Synthesis, characterization and in vitro cytotoxicity of gallium(III)-dithiocarbamate complexes.

A library of homoleptic mononuclear Ga(III) complexes of the general formula [Ga(DTC)3], where DTC is an alicyclic or a linear dithiocarbamate chelator, is reported. The complexes were prepared in high yields starting from Ga(NO3)3·6H2O and fully characterized by elemental analysis and IR and NMR spectroscopy. Crystals of five of these complexes were obtained. The antitumor activity of the newly synthesized compounds against a panel of human cancer cell lines was evaluated. The chemical nature of the DTC does not have a marked impact on the structural features of the final compound. X-ray crystal structure analyses revealed that all these complexes have a trigonal prismatic geometry with three identical chelating DTCs coordinating the Ga(III) ion. It is noteworthy that in complex 22, [Ga(NHEt)3] (NHEt = N-ethyldithiocarbamate), the asymmetric unit is formed by two independent and structurally different molecules. Cellular studies showed that all the synthesized Ga-DTC complexes exhibit marked cytotoxic activity, even against human colon cancer cells that are less sensitive to cisplatin. Among the tested compounds, 6 ([Ga(CEPipDTC)3], CEPipDTC = (ethoxycarbonyl)-piperidinedithiocarbamate) and 21 ([Ga(Pr-13)3], PR13 = 4 and N-(2-ethoxy-2-oxoethyl)-N-methyldithiocarbamate) are very promising derivatives, but they have no selectivity towards cancer cells. Nevertheless, the obtained data provide a foundation for developing gallium-dithiocarbamate complexes as anticancer agents.

Fundamentals of Rhenium-188 Radiopharmaceutical Chemistry.

The ß- emitter, rhenium-188 (188Re), has long been recognized as an attractive candidate for targeted cancer radionuclide therapy (TRNT). This transition metal shares chemical similarities with its congener element technetium, whose nuclear isomer technetium-99m (99mTc) is the current workhorse of diagnostic nuclear medicine. The differences between these two elements have a significant impact on the radiolabelling methods and should always receive critical attention. This review aims to highlight what needs to be considered to design a successful radiopharmaceutical incorporating 118Re. Some of the most effective strategies for preparing therapeutic radiopharmaceuticals with 188Re are illustrated and rationalized using the concept of the inorganic functional group (core) and a simple ligand field theoretical model combined with a qualitative definition of frontiers orbitals. Of special interest are the Re(V) oxo and Re(V) nitrido functional groups. Suitable ligands for binding to these cores are discussed, successful clinical applications are summarized, and a prediction of viable future applications is presented. Rhenium-188 decays through the emission of a high energy beta particle (2.12 MeV max energy) and a half-life of 16.9 h. An ideal biological target would therefore be a high-capacity target site (transporters, potential gradients, tumour microenvironment) with less emphasis on saturable targets such as overexpressed receptors on smaller metastases.

Development and Characterization of 99mTc-scFvD2B as a Potential Radiopharmaceutical for SPECT Imaging of Prostate Cancer.

