The purpose of this study was to examine whether the replacement of the positively-charged Lys or Arg linker with a neutral linker could reduce the renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys3,4,10, D-Phe7, Arg11]α-MSH3-13 {(Arg11)CCMSH} through the neutral βAla or Ahx {aminohexanoic acid} linker (replacing the Lys or Arg linker) to generate novel RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH hybrid peptides. The receptor-binding affinity and cytotoxicity of RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH were determined in B16/F1 melanoma cells. The melanoma targeting and imaging properties of 99mTc-RGD-βAla-(Arg11)CCMSH and 99mTc-RGD-Ahx-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The replacement of the Lys or Arg linker with the βAla or Ahx linker retained nanomolar receptor-binding affinities and remarkable cytotoxicity of RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH. The receptor-binding affinities of RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH were 0.8 ± 0.05 and 1.3 ± 0.1 nM. Three-hour incubation with 0.1 µM of RGD-βAla-(Arg11)CCMSH and RGD-Ahx-(Arg11)CCMSH decreased the survival percentages of B16/F1 cells by 71 and 67 % as compared to the untreated control cells 5 days post the treatment. The replacement of the Arg linker with the βAla or Ahx linker reduced the non-specific renal uptake of 99mTc-RGD-βAla-(Arg11)CCMSH and 99mTc-RGD-Ahx-(Arg11)CCMSH by 62 and 61 % at 2 h post-injection. 99mTc-RGD-βAla-(Arg11)CCMSH displayed higher melanoma uptake than 99mTc-RGD-Ahx-(Arg11)CCMSH at 0.5, 2, 4, and 24 h post-injection. Enhanced tumor to kidney uptake ratio of 99mTc-RGD-βAla-(Arg11)CCMSH warranted the further evaluation of 188Re-labeled RGD-βAla-(Arg11)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.

The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg11)CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg11)CCMSH (2), RVD-β-Ala-(Arg11)CCMSH (3), RAD-β-Ala-(Arg11)CCMSH (4), NAD-β-Ala-(Arg11)CCMSH (5), and EAD-β-Ala-(Arg11)CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their 99mTc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six 99mTc-peptides. 99mTc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these 99mTc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using 99mTc-4 as an imaging probe.

The purpose of this study was to examine the biodistribution of 99mTc-RAD-Arg-(Arg11)CCMSH in B16/F1 melanoma-bearing C57 mice to determine whether the replacement of the Lys linker with an Arg linker could decrease the renal uptake of 99mTc-RAD-Arg-(Arg11)CCMSH. 99mTc-RAD-Arg-(Arg11)CCMSH exhibited rapid and high tumor uptake (17.98 ± 4.96% ID/g at 2 h post-injection) in B16/F1 melanoma-bearing C57 mice. As compared to 99mTc-RAD-Lys-(Arg11)CCMSH, the replacement of the Lys linker with an Arg linker dramatically decreased the renal uptake of 99mTc-RAD-Arg-(Arg11)CCMSH by 68%, 62%, 73% and 64% at 0.5, 2, 4 and 24 h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2 h post-injection using 99mTc-RAD-Arg-(Arg11)CCMSH as an imaging probe.

The purpose of this study was to examine whether the introduction of d-Phe could improve the GnRH receptor binding affinities of DOTA-conjugated d-Lys6-GnRH peptides. Building upon the construct of DOTA-Ahx-(d-Lys6-GnRH1) we previously reported, an aromatic amino acid of d-Phe was inserted either between the DOTA and Ahx or between the Ahx and d-Lys6 to generate new DOTA-d-Phe-Ahx-(d-Lys6-GnRH) or DOTA-Ahx-d-Phe-(d-Lys6-GnRH) peptides. Compared to DOTA-Ahx-(d-Lys6-GnRH1) (36.1 nM), the introduction of d-Phe improved the GnRH receptor binding affinities of DOTA-d-Phe-Ahx-(d-Lys6-GnRH) (16.3 nM) and DOTA-Ahx-d-Phe-(d-Lys6-GnRH) (7.6 nM). The tumor targeting and pharmacokinetic properties of 111In-DOTA-Ahx-d-Phe-(d-Lys6-GnRH) was determined in MDA-MB-231 human breast cancer-xenografted nude mice. Compared to 111In-DOTA-Ahx-(d-Lys6-GnRH1), 111In-DOTA-Ahx-d-Phe-(d-Lys6-GnRH) exhibited comparable tumor uptake with faster renal and liver clearance. The MDA-MB-231 human breast cancer-xenografted tumors were clearly visualized by single photon emission computed tomography (SPECT) using 111In-DOTA-Ahx-d-Phe-(d-Lys6-GnRH) as an imaging probe, providing a new insight into the design of new GnRH peptides in the future.

