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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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Cy5.5-3-Benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-methoxyphenyl)-4-(3,4,5-trimethoxybenzyl)-5H-furan-2-one)

Cy5.5-PD156707
, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: September 30, 2009.

Chemical name:Cy5.5-3-Benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-methoxyphenyl)-4-(3,4,5-trimethoxybenzyl)-5H-furan-2-one)
image 81080216 in the ncbi pubchem database
Abbreviated name:Cy5.5-PD156707, ETA-Cy5.5
Synonym:
Agent category:Compound
Target:Endothelin A receptor
Target category:Receptor
Method of detection:Optical, near-infrared (NIR) fluorescence imaging
Source of signal:Cy5.5
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about endothelin A receptor.

Background

[PubMed]

Optical fluorescence imaging is increasingly being used to monitor biological functions of specific targets in small animals (1-3). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues in small animals. NIR fluorophores have a wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. NIR fluorophores also have high sensitivity, attributable to low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a non-invasive alternative to radionuclide imaging in small animals.

Endothelin (ET) plays an important role in a variety of physiological processes and contributes to the development of diseases such as acute and chronic renal failure, atherosclerosis, hypertension, chronic heart failure, and pulmonary hypertension (4-6). All these effects are mediated through G-protein–coupled receptor binding. Three isoforms of ET (ET-1, ET-2, and ET-3) exist in mammalian tissues, each of which has 21 amino acids with two disulfide bonds. Two ET receptors (ETRs), ETAR and ETBR, have been identified (7, 8). Each receptor subtype is expressed in a variety of tissues, with some tissues expressing both types (9). Various growth factors and cytokines are known to regulate ET expression under physiological conditions (4). Stimulation of the ETAR by ET on underlying smooth muscle cells of the endothelium causes vasoconstriction that leads to an elevation of blood pressure and the development of hypertension (10, 11). Stimulation of the ETBR on the smooth muscle cells and endothelial cells results in the release of nitric oxide and prostacyclin resulting in vasodilation (12).

Positron emission tomography (13) is a sensitive imaging technique for investigation of the ETRs in a small animal model (12, 14-16). 18F-Labeled ET-1 was among the first ligands developed for in vivo PET study of these receptors (14, 17). ETAR has been found to be associated with tumor growth of various cancers (18, 19). As a result of the involvement of ETAR in cancers, it is necessary to assess the ETAR expression in tumor tissues. PD156707 is a potent and selective non-peptide ETAR antagonist with high affinity (20, 21). Holtke and colleagues (22, 23) prepared Cy5.5-labeled PD156707 (Cy5.5-PD156707) for initial NIR fluorescence imaging of ETAR expression in normal mice.

Synthesis

[PubMed]

Cy5.5 Monofunctional N-hydroxysuccinimide ester (1.3 µmol) was used to conjugate the amino PD156707 precursor (1.3 µmol) to form Cy5.5-PD156707, which was purified with high-performance liquid chromatography with 60% labeling yield and >95% chemical purity (22). The reaction was carried out for 1 h at room temperature. Cy5.5-PD156707 was identified with mass spectrometry. Cy5.5-PD156707 was adjusted to a final concentration of 800 nM. Cy5.5 has an absorbance maximum at 675 nm and an emission maximum at 694 nm with a high extinction coefficient of 250,000 M-1cm-1.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Doherty and Patt et al. (20) showed that PD156707 inhibited 125I-labeled ET-1 and 125I-labeled ET-3 binding to human cloned ETAR and ETBR with 50% inhibition concentration values of 0.3 and 420 nM, respectively. Cy5.5-PD156707 (12 nmol (0.02 ml)) was incubated in mouse plasma (0.075 ml) at 37°C (22). The amount of intact probe was 80% at 1 h, 40% at 5 h, and 20% at 24 h. Holtke et al. (22) used Cy5.5-PD156707 (2 nmol) to perform receptor binding assays using ETAR-positive MCF-7 human breast adenocarcinoma, HT-1080 human fibrosarcoma cells and ETAR-negative MDAMB-435 human breast carcinoma cells. HT-1080 cells exhibited a higher fluorescence signal than MCF-7 cells, whereas no fluorescence signal was detected in the MDAMB-435 cells. These results correlate with the ETAR expression in these cells. The fluorescence signal of HT-1080 cells was blocked by 20 nmol of PD156707.

Animal Studies

Rodents

[PubMed]

Holtke et al. (23) performed ex vivo biodistribution studies of Cy5.5-PD156707 in normal mice (n = 6–8/group). Near-infrared fluorescence was measured in brain, muscle, heart, lung, spleen, kidney, and liver at 10 min to 48 h after injection of 2 nmol Cy5.5-PD156707. The organ with the highest signal intensity at 10 min after injection was the lung (133,100 ± 14,554 photon counts (pc)/mg), followed by the kidney (83,012 ± 4,085 pc/mg), liver (78,616 ± 6,208 pc/mg), heart (50,142 ± 5,190 pc/mg), spleen (44,004 ± 1,546 pc/mg), and muscle (31,169 ± 1,600 pc/mg). The signal intensity was the lowest in the brain (13,749 ± 1,185 pc/mg). Subsequently, all organs measured showed increases in signal intensities at 3 and 5 h after injection with the highest increase in the lung. After 24–48 h, signal intensities decreased markedly from their peaks in all organs except the lung (243,169 pc/mg) with only slight attenuation. Pretreatment with PD156707 (2,000 nmol/mouse) 10 min before Cy5.5-PD156707 injection resulted in signal intensities decreases of ~50% in only the lung, heart, and liver at 1–5 h after injection. The authors mentioned that studies of Cy5.5-PD156707 in tumor tissues in vivo are in progress.

