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64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid- 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 20, 2008.

Chemical name:64Cu-1,4,7,10-Tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid-cyclic arginine-glycine-aspartic acid peptideimage 24697457 in the ncbi pubchem database
Abbreviated name:64Cu-DOTA-c(RGDyK)
Agent Category:Peptide
Target:Integrin αvβ3
Target Category:Receptor binding
Method of detection:Positron Emission Tomography (PET)
Source of signal/contrast:64Cu
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64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid -cyclic arginine-glycine-aspartic acid peptide [64Cu-DOTA-c(RGDyK)] is an integrin-targeted molecular imaging agent developed for positron emission tomography (PET) of tumor vasculature, tumor angiogenesis, and osteoclasts (1).

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. 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 have been developed for in vivo binding to integrin αvβ3 (8). Chen et al. (1) evaluated a cyclic RGD peptide [c(RGDyK)] labeled with 64Cu or 18F in nude mice bearing breast tumor. They used DOTA for c(RGDyK) conjugation with 64Cu. 64Cu-DOTA-c(RGDyK) showed prolonged tumor radioactivity retention but persistent liver radioactivity.



Chen et al. (1) reported the synthesis of 64Cu-DOTA-c(RGDyK). The cyclic peptide was first prepared via solution cyclization of fully protected linear pentapeptide H-Gly-Asp(OtBu)-D-Tyr(OtBu)-Lys(Boc)-Arg(Pbf)-OH, followed by trifluoroacetic acid deprotection in the presence of the free radical scavenger triisopropylsilane. DOTA was obtained commercially. DOTA-c(RGDyk) conjugate was prepared with the use of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and N-hydroxysulfonosuccinimide (SNHS) to activate DOTA at pH 5.5 for 30 min at 4ºC. The c(RGDyk) peptide in water at 4ºC was added to the reaction mixture, and the pH was adjusted to 8.5. The reaction was allowed to incubate overnight at 4ºC. The resultant DOTA-c(RGDyk) conjugate was purified by semi-preparative high-performance liquid chromatography (HPLC) with a yield of 79%. The purified product was labeled with 64Cu by addition of 64Cu in the ratio of 2–5 μg peptide per 37 MBq (1 mCi) 64Cu in 0.1 N sodium acetate (pH 5.5) buffer. This was followed by incubation at 50ºC for 45 minutes. 64Cu-DOTA-c(RGDyK) was purified with a C-18 Sep-Pak cartridge using 85% ethanol as the elution solvent. The radiochemical yield was ≥90%, and the radiochemical purity was ≥98%. The specific activity ranged from 7.4–18.5 GBq/μmol (200–500 Ci/μmol).

In Vitro Studies: Testing in Cells and Tissues


No publication is currently available.

Animal Studies



Biodistribution studies of 64Cu-DOTA-c(RGDyK) were conducted in mice bearing MDA-MB-435 human breast cancer (1). Each mouse received an i.v. dose of 370 kBq (10 μCi) 64Cu-DOTA-c(RGDyK). Rapid blood clearance of the radioactivity from the blood circulation was observed. The tumor radioactivity levels (n = 5) in percent injected dose per g (% ID/g) for the tumor were 2.02 ± 0.15 (0.25 h), 1.98 ± 0.10 (0.5 h), 1.90 ± 0.36 (1 h), 1.36 ± 0.10 (2 h), and 1.44 ± 0.09 (4 h). The tumor/blood ratios were 1.67 ± 0.11 (0.25 h), 3.72 ± 0.18 (0.5 h), 8.14 ± 0.88 (1 h), 6.94 ± 0.51 (2 h), and 6.00 ± 0.06 (4 h). The liver, kidneys, and intestine were the organs with similar or slightly higher radioactivity levels than the tumor. A co-injection dose of 10 mg/kg c(RGDyK) decreased the tumor radioactivity level to 0.34 ± 0.02% ID/g (n = 4) at 1 h. MicroPET imaging of tumor-bearing mice injected with 14.8 MBq (0.4 mCi) 64Cu-DOTA-c(RGDyK) visualized the tumor with clear contrast. The tumor/muscle ratio at 2 h was 5.6 ± 0.4 (n = 3). In comparison, the tumor/muscle ratios from the biodistribution and autoradiography studies were 6.0 ± 0.6 and 6.9 ± 0.8 (n = 3), respectively. Co-injection with different amounts of c(RGDyK) revealed a dose-dependent blocking effect on the tumor radioactivity level.

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, R24 CA86307.


Chen X., Park R., Tohme M., Shahinian A.H., Bading J.R., Conti P.S. MicroPET and autoradiographic imaging of breast cancer alpha v-integrin expression using 18F- and 64Cu-labeled RGD peptide. Bioconjug Chem. 2004;15(1):41–9. [PubMed: 14733582]
Jin H., Varner J. Integrins: roles in cancer development and as treatment targets. Br J Cancer. 2004;90(3):561–5. [PMC free article: PMC2410157] [PubMed: 14760364]
Paulhe F., Manenti S., Ysebaert L., Betous R., Sultan P., Racaud-Sultan C. Integrin function and signaling as pharmacological targets in cardiovascular diseases and in cancer. Curr Pharm Des. 2005;11(16):2119–34. [PubMed: 15974963]
Hood J.D., Cheresh D.A. Role of integrins in cell invasion and migration. Nat Rev Cancer. 2002;2(2):91–100. [PubMed: 12635172]
Hwang R., Varner J. The role of integrins in tumor angiogenesis. Hematol Oncol Clin North Am. 2004;18(5):991–1006. [PubMed: 15474331]
Cai W., Shin D.W., Chen K., Gheysens O., Cao Q., Wang S.X., Gambhir S.S., Chen X. Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects. Nano Lett. 2006;6(4):669–76. [PubMed: 16608262]
Massoud T.F., Gambhir S.S. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 2003;17(5):545–80. [PubMed: 12629038]
Haubner R., Wester H.J. Radiolabeled tracers for imaging of tumor angiogenesis and evaluation of anti-angiogenic therapies. Curr Pharm Des. 2004;10(13):1439–55. [PubMed: 15134568]


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