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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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Mesenchymal-epithelial transition factor binding peptide-8-aminooctanoic acid conjugated to Cy5.5

cMBP-AOC-Cy5.5
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894

Created: ; Last Update: September 10, 2009.

Chemical name:Mesenchymal-epithelial transition factor binding peptide-8-aminooctanoic acid conjugated to Cy5.5
Image cMBPAOCCy55.jpg
Abbreviated name:c-MBP-AOC-Cy5.5
Synonym:
Agent Category:Ligand
Target:Mesenchymal-epithelial transition factor receptor (c-Met); hepatocyte growth factor receptor (HGFR)
Target Category:Receptor
Method of detection:Near-infrared fluorescence (NIR) imaging
Source of signal / contrast:Cy5.5
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure of cMBP-AOC-Cy5.5.

Background

[PubMed]

The hepatocyte growth factor (HGF) is the only known ligand of the mesenchymal-epithelial transition factor (c-Met) (also known as the HGF receptor) that has an important function, including cell development, proliferation, scattering, migration, and angiogenesis, all of which are necessary for the survival and repair of tissues (1). c-Met is known to mediate its activity through an intracellular tyrosine kinase (TK) that activates different signal transduction pathways, leading to initiation of the various cell processes (2). Also, deregulation of c-Met activity due to paracrine/autocrine activation, overexpression, or a mutation promotes the development of a neoplastic cell phenotype (2). Overexpression of c-Met and HGF has been associated with a poor prognosis and survival of the patient (3), suggesting that the c-Met receptor may be a suitable target for the detection of cancers at an early stage, which could help in the development of an appropriate treatment regimen to improve patient prognosis (4). c-Met is the target of several drugs under evaluation in clinical trials approved by the United States Food and Drug Administration. Investigators recently generated a monoclonal antibody (mAb) that inhibits the HGF–c-Met interaction and identified the active mAb epitope peptide as KSLSRHDHIHHH, which was designated as the c-Met binding peptide (cMBP) (5). The cMBP and its derivatives (containing either a tri-amino acid linker, Gly-Gly-Gly (GGG), or an aliphatic carbon linker, 8-aminooctanoic acid (AOC)) were labeled with 125I to generate 125I-cMBP, 125I-cMBP-GGG, and 125I-cMBP-AOC, and these agents were used to detect c-Met TK-positive tumor xenografts in mice (6). From this study the investigators concluded that although 125I-cMBP-AOC was a suitable imaging agent , and superior to 125I-cMBP-GGG for the imaging of c-Met–expressing tumors, the imaging signal to background ratio could probably be improved further either by changing the linker in the molecule or by using a different nuclide or source of signal (such as an optical imaging agentIn an attempt to generate a c-Met–positive tumor imaging agent superior to 125I-cMBP-AOC, investigators conjugated a near-infrared (NIR) fluorescent dye, cyanine 5.5 (Cy5.5), to cMBP-GGG and cMBP-AOC to obtain cMBP-GGG-Cy5.5 and cMBP-AOC-Cy5.5 (4). The Cy5.5-conjugated cMBP derivatives were then evaluated for the NIR imaging of c-Met–positive xenograft tumors in a mouse model.

This chapter describes the characterization of only cMBP-AOC-Cy5.5. The other c-Met imaging agents (125I-cMBP, 125I-cMBP-GGG, 125I-cMBP-AOC, and cMBP-GGG-Cy5.5) are described in separate MICAD chapters (7-10).

Synthesis

[PubMed]

The synthesis of cMBP and cMBP-AOC-Cy5.5 has been described previously (4). Briefly, the cMBP-AOC peptide was synthesized using Fmoc chemistry, and it was mixed with Cy5.5-maleimide for 6 h in the dark at room temperature. The AOC linker was bound to the C-terminus of cMBP and Cy5.5 was linked to the C-terminus of AOC. The crude cMBP-AOC-Cy5.5 product was purified with dialysis using a 2,000 molecular weight cut-off membrane. The typical yield of cMBP-AOC-Cy5.5 was reported to be >65%. The molecular mass of the purified cMBP-AOC-Cy5.5 was determined with matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. The final product was analyzed with reverse-phase high-performance liquid chromatography (RP-HPLC). The purity of cMBP-AOC-Cy5.5 was reported to be >90% as determined with RP-HPLC. The absorption and fluorescence characteristics of cMBP-AOC-Cy5.5 were determined in water with the use of an ultraviolet-visible spectrophotometer, and the agent’s spectra were reported to be similar to that of free Cy5.5. Stability of the Cy5.5-labeled peptide was not reported.

For some in vitro studies, 125I-labeled cMBP was also used, and this radiolabeled compound was prepared as described before (6).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

A soluble human c-Met/Fc chimeric protein was purchased from a commercial source and used for an in vitro 125I-cMBP displacement binding assay using cMBP and cMBP-AOC-Cy5.5, respectively (4). The 50% inhibitory concentration values of cMBP and cMBP-AOC-Cy5.5 were determined to be 1.5 μM and 342 nM, respectively.

