Effect of Ibuprofen as an Albumin Binder on Melanoma-Targeting Properties of 177Lu-Labeled Ibuprofen-Conjugated Alpha-Melanocyte-Stimulating Hormone Peptides.

The purpose of this study was to examine how the introduction of ibuprofen (IBU) affected tumor-targeting and biodistribution properties of 177Lu-labeled IBU-conjugated alpha-melanocyte-stimulating hormone peptides. The IBU was used as an albumin binder and conjugated to the DOTA-Lys moiety without or with a linker to yield DOTA-Lys(IBU)-GG-Nle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys(IBU)-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2}, DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex, DOTA-Lys(Asn-IBU)-GGNle-CycMSHhex, and DOTA-Lys(Dab-IBU)-GGNle-CycMSHhex peptides. Their melanocortin-receptor 1 (MC1R) binding affinities were determined on B16/F10 melanoma cells first. Then the biodistribution of 177Lu-labeled peptides was determined on B16/F10 melanoma-bearing C57 mice at 2 h postinjection to choose the lead peptide for further examination. The full biodistribution and melanoma imaging properties of 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex were further evaluated using B16/F10 melanoma-bearing C57 mice. DOTA-Lys(IBU)-GG-Nle-CycMSHhex, DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex, DOTA-Lys(Asn-IBU)-GGNle-CycMSHhex, and DOTA-Lys(Dab-IBU)-GGNle-CycMSHhex displayed the IC50 values of 1.41 ± 0.37, 1.52 ± 0.08, 0.03 ± 0.01, and 0.58 ± 0.06 nM on B16/F10 melanoma cells, respectively. 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex exhibited the lowest liver and kidney uptake among all four designed 177Lu peptides. Therefore, 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex was further evaluated for its full biodistribution and melanoma imaging properties. The B16/F10 melanoma uptake of 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex was 19.5 ± 3.12, 24.12 ± 3.35, 23.85 ± 2.08, and 10.80 ± 2.89% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. Moreover, 177Lu-DOTA-Lys(Asp-IBU)-GGNle-CycMSHhex could clearly visualize the B16/F10 melanoma lesions at 2 h postinjection. The conjugation of IBU with or without a linker to GGNle-CycMSHhex affected the MC1R binding affinities of the designed peptides. The charge of the linker played a key role in the liver and kidney uptake of 177Lu-Asp-IBU, 177Lu-Asn-IBU, and 177Lu-Dab-IBU. 177Lu-Asp-IBU exhibited higher tumor/liver and tumor/kidney uptake ratios than those of 177Lu-Asn-IBU and 177Lu-Dab-IBU, underscoring its potential evaluation for melanoma therapy in the future.

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.

Biodistribution and Multicompartment Pharmacokinetic Analysis of a Targeted α Particle Therapy.

Targeted α particle therapy (TAT) is ideal for treating disease while minimizing damage to surrounding nontargeted tissues due to short path length and high linear energy transfer (LET). We developed a TAT for metastatic uveal melanoma, targeting the melanocortin-1 receptor (MC1R), which is expressed in 94% of uveal melanomas. Two versions of the therapy are being investigated: 225Ac-DOTA-Ahx-MC1RL (225Ac-Ahx) and 225Ac-DOTA-di-d-Glu-MC1RL (225Ac-di-d-Glu). The biodistribution (BD) from each was studied and a multicompartment pharmacokinetic (PK) model was developed to describe drug distribution rates. Two groups of 16 severe combined immunodeficient (SCID) mice bearing high MC1R expressing tumors were intravenously injected with 225Ac-Ahx or 225Ac-di-d-Glu. After injection, four groups (n = 4) were euthanized at 24, 96, 144, and 288 h time points for each cohort. Tumors and 13 other organs were harvested at each time point. Isomeric γ spectra were measured in tissue samples using a scintillation γ detector and converted to α activity using factors for γ ray abundance per α decay. Time activity curves were calculated for each organ. A five-compartment PK model was built with the following compartments: blood, tumor, normal tissue, kidney, and liver. This model is characterized by a system of five ordinary differential equations using mass action kinetics, which describe uptake, intercompartmental transitions, and clearance rates. The ordinary differential equations were simultaneously solved and fit to experimental data using a genetic algorithm for optimization. The BD data show that both compounds have minimal distribution to organs at risk other than the kidney and liver. The PK parameter estimates had less than 5% error. From these data, 225Ac-Ahx showed larger and faster uptake in the liver. Both compounds had comparable uptake and clearance rates for other compartments. The BD and PK behavior for two targeted radiopharmaceuticals were investigated. The PK model fit the experimental data and provided insight into the kinetics of the compounds systematically.