Previously, we demonstrated that the 177Lu-labeled single-chain variable fragment of an anti-prostate-specific membrane antigen (PSMA) IgG D2B antibody (scFvD2B) showed higher prostate cancer (PCa) cell uptake and tumor radiation doses compared to 177Lu-labeled Glu-ureide-based PSMA inhibitory peptides. To obtain a 99mTc-/177Lu-scFvD2B theranostic pair, this research aimed to synthesize and biochemically characterize a novel 99mTc-scFvD2B radiotracer. The scFvD2B-Tag and scFvD2B antibody fragments were produced and purified. Then, two HYNIC derivatives, HYNIC-Gly-Gly-Cys-NH2 (HYNIC-GGC) and succinimidyl-HYNIC (S-HYNIC), were used to conjugate the scFvD2B-Tag and scFvD2B isoforms, respectively. Subsequently, chemical characterization, immunoreactivity tests (affinity and specificity), radiochemical purity tests, stability tests in human serum, cellular uptake and internalization in LNCaP(+), PC3-PIP(++) or PC3(-) PCa cells of the resulting unlabeled HYNIC-scFvD2B conjugates (HscFv) and 99mTc-HscFv agents were performed. The results showed that incorporating HYNIC as a chelator did not affect the affinity, specificity or stability of scFvD2B. After purification, the radiochemical purity of 99mTc-HscFv radiotracers was greater than 95%. A two-sample t-test of 99mTc-HscFv1 and 99mTc-HscFv1 uptake in PC3-PIP vs. PC3 showed a p-value < 0.001, indicating that the PSMA receptor interaction of 99mTc-HscFv agents was statistically significantly higher in PSMA-positive cells than in the negative controls. In conclusion, the results of this research warrant further preclinical studies to determine whether the in vivo pharmacokinetics and tumor uptake of 99mTc-HscFv still offer sufficient advantages over HYNIC-conjugated peptides to be considered for SPECT/PSMA imaging.

A Review on the Current State and Future Perspectives of [99mTc]Tc-Housed PSMA-i in Prostate Cancer.

Recently, prostate-specific membrane antigen (PSMA) has gained momentum in tumor nuclear molecular imaging as an excellent target for both the diagnosis and therapy of prostate cancer. Since 2008, after years of preclinical research efforts, a plentitude of radiolabeled compounds mainly based on low molecular weight PSMA inhibitors (PSMA-i) have been described for imaging and theranostic applications, and some of them have been transferred to the clinic. Most of these compounds include radiometals (e.g., 68Ga, 64Cu, 177Lu) for positron emission tomography (PET) imaging or endoradiotherapy. Nowadays, although the development of new PET tracers has caused a significant drop in single-photon emission tomography (SPECT) research programs and the development of new technetium-99m (99mTc) tracers is rare, this radionuclide remains the best atom for SPECT imaging owing to its ideal physical decay properties, convenient availability, and rich and versatile coordination chemistry. Indeed, 99mTc still plays a relevant role in diagnostic nuclear medicine, as the number of clinical examinations based on 99mTc outscores that of PET agents and 99mTc-PSMA SPECT/CT may be a cost-effective alternative for 68Ga-PSMA PET/CT. This review aims to give an overview of the specific features of the developed [99mTc]Tc-tagged PSMA agents with particular attention to [99mTc]Tc-PSMA-i. The chemical and pharmacological properties of the latter will be compared and discussed, highlighting the pros and cons with respect to [68Ga]Ga-PSMA11.

Impact of Different [Tc(N)PNP]-Scaffolds on the Biological Properties of the Small cRGDfK Peptide: Synthesis, In Vitro and In Vivo Evaluations.

Background: The [99mTc][Tc(N)(PNP)] system, where PNP is a bisphosphinoamine, is an interesting platform for the development of tumor 'receptor-specific' agents. Here, we compared the reactivity and impact of three [Tc(N)(PNP)] frameworks on the stability, receptor targeting properties, biodistribution, and metabolism of the corresponding [99mTc][Tc(N)(PNP)]-tagged cRGDfK peptide to determine the best performing agent and to select the framework useful for the preparation of [99mTc][Tc(N)(PNP)]-housing molecular targeting agents. Methods: cRGDfK pentapeptide was conjugated to Cys and labeled with each [Tc(N)(PNP)] framework. Radioconjugates were assessed for their lipophilicity, stability, in vitro and in vivo targeting properties, and performance. Results: All compounds were equally synthetically accessible and easy to purify (RCY ≥ 95%). The main influences of the synthon on the targeting peptide were observed in in vitro cell binding and in vivo. Conclusions: The variation in the substituents on the phosphorus atoms of the PNP enables a fine tuning of the biological features of the radioconjugates. ws[99mTc][Tc(N)(PNP3OH)]- and [99mTc][Tc(N)(PNP3)]- are better performing synthons in terms of labeling efficiency and in vivo performance than the [99mTc][Tc(N)(PNP43)] framework and are therefore more suitable for further radiopharmaceutical purposes. Furthermore, the good labeling properties of the ws[99mTc][Tc(N)(PNP3OH)]- framework can be exploited to extend this technology to the labeling of temperature-sensitive biomolecules suitable for SPECT imaging.