The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new 99mTc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg11)CCMSH {c[Arg-Ser-Asp-DTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg11)CCMSH, RPheD-Lys-(Arg11)CCMSH, and RdPheD-Lys-(Arg11)CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of 99mTc-RSD-Lys-(Arg11)CCMSH, 99mTc-RFD-Lys-(Arg11)CCMSH, and 99mTc-RfD-Lys-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The substitution of Gly with Ser, Phe, and dPhe increased the MC1 receptor binding affinities of the peptides, whereas the substitution of Gly with Nle decreased the MC1 receptor binding affinity of the peptide. 99mTc-RSD-Lys-(Arg11)CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these 99mTc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using 99mTc-RSD-Lys-(Arg11)CCMSH as an imaging probe. It is desirable to reduce the renal uptake of 99mTc-RSD-Lys-(Arg11)CCMSH to facilitate its potential therapeutic application.

The purpose of this study was to examine the melanoma targeting and imaging properties of two new 99mTc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RTD-Lys-(Arg11)CCMSH {c[Asp-Arg-Thr-Asp-DTyr]-Lys-Cys-Cys-Glu-His-DPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2} and RVD-Lys-(Arg11)CCMSH peptides were synthesized, and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution and melanoma imaging properties of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The IC50 values of RTD-Lys-(Arg11)CCMSH and RVD-Lys-(Arg11)CCMSH were 0.7 ± 0.07 and 1.0 ± 0.3 nM in B16/F1 melanoma cells. Both 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH displayed high melanoma uptake. 99mTc-RTD-Lys-(Arg11)CCMSH exhibited the highest tumor uptake of 18.77 ± 5.13% ID/g at 2 h postinjection, whereas 99mTc-RVD-Lys-(Arg11)CCMSH reached the highest tumor uptake of 19.63 ± 4.68% ID/g at 4 h postinjection. Both 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH showed low accumulation in normal organs (<1.7% ID/g) except for the kidneys at 2 h postinjection. The renal uptake of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH was 135.14 ± 23.62 and 94.01 ± 18.31% ID/g at 2 h postinjection, respectively. The melanoma lesions were clearly visualized by single-photon emission computed tomography (SPECT)/CT using either 99mTc-RTD-Lys-(Arg11)CCMSH or 99mTc-RVD-Lys-(Arg11)CCMSH as an imaging probe at 2 h postinjection. Overall, the introduction of Thr or Val residue retained high melanoma uptake of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH. However, high renal uptake of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH need to be reduced to facilitate their future applications.Keywords: melanoma imaging; receptor-targeting; α-melanocyte stimulating hormone peptide;

