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68Ga-1,4,7-Triazacyclononane-1,4-7-triacetic acid-Glu-c(RGDyK)-bombesin[7-14]

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

Created: ; Last Update: January 28, 2010.

Chemical name:68Ga-1,4,7-Triazacyclononane-1,4-7-triacetic acid-Glu-c(RGDyK)-bombesin[7-14]
Abbreviated name:68Ga-NOTA-RGD-BBN
Agent category:Peptide
Target:Gastrin-releasing peptide receptor (GRPR), integrin αvβ3
Target category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal\contrast:68Ga
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about integrin αvβ3.



The amphibian bombesin (BBN or BN, a peptide of 14 amino acids) is an analog of human gastrin-releasing peptide (GRP, a peptide of 27 amino acids) that binds to GRP receptors (GRPR) with high affinity and specificity (1, 2). Both GRP and BBN share an amidated C-terminus sequence homology of seven amino acids, Trp-Ala-Val-Gly-His-Leu-Met-NH2. BBN-Like peptides have been shown to induce various biological responses in diverse tissues, including the central nervous system (CNS) and the gastrointestinal (GI) system. They also act as potential growth factors for both normal and neoplastic tissues (3). Specific BBN receptors (BBN-R) have been identified on CNS and GI tissues and on a number of tumor cell lines (4). The BBN-R superfamily includes at least four different subtypes, namely the GRPR subtype (BB2), the neuromedin B (NMB) receptor subtype (BB1), the BB3 subtype, and the BB4 subtype. The findings of GRPR overexpression in various human tumors, such as breast, prostate, lung, colon, ovarian, and pancreatic cancers, provide opportunities for tumor imaging by designing specific molecular imaging agents to target the GRPR (5, 6).

Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (7). 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 (8-13). 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. A peptide 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. Various ligands have been introduced for imaging of tumors and tumor angiogenesis (14).

Because breast and prostate cancers express GRPR and αvβ3, Liu et al. (15) designed an RGD-BBN heterodimer in which BBN[7-14] and c(RGDyK) are connected with a glutamate linker (BBN on the Glu side chain γ-carboxylate group and RGD on the Glu side chain α-carboxylate group). A spacer, 11-amino-3,6,9-trioxaundecanoic acid (PEG3), was put onto the glutamate α-amino group of Glu-RGD-BBN to increase the hydrophilicity and to relieve the steric hindrance. N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was used to synthesize [18F]FB-PEG3-RGD-BBN for tumor targeting. Liu et al. (16) used 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA) as a bifunctional chelator for labeling RGD-BBN to form 68Ga-NOTA-RGD-BBN for positron emission tomography (PET) imaging of αvβ3 and GRPR in nude mice bearing human tumors (15, 17).

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RGD-BBN was prepared with solid-phase peptide synthesis with subsequent addition of a NOTA group to form NOTA-RGD-BBN (15). Addition of a NOTA group to RGD-BBN was performed by mixing 2 µmol RGD-BBN with 6 µmol p-SCN-Bz-NOTA in sodium bicarbonate buffer (pH 9) for 5 h at room temperature. NOTA-RGD-BBN was isolated with high-performance liquid chromatography (HPLC) with 52% yield. Measurement with matrix-assisted laser desorption ionization time of flight mass spectrometry indicated the molecular mass to be m/z 2,235.3 (calculated molecular weight, 2,234.6). For 68Ga labeling, a solution of 148 MBq (4 mCi) 68Ga and 5 nmol NOTA-RGD-BBN in acetate buffer (pH 5) was heated for 15 min at 40°C (16). 68Ga-NOTA-RGD-BBN was purified with HPLC with a yield of 90% and a radiochemical purity of >98%. The specific activity was 7.4–14.8 GBq/µmol (0.2–0.4 Ci/µmol) at the end of synthesis. The total preparation time was ~45 min. 68Ga-NOTA-BBN and 68Ga-NOTA-RGD as controls were also prepared.

In Vitro Studies: Testing in Cells and Tissues


Liu et al. (16) performed in vitro inhibition studies of NOTA-RGD-BBN in cultured U87MG cells with 125I-c(RGDyK). The 50% inhibition concentration (IC50) value was 22.6 ± 6.7 nM. The IC50 values for RGD-BBN and c(RGDyK) were 17.9 ± 5.7 and 11.2 ± 4.2 nM, respectively. In vitro inhibition studies of NOTA-RGD-BBN were also performed in cultured PC-3 cells with 125I-BBN, with an IC50 value of 55.9 ± 4.2 nM. The IC50 values for RGD-BBN and BBN were 67.9 ± 5.0 and 79.0 ± 4.9 nM, respectively. PC-3 cells exhibited 6.7%, 1.1% and 0.1% uptake of the incubation dose for 68Ga-NOTA-BBN, 68Ga-NOTA-RGD-BBN, and 68Ga-NOTA-RGD respectively, within 60 min of incubation at 37°C.

Animal Studies



Liu et al. (16) performed PET imaging of PC-3 tumor-bearing mice (n = 4/group) after injection of 3.7 MBq (100 uCi) of 68Ga-NOTA-BBN, 68Ga-NOTA-RGD-BBN, or 68Ga-NOTA-RGD. The estimated accumulation of 68Ga-NOTA-RGD-BBN in the tumors was 6.55 ± 0.83% injected dose per gram (ID/g) at 30 min, and it decreased to 5.26 ± 0.32% ID/g at 1 h and 4.04 ± 0.28% ID/g at 2 h after injection. The tumor accumulation of 68Ga-NOTA-RGD-BBN was higher that of 68Ga-NOTA-BBN, and 68Ga-NOTA-RGD at these time points (P < 0.05). The liver uptake of the three tracers were <2% ID/g at these time points. The kidney uptake of 68Ga-NOTA-RGD-BBN, and 68Ga-NOTA-BBN (9-10% ID/g) at 30 min after injection was higher than 68Ga-NOTA-RGD (5% ID/g) (P < 0.05). The tumor/muscle ratios were 6, 4, and 3 for 68Ga-NOTA-BBN, 68Ga-NOTA-RGD-BBN, and 68Ga-NOTA-RGD at 60 min after injection, respectively. In another study, BBN, c(RGDyK), or BBN+c(RGDyK) were co-injected with 68Ga-NOTA-RGD-BBN in mice bearing PC-3 tumors (n = 4/group). Tumor accumulation at 1 h after injection (5.26% ID/g) was only partially inhibited by either BBN (1.61% ID/g) or c(RGDyK) (2.08% ID/g) alone. However, BBN+c(RGDyK) reduced tumor accumulation to the background level (0.42% ID/g). PET scans were also performed after intravenous injection of 3.7 MBq (100 μCi) 68Ga-NOTA-BBN, 68Ga-NOTA-RGD-BBN, or 68Ga-NOTA-RGD in nude mice (n = 4/group) bearing MDA-MB-435 human breast tumors with moderate expression of including αvβ3 and undetectable expression of GRPR at 60 min. The estimated accumulation in the MDA-MB-435 tumors was 0.38%, 3.23% and 1.63% ID/g at 60 min, respectively. Ex vivo biodistribution studies showed that the tissue with the highest accumulation was the pancreas, followed by the kidney, PC-3 tumor, intestine, stomach, liver, blood, and muscle at 0.5 and 1 h after injection.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

R01 CA119053, R21 CA121842, P50 CA114747, U54 CA119367


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