Evaluation of 64 Cu-DOTA-and 64 Cu-CBTE 2 A-Galectin-3 Peptide as a PET Radiotracer for Breast Carcinoma

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 binding 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 radiolabeled with Cu. The synthesized peptides Cu-DO3A-(GSG)-ANTPCGPYTHDCPVKR (Cu-DO3Apep) and Cu-CB-TE2A-(GSG)-ANTPCGPYTHDCPVKR(Cu-CB-TE2A-pep) demonstrated an IC50 value of (97 ± 6.7) nM and (130 ± 10.2) nM, respectively, to cultured MDA-MB-435 breast carcinoma cells in vitro in a competitive displacement binding study. The tumor tissue uptake in SCID mice bearing MDA-MB435 tumors was (1.2 ± 0.18) %ID/g (Cu-DO3A-pep) and (0.85 ± 0.0.9) %ID/g (Cu-CB-TE2A-pep) at 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 with both Cu-DO3Aand Cu-CB-TE2A-(GSG)-ANTPCGPYTHDCPVKR after 2 h post injection. These studies suggest that gal-3 binding peptide could be developed into a PET imaging agent for galectin-3-expressing breast tumors.


Introduction
Galectin-3 (Gal-3) is a 31 kDa protein, which has high affinity for β-galactosides, and possesses a highly conserved 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 biological processes such as adhesion, apoptosis, inflammation, 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 increased adhesion to monolayers of endothelial cells compared with their non-metastatic 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 demonstrated that gal-3 is involved in heterotypic (carcinoma-endothelial cells) and homotypic (carcinoma-carcinoma) cellular adhesion via interactions with the tumor-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 surface of endothelial cells [11,12].Thus, gal-3-carbohydrate interactions are crucial in cancer cell adhesion to the endothelium.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 inhibitors bound other galectin family members due to their conserved carbohydrate binding residues which 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.
The bifunctional chelator DOTA has been used for chelating 64 Cu, however, its ability to chelate many +2 and +3 metal ions, and its relative instability compared to other chelators such as CB-TE2A, have made it less favourable [19,20].CB-TE2A has been reported to form very stable complexes with 64 Cu with less tranchelation of the metal in vivo [21].In the present study, we compared both 64 Cu-DO3A-GSG-G3-C12( 64 Cu-DO3A-peptide) and 64 Cu-CB-TE2A-GSG-G3-C12 ( 64 Cu-CB-TE2Apeptide) to evaluate the biodistrubtion and PET imaging properties of the peptides in a mouse model of human breast carcinoma.

Chemicals and Reagents
Amino acids and resin were purchased from Advanced Chem Tech (Louisville, KY).The bifunctional chelator CB-TE2A was synthesized as previously described [22].DOTA was purchased from Macrocyclics (Richardson, TX).Copper-64 was purchased from Nuclear Reactor Laboratory, University of Wisconsin (Madison, WI).All other reagents in this study were obtained from Fisher Scientific Company (Pittsburgh, PA) unless otherwise specified.

Cell Lines
Human breast carcinoma cell line MDA-MB-435 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% CO 2 humidified incubator.

Peptide 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 amino terminus of the peptide was carried out using an Advanced ChemTech 396 multiple peptide synthesizer (Advanced Chem Tech, Louisville, KY).The chelator CB-TE2A was coupled to the amino terminus of the linear GSG-G3-C12 through an intermediate preparation of the activated mixed anhydride by use of DIC/DIEA/ DMF as described earlier [22].Reverse phase high pressure liquid chromatography (RP-HPLC) analysis of synthesized 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% acetonitrile with 0.1% TFA.Identities of the peptides were confirmed by electrospray ionization mass spectrometry (ESI-MS) (Mass Consortium Corporation, San Diego, CA).
For concentrating, the peak-purified peptides were percolated 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 evaporated under a stream of nitrogen and was diluted to the appropriate volume with sterile saline.Radiochemical yields for 64 Cu-DO3A-peptide and 64 Cu-CB-TE2Apeptide averaged 45% and 40%, respectively.Radiochemical purity of both 64 Cu-labeled peptides was found to 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-pentahydrate solution.The solutions were heated at identical conditions as mentioned above for radiolabeling, and allowed to cool to room temperature.Native copper ( nat Cu)-peptides were then peak purified by RP-HPLC and determined to be ≥ 97% pure.ESI -MS was performed to check the integrity of the peptides.

