Fig 1.
DOTA-NAPamide and Ga-DOTA-NAPamide are high affinity ligands for the melanocortin 1 receptor (MC1R): 125I-NDP-MSH ligand displacement and saturation assays performed with whole B16/F1 cells.
A Various concentrations of NAPamide were used to displace 125I-NDP-MSH. The inset shows a saturation experiment to determine the dissociation constant. B 125I-NDP-MSH ligand displacement assay to assess the impact of gallium chelation on DOTA-NAPamide. Curve fits were performed in GraphPad Prism by applying a one‐site binding equation. (n = 3; mean +/‐ SEM).
Fig 2.
A Reverse-phase HPLC chromatogram of a 68Ga-DOTA-NAPamide purification. The dashed line shows the radiodetector signal and the peak there represents the 68Ga-DOTA-NAPamide fraction, while the solid line shows the 280 nm absorption signal with the peak of DOTA-NAPamide. The area surrounded by dotted lines represents the purified fraction used in in vivo experiments. B In vitro displacement of 68Ga-DOTA-NAPamide from B16/F1 cells by an excess of unlabeled DOTA-NAPamide (n = 3; mean +/- SEM). Differences were not statistically significant (p = 0.12, R2 = 0.51, one-way ANOVA).
Fig 3.
Biodistribution at 1 h after i.v. injection of approximately 5 MBq radiotracer.
Results of 68Ga-DOTA-NAPamide produced by the standard protocol (n = 7) are shown in light gray, and results obtained with the purification protocol (n = 7) are shown in dark gray (mean +/‐ SEM; ****p = 0.0001, two-way ANOVA followed by Bonferroni post-test).
Fig 4.
68Ga-DOTA-NAPamide biodistribution at 1 h after i.v. injection of approximately 5 MBq radiotracer.
Purified 68Ga-DOTA-NAPamide was injected either alone or in parallel with an excess of unlabeled DOTA-NAPamide (1,000-fold or 10,000-fold excess over radiotracer) (n = 3 per group; mean +/- SEM; ****p = 0.0001, two-way ANOVA followed by Bonferroni post-test).
Fig 5.
Coronal sections from MRI and PET scans of mice bearing B16/F1 tumors on the right shoulder at 1 h after tail vein injection of 68Ga-NAPamide with various ratios of labeled and unlabeled peptide.
A T2-weighted MRI (corresponding to PET image in B) B unpurified 68Ga-DOTA-NAPamide C HPLC-purified 68Ga-NAPamide D HPLC-purified 68Ga-NAPamide + 1,000-fold excess of DOTA-NAPamide, and E HPLC-purified 68Ga-NAPamide + 10,000-fold excess of DOTA-NAPamide.
Fig 6.
A Short-interval reconstruction from dynamic PET data of a mouse bearing a B16/F1 tumor on the right shoulder after tail vein injection of 5 MBq purified 68Ga-NAPamide. Injected peptide amounts: 500 pmol (unpurified and 10,000-fold excess), 50 pmol (1,000-fold excess) and 0.05 pmol (purified). B Time-activity relationships of 68Ga-DOTA-NAPamide B16/F1 tumor accumulation derived from dynamic PET data obtained after injection of four different tracer formulations (n = 1).