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Radiolabeled α-melanocyte stimulating hormone (α-MSH) derivatives have a high potential for diagnosis and treatment of melanoma, due to high specificity and binding affinity to the melanocortin-1 receptor (MC1R). Hence, the α-MSH-derived peptide NAP-NS1 with a β-Ala linker (ε-Ahx-beta-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 64Cu and investigated in terms of radiochemical and radiopharmacological properties. For the three 64Cu-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 [64Cu]Cu-bispidine-NAP-NS1 ([64Cu]Cu-2) and [64Cu]Cu-DMPTACN-NAP-NS1 ([64Cu]Cu-3) were higher than those of [64Cu]Cu-NOTA-NAP-NS1 ([64Cu]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 [64Cu]Cu-1 and [64Cu]Cu-3 to be renal, while that of [64Cu]Cu-2 seemed to be both renal and hepatobiliary. An initial moderate uptake in the kidney decreased clearly after 60 min. All three 64Cu-labeled conjugates should be considered for further in vivo investigations using a suitable xenograft mouse model.

α-Melanocyte stimulating hormone (α-MSH) derivatives target the melanocortin-1 receptor (MC1R) specifically and selectively. In this study, the α-MSH derived peptide NAP-NS1 (Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH₂) with and without linkers was conjugated with 5-(bis(pyridin-2-yl)methyl)amino)pentanoic acid (DPA-COOH) and labeled with [⁹⁹ᵐTc]Tc-tricarbonyl by two methods. With the one-pot method the labeling was faster than with the two-pots method, however obtaining similarly high yields. Negligible transchelation and high stability in physiological solutions was determined for the [⁹⁹ᵐTc]Tc-tricarbonyl-peptide conjugates. Coupling an ethylenglycol (EG)-based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenate. Preliminary in vivo studies with one of the [⁹⁹ᵐTc]Tc-tricarbonyl-peptide conjugates showed good stability in blood and both a renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insights into the potential relevance of the ⁹⁹ᵐTc-labeled peptides for in vivo imaging.

The melanocortin 1 receptor (MC1R), since overexpressed in melanoma cells, is an attractive target for imaging or treatment of this type of malignancies. The α-melanocyte stimulating hormone (α-MSH) derived peptide NAP-NS1 (Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2) binds with high affinity [1] and was engineered in three different ways for labeling with [Tc-99m]Tc-tricarbonyl. ɛ-Ahx-β-Ala-NAP-NS1, ethylenglycol (EG)-based linker NAP-NS1 and NAP-NS1 without linker were conjugated with bis(pyridin-2-ylmethyl)amine (DPA) obtaining yields of 42%, 38% and 45%, respectively. For standard purpose non-radioactive Re-DPA-peptides were prepared [2] with yields > 55%. The labeling with a one-pot method was faster than with a two steps method, and even receiving slightly higher radiochemical yields. High stability as well as negligible transschelation was observed for the [Tc-99m]Tc-tricarbonyl-peptide conjugates. The ethylenglycol based linker increased the hydrophilicity. Binding results, first of competition assays with the non-radioactive α-MSH conjugates, and second of saturation assays with the [Tc-99m]Tc-tricarbonyl-DPA peptides resulted in high affinity (low nanomolar range) where the labeled peptides showed a marginal loss of affinity; that was relevant for murine B16F10, as well as for human MeWo and TXM13 cells. The number of binding sites was considerably higher in murine melanoma cells. The three [Tc-99m]Tc-tricarbonyl-peptide conjugates exhibited similar in vitro properties. Preliminary in vivo studies were performed with [Tc-99m]Tc-tricarbonyl-DPA-ɛ-Ahx-β-Ala-NAP-NS1 in rats and showed good metabolic stability in blood and both a renal and hepatobiliary excretion. Hence, it displayed prospective radiochemical and radiopharmacological properties, suggesting a promising candidate for further investigation in a melanoma xenograft model. [1] Gao et al. J. Amino Acids 2016; 48: 833-847. [2] Alberto et al. J. Chem. Soc. Dalton 1994; 19: 2815-2820.

