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64Cu-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-TNYLFSPNGPIARAW (TNYL-RAW)

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

Created: ; Last Update: December 15, 2011.

Chemical name:64Cu-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid- TNYLFSPNGPIARAW (TNYL-RAW)
Abbreviated name:64Cu-DOTA-TNYL-RAW
Agent category:Peptide
Target:Ephrin receptor B4 (EphB4)
Target category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal:64Cu
  • Checkbox In vitro
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Structure is not available in PubChem.



The ephrin (Eph) receptors constitute the largest member of the receptor tyrosine kinase family (1, 2). The Eph receptors and their ligands (ephrins) mediate numerous biological processes in normal development, particularly in the nervous and cardiovascular systems (3-5). Based on their structures and sequence relationships, ephrins are divided into the ephrin-A class, which are anchored to the cell membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B class, which are transmembrane proteins. The Eph family of receptors is divided into two groups, EphA and EphB, on the basis of the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. The Eph receptors transmit forward signals via their kinase domain and reverse signals via their transmembrane ephrin ligands (6). EphB–ephrin-B interactions are capable of mediating bi-directional signaling events upon cell–cell contact, either into the receptor-expressing cell as "forward signaling" or into the ligand-expressing cell as "reverse signaling" (7).

Ephrin-2 is expressed on arterial and activated endothelial cells, whereas EphB4 is normally expressed on venous endothelial cells and various blood cells (8). EphB4 selectively binds to ephrin-2 to promote cell signaling and angiogenesis. EphB4 has been implicated in cancer progression and in pathological forms of angiogenesis. Overexpression of EphB4 has been observed in cancer cells and is associated with tumorigenesis via forward signaling and angiogenesis via reverse signaling through ephrin-2 interaction (9). EphB4 forward signaling stimulates cellular proliferation. Koolpe et al. (10) identified a 15-mer peptide, Tyr-Asn-Tyr-Leu-Phe-Ser-Pro-Asn-Gly-Pro-Ile-Ala-Arg-Ala-Trp (TNYL-RAW), to be a selective antagonist of EphB4 using phage display screening. Xiong et al. (11) reported the development of 64Cu-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-TNYL-RAW (64Cu-DOTA-TNYL-RAW) for positron emission tomography (PET) imaging of EphB4 in nude mice bearing tumor xenografts.



TNYL-RAW peptide was prepared using solid-phase synthesis and purified with high-performance liquid chromatography (HPLC) (11). DOTA-TNYL-RAW was prepared by reaction of TNYL-RAW with DOTA-N-hydroxysuccinate, followed by HPLC purification. DOTA-TNYL-RAW was eluted as a single peak, and its mass (2,092.0554 for [M+H]+) was confirmed with mass spectroscopy. DOTA-TNYL-RAW (~5 nmol) was incubated with 74–148 MBq (2–4 mCi) 64CuCl2 in sodium acetate buffer (pH 5.2) for 1 h at 70°C. 64Cu-DOTA-TNYL-RAW exhibited a radiochemical purity of >95% as determined with HPLC. The specific activity of 64Cu-DOTA-TNYL-RAW was 7.4–14.8 MBq/nmol (0.2–0.4 mCi/nmol) at the end of synthesis. natCu-DOTA-TNYL-RAW was prepared similarly. 64Cu-DOTA-TNYL-RAW was stable in medium containing 10% fetal bovine serum for up to 24 h at 37°C, whereas only 70% of 64Cu-DOTA-TNYL-RAW was intact in mouse serum after 2 h at 37°C.

