Effect of a Negatively Charged Amino Acid Linker on the Tumor-Targeting Properties of [¹⁷⁷Lu]Lu-Labeled 4-p-(Tolyl)butyric Acid-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides

This study aimed to examine how negatively charged amino acid linkers influence the tumor-targeting and biodistribution properties of [¹⁷⁷Lu]Lu-labeled 4-p-(tolyl)butyric acid (p-TBA)-conjugated alpha-melanocyte-stimulating hormone peptides. The p-TBA moiety functions as an albumin binder (ALB), either directly attached to the peptide or through a negatively charged aspartic acid (Asp) or glutamic acid (Glu) linker. DOTA-Lys(p-TBA)-GGNle-CycMSH_hex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(4-p-(tolyl)butyric acid)-GlyGly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂}, DOTA-Lys(Asp-p-TBA)-GGNle-CycMSH_hex, and DOTA-Lys(Glu-p-TBA)-GGNle-CycMSH_hex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. Their melanocortin-1 receptor (MC1R) binding affinities were measured in B16/F10 melanoma cells, and the biodistribution of [¹⁷⁷Lu]Lu-labeled peptides was examined in B16/F10 melanoma-bearing mice at 0.5, 2, 4, and 24 h postinjection. Additionally, the melanoma imaging capability of [¹⁷⁷Lu]Lu-DOTA-Lys(Asp-p-TBA)-GGNle-CycMSH_hex was examined in B16/F10 melanoma-bearing mice. The IC₅₀ values were 0.5 ± 0.09 (p-TBA), 0.03 ± 0.03 (Asp-p-TBA), and 7.2 ± 0.07 nM (Glu-p-TBA). Among the three, [¹⁷⁷Lu]Lu-DOTA-Lys(Asp-p-TBA)-GGNle-CycMSH_hex displayed the highest tumor/kidney and tumor/liver uptake ratios at 2 and 4 h postinjection. The B16/F10 melanoma uptake of [¹⁷⁷Lu]Lu-DOTA-Lys(Asp-p-TBA)-GGNle-CycMSH_hex was 20.40 ± 1.91, 45.24 ± 1.72, 31.30 ± 2.85, and 11.16 ± 1.79%ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using [¹⁷⁷Lu]Lu-DOTA-Lys(Asp-p-TBA)-GGNle-CycMSH_hex as an imaging probe at 2 h postinjection. Overall, the presence and charge of the linker significantly affected MC1R binding and tumor uptake. The linker charge played a key role in the liver and kidney uptake of [¹⁷⁷Lu]Lu-DOTA-Lys(Asp-p-TBA)-GGNle-CycMSH_hex and [¹⁷⁷Lu]Lu-DOTA-Lys(Glu-p-TBA)-GGNle-CycMSH_hex. [¹⁷⁷Lu]Lu-DOTA-Lys(Asp-p-TBA)-GGNle-CycMSH_hex exhibited the highest tumor/liver and tumor/kidney uptake ratios among the three [¹⁷⁷Lu]Lu-peptides, highlighting its potential for future melanoma therapy.