Water-Soluble [Tc(N)(PNP)] Moiety for Room-Temperature 99mTc Labeling of Sensitive Target Vectors.

The incorporation of bioactive molecules into a water-soluble [99mTc][Tc(N)(PNP)]-based mixed compound is described. The method, which exploits the chemical properties of the new [99mTc][Tc(N)(PNP3OH)]2+ synthon [PNP3OH = N,N-bis(di-hydroxymethylenphosphinoethyl)methoxyethylamine], was successfully applied to the labeling of small, medium (cysteine-functionalized biotin and c-RGDfK pentapeptide), and large molecules. Apomyoglobin was chosen as a model protein and derivatized via site-specific enzymatic reaction catalyzed by transglutaminase (TGase) with the H-Cys-Gly-Lys-Gly-OH tetrapeptide for the insertion in the protein sequence of a reactive N-terminal Cys for 99mTc chelation. Radiosyntheses were performed under physiological conditions at room temperature within 30 min. They were reproducible, highly specific, and quantitative. Heteroleptic complexes are hydrophilic and stable. Biodistributions of the selected compounds show favorable pharmacokinetics within 60 min post-injection and predominant elimination through the renal-urinary pathway. In a wider perspective, these data suggest a role of the [99mTc][Tc(N)(PNP)] technology in the labeling of temperature-sensitive biomolecules, especially targeting proteins for SPECT imaging.

Enzymatic Methods for the Site-Specific Radiolabeling of Targeting Proteins.

Site-specific conjugation of proteins is currently required to produce homogenous derivatives for medicine applications. Proteins derivatized at specific positions of the polypeptide chain can actually show higher stability, superior pharmacokinetics, and activity in vivo, as compared with conjugates modified at heterogeneous sites. Moreover, they can be better characterized regarding the composition of the derivatization sites as well as the conformational and activity properties. To this aim, several site-specific derivatization approaches have been developed. Among these, enzymes are powerful tools that efficiently allow the generation of homogenous protein-drug conjugates under physiological conditions, thus preserving their native structure and activity. This review will summarize the progress made over the last decade on the use of enzymatic-based methodologies for the production of site-specific labeled immunoconjugates of interest for nuclear medicine. Enzymes used in this field, including microbial transglutaminase, sortase, galactosyltransferase, and lipoic acid ligase, will be overviewed and their recent applications in the radiopharmaceutical field will be described. Since nuclear medicine can benefit greatly from the production of homogenous derivatives, we hope that this review will aid the use of enzymes for the development of better radio-conjugates for diagnostic and therapeutic purposes.

Preclinical dosimetric studies of 177 Lu-scFvD2B and comparison with 177 Lu-PSMA-617 and 177 Lu-iPSMA endoradiotherapeutic agents.