In another study, human U87MG glioma cells were exposed to cMBP-AOC-Cy5.5 (2 nmol) in the presence of excess unlabeled cMBP (86 nmol) and fluorescence was acquired 1 h later (4).Cells exposed to cMBP-AOC-Cy5.5 alone were reported to show fluorescence that was inhibited by the presence of excess unlabeled cMBP. Confocal microscopy revealed that cMBP-AOC-Cy5.5 bound only to the cell surface and was not internalized by the cells.

Animal Studies

Rodents

[PubMed]

NIR fluorescence imaging was performed on athymic mice (the number of animals used per time point was not reported) bearing U87MG cell xenograft tumors at predetermined time points for up to 24 h after injection of cMBP-AOC-Cy5.5 through the tail vein (4). For comparison, another group of animals was injected with cMBP-GGG-Cy5.5. Fluorescence imaging of the two groups of animals showed that the signals from both conjugates were visible primarily in the tumor, kidney, and the bladder regions of the animals. The tumor/muscle (T/M) ratios for the two conjugates were generated for the different time points after imaging. With cMBP-AOC-Cy5.5, the ratio was observed to increase from ~3 at 10 min to ~33 at 17 h, and it remained stable thereafter for up to 24 h. A similar trend was noticed with cMBP-GGG-Cy5.5, but the fluorescence with this conjugate was consistently approximately three-fold lower than that obtained with cMBP-AOC-Cy5.5, indicating that the latter was a superior imaging agent for c-Met-expressing tumors compared with cMBP-GGG-Cy5.5. Blocking studies were performed by co-injecting the mice with cMBP-AOC-Cy5.5 in the absence or presence of excess cMBP (0.13 μmol) followed by imaging at designated time points as before. Compared with animals injected with cMBP-AOC-Cy5.5 alone, a low fluorescence signal was obtained (at all time points) from animals injected with the peptide mixture. This indicated that cMBP blocked the binding of cMBP-AOC-Cy5.5 to the xenograft tumors. To study the biodistribution of cMBP-AOC-Cy5.5 in the xenograft tumor mice, animals from the blocking study were euthanized after imaging, and all the major organs were collected to determine the amount of fluorescent dye accumulated in the tissues (4). Compared with all the harvested organs, a high signal was obtained from the tumor and kidneys of the animals, but the signal was reduced only in tumors from the blocked animals. From these studies the investigators concluded that cMBP-AOC-Cy5.5 was a superior imaging agent for c-Met-expressing tumors compared with cMBP-GGG-Cy5.5.

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

Supplemental Information

[Disclaimer]

No information is currently available.

References

1.
Naran S., Zhang X., Hughes S.J. Inhibition of HGF/MET as therapy for malignancy. Expert Opin Ther Targets. 2009;13(5):569–81. [PubMed: 19397476]
2.
Abounader R., Laterra J. Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis. Neuro Oncol. 2005;7(4):436–51. [PMC free article: PMC1871724] [PubMed: 16212809]
3.
Birchmeier C., Birchmeier W., Gherardi E., Vande Woude G.F. Met, metastasis, motility and more. Nat Rev Mol Cell Biol. 2003;4(12):915–25. [PubMed: 14685170]
4.
Kim E.M., Park E.H., Cheong S.J., Lee C.M., Jeong H.J., Kim D.W., Lim S.T., Sohn M.H. In vivo imaging of mesenchymal-epithelial transition factor (c-Met) expression using an optical imaging system. Bioconjug Chem. 2009;20(7):1299–306. [PubMed: 19534520]
5.
Kim K., Hur Y., Ryu E.K., Rhim J.H., Choi C.Y., Baek C.M., Lee J.H., Chung J. A neutralizable epitope is induced on HGF upon its interaction with its receptor cMet. Biochem Biophys Res Commun. 2007;354(1):115–21. [PubMed: 17214965]
6.
Kim E.M., Park E.H., Cheong S.J., Lee C.M., Kim D.W., Jeong H.J., Lim S.T., Sohn M.H., Kim K., Chung J. Characterization, biodistribution and small-animal SPECT of I-125-labeled c-Met binding peptide in mice bearing c-Met receptor tyrosine kinase-positive tumor xenografts. Nucl Med Biol. 2009;36(4):371–8. [PubMed: 19423004]
7.
Chopra, A., 125I labeled mesenchymal-epithelial transition factor binding peptide [125I-cMBP]. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -to current.
8.
Chopra, A., 125I labeled mesenchymal-epithelial transition factor binding peptide-Gly-Gly-Gly. [125I-cMBP-GGG]. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -to current.
9.
Chopra, A., 125I labeled mesenchymal-epithelial transition factor binding peptide-8-aminooctanoic acid linker. [125I-cMBP-AOC]. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -to current.
10.
Chopra, A., Mesenchymal-epithelial transition factor binding peptide-Gly-Gly-Gly-conjugated to Cy5.5 [cMBP-GGG-Cy5.5]. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -to current.
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