Effects of Polyethylene Glycol and 8-Aminooctanoic Acid Linkers on Melanoma Uptake of [99mTc]Tc-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized α-MSH Peptides.

The purpose of this study was to evaluate the effect of linkers on tumor targeting and biodistribution of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex {[99mTc]Tc(CO)3-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-d-Phe-Arg-Trp-Lys]-CONH2} and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex {[99mTc]Tc(CO)3-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on B16/F10 melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized and radiolabeled with [99mTc]Tc via the {[99mTc]Tc(CO)3(OH2)3}+ intermediate. The biodistribution of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex and [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex were readily prepared with more than 90% radiochemical yields and exhibited MC1R-specific binding on B16/F10 melanoma cells. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex exhibited a higher tumor uptake than [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex at 2, 4, and 24 h postinjection. The tumor uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was 13.63 ± 1.13, 31.93 ± 2.57, 20.31 ± 3.23, and 1.33 ± 0.15% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The tumor uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was 1.6 and 3.4 times the tumor uptake of [99mTc]Tc(CO)3-NOTA-AocNle-CycMSHhex at 2 and 4 h postinjection, respectively. Meanwhile, the normal organ uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was lower than 1.8% ID/g at 2 h postinjection. The renal uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex was only 1.73 ± 0.37, 0.73 ± 0.14, and 0.03 ± 0.01% ID/g at 2, 4, and 24 h postinjection, respectively. [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios at 2 h postinjection. Single-photon emission computed tomography imaging revealed that the B16/F10 melanoma lesions could be clearly visualized by [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex at 2 h postinjection. Overall, the high tumor uptake and low kidney uptake of [99mTc]Tc(CO)3-NOTA-PEG2Nle-CycMSHhex highlighted its potential for melanoma imaging and warranted the future evaluation of [188Re]Re(CO)3-NOTA-PEG2Nle-CycMSHhex for melanoma therapy.

61Cu-Labelled radiodiagnostics of melanoma with NAPamide-targeted radiopharmaceutical.

Malignant melanoma is a major public health problem with an increasing incidence and mortality in the Caucasian population due to its significant metastatic potential. The early detection of this cancer type by imaging techniques like positron emission tomography acts as an important contributor to the long-term survival. Based on literature data, the radio labelled alpha-MSH analog NAPamide molecule is an appropriate diagnostic tool for the detection of melanoma tumors. Inspired by these facts, a new radiotracer, the [61Cu]Cu-KFTG-NAPamide has been synthesized to exploit the beneficial features of the positron emitter 61Cu and the melanoma specificity of the NAPamide molecule. In this work, we report a new member of the CB-15aneN5 ligand family (KFTG) as the chelator for 61Cu(II) complexation. On the basis of the thorough physico-chemical characterization, the rigid [Cu(KFTG)]+ complex exhibits fast complex formation (t1/2 = 155 s at pH 5.0 and 25 °C) and high inertness (t1/2 = 2.0 h in 5.0 M HCl at 50 °C) as well as moderate superoxide dismutase activity (IC50 = 2.3 µM). Furthermore, the [61Cu]Cu-KFTG-NAPamide possesses outstanding features in the diagnostics of B16-F10 melanoma tumors by PET imaging: (T/M(SUVs) (in vivo): appr. 14, %ID/g: 7 ± 1 and T/M (ex vivo): 315 ± 24 at 180 min).

Novel Al18F-NOTA-Conjugated Lactam-Cyclized α-Melanocyte-Stimulating Hormone Peptides with Enhanced Melanoma Uptake.

The purpose of this study was to evaluate the effect of linker on tumor targeting and biodistribution of Al18F-NOTA-PEG2Nle-CycMSHhex {Al18F-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-poly(ethylene glycol)-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and Al18F-NOTA-AocNle-CycMSHhex {Al18F-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of Al18F-NOTA-PEG2Nle-CycMSHhex and Al18F-NOTA-AocNle-CycMSHhex was determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of Al18F-NOTA-PEG2Nle-CycMSHhex was further examined on B16/F10 melanoma-bearing C57 mice because of its higher melanoma uptake and lower renal uptake than that of Al18F-NOTA-AocNle-CycMSHhex. The IC50 values of NOTA-PEG2/AocNle-CycMSHhex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 cells. Al18F-NOTA-PEG2Nle-CycMSHhex and Al18F-NOTA-AocNle-CycMSHhex were readily prepared with more than 55% of radiolabeling yields and displayed melanocortin-1 receptor (MC1R)-specific binding on B16/F10 cells. Al18F-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake and lower kidney and liver uptake than Al18F-NOTA-AocNle-CycMSHhex at 1 and 2 h post injection. The tumor and renal uptakes of Al18F-NOTA-PEG2Nle-CycMSHhex were 17.44 ± 0.76 and 2.07 ± 0.43% ID/g at 1 h post injection, respectively. Al18F-NOTA-PEG2Nle-CycMSHhex showed the high tumor to normal organ uptake ratios after 1 h post injection. The B16/F10 melanoma lesions could be clearly visualized by positron emission tomography (PET) using Al18F-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 1 and 2 h post injection. Overall, high tumor uptake, low kidney and liver uptake, and fast urinary clearance of Al18F-NOTA-PEG2Nle-CycMSHhex highlighted its potential as an MC1R-targeted imaging probe for melanoma detection.