Objectives: Melanocortin-1 receptor (MC1R) is well known to be overexpressed in melanoma. Thus, it has been a great interest in targeting this receptor for diagnosis of human metastasized melanoma. We aimed at investigating (radio)pharmacological properties of novel derivatives of the α-melanocyte-stimulating hormone (α-MSH) and selecting most promising candidates for further studies in melanoma models in vivo. Methods: Linear and cyclic α-MSH derivatives (NAP-NS1(1), NOTA-NAP-NS1(2), natCu-NOTA-NAP-NS1(3), NAP-NS2(4), NOTA-NAP-NS2(5), natCu-NOTA-NAP-NS2(6), DPA-NAP-NS1(7) and Re-tricarbonyl-DPA-NAPNS1(8)) were investigated in competition assays in both murine B16F10 and human MeWo cells. In vitro stabilities of [64Cu]Cu-2, [64Cu]Cu-5 and 99mTc-tricarbonyl-7 were tested in phosphate buffer (pH=7.4) and human serum at 37°C for 1h and 24h. Transchelation and octanol/water partition coefficients of radiolabeled peptides were also investigated. Additionally, [64Cu]Cu-2, [64Cu]Cu-5 and 99mTc-tricarbonyl-7 with high radiochemical purities and specific activities were applied in saturation assays and kinetic studies. Results: Linear α-MSH derivatives (1, 2, 3, 7 and 8) showed higher affinities on both murine and human cells than cyclic α-MSH derivatives (4, 5, 6). Linking the chelator to the peptide and coordinating the chelator-peptide with natCu or Re were accompanied by some loss of affinity. [64Cu]Cu-2, [64Cu]Cu-5 and 99mTc-tricarbonyl-7 were stable in phosphate buffer and serum at 37°C after incubation for 1h and 24h. No transchelation of radiolabeled peptides was observed in cysteine and histidine challenge experiments. LogD values suggested that [64Cu]Cu-2 (-2.30±0.01) and [64Cu]Cu-5 (-3.39±0.04) had higher hydrophilicity than 99mTc-tricarbonyl-7 (-0.43±0.01). Saturation studies in both cell lines resulted in Kd values (nM) in the lower nanomolar ranges for [64Cu]Cu-2 (B16F10: 1.7±0.2; MeWo: 2.6±0.5) and 99mTc-tricarbonyl-7 (B16F10: 6.0±0.5; MeWo: 4.5±0.8). But Bmax (fmol/mg protein) of [64Cu]Cu-2 on murine and human cells (B16F10: 46.6±3.9; MeWo: 16.6±1.6) was notably lower than that of 99mTc-tricarbonyl-7 (B16F10: 403.5±46.1; MeWo: 50.3±6.4). Kinetic study of [64Cu]Cu-2 in murine cells showed rapid cellular association and dissociation in vitro. Conclusions: [64Cu]Cu-2 showed high stability, hydrophilicity, binding affinities and rapid cellular association and dissociation in vitro, which made it promising for further investigations in melanoma models.

