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

College of Pharmacy, University of New Mexico.

Department of Internal Medicine, University of New Mexico.

Cancer Research Treatment Center, University of New Mexico.

Department of Dermatology, University of New Mexico.

University of Missouri.

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 [Cys^3,4,10, 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.