Background

[PubMed]

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 α_vβ₃ integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (2-7). Expression of the α_vβ₃ integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. The α_vβ₃ antagonists are being studied as antitumor and antiangiogenic agents, and the agonists are being studied as angiogenic agents for coronary angiogenesis (6, 8, 9). The peptide sequence 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 α_vβ₃. 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. Various cyclic RGD peptides exhibit selective inhibition of binding to α_vβ₃ (50% inhibition concentration (IC₅₀), 7–40 nM) but not to α_vβ₅ (IC₅₀, 600–4,000 nM) or α_IIbβ₃ (IC₅₀, 700–5,000 nM) integrins (11). Various radiolabeled cyclic RGD peptides and peptidominetics have been found to have high accumulation in tumors in mice (12, 13). Out of these developments [¹⁸F]Galacto-c(RGDfK) has been evaluated in a number of clinical studies for imaging of α_vβ₃ in cancer patients (14-19). Liu et al. (20) used 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA) as a bifunctional chelator for labeling Glu-[15-amino-4,7,10,13-tetraoxapentadecanoic acid-cyclo(RGDfK)]₂ (P₄-RGD2) to form (⁶⁸Ga-NOTA-P₄-RGD2) for positron emission tomography (PET) imaging of α_vβ₃ receptors in nude mice bearing human glioblastoma U87MG tumors.

Synthesis

[PubMed]

P₄-RGD2 was prepared with solid-phase peptide synthesis (20). Addition of the NOTA group to P₄-RGD2 was performed by mixing 2 µmol P₄-RGD2 with 6 μmol S-2-(4-isothiocyanatobenzyl)-NOTA in sodium bicarbonate buffer (pH 9) for 5 h at room temperature. NOTA-P₄-RGD2 was isolated with high-performance liquid chromatography (HPLC) with 42% yield and >95% purity. Molecular weight was determined with MALDI-TOF-MS to be m/z 2,265.80 Da (calculated molecular weight, 2,263.52 Da). For ⁶⁸Ga labeling, a solution of 185 MBq (5 mCi) ⁶⁸GaCl₃ and 10 nmol RGD2 was heated for 10 min at 42°C. ⁶⁸Ga-NOTA-P₄-RGD2 was purified with HPLC with a yield of 92% and a radiochemical purity of >98%. The specific activity was 9.8–11.8 MBq/nmol (0.26–0.32 mCi/nmol).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Liu et al. (20) performed in vitro inhibition studies of NOTA-P₄-RGD2 in cultured human U87MG cells with ¹²⁵I-echistatin. The IC₅₀ values were 88.8, 41.8, 100.0, and 34.0 nM for RGD2, P₄-RGD2, NOTA-RGD2, and NOTA-P₄-RGD2, respectively. These comparable IC₅₀ values indicated that NOTA and P₄ conjugation had little effect on the receptor binding affinity.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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