In Vitro Competitive Cell Binding Affinity and Serum Stability Studies
A competitive displacement binding assay was used to determine the fifty-percent inhibitory concentration (IC 50 ) for radiolabeled G3-C12 peptide using nat Cu-DO3A-or nat Cu-CB-TE2A-peptide as the displacement ligand.Approximately, 2.0 × 10 6 MDA-MB-435 cells/tube were suspended in cell binding media along with 2.5 × 10 4 cpm of each 64 Cu-DO3A or 64 Cu-CB-TE2A-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 pelleting the cells by centrifugation.The cell pellet was further washed twice with 0.5 mL of ice-cold binding buffer.Cell-associated radioactivity was measured in a γ counter and the binding affinity was determined using Grafit software (Erithacus Software Limited, Surrey, UK).
In vitro serum stability of the radiolabeled peptide conjugates was tested by incubating 30.0 MBq (810 μCi) of 64 Cu-DO3A-or 64 Cu-CB-TE2A-peptide in 0.3 mL of mouse serum at 37˚C for 0.5, 1, 2, 4 and 24 h, respectively.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.

Pharmacokinetic Studies in Mice Bearing Human Breast Tumors
All animal studies were conducted in accordance with highest standards of care according to the National Institute of Health guide for care and use of laboratory animals and the policy and procedure for animal research 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 subcutaneously in the right shoulder with 1 × 10 7 MDA-MB-435 human breast carcinoma cells.Fully grown tumors ranged from 0.28 -0.85 g (3 -4 wk after inoculation).Each mouse (three mice per time point) was injected in the tail vein with 0.185 MBq (5 μCi) of 64 Cu-DO3A-or 64 Cu-CB-TE2A-peptide in 100 μl saline.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 excised tissues were weighed, and the tissue activity was measured in a γ counter.The percent injected dose (%ID) and percent injected dose per gram (%ID/g) were determined for each tissue.Whole blood %ID and %ID/g were determined assuming the blood accounted for 6.5% of the body weight of the mouse.Tumor blocking studies was performed by administering nat Cu-DO3A-or nat Cu-CB-TE2A-peptide (120 μg) in MDA-MB-435 tumor mice (n = 3) 20 min before administering respective radiolabeled counterparts.After 2 h, a biodistribution study were performed as describe above and %ID/g was determined for each tissue.Inhibition experiments with bovine serum albumin fragments (BSA-f) were performed to reduce the kidney retention of the radiolabeled peptides during biodistribution studies [23].Briefly, 3.0 g of BSA in 50 mM ammonium carbonate (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 fragments with molecular weight range of 3 -50 kDa (residue).The 3 -50 kDa albumin fragment(s) (200  g) sample in normal saline (100 μL) was pre-injected into the MDA-MB-435 tumor bearing mice (n = 3) via the tail vein.After 5 min, 0.185 MBq (5 μCi) of 64 Cu-DO3A-or 64 Cu-CB-TE2A-peptide was administered into the mice.The mice were sacrificed after 2 h, and the kidney uptake of the radiolabeled peptides was measured in a γ-counter.Control experiments (n = 3) were run in parallel with pre-injection of normal saline instead of albumin fragments.

Dosimetry Studies
The biodistribution data was processed with a dosimetry model to determine doses to all organs and to the tumor in the mouse model.The dosimetry model being used is a derivation on that developed by Hui et al. [24] to provide realistic beta dose estimates for organs in mice that received therapeutic radiopharmaceuticals for high energy betas.This model is required because the beta range of candidate radioisotopes such as 64 Cu is large relative to the size of many of the organs.This model has already been used to calculate the necessary dosimetry values for several isotopes including 64 Cu [25].

MicroPET/MicroCT Studies
In vivo microPET/CT of MDA-MB-435 tumor-bearing mice with 64 Cu-DO3A-or 64 Cu-CB-peptides were performed.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 operates in a 3-dimensional mode (3D).The mouse to be 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-likelyhood 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 Communication 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 visualization.

Statistical Analysis
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.