PURPOSE: Internal dosimetry has become a very important tool to evaluate the risks and benefits of new endoradiotherapeutic agents. Nowadays, some of the most successful targeted radionuclide therapy (TRT) agents are 177 Lu-DOTA conjugates based on low molecular weight (LMW) Glu-ureido PSMA inhibitors. It has, however, been demonstrated that the DOTA chelating moiety reduces the internalization of the LMW-PSMA agent and its radiation dose to the tumor. Previously, we reported that 177 Lu-scFvD2B, an antibody-based construct, demonstrated statistically significant higher cell uptake and internalization in LNCaP prostate cancer (PCa) cells (PSMA-positive) when compared to the LMW-PSMA agents, 177 Lu-PSMA-617 and 177 Lu-iPSMA, two of the endoradiotherapeutic agents which currently are the most used in PCa therapy. The aim of this study is to estimate the preclinical 177 Lu-scFvD2B organ and tumor-absorbed doses, and to compare the values with those of 177 Lu-PSMA-617 and 177 Lu-iPSMA. METHODS: 177 Lu-scFvD2B, 177 Lu-PSMA-617, and 177 Lu-iPSMA were prepared and their radiochemical purity determined. Biodistribution studies of each radiopharmaceutical were then carried out in healthy mice to define the main source organs (SO) and to calculate the number of disintegrations in each source organs per unit of administered activity (NSO ). Absorbed dose in the main organs were then calculated for each 177 Lu-conjugate by means of OLINDA/EXM 2.1.1 software, using the calculated NSO for both the adult male and the mouse phantoms as program inputs. Images of mice bearing micropulmonary tumors injected with 177 Lu-conjugates were also obtained. Tumor standardized uptake values (SUV) for the different conjugates, obtained from the 3D SPECT image reconstruction of these mice, were used as the number of disintegrations in a tumor site per unit of administered activity (NT ). The tumor-absorbed dose was calculated using the published electron dose S-values for sphere models with diameters ranging from 10 µm to 10 mm and considering a uniform activity distribution and tumor density equivalent to water density. RESULTS: All 177 Lu-labeled agents were obtained in high yield (98%). Dosimetric studies carried out using mouse phantoms demonstrated that organ absorbed doses of 177 Lu-scFvD2B were from 1.4 to 2.3 times higher than those for 177 Lu-iPSMA and from 1.5 to 2.6 times higher than those for 177 Lu-PSMA-617. However, the 177 Lu-scFvD2B values of tumor-absorbed doses for all investigated tumor sizes were from 2.8 to 3.0 times greater than those calculated for 177 Lu-iPSMA and 177 Lu-PSMA-617, respectively. Moreover, 177 Lu-scFvD2B showed the highest tumor/kidney ratio when compared to those reported for 177 Lu-albumin conjugates. CONCLUSIONS: In this preclinical study, we demonstrated the potential of 177 Lu-scFvD2B as a therapeutic agent for PSMA-expressing tumors, due to its higher tumor-absorbed dose when compared with 177 Lu-LMW agents.

The emerging value of 64Cu for molecular imaging and therapy.

Along with other novel metallic radionuclides, copper-64 (64Cu) is currently being investigated as an alternative option to the gallium-68 (68Ga) and lutetium-177 (177Lu) radiopharmaceuticals widely used for targeting somatostatin receptors, expressed by neuroendocrine tumors (NETs), and recently prostate specific membrane antigen (PSMA), expressed by prostate cancer cells. This interest is mostly driven by the peculiar nuclear properties of 64Cu that make it an almost ideal example of theranostic radionuclide. In fact, 64Cu emits both low-energy positrons, ß- particles and a swarm of Auger electrons. This combination of different emissions may allow to collect high-resolution PET images, but also to use the same radiopharmaceutical for eliciting a therapeutic effect. Another unique behavior of 64Cu originates from the fundamental biological role played in organisms by the ionic forms of the copper element, which is naturally involved in a multitude of cellular processes including cell replication. These intrinsic biological characteristics has led to the discovery that 64Cu, under its simplest dicationic form Cu2+, is able to specifically target a variety of cancerous cells and to detect the onset of a metastatic process in its initial stage. This short review reports an outline of the status of 64Cu radiopharmaceuticals and of the most relevant results that are constantly disclosed by preclinical and investigational clinical studies.

Development of 177Lu-scFvD2B as a Potential Immunotheranostic Agent for Tumors Overexpressing the Prostate Specific Membrane Antigen.

