported previously for 111In-DO3A-( GSG) -
G3-C12 ((200.0 ± 6.7) nM) [17]. The affinity values
were also in keeping with that of G3-C12 (88 ± 23.0 nM )
toward recombinant gal-3 protein, as determined by flu-
orescent titration experiments [16].
The metabolic stability of the 64Cu-la beled peptides in
serum was analyzed at different time points at 37˚C by
RP-HPLC. Both the peptides were stable in serum for
periods up to 4 h. However, at 24 h an additional peak
was ob s e rved wi th both radiolabel e d p e ptides prior t o t he
original radiolabeled peptide peak suggesting the degra-
dation of peptides during prolonged serum incubation. A
representative RP-HPLC profile of 64Cu-CB-TE2A-
peptide is shown in Figure 3. A similar profile was ob-
served with 64Cu-DO3A-peptide.
Detailed in vivo studies in SCID mice bearing human
MDA-MB-435 tumor xenografts at different time inter-
vals indicated 64Cu-DO3A-peptide tumor accumulation
of (1.2 ± 0.18) %ID/g, (0.7 ± 0.16) %ID/g, (0.61 ±
0.04) %ID/g, and (0.59 ± 0.03) %ID/g; and
CB-TE2A-peptide, (0.85 ± 0.62) %ID/g, (0.72 ±
0.17) %ID/g, (0.68 ± 0.07) %ID/g and (0.19 ±
0.03) %ID/g in tumor tissue at 30 min, 1 h, 2 h, and 4 h,
respectively (Tables 1 and 2). For tumor blocking stu-
dies, natCu-DO3A- or natCu-CB-TE2A-peptide (120 μg)
was administered in MDA-MB-435 tumor mice (n = 3)
20 min before administering respective radiolabeled
Figure 2. Competitive binding assays using MDA-MB-435
cells. Displacement of (a) 64 Cu -DO3A-peptide and (b) 64Cu-
CB -TE2A-peptide by respective natCu-peptide counterpart
is shown. MDA-MB-435 cells (2 × 106/tube) were incubated
with 2.5 × 104 cpm radioligand and increasing concentra-
tions of the natCu-peptide. Each data point represents the
mean ± SD of three replicates. IC50 value obtained was (97
± 6.7) nM (64 C u-CB-TE2A-peptde), re spec tively.
counterparts. This approach reduced ~ 43% and ~ 47%
of tumor uptake of the radiolabeled peptides (Tables 1
and 2) compared to mice injected with radioactive pep-
tide alone (P = 0.03). Overall, the radiolabeled peptide
conjugate showed fast whole body clearance from the
mouse. The observed partial blocking upon injection of
the non-radiolabeled peptide could be due to faster
Copyright © 2011 SciRes. AMI
(a) (b)
Figure 3. The serum stability of 64Cu-CB-TE2A-peptide at 0 and 24 h. (a) Elution of 64Cu-CB-TE2A-
peptide at 0 t i me point. (b) At 24 h, an additional peptide peak (arrow) was observed prior to the orig-
inal radiolabeled peptide suggesting the degradation of peptide during prolonged serum incubation.
Ta b le 1 . Biod istribution studies of 64Cu-DO3A-peptide using MDA-MB-435 tumor bearing SCID mice.
