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In vivo imaging of GLP-1R with a targeted bimodal PET/fluorescence imaging agent.

Brand C, Abdel-Atti D, Zhang Y, Carlin S, Clardy SM, Keliher EJ, Weber WA, Lewis JS, Reiner T - Bioconjug. Chem. (2014)

Bottom Line: The bimodal imaging agent (64)Cu-E4-Fl was synthesized in good radiochemical yield and specific activity (RCY = 36%, specific activity: 141 μCi/μg, >98% radiochemical purity).The agent showed good performance in vivo and ex vivo, visualizing small xenografts (<2 mm) with PET and pancreatic β-cell mass by phosphor autoradiography.We believe that our procedure could become relevant as a protocol for the development of bimodal imaging agents.

View Article: PubMed Central - PubMed

Affiliation: Radiochemistry and Imaging Sciences Service and §Molecular Imaging and Therapy Service, Department of Radiology, ∥Molecular Pharmacology and Chemistry Program, and ⊥Center for Molecular Imaging and Nanotechnology, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.

ABSTRACT
Accurate visualization and quantification of β-cell mass is critical for the improved understanding, diagnosis, and treatment of both type 1 diabetes (T1D) and insulinoma. Here, we describe the synthesis of a bimodal imaging probe (PET/fluorescence) for imaging GLP-1R expression in the pancreas and in pancreatic islet cell tumors. The conjugation of a bimodal imaging tag containing a near-infrared fluorescent dye, and the copper chelator sarcophagine to the GLP-1R targeting peptide exendin-4 provided the basis for the bimodal imaging probe. Conjugation was performed via a novel sequential one-pot synthetic procedure including (64)Cu radiolabeling and copper-catalyzed click-conjugation. The bimodal imaging agent (64)Cu-E4-Fl was synthesized in good radiochemical yield and specific activity (RCY = 36%, specific activity: 141 μCi/μg, >98% radiochemical purity). The agent showed good performance in vivo and ex vivo, visualizing small xenografts (<2 mm) with PET and pancreatic β-cell mass by phosphor autoradiography. Using the fluorescent properties of the probe, we were able to detect individual pancreatic islets, confirming specific binding to GLP-1R and surpassing the sensitivity of the radioactive label. The use of bimodal PET/fluorescent imaging probes is promising for preoperative imaging and fluorescence-assisted analysis of patient tissues. We believe that our procedure could become relevant as a protocol for the development of bimodal imaging agents.

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In vitro binding andinhibition studies. (A) IC50 valueof Cu-E4-Fl 6, measured via a competitive binding assayusing HEK-hGLP1R cells. (B) Confocal microscopy imaging experimentsusing 916–1 insulinoma cells.
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fig2: In vitro binding andinhibition studies. (A) IC50 valueof Cu-E4-Fl 6, measured via a competitive binding assayusing HEK-hGLP1R cells. (B) Confocal microscopy imaging experimentsusing 916–1 insulinoma cells.

Mentions: We evaluated the affinityof Cu-E4-Fl 6 to GLP-1Rusing an 125I-exendin-4 (9–39) competitive bindingassay (Figure 2A).18 In comparison to exendin-4 with an IC50 of 4.7 ±0.8 nM, we observed a slightly higher IC50 value of 50.3± 3.7 nM for our bimodal imaging tracer Cu-E4-Fl 6. The binding affinity of Cu-E4-Fl 6 was confirmed inconfocal cell imaging, where GLP-1R positive 916–1 insulinomacells showed strong uptake (Figure 2B). Afterincubation with Cu-E4-Fl (10 nM or 100 nM, 90 min), cells were fixedand stained with Cellomics blue whole cell stain (Thermo Scientific,MA, USA), indicating internalization of the fluorescent imaging probe,similar to what was seen previously.12 Toshow GLP-1R specificity of Cu-E4-Fl, we preincubated 916–1cells with an excess of unmodified peptide E4x12 (1 μM)before incubation with Cu-E4-Fl and observed suppressed fluorescentsignal in the NIR (Figure 2B).