The clinical translation of theranostic 177Lu-radiopharmaceuticals based on inhibitors of the prostate-specific membrane antigen (PSMA) has demonstrated positive clinical responses in patients with advanced prostate cancer (PCa). However, challenges still remain, particularly regarding their pharmacokinetic and dosimetric properties. We developed a potential PSMA-immunotheranostic agent by conjugation of a single-chain variable fragment of the IgGD2B antibody (scFvD2B) to DOTA, to obtain a 177Lu-labelled agent with a better pharmacokinetic profile than those previously reported. The labelled conjugated 177Lu-scFvD2B was obtained in high yield and stability. In vitro, 177Lu-scFvD2B disclosed a higher binding and internalization in LNCaP (PSMA-positive) compared to PC3 (negative control) human PCa cells. In vivo studies in healthy nude mice revealed that 177Lu-scFvD2B present a favorable biokinetic profile, characterized by a rapid clearance from non-target tissues and minimal liver accumulation, but a slow wash-out from kidneys. Micro-SPECT/CT imaging of mice bearing pulmonary microtumors evidenced a slow uptake by LNCaP tumors, which steadily rose up to a maximum value of 3.6 SUV at 192 h. This high and prolonged tumor uptake suggests that 177Lu-scFvD2B has great potential in delivering ablative radiation doses to PSMA-expressing tumors, and warrants further studies to evaluate its preclinical therapeutic efficacy.

Radiation effective dose assessment of [51Mn]- and [52Mn]-chloride.

In order to establish the potential of [51/52Mn]Cl2 as safe PET brain tracers, the radiation effective dose (ED) of [51Mn]- and [52Mn]-chloride has been assessed by using biokinetic models in anthropomorphic phantoms. Results showed that [52Mn]-chloride releases one hundred thirty times more radiation dose (ED = 1.35 mSv/MBq) than [51Mn]-chloride (ED = 1.02E-02 mSv/MBq). Although the maximum positron energy of 52Mn allows a PET image resolution similar to that of 18F, activities below 15 MBq should be administered.

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.

Improved synthesis on solid phase of dithiocarbamic cRGD-derivative and 99m Tc-radiolabelling.

In the field of angiogenesis, small cyclic pentapeptides containing the RGD motif are playing a relevant role for their high affinity and specificity for integrin receptors and for the possibility to act at both therapeutic and diagnostic level by inhibiting pathological angiogenesis and by serving as shuttles to deliver imaging-probe including SPECT/PET radionuclides to specific tissues. In the last decade, several new protocols were reported in literature for the direct synthesis of cyclic RDG either in solution or by SPPS. Here, we have elaborated and tested some alternative approaches using different resins and different protective groups. The introduction of the dithiocarbamate function, useful to complex radio-metals suitable for nuclear medicine applications, has also been considered and achieved.

[99mTc][Tc(N)(DASD)(PNP n)]+ (DASD = 1,4-Dioxa-8-azaspiro[4,5]decandithiocarbamate, PNP n = Bisphosphinoamine) for Myocardial Imaging: Synthesis, Pharmacological and Pharmacokinetic Studies.

[99mTc][TcN-DBODC(5) is the lead candidate of a class of cationic complexes proposed as myocardial imaging agents (MPIAs). Phase I clinical studies showed that its clinical properties were comparable to those of the commercially available agents. Thus, modification of [99mTc]TcN-DBODC(5), directed to obtain an ideal myocardial imaging without interference from the adjacent organ activities, is desirable. This work describes the pharmacological and pharmacokinetic development of four new complexes of general formula [99mTc][Tc(N)(DASD)(PNP n)]+, [99mTc]TcN-DASD( n) (DASD = 1,4-dioxa-8-azaspiro[4,5]decandithiocarbamate; PNP n = bisphosphinoamine), proposed as improved MPIAs. Among the tested compounds, [99mTc]TcN-DASD(5) and [99mTc]TcN-DASD(7) showed enhanced heart uptake compared with the gold standards, with a rapid liver washout and superior heart-to-liver ratio. These features might shorten the duration of imaging procedures below 30 min, consenting the early acquisition of high-quality images. In addition, mechanistic studies were performed in cellulo by using human drug-sensitive and drug-resistant cancer cell lines, obtaining results which might be conveniently applied to tumor imaging.

