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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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Pamidronate-IRDye78

PAM78
, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: January 5, 2012.

Chemical name:Pamidronate-IRDye78image 3131787 in the ncbi pubchem database
Abbreviated name:Pam78
Synonym:
Agent category:Compound
Target:Hydroxyapatite
Target category:Acceptor
Method of detection:Optical, near-infrared (NIR) fluorescence imaging
Source of signal\contrast:IRDye78
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on the above structure for additional information in PubChem.

Background

[PubMed]

Optical fluorescence imaging is increasingly used to obtain biological functions of specific targets in vitro and in small animals (1, 2). However, the intrinsic fluorescence of biomolecules poses a problem when visible light (350-700 nm) absorbing fluorophores are used because of high tissue absorption and scatter. Near-infrared (NIR) fluorescence (700-900 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background as a result of reduced scattering compared to visible fluorescence detection. They also have high sensitivity resulting from low infrared background, and high extinction coefficients, which provide high quantum yields. The NIR region was also compatible with solid-state optical components such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive alterative to radionuclide imaging in vitro and in small animals.

IRDye78 is a heptamethine indocyanine-type NIR fluorophore with peak absorption at 771 nm, and peak excitation emission at 806 nm. It provides a quantum yield of 14.2%. It has a molecular weight of 1083 Da. IRDye78 is a highly charged IR-786 derivative, which localized to mitochondria at low concentrations and endoplasmic reticulum (ER) at high concentrations in vitro (3). IRDye78 was shown to be a useful perfusion agent in myocardium. IRDye78 N-hydroxysuccinimide (NHS) ester can be conjugated to antibodies and low-molecular weight ligands with one or more free primary amines.

Osteoblasts (mineralization) and osteoclasts (demineralization) are two importance cell types in development and integrity of vertebral skeleton (4, 5). Osteoblast-like cells are present in vascular tissues and play a role in arteriosclerosis (6). Microcalcifications are found in breast tissue (7, 8). Hydroxyapatite (HA) is a mineral product deposited in the bone and vascular tissue by the osteoblast. HA binds pyrophosphates and phosphonates with high affinity. Nitrogen-containing synthetic diphosphonates are inhibitors of farnesyl diphosphate synthase (FDPS) of osteoclast and are used for treatment of osteoporosis (bone resorption) (9). Inhibition of FDPS inhibits osteoclast bone-resorption activity and induces osteoclast apoptosis (10, 11). Diphosphonates bind to bone mineral with high affinity for their long duration of action. For example, [99mTc]Methylene diphosphonate (MDP) was developed for bone scanning (12) and it is believed that it binds to HA. Pamidronate (Pam) is a diphosphonate derivative with a single primary amine for conjugation with IRDye78-NHS ester to form Pam78. Pam78 exhibits rapid and specific binding to HA in vitro and in vivo. Zaheer et al. (13) demonstrated NIR fluorescence detection of bone with Pam78 in nude mice. A simple and rapid rat model of focal calcification in breast cancer tumors was validated by Pam78 NIR imaging (14).

Synthesis

[PubMed]

IRDye78 monofunctional NHS ester reacted with the single primary amino group of Pam to form Pam78, which was purified using thin-layer chromatography and confirmed by mass spectroscopy. Pam78 had peak absorption at 771 nm and peak emission at 796 nm. The conjugation yields were 18-21% (13).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In a kinetic binding assay, Pam78 (5 μm) exhibited rapid binding to 1.5 mm HA, with 37% binding capacity achieved in 5 min and a start to plateau by 1 h. A similar concentration of IRDye78 showed no detectable binding to HA. Pam78 binding to HA was inhibited by Pam with an IC90 of 800 μm. HA was estimated to have a maximum binding capacity of 0.84 mmol of Pam78 per gram of HA (13).

Animal Studies

Rodents

[PubMed]

Nude mice received injections intravenously with 2.6 nmol of Pam78 or IRDye78. Peak serum concentration of 2.6 μm was reached within 1 min for both. IRDye78 exhibited a two-phase elimination from the plasma, with early and late half-lives of 7.2 and 24.7 min, respectively (3). Pam78 also showed a faster two-phase elimination, having early and late half-lives of 5.0 and 15.4 min, respectively (13).