Tissues 0. 5 h 1 h 2 h 2 h block 4 h 24 h
Perrcent inject ed dose/gram (%ID/g)a
Tumor 1.20 ± 0.18 0.70 ± 0.16 0.61 ± 0.04 0.35 ± 0.07b 0.59 ± 0.04 0.22 ± 0.12
Blood 1.59 ± 0.24 0.47 ± 0.05 0.28 ± 0.08 0.25 ± 0.05 0.17 ± 0.05 0.15 ± 0.02
Brain 0.05 ± 0.01 0.04 ± 0.01 0.03 ± 0.01 0.02 ± 0.00 0.05 ± 0.01 0.01 ± 0.00
Heart 0.64 ± 0.09 0.36 ± 0.01 0.36 ± 0.02 0.29 ± 0.07 0.12 ± 0.03 0.08 ± 0.01
Lu ng 1.25 ± 0.07 0.89 ± 0.23 0.78 ± 0.11 0.75 ± 0.10 0.27 ± 0.20 0.19 ± 0.12
Liver 1.78 ± 0.16 2.41 ± 0.36 2.40 ± 0.14 2.30 ± 0.17 1.43 ± 0.21 0.95 ± 0.11
Spleen 0.51 ± 0.04 0.40 ± 0.21 0.55 ± 0.10 0.57 ± 0.20 0.23 ± 0.15 0.13 ± 0.02
Stomach 0.52 ± 0.17 0.59 ± 0.07 0.63 ± 0.04 0.69 ± 0.00 0.17 ± 0.05 0.10 ± 0.02
Kidneys 85.31 ± 10.63 96.0 ± 6.70 75.3 ± 5.88 79.2 ± 3.57 77.13 ± 8.26 18.2 ± 4.40
Mus le 0.29 ± 0.06 0.11 ± 0.01 0.07 ± 0.00 0.05 ± 0.02 0.05 ± 0.00 0.01 ± 0.00
Pancreas 0.41 ± 0.11 0.23 ± 0.08 0.22 ± 0.07 0.19 ± 0.02 0.12 ± 0.01 0.06 ± 0.00
Bone 0.28 ± 0.04 0.19 ± 0.07 0.11 ± 0.03 0.09 ± 0.01 0.09 ± 0.02 0.03 ± 0.01
Percent inject ed dose (%ID)
Intestines 1.54 ± 0.08 1.65 ± 0.14 2.20 ± 0.30 2.09 ± 0.46 3.48 ± 0.47 2.70 ± 0.70
Urine 55.22 ± 8.70 64.5 ± 1.60 69.8 ± 3.10 77.41 ± 4.80 66.4 ± 3.70 84.4 ± 4.0
Up tak e r atio of tum or/normal tis su e
T umor/ blood 0.75 1.50 2.18 --- 3.47 1.46
Tumor /Muscle 4.13 4.27 8.70 11.8 22.0
Tumor /liver 0.67 0.29 0.25 0.41 0.23
aData are pre sented as %ID/g ± SD except f or intest ine s and urine, values for which are expressed as % I D/g ± SD (n = 3); bP = 0.03,
significance comparison between the tumor uptake of radiolabeled peptide in the absence and presence of its non-radiolabeled
counter part at 2 h p.i.
Copyright © 2011 SciRes. AMI
Tab le 2. Biodistribution studies of 64Cu-CB-TE2A-pep t ide usi ng MD A-MB-435 tumor bearing S C ID mic e.
Tissues 0. 5 h 1 h 2 h 2 h block 4 h 24 h
Percent inject e d d ose/gram (%ID/g)a
Tumor 0. 85 ± 0. 12 0. 72 ± 0. 10 0.68 ± 0.07 0.36 ± 0. 08b 0.25 ± 0.10 0.10 ± 0.01
Blood 1.57 ± 0.26 0.42 ± 0. 15 0.07 ± 0.00 0.08 ± 0.03 0.06 ± 0.00 0.04 ± 0.01
Brain 0.05 ± 0.01 0.03 ± 0.01 0. 01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0. 00 ± 0. 00
Heard 0.56 ± 0.07 0.20 ± 0. 10 0.06 ± 0.01 0.10 ± 0. 04 0.08 ± 0.02 0. 07 ± 0. 01
Lu ng 1.33 ± 0. 20 0.61 ± 0.12 0. 29 ± 0. 05 0.27 ± 0.08 0. 25 ± 0.11 0.22 ± 0. 05
Liver 1. 34 ± 0. 23 1. 23 ± 0.30 0.97 ± 0.10 1. 02 ± 0.21 1.21 ± 0.11 0.79 ± 0.07
Spleen 0.50 ± 0.11 0.35 ± 0. 06 0.20 ± 0.04 0.19 ± 0.02 0.24 ± 0.11 0.15 ± 0.05
Stomach 0.44 ± 0.12 0.18 ± 0.06 0. 15 ± 0. 04 0.15 ± 0.01 0. 17 ± 0. 08 0.13 ± 0.06
Kidneys 91 .5 ± 21.0 98.0 ± 24.2 83 .31 ± 19.0 81. 2 ± 7.20 82 .0 ± 8.65 39.03 ± 3.0
Muscle 0.23 ± 0.02 0.08 ± 0.03 0. 06 ± 0. 01 0.02 ± 0.00 0. 07 ± 0.04 0.05 ± 0.01
Pancreas 0.31 ± 0. 10 0.14 ± 0.09 0. 16 ± 0. 05 0.14 ± 0.03 0. 18 ± 0. 09 0.17 ± 0.08
Bone 0.16 ± 0. 09 0.12 ± 0.05 0. 10 ± 0. 08 0.12 ± 0.05 0. 10 ± 0. 02 0.03 ± 0.01
Percent inject e d d ose (%ID)
Intestines 1.54 ± 0.08 0.62 ± 0. 12 0.69 ± 0.06 0.66 ± 0. 08 0.90 ± 0.14 0. 59 ± 0. 23
Urine 51.3 ± 4.50 64.2 ± 8.20 73.2 ± 2.60 76. 1 ± 4.90 70.6 ± 4.99 86.5 ± 0.70
Up tak e r ation of tum or /n ormal tissue
T umor/ blood 0.54 1.71 9.70 ---- 4.2 2 .5
Tumor /muscl e 3.70 9.00 11.33 3.6 2.0
Tumor /liver 0.60 0.58 0.70 0.21 0.13
aData are presented as %ID/g ± SD exce pt for intinte stine s and uri ne, values for whic h are expre sse d a s % ID ± SD (n = 3); bP = 0.032,
significance comparison between the tumor uptake of radiolabeled peptide in the absence and presence pf its non-radiolabeled
counter part at 2 h p.i.