Objectives: The purpose of this study was to examine the effect of cyclization on the biological profiles of [99mTcN(PNP3)]-labeled α-MSH analogs. Methods: The linear peptide H-Cys-Ahx-bAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) (1) and a corresponding lactam bridge-cyclized peptide, H-Cys-Ahx-bAla3-c[Lys4-Glu-His-D-Phe-Arg-Trp-Glu10]-Arg11-Pro-Val-NH2 (NAP-NS2) (2), were synthesized, characterized by ESI-MS, and their melanocortin-1 receptor (MC1R) binding affinity was determined in B16F10 melanoma cells. In vitro stability and pharmacological parameters of [99mTc(N)(NAP-NS1)(PNP3)]+ (1a) and [99mTc(N)(NAP-NS2)(PNP3)]+ (2a) were assessed. Challenges with an excess of glutathione and cysteine and LogD values were also investigated. Furthermore, 1a and 2a were applied to study in vivo stability and the pharmacokinetic profiles on healthy rats. Results: 1a and 2a were obtained in high yield (RCY > 90%). LogD values demonstrated the hydrophilic nature of the radiolabeled peptides: -1.43 for 1a; - 2.09 for 2a. No significant variations in RCPs of both the complexes were observed. Both complexes showed high stability after incubation in human and rat sera as well as in rat liver homogenate. A fast degradation of 2a was detected in kidneys homogenate. 1a retained a high receptor affinity (Kd: 7.1±0.5 nM). Biodistribution of 1a displayed a favorable pharmacokinetic profile with fast blood clearance and elimination from normal tissues. Rapid renal excretion of 1a was observed due to the high hydrophilic character. The pharmacokinetic profile of 2a was reflected in reduction of the blood clearance and the elimination from the other organs; especially the kidneys showed restraint elimination. Conclusions: Compared with the linear peptide 1, cyclization affected the pharmacological properties of 2 negatively by reducing its stability, its binding affinity to MC1Rs (Ki: 0.9±0.3 nM for 1; 7.1±2.4 nM for 2) and decreasing the overall excretion rate of the corresponding [99mTcN(PNP3)]-labeled peptide from the body. Thus, only the linear labeled peptide 1a will be considered for further investigations in tumor bearing mice.

Succinic dihydrazide (SDH), N-methyl-S-methyl dithiocarbazate (HDTCZ) and PEGylated N-methyl-S-methyl dithiocarbazate (HO2C-PEG600-DTCZ) are nitrido nitrogen atom donors employed for the preparation of nitride [M(N)]‐complexes (M = 99mTc and 188Re). This study aims to compare the capability and the efficiency of these three N3− group donors, in the preparation of [M(N)PNP]-based target-specific compounds (M = 99mTc, 188Re; PNP = aminodiphosphine). For this purpose, three different kit formulations (SDH kit; HO2C-PEG600-DTCZ kit; HDTCZ kit) were assembled and used in the preparation of [M(N)(cys~)(PNP3)]0/+ complexes (cys~ = cysteine derivate ligands). For each formulation, the radiochemical yield (RCY) of the [M(N)(~cys)(PNP3)] compounds, was determined by HPLC. The deviation of the percentage of RCY, due to changes in concentration of the N3− donors and of the exchanging ligand, was determined. For 99mTc, data clearly show that HDTCZ is the most efficient donor of N3−; however, SDH is the most suitable nitrido nitrogen atom donor for the preparation of [99mTc(N)(PNP)]-based target-specific agents with high specific activity. When HO2C-PEG600-DTCZ or HDTCZ are used in N3− donation, high amounts of the exchanging ligand (10−4 M) were required for the formation of the final complex in acceptable yield. The possibility to usemicrogram amounts of HDTCZ also in [188Re(N)] preparation (0.050 mg) reduces its ability to compete in ligand exchange reactions, minimizing the quantity of chelators required to obtain the final complex in high yield. This finding can be exploit for increasing the radiolabeling efficiency in [188Re(N)]-radiopharmaceutical preparations compared to the previously reported HDTCZ-based procedure, notwithstanding a purification process could be necessary to improve the specific activity of the complexes.