[99mTc][Tc(N)PNP43]-Labeled RGD Peptides As New Probes for a Selective Detection of αvß3 Integrin: Synthesis, Structure-Activity and Pharmacokinetic Studies.

New integrin-selective molecules suitable for therapeutic or imaging purposes are currently of interest in development of effective personalized medical platforms. RGDechi is a bifunctional peptide selective for integrin αvß3. Herein, RGDechi and three truncated derivatives functionalized with a cysteine (1-4) were synthesized and labeled with the [99mTc][Tc(N)PNP43]-synthon ([PNP43 = (CH3)2P(CH2)2N(C2H4OCH3)(CH2)2P(CH3)2]) (99mTc1-4) as a basis for selective integrin recognition. The pharmacological parameters of all radiolabeled peptides were assessed along with the pharmacokinetic profiles of the most promising 99mTc1 and 99mTc2 compounds both on healthy and melanoma-bearing mice. Their metabolism and metabolite identification are also reported. 99mTc1-2 are able to discriminate between endogenously expressed integrins αvß3 and αvß5 and possess favorable pharmacokinetics characterized by low liver uptake and rapid elimination from nontarget tissues resulting in positive target-to-nontarget ratios. Results are encouraging; the presented construct can be considered the starting point for the development of agents for the selective detection of αvß3 expression by SPECT.

Synthesis, Characterization, and Initial Biological Evaluation of [99m Tc]Tc-Tricarbonyl-labeled DPA-α-MSH Peptide Derivatives for Potential Melanoma Imaging.

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-ylmethyl)amino)pentanoic acid (DPA-COOH) and labeled with [99m Tc]Tc-tricarbonyl by two methods. With the one-pot method the labeling was faster than with the two-pot method, while obtaining similarly high yields. Negligible trans-chelation and high stability in physiological solutions was determined for the [99m Tc]Tc-tricarbonyl-peptide conjugates. Coupling an ethylene glycol (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 homogenates. Preliminary in vivo studies with one of the [99m Tc]Tc-tricarbonyl-peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the 99m Tc-labeled peptides for in vivo imaging.

Transglutaminase-mediated conjugation and nitride-technetium-99m labelling of a bis(thiosemicarbazone) bifunctional chelator.

An assessment study involving the use of the transglutaminase (TGase) conjugation method and the nitride-technetium-99m labelling on a bis(thiosemicarbazone) (BTS) bifunctional chelating agent is presented. The previously described chelator diacetyl-2-(N4-methyl-3-thiosemicarbazone)-3-(N4-amino-3-thiosemicarbazone), H2ATSM/A, has been functionalized with 6-aminohexanoic acid (ε-Ahx) to generate the bifunctional chelating agent diacetyl-2-(N4-methyl-3-thiosemicarbazone)-3-[N4-(amino)-(6-aminohexanoic acid)-3-thiosemicarbazone], H2ATSM/A-ε-Ahx (1), suitable for conjugation to glutamine (Gln) residues of bioactive molecules via TGase. The feasibility of the TGase reaction in the synthesis of a bioconjugate derivative was investigated using Substance P (SP) as model peptide. Compounds 1 and H2ATSM/A-ε-Ahx-SP (2) were labelled with nitride-technetium-99m, obtaining the complexes [99mTc][Tc(N)(ATSM/A-ε-Ahx)] (99mTc1) and [99mTc][Tc(N)(ATSM/A-ε-Ahx-SP)] (99mTc2). The chemical identity of 99mTc1 and 99mTc2 was confirmed by radio/UV-RP-HPLC combined with ESI-MS analysis on the respective carrier-added products 99g/99mTc1 and 99g/99mTc2. The stability of the radiolabelled complexes after incubation in various environments was investigated. All the results were compared with those obtained for the corresponding 64Cu-analogues, 64Cu1 and 64Cu2. The TGase reaction allows the conjugation of 1 with the peptide, but it is not highly efficient due to instability of the chelator in the required conditions. The SP-conjugated complexes are unstable in mouse and human sera. However, indeed the BTS system can be exploited as nitride-technetium-99m chelator for highly efficient technetium labelling, thus making compound 1 worthy of further investigations for new targeted technetium and copper radiopharmaceuticals encompassing Single Photon Emission Computed Tomography and Positron Emission Tomography imaging.