clea ran ce of th e competin g peptide fr om circulation, and
longer residence time of the competing molecule might
aid in more efficient blocking of the radioligand. Wheth-
er increasing the dose of the non-radiolabeled peptide
further could compete off the radiolabeled peptide bind-
ing to the tumor is not clear. In the future, a dose-ranging
study with natCu-labeled peptides could shed some light
on the blocking efficiency of this peptide.
The majority of the peptides in circulation cleared
through the renal system (DO3A-peptide—(69.9 ±
3.1) %ID; CB-TE2A peptide—(73.3 ± 2.64) %ID) and
less through the hepatobiliary system (DO3A-peptide—
(2.45 ± 0.27) %ID; CB-TE2A peptide—(0.96 ± 0.06) %ID)
at the en d of 2 h. The disappearance of radiolabeled pep-
tides from the blood was comparable between the two
peptides, (1.59 ± 0.24) %ID/g (DO3A-peptide) and (1.57
± 0.26) %ID/g (CB-TE2A-peptide) at 30 min p.i. How-
ever, at 2 h and beyond a rapid disappearance of
CB-TE2A-peptide from blood was obse rved compa red to
DOTA-peptide (P = 0.015). Variable radiolabeled pep-
tide uptake was obser ved in normal tissues. Most of the
tissues exhibited minimal uptake, while modest radioac-
tivity was observed mainly in lungs and liver. Lung ra-
dioactivity with DO3A-peptide was higher at all time
points beyond 1 h p.i., compared to CB-TE2A-peptide
(P = 0.048). While the washout of radioactivity in the
lun g s with DO3A-peptide w as not promine nt, CB -TE2A-
peptide disappearan ce was obser ved a fter 1 h ti me point .
Radioactivity uptake in the liver for DO3A-peptide and
CB-TE2A-peptide was similar at 30 min p.i., with values
of (1.78 ± 0.16) %ID/g and (1.34 ± 0.23) %ID/g, r espec-
tively. However, at time points 1 h and later the DO3A-
peptide accumulation in liver was higher than that of the
CB-TE2A-peptide (P = 0.01). Copper radioisotopes have
been shown to transchelate from the chelation systems
such a s 1,4, 7,10-tetra-azacyclodode can e-N,N’,N’ ’, N’’’-
tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotet ra -
decane-, N,N’,N’’ N’’’-tetracetic acid (TETA) [30]. The
transchelated metals bind to proteins in liver (e.g. supe-
roxide dismutase) and to serum albumin [31]. Earlier
Copyright © 2011 SciRes. AMI
work by Weisman and coworkers suggested that
CB-TE2A chelator can stabilize copper due to the rigidi-
ty of its crossbridge system which offers increased ki-
netic stability compared to DOTA or TETA, thereby
reducing tr a nschelation of 64Cu [30].
