The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of 111 In-labeled lactam bridge-cyclized DOTA-[X]-CycMSH hex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH 2 ; X 5 GGNle, GENle, or NleGE; GG 5 -Gly-Gly-and GE 5 -Gly-Glu-} peptides. Methods: Three novel peptides (DOTA-GGNleCycMSH hex , DOTA-GENle-CycMSH hex , and DOTA-NleGECycMSH hex ) were designed and synthesized. The melanocortin-1 (MC1) receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of 111 In-DOTA-GGNle-CycMSH hex and 111 In-DOTA-GENle-CycMSH hex were determined in B16/F1 melanoma-bearing C57 mice. Results: DOTA-GGNleCycMSH hex and DOTA-GENle-CycMSH hex displayed 2.1 and 11.5 nM MC1 receptor-binding affinities, whereas DOTA-NleGECycMSH hex showed 873.4 nM MC1 receptor-binding affinity. The introduction of the -GG-linker maintained high melanoma uptake while decreasing kidney and liver uptake of 111 In-DOTAGGNle-CycMSH hex . The tumor uptake of 111 In-DOTA-GGNleCycMSH hex was 19.05 6 5.04 and 18.6 6 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. 111 In-DOTA-GGNle-CycMSH hex exhibited 28%, 32%, and 42% less kidney uptake than 111 In-DOTA-Nle-CycMSH hex we reported previously, and 61%, 65%, and 68% less liver uptake than 111 In-DOTA-Nle-CycMSH hex at 2, 4, and 24 h after injection, respectively. Conclusion: The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the 111 In-labeled lactam bridge-cyclized a-melanocyte-stimulating hormone peptides. Introduction of the -GG-linker maintained high melanoma uptake while reducing kidney and liver uptake of 111 In-DOTA-GGNleCycMSH hex , highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide. Over the last decade, both radiolabeled linear and cyclized a-melanocyte-stimulating hormone (a-MSH) peptides have been designed to target G protein-coupled melanocortin-1 (MC1) receptors (1-5) for melanoma radioimaging and radiotherapy (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Because of the stabilization of secondary structures (i.e., b-turns), the cyclic peptides possess less conformational freedom and higher stability than the linear peptides. Furthermore, the stabilization of secondary structures makes the cyclic peptides better fit the receptor-binding pocket, thus enhancing their receptor-binding affinities. Presently, disulfide bond, metal, and lactam bridge have been successfully used to cyclize the radiolabeled a-MSH peptides (9)(10)(11)(12)(13)(15)(16)(17)(18)(19)(20). Among these cyclization strategies, metal and lactam bridge cyclization resulted in greater tumor uptake and lower kidney uptake of the radiolabeled a-MSH peptides than the disulfide bridge cycli...

The purpose of this study was to examine the profound effect of the ring size of the radiolabeled lactam bridge-cyclized a-melanocyte-stimulating hormone (a-MSH) peptide on its melanoma-targeting properties. Methods: A novel cyclic a-MSH peptide, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-CONH 2 (DOTA-NleCycMSH hex ), was synthesized and radiolabeled with 111 In. The melanocortin-1 receptor-binding affinity of DOTA-Nle-CycMSH hex was determined in B16/F1 melanoma cells. The internalization and efflux of 111 In-DOTA-Nle-CycMSH hex were examined in B16/F1 cells. The melanoma-targeting properties and SPECT/ CT characteristics of 111 In-DOTA-Nle-CycMSH hex were determined in B16/F1 melanoma-bearing C57 mice. Results: DOTA-Nle-CycMSH hex displayed 1.77 nM receptor-binding affinity. 111 In-DOTA-Nle-CycMSH hex exhibited rapid internalization and extended retention in B16/F1 cells. The tumor uptake of 111 In-DOTA-Nle-CycMSH hex was 24.94% 6 4.58% and 10.53% 6 1.11% injected dose per gram at 0.5 and 24 h after injection, respectively. Greater than 82% of the injected radioactivity was cleared through the urinary system by 2 h after injection. The tumor-to-kidney uptake ratios reached 2.04 and 1.70 at 2 and 4 h after injection, respectively. Flank melanoma tumors were clearly visualized by SPECT/CT using 111 In-DOTA-NleCycMSH hex as an imaging probe at 2 and 24 h after injection. The radioactivity accumulation in normal organs, except for the kidneys, was low at 2, 4, and 24 h after injection. Conclusion: The reduction of the peptide ring size dramatically increased the melanoma uptake and decreased the renal uptake of 111 In-DOTA-Nle-CycMSH hex , providing a new insight into the design of a novel radiolabeled lactam bridge-cyclized a-MSH peptide for melanoma imaging and treatment.Key Words: melanoma imaging; radiolabeled cyclic peptide; a-melanocyte-stimulating hormone; small-animal imaging Ski n cancer is the most commonly diagnosed cancer in the United States. Melanoma accounts for less than 5% of skin cancer cases but causes greater than 75% of deaths from skin cancer. It is predicted that 68,720 new cases will be diagnosed and 8,650 deaths will occur in 2009 (1). Early diagnosis and prompt surgical removal are the best opportunities for a patient's cure, because no curative treatment exists for metastatic melanoma. Despite the clinical use of 18 F-FDG for PET diagnosis and staging of melanoma, 18 F-FDG is not a melanoma-specific imaging agent and is also not effective in imaging small melanoma metastases (,5 mm) and melanomas that have primary energy sources other than glucose (2-4). Alternatively, melanocortin-1 (MC1) receptor is a distinct molecular target because of its overexpression on both human and mouse melanoma cells (5-9). Radiolabeled a-melanocytestimulating hormone (a-MSH) peptides can bind the MC1 receptors with nanomolar binding affinities (10-20) and represent a class of promising melanoma-specific radiopharmaceuticals for melanoma imaging and therapy.Rece...

The purpose of this study was to determine whether Arg-Gly-Asp (RGD)-conjugated alpha-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, D-Phe7, 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 post-injection) and prolonged tumor retention (7.59±2.04 %ID/g 24 h post-injection) in B16/F1 melanoma-bearing mice. Non-target 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 post-injection. Flank melanoma tumors were clearly imaged by small animal SPECT/CT using 99mTc-RGD-Lys-(Arg11)CCMSH as an imaging probe 2 h post-injection. 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 co-injection or structural modification of peptide sequence substantially reduce the renal uptake.

A small subpopulation of cancer cells with stem cell-like features might be responsible for tumour generation, progression, and chemoresistance. Hes1 influences the maintenance of certain stem cells and progenitor cells and the digestive systems. We found upregulated Hes1 in poorly differentiated cancer samples compared with well-differentiated tumour samples, and most of the adenocarcinomas exhibited significantly higher levels of Hes1 mRNA compared with that observed in matched normal colon samples. Moreover, Hes1 expression was found to be correlated with the expression of stem cell markers in colon cancer samples, and Hes1 upregulates the expression of stemness-related genes in colon cancer cells. In addition, Hes1 enhances the self-renewal properties of the stem-like cells by increasing the sizes of CD133+ cells and SP cells and the ability of tumour sphere formation. Additionally, the Hes1-overexpressing cells formed significantly larger and higher number of colonies, as determined through the colony and the soft agar assays. More importantly, Hes1 enhances the tumourigenicity of colon cancer cell lines in nude mice and exhibits a strong tumour-formation ability at a cell density of 1 × 103. Taken together, our data indicate that Hes1 induces stem-like cell self-renewal and increases the number of tumour-initiating cells in colon cancer.