Novel [67Cu]Cu-Labeled 4- p -(Tolyl)butyric Acid-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptides with Glycine and Polyethylene Glycol Linkers for Melanoma Targeting

This study managed to examine how the 4-p-(tolyl)butyric acid (p-TBA) albumin binder and the aspartic acid linker affect the tumor targeting and biodistribution properties of [67Cu]Cu-labeled p-TBA-conjugated alpha-melanocyte-stimulating hormone (α-MSH) peptides. p-TBA was attached to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-Lys-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2 (NOTA-Lys-GGNle-CycMSHhex) and NOTA-Lys-PEG2Nle-CycMSHhex with or without the Asp linker to generate NOTA-Lys(p-TBA/Asp-p-TBA)-GGNle-CycMSHhex and NOTA-Lys(p-TBA/Asp-p-TBA)-PEG2Nle-CycMSHhex. The melanocortin-1 receptor (MC1R) binding affinities of the designed peptides were determined on B16/F10 melanoma cells. The biodistribution properties of [67Cu]Cu-NOTA-Lys(p-TBA/Asp-p-TBA)-GGNle-CycMSHhex and [67Cu]Cu-NOTA-Lys(p-TBA/Asp-p-TBA)-PEG2Nle-CycMSHhex were evaluated on B16/F10 melanoma-bearing C57BL/6 mice at 2 h post-injection to select a lead peptide. Then, the melanoma targeting and imaging properties of [67Cu]Cu-NOTA-Lys(Asp-p-TBA)-PEG2Nle-CycMSHhex were determined on B16/F10 melanoma-bearing C57BL/6 mice. The IC50 values of NOTA-Lys(p-TBA/Asp-p-TBA)-GGNle-CycMSHhex and NOTA-Lys(p-TBA/Asp-p-TBA)-PEG2Nle-CycMSHhex were 0.26 ± 0.04, 0.54 ± 0.03, 0.46 ± 0.08, and 0.32 ± 0.04 nM, respectively. [67Cu]Cu-NOTA-Lys(Asp-p-TBA)-PEG2Nle-CycMSHhex was selected as a lead peptide due to its highest tumor/kidney and tumor/liver uptake ratios among the [67Cu]Cu-peptides at 2 h post-injection. The B16/F10 melanoma uptake of [67Cu]Cu-NOTA-Lys(Asp-p-TBA)-PEG2Nle-CycMSHhex was 29.31 ± 3.22, 39.05 ± 1.38, 25.07 ± 2.03, and 11.77 ± 1.61% ID/g at 0.5, 2, 4, and 24 h post-injection, respectively. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using [67Cu]Cu-NOTA-Lys(Asp-p-TBA)-PEG2Nle-CycMSHhex as an imaging probe at 2 h post-injection. The utilization of p-TBA as an albumin binder prolonged the blood circulation and enhanced the tumor uptake of [67Cu]Cu-Asp-p-TBA-PEG as compared to [67Cu]Cu-NOTA-PEG2Nle-CycMSHhex without an albumin-binding moiety. The Asp linker expedited the blood clearance of [67Cu]Cu-Asp-p-TBA-PEG as compared to [67Cu]Cu-p-TBA-PEG. The combination of p-TBA, Asp, and PEG linkers resulted in the highest tumor/blood, tumor/kidney, and tumor/liver uptake ratios of [67Cu]Cu-Asp-p-TBA-PEG among the designed [67Cu]Cu-labeled p-TBA-conjugated CycMSHhex peptides. The favorable tumor targeting and blood clearance properties of [67Cu]Cu-Asp-p-TBA-PEG underscore its potential for future melanoma therapy.