Previous studies indicated that the injection of [64Cu]
Cu2+ alone in mice, resulted in half the injected dose (~
54%) concentrated in the liver after 2 h [32]. Morever,
oral administration of copper acetate in tumor bearing
mice r evea led th e accumulation of copper in liver (~ 62.1
μg/g tissue) and kidneys (~ 4.8 μg/g tissue) [32] Though
we observed serum stability of the radiolabeled pep-
tides in vitro, radioactivity in the liver suggests that tran-
schelation of 64Cu could occur from the radiolabeled
peptides, in vivo. Such a scen ar io wa s reported earlier for
64Cu-DOTA [30], and for 64Cu-TETA, where in vitro
serum stability was high [33] while in vivo there was
tr ansch elati on of 64Cu to superoxide dismutase and other
proteins [31].
Substantial uptake in kidneys was observed at 2 h p.i.,
for 64Cu-DO3A-peptide ((75.32 ± 5.88) %ID/g) and
64Cu-CB-TE2A-peptide ((83.31 ± 19.0) %ID/g) which
declined to (18. 2 ± 4. 4 ) ID/g an d (3 9.0 3 ± 3. 0) %ID/g at
th e end of 24 h, respectivel y. Th is high renal upta ke and
slow excretion could be due to the overall charge of the
peptide. Previous studies h ave reported the effect of pep-
tide charge on kidney uptake of both 64Cu and
111In-labeled compounds [34,35]. Indium-111-DTPA-
conjugated peptides are retained in the kidney after
reabsorption by renal tubular cells and lysosomal pro-
t e olys is [ 34]. Renal retention of 111In activity was highest
with positively charged and lowest for negatively
charged particles, respectively [36]. Similar charge ef-
fects were also seen with 64Cu-labeled azamacrocycles
[31]. Although studies including ours have reported that
blockin g the cationi c bind ing sit es in th e kidney with D-
or L-lysine [17,24,37] could decrease the peptide reten-
ti on in th e k idn ey, complete inhibition of uptake was not
The absorbed radiation doses to tumor and normal or-
gans from the radiolabeled peptides were estimated in
thi s study based on the biodistribution data in MD A-MB-
435 human breast xenografted SCID mice (Table 3).
Dosimetry calculations were based on energy deposition
from the β-decay of 64Cu. The absorbed dose from
CB-TE2A and DO3A peptide to the tumor was 42 mGy/
mCi an d 129 mGy/mCi, r espe ctivel y. Normal tissu e doses
were low except for th e ki dneys, which were estimated at
16,281 mGy/mCi (CB-TE2A) and 7381 mGy/mCi
(DO3A). These results suggest that the kidneys will be
the d os e -limiting normal organ.
In order to reduce the kidney uptake of the radiola-
beled peptides, BSA-f (3 - 50 kDa) generated by tryptic
digestion was used as a blocking agent. The renal uptake
of radiolabeled peptides without and with co-infusion of
200 μg of BSA-f was performed in SCID mice (n = 3
Table 3. Absorbed radiation doses from radiolabeled pep-
tides in MDA-MB-435 t u m or bea r ing S C ID mic e.
Or gan CB-TE2 A -peptide DO3A-peptide
Tumor 42 129
Blood 40 116
Brain 02 60
Heard 26 158
Lu ng 71 235
Liver 302 546
Spleen 49 156
Stomach 46 166
Kidneys 16,289 7381
Muscle 04 15
Pancreas 38 110
Bone 27 29
Dosimetry calculations were based on energy deposition from the β-decay
of 64Cu. The value s ar e gi ven in mGy/mCi.
each) (Figure 4). Results indicated a ~ 43% (64Cu-
DO3A-peptide, P = 0.032) and ~ 47% (64Cu-CB-TE2A-
peptide, P = 0.037) inhibition of r enal radioligan d uptake
compared to the radiotracer only group. However, no
notable difference in the uptake of radioligand in other
ti ssu es an d organ s was observed.
MicroPET imaging studies of 64Cu-DO3A- or CB-
TE2A-pepti d e in M D A-MB-4 35 t um or bea r in g m i ce at 2
h p.i., demonstrated their use as a PET targeting agent for
primary breast tumors. Fused microPET/CT of MDA-
MB-435 tumor-bear in g SC ID mi ce a t 2 h p.i., are shown
in F i g 5. In ord er bet t er vi su a li z e th e t um or up ta ke of the
radiolabeled peptides along with reduced background,
the imagi ng wa s perform ed a t 2 h p.i .
As demonstrated in Figure 5, the tumor uptake and
retention of the radiolabeled conjugate was easily visua-
lized in the microPET/CT image and appeared to corre-
late well with the biodistribution studies observed at 2 h
p.i. However, some accumulation of radioactivity was
observed in the liver and is in keeping with the biodi-
stribution studies.
