⁶⁴Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-Asp-cyclohexylalanine-Phe-D-Ser-D-Arg-Tyr-Leu-Trp-Ser

[⁶⁴Cu-DOTA-AE105]

Background

[PubMed]

Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (1). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play an important role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). Invasive tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degradation of the basement membrane and ECM (3). Several families of proteases are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastasis (4-7).

Urokinase-type plasminogen activator (uPA) is a serine protease (8, 9). The uPA and uPA receptor (uPAR) system is responsible for tissue degradation after plasminogen activation to plasmin, which leads to a cascade of proteolysis or thrombolysis depending on the physiological conditions. uPA also directly activates MMPs, vascular endothelial growth factor, and human growth factor (10). Malignant tumors often express high levels of uPA and uPAR (11); therefore, the uPA/uPAR system is linked to vascular diseases and cancer. A synthetic peptide, Asp-cyclohexylalanine-Phe-D-Ser-D-Arg-Tyr-Leu-Trp-Ser (AE105), has been identified to have high affinity (dissociation constant (K_d) = 0.4 nM) for human uPAR (12), and it has been labeled with ⁶⁴Cu as ⁶⁴Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-AE105 (⁶⁴Cu-DOTA-AE105) for use in positron emission tomography (PET) imaging of uPAR expression in tumors (13).

Synthesis

[PubMed]

Li et al. (13) performed ⁶⁴Cu labeling of AE105 through DOTA chelation. DOTA was activated by a five-fold molar excess of N-hydroxysuccinimide (pH 8) for 60 min. A mixture of AE105 and DOTA-OSSu in a molar ratio of 1:5 was incubated for 18 h at 4ºC. The DOTA-AE105 conjugate was purified with high-performance liquid chromatography (HPLC) with a 52% yield. The number of DOTA molecules per peptide was ~1. For radiolabeling, 20 μg DOTA-AE105 was added to 74 MBq (2 mCi) ⁶⁴CuCl₂ diluted in 0.1 M sodium acetate buffer (pH 6.5). The reaction mixture was incubated for 1 h at 50ºC. ⁶⁴Cu-DOTA-AE105 was purified with HPLC (>98% radiochemical purity) with a radiolabeling yield of 80–90% (decay-corrected). ⁶⁴Cu-DOTA-AE105_mut, used as an inactive control, was prepared similarly. The specific activity of ⁶⁴Cu-DOTA-AE105 and ⁶⁴Cu-DOTA-AE105_mut was ~23 GBq/μmol (0.62 Ci/μmol).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Li et al. (13) performed binding experiments with AE105, DOTA-AE105, and DOTA-AE105_mut with the use of a Biacore sensor chip immobilized with uPAR. The K_d value of AE105 was calculated to be 15 nM compared with the K_d value of 53 nM for DOTA-AE105; therefore, the binding affinity of DOTA-AE105 was 3.5-fold lower than the parent AE105. DOTA-AE105_mut binding was not detected up to 1,000 nM. AE105 and DOTA-AE105 inhibited the binding of pro-uPA to the immobilized uPAR with 50% inhibition concentration values of 16 and 130 nM, respectively. DOTA-AE105_mut exhibited little inhibition.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

R01 CA119053, R21 CA121842, R21 CA102123, P50 CA114747, U54 CA119367, R24 CA93862