Results and Discussion
The DO3A-and CB-TE2A-peptides was prepared by solid-phase peptide synthesis using standard F-moc procedures [26].In order to enhance the conformational freedom, a GSG spacer was introduced between the chelators and the amino-terminus of the peptides [24].nat Culabeled peptides was prepared by heating aqueous solutions of the peptides with CuSO 4 .5H 2 O at 85˚C for 60 min.nat Cu-labeled peptides were purified and analyzed by mass spectrometry before cell binding assays.The calculated and observed molecular weights of the nat Cupeptides were 2405.0Da and 2405.3Da (DO3A-peptide) and 2342.0 and 2342.5 Da (CB-TE2A-peptide), respectively.For radiolabeling, the peptides were labeled with 64 Cu in ammonium acetate buffer (pH 7.0) at 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 64 Cu-CB-TE2A-peptide eluted with a retention time (t r ) of 13.2 min and 13.8 min, respectively, a minute after the elution of their respective non-radiolabeled counterparts.The radiolabeled peptides were obtained at a purity of ≥ 98%.
Specific binding of radiolabeled peptides was demonstrated with MDA-MB-435 cells.Minimal or no binding was observed with 184A-1 control cells.The time course experiments (Figure 1) revealed the association of radiolabeled 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 radioactivity was observed.Total cell binding capacity 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 carcinoma [27].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 melanoma cell line [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 speculations, the MDA-MB-435 cell line has been widely used as a breast carcinoma model and remains one of the most reliable in vivo models of human breast cancer.
The metabolic stability of the 64 Cu-labeled 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 observed with both radiolabeled peptides prior to the original radiolabeled peptide peak suggesting the degradation of peptides during prolonged serum incubation.A representative RP-HPLC profile of 64 Cu-CB-TE2Apeptide is shown in Figure 3.A similar profile was observed with 64 Cu-DO3A-peptide.
Detailed in vivo studies in SCID mice bearing human MDA-MB-435 tumor xenografts at different time intervals indicated 64 Cu-DO3A-peptide tumor accumulation of (1.2 ± 0.18) %ID/g, (0.7 ± 0.16) %ID/g, (0.61 ± 0.04) %ID/g, an d (0.59 ± 0.03) %ID/g; an d 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).counterparts.This approach reduced ~ 43% and ~ 47% of tumor uptake of the radiolabeled peptides (Tables 1  and 2) compared to mice injected with radioactive peptide 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  a Data are presented as %ID/g ± SD except for intestines and urine, values for which are expressed as %ID/g ± SD (n = 3); b P = 0.03, significance comparison between the tumor uptake of radiolabeled peptide in the absence and presence of its non-radiolabeled counterpart at 2 h p.i. a Data are presented as %ID/g ± SD except for intintestines and urine, values for which are expressed as %ID ± SD (n = 3); b P = 0.032, significance comparison between the tumor uptake of radiolabeled peptide in the absence and presence pf its non-radiolabeled counterpart at 2 h p.i.
clearance of the competing peptide from circulation, and longer residence time of the competing molecule might aid in more efficient blocking of the radioligand.Whether increasing the dose of the non-radiolabeled peptide further could compete off the radiolabeled peptide binding to the tumor is not clear.In the future, a dose-ranging study with nat Cu-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 end of 2 h.The disappearance of radiolabeled peptides 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.However, at 2 h and beyond a rapid disappearance of CB-TE2A-peptide from blood was observed compared to DOTA-peptide (P = 0.015).Variable radiolabeled pep-tide uptake was observed in normal tissues.Most of the tissues exhibited minimal uptake, while modest radioactivity was observed mainly in lungs and liver.Lung radioactivity 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 lungs with DO3A-peptide was not prominent, CB-TE2Apeptide disappearance was observed after 1 h time 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, respectively.However, at time points 1 h and later the DO3Apeptide 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 as 1,4, 7,10-tetra-azacyclododecane-N,N',N'', N'''tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradecane-, N,N',N'' N'''-tetracetic acid (TETA) [30].The transchelated metals bind to proteins in liver (e.g.superoxide dismutase) and to serum albumin [31].Earlier work by Weisman and coworkers suggested that CB-TE2A chelator can stabilize copper due to the rigidity of its crossbridge system which offers increased kinetic stability compared to DOTA or TETA, thereby reducing transchelation of 64 Cu [30].
Previous studies indicated that the injection of [64Cu] Cu 2+ 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 revealed the 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 peptides in vitro, radioactivity in the liver suggests that transchelation of 64 Cu could occur from the radiolabeled peptides, in vivo.Such a scenario was reported earlier for 64 Cu-DOTA [30], and for 64 Cu-TETA, where in vitro serum stability was high [33] while in vivo there was transchelation of 64 Cu to superoxide dismutase and other proteins [31].
Substantial uptake in kidneys was observed at 2 h p.i., for 64 Cu-DO3A-peptide ((75.32 ± 5.88) %ID/g) and 64 Cu-CB-TE2A-peptide ((83.31 ± 19.0) %ID/g) which declined to (18.2 ± 4.4) ID/g and (39.03 ± 3.0) %ID/g at the end of 24 h, respectively.This high renal uptake and slow excretion could be due to the overall charge of the peptide.Previous studies have reported the effect of peptide charge on kidney uptake of both 64 Cu and 111 In-labeled compounds [34,35].Indium-111-DTPAconjugated peptides are retained in the kidney after reabsorption by renal tubular cells and lysosomal proteolysis [34].Renal retention of 111 In activity was highest with positively charged and lowest for negatively charged particles, respectively [36].Similar charge effects were also seen with 64 Cu-labeled azamacrocycles [31].Although studies including ours have reported that blocking the cationic binding sites in the kidney with Dor L-lysine [17,24,37] could decrease the peptide retention in the kidney, complete inhibition of uptake was not achievable.
The absorbed radiation doses to tumor and normal organs from the radiolabeled peptides were estimated in this study based on the biodistribution data in MDA-MB-435 human breast xenografted SCID mice (Table 3).Dosimetry calculations were based on energy deposition from the β-decay of 64 Cu.The absorbed dose from CB-TE2A and DO3A peptide to the tumor was 42 mGy/ mCi and 129 mGy/mCi, respectively.Normal tissue doses were low except for the kidneys, which were estimated at 16,281 mGy/mCi (CB-TE2A) and 7381 mGy/mCi (DO3A).These results suggest that the kidneys will be the dose-limiting normal organ.
In order to reduce the kidney uptake of the radiolabeled 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  4).Results indicated a ~ 43% ( 64 Cu-DO3A-peptide, P = 0.032) and ~ 47% ( 64 Cu-CB-TE2Apeptide, P = 0.037) inhibition of renal radioligand uptake compared to the radiotracer only group.However, no notable difference in the uptake of radioligand in other tissues and organs was observed.
MicroPET imaging studies of 64 Cu-DO3A-or CB-TE2A-peptide in MDA-MB-435 tumor bearing mice 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-bearing SCID mice at 2 h p.i., are shown in Fig 5 .In order better visualize the tumor uptake of the radiolabeled peptides along with reduced background, the imaging was performed at 2 h p.i.
As demonstrated in Figure 5, the tumor uptake and retention of the radiolabeled conjugate was easily visualized in the microPET/CT image and appeared to correlate 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 biodistribution studies.
Also, substantial radioactivity in the kidneys was clearly evident which correlated well with the biodistribution pattern of the radiolabeled peptide.Since the charge of a peptide can influence renal uptake of a radiolabeled peptide, Sprague et al, suggested that modifica-tion of the CB-TE2A backbone by addition of a carboxylate arm could improve the distribution kinetics of64 Cu-CB-TE2A-Y3-TATE by changing the net charge from positive to neutral or negative [22].Alternate strategies reported to block the kidney retention of peptides include the use of gelofusine [38], and blocking the uptake by albumin fragments [23].Our studies 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 the kidneys could be to use a combination of BSA-f and 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 the bifunctional-chelator and the amino terminus of the peptide, which might influence the kidney clearance of the peptide, as reported previously for bombesin peptide analogs [39].Overall, our study indicates that the 64 Cu-DO3A-and 64 Cu CB-TE2A-peptides could be developed into a radioimaging agent for tumors expressing gal-3, and further improvements with respect to the bifunctional chelator or the linkers might improve the 64 Cu-chelate in vivo stability and clearance in the circulation.