The purpose of this study was to examine the melanoma targeting and imaging properties of new 99mTc-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (α-MSH) peptides using bifunctional chelating agents. MAG3-GGNle-CycMSHhex, AcCG3-GGNle-CycMSHhex, and HYNIC-GGNle-CycMSHhex peptides were synthesized, and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution of 99mTc-MAG3-GGNle-CycMSHhex, 99mTc-AcCG3-GGNle-CycMSHhex, 99mTc(CO)3-HYNIC-GGNle-CycMSHhex, and 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex were further examined because of its high melanoma uptake and fast urinary clearance. The IC50 values of MAG3-GGNle-CycMSHhex, AcCG3-GGNle-CycMSHhex, and HYNIC-GGNle-CycMSHhex were 1.0 ± 0.05, 1.2 ± 0.19, and 0.6 ± 0.04 nM in B16/F1 melanoma cells, respectively. Among these four 99mTc-peptides, 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex exhibited the highest melanoma uptake (14.14 ± 4.90% ID/g) and fastest urinary clearance (91.26 ± 1.96% ID) at 2 h postinjection. 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex were 2.50 and 3.55 at 4 and 24 h postinjection. The melanoma lesions were clearly visualized by SPECT/CT using 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex as an imaging probe at 2 h postinjection. Overall, high melanoma uptake coupled with fast urinary clearance of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex highlighted its potential for metastatic melanoma detection in the future.Keywords: 99mTc-labeled lactam bridge-cyclized peptide; alpha-melanocyte stimulating hormone; melanoma SPECT imaging;

The purpose of this study was to examine the effects of the -Arg-Pro- (RP) and -Gly-Gly-Nle- (GGNle) moieties on the melanoma targeting and clearance properties of 99mTc-peptides. We synthesized four new peptides {Ac-GGNle-CCEHdFRWC-NH2, Ac-GGNle-CCEHdFRWCRP-NH2, Ac-CCEHdFRWC-NleGG-NH2, and Ac-CCEHdFRWCRP-NleGG-NH2} and determined their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. Then we further examined the biodistribution properties of 99mTc-Ac-GGNle-CCEHdFRWCRP-NH2 and 99mTc-Ac-CCEHdFRWCRP-NleGG-NH2 in B16/F1 melanoma-bearing C57 mice. Overall, the -RP- moiety was critical for retaining low nanomolar MC1 receptor binding affinity. The deletion of the -RP- moiety dramatically reduced the receptor binding affinities of the peptides. The N-terminus was a better position than C-terminus for the -GGNle- moiety in retaining the lower renal and liver uptake. High melanoma uptake coupled with fast urinary clearance of 99mTc-Ac-GGNle-CCEHdFRWCRP-NH2 provided a new insight into the design of new α-melanocyte stimulating hormone (α-MSH) peptides.Keywords: 99mTc-labeled; alpha-MSH peptide; Melanocortin-1 receptor; melanoma;

Three new DOTA-conjugated GnRH peptides with various hydrocarbon linkers were synthesized to evaluate the influences of the linkers on their receptor binding affinities. The hydrocarbon linker displayed a profound impact on the receptor binding affinities of DOTA-conjugated GnRH peptides. The Aun linker was better than Gaba, Ahx and Aoc linkers in retaining strong receptor binding affinity of the GnRH peptide. DOTA-Aun-(d-Lys6-GnRH) displayed 22.8 nM GnRH receptor binding affinity. 111In-DOTA-Aun-(d-Lys6-GnRH) exhibited fast tumor uptake and urinary clearance in MDA-MB-231 human breast cancer-xenografted nude mice. The cellular and biological results provided an insight into the design of new GnRH peptides in the future.

The purpose of this study was to determine the melanoma targeting property of 177Lu-DOTA-GGNle-CycMSHhex in B16/F1 melanoma-bearing C57 mice. 177Lu-DOTA-GGNle-CycMSHhex exhibited high receptor-mediated melanoma uptake and fast urinary clearance. The tumor uptake of 177Lu-DOTA-GGNle-CycMSHhex was 20.25 ± 4.59 and 21.63 ± 6.27% ID/g at 0.5 and 2 h post-injection, respectively. Approximately 83% of injected dose cleared out the body via urinary system at 2 h post-injection. 177Lu-DOTA-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of 177Lu-DOTA-GGNle-CycMSHhex were 2.76 and 1.74 at 2 and 24 h post-injection. The melanoma lesions were clearly visualized by SPECT/CT using 177Lu-DOTA-GGNle-CycMSHhex as an imaging probe at 2 h post-injection. Overall, high melanoma uptake coupled with fast urinary clearance of 177Lu-DOTA-GGNle-CycMSHhex underscored its potential for melanoma treatment in the future.Visualization of B16/F1 melanoma lesions using 177Lu-DOTA-GGNle-CycMSHhex as an imaging probe.

