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64Cu-1,4,7-Triazacyclononane-1,4-7-triacetic acid-Glu-c(RGDyK)-bombesin[7-14]

64Cu-NOTA-RGD-BBN
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: January 28, 2010.

Chemical name:64Cu-1,4,7-Triazacyclononane-1,4-7-triacetic acid-Glu-c(RGDyK)-bombesin[7-14]
Abbreviated name:64Cu-NOTA-RGD-BBN
Synonym:
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:64Cu
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about integrin αvβ3.

Background

[PubMed]

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 both 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]SFB-PEG3-RGD-BBN for tumor targeting. Liu et al. used 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA) as a bifunctional chelator for labeling RGD-BBN to form 64Cu-NOTA-RGD-BBN for positron emission tomography (PET) imaging of αvβ3 and GRPR in nude mice bearing human tumors (15, 16).

Synthesis

[PubMed]

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 64Cu labeling, a solution of 74 MBq (2 mCi) 64CuCl2 and 5 nmol NOTA-RGD-BBN in acetate buffer (pH 5) was heated for 15 min at 40°C. 64Cu-NOTA-RGD-BBN was purified with HPLC with a yield of 92% and a radiochemical purity of >98%. The specific activity was 7.4–14.8 GBq/µmol (0.2–0.4 Ci/µmol). The total preparation time was ~40 min.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Liu et al. (15) performed in vitro inhibition studies of NOTA-RGD-BBN in cultured U87MG cells with 125I-c(RGDyK). The 50% inhibition concentration (IC50) value was 16.2 ± 2.8 nM. In vitro inhibition studies of NOTA-RGD-BBN were also performed in cultured PC-3 cells with 125I-BBN, with an IC50 value of 92.8 ± 3.5 nM. The NOTA-RGD-BBN heterodimer exhibited comparable binding affinities for αvβ3 integrin with GRPR and the αvβ3 integrin receptor as the parent RGD and BBN peptides. T47D breast cancer cells were shown to express a moderate level of GRPR and a high level of the αvβ3 integrin. On the other hand, MDA-MB-435 breast cancer cells exhibited a very low level of GRPR and a high level of the αvβ3 integrin. The bindings to both cell lines were specific as the respective cold ligands decreased cell-bound radioactivity to background level. T47D and MDA-MB-435 cells exhibited 5.3% and 2.7% uptake of the incubation dose of 64Cu-NOTA-RGD-BBN, respectively, within 120 min of incubation at 37°C.

Animal Studies

Rodents

[PubMed]

Liu et al. (16) performed ex vivo biodistribution studies in normal mice (n = 4/group) at 1 h after injection of 0.37 MBq (0.01 mCi) 64Cu-NOTA-RGD-BBN. The organ with the highest accumulation was the pancreas, followed by the liver, kidney, intestine, stomach, blood, and muscle. Co-injection with BBN, c(RGDyK), or BBN+c(RGDyK) experiments were also performed. Little inhibition with c(RGDyK) was observed in most tissues, with the exception of moderate inhibition in the pancreas. BBN exhibited inhibition only in the pancreas, stomach, and intestine. BBN+c(RGDyK) showed inhibition similar to BBN but also showed strong inhibition in the kidney. PET scans were performed after intravenous injection of 3.7 MBq (100 μCi) 64Cu-NOTA-RGD-BBN in nude mice (n = 4/group) bearing T47D and MDA-MB-435 human breast tumors at 30, 60, and 120 min (15). The estimated accumulation in the T47D tumors was 3.73 ± 0.81% injected dose per gram (ID/g) at 30 min, and it decreased to 2.33 ± 0.59% ID/g at 1 h and 1.97 ± 0.32% ID/g at 2 h after injection. The estimated accumulation in the MDA-MB-435 tumors was 3.42 ± 1.23% ID/g at 30 min, and it decreased to 1.84 ± 0.44% ID/g at 1 h and 1.47 ± 0.19% ID/g at 2 h after injection. The organs with the highest radioactivity were the kidney (4.32% ID/g at 60 min) and liver (3.28% ID/g at 60 min). The tumor/blood, tumor/liver, tumor/kidney, and tumor/muscle ratios were 2, <1, <1, and 3, respectively. On the other hand, [18F]FB-PEG3-RGD-BBN exhibited higher tumor/organ ratios than 64Cu-NOTA-RGD-BBN. In later PET experiments, Liu et al. (16) showed that 64Cu-NOTA-RGD-BBN exhibited significantly higher tumor uptake than did 64Cu-NOTA-RGD, 64Cu-NOTA-BBN, the mixture of 64Cu-NOTA-RGD and 64Cu-NOTA-BBN, or 64Cu-DOTA-RGD-BBN in PC-3 prostate tumors (n = 4/group). The organs with the highest radioactivity were the kidney and liver. However, 64Cu-NOTA-RGD-BBN showed lower liver and intestinal activity accumulation than did the BBN tracers. BBN, c(RGDyK), or BBN+c(RGDyK) were co-injected with 64Cu-NOTA-RGD-BBN in mice bearing 4T1 breast tumors (n = 3/group). Tumor accumulation at 1 h after injection (2.78% ID/g) was only partially inhibited by either BBN (0.76% ID/g) or c(RGDyK) (1.64% ID/g) alone. However, BBN+c(RGDyK) reduced tumor accumulation to the background level (0.54% ID/g).

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

R01 CA120188, R01 CA119053, R21 CA121842, R21 CA102123, P50 CA114747, U54 CA119367, R24 CA93862

References

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Chung D.H., Evers B.M., Beauchamp R.D., Upp J.R. Jr, Rajaraman S., Townsend C.M. Jr, Thompson J.C. Bombesin stimulates growth of human gastrinoma. Surgery. 1992;112(6):1059–65. [PubMed: 1455308]
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Hynes R.O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69(1):11–25. [PubMed: 1555235]
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Jin H., Varner J. Integrins: roles in cancer development and as treatment targets. Br J Cancer. 2004;90(3):561–5. [PMC free article: PMC2410157] [PubMed: 14760364]
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Varner J.A., Cheresh D.A. Tumor angiogenesis and the role of vascular cell integrin alphavbeta3. Important Adv Oncol. 1996:69–87. [PubMed: 8791129]
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Wilder R.L. Integrin alpha V beta 3 as a target for treatment of rheumatoid arthritis and related rheumatic diseases. Ann Rheum Dis. 2002;61 Suppl 2:ii96–9. [PMC free article: PMC1766704] [PubMed: 12379637]
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Grzesik W.J. Integrins and bone--cell adhesion and beyond. Arch Immunol Ther Exp (Warsz) 1997;45(4):271–5. [PubMed: 9523000]
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Kumar C.C. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets. 2003;4(2):123–31. [PubMed: 12558065]
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Ruegg C., Dormond O., Foletti A. Suppression of tumor angiogenesis through the inhibition of integrin function and signaling in endothelial cells: which side to target? Endothelium. 2002;9(3):151–60. [PubMed: 12380640]
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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]
15.
Liu Z., Yan Y., Chin F.T., Wang F., Chen X. Dual integrin and gastrin-releasing peptide receptor targeted tumor imaging using 18F-labeled PEGylated RGD-bombesin heterodimer 18F-FB-PEG3-Glu-RGD-BBN. J Med Chem. 2009;52(2):425–32. [PubMed: 19113865]
16.
Liu Z., Li Z.B., Cao Q., Liu S., Wang F., Chen X. Small-animal PET of tumors with (64)Cu-labeled RGD-bombesin heterodimer. J Nucl Med. 2009;50(7):1168–77. [PubMed: 19525469]
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