Aim. 99mTc(N)-DBODC(5) [DBODC = bis(N -ethoxyethyl)dithiocarbamato; and 5 = bis(dimethoxypropylphosphinoethyl)ethoxyethylamine], is a lipophilic cationic mixed-compound originally investigated as myocardial imaging agent. This compound has been recently identified as a suitable scaffold for the design of new 99mTc-based agents for the noninvasive imaging of P-gp and closely related transporter activities [1]. The possibility to control the hydrophobicity and pharmacological activity of this heterocomplex through the independent variation of the substituents on the two ligand backbones, without affecting the P2S2 coordinating sphere, makes 99mTc(N)-DBODC(5) an appropriate platform for the preparation of different molecular probes for SPECT of MDR. To evaluate the impact of the [99mTc(N)PNP]-moiety on the tumor cell accumulation and on the MDR recognition different 99mTc(N)-DBODC(5) like complexes were synthesized varying the substituents on the PNP ligand and their in-vitro biological behaviors on selected human cancer cell lines and in the corresponding sub-lines were investigated. Method. 99mTc(N)-DBODC(n) complexes were efficiently prepared as previously described [2], [n is PNP3 bis(dimethoxypropylphosphinoethyl)- methoxyethylamine, PNP7 (bis(dimethoxyethylphosphinoethyl)-ethoxyethylamine) and PNP10 (bis(dimethoxyethylphosphinoethyl)-methoxyethylamine). Basic parameters such as the kinetic of uptake, at 4 and 37 °C, of the 99mTc(N)-DBODC(n) complexes were evaluated in-vitro in selected cell lines such us MCF7 (human breast cancer) and 2008 (human ovarian cancer), and in the corresponding sublines by using 99mTc-sestamibi and 99mTc(N)-DBODC(5) as references. Results. In drug-sensitive cell lines a significant increase of the % of cell accumulation of 99mTc(N)-DBODC(7) and 99mTc(N)-DBODC(10) complexes was observed with respect to 99mTc-sestamibi and 99mTc(N)DBODC(5). At steady-state level, this amount was quantified to be two or three times the % of uptake value of the reference compounds. At 4 °C a significant reduction of cellular accumulation was observed for all 99mTc agents. After 60 min, the nonspecific uptake was assessed as around 2 % (p

Synthetic peptides represent first choice biomelecules, with respect to proteins and mAb, for the monitoring of population variability and receptor functionality associated to a tumor pathology due to their favorable pharmacokinetic profile. It has been demonstrated that the surface molecule integrin alfa-V beta-3 could be an ideal target for the interaction with melanoma directed radiolabeled peptides.....

Currently, receptor based radiopharmaceuticals have received great attention in molecular imaging and radiotherapy of cancer, and provide a unique tool for target-specific delivery of radionuclides to pathological tissues. In this context, receptor binding peptides represent an attractive class of target vectors for Nuclear Medicine purposes. The rich chemistry of the group 7 elements elaborated in past years, has allowed the development of different procedures for the preparation of radiolabeled peptides in high yield. This, joint to the use of solid-phase peptide synthesis, has opened the possibility to explore new strategies for approaching the design of new class of radiolabeled receptor-targeted peptides, and to create new versatilities in targeting vehicle design e.g. in synthesis of metal-cyclized peptides or of multivalent targeting agents. This review provides an overview on several aspects of the development of new 99mTc/ 188Re-peptide based target specific radiopharmaceuticals, in particular on the synthetic strategies employed for modifying molecular vectors, and the application of the different metal-cores and/or building block for preparing high specific activity agents.

We report on an efficient procedure for the preparation of [188Re(N)(PNP)]-based complexes (where PNP is diphosphinoamine) useful in the development of target-specific radiopharmaceuticals. The radiochemical yield of the compounds was optimized considering such reaction parameters as nature of the nitrido nitrogen donor, reaction times and pH level. The chemical identity of the 188Re agents was determined by high-performance liquid chromatography comparison with the corresponding well-characterized cold Re compounds. 188Re(N) mixed compounds have been evaluated with regard to stability toward transchelation with GSH and degradation by serum enzymes. The clearance of selected radiocompounds from normal tissues and their in vivo stability were evaluated in rats by biodistribution and imaging studies. [188Re(N)(cys∼)(PNP)]+/0 mixed-ligand compounds were efficiently prepared in aqueous solution from perrhenate using a multistep procedure based on the preliminary formation of the labile 188ReIII–EDTA species, which easily undergo oxidation/ligand exchange reaction to afford the [188ReV≡N]2+ core in the presence of dithiocarbazate. The final mixed-ligand compounds were obtained, at 100°C, by adding the two bidentate ligands to the buffered [188ReV≡N]2+ solution (pH 3.2–3.6). However, a relatively high amount of cys∼ ligand was required to obtain a quantitative radiochemical yield. The complexes were stable toward reoxidation to perrhenate and ligand exchange reactions. In vivo studies showed rapid distribution and elimination of the complexes from the body. No specific uptakes in sensitive tissues/organs were detected. A positive correlation of the distribution of the complexes estimated with biodistribution studies (%ID) and with micro-SPECT semiquantification imaging analysis (standard uptake values) was observed. These results support the possibility of applying [188Re(N)(PNP)] technology to the preparation of target-specific agents.