Novel 64Cu-Labeled NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptides with Enhanced Tumor to Kidney Uptake Ratios.

The aim of this study was to evaluate the effect of linker on tumor targeting and biodistribution of 64Cu-NOTA-PEG2Nle-CycMSHhex {64Cu-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 64Cu-NOTA-AocNle-CycMSHhex {64Cu-NOTA-8-aminooctanoic acid-Nle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were synthesized and purified by HPLC. The melanocortin-1 (MC1) receptor binding affinities of the peptides were examined on B16/F10 melanoma cells. The biodistributions of 64Cu-NOTA-PEG2Nle-CycMSHhex and 64Cu-NOTA-AocNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of 64Cu-NOTA-PEG2Nle-CycMSHhex was further examined on B16/F10 melanoma-bearing C57 mice because of its higher melanoma uptake than 64Cu-NOTA-AocNle-CycMSHhex. The IC50 values of NOTA-PEG2Nle-CycMSHhex and NOTA-AocNle-CycMSHhex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 melanoma cells. 64Cu-NOTA-PEG2Nle-CycMSHhex and 64Cu-NOTA-AocNle-CycMSHhex were readily prepared with more than 90% radiolabeling yields and showed MC1R-specific binding on B16/F10 cells. 64Cu-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake than 64Cu-NOTA-AocNle-CycMSHhex at 0.5, 2, 4, and 24 h post-injection. The tumor uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 16.23 ± 0.42, 19.59 ± 1.48, 12.83 ± 1.69, and 8.78 ± 2.29% ID/g at 0.5, 2, 4, and 24 h post-injection, respectively. Normal organ uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was lower than 2% ID/g at 2 h post-injection except for kidney uptake. The renal uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 3.66 ± 0.52, 3.27 ± 0.52, and 1.47 ± 0.56 ID/g at 2, 4, and 24 h post-injection, respectively. 64Cu-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h post-injection. The B16/F10 melanoma lesions could be clearly visualized by positron emission tomography (PET) using 64Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 2 h post-injection. High tumor uptake and low kidney uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex underscored its potential as an MC1R-targeted theranostic peptide for melanoma imaging and therapy.

[213Bi]Bi3+/[111In]In3+-neunpa-cycMSH: Theranostic Radiopharmaceutical Targeting Melanoma─Structural, Radiochemical, and Biological Evaluation.

With the emergence of [225Ac]Ac3+ as a therapeutic radionuclide for targeted α therapy (TAT), access to clinical quantities of the potent, short-lived α-emitter [213Bi]Bi3+ (t1/2 = 45.6 min) will increase over the next decade. With this in mind, the nonadentate chelator, H4neunpa-NH2, has been investigated as a ligand for chelation of [213Bi]Bi3+ in combination with [111In]In3+ as a suitable radionuclidic pair for TAT and single photon emission computed tomography (SPECT) diagnostics. Nuclear magnetic resonance (NMR) spectroscopy was utilized to assess the coordination characteristics of H4neunpa-NH2 on complexation of [natBi]Bi3+, while the solid-state structure of [natBi][Bi(neunpa-NH3)] was characterized via X-ray diffraction (XRD) studies, and density functional theory (DFT) calculations were performed to elucidate the conformational geometries of the metal complex in solution. H4neunpa-NH2 exhibited fast complexation kinetics with [213Bi]Bi3+ at RT achieving quantitative radiolabeling within 5 min at 10-8 M ligand concentration, which was accompanied by the formation of a kinetically inert complex. Two bioconjugates incorporating the melanocortin 1 receptor (MC1R) targeting peptide Nle-CycMSHhex were synthesized featuring two different covalent linkers for in vivo evaluation with [213Bi]Bi3+ and [111In]In3+. High molar activities of 7.47 and 21.0 GBq/µmol were achieved for each of the bioconjugates with [213Bi]Bi3+. SPECT/CT scans of the [111In]In3+-labeled tracer showed accumulation in the tumor over time, which was accompanied by high liver uptake and clearance via the hepatic pathway due to the high lipophilicity of the covalent linker. In vivo biodistribution studies in C57Bl/6J mice bearing B16-F10 tumor xenografts showed good tumor uptake (5.91% ID/g) at 1 h post-administration with [213Bi][Bi(neunpa-Ph-Pip-Nle-CycMSHhex)]. This study demonstrates H4neunpa-NH2 to be an effective chelating ligand for [213Bi]Bi3+ and [111In]In3+, with promising characteristics for further development toward theranostic applications.

