NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

Cover of Molecular Imaging and Contrast Agent Database (MICAD)

Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

Show details

64Cu-1,4,8,11-Tetraazacyclotetradecane-regioselectively addressable functionalized template-[cyclo-(Arg-Gly-Asp-d-Phe-Lys)]4

, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: December 1, 2011.

Chemical name:64Cu-1,4,8,11-Tetraazacyclotetradecane-regioselectively addressable functionalized template-[cyclo-(Arg-Gly-Asp-d-Phe-Lys)]4
Abbreviated name:64Cu-Cyclam-RAFT-c(-RGDfK-)4
Agent category:Peptide
Target:Integrin αvβ3
Target category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal:64Cu
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about integrin αvβ3.



Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (1). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The αvβ3 integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (2-7). Expression of αvβ3 integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. Antagonists of αvβ3 are being studied as antitumor and antiangiogenic agents, and the agonists of αvβ3 are being studied as angiogenic agents for coronary angiogenesis (6, 8, 9). A peptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including αvβ3. Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (10).

Most of the cyclic RGD peptides are composed of five amino acids. Haubner et al. (11) reported that various cyclic RGD peptides exhibit selective inhibition of binding to αvβ3 (IC50, 7–40 nM) but not to αvβ5 (IC50, 600–4,000 nM) or αIIbβ3 (IC50, 700–5,000 nM) integrins. Various radiolabeled cyclic RGD peptides have been found to have high accumulation in tumors in nude mice (12). Hydrazinonicotinic acid (HYNIC) is a coupling agent for 99mTc labeling of peptides that can achieve high specific activities without affecting receptor-binding ability of the amino acid sequence. 99mTc is bound to the hydrazine group, and other coordination sites could be occupied by one or more coligands. Liu et al. (13) reported the success of radiolabeling cylco(Arg-Gly-Asp-d-Phe-Lys) (c(RGDfK)) tetramer linked by glutamic acid that was conjugated with HYNIC, which showed high tumor accumulation in nude mice bearing human tumor xenografts. Boturyn et al. (14) generated a versatile molecular “Regioselectively Addressable Functionalized Template” (RAFT) platform with a cyclic decapeptide [c(-Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-)] with two attachment sides. The upper side is linked to four copies of the c(RGDfK) peptide for targeting of integrin αvβ3, and the bottom side is linked to 99mTc (15) or 111In (16) for single-photon emission computed tomography imaging or to other labels for other imaging modalities. 99mTc-RAFT-c(-RGDfK-)4 and 111In-RAFT-c(-RGDfK-)4 efficiently accumulated in tumors in mice. Jin et al. (17) conjugated RAFT-c(-RGDfK-)4 with a bifunctional chelator, 1,4,8,11-tetraazacyclotetradecane (cyclam), for radiolabeling with 64Cu. 64Cu-Cyclam-RAFT-c(-RGDfK-)4 is an integrin-targeted molecular imaging agent developed for positron emission tomography (PET) imaging of tumor vasculature and tumor angiogenesis.



Cyclam-RAFT-c(-RGDfK-)4 was prepared with solid-phase and solution-phase syntheses (17). Radiolabeling of cyclam-RAFT-c(-RGDfK-)4 was performed by heating 64CuCl2 (0.74 GBq/ml, 20 mCi/ml) and RAFT-c(-RGDfK-)4 (1 mM) in a 1:1 (volume:volume) ratio for 60 min at 37°C. 64Cu-Cyclam-RAFT-c(-RGDfK-)4 had a radiochemical purity of >99% with a labeling efficiency of >99%. 64Cu-Cyclam-RAFT-c(-RGDfK-)4 exhibited a specific activity of ~0.74 MBq/nmol (0.02 mCi/nmol) 64Cu-Cyclam-RAFT-c(-RGDfK-)4 was 93% intact in mouse serum after incubation for 60 min at 37°C with little binding to serum proteins.

In Vitro Studies: Testing in Cells and Tissues


Jin et al. (17) performed in vitro assays using the human embryonic kidney (HEK) cell line stably transfected with human integrin β1 or β3 and the human glioblastoma U87MG cell line expressing β3. Flow cytometry analysis showed that HEK(β3), U87MG, and HEK(β1) cells exhibited high, medium, and low levels of αvβ3, respectively. A cell-binding assay (n = 3) was performed using 5 nM 64Cu-cyclam-RAFT-c(-RGDfK-)4 for 1 h at 4°C. The relative binding for HEK(β1), HEK(β3), and U87MG cells were 1.8%, 6.1%, and 1.0% incubation dose/100 ug protein, respectively. Another cell-binding study (n = 6) was performed using 10 nM 64Cu-cyclam-RAFT-c(-RGDfK-)4 for 1 h at 37°C. The relative accumulation of radioactivity for HEK(β1), HEK(β3), and U87MG cells were 0.3%, 15.7%, and 0.6% incubation dose/100 ug protein, respectively. Competitive inhibition assay was performed using HEK(β3) and 64Cu-cyclam-RAFT-c(-RGDfK-)4. Cyclam-RAFT-c(-RGDfK-)4 and c(RGDfV) showed a dose-dependent inhibition of 64Cu-cyclam-RAFT-c(-RGDfK-)4 binding to HEK(β3), with 50% inhibition concentration values of 38.9 ± 1.4 and 2,642 ± 221 nM, respectively.