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.

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.

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.

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.

Design of MC1R Selective γ-MSH Analogues with Canonical Amino Acids Leads to Potency and Pigmentation.

Melanoma is a lethal form of skin cancer. Skin pigmentation, which is regulated by the melanocortin 1 receptor (MC1R), is an effective protection against melanoma. However, the endogenous MC1R agonists lack selectivity for the MC1R and thus can have side effects. The use of noncanonical amino acids in previous MC1R ligand development raises safety concerns. Here we report the development of the first potent and selective hMC1R agonist with only canonical amino acids. Using γ-MSH as a template, we developed a peptide, [Leu3, Leu7, Phe8]-γ-MSH-NH2 (compound 5), which is 16-fold selective for the hMC1R (EC50 = 4.5 nM) versus other melanocortin receptors. Conformational studies revealed a constrained conformation for this linear peptide. Molecular docking demonstrated a hydrophobic binding pocket for the melanocortin 1 receptor. In vivo pigmentation study shows high potency and short duration. [Leu3, Leu7, Phe8]-γ-MSH-NH2 is ideal for inducing short-term skin pigmentation without sun for melanoma prevention.

Preclinical evaluation of melanocortin-1 receptor (MC1-R) specific 68Ga- and 44Sc-labeled DOTA-NAPamide in melanoma imaging.

PURPOSE: Alpha melanocyte stimulating hormone (α-MSH) enhances melanogenesis in melanoma malignum by binding to melanocortin-1 receptors (MC1-R). Earlier studies demonstrated that alpha-MSH analog NAPamide molecule specifically binds to MC1-R receptor. Radiolabeled NAPamide is a promising radiotracer for the non-invasive detection of melanin producing melanoma tumors by Positron Emission Tomography (PET). In this present study the MC1-R selectivity of the newly developed Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using melanoma tumors. METHODS: DOTA-NAPamide was labeled with Ga-68 and Sc-44 radionuclides. The MC1-R specificity of Ga-68- and Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using MC1-R positive (B16-F10) and negative (A375) melanoma cell lines. For in vivo imaging studies B16-F10 and A375 tumor-bearing mice were injected with 44Sc/68Ga-DOTA-NAPamide (in blocking studies with α-MSH) and whole body PET/MRI scans were acquired. Radiotracer uptake was expressed in terms of standardized uptake values (SUVs). RESULTS: 44Sc/68Ga-labeled DOTA-NAPamide were produced with high specific activity (approx. 19 GBq/µmol) and with excellent radiochemical purity (99%<). MC1-R positive B16-F10 cells showed significantly (p≤0.01) higher in vitro radiotracer accumulation than that of receptor negative A375 melanoma cells. In animal experiments, also significantly (p≤0.01) higher Ga-68-DOTA-NAPamide (SUVmean: 0.38±0.02), and Sc-44-DOTA-NAPamide (SUVmean: 0.52±0.13) uptake was observed in subcutaneously growing B16-F10 tumors, than in receptor negative A375 tumors, where the SUVmean values of Ga-68-DOTA-NAPamide and Sc-44-DOTA-NAPamide were 0.04±0.01 and 0.07±0.01, respectively. Tumor-to-muscle (T/M SUVmean) ratios were approximately 15-fold higher in B16-F10 tumor-bearing mice, than that of A375 tumors, and this difference was also significant (p≤0.01) using both radiotracers after 60 min incubation time. CONCLUSION: Our newly synthesized 44Sc-labeled DOTA-NAPamide probe showed excellent binding properties to melanocortin-1 receptor (MC1-R) positive melanoma cell and tumors. Due to its high specificity and sensitivity 44Sc-DOTA-NAPamide is a promising radiotracer in molecular imaging of malignant melanoma.

The Effect of A Hexanoic Acid Linker Insertion on the Pharmacokinetics and Tumor Targeting Properties of the Melanoma Imaging Agent 99mTc-HYNIC-cycMSH.

