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

Created: ; Last Update: January 5, 2012.

Chemical name:64Cu-{N-[1,4,8,11-Tetraazacyclotetradecanyl-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine}
Abbreviated name:64Cu-AMD3465
Agent category:Compound
Target:Chemokine receptor 4 (CXCR4)
Target category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal:64Cu
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.



Chemokine receptors are G-protein–coupled receptors that direct cell movement (when activated by a ligand) toward higher concentrations of chemokines. Chemokine receptor 4 (CXCR4) and its ligand, stromal cell–derived factor-1 (SDF-1 or CXCL12), are known to play a major role in the migration of progenitor cells during embryonic development of the central nervous, cardiovascular, and hematopoietic systems (1, 2). In addition, this CXCR4/SDF-1 receptor system has a function in the development, progression, and spread of various cancers (3), and the CXCR4 acts as a co-receptor for human immunodeficiency virus (HIV) on CD4+ T cells (4). It has also been suggested that CXCR4/SDF-1 interaction participates in the pathogenesis of neurodegenerative and inflammatory conditions (5). CXCR4 is expressed by many different types of cancers, and overexpression of CXCR4 in cancers indicates poor prognosis with aggressive and metastatic tumors and resistance to chemotherapy (6).

CXCR4 is considered to play an important role in HIV infections and cancers (4). It is critical to perform imaging studies to measure CXCR4 levels under in vivo conditions for various pathological and physiological conditions (7). 99mTc-SDF-1 has been used with single-photon emission computed tomography (SPECT) to determine changes in CXCR4 expression in the heart after a myocardial infarction. 64Cu-1,1'-[1,4-Phenylenebis(methylene)]-bis[1,4,8,11-tetraazacyclotetradecane] (64Cu-AMD3100), a bicyclam inhibitor of CXCR4 activity, has been studied in mice bearing orthotopic breast tumors and in mice with lung metastases with positron emission tomography (PET) (8). {N-[1,4,8,11-Tetraazacyclotetradecanyl-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine} (AMD3465) is a monocyclam CXCR4 inhibitor with a ~15-fold higher affinity for CXCR4 than AMD3100. De Silva et al. (9) radiolabeled AMD3465 with 64Cu to form 64Cu-AMD3465 for PET imaging of CXCR4 expression in xenograft tumors in mice.



AMD3465 (0.2 mg) was incubated with 370–740 MBq (10–20 mCi) 64CuCl2 in sodium acetate buffer (pH 5) for 45 min at 60°C (9). 64Cu-AMD3465 was purified with high-performance liquid chromatography. This procedure provided a radiochemical purity of >98% with a specific activity of 6.0 ± 3.1 GBq/µmol (162 ± 84 mCi/µmol). The log P value for 64Cu-AMD3465 was −2.71.

In Vitro Studies: Testing in Cells and Tissues


A receptor-binding assay was performed with 125I-SDF-1 (9). AMD3465 exhibited a 50% inhibition concentration (IC50) value of 41.7 ± 1.2 nM. Cellular accumulation was performed using human glioblastoma U87 cells transfected with human CD4 and CXCR4 (U87-stb-CXCR4, 95% of cells positive for CXCR4), U87 cells (2% positive for CXCR4) and human colon HT-29 cells (low CXCR4 expression, 30% positive for CXCR4). These cells were incubated with 64Cu-AMD3465 for 30 min at 4°C and then washed four times with cold binding medium. The amounts of radioactivity bound to U87-stb-CXCR4 cells, U87 cells, and HT-29 cells were 11%, 1%, and 2% incubation dose/106 cells, respectively. Incubation of 64Cu-AMD3465 with U87-stb-CXCR4 cells at 37°C showed a linear accumulation of radioactivity with the time of incubation. At 240 min of incubation, ~50% of radioactivity was internalized. No blocking studies were performed.

Animal Studies



De Silva et al. (9) performed ex vivo biodistribution studies of 0.74 MBq (0.020 mCi) 64Cu-AMD3465 in nude mice (n = 4–6/group) bearing U87-stb-CXCR4 tumor xenografts at 30, 60, 90, 240, and 600 min after injection. Tumor accumulation was 106.6 ± 6.0% injected dose/gram (ID/g), 96.3 ± 14.0% ID/g, and 51.0 ± 6.4% ID/g at 30, 90, and 240 min after injection, respectively. The maximum tumor/blood and tumor/muscle ratios of 139 and 363, respectively, occurred at 90 min after injection. Accumulation values in the liver, kidneys, bone marrow, and spleen were noticeable with 36.2% ID/g, 38.0% ID/g, 9.2% ID/g, and 4.3% ID/g, respectively, at 90 min after injection. Pretreatment with AMD3465 (25 mg/kg, 60 min before tracer injection) blocked the accumulation of 64Cu-AMD3465 in the tumor by >90%, whereas the accumulation in the liver, kidneys, bone marrow, and spleen was reduced by ~50% at 90 min after injection. In separate experiments, 64Cu-AMD3465 accumulation in U87 and HT-29 tumors was ~4% ID/g and 5.6% ID/g at 90 min after injection, respectively. Immunohistochemistry studies showed the expression of CXCR4 on tumor cells in the HT-29 tumor sections.

Biodistribution of 64Cu-AMD3465 was also assessed with whole-body and dynamic PET imaging in mice bearing U87-stb-CXCR4, U87, or HT-29 tumor xenografts. The accumulation in the U87-stb-CXCR4 tumors was two-fold greater than that in the kidneys and liver and ~17-fold greater than that in the U87 and HT-29 tumors. The best image contrast for U87-stb-CXCR4 tumors was observed at 90 min after injection. The liver and kidneys were also clearly visualized. Pretreatment with AMD3465 (25 mg/kg, 60 min before tracer injection) blocked the accumulation of radioactivity levels in all three tumors.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

U24 CA92871


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Nimmagadda S., Pullambhatla M., Stone K., Green G., Bhujwalla Z.M., Pomper M.G. Molecular imaging of CXCR4 receptor expression in human cancer xenografts with [64Cu]AMD3100 positron emission tomography. Cancer Res. 2010;70(10):3935–44. [PMC free article: PMC2874192] [PubMed: 20460522]
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