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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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4-[18F]Fluorobenzoyl-knottin 2.5D

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD

Created: ; Last Update: September 23, 2010.

Chemical name:4-[18F]Fluorobenzoyl-knottin 2.5D
Abbreviated name:[18F]FB-2.5D
Agent Category:Peptide
Target:αvβ3, αvβ5, and α5β1 integrin receptors
Target Category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:18F
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.



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). 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 αvβ3. Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (10).

Most cyclic RGD peptides are composed of five amino acids. Haubner et al. (11) reported that various cyclic RGD peptides exhibit selective inhibition of binding (expressed as 50% inhibitory concentration (IC50)) to αvβ3 (IC50, 7–40 nM) but not to αvβ5 (IC50, 600–4,000 nM) or αIIbβ3 (IC50, 700–5,000 nM) integrins. Various radiolabeled cyclic RGD peptides have been found to have high accumulation in tumors in nude mice (12). Only one cyclic RGD peptide used for imaging, [18F]fluoropropionyl-galacto-c(Arg-Gly-Asp-d-Phe-Lys) ([18F]-galacto-RGD), has been investigated for measuring expression of αvβ3 integrin in cancer patients with tumors. However, [18F]-galacto-RGD has been shown to have low tumor accumulation and low signal/background ratios. Cystine knot peptides (knottins) share a common disulfide-bonded framework and a triple-stranded β-sheet fold (13). The integrin-binding RGD motif was grafted into a knottin from trypsin inhibitor II of the squash plant (Ecballium elaterium). Knottin 2.5D (with three disulfide bonds; (GCPQGRGDWAPTSCSQDSDCLAGCVCGPNGFCG-NH2) was identified from a series of genetically engineered knottin peptides to have nanomolar binding to the αvβ3, αvβ5, and α5β1 integrin receptors (14, 15). Knottin 2.5D was labeled with N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) to form 4-[18F]fluorobenzoyl-knottin 2.5D ([18F]FB-2.5D) and evaluated for positron emission tomography (PET) imaging of tumor xenografts in mice (16).



Knottin 2.5D was prepared with solid-phase peptide synthesis (16). The linear peptide was folded in the presence of 2.5 mM reduced glutathione and 20% dimethylsulfoxide (DMSO) in 0.1 M ammonium bicarbonate (pH, 9). Knottin 2.5D was purified with high-performance liquid chromatography. Peptide purity and molecular mass confirmed with MALDI-TOF mass spectroscopy and electrospray ionization mass spectrometery. To label with 18F, knottin 2.5D was incubated with 18F-SFB in DMSO for 1 h at 60°C. [18F]FB-2.5D was purified with HPLC. The total synthesis and purification time was 3 h with a radiochemical yield of 28% at the end of synthesis (EOS). The radiochemical purity and specific activity were >95% and ~100 GBq/µmol (2.7 Ci/µmol) at EOS, respectively.

In Vitro Studies: Testing in Cells and Tissues


To determine the relative integrin-binding affinities (expressed as IC50 values) of 2.5D, FB-2.5D, and echistatin, a competition binding assay was performed with 125I-echistatin using U87MG human glioblastoma cells (16). The IC50 values for 2.5D, FB-2.5D, and echistatin were determined to be 20.3 ± 7.3, 13.2 ± 5.4, and 3.7 ± 1.1 nM, respectively.

Animal Studies



Miao et al. (16) performed ex vivo biodistribution studies of [18F]FB-2.5D (0.74 MBq (20 μCi)) in nude mice (n = 3/group) bearing U87MG xenografts at 0.5 h after intravenous injection. The tracer accumulation in the tumors was 1.9 ± 1.2% injected dose per gram (ID/g). The organ with the highest radioactivity was the kidney (4.2 ± 0.2% ID/g), followed by the liver (1.4 ± 0.6% ID/g) and blood (1.3 ± 0.1% ID/g). The radioactivity in the bone was <0.5% ID/g. Co-administration of excess c(RGDyK) decreased tumor accumulation by 70% at 0.5 h after injection (P < 0.05), whereas little inhibition was observed in the kidneys, liver, and bone. PET analysis was performed in nude mice bearing U87MG tumors after intravenous injection of ~4.4 MBq (120 μCi) [18F]FB-2.5D. Tumors were clearly visualized at 0.5–1 h along with the kidneys, gall bladder, and urinary bladder. Tumor accumulation was determined to be 2.6% ID/g at 0.5 h and 1.5% ID/g at 1 h. Co-administration of excess c(RGDyK) decreased tumor accumulation by 75% at 0.5 h after injection (P < 0.05).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.

NIH Support

01 CA104706, 5R01 CA119053, R24 CA93862, P50 CA114747.


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