This study presents the first application of a general procedure based on the use of the [Tc(N)Cl(PS) (PPh3)] species (PS is an alkyl phosphinothiolate ligand) for the preparation of Tc(N) target-specific compounds. [Tc(N)Cl(PS)(PPh3)] selectively reacts with an appropriate dithiocarbamate ligand (S^Y) to give [Tc(N)(PS)(S^Y)] compounds. 1-(2-Methoxyphenyl)piperazine, which displays a potent and specific affinity for 5HT1A receptors, was selected as a functional group and conjugated to the dithiocarbamate unit through different spacers (Ln). [99mTc(N)(PS)(Ln)] complexes were prepared in high yield (more than 90%). The chemical identity of 99mTc complexes was determined by high performance liquid chromatography comparison with the corresponding 99gTc complexes. All complexes were found to be inert toward transchelation with an excess of glutathione and cysteine. No notable biotransformation of the native compound into different species by the in vitro action of the serum and liver enzymes was shown. Nanomolar affinity for the 5HT1A receptor was obtained for [99mTc(N)(PSiso)L3] (IC50 = 1.5 nM); a reduction of the affinity was observed for the other complexes as a function of the shortening of the alkyl chain interposed between the dithiocarbamate and the pharmacophore. Negligible brain uptake was found from in vivo distribution data of [99mTc(N)(PSiso)L3]. The key finding of this study is that the complexes maintained good affinity and selectivity for 5HT1A receptors, and the IC50 value for [99gTc(N)(PSiso)L3] being comparable to the IC50 value found for WAY 100635. This result confirmed the possibility of preparing [99mTc(N)(PS)]-based targetspecific compounds without affecting the affinity and selectivity of the bioactive molecules for the corresponding receptors.

An efficient procedure for the preparation of [188Re(N)(PNP)]-based complexes is reported. The radiochemical yield (RCY) of the compounds was optimized considering reaction parameters such as: nature of nitrido nitrogen donors, reaction times and pH. The chemical identity of 188Re-agents was determined by high-performance liquid chromatography comparison with the corresponding well characterized cold Re-compounds. The stability of the [188Re(N)(PNP)] complexes toward transchelation with glutathione and degradation by serum enzymes was assessed. The clearance of selected radiolabeled agents from normal tissues was evaluated in rats. [188Re(N)(cys∼)(PNP)]+/0 asymmetrical compounds were prepared in aqueous solution from perrhenate using a multistep procedure based on the preliminary formation of the labile 188ReIIIEDTA species which easily undergo to oxidation/ligand exchange reaction to afford in presence of DTCZ the [188ReV≡N]2+core. The final compounds were obtained by adding to the [188ReV≡N]int 2+ the two bidentate ligands. However, a relative high amount of cys∼ ligand was required to obtain a quantitative RCY. The complexes were stable toward reoxidation to perrhenate and ligand exchange reactions. In vivo studies showed a rapid distribution and elimination of the complexes from the body. No specific uptakes in radiation sensitive tissues/organs were detected. A positive correlation of the distribution of the complexes estimated with biodistribution studies (% injected dose) andwithmicro-single photon emission computed tomography semiquantification imaging analysis was observed. The low labeling efficiency limits but not excludes the possibility to apply [188Re(N)(PNP)]-technology to the preparation of target specific radiotherapeutical agents.