The Effect of Albumin-Binding Moiety on Tumor Targeting and Biodistribution Properties of 67Ga-Labeled Albumin Binder-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides.

Background: The purpose of this study was to examine the effect of 4-p-(tolyl)butyric acid as an albumin-binding (ALB) moiety on tumor targeting and biodistribution properties of 67Ga-labeled albumin binder-conjugated alpha-melanocyte-stimulating hormone peptides. Materials and Methods: DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(ALB)-Gly/GlyGly/GlyGlyGly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} were synthesized with 4-p-(tolyl)butyric acid serving as an ALB moiety. The melanocortin-1 receptor (MC1R)-binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of 67Ga-DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex was examined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 67Ga-DOTA-Lys(ALB)-GGNle-CycMSHhex {67Ga-ALB-G2} were determined on B16/F10 melanoma-bearing C57 mice. Results: The IC50 value of DOTA-Lys(ALB)-G/GG/GGG-Nle-CycMSHhex {ALB-G1, ALB-G2, ALB-G3} was 0.67 ± 0.07, 0.5 ± 0.09 and 0.51 ± 0.03 nM on B16/F10 cells, respectively. 67Ga-ALB-G2 was further evaluated as a lead peptide because of its higher tumor uptake (30.25 ± 3.24%ID/g) and lower kidney uptake (7.09 ± 2.22%ID/g) than 67Ga-ALB-G1 and 67Ga-ALB-G3 at 2 h postinjection. The B16/F10 melanoma uptake of 67Ga-ALB-G2 was 15.64 ± 4.55, 30.25 ± 3.24, 26.76 ± 3.23, and 10.71 ± 1.21%ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 67Ga-ALB-G2 as an imaging probe at 2 h postinjection. Conclusions: The introduction of 4-p-(tolyl)butyric acid as an ALB moiety increased the blood retention, and resulted in higher tumor/kidney ratio of 67Ga-ALB-G2 as compared with its counterpart without an albumin binder. However, the resulting high uptake of 67Ga-ALB-G2 in blood and liver need to be further reduced to facilitate its therapeutic application when replacing 67Ga with therapeutic radionuclides.

Facile preparation of a novel Ga-67-labeled NODAGA-conjugated lactam-cyclized alpha-MSH peptide at room temperature for melanoma targeting.

In this study, the melanoma targeting property of 67Ga-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-D-Phe-Arg-Trp-Lys]-CONH2} was determined on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NODAGA as a radiometal chelator for facile room temperature radiolabeling of NODAGA-GGNle-CycMSHhex. The IC50 value of NODAGA-GGNle-CycMSHhex was 0.87 ± 0.12 nM on B16/F10 melanoma cells. 67Ga-NODAGA-GGNle-CycMSHhex was readily prepared at room temperature with greater than 98% radiolabeling yield and displayed MC1R-specific binding on B16/F10 melanoma cells. The B16/F10 melanoma uptake of 67Ga-NODAGA-GGNle-CycMSHhex was 10.31 ± 0.78, 14.96 ± 1.34, 13.7 ± 3.33 and 10.4 ± 2.2% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Approximately 85% of the injected dose was cleared out the body via urinary system at 2 h post-injection. 67Ga-NODAGA-GGNle-CycMSHhex showed high tumor/blood, tumor/muscle and tumor/skin uptake ratios after 2 h post-injection. Overall, 67Ga-NODAGA-GGNle-CycMSHhex could be easily prepared at room temperature and exhibited favorable melanoma targeting property, suggesting the potential use of NODAGA as a radiometal chelator for facile room temperature radiolabeling of α-MSH peptides.

Novel [99mTc]-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized Alpha-MSH Peptide with Enhanced Melanoma Uptake and Reduced Renal Uptake.