BACKGROUND: Lactam cyclized alpha-melanocyte stimulating hormone (α-MSH) analogues exhibit high stability and affinity for the MC1-R receptors over expressed in melanoma cells. Recently, we reported a novel 99mTc-HYNIC-cycMSH4-13 analogue with the HYNIC chelator directly attached to the lactam cyclized ring. OBJECTIVE: In this study we proposed the introduction of a 6-aminohexanoic acid (Ahx) linker between the HYNIC chelator and lactam cyclized peptide cycMSH4-13 to reduce steric hindrance and improve the melanoma targeting and imaging proprieties of the radiolabeled peptide. METHOD: HYNIC-Ahx-cycMSH4-13 peptide was synthesized on an automated peptide synthesizer and displayed an IC50 of 0.3 nM using B16/F1 cells. The 99mTc/tricine radiolabeled peptide was examined for radiochemical purity, stability and cell binding. In vivo, biodistribution and planar gamma imaging studies were performed in B16/F1 melanoma tumor bearing C57BK mice. RESULTS: 99mTc-HYNIC-Ahx-cycMSH4-13 was obtained with a radiochemical purity > 95%, was stable up to 24 h at room temperature and exhibited high binding and rapid internalization in B16/F1 cells. In vivo biodistribution studies showed a tumor uptake of 4.92 ± 0.92 % ID/g and 2.78 ± 1.48 % ID/g at 2 h and 4 h post injection, respectively. Whole-body clearance was rapid through urinary excretion. The melanoma tumors were clearly visualized by planar gamma imaging. CONCLUSION: 99mTc-HYNIC-Ahx-cycMSH4-13 was shown radiochemically stability and exhibited rapid and selective uptake in melanoma cells and tumors. Imaging studies yielded promising preclinical results, warranting further evaluation of 99mTc-HYNIC-cycMSH analogs as melanoma specific imaging agents.

TAT-HSA-α-MSH fusion protein with extended half-life inhibits tumor necrosis factor-α in brain inflammation of mice.

Neuroinflammation constitutes a principal process involved in the progression of various central nervous system (CNS) disorders, including Parkinson's disease, Alzheimer's disease, ischemic stroke, and traumatic brain injury. The safety and efficacy of potential neuroprotective therapeutic agents is controversial and limited. Alpha-melanocyte-stimulating hormone (α-MSH) as a tridecapeptide derived from pro-opiomelanocortin displays potent anti-inflammatory and protective effects with a wide therapeutic window in brain damage. However, it is difficult to deliver effective concentrations of α-MSH into brain tissue via nondirect application. Besides, the half-life of the tridecapeptide is only a few minutes. In the present study, we generated a novel TAT-HSA-α-MSH by genetically fusing α-MSH with N-terminus 11-amino acid protein transduction domain of the human immunodeficiency virus Tat protein (TAT) and human serum albumin (HSA), which showed favorable pharmacokinetic properties and can effectively cross the blood brain barrier (BBB). The findings showed that TAT-HSA-α-MSH significantly inhibits NF-κB activation in human glioma cells A172 and tumor necrosis factor-α (TNF-α) production in experimental brain inflammation. These results indicate that TAT-HSA-α-MSH may be a potential therapeutic agent for treating neuroinflammation which plays a fundamental role in CNS disorders.

Radiopharmacological characterization of 64Cu-labeled α-MSH analogs for potential use in imaging of malignant melanoma.

The melanocortin-1 receptor (MC1R) plays an important role in melanoma growth, angiogenesis and metastasis, and is overexpressed in melanoma cells. α-Melanocyte stimulating hormone (α-MSH) and derivatives are known to bind with high affinity at this receptor that provides the potential for selective targeting of melanoma. In this study, one linear α-MSH-derived peptide Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) without linker and with εAhx-ß-Ala linker, and a cyclic α-MSH derivative, [Lys-Glu-His-D-Phe-Arg-Trp-Glu]-Arg-Pro-Val-NH2 (NAP-NS2) with εAhx-ß-Ala linker were conjugated with p-SCN-Bn-NOTA and labeled with (64)Cu. Radiochemical and radiopharmacological investigations were performed with regard to transchelation, stability, lipophilicity and in vitro binding assays as well as biodistribution in healthy rats. No transchelation reactions, but high metabolic stability and water solubility were demonstrated. The linear derivatives showed higher affinity than the cyclic one. [(64)Cu]Cu-NOTA-εAhx-ß-Ala-NAP-NS1 ([(64)Cu]Cu-2) displayed rapid cellular association and dissociation in murine B16F10 cell homogenate. All [(64)Cu]Cu-labeled conjugates exhibited affinities in the low nanomolar range in B16F10. [(64)Cu]Cu-2 showed also high affinity in human MeWo and TXM13 cell homogenate. In vivo studies suggested that [(64)Cu]Cu-2 was stable, with about 85 % of intact peptide in rat plasma at 2 h p.i. Biodistribution confirmed the renal pathway as the major elimination route. The uptake of [(64)Cu]Cu-2 in the kidney was 5.9 % ID/g at 5 min p.i. and decreased to 2.0 % ID/g at 60 min p.i. Due to the prospective radiochemical and radiopharmacological properties of the linear α-MSH derivative [(64)Cu]Cu-2, this conjugate is a promising candidate for tracer development in human melanoma imaging.