Animal Studies



Jin et al. (17) performed biodistribution studies of 64Cu-cyclam-RAFT-c(-RGDfK-)4 (1.0 nmol) in mice (n =5–6/group) bearing HEK(β1), HEK(β3), or U87MG tumors at 3 h after injection. 64Cu-Cyclam-RAFT-c(-RGDfK-)4 uptake was significantly higher in HEK(β3) (9.4 ± 1.2% injected dose/gram (ID/g)) than in U87MG (3.5 ± 0.5% ID/g) and HEK(β1) (1.2 ± 0.3% ID/g) at 3 h after injection (P < 0.05 between any two of the three tumor models). Prominent accumulation was observed in the kidney (20%–25% ID/g) and liver (3%–4% ID/g). The other examined normal tissues exhibited low or negligible radioactivity accumulation. The HEK(β3) tumor/blood and tumor/muscle ratios were ~165 and ~19, respectively. Co-injection of excess cyclam-RAFT-c(-RGDfK-)4 (150 nmol) in mice (n = 3) bearing HEK(β3) tumors showed >85% reduction of radioactivity in the tumors. Some inhibition was also observed in all normal tissues examined except the kidney. Autoradiography studies indicated that there is a linear correlation between the αvβ3 expression and the tumor radioactivity accumulation of 64Cu-cyclam-RAFT-c(-RGDfK-)4 (R = 0.967).

PET imaging scans were performed at 1, 3, 6, and 20 h after injection in tumor-bearing mice. HEK(β3) and U87MG tumors were clearly visualized at all the time points in comparison to HEK(β1) tumors (17). The highest tumor contrast was observed at 1 h after injection for the three tumors; this was followed by a gradual washout with time. The kidneys, urinary bladder, and liver showed high radioactivity at the early time points. No blocking studies were reported.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


Hynes R.O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69(1):11–25. [PubMed: 1555235]
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]
Varner J.A., Cheresh D.A. Tumor angiogenesis and the role of vascular cell integrin alphavbeta3. Important Adv Oncol. 1996:69–87. [PubMed: 8791129]
Wilder R.L. Integrin alpha V beta 3 as a target for treatment of rheumatoid arthritis and related rheumatic diseases. Ann Rheum Dis. 2002;61 Suppl 2:ii96–9. [PMC free article: PMC1766704] [PubMed: 12379637]
Grzesik W.J. Integrins and bone--cell adhesion and beyond. Arch Immunol Ther Exp (Warsz) 1997;45(4):271–5. [PubMed: 9523000]
Kumar C.C. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets. 2003;4(2):123–31. [PubMed: 12558065]
Ruegg C., Dormond O., Foletti A. Suppression of tumor angiogenesis through the inhibition of integrin function and signaling in endothelial cells: which side to target? Endothelium. 2002;9(3):151–60. [PubMed: 12380640]
Kerr J.S., Mousa S.A., Slee A.M. Alpha(v)beta(3) integrin in angiogenesis and restenosis. Drug News Perspect. 2001;14(3):143–50. [PubMed: 12819820]
Mousa S.A. alphav Vitronectin receptors in vascular-mediated disorders. Med Res Rev. 2003;23(2):190–9. [PubMed: 12500288]
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]
Haubner R., Wester H.J., Burkhart F., Senekowitsch-Schmidtke R., Weber W., Goodman S.L., Kessler H., Schwaiger M. Glycosylated RGD-containing peptides: tracer for tumor targeting and angiogenesis imaging with improved biokinetics. J Nucl Med. 2001;42(2):326–36. [PubMed: 11216533]
Chen X., Park R., Shahinian A.H., Tohme M., Khankaldyyan V., Bozorgzadeh M.H., Bading J.R., Moats R., Laug W.E., Conti P.S. 18F-labeled RGD peptide: initial evaluation for imaging brain tumor angiogenesis. Nucl Med Biol. 2004;31(2):179–89. [PubMed: 15013483]
Liu S., Hsieh W.Y., Jiang Y., Kim Y.S., Sreerama S.G., Chen X., Jia B., Wang F. Evaluation of a (99m)Tc-labeled cyclic RGD tetramer for noninvasive imaging integrin alpha(v)beta3-positive breast cancer. Bioconjug Chem. 2007;18(2):438–46. [PubMed: 17341108]
Boturyn D., Coll J.L., Garanger E., Favrot M.C., Dumy P. Template assembled cyclopeptides as multimeric system for integrin targeting and endocytosis. J Am Chem Soc. 2004;126(18):5730–9. [PubMed: 15125666]
Sancey L., Ardisson V., Riou L.M., Ahmadi M., Marti-Batlle D., Boturyn D., Dumy P., Fagret D., Ghezzi C., Vuillez J.P. In vivo imaging of tumour angiogenesis in mice with the alpha(v)beta (3) integrin-targeted tracer (99m)Tc-RAFT-RGD. Eur J Nucl Med Mol Imaging. 2007;34(12):2037–47. [PubMed: 17674000]
Ahmadi M., Sancey L., Briat A., Riou L., Boturyn D., Dumy P., Fagret D., Ghezzi C., Vuillez J.P. Chemical and biological evaluations of an (111)in-labeled RGD-peptide targeting integrin alpha(V) beta(3) in a preclinical tumor model. Cancer Biother Radiopharm. 2008;23(6):691–700. [PubMed: 19111043]
Jin Z.H., Furukawa T., Galibert M., Boturyn D., Coll J.L., Fukumura T., Saga T., Dumy P., Fujibayashi Y. Noninvasive visualization and quantification of tumor alphaVbeta3 integrin expression using a novel positron emission tomography probe, 64Cu-cyclam-RAFT-c(-RGDfK-)4. Nucl Med Biol. 2011;38(4):529–40. [PubMed: 21531290]


  • PubReader
  • Print View
  • Cite this Page
  • PDF version of this page (86K)
  • MICAD Summary (CSV file)

Search MICAD

Limit my Search:

Related information

  • PMC
    PubMed Central citations
  • PubMed
    Links to PubMed

Similar articles in PubMed

See reviews...See all...

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...