The purpose of this study was to examine the melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex {1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NOTA/NODAGA as metal chelators for 99mTc(CO)3+ radiolabeling. NOTA/NODAGA-GGNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice. The IC50 values of NOTA/NODAGA-GGNle-CycMSHhex were 0.8 ± 0.1 and 0.9 ± 0.1 nM on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex and 99mTc(CO)3-NODAGA-GGNle-CycMSHhex were readily prepared via the [99mTc(CO)3(OH2)3]+ intermediate and displayed MC1R-specific binding on B16/F10 cells. 99mTc(CO)3-NOTA-GGNle-CycMSHhex was further evaluated as a lead peptide because of its higher tumor uptake (19.76 ± 3.62% ID/g) and lower kidney uptake (1.59 ± 0.52% ID/g) at 2 h postinjection than 99mTc(CO)3-NODAGA-GGNle-CycMSHhex. The B16/F10 melanoma uptake of 99mTc(CO)3-NOTA-GGNle-CycMSHhex was 16.07 ± 4.47, 19.76 ± 3.62, 11.30 ± 2.81, and 3.16 ± 2.28% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. 99mTc(CO)3-NOTA-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h postinjection. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using 99mTc(CO)3-NOTA-GGNle-CycMSHhex as an imaging probe at 2 h postinjection. High tumor uptake, low kidney uptake, and fast urinary clearance of 99mTc(CO)3-NOTA-GGNle-CycMSHhex highlighted its potential for melanoma imaging and facilitated the evaluation of 188Re(CO)3-NOTA-GGNle-CycMSHhex for melanoma therapy.

Melanoma-Targeting Property of Y-90-Labeled Lactam-Cyclized α-Melanocyte-Stimulating Hormone Peptide.

Purpose: The purpose of this study was to evaluate melanoma-targeting property of 90Y-DOTA-GGNle-CycMSHhex to facilitate its potential therapeutic application. Materials and Methods: DOTA-GGNle-CycMSHhex was synthesized and readily labeled with 90Y in 0.25 M NH4Ac-buffered solution to generate 90Y-DOTA-GGNle-CycMSHhex. The specific receptor binding, internalization, and efflux of 90Y-DOTA-GGNle-CycMSHhex were determined on B16/F10 murine melanoma cells. The biodistribution property of 90Y-DOTA-GGNle-CycMSHhex was examined on B16/F10 melanoma-bearing C57 mice. Results: 90Y-DOTA-GGNle-CycMSHhex displayed receptor-specific binding, rapid internalization, and prolonged efflux on B16/F10 melanoma cells. 90Y-DOTA-GGNle-CycMSHhex exhibited high uptake and prolonged retention in melanoma, and fast urinary clearance on B16/F10 melanoma-bearing C57 mice. The B16/F10 tumor uptake was 20.73% ± 7.99%, 19.93% ± 5.73%, 14.8% ± 4.61%, and 6.69% ± 1.85% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. Conclusions: 90Y-DOTA-GGNle-CycMSHhex displayed melanocortin-1 receptor (MC1R) targeting and specificity on B16/F10 melanoma cells and tumors. The favorable melanoma-targeting property and fast urinary clearance of 90Y-DOTA-GGNle-CycMSHhex warranted its evaluation for melanoma therapy in future studies.

18F-Labeled Cyclized α-Melanocyte-Stimulating Hormone Derivatives for Imaging Human Melanoma Xenograft with Positron Emission Tomography.

Since metastatic melanoma is deadly, early diagnosis thereof is crucial for managing the disease. We recently developed α-melanocyte-stimulating hormone (αMSH) derivatives, [68Ga]Ga-CCZ01048 and [18F]CCZ01064, that target the melanocortin 1 receptor (MC1R) for mouse melanoma imaging. In this study, we aim to evaluate [18F]CCZ01064 as well as a novel dual-ammoniomethyl-trifluoroborate (AmBF3) derivative, [18F]CCZ01096, for targeting human melanoma xenograft using µPET imaging. The peptides were synthesized on solid phase using Fmoc chemistry. Radiolabeling was achieved in a one-step 18F-19F isotope-exchange reaction. µPET imaging and biodistribution studies were performed in NSG mice bearing SK-MEL-1 melanoma xenografts. The MC1R density on the SK-MEL-1 cell line was determined to be 972 ± 154 receptors/cell (n = 4) via saturation assays. Using [18F]CCZ01064, moderate tumor uptake (3.05 ± 0.47%ID/g) and image contrast were observed at 2 h post-injection. Molar activity was determined to play a key role. CCZ01096 with two AmBF3 motifs showed comparable sub-nanomolar binding affinity to MC1R and much higher molar activity. This resulted in improved tumor uptake (6.46 ± 1.42%ID/g) and image contrast (tumor-to-blood and tumor-to-muscle ratios were 30.6 ± 5.7 and 85.7 ± 11.3, respectively) at 2 h post-injection. [18F]CCZ01096 represents a promising αMSH-based µPET imaging agent for human melanoma and warrants further investigation for potential clinical translation.

