Evaluation of a Novel Arg-Gly-Asp-Conjugated α-Melanocyte Stimulating Hormone Hybrid Peptide for Potential Melanoma Therapy

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Abstract
The purpose of this study was to determine whether Arg-Gly-Asp (RGD)-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptide could be employed to target melanocortin-1 (MC1) receptor for potential melanoma therapy. Methods: The RGD motif {cyclic(Arg-Gly-Asp-dTyr-Asp)} was coupled to [Cys3,4,10, dPhe7, Arg11]α-MSH3−13 {(Arg11)CCMSH} to generate RGD-Lys-(Arg11)CCMSH hybrid peptide. The MC1 receptor binding affinity of RGD-Lys-(Arg11)CCMSH was determined in B16/F1 melanoma cells. The internalization and efflux, melanoma targeting and pharmacokinetic properties and single photon emission computed tomography/CT (SPECT/CT) imaging of 99mTc-RGD-Lys-(Arg11)CCMSH were determined in B16/F1 melanoma cells and melanoma-bearing C57 mice. Clonogenic cytotoxic effect of RGD-Lys-(Arg11)CCMSH was examined in B16/F1 melanoma cells. Results: RGD-Lys-(Arg11)CCMSH displayed 2.1 nM MC1 receptor binding affinity. 99mTc-RGD-Lys-(Arg11)CCMSH showed rapid internalization and extended retention in B16/F1 cells. The cellular uptake of 99mTc-RGD-Lys-(Arg11)CCMSH was MC1 receptor-mediated. 99mTc-RGD-Lys-(Arg11)CCMSH exhibited high tumor uptake (14.83 ± 2.94% ID/g 2 h postinjection) and prolonged tumor retention (7.59 ± 2.04% ID/g 24 h postinjection) in B16/F1 melanoma-bearing mice. Nontarget organ uptakes were generally low except for the kidneys. Whole-body clearance of 99mTc-RGD-Lys-(Arg11)CCMSH was rapid, with approximately 62% of the injected radioactivity cleared through the urinary system by 2 h postinjection. Flank melanoma tumors were clearly imaged by small animal SPECT/CT using 99mTc-RGD-Lys-(Arg11)CCMSH as an imaging probe 2 h postinjection. Single treatment (3 h incubation) with 100 nM of RGD-Lys-(Arg11)CCMSH significantly (p < 0.05) decreased the clonogenic survival of B16/F1 cells by 65% compared to the untreated control cells. Conclusion: Favorable melanoma targeting property of 99mTc-RGD-Lys-(Arg11)CCMSH and remarkable cytotoxic effect of RGD-Lys-(Arg11)CCMSH in B16/F1 cells warranted the further evaluation of 188Re-labeled α-MSH hybrid peptides as novel therapeutic peptides for melanoma treatment once the strategies of amino acid coinjection or structural modification of peptide sequence substantially reduce the renal uptake.