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The purpose of this study was to reduce the non-specific renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (alpha-MSH) hybrid peptide through structural modification or L-lysine co-injection. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys(3,4,10), D-Phe7, Arg11] alpha-MSH3-13 {(Arg11)CCMSH} through the Arg linker (substituting the Lys linker) to generate a novel RGD-Arg-(Arg11)CCMSH hybrid peptide. The melanoma targeting and pharmacokinetic properties of 99mTc-RGD-Arg-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The effect of L-lysine co-injection on the renal uptake was determined through the co-injection of L-lysine with 99mTc-RGD-Arg-(Arg11)CCMSH or 99mTc-RGD-Lys-(Arg11)CCMSH. Replacement of the Lys linker with an Arg linker exhibited a profound effect in reducing the non-specific renal uptake of 99mTc-RGD-Arg-(Arg11)CCMSH, as well as increasing the tumor uptake of 99mTc-RGD-Arg-(Arg11)CCMSH compared to 99mTc-RGD-Lys-(Arg11)CCMSH. 99mTc-RGD-Arg-(Arg11)CCMSH exhibited high tumor uptake (21.41+/-3.74% ID/g at 2 h post-injection) and prolonged tumor retention (6.81+/-3.71% ID/g at 24 h post-injection) in B16/F1 melanoma-bearing mice. The renal uptake values of 99mTc-RGD-Arg-(Arg11)CCMSH were 40.14-64.08% of those of 99mTc-RGD-Lys-(Arg11)CCMSH (p0.05) at 0.5, 2, 4 and 24 h post-injection. Co-injection of L-lysine was effective in decreasing the renal uptakes of 99mTc-RGD-Arg-(Arg11)CCMSH by 27.7% and 99mTc-RGD-Lys-(Arg11)CCMSH by 52.1% at 2 h post-injection. Substitution of the Lys linker with an Arg linker dramatically improved the melanoma uptake and reduced the renal uptake of 99mTc-RGD-Arg-(Arg11)CCMSH, warranting the further evaluation of 188Re-labeled RGD-Arg-(Arg11)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.

To explore the response of β globin locus with established chromatin domains upon their exposure to new transcriptional environments, we transferred the chromatin-packaged β globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroblasts into an adult transcriptional environment. Distinct globin expression patterns were observed. In hESC-derived erythroblasts where both ε and γ globin were active and marked by similar chromatin modifications, ε globin was immediately silenced upon transfer, whereas γ globin continued to be expressed for months, implying that different transcriptional environments were required for their continuing expression. Whereas β globin was silent both in hESCs and in hESC-derived erythroblasts, β globin was only activated upon transfer from hESCs, but not in the presence of dominant γ globin transferred from hESC-derived erythroblasts, confirming the competing nature of γ versus β globin expression. With time, however, silencing of γ globin occurred in the adult transcriptional environment with concurrent activation of β-globin, accompanied by a drastic change in the epigenetic landscape of γ and β globin gene regions without apparent changes in the transcriptional environment. This switching process could be manipulated by overexpression or downregulation of certain transcription factors. Our studies provide important insights into the interplay between the transcription environment and existing chromatin domains, and we offer an experimental system to study the time-dependent human globin switching.