Al18F-labeled alpha-melanocyte-stimulating hormone (α-MSH) peptide derivative for the early detection of melanoma.

OBJECTIVE: Early detection plays a role in the prognosis of melanoma, the most aggressive skin cancer. 64Cu- and 68Ga-labeled alpha-melanocyte-stimulating hormone (α-MSH) analogs targeting the melanocortin-1 receptor are promising positron emission tomography (PET) tracers for detecting melanoma, and the use of 18F-labeling will further contribute to the detectability and availability. However, the high radiochemistry demand related to the conventional 18F-labeling methods has restricted the development of 18F-labeled α-MSH analogs. A recently developed radiofluorination method using aluminum-fluoride (Al18F) offers a simple, efficient, and time-saving labeling procedure compared to the conventional 18F-labeling methods. Herein, we sought to establish a simple preparation method for an 18F-labeled α-MSH analog using Al18F, and we examined its potential for the early detection of melanoma. METHODS: A 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA)-conjugated α-MSH analog (NOTA-GGNle-CycMSHhex) was prepared by the Fmoc solid-phase strategy. NOTA-GGNle-CycMSHhex was labeled with Al18F by heating at 105 °C using a microwave synthesizer for 15 min. Biodistribution study was conducted on B16/F10-luc melanoma-bearing mice at 30 min, 1 h and 3 h after injection of Al18F-NOTA-GGNle-CycMSHhex. PET imaging was conducted on melanoma-bearing mice at 1 h post-injection. One day prior to the PET imaging, bioluminescence imaging was also performed. RESULTS: Al18F-NOTA-GGNle-CycMSHhex was readily prepared with a high radiochemical yield (94.0 ± 2.8%). The biodistribution study showed a high accumulation of Al18F-NOTA-GGNle-CycMSHhex in the tumor at 30 min and 1 h post-injection (6.69 ± 1.49 and 7.70 ± 1.71%ID/g, respectively). The tumor-to-blood ratio increased with time: 3.46 ± 0.89, 12.67 ± 1.29, and 35.27 ± 9.12 at 30 min, 1 h, and 3 h post-injection, respectively. In the PET imaging, Al18F-NOTA-GGNle-CycMSHhex clearly visualized the tumors and depicted very small tumors (< 3 mm). CONCLUSIONS: We successfully prepared Al18F-NOTA-GGNle-CycMSHhex in a simple and efficient manner. Al18F-NOTA-GGNle-CycMSHhex showed high tumor accumulation and clearly visualized very small tumors in melanoma-bearing mice. These findings suggest that Al18F-NOTA-GGNle-CycMSHhex will be a promising PET tracer for melanoma imaging at an earlier stage.

A tumor-targeted immune checkpoint blocker.

To direct checkpoint inhibition to the tumor microenvironment, while avoiding systemic immune activation, we have synthesized a bispecific antibody [norleucine4, d-Phe7]-melanocyte stimulating hormone (NDP-MSH)-antiprogrammed cell death-ligand 1 antibody (αPD-L1) by conjugating a melanocyte stimulating hormone (α-MSH) analog to the antiprogrammed cell death-ligand 1 to (αPD-L1) antibody avelumab. This bispecific antibody can bind to both the melanocortin-1 receptor (MC1R) and to PD-L1 expressed on melanoma cells and shows enhanced specific antitumor efficacy in a syngeneic B16-SIY melanoma mouse model compared with the parental antibody at a 5 mg/kg dose. Moreover, the bispecific antibody showed increased infiltrated T cells in the tumor microenvironment. These results suggest that a tumor-targeted PD-L1-blocking bispecific antibody could have a therapeutic advantage in vivo, especially when used in combination with other checkpoint inhibitors.

Increasing molar activity by HPLC purification improves 68Ga-DOTA-NAPamide tumor accumulation in a B16/F1 melanoma xenograft model.