In Vivo Targeting of Cutaneous Melanoma Using an Melanoma Stimulating Hormone-Engineered Human Protein Cage with Fluorophore and Magnetic Resonance Imaging Tracers.

Nanoparticle (NP)-based materials are promising agents for enhancing cancer diagnosis and treatment. Once functionalized for selective targeting of tumor-expressed molecules, they can specifically deliver drugs and diagnostic molecules inside tumor cells. In the present work, we evaluated the in vivo melanoma-targeting ability of a nanovector (HFt-MSH-PEG) based on human protein ferritin (HFt), functionalized with both melanoma-targeting melanoma stimulating hormone (α-MSH) and stabilizing poly(ethylene glycol) (PEG) molecules. Independent and complementary techniques, such as whole-specimen confocal microscopy and magnetic resonance imaging, were used to detect in vivo localization of NP constructs with suitable tracers (i.e., fluorophores or magnetic metals). Targeted HFt-MSH-PEG NPs accumulated persistently at the level of primary melanoma and with high selectivity with respect to other organs. Melanoma localization of untargeted HFt-PEG NPs, which lack the α-MSH moiety, was less pronounced. Furthermore, HFt-MSH-PEG NPs accumulated to a significantly lower extent and with a different distribution in a diverse type of tumor (TS/A adenocarcinoma), which does not express α-MSH receptors. Finally, in a spontaneous lung metastasis model, HFt-MSH-PEG NPs localized at the metastasis level as well. These results suggest that HFt-MSH-PEG NPs are suitable carriers for selective in vivo delivery of diagnostic or therapeutic agents to cutaneous melanoma.

Tc-99m-labeled RGD-conjugated alpha-melanocyte stimulating hormone hybrid peptides with reduced renal uptake.

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 [Cys(3,4,10), D-Phe(7), Arg(11)]α-MSH3-13 {(Arg(11))CCMSH} through the neutral ßAla or Ahx {aminohexanoic acid} linker (replacing the Lys or Arg linker) to generate novel RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))CCMSH hybrid peptides. The receptor-binding affinity and cytotoxicity of RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))CCMSH were determined in B16/F1 melanoma cells. The melanoma targeting and imaging properties of (99m)Tc-RGD-ßAla-(Arg(11))CCMSH and (99m)Tc-RGD-Ahx-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The replacement of the Lys or Arg linker with the ßAla or Ahx linker retained nanomolar receptor-binding affinities and remarkable cytotoxicity of RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))CCMSH. The receptor-binding affinities of RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))CCMSH were 0.8 ± 0.05 and 1.3 ± 0.1 nM. Three-hour incubation with 0.1 µM of RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))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 ßAla or Ahx linker reduced the non-specific renal uptake of (99m)Tc-RGD-ßAla-(Arg(11))CCMSH and (99m)Tc-RGD-Ahx-(Arg(11))CCMSH by 62 and 61 % at 2 h post-injection. (99m)Tc-RGD-ßAla-(Arg(11))CCMSH displayed higher melanoma uptake than (99m)Tc-RGD-Ahx-(Arg(11))CCMSH at 0.5, 2, 4, and 24 h post-injection. Enhanced tumor to kidney uptake ratio of (99m)Tc-RGD-ßAla-(Arg(11))CCMSH warranted the further evaluation of (188)Re-labeled RGD-ßAla-(Arg(11))CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.

Linker modification reduced the renal uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide.

The purpose of this study was to examine the biodistribution of (99m)Tc-RAD-Arg-(Arg(11))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 (99m)Tc-RAD-Arg-(Arg(11))CCMSH. (99m)Tc-RAD-Arg-(Arg(11))CCMSH exhibited rapid and high tumor uptake (17.98±4.96% ID/g at 2h post-injection) in B16/F1 melanoma-bearing C57 mice. As compared to (99m)Tc-RAD-Lys-(Arg(11))CCMSH, the replacement of the Lys linker with an Arg linker dramatically decreased the renal uptake of (99m)Tc-RAD-Arg-(Arg(11))CCMSH by 68%, 62%, 73% and 64% at 0.5, 2, 4 and 24h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2h post-injection using (99m)Tc-RAD-Arg-(Arg(11))CCMSH as an imaging probe.

