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 the αvβ3 integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. The αvβ3 antagonists are being studied as antitumor and antiangiogenic agents, and the agonists are being studied as angiogenic agents for coronary angiogenesis (6, 8, 9). A tripeptide 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 and αvβ6. Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (10).
The αvβ6 integrin plays an important role in the development of epithelial cells and is nearly undetectable in adult normal tissues. However, levels of the αvβ6 integrin can be upregulated during tissue remodeling and wound healing (11). On the other hand, αvβ6 integrin is strongly expressed on tumor cells of the oral cavity, pancreas, breast, ovary, colon, and stomach (12-14). The αvβ6 integrin affects tumor growth, tumor invasiveness, and metastasis (13). αvβ6 binds to the RGD motif in fibronectin, tenascin, and the viral protein 1 (VP1) of the foot-and-mouth disease virus (FMDV) (15). FMDV binds to cells through the RGD motif of the GH loop of the VP1. A consensus αvβ6-binding motif DLXXL was identified with phage display screening with minimal binding to αvβ3, αIIbβ3, and αvβ5 (16). A peptide of 20 amino acids, NAVPNLRGDLQVLAQKVART (A20FMDV2), was identified as a ligand binding to αvβ6 integrin (15). A20FMDV2 was radiolabeled with N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) to study in vivo biodistribution of the tracer in tumor-bearing mice (17). [18F]FB-A20FMDV2 was found to have specific accumulation in αvβ6-positive tumor.
Hausner et al. (17) reported that of [18F]FB-A20FMDV2. A20FMDV2 was prepared by standard solid-phase peptide synthesis. [18F]SFB was synthesized in two steps from ethyl 4-(trimethylammoniumtrifluoromethanesulfonate) benzoate by nucleophilic fluorination ([18F]KF/Kryptofix 2.2.2./K2CO3), followed by saponification of the ethyl ester protecting group in 50 min using a chemistry process control unit with >99% radiochemical purity. The decay-corrected yields of [18F]SFB were 70% on the basis of starting [18F]KF. [18F]SFB was used to conjugate the N-terminus of A20FMDV2 in solid phase to form [18F]FB-A20FMDV2. Radiochemical purities of >98% were obtained with a decay-corrected radiochemical yield of 3.6% after high-performance liquid chromatography purification. The specific activity of [18F]FB-A20FMDV2 (n = 13) was 37 GBq/μmol (1 Ci/μmol) at end of synthesis with a total synthesis time of 130 min from the end of bombardment.
In Vitro Studies: Testing in Cells and Tissues
Using competitive binding to immobilized αvβ6, A20FMDV2 and FB-A20FMDV2 exhibited similar 50% inhibition concentration values (3 ± 1 nM) to inhibit αvβ6-fibronetin binding, >1,000-fold more selective for αvβ6 than for other RGD-directed integrins (α5β1, αvβ3, and αvβ5) (17). A receptor-binding assay with biotinylated A20FMDV2 using human melanoma DX3puro (αvβ6-negative) and DX3puroβ6 (αvβ6-positive) cell lines was analyzed with flow cytometry. The α5β1, αvβ3, αvβ5, αvβ6, and αvβ8 integrins were present on both cell lines. A20FMDV2 bound well at 1–100 nM to DX3puroβ6 cells but not to DX3puro, which suggests that A20FMDV2 is highly selective for αvβ6.
Hausner et al. (17) performed biodistribution studies of [18F]FB-A20FMDV2 in nude mice (n = 3/group) bearing DX3puroβ6 and DX3puro xenografts on opposite flanks in the shoulder region. Data were obtained at 1, 2, and 4 h after injection. [18F]FB-A20FMDV2 had a rapid blood clearance with 0.27, 0.06, and 0.03% injected dose (ID)/g at 1, 2, and 4 h. The tracer radioactivity accumulation in the αvβ6-expressing DX3puroβ6 tumor was ~0.66% ID/g at 1 h and decreased to 0.28% ID/g at 2 h and 0.06% ID/g at 4 h. The tracer radioactivity accumulation in the αvβ6-negative DX3puro tumor was ~0.21% ID/g at 1 h and decreased to 0.07% ID/g at 2 h and 0.02% ID/g at 4 h. At 60 min, the highest radioactive concentration was found in the urine (1,082% ID/g), followed by the gallbladder (15% ID/g) and kidneys (3.6% ID/g). The spleen, bone, and liver showed low levels of radioactivity. [18F]FB-A20FMDV2 positron emission tomography imaging studies were performed at 15 min to 5 h after injection in mice (n = 6) bearing both tumors. The DX3puroβ6 tumor was clearly visualized as early as 15 min and as late as 5 h after injection. Considerable radioactivity accumulation was observed in the urinary bladder (urine), gallbladder, and kidneys. Tumor/background ratios were ~1.0 (DX3puro) and 2.2 (DX3puroβ6) at 1 h after injection. Pre-administration of unlabeled FB-A20FMDV2 blocked tumor uptake of radioactivity by 32% in the DX3puroβ6 tumor and <1% in the DX3puro tumor. The partial inhibition exhibited by the DX3puroβ6 tumor may be caused by the rapid blood and renal clearance of FB-A20FMDV2. Both tumors showed a similar accumulation of the metabolic tracer 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG).
Other Non-Primate Mammals
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Created: March 16, 2008; Last Update: May 12, 2008.
National Center for Biotechnology Information (US), Bethesda (MD)
Leung K. 4-[18F]Fluorobenzoyl-NAVPNLRGDLQVLAQKVART. 2008 Mar 16 [Updated 2008 May 12]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.