PURPOSE: Melanocortin receptor 1 (MC1R) is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium (68Ga) labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography (PET) imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide. PROCEDURES: The MC1R ligand DOTA-NAPamide was labeled with 68Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68Ga (95°C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors. RESULTS: In biodistribution studies, non-purified 68Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracer preparations. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. CONCLUSIONS: We demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.

Radiochemical and radiopharmacological characterization of a 64 Cu-labeled α-MSH analog conjugated with different chelators.

Radiolabeled α-melanocyte-stimulating hormone (α-MSH) derivatives have a high potential for diagnosis and treatment of melanoma, because of high specificity and binding affinity to the melanocortin-1 receptor (MC1R). Hence, the α-MSH-derived peptide NAP-NS1 with a ß-Ala linker (ε-Ahx-ß-Ala-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 ) was conjugated to different chelators: either to NOTA (p-SCN-Bn-1,4,7-triazacyclononane-1,4,7-triacetic acid), to a hexadentate bispidine carbonate derivative (dimethyl-9-(((4-nitrophenoxy)carbonyl)oxy)-2,4-di(pyridin-2-yl)-3,7-bis(pyridin-2-ylmethyl)-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate), or to DMPTACN (p-SCN-Ph-bis(2-pyridyl-methyl)-1,4,7-triaza-cyclononane), labeled with 64 Cu, and investigated in terms of radiochemical and radiopharmacological properties. For the three 64 Cu-labeled conjugates negligible transchelation, suitable buffer and serum stability, as well as appropriate water solubility, was determined. The three conjugates exhibited high binding affinity (low nanomolar range) in murine B16F10, human MeWo, and human TXM13 cells. The Bmax values of [64 Cu]Cu-bispidine-NAP-NS1 ([64 Cu]Cu-2) and [64 Cu]Cu-DMPTACN-NAP-NS1 ([64 Cu]Cu-3) were higher than those of [64 Cu]Cu-NOTA-NAP-NS1 ([64 Cu]Cu-1), implying that different charged chelate units might have an impact on binding capacity. Preliminary in vivo biodistribution studies suggested the main excretion pathway of [64 Cu]Cu-1 and [64 Cu]Cu-3 to be renal, while that of [64 Cu]Cu-2 seemed to be both renal and hepatobiliary. An initial moderate uptake in the kidney decreased clearly after 60 minutes. All three 64 Cu-labeled conjugates should be considered for further in vivo investigations using a suitable xenograft mouse model.

The pharmacological properties of 3-arm or 4-arm DOTA constructs for conjugation to α-melanocyte-stimulating hormone analogues for melanoma imaging.

BACKGROUND: Although a 3-arm DOTA construct, which has three carboxylic acids, h has been applied for conjugation to many peptides, we investigated if a 4-arm DOTA construct conjugated to peptides improves chemical properties for melanoma imaging of the melanocortin 1 receptor compared to 3-arm DOTA-conjugated peptides. METHODS: Specific activities, radiolabeling efficiencies, and partition coefficients were evaluated using 111In-labeled 3-arm and 4-arm DOTA-α-melanocyte-stimulating hormone (MSH). For assessment of MC1-R affinity and accumulation in tumor cells in vitro, B16-F1 melanoma and/or 4T1 breast cancer cells were incubated with 111In-labeled 3-arm and 4-arm DOTA-α-MSH with and without α-MSH as a substrate. The stability was evaluated using mouse liver homogenates and plasma. Biological distribution and whole-body single photon emission computed tomography imaging of 111In-labeled 3-arm and 4-arm DOTA-α-MSH were obtained using B16-F1 melanoma-bearing mice. RESULTS: Specific activities and radiolabeling efficiencies of both radiotracers were about 1.2 MBq/nM and 90-95%, respectively. The partition coefficients were -0.28 ± 0.03 for 111In-labeled 3-arm DOTA-α-MSH and -0.13 ± 0.04 for 111In-labeled 4-arm DOTA-α-MSH. Although accumulation was significantly inhibited by α-MSH in B16-F1 cells, the inhibition rate of 111In-labeled 4-arm DOTA-α-MSH was lower than that of 111In-labeled 3-arm DOTA-α-MSH. 111In-labeled 4-arm DOTA-α-MSH was taken up early into B16-F1 cells and showed higher accumulation than 111In-labeled 3-arm DOTA-α-MSH after 10 min of incubation. Although these stabilities were relatively high, the stability of 111In-labeled 4-arm DOTA-α-MSH was higher than that of 111In-labeled 3-arm DOTA-α-MSH. Regarding biological distribution, 111In-labeled 4-arm DOTA-α-MSH showed significantly lower average renal accumulation (1.38-fold) and significantly higher average melanoma accumulation (1.32-fold) than 111In-labeled 3-arm DOTA-α-MSH at all acquisition times. 111In-labeled 4-arm DOTA-α-MSH showed significantly higher melanoma-to-kidney, melanoma-to-blood, and melanoma-to-muscle ratios than 111In-labeled 3-arm DOTA-α-MSH. CONCLUSIONS: The 4-arm DOTA construct has better chemical properties for peptide radiotracers than the 3-arm DOTA construct.