Investigation of safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple doses of a long-acting α-MSH analog in healthy overweight and obese subjects.

MC4-NN2-0453 is a novel, long-acting, selective, melanocortin-4-receptor agonist developed for treatment of obesity. This first-human-dose, randomized, double-blind, placebo-controlled trial investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple doses of MC4-NN2-0453 in overweight to obese but otherwise healthy subjects. The trial included a single-dose part of ascending subcutaneous 0.03-1.50 mg/kg doses in overweight to obese but otherwise healthy men, and a multiple-dose part of ascending subcutaneous 0.75-3.0 mg/day doses in obese but otherwise healthy men/women. The single-dose part included 7 cohorts of 8 subjects, randomized 6:2 to active drug/placebo; the multiple-dose part included 4 cohorts of 20 subjects, randomized 16:4 to active drug/placebo. MC4-NN2-0453 was well tolerated and raised no safety concerns except for nonserious skin-related adverse events, this along with lack of weight loss effect led to premature termination of the trial. Headache, sexual-arousal disturbance, and penile erection were also reported. Single-dose pharmacokinetics showed dose-linearity and dose-proportionality. Maximum plasma concentration was observed after 50-100 hours, which then declined with a of approximately 250 hours. Plasma concentration reached steady state after 4 weeks for 0.75 and 1.5 mg/day multiple-dose cohorts, and the was similar to single dose. There were no significant pharmacodynamic effects, including effect on body weight.

Effects of amino acids on melanoma targeting and clearance properties of Tc-99m-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone peptides.

The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new (99m)Tc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg(11))CCMSH {c[Arg-Ser-Asp-DTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg(11))CCMSH, RPheD-Lys-(Arg(11))CCMSH, and RdPheD-Lys-(Arg(11))CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of (99m)Tc-RSD-Lys-(Arg(11))CCMSH, (99m)Tc-RFD-Lys-(Arg(11))CCMSH, and (99m)Tc-RfD-Lys-(Arg(11))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. (99m)Tc-RSD-Lys-(Arg(11))CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these (99m)Tc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using (99m)Tc-RSD-Lys-(Arg(11))CCMSH as an imaging probe. It is desirable to reduce the renal uptake of (99m)Tc-RSD-Lys-(Arg(11))CCMSH to facilitate its potential therapeutic application.

Melanoma targeting property of a Lu-177-labeled lactam bridge-cyclized alpha-MSH peptide.

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

Influence of the bifunctional chelator on the pharmacokinetic properties of 99mTc(CO)3-labeled cyclic α-melanocyte stimulating hormone analog.

Aiming at the design of specific melanocortin-1 receptor (MC1R) targeted imaging probes, we report on the effect of different azolyl-ring substitution patterns (carboxylate at the 4-position and/or methyl groups at the 3,5 positions) of pyrazolyl-diamine bifunctional chelators (Pz(2)-Pz(4)) on the pharmacokinetic profile of the (99m)Tc(CO)3-labeled lactam bridge-cyclized α-melanocyte stimulating hormone derivative, ßAlaNleCycMSH(hex). Three pyrazolyl-diamine-containing chelators were conjugated to ßAlaNleCycMSHhex, with the resulting peptide conjugates displaying subnanomolar MC1R binding affinity. Biodistribution studies in B16F1 melanoma-bearing mice show that all radiopeptides present a good melanoma uptake. The introduction of a carboxylate group in the azolyl-ring leads to a remarkable reduction of the kidney (>89%) and liver (>91%) accumulation for (99m)Tc(CO)3-Pz(3)-ßAlaNleCycMSH(hex) and (99m)Tc(CO)3-Pz(4)-ßAlaNleCycMSH(hex) when compared to the radiopeptide (99m)Tc(CO)3-Pz(1)-ßAlaNleCycMSH(hex), where that group is absent. The good tumor uptake and favorable tumor-to-nontarget-organs ratios of (99m)Tc(CO)3-Pz(3)-ßAlaNleCycMSH(hex) and (99m)Tc(CO)3-Pz(4)-ßAlaNleCycMSH(hex) highlights the potential of both compounds as melanoma imaging agents.