The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GGNle-CycMSHhex {67Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 67Ga-NOTA-GGNle-CycMSHhex {67Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and compare with 67Ga-DOTA-GlyGlu-CycMSH {67Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM) in B16/F1 melanoma cells. Both 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than 67Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, 67Ga-NOTA-GGNle-CycMSHhex exhibited more favorable radiolabeling conditions (>85% radiolabeling yields started at 37 °C), as well as higher tumor/kidney uptake ratios than 67Ga-DOTA-GGNle-CycMSHhex at 0.5, 2, and 24 h postinjection. High melanoma uptake coupled with low renal uptake highlighted the potential of 67Ga-NOTA-GGNle-CycMSHhex for melanoma imaging and therapy.

The purpose of this study was to examine whether the structural modification on the positively charged Lys linker could reduce the kidney uptake of 99mTc-labeled Arg-Gly-Asp (RGD)-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptides. The RGD motif {cyclic(Arg-Gly-Asp-d-Tyr-Asp)} was coupled to [Cys3,4,10, d-Phe7, Arg11]α-MSH3–13 {(Arg11)CCMSH} through a neutral glycine linker to eliminate the positively charged amino side chain of the Lys linker or without a linker to delete the Lys linker. The receptor binding affinity of RGD-Gly-(Arg11)CCMSH and RGD-(Arg11)CCMSH was determined in B16/F1 melanoma cells. The melanoma targeting and imaging properties of 99mTc-RGD-Gly-(Arg11)CCMSH and 99mTc-RGD-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The structural modification on the Lys linker retained a low nanomolar receptor binding affinity of RGD-Gly-(Arg11)CCMSH and RGD-(Arg11)CCMSH (1.5 and 1.0 nM, respectively). The structural modification on the Lys linker dramatically decreased the renal uptake of 99mTc-RGD-Gly-(Arg11)CCMSH and 99mTc-RGD-(Arg11)CCMSH by 79% and 77% at 4 h postinjection compared to 99mTc-RGD-Lys-(Arg11)CCMSH. 99mTc-RGD-(Arg11)CCMSH displayed a higher melanoma uptake (16.12 ± 3.09% ID/g) than 99mTc-RGD-Gly-(Arg11)CCMSH (11.50 ± 1.01% ID/g) at 2 postinjection. The tumor uptake of 99mTc-RGD-(Arg11)CCMSH was 1.4 times the tumor uptake of 99mTc-RGD-Gly-(Arg11)CCMSH at 2 postinjection. A dramatically enhanced tumor-to-kidney uptake ratio of 99mTc-RGD-(Arg11)CCMSH suggests that 188Re-RGD-(Arg11)CCMSH may behave in a similar fashion warranting future evaluation for melanoma treatment.Keywords: melanoma imaging; RGD-conjugated α-MSH hybrid peptide; structural modification;