Insights into the Allosteric Mechanism of Setmelanotide (RM-493) as a Potent and First-in-Class Melanocortin-4 Receptor (MC4R) Agonist To Treat Rare Genetic Disorders of Obesity through an in Silico Approach.

Human melanocortin-4 receptor (hMC4R) mutations have been implicated as the cause for about 6-8% of all severe obesity cases. Drug-like molecules that are able to rescue the functional activity of mutated receptors are highly desirable to combat genetic obesity among this population of patients. One such molecule is the selective MC4R agonist RM-493 (setmelanotide). While this molecule has been shown to activate mutated receptors with 20-fold higher potency over the endogenous agonist, little is known about its binding mode and how it effectively interacts with hMC4R despite the presence of mutations. In this study, a MC4R homology model was constructed based on the X-ray crystal structure of the adenosine A2A receptor in the active state. Four MC4R mutations commonly found in genetically obese patients and known to effect ligand binding in vitro were introduced into the constructed model. RM-493 was then docked into the wild-type and mutated models in order to better elucidate the possible binding modes for this promising drug candidate and assess how it may be interacting with MC4R to effectively activate receptor polymorphisms. The results reflected the orthosteric interactions of both the endogenous and synthetic ligands with the MC4R, which is supported by the site-directed mutagenesis studies. Meanwhile it helped explain the decremental affinity and potency of these ligands with the receptor polymorphisms. More significantly, our findings indicated that the structural characteristics of RM-493 may allow for enhanced receptor-ligand interactions, particularly through those with the putative allosteric binding sites, which facilitated the ligand to stabilize the active state of native and mutant MC4Rs to maintain reasonably high affinity and potency.

MC1R: Front and Center in the Bright Side of Dark Eumelanin and DNA Repair.

Melanin, the pigment produced by specialized cells, melanocytes, is responsible for skin and hair color. Skin pigmentation is an important protective mechanism against the DNA damaging and mutagenic effects of solar ultraviolet radiation (UV). It is acknowledged that exposure to UV is the main etiological environmental factor for all forms of skin cancer, including melanoma. DNA repair capacity is another major factor that determines the risk for skin cancer. Human melanocytes synthesize eumelanin, the dark brown form of melanin, as well as pheomelanin, which is reddish-yellow in color. The relative rates of eumelanin and pheomelanin synthesis by melanocytes determine skin color and the sensitivity of skin to the drastic effects of solar UV. Understanding the complex regulation of melanocyte function and how it responds to solar UV has a huge impact on developing novel photoprotective strategies to prevent skin cancer, particularly melanoma, the most fatal form, which originates from melanocytes. This review provides an overview of the known differences in the photoprotective effects of eumelanin versus pheomelanin, how these two forms of melanin are regulated genetically and biochemically, and their impact on the DNA damaging effects of UV exposure. Additionally, this review briefly discusses the role of paracrine factors, focusing on α-melanocortin (α-melanocyte stimulating hormone; α-MSH), in regulating melanogenesis and the response of melanocytes to UV, and describes a chemoprevention strategy based on targeting the melanocortin 1 receptor (MC1R) by analogs of its physiological agonist α-MSH.

Melanocortin 1 Receptor Targeted Imaging of Melanoma With Gold Nanocages and Positron Emission Tomography.

PURPOSE: Melanoma is a lethal skin cancer with unmet clinical needs for targeted imaging and therapy. Nanoscale materials conjugated with targeting components have shown great potential to improve tumor delivery efficiency while minimizing undesirable side effects in vivo. Herein, we proposed to develop targeted nanoparticles for melanoma theranostics. METHOD: In this work, gold nanocages (AuNCs) were conjugated with α-melanocyte-stimulating hormone (α-MSH) peptide and radiolabeled with 64Cu for melanocortin 1 receptor-(MC1R) targeted positron emission tomography (PET) in a mouse B16/F10 melanoma model. RESULTS: Their controlled synthesis and surface chemistry enabled well-defined structure and radiolabeling efficiency. In vivo pharmacokinetic evaluation demonstrated comparable organ distribution between the targeted and nontargeted AuNCs. However, micro-PET/computed tomography (CT) imaging demonstrated specific and improved tumor accumulation via MC1R-mediated delivery. By increasing the coverage density of α-MSH peptide on AuNCs, the tumor delivery efficiency was improved. CONCLUSION: The controlled synthesis, sensitive PET imaging, and optimal tumor targeting suggested the potential of targeted AuNCs for melanoma theranostics.