NIR fluorescence reflectance imaging showed negligible autofluorescence of the mice. IRDye78 fluorescence was homogeneous in the entire mouse within 1 min and stable for the next 15 min (13). The dye was eliminated to near background by the urinary and biliary systems over the next 6 h. Pam78 showed a similar rapid distribution. However, Pam78 uptake in the spine, ribs, paws, and knees could be detected as early as 15 min. By 3 h, most bony structures were visible. At 6 h, most of the bones of the mouse could be visualized by high-resolution imaging. However, deep bone structures were either poorly visualized or not visualized at all as a result of skin and soft-tissue attenuation and scatter. MRI T1-weighted scans were used to measure the distance between the skin surface and the target bone. An estimation of intensity attenuation was 32% as compared with 44% obtained by actual NIR measurements with and without skin. Optical scatter is a major limitation to NIR planar imaging. There was a 57% uptake of Pam78 in the ribs as compared with 52% uptake of 99mTc-MDP.

Female rats were implanted with the R3230 mammary adenocarcinoma cell line in the mammary fat pad. After growth to 1-2 cm in diameter, tumors were implanted with 100 μm HA crystals (positive control) or calcium oxalate crystals (negative control). Twenty-four hours after crystal implantation, rats were injected intravenously with Pam78 (0.1 μmol/kg), and the tumors were imaged using a reflectance optical imaging system. Tumors implanted with HA crystals displayed bright, focal, NIR fluorescence in the area of crystal implantation. Control tumors, grown in the same animal and implanted with calcium oxalate, did not display any NIR fluorescence. A simple and rapid animal model of focal calcification in breast cancer tumors was validated by a NIR fluorescent agent specific for HA (14).

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

R21 CA88245, R21 CA88870, CA70362

References

1.
Achilefu S. Lighting up tumors with receptor-specific optical molecular probes. Technol Cancer Res Treat. 2004;3(4):393–409. [PubMed: 15270591]
2.
Ntziachristos V., Bremer C., Weissleder R. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur Radiol. 2003;13(1):195–208. [PubMed: 12541130]
3.
Nakayama A., del Monte F., Hajjar R.J., Frangioni J.V. Functional near-infrared fluorescence imaging for cardiac surgery and targeted gene therapy. Mol Imaging. 2002;1(4):365–77. [PubMed: 12940233]
4.
Marks S.C. Jr, Popoff S.N. Bone cell biology: the regulation of development, structure, and function in the skeleton. Am J Anat. 1988;183(1):1–44. [PubMed: 3055928]
5.
Seibel M.J. Bone metabolism, mineral homeostasis and its pharmacological modulation. Clin Lab. 2004;50(5-6):255–64. [PubMed: 15209433]
6.
Demer L.L., Tintut Y. Mineral exploration: search for the mechanism of vascular calcification and beyond: the 2003 Jeffrey M. Hoeg Award lecture. Arterioscler Thromb Vasc Biol. 2003;23(10):1739–43. [PubMed: 12958041]
7.
Bassett L.W. Mammographic analysis of calcifications. Radiol Clin North Am. 1992;30(1):93–105. [PubMed: 1732937]
8.
Bassett L.W. Digital and Computer-Aided Mammography. Breast J. 2000;6(5):291–293. [PubMed: 11348384]
9.
Body J.J. Rationale for the use of bisphosphonates in osteoblastic and osteolytic bone lesions. Breast. 2003;12 Suppl 2:S37–44. [PubMed: 14659142]
10.
Drake M.T., Clarke B.L., Khosla S. Bisphosphonates: mechanism of action and role in clinical practice. Mayo Clin Proc. 2008;83(9):1032–45. [PMC free article: PMC2667901] [PubMed: 18775204]
11.
Russell R.G., Rogers M.J. Bisphosphonates: from the laboratory to the clinic and back again. Bone. 1999;25(1):97–106. [PubMed: 10423031]
12.
Subramanian G., McAfee J.G., Blair R.J., Kallfelz F.A., Thomas F.D. Technetium-99m-methylene diphosphonate--a superior agent for skeletal imaging: comparison with other technetium complexes. J Nucl Med. 1975;16(8):744–55. [PubMed: 170385]
13.
Zaheer A., Lenkinski R.E., Mahmood A., Jones A.G., Cantley L.C., Frangioni J.V. In vivo near-infrared fluorescence imaging of osteoblastic activity. Nat Biotechnol. 2001;19(12):1148–54. [PubMed: 11731784]
14.
Lenkinski R.E., Ahmed M., Zaheer A., Frangioni J.V., Goldberg S.N. Near-infrared fluorescence imaging of microcalcification in an animal model of breast cancer. Acad Radiol. 2003;10(10):1159–64. [PubMed: 14587634]

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