The purpose of this study was to examine and compare the melanoma targeting and imaging properties of 64Cu-NOTA-GGNle-CycMSHhex {64Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 64Cu-DOTA-GGNle-CycMSHhex {64Cu-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-GGNle-CycMSHhex}. Two lactam bridge-cyclized peptides, NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex, were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and imaging properties of 64Cu-NOTA-GGNle-CycMSHhex and 64Cu-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM). The substitution of DOTA with NOTA dramatically increased the melanoma uptake and decreased the renal and liver uptake of 64Cu-NOTA-GGNle-CycMSHhex. The tumor uptake of 64Cu-NOTA-GGNle-CycMSHhex was between 12.39 ± 1.61 and 12.71 ± 2.68% ID/g at 0.5, 2, and 4 h postinjection. The accumulation of 64Cu-NOTA-GGNle-CycMSHhex activity in normal organs was lower than 1.02% ID/g except for the kidneys 2, 4, and 24 h postinjection. The tumor/liver uptake ratios of 64Cu-NOTA-GGNle-CycMSHhex were 17.96, 16.95, and 8.02, whereas the tumor/kidney uptake ratios of 64Cu-NOTA-GGNle-CycMSHhex were 2.52, 3.60, and 5.74 at 2, 4, and 24 h postinjection, respectively. Greater than 91% of the injected radioactivity cleared through the urinary system by 2 h postinjection. The substitution of DOTA with NOTA resulted in a dramatic increase in melanoma uptake and decrease in renal and liver uptake of 64Cu-NOTA-GGNle-CycMSHhex as compared to 64Cu-DOTA-GGNle-CycMSHhex. High melanoma uptake coupled with low accumulation in nontarget organs suggested 64Cu-NOTA-GGNle-CycMSHhex as a lead radiolabeled peptide for melanoma imaging and therapy.Keywords: 64Cu-labeled peptide; lactam bridge-cyclized peptide; melanoma imaging; positron emission tomography; α-melanocyte stimulating hormone;

The purpose of this study was to determine the melanoma targeting property of 99mTc-RAD-Lys-(Arg11)CCMSH in B16/F1 melanoma-bearing C57 mice and compare with 99mTc-RGD-Lys-(Arg11)CCMSH we previously reported. 99mTc-RAD-Lys-(Arg11)CCMSH exhibited rapid and high tumor uptake (19.91 ± 4.02% ID/g at 2 h post-injection) in B16/F1 melanoma-bearing C57 mice. The tumor uptake of 99mTc-RAD-Lys-(Arg11)CCMSH was 1.51, 1.34 and 1.43 times the tumor uptake of 99mTc-RGD-Lys-(Arg11)CCMSH at 0.5, 2 and 4 h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2 h post-injection using 99mTc-RAD-Lys-(Arg11)CCMSH as an imaging probe. The substitution of Gly with Ala significantly enhanced the melanoma uptake of 99mTc-RAD-Lys-(Arg11)CCMSH compared to 99mTc-RGD-Lys-(Arg11)CCMSH in B16/F1 melanoma-bearing C57 mice, providing a new insight into the design of α-MSH peptides for melanoma targeting.Visualization of B16/F1 melanoma lesions using 99mTc-RAD-Lys-(Arg11)CCMSH as an imaging probe.

The purpose of this study was to develop novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor-targeting peptides for breast cancer imaging. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated GnRH peptides were designed and synthesized. The radiometal chelator DOTA was conjugated to the epsilon or alpha amino group of d-lysine, or the epsilon amino group of l-lysine via an Ahx {aminohexanoic acid} linker to generate DOTA–Ahx–(d-Lys6-GnRH1), DOTA–Ahx–(d-Lys6-GnRH2) and DOTA–Ahx–(l-Lys6-GnRH3), respectively. The conjugation of the DOTA to the epsilon amino group of d-lysine (rather than alpha amino group of d-lysine nor epsilon amino group of l-lysine) maintained the nanomolar GnRH receptor binding affinity. The IC50 values of DOTA–Ahx–(d-Lys6-GnRH1), DOTA–Ahx–(d-Lys6-GnRH2) and DOTA–Ahx–(l-Lys6-GnRH3) were 36.1 nM, 10.6 mM and 4.3 mM, respectively. Since only DOTA–Ahx–(d-Lys6-GnRH1) displayed nanomolar receptor binding affinity, the specific GnRH receptor binding of 111In-DOTA–Ahx–(d-Lys6-GnRH1) was determined in human GnRH receptor membrane preparations. Furthermore, the biodistribution and tumor imaging properties of 111In-DOTA–Ahx–(d-Lys6-GnRH1) were examined in MDA-MB-231 human breast cancer-xenografted nude mice. 111In-DOTA–Ahx–(d-Lys6-GnRH1) exhibited specific GnRH receptor binding and rapid tumor uptake (1.76 ± 0.58% ID/g at 0.5 h postinjection) coupled with fast whole-body clearance through the urinary system. The MDA-MB-231 human breast cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 1 h postinjection of 111In-DOTA–Ahx–(d-Lys6-GnRH1). The profound impact of DOTA position on the binding affinity of the GnRH peptide provided a new insight into the design of novel radiolabeled GnRH peptides. The successful imaging of MDA-MB-231 human breast cancer-xenografted tumor lesions using 111In-DOTA–Ahx–(d-Lys6-GnRH1) suggested its potential as a novel imaging probe for human breast cancer imaging.