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.

References

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Becker A., Hessenius C., Licha K., Ebert B., Sukowski U., Semmler W., Wiedenmann B., Grotzinger C. Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands. Nat Biotechnol. 2001;19(4):327–31. [PubMed: 11283589]
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Arai H., Hori S., Aramori I., Ohkubo H., Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature. 1990;348(6303):730–2. [PubMed: 2175396]
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Sakurai T., Yanagisawa M., Takuwa Y., Miyazaki H., Kimura S., Goto K., Masaki T. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 1990;348(6303):732–5. [PubMed: 2175397]
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Davenport, A.P., Distribution of endothelin receptors. Endothelins in biology and medicine., ed. J.P. Huggins and J.T. Pelton. 1997, Boca Raton: CRC Press, Inc. 45-68.
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de Nucci G., Thomas G.R., D'Orleans-Juste P., Antunes E., Walder C., Warner T.D., Vane J.R. Pressor effects of circulating endothelin are limited by its removal in primary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc. Natl. Acad. Sci. USA. 1988;85(24):9797–800. [PMC free article: PMC282868] [PubMed: 3059352]
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Yokokawa K., Tahara H., Kohno M., Murakawa K., Yasunari K., Nakagawa K., Hamada T., Otani S., Yanagisawa M., Takeda T. Hyperstension associated with endothelin secreting malignant hemangioendothelioma. Ann Intern Med. 1991;114(3):213–5. [PubMed: 1984746]
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Johnstrom P., Fryer T.D., Richards H.K., Barret O., Clark J.C., Ohlstein E.H., Pickard J.D., Davenport A.P. In Vivo Imaging of Cardiovascular Endothelin Receptors Using the Novel Radiolabelled Antagonist [18F]-SB209670 and Positron Emission Tomography (microPET). J Cardiovasc Pharmacol. 2004;44:S34–S38. [PubMed: 15838315]
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Gao X., Yang L., Petros J.A., Marshall F.F., Simons J.W., Nie S. In vivo molecular and cellular imaging with quantum dots. Curr Opin Biotechnol. 2005;16(1):63–72. [PubMed: 15722017]
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Johnstrom P., Fryer T.D., Richards H.K., Harris N.G., Barret O., Clark J.C., Pickard J.D., Davenport A.P. Positron emission tomography using 18F-labelled endothelin-1 reveals prevention of binding to cardiac receptors owing to tissue-specific clearance by ET B receptors in vivo. Br J Pharmacol. 2005;144(1):115–22. [PMC free article: PMC1575985] [PubMed: 15644875]
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Johnstrom P., Harris N.G., Fryer T.D., Barret O., Clark J.C., Pickard J.D., Davenport A.P. (18)F-Endothelin-1, a positron emission tomography (PET) radioligand for the endothelin receptor system: radiosynthesis and in vivo imaging using microPET. Clin Sci (Lond) 2002;103 Suppl 48:4S–8S. [PubMed: 12193043]
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Johnstrom P., Rudd J.H., Richards H.K., Fryer T.D., Clark J.C., Weissberg P.L., Pickard J.D., Davenport A.P. Imaging endothelin ET(B) receptors using [18F]-BQ3020: in vitro characterization and positron emission tomography (microPET). Exp Biol Med (Maywood) 2006;231(6):736–40. [PubMed: 16740990]
17.
Johnstrom P., Aigbirhio F.I., Clark J.C., Downey S.P., Pickard J.D., Davenport A.P. Syntheses of the first endothelin-A- and -B-selective radioligands for positron emission tomography. J Cardiovasc Pharmacol. 2000;36(5) Suppl 1:S58–60. [PubMed: 11078336]
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Bagnato A., Rosano L. The endothelin axis in cancer. Int J Biochem Cell Biol. 2008;40(8):1443–51. [PubMed: 18325824]
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Smollich M., Wulfing P. Targeting the endothelin system: novel therapeutic options in gynecological, urological and breast cancers. Expert Rev Anticancer Ther. 2008;8(9):1481–93. [PubMed: 18759699]
20.
Doherty A.M., Patt W.C., Edmunds J.J., Berryman K.A., Reisdorph B.R., Plummer M.S., Shahripour A., Lee C., Cheng X.M., Walker D.M. et al. Discovery of a novel series of orally active non-peptide endothelin-A (ETA) receptor-selective antagonists. J Med Chem. 1995;38(8):1259–63. [PubMed: 7731010]
21.
Doherty A.M., Uprichard A.C. Discovery and development of an endothelin A receptor-selective antagonist PD 156707. Pharm Biotechnol. 1998;11:81–112. [PubMed: 9760677]
22.
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23.
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