Melanoma Imaging Using 18F-Labeled α-Melanocyte-Stimulating Hormone Derivatives with Positron Emission Tomography.

Melanocortin 1 receptor (MC1R) is specifically expressed in the majority of melanomas, a leading cause of death related to skin cancers. Accurate staging and early detection is crucial in managing melanoma. Based on the α-melanocyte-stimulating hormone (αMSH) sequence, MC1R-targeted peptides have been studied for melanoma imaging, predominately for use with single-photon emission computed tomography, with few attempts made for positron emission tomography (PET). 18F is a commonly used PET isotope due to readily available cyclotron production, pure positron emission, and a favorable half-life (109.8 min). In this study, we aim to design and evaluate αMSH derivatives that enable radiolabeling with 18F for PET imaging of melanoma. We synthesized three imaging probes based on the structure of Nle4-cyclo[Asp5-His-d-Phe7-Arg-Trp-Lys10]-NH2 (Nle-CycMSHhex), with a Pip linker (CCZ01064), an Acp linker (CCZ01070), or an Aoc linker (CCZ01071). 18F labeling was enabled by an ammoniomethyl-trifluoroborate (AmBF3) moiety. In vitro competition binding assays showed subnanomolar inhibition constant ( Ki) values for all three peptides. The 18F radiolabeling was performed via a one-step 18F-19F isotope exchange reaction that resulted in high radiochemical purity (>95%) and good molar activity (specific activity) ranging from 40.7 to 66.6 MBq/nmol. All three 18F-labeled peptides produced excellent tumor visualization with PET imaging in C57BL/6J mice bearing B16-F10 tumors. The tumor uptake was 7.80 ± 1.77, 5.27 ± 2.38, and 5.46 ± 2.64% injected dose per gram of tissue (%ID/g) for [18F]CCZ01064, [18F]CCZ01070, and [18F]CCZ01071 at 1 h post-injection (p.i.), respectively. Minimal background activity was observed except for kidneys at 4.99 ± 0.20, 4.42 ± 0.54, and 13.55 ± 2.84%ID/g, respectively. The best candidate [18F]CCZ01064 was further evaluated at 2 h p.i., which showed increased tumor uptake at 11.96 ± 2.31%ID/g and further reduced normal tissue uptake. Moreover, a blocking study was performed for CCZ01064 at 1 h p.i., where tumor uptake was significantly reduced to 1.97 ± 0.60%ID/g, suggesting the tumor uptake was receptor mediated. In conclusion, [18F]CCZ01064 showed high tumor uptake, low normal tissue uptake, and fast clearance and is therefore a suitable and promising candidate for PET imaging of melanoma.

Molecular Imaging and Radionuclide Therapy of Melanoma Targeting the Melanocortin 1 Receptor.

Melanoma is a deadly disease at late metastatic stage, and early diagnosis and accurate staging remain the key aspects for managing melanoma. The melanocortin 1 receptor (MC1 R) is overexpressed in primary and metastatic melanomas, and its endogenous ligand, the α-melanocyte-stimulating hormone (αMSH), has been extensively studied for the development of MC1 R-targeted molecular imaging and therapy of melanoma. Natural αMSH is not well suited for this purpose due to low stability in vivo. Unnatural amino acid substitutions substantially stabilized the peptide, while cyclization via lactam bridge and metal coordination further improved binding affinity and stability. In this study, we summarized the development and the in vitro and in vivo characteristics of the radiolabeled αMSH analogues, including 99mTc-, 111In-, 67 Ga-, or 125I-labeled αMSH analogues for imaging with single-photon emission computed tomography; 68Ga-, 64Cu-, or 18F-labeled αMSH analogues for imaging with positron emission tomography; and 188Re-, 177Lu-, 90Y-, or 212Pb-labeled αMSH analogues for radionuclide therapy. These radiolabeled αMSH analogues showed promising results with high tumor uptake and rapid normal tissue activity clearance in the preclinical model of B16F1 and B16F10 mouse melanomas. These results highlight the potential of using radiolabeled αMSH analogues in clinical applications for molecular imaging and radionuclide therapy of melanoma.