The purpose of this study was to evaluate the tumor targeting and imaging properties of a novel 111In-labeled gonadotropin-releasing hormone (GnRH) peptide {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-Ahx-(d-Lys6-GnRH1)} for human prostate cancer. The biodistribution and tumor imaging properties of 111In-DOTA-Ahx-(d-Lys6-GnRH1) were determined in DU145 human prostate cancer-xenografted nude mice. 111In-DOTA-Ahx-(d-Lys6-GnRH1) exhibited rapid tumor uptake (1.27 ± 0.40% ID/g at 0.5 h post-injection) coupled with fast whole-body clearance through the urinary system. The DU145 human prostate cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 0.5 h post-injection of 111In-DOTA-Ahx-(d-Lys6-GnRH1). The successful imaging of DU145 human prostate cancer-xenografted tumor lesions using 111In-DOTA-Ahx-(d-Lys6-GnRH1) highlighted its potential as a novel imaging probe for human prostate cancer imaging.Visualization of DU145 human prostate cancer-xenografted tumor lesions using 111In-DOTA-Ahx-(d-Lys6-GnRH1) as an imaging probe.

The purpose of this study was to reduce the non-specific renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide through structural modification or l-lysine co-injection. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys3,4,10, d-Phe7, Arg11] α-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 (p <0.05) 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.The 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.

The purpose of this study was to examine the effect of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) position on melanoma targeting and pharmacokinetics of radiolabeled lactam bridge-cyclized α-melanocyte stimulating hormone (α-MSH) peptide. Method: A novel lactam bridge-cyclized α-MSH peptide, Ac-GluGlu-CycMSH[DOTA] {Ac-Glu-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Lys(DOTA)]}, was synthesized using standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. DOTA was directly attached to the α-amino group of Lys in the cyclic ring, while the N-terminus of the peptide was acetylated to generate Ac-GluGlu-CycMSH[DOTA]. The MC1 receptor binding affinity of Ac-GluGlu-CycMSH[DOTA] was determined in B16/F1 melanoma cells. Melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In were determined in B16/F1 melanoma-bearing C57 mice and compared to that of 111In-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp] (111In-DOTA-GlyGlu-CycMSH; DOTA was coupled to the N-terminus of the peptide). Results: Ac-GluGlu-CycMSH[DOTA] displayed 0.6 nM MC1 receptor binding affinity in B16/F1 cells. Ac-GluGlu-CycMSH[DOTA]-111In was readily prepared with greater than 95% radiolabeling yield. Ac-GluGlu-CycMSH[DOTA]-111In exhibited high tumor uptake (11.42 ± 2.20% ID/g 2 h postinjection) and prolonged tumor retention (9.42 ± 2.41% ID/g 4 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<1.3% ID/g) except for the kidneys 2, 4, and 24 h postinjection. Conclusions: DOTA position exhibited profound effect on melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In, providing a new insight into the design of lactam bridge-cyclized peptide for melanoma imaging and therapy.