Synthesis and characterization of rhenium(iii) complexes with (Ph2PCH2CH2)2NR diphosphinoamine ligands.

The synthesis and characterization of a new series of neutral, six-coordinated compounds [ReIIIX3(PNPR)], where X is Cl or Br and PNPR is a diphosphinoamine having the general formula (Ph2PCH2CH2)2NR (R = H, CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3 and CH2CH2OCH3) are reported. Stable [ReIIIX3(PNPR)] complexes were synthesized, in variable yields, starting from precursors where the metal was in different oxidation states (iii and v), by ligand-exchange and/or redox-substitution reactions. The compounds were characterized by elemental analysis, proton NMR spectroscopy, cyclic voltammetry, UV/vis spectroscopy, positive-ion electrospray ionization mass spectrometry (ESI(+)-MS) and X-ray diffraction analysis. Although the formulation of the complexes allows either meridional or facial isomers, the latter arrangement was prevalent both in the solid and solution states. Only [ReCl3(PNPH)] showed a meridional configuration both in solution and in the crystalline state. [ReBr3(PNPme)] prefers the meridional configuration in the crystalline state and the facial one in solution. While ESI(+)-MS and voltammetric data seem to indicate some dependency from the nature of the alkyl substituent at the nitrogen, the available structural data of the complexes show only slight differences both for angles and bond lengths upon change of the alkyl chain tethered to the nitrogen.

Melanoma targeting with [99mTc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analogs: Effects of cyclization on the radiopharmaceutical properties.

The purpose of this study was to evaluate the effect of cyclization on the biological profile of a [99mTc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analog. A lactam bridge-cyclized H-Cys-Ahx-ßAla3-c[Lys4-Glu-His-D-Phe-Arg-Trp-Glu10]-Arg11-Pro-Val-NH2 (NAP-NS2) and the corresponding linear H-Cys-Ahx-ßAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) peptide were synthetized, characterized by ESI-MS spectroscopy and their melanocortin-1 receptor (MC1R) binding affinity was determined in B16/F10 melanoma cells. The consistent [99mTc(N)(PNP3)]-labeled compounds were readily obtained in high specific activity and their stability and biological properties were assessed. As an example, the chemical identity of [99mTc(N)(NAP-NS1)(PNP3)]+ was confirmed by carrier added experiments supported by radio/UV HPLC analysis combined with ESI(+)-MS. Compared with the linear peptide, cyclization negatively affected the biological properties of NAP-NS2 peptide by reducing its binding affinity for MC1R and by decreasing the overall excretion rate of the corresponding [99mTc(N)(PNP3)]-labeled peptide from the body as well as its in vivo stability. [99mTc(N)(NAP-NS1)(PNP3)]+ was evaluated for its potential as melanoma imaging probe in murine melanoma model. Data from in vitro and in vivo studies on B16/F10 melanoma model of [99mTc(N)(NAP-NS1)(PNP3)]+ clearly evidenced that the radiolabeled linear peptide keeps its biological properties up on the conjugation to the [99mTc(N)(PNP3)]-building block. The progressive increase of the tumor-to-nontarget ratios over the time indicates a quite stable interaction between the radio-complex and the MC1R.

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.