Also, substantial radioactivity in the kidneys was
clearly evident which correlated well with the biodistri-
bution pattern of the radiolabeled peptide. Since the
charge of a peptide can influence renal uptake of a radi-
olabeled peptide, Sprague et al, suggested that modifica-
Copyright © 2011 SciRes. AMI
tion of the CB-TE2A backbone by addition of a carbox-
ylate arm could improve the distribution kinetics of
64Cu-CB-TE2A-Y3-TATE by changing the net charge
from positive to neutral or negative [22].
Figure 4. Blocking studies with bovine serum albumin
fragments, albumin fragments (BSA-F, 200 mg) were
preinjected in the mice 5 min prior to the injection of radi-
olabeled peptides. The kidney uptake of the radioli ga nds i n
mice with and without BSA-f was me a sured i n a γ-counter.
(a) A 43% (P = 0.32) and 47% (P = 0.037) decrease in the
uptake of 64Cu-DO3A-peptide and (b) 64Cu -CB-TE2A-
peptides was observed, respectively.
Al ter nat e str at egi es rep or ted to bl ock th e ki dn ey r eten-
tion of peptides include the use of gelofusine [38], and
blockin g th e uptake by albumin fragments [23]. Our stu-
dies with BSA-f indicated a partial kidney block of the
radiolabeled peptide in vivo. An alternate approach for
efficiently blocking the radiolabeled peptide uptake by
th e kidn eys c oul d be t o us e a com bi nati on of BSA-f an d
the amino acid lysine, simultaneously. Another possibil-
ity would be to replace the amino acid spacers with
chemical linkers such as 5-amino-3-oxapentyl-succinamic
acid (5-ADS), 8-amino-3,6-dioxaoctyl-succinamic acid
(8-AOS), and p-aminobenzoic acid (AMBA), between
(a) (b)
Figure 5. MicorPET/CT of MDA-ME-435 tumor-bearing
mice. (a) 64Cu-DO3A-peptide or (b) 64Cu-DO3A-peptide or
(b) 64 Cu -CB-TE2A-pept i de 12 MB q (3 24 mC i) was i nj e ct e d
into the tail vein of SCID mice bearing MDA-MB-435 tu-
mor xenograft. Imaging was acquired 2 h post injection of
the fadiolabeled in a PET scanner, The PET images were
fused with Conventional microCT images to validate re-
gions of increased radiolabel ed peptide uptake, Left l ateral
view images are shown which depicts the distribution of
radiolabeled peptides in tumor and other organs. T-tumor,
K-kidneys .
the bifunction al-chelator and the amino terminus of the
peptide, which might influence the kidney clearance of
the peptide, a s report ed previously for bombesin peptide
analogs [39]. Overall, our study indicates that the
64Cu-DO3A- and 64 Cu CB-TE2A-peptides could be de-
vel oped into a ra dioi mag in g ag en t for tumors expr essin g
gal-3, and further improvements with respect to the bi-
functional chelator or the linkers might improve the
64Cu-chelate in vivo sta bili t y and cl earan ce in th e circu la-
4. Conclusions
64Cu-DO3A-and 64Cu-CB-TE2A-( GSG) -G3-C12 were
evaluated for MDA-MB-435 breast carcinoma cell bind-
ing capacity, in vivo biodistribution and microPET im-
aging of gal-3 expressing breast tumors in hetero-
transplanted mice. The radiolabeled peptides exhibited
specific binding to the carcinoma cells, and targeted the
tumors in vivo which was clearly demonstrated in mi-
croPET imaging. The rapid clearance of the peptides
Copyright © 2011 SciRes. AMI
reduced non-target background except for the liver and
ki dn eys. I t r em ain s to be se en wheth er chela t or or spa cer
modification will further help to improve the renal han-
dling of the radiometallo-peptides.
5. Acknow ledgements
We thank Dr. Said Figueroa for help with the imaging
experiments at the Harry S. Truman Veterans Hospital.
We acknowledge Dr. Carolyn Anderson, Washington
University School of Medicine, St Louis, for providing
the chelator CB-TE2A. The authors also acknowledge
Lisa Watkinson and Terry Carmack for performing ani-
mal ex peri m en ts an d Mari e Di cker s on for t echn ica l h elp.
This work was supported in part by a Merit Review
Award from the Veterans Administration and NIH (P50
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