In vivo imaging of GLP-1R with a targeted bimodal PET/fluorescence imaging agent.

Brand C, Abdel-Atti D, Zhang Y, Carlin S, Clardy SM, Keliher EJ, Weber WA, Lewis JS, Reiner T - Bioconjug. Chem. (2014)

In vitro binding andinhibition studies. (A) IC50 valueof Cu-E4-Fl 6, measured via a competitive binding assayusing HEK-hGLP1R cells. (B) Confocal microscopy imaging experimentsusing 916–1 insulinoma cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215873&req=5

fig2: In vitro binding andinhibition studies. (A) IC50 valueof Cu-E4-Fl 6, measured via a competitive binding assayusing HEK-hGLP1R cells. (B) Confocal microscopy imaging experimentsusing 916–1 insulinoma cells.
Mentions: We evaluated the affinityof Cu-E4-Fl 6 to GLP-1Rusing an 125I-exendin-4 (9–39) competitive bindingassay (Figure 2A).18 In comparison to exendin-4 with an IC50 of 4.7 ±0.8 nM, we observed a slightly higher IC50 value of 50.3± 3.7 nM for our bimodal imaging tracer Cu-E4-Fl 6. The binding affinity of Cu-E4-Fl 6 was confirmed inconfocal cell imaging, where GLP-1R positive 916–1 insulinomacells showed strong uptake (Figure 2B). Afterincubation with Cu-E4-Fl (10 nM or 100 nM, 90 min), cells were fixedand stained with Cellomics blue whole cell stain (Thermo Scientific,MA, USA), indicating internalization of the fluorescent imaging probe,similar to what was seen previously.12 Toshow GLP-1R specificity of Cu-E4-Fl, we preincubated 916–1cells with an excess of unmodified peptide E4x12 (1 μM)before incubation with Cu-E4-Fl and observed suppressed fluorescentsignal in the NIR (Figure 2B).

Bottom Line: The bimodal imaging agent (64)Cu-E4-Fl was synthesized in good radiochemical yield and specific activity (RCY = 36%, specific activity: 141 μCi/μg, >98% radiochemical purity).The agent showed good performance in vivo and ex vivo, visualizing small xenografts (<2 mm) with PET and pancreatic β-cell mass by phosphor autoradiography.We believe that our procedure could become relevant as a protocol for the development of bimodal imaging agents.

View Article: PubMed Central - PubMed

Affiliation: Radiochemistry and Imaging Sciences Service and §Molecular Imaging and Therapy Service, Department of Radiology, ∥Molecular Pharmacology and Chemistry Program, and ⊥Center for Molecular Imaging and Nanotechnology, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.

ABSTRACT
Accurate visualization and quantification of β-cell mass is critical for the improved understanding, diagnosis, and treatment of both type 1 diabetes (T1D) and insulinoma. Here, we describe the synthesis of a bimodal imaging probe (PET/fluorescence) for imaging GLP-1R expression in the pancreas and in pancreatic islet cell tumors. The conjugation of a bimodal imaging tag containing a near-infrared fluorescent dye, and the copper chelator sarcophagine to the GLP-1R targeting peptide exendin-4 provided the basis for the bimodal imaging probe. Conjugation was performed via a novel sequential one-pot synthetic procedure including (64)Cu radiolabeling and copper-catalyzed click-conjugation. The bimodal imaging agent (64)Cu-E4-Fl was synthesized in good radiochemical yield and specific activity (RCY = 36%, specific activity: 141 μCi/μg, >98% radiochemical purity). The agent showed good performance in vivo and ex vivo, visualizing small xenografts (<2 mm) with PET and pancreatic β-cell mass by phosphor autoradiography. Using the fluorescent properties of the probe, we were able to detect individual pancreatic islets, confirming specific binding to GLP-1R and surpassing the sensitivity of the radioactive label. The use of bimodal PET/fluorescent imaging probes is promising for preoperative imaging and fluorescence-assisted analysis of patient tissues. We believe that our procedure could become relevant as a protocol for the development of bimodal imaging agents.

Show MeSH
Related in: MedlinePlus