Melanoma imaging with ⁶⁸Ga-labeled cyclized alpha-MSH peptides using positron emission tomography

Objectives The melanocortin-1 receptor (MC1R) is overexpressed in most cutaneous and uveal melanomas, and alpha-melanocyte stimulating hormone (alpha-MSH) derivatives have been proposed to target MC1R for imaging or radionuclide therapy of metastatic melanoma. The purpose of this study was to develop improved MC1R ligands for positron emission tomography (PET) imaging of melanoma, in a preclinical model of melanoma.

Methods Three DOTA-conjugated alpha-MSH peptides were synthesized using solid phase peptide synthesis, based on the sequence Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂ (Nle-CycMSH_hex) using PEG₂ (CCZ01047), a single cationic (4-amino-1-carboxymethyl-piperidine) linker (CCZ01048), or dual cationic linkers (CCZ01056). Radiolabeling with ⁶⁸Ga was carried out under microwave for 1 min in HEPES buffer (2 M, pH 5.0), followed by HPLC purification to ensure high specific activity. MC1R binding affinity was measured using an in vitro competition binding assay with B16-F10 cells. PET/CT imaging and biodistribution studies were performed in C57BL/6J mice bearing B16-F10 tumors.

Results All three peptides, CCZ01047, CCZ01048 and CCZ01056 showed excellent binding affinity to MC1R with K_i = 0.73 ± 0.08, 0.31 ± 0.06 and 0.16 ± 0.10 nM (n = 3), respectively. ⁶⁸Ga-labeled peptides were prepared with > 96% radiochemical purity in average decay-corrected radiochemical yields ranging from 58-61% with specific activity > 6 Ci / μmol. PET imaging and biodistribution studies for ⁶⁸Ga-labeled CCZ01047, CCZ01048 and CCZ01056 showed high tumor take at 1 h p.i. at 8.02 ± 3.04, 12.28 ± 3.30 and 6.51 ± 1.44 %ID/g (n 蠅 6), respectively. Minimal background activity was observed besides the kidneys at 5.64 ± 1.17, 4.65 ± 0.50 and 6.15 ± 1.44 %ID/g, respectively. On dynamic PET imaging, sustained and increased tumor uptake was observed for CCZ01047 and CCZ01048, but not CCZ01056. For [⁶⁸Ga]CCZ01048 at 2 h p.i., tumor uptake increased to 21.92 ± 4.63 %ID/g (n = 6), with a further reduction in background and kidney activity. Excellent tumor-to-blood and tumor-to-muscle contrast ratios were achieved at 96.42 ± 13.85 and 210.90 ± 20.92, respectively. Tumor uptake of [⁶⁸Ga]CCZ01048 was effectively blocked by co-injection of 100 μg of non-radioactive Ga-CCZ01048 (n = 8).

Conclusions Excellent tumor visualization and exceptional contrast was achieved with all three radiopeptides. The use of a cationic linker led to high and sustained tumor uptake, excellent image contrast and lower kidney accumulation in a preclinical model of MC1R-positive tumor.