In Vitro Studies: Testing in Cells and Tissues


Xiong et al. (11) performed binding experiments with TNYL-RAW, DOTA-TNYL-RAW, and natCu-DOTA-TNYL-RAW using a Biacore sensor chip immobilized with EphB4-Fc. The dissociation constant (Kd) values of TNYL-RAW, DOTA-TNYL-RAW, and natCu-DOTA-TNYL-RAW were calculated to be 3.06 nM, 23.3 nM, and 1.98 nM, respectively. No binding to EphB4 was detected with a scrambled TNYL-RAW peptide. In vitro binding of FITC-TNYL-RAW to PC-3M human prostate cancer cells (EphB4-expressing), CT26 human colon cancer cells (EphB4-expressing), and A549 human lung adenocarcinoma cells (EphB4-negative) was determined with fluorescence microscopy. PC-3M and CT26 cells, but not A549 cells, were readily stained with FITC-TNYL-RAW. The binding of FITC-TNYL-RAW to PC-3M and CT26 cells was almost completely blocked by co-incubation with 100-fold excess TNYL-RAW. No binding of FITC-scrambled TNYL-RAW to PC-3M and CT26 cells was observed. 64Cu-DOTA-TNYL-RAW exhibited increased cellular uptake with time in PC-3M and CT26 cells but not in A549 cells. Incubation with 100-fold excess of TNYL-RAW almost completely inhibited the radioactivity in PC-3M cells at 30, 60, and 120 min of incubation.

Animal Studies



Xiong et al. (11) performed PET imaging and ex vivo biodistribution studies of 64Cu-DOTA-TNYL-RAW in nude mice (n = 4/group) bearing CT26, PC-3M, and A549 tumors at 1, 4, and 24 h after intravenous injection. Tumors were clearly visualized at 4 h for CT26 tumors and at 4 h and 24 h for PC-3M tumors. PET imaging estimated tumor accumulation values of 1.3% injected dose/gram (ID/g), 2.6% ID/g, and <0.5% ID/g at 1, 4, and 24 h after injection in CT26 tumors, respectively. Tumor accumulation values in PC-3M tumors were 1.4% ID/g, 3.2% ID/g, and 3.6% ID/g at 1, 4, and 24 h after injection, respectively. A549 tumor accumulation values were low, with 1.7% ID/g, 1.5% ID/g, and 1.2% ID/g at 1, 4, and 24 h after injection, respectively. Co-injection of excess TNYL-RAW reduced the radioactivity accumulation by 77% and 81% in CT26 (4 h) and PC-3M (24 h) tumors, respectively.

Ex vivo tumor accumulation was 0.84% ID/g for PC-3M tumors and 0.44% ID/g for A549 tumors at 24 h after injection. Accumulation in the liver, spleen, and kidneys was higher than in the tumors. Co-injection of excess TNYL-RAW inhibited tumor/muscle ratios (~10) for CT26 tumors by 57% and PC-3M tumors by 48% at 4 h and 24 h after injection, respectively.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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Pasquale E.B. The Eph family of receptors. Curr Opin Cell Biol. 1997;9(5):608–15. [PubMed: 9330863]
Adams R.H., Klein R. Eph receptors and ephrin ligands. essential mediators of vascular development. Trends Cardiovasc Med. 2000;10(5):183–8. [PubMed: 11282292]
Cheng N., Brantley D.M., Chen J. The ephrins and Eph receptors in angiogenesis. Cytokine Growth Factor Rev. 2002;13(1):75–85. [PubMed: 11750881]
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Noren N.K., Lu M., Freeman A.L., Koolpe M., Pasquale E.B. Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth. Proc Natl Acad Sci U S A. 2004;101(15):5583–8. [PMC free article: PMC397426] [PubMed: 15067119]
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Pfaff D., Heroult M., Riedel M., Reiss Y., Kirmse R., Ludwig T., Korff T., Hecker M., Augustin H.G. Involvement of endothelial ephrin-B2 in adhesion and transmigration of EphB-receptor-expressing monocytes. J Cell Sci. 2008;121(Pt 22):3842–50. [PubMed: 18957513]
Noren N.K., Pasquale E.B. Paradoxes of the EphB4 receptor in cancer. Cancer Res. 2007;67(9):3994–7. [PubMed: 17483308]
Koolpe M., Burgess R., Dail M., Pasquale E.B. EphB receptor-binding peptides identified by phage display enable design of an antagonist with ephrin-like affinity. J Biol Chem. 2005;280(17):17301–11. [PubMed: 15722342]
Xiong C., Huang M., Zhang R., Song S., Lu W., Flores L. 2nd, Gelovani J., Li C. In vivo small-animal PET/CT of EphB4 receptors using 64Cu-labeled peptide. J Nucl Med. 2011;52(2):241–8. [PubMed: 21233177]


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