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64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-PEGylated dimeric cyclic arginine-glycine-aspartic acid peptide

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: May 8, 2008.

Chemical name:64Cu-1,4,7,10-Tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid-pegylated dimeric cyclic arginine-glycine-aspartic acid peptideimage 26522134 in the ncbi pubchem database
Abbreviated name:64Cu-DOTA-PEG-E[c(RGDyK)]2
Synonym:64Cu-pegylated dimeric c(RGDyK), 64Cu-PEG-dimeric RGD
Agent Category:Peptide
Target:Integrin αvβ3
Target Category:Receptor binding
Method of detection:Positron Emission Tomography (PET)
Source of signal:64Cu
  • Checkbox In vitro
  • Checkbox Rodents
PEG is a heterofunctional polyethylene glycol with a molecular weight of 3,400.
Click on the above structure for additional information in PubChem.
Click on protein, nucleotide (RefSeq), and gene for more information about integrin αvβ3.



64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid –PEGylated dimeric cyclic arginine-glycine-aspartic acid peptide {64Cu-DOTA-PEG-E[c(RGDyK)]2} is an integrin-targeted molecular imaging agent developed for positron emission tomography (PET) of tumor vasculature and tumor angiogenesis (1). 64Cu is a positron emitter with a physical half-life (t½) of 12.8 h.

Cellular survival, invasion, and migration control embryonic development, angiogenesis, tumor metastasis, and other physiologic processes (2, 3). Among the molecules that regulate angiogenesis are integrins, which comprise a superfamily of cell adhesion proteins that form heterodimeric receptors for extracellular matrix (ECM) molecules (4, 5). These transmembrane glycoproteins consist of two noncovalently associated subunits, α and β (18 α- and 8 β-subunits in mammals), which are assembled into at least 24 α/β pairs. Several integrins, such as integrin αvβ3, have affinity for the arginine-glycine-aspartic acid (RGD) tripeptide motif, which is found in many ECM proteins. Expression of integrin αvβ3 receptors on endothelial cells is stimulated by angiogenic factors and environments. The integrin αvβ3 receptor is generally not found in normal tissue, but it is strongly expressed in vessels with increased angiogenesis, such as tumor vasculature. It is significantly upregulated in certain types of tumor cells and in almost all tumor vasculature (1).

Molecular imaging probes carrying the RGD motif that binds to the integrin αvβ3 can be used to image tumor vasculature and evaluate angiogenic response to tumor therapy (6, 7). Various RGD peptides in both linear and cyclic forms (RGDfK or RGDyK) have been developed for in vivo binding to integrin αvβ3 (8). It has been hypothesized that cyclic RGD peptide may have a faster rate of receptor binding or a slower rate of dissociation from the integrin αvβ3 than linear single RGD peptides (9). They also suggested that the increase in molecular size might prolong circulation time and reduce tumor washout rate. Chen et al. (10) prepared a cyclic RGD d-Tyr analog [64Cu-DOTA-c(RGDyK)] and showed the cyclic peptide had prolonged tumor radioactivity retention but persistent liver radioactivity. To improve the pharmacokinetics and tumor retention of the radiolabeled peptide, Chen et al. (11) modified c(RGDyK) with monofunctional methoxy-polyethylene glycol (mPEG; molecular weight = 2,000) and showed that the modified PEGylated RGD peptide had faster blood clearance, lower kidney uptake, and prolonged tumor uptake. Using the same strategy, Chen et al. (12) inserted a heterobifunctional PEG linker (molecular weight = 3,400) between DOTA and c(RGDyK) to produce 64Cu-DOTA-PEG-c(RGDyK). The PEG moiety appeared to improve the in vivo kinetics of this PET radioligand. Chen et al. (13) also observed that a dimeric RGD peptide E[c(RGDyK)]2 had higher binding affinity than the monomeric analog. Subsequently, the same research group also reported the successful use of a heterobifunctional PEG to prepare 64Cu-lableled PEGylated E[c(RGDyK)]2 peptide (1).