The purpose of this study was to examine the melanoma imaging properties of a novel 67Ga-labeled lactam bridge-cyclized α-melanocyte stimulating hormone (α-MSH) peptide. A lactam bridge-cyclized α-MSH peptide, DOTA-GlyGlu-CycMSH {DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]}, was synthesized and radiolabeled with 67Ga. The melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GlyGlu-CycMSH were determined in B16/F1 flank primary melanoma-bearing and B16/F10 pulmonary metastatic melanoma-bearing C57 mice. Flank primary melanoma and pulmonary metastatic melanoma imaging were performed by small animal single photon emission computed tomography (SPECT)/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe. 67Ga-DOTA-GlyGlu-CycMSH was readily prepared with greater than 95% radiolabeling yield. 67Ga-DOTA-GlyGlu-CycMSH exhibited substantial tumor uptake (12.93 ± 1.63%ID/g at 2 h postinjection) and prolonged tumor retention (5.02 ± 1.35%ID/g at 24 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<0.30%ID/g) except for the kidneys at 2, 4, and 24 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited significantly (p < 0.05) higher uptakes (1.44 ± 0.75%ID/g at 2 h postinjection and 1.49 ± 0.69%ID/g at 4 h postinjection) in metastatic melanoma-bearing lung than those in normal lung (0.15 ± 0.10%ID/g and 0.17 ± 0.11%ID/g at 2 and 4 h postinjection, respectively). Both flank primary B16/F1 melanoma and B16/F10 pulmonary melanoma metastases were clearly visualized by SPECT/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe 2 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited favorable melanoma targeting and imaging properties, highlighting its potential as an effective imaging probe for early detection of primary and metastatic melanoma.

The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized α-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with 111In. The internalization and efflux of 111In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics, and SPECT/CT imaging of 111In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both 111In-DOTA-CycMSH and 111In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of 111In-DOTA-CycMSH and 111In-DOTA-GlyGlu-CycMSH were 9.53 ± 1.41% injected dose/gram (% ID/g) and 10.40 ± 1.40% ID/g at 2 h postinjection, respectively. Flank melanoma tumors were clearly visualized with 111In-DOTA-CycMSH and 111In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h postinjection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h postinjection. There was low radioactivity (<0.8% ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h postinjection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using 111In-labeled lactam bridge-cyclized α-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.

IntroductionThe aim of this study was to examine whether the substitution of the Lys linker with the aminooctanoic acid (Aoc) and polyethylene glycol (PEG) linker could substantially decrease the non-specific renal uptake of 99mTc-labeled Arg-Gly-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptides.MethodsThe RGD motif {Arg-Gly-Asp-DTyr-Asp} was coupled to [Cys3,4,10, D-Phe7, Arg11]α-MSH3–13 via the Aoc or PEG2 linker to generate RGD-Aoc-(Arg11)CCMSH and RGD-PEG-(Arg11)CCMSH. The biodistribution results of 99mTc-RGD-Aoc-(Arg11)CCMSH and 99mTc-RGD-PEG2-(Arg11)CCMSH were examined in M21 human melanoma-xenografted nude mice.ResultsThe substitution of Lys linker with Aoc and PEG2 linker significantly reduced the renal uptake of 99mTc-RGD-Aoc-(Arg11)CCMSH and 99mTc-RGD-PEG2-(Arg11)CCMSH by 58% and 63% at 2 h post-injection. The renal uptake of 99mTc-RGD-Aoc-(Arg11)CCMSH and 99mTc-RGD-PEG2-(Arg11)CCMSH was 27.93 ± 3.98 and 22.01 ± 9.89% ID/g at 2 h post-injection. 99mTc-RGD-Aoc-(Arg11)CCMSH displayed higher tumor uptake than 99mTc-RGD-PEG2-(Arg11)CCMSH (2.35 ± 0.12 vs. 1.71 ± 0.25% ID/g at 2 h post-injection). The M21 human melanoma lesions could be clearly visualized by SPECT/CT using 99mTc-RGD-Aoc-(Arg11)CCMSH as an imaging probe.ConclusionsThe favorable effect of Aoc and PEG2 linker in reducing the renal uptake provided a new insight into the design of novel dual receptor-targeting radiolabeled peptides.