Background

[PubMed]

⁶⁴Cu-4,11-Bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-cyclic arginine-glycine-aspartic acid peptide [⁶⁴Cu-CD-TE2A-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 α_vβ₃, have affinity for the arginine-glycine-aspartic acid (RGD) tripeptide motif, which is found in many ECM proteins. Expression of integrin α_vβ₃ receptors on endothelial cells is stimulated by angiogenic factors and environments. The integrin α_vβ₃ 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 α_vβ₃ 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 α_vβ₃ (8). Chen et al. (9) evaluated a cyclic RGD peptide [c(RGDyK)] labeled with ⁶⁴Cu or ¹⁸F in nude mice bearing breast tumor. They used 1,4,7,10-tetrazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) for c(RGDyK) conjugation with ⁶⁴Cu. ⁶⁴Cu-DOTA-c(RGDyK) showed prolonged tumor radioactivity retention but persistent liver radioactivity.

Osteoclasts express high levels of α_vβ₃ (1, 10), and α_vβ₃ ligands have also been shown to inhibit osteoclastic bone resorption (11). The skeleton is one of the most common organs of cancer metastasis. Osteolytic bone lesions are difficult to detect, and molecular imaging agents that target osteoclasts in vivo can be useful for imaging osteolytic bone metastases and monitoring their responses to therapy. Boswell et al. (12) showed that the ⁶⁴Cu complex of the cross-bridged macrocyclic chelator 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) had less in vivo transchelation and improved liver clearance in normal rats than other cyclen derivatives. Sprague et al. (1) prepared ⁶⁴Cu-CB-TE2A-c(RGDyK)] and showed that this radioligand selectively bound to osteoclasts in mice with induced osteoclastogenesis.

Synthesis

[PubMed]

Sprague et al. (1) described the synthesis of ⁶⁴Cu-CB-TE2A-c(RGDyK). The cyclic peptide was prepared in three steps: solid-phase peptide synthesis, intramolecular cyclization in solution, and conjugation of peptide with CB-TE2A. Briefly, the orthogonally protected linear peptide (DTyr(Bu^t)-Lys(Dde)-Arg(Pbf)-Gly-Asp(OBu^t)-O-resin was prepared on a 2-chlorotrityl resin and cleaved with 1% trifluoroacetic acid (TFA) in dichloromethane. After cyclizing the protected peptide in solution, the Dde group was selectively removed with 1% hydrazine in methanol. CB-TE2A was conjugated to the free ε-amino lysine group of the peptide in the presence of diisopropylcarbodiimide and N-hydroxybenzotriazole in anhydrous N,N-dimethylformamide. All side-chain–protecting groups were removed with 95% aqueous TFA solution, and the final product was purified by HPLC. CB-TE2A-c(RGDyK) was radiolabeled by adding ⁶⁴Cu chloride to the conjugated peptide in 0.1 M ammonium acetate (pH 8.0), which was then heated for 45–60 min. The prepared ⁶⁴Cu-CB-TE2A-c(RGDyK) had >95% radiochemical purity. The specific activity was ≤103.6 MBq/μg (2.8 mCi/μg).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Sprague et al. (1) performed in vitro integrin binding assays with Cu(II)-CB-TE2A-c(RGDyK) to assess the effect of conjugation of CB-TE2A to c(RGDyK). The affinity of the peptide was assessed by a heterologous competitive binding assay with biotinylated vitronectin, which was recognized by α_vβ₃ and α_vβ₅ integrins as a competing ligand. The inhibition concentration (IC₅₀) values of Cu(II)-CB-TE2A-c(RGDyK) were 6.0 nM and 171 nM for α_vβ₃ and α_vβ₅, respectively. In comparison, the IC₅₀ values of unconjugated c(RGDyK) were 3.7 nM and 194 nM for α_vβ₃ and α_vβ₅, respectively. Cell uptake of ⁶⁴Cu-CB-TE2A-c(RGDyK) by α_vβ₅-positive bone marrow macrophages (BMMs) and α_vβ₃-positive osteoclasts were studied (1). It was found that osteoclast uptake of ⁶⁴Cu-CB-TE2A-c(RGDyK) was 2.6 ± 0.7 times higher than BMM uptake. Uptake of ⁶⁴Cu-CB-TE2A-c(RGDyK) by osteoclasts was blocked by increasing concentrations of c(RGDyK), whereas the uptake by BMMs was not blocked.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NCI R21 CA098698, AR03278, AR046523, AR048853, P30 CA91842, R24 CA86307, R24 CA83060, P30 CA91842.

References

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2.

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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]

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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]

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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]

9.

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]

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Horton M.A. The alpha v beta 3 integrin "vitronectin receptor". Int J Biochem Cell Biol. 1997; 29 (5):721–5. [PubMed: 9251239]

11.

Fisher J.E. , Caulfield M.P. , Sato M. , Quartuccio H.A. , Gould R.J. , Garsky V.M. , Rodan G.A. , Rosenblatt M. Inhibition of osteoclastic bone resorption in vivo by echistatin, an "arginyl-glycyl-aspartyl" (RGD)-containing protein. Endocrinology. 1993; 132 (3):1411–3. [PubMed: 8440195]

12.

Boswell C.A. , Sun X. , Niu W. , Weisman G.R. , Wong E.H. , Rheingold A.L. , Anderson C.J. Comparative in vivo stability of copper-64-labeled cross-bridged and conventional tetraazamacrocyclic complexes. J Med Chem. 2004; 47 (6):1465–74. [PubMed: 14998334]