Conclusions
reduced non-target background except for the liver and kidneys.It remains to be seen whether chelator or spacer modification will further help to improve the renal handling of the radiometallo-peptides.

Figure 3 .
Figure 3.The serum stability of 64 Cu-CB-TE2A-peptide at 0 and 24 h.(a) Elution of 64 Cu-CB-TE2Apeptide at 0 time point.(b) At 24 h, an additional peptide peak (arrow) was observed prior to the original radiolabeled peptide suggesting the degradation of peptide during prolonged serum incubation.

Figure 4 .
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 radiolabeled peptides.The kidney uptake of the radioligands in mice with and without BSA-f was measured in a γ-counter.(a) A 43% (P = 0.32) and 47% (P = 0.037) decrease in the uptake of 64 Cu-DO3A-peptide and (b) 64 Cu-CB-TE2Apeptides was observed, respectively.

Figure 5 .
Figure 5. MicorPET/CT of MDA-ME-435 tumor-bearing mice.(a) 64 Cu-DO3A-peptide or (b) 64 Cu-DO3A-peptide or (b) 64 Cu-CB-TE2A-peptide 12 MBq (324 mCi) was injected into the tail vein of SCID mice bearing MDA-MB-435 tumor 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 regions of increased radiolabeled peptide uptake, Left lateral view images are shown which depicts the distribution of radiolabeled peptides in tumor and other organs.T-tumor, K-kidneys.

Table 3 . Absorbed radiation doses from radiolabeled pep- tides in MDA-MB-435 tumor bearing SCID mice.
Dosimetry calculations were based on energy deposition from the β-decay of64Cu.The values are given in mGy/mCi.