Chen et al. (1) reported the synthesis of 64Cu-DOTA-PEG-E[c(RGDyK)]2. The E[c(RGDyK)]2 peptide was prepared by coupling Boc-Glu-OH with two equivalents of monomeric RGD peptide followed by trifluoroacetic acid (TFA) cleavage. The monomeric c(RGDyK) was first prepared via solution cyclization of the fully protected linear pentapeptide H-Gly-Asp(OtBu)-D-Tyr(OtBu)-Lys(Boc)-Arg(Pbf)-OH, followed by TFA deprotection in the presence of the free radical scavenger triisopropylsilane. A bifunctional PEG (molecular weight 3,400) was used. The DOTA-PEG-E[c(RGDyK)]2 conjugate was prepared from t-butoxycarbonyl (t-Boc)-NH-PEG-E[c(RGDyK)]2 which was synthesized by reacting t-Boc-protected PEG succinimidyl ester with the E[c(RGDyK)]2 peptide in 0.2 M Na2B4O7 buffer (pH 8.3) overnight at 4ºC. The t-Boc-NH-PEG-E[c(RGDyK)]2 was then dissolved in 95% TFA at room temperature for 2 h to produce the NH2-PEG-E[c(RGDyK)]2 conjugate. DOTA was obtained commercially. The DOTA-PEG-c(RGDyk) conjugate was prepared by activating DOTA in situ by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and N-hydroxysulfonosuccinimide (SNHS) in a molar ratio of 10:5:4 (DOTA:EDC:SNHS).The NH2-PEG-E[c(RGDyk)]2 conjugate in water at 4ºC was added to the resultant DOTA-sulfosuccinimydyl ester (OSSu), and the pH was adjusted to 8.5. The reaction mixture was allowed to incubate overnight at 4ºC. The final product DOTA-PEG-E[c(RGDyk)]2 conjugate was then purified by semi-preparative high-performance liquid chromatography (HPLC) with a final yield of ~60%. The molecular weight of the DOTA-PEG-E[c(RGDyk)]2 conjugate was determined to be 5,100 by matrix-assisted desorption/ionization time-of-flight mass spectroscopy and appeared to agree with the theoretical value. The purified product was labeled with 64Cu by addition of 64Cu chloride in the ratio of 25 μg peptide per 5 MBq (0.135 mCi) 64Cu in 0.1 N sodium acetate (pH 5.5) buffer. This was followed by incubation at 50ºC for 50 minutes. The reaction was terminated by addition of ethylenediaminetetraacetic acid. 64Cu-DOTA-PEG-E[c(RGDyK)]2 was purified with a C-18 Sep-Pak cartridge using 85% ethanol as the elution solvent. The radiochemical yield was >95%. After Sep-Pak purification, the radiochemical purity was >99%. The specific activity of 64Cu-DOTA-PEG-E[c(RGDyK)]2 was ~18,500 GBq/mmol (500 Ci/mmol, decay-corrected).

In Vitro Studies: Testing in Cells and Tissues


Competitive binding experiments of unlabeled DOTA-PEG-E[c(RGDyK)]2 for integrin αvβ3 on the surface of NSCLC NCI-H1975 human female lung adenocarcinoma cells were conducted by the use of 125I-echistatin as a radioligand (1). The 50% inhibitory concentration (IC50) of DOTA-PEG-E[c(RGDyK)]2 was 84.9 nM. In comparison, the IC50 for unpegylated E[c(RGDyK)]2 was 11.1 nM.

Animal Studies



Biodistribution studies of 64Cu-DOTA-PEG-E[c(RGDyK)]2 were conducted in severe combined immunodeficient (SCID-bg) mice bearing s.c. NCI-H1975 (1). Each mouse received 370 kBq (10 μCi) of 64Cu-DOTA-PEG-E[c(RGDyK)]2 by i.v. administration. The radioligand was rapidly cleared from the blood. The radioactivity levels (n = 3) in percent dose per g (% ID/g) in the tumor were 1.87 ± 0.05 (0.5 h), 2.64 ± 0.14 (1 h), 1.35 ± 0.06 (2 h), 1.46 ± 0.12 (4 h), and 1.15 ± 0.07 (24 h). The radioactivity levels in the blood were 0.57 ± 0.19 (0.5 h), 0.17 ± 0.02 (1 h), 0.05 ± 0.01 (2 h), 0.04 ± 0.02 (4 h), and 0.04 ± 0.01 (24 h). The tumor/blood ratios were 3.58 ± 1.43 (0.5 h), 15.3 ± 1.06 (1 h), 27.9 ± 6.37 (2 h), 36.9 ± 13.1 (4 h), and 28.7 ± 3.71 (24 h). The radioligand was primarily excreted by the kidneys. The radioactivity levels in the kidney were 3.50 ± 0.54 (0.5 h), 1.92 ± 0.26 (1 h), 1.63 ± 0.04 (2 h), 1.61 ± 0.17 (4 h), and 1.15 ± 0.11 (24 h). Both liver and lung radioactivity levels were relatively low. There was minimal radioactivity in other organs. With coinjection of 10 mg/kg unlabeled c(RGDyK) peptide, the tumor radioactivity was decreased by four times to 0.68 ± 0.10 at 1 h. When compared to the unpegylated radioligand, the authors suggested that the pegylated radioligand had a relatively more rapid renal clearance and a lower liver accumulation. However, the tumor radioactivity was also lowered because the pegylated peptide had a lower receptor binding affinity than the unpegylated peptide.

Imaging of 11.1 MBq (300 μCi) 64Cu-DOTA-PEG--E[c(RGDyK)]2 with microPET was conducted in SCID-bg mice bearing the s.c. tumors (1). In addition, orthotopic lung cancer tumors (NCI-H1975 cells injected directly into the upper lung lobe) and its metastases were also induced in the same mice. At 2 h after injection, PET imaging clearly visualized the s.c. tumor, the primary orthotopic lung tumor and its metastases to the contralateral lung, mediastinum, and diapghragm. The kidneys, liver and urinary bladder were also visible on the image. In comparison, a [18F]FDG scan on the same mouse visualized the primary lung cancer but failed to visualize the metastases because of the high cardiac and lung radioactivity background. Whole-body autoradiography of the mice after PET imaging showed a distribution pattern similar to those of the biodistribution and imaging studies. Chen et al. (1) concluded that 64Cu-DOTA-PEG--E[c(RGDyK)]2 had similar blood clearance, more rapid renal clearance, reduced hepatic uptake, but also lower tumor uptake when compared with the unpegylated peptide.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

NIBIB R21 EB001785, NCI P20 CA86532.


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