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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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Amino acid sequence oftargeting peptide, synthesis of imagingtracer Cu-E4-Fl 6 (cold peptide) as well as bimodal imagingtracer 64Cu-E4-Fl 6 (radiolabeled peptide),and stability studies of 64Cu-5. (A) Abbreviationsand amino acid sequences of modified exendin-4 peptides. (B) Azide–alkyneHuisgen cycloaddition yielding imaging agent Cu-E4-Fl 6 and sequential one-pot synthesis (radiolabeling and copper-catalyzedazide–alkyne cycloaddition) of the bimodal imaging tracer 64Cu-E4-Fl 6. (C) HPLC chromatograms of 64Cu-5 and 64Cu-E4-Fl 6. (D) Competitionand stability of 64Cu-5 in the presence ofeither CuSO4 or ascorbic acid over 4 h.
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fig1: Amino acid sequence oftargeting peptide, synthesis of imagingtracer Cu-E4-Fl 6 (cold peptide) as well as bimodal imagingtracer 64Cu-E4-Fl 6 (radiolabeled peptide),and stability studies of 64Cu-5. (A) Abbreviationsand amino acid sequences of modified exendin-4 peptides. (B) Azide–alkyneHuisgen cycloaddition yielding imaging agent Cu-E4-Fl 6 and sequential one-pot synthesis (radiolabeling and copper-catalyzedazide–alkyne cycloaddition) of the bimodal imaging tracer 64Cu-E4-Fl 6. (C) HPLC chromatograms of 64Cu-5 and 64Cu-E4-Fl 6. (D) Competitionand stability of 64Cu-5 in the presence ofeither CuSO4 or ascorbic acid over 4 h.

Mentions: In ourcurrent work, we used the GLP-1R targeting peptide exendin-4(E4x12), which was modified at the K12 position with the unnatural alkyne-amino acid (S)-2-amino-4-pentynoic acid (Figure 1A).11,12,17 With the site-specific modificationof the peptide with an alkyne and the attachment of an azide functionalgroup to the chelator, we were able to perform a copper-catalyzedazide–alkyne cycloaddition (CuAAC) between E4x12 and chelator 5, using standard cycloaddition reactionconditions (buffered water, copper sulfate, ascorbic acid, room temperature)(Figure S1A). We obtained Cu-E4-Fl 6 in 20% yield over 1 h. HPLC and ESI-MS confirmed the identityof Cu-E4-Fl (Figure S1).


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)

Amino acid sequence oftargeting peptide, synthesis of imagingtracer Cu-E4-Fl 6 (cold peptide) as well as bimodal imagingtracer 64Cu-E4-Fl 6 (radiolabeled peptide),and stability studies of 64Cu-5. (A) Abbreviationsand amino acid sequences of modified exendin-4 peptides. (B) Azide–alkyneHuisgen cycloaddition yielding imaging agent Cu-E4-Fl 6 and sequential one-pot synthesis (radiolabeling and copper-catalyzedazide–alkyne cycloaddition) of the bimodal imaging tracer 64Cu-E4-Fl 6. (C) HPLC chromatograms of 64Cu-5 and 64Cu-E4-Fl 6. (D) Competitionand stability of 64Cu-5 in the presence ofeither CuSO4 or ascorbic acid over 4 h.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Amino acid sequence oftargeting peptide, synthesis of imagingtracer Cu-E4-Fl 6 (cold peptide) as well as bimodal imagingtracer 64Cu-E4-Fl 6 (radiolabeled peptide),and stability studies of 64Cu-5. (A) Abbreviationsand amino acid sequences of modified exendin-4 peptides. (B) Azide–alkyneHuisgen cycloaddition yielding imaging agent Cu-E4-Fl 6 and sequential one-pot synthesis (radiolabeling and copper-catalyzedazide–alkyne cycloaddition) of the bimodal imaging tracer 64Cu-E4-Fl 6. (C) HPLC chromatograms of 64Cu-5 and 64Cu-E4-Fl 6. (D) Competitionand stability of 64Cu-5 in the presence ofeither CuSO4 or ascorbic acid over 4 h.
Mentions: In ourcurrent work, we used the GLP-1R targeting peptide exendin-4(E4x12), which was modified at the K12 position with the unnatural alkyne-amino acid (S)-2-amino-4-pentynoic acid (Figure 1A).11,12,17 With the site-specific modificationof the peptide with an alkyne and the attachment of an azide functionalgroup to the chelator, we were able to perform a copper-catalyzedazide–alkyne cycloaddition (CuAAC) between E4x12 and chelator 5, using standard cycloaddition reactionconditions (buffered water, copper sulfate, ascorbic acid, room temperature)(Figure S1A). We obtained Cu-E4-Fl 6 in 20% yield over 1 h. HPLC and ESI-MS confirmed the identityof Cu-E4-Fl (Figure S1).

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