64Cu PET Imaging of the CXCR4 Chemokine Receptor Using a Cross-Bridged Cyclam Bis-Tetraazamacrocyclic Antagonist

Abstract

Expression of the chemokine receptor chemokine C-X-C motif receptor 4 (CXCR4) plays an important role in cancer metastasis, in autoimmune diseases, and during stem cell-based repair processes after stroke and myocardial infarction. Previously reported PET imaging agents targeting CXCR4 suffer from either high nonspecific uptake or bind only to the human form of the receptor. The objective of this study was to develop a high-stability ⁶⁴Cu-labeled small-molecule PET agent for imaging both human and murine CXCR4 chemokine receptors. Methods: Synthesis, radiochemistry, stability and radioligand binding assays were performed for the novel tracer ⁶⁴Cu-CuCB-bicyclam. In vivo dynamic PET studies were performed on mice bearing U87 (CXCR4 low-expressing) and U87.CXCR4 (human-CXCR4 high-expressing) tumors. Biodistribution and receptor blocking studies were performed on CD1-IGS immunocompetent mice. CXCR4 expression on tumor and liver disaggregates was confirmed using a combination of immunohistochemistry, quantitative polymerase chain reaction, and Western blot. Results:⁶⁴Cu-CuCB-bicyclam has a high affinity for both the human and the murine variants of the CXCR4 receptor (half-maximal inhibitory concentration, 8 nM [human]/2 nM [murine]) and can be obtained from the parent chelator that has low affinity. In vitro and in vivo studies demonstrate specific uptake in CXCR4-expressing cells that can be blocked by more than 90% using a higher-affinity antagonist, with limited uptake in non-CXCR4-expressing organs and high in vivo stability. The tracer was also able to selectively displace the CXCR4 antagonists AMD3100 and AMD3465 from the liver. Conclusion: The tetraazamacrocyclic small molecule ⁶⁴Cu-CuCB-bicyclam has been shown to be an imaging agent for the CXCR4 receptor that is likely to be applicable across a range of species. It has high affinity and stability and is suitable for preclinical research in immunocompetent murine models.

Keywords: 64Cu; AMD3100; CXCR4; azamacrocycle; cyclam.

FIGURE 1.

In vitro assessment of ⁶⁴Cu-CuCB-bicyclam. (A) Comparative affinity values vs. CXCL12 determined by calcium flux assay. (B) Chemical structures of ⁶⁴Cu-CuAMD3100 and ⁶⁴Cu-CuCB-bicyclam. (C) Fluorescence-activated cell sorting determination of CXCR4 expression. (D) Binding of ⁶⁴Cu-CuCB-bicyclam on U87 and U87.CXCR4-expressing cell lines.

FIGURE 2.

In vivo PET/CT evaluation of ⁶⁴Cu-CuCB-bicyclam. (A) Fused PET/CT maximum-intensity projections at 70–90 min after injection from CD1 mice bearing U87-CXCR4 (n = 3), U87 (n = 3), and U87.CXCR4 block (n = 2). Animals were injected with 9.6 ± 0.7 MBq of ⁶⁴Cu-CuCB-bicyclam. A 5 mg/kg blocking dose of Cu₂CB-bicyclam was given 1 h before ⁶⁴Cu-CuCB-bicyclam injection where indicated. Arrows denote position of tumor and liver. (B) Dynamic tumor time–activity curve for showing uptake of ⁶⁴Cu-CuCB-bicyclam in tumor (top) and liver (bottom) during 90 min. Data represent mean uptake of 2–3 animals ± SEM. *U87.CXCR4 vs. U87, P < 0.01. **Block vs. naïve, P < 0.01. Supplemental Figures 6 and 7 show all images at 70–90 min and time–activity curves for bladder, kidney, and vena cava.

FIGURE 3.

Assessment of in vivo uptake of ⁶⁴Cu-CuCB-bicyclam after blocking with range of CXCR4 antagonists. Biodistribution of selected tissues from CD1-IGS mice given 5 mg/kg dose of Cu₂CB-bicyclam, CuCB-bicyclam, AMD3100, or AMD3465 60 min (top) or 12 h (bottom) before ⁶⁴Cu-CuCB-bicyclam injection. Tissue was harvested 90 min after tracer administration (n = 4). *Block vs. naïve, P < 0.05.