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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010NAP-NS1: H-Cys-Ahx-ßAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Val-NH2NAP-NS2: H-Cys-Ahx-ßAla-c[Lys-Glu-His-D-Phe-Arg-Trp-Glu]-Arg-Pro-Val-NH2RRR[Tc(N)PNP] Metal Fragment Labeled Peptide for MC1Receptors Imaging: Preliminary StudiesBarbara Biondi 1 , Cristina Bolzati 2 , Davide Carta 3 , Nicola Salvarese 3 ,Fiorenzo Refosco 2 , Andrea Calderan 1 , and Paolo Ruzza 11 Institute of Biomolecular Chemistry of CNR, Padova Unit and Department of ChemicalSciences, University of Padova, Padova, 35131,Italy; 2 Institute of Inorganic Chemistry andSurfaces of CNR, Padova, 35020, Italy; 3 Department of Pharmaceutical Sciences,University of Padova, Padova, 35131, ItalyIntroductionMalignant melanoma is the most lethal form of skin cancer: melanoma metastases are veryaggressive and no curative treatment exists for it due to its resistance to chemotherapy andimmunotherapy regimens. Therefore, development of new melanoma-specific radiopharmaceuticalsfor early detection of primary melanoma tumours or internal radiotherapyis a subject of great interest. In this perspective, melanocortin type-1 receptors (MC1R)represent a promising target for the development of effective molecular probes fordiagnosis or therapy. MC1R are overexpressed on the surface of melanoma cells and ableto selectively recognise the peptide sequence His-D-Phe-Arg-Trp mimicking themelanocyte stimulating hormone ( MSH). The literature described several linear andcyclic radiolabeled MSH analogues. In particular, the introduction of a cyclic constraint ina lead peptide restricts the flexibility and may favour peptide–receptor interactions,receptor selectivity and enzymatic stability [1].In this work we synthesised linear and cyclic NAPamide analogues (Figure 1), inorder to improve their affinity toward MC1R and to be used as ligands in the formation of99m Tc complexes with the [ 99m Tc(N)(PNPn)] 2+ metal fragment [2]. We investigated the invitro stability in phosphate buffer solutions (PBS), in cysteine and glutathione solutions aswell as in rat liver (RLH) and kidney homogenates (RKH). Biodistribution studies wereperformed in female Sprague Dowley (S.D.) rats with [ 99m Tc(N)(NAP-NS1/2)(PNPn)] + inorder to evaluate their organ uptake and excretion pathways.Results and DiscussionPeptides were synthesised according to standard Fmoc/tBu method using HBTU/HOBt asPPRNNTcR'SH 2 NFig. 1. Peptide sequences and structure of complexes.CONHC-activation procedure. TheAhx- Ala sequence wasconjugated to the N-terminusof both peptides as a spacerbetween the radiolabeledmoiety and the binding regionto avoid interference betweenthe different portions of themolecule, while Cys residue isinvolved in complexformation. NAP-NS2 cyclizationwas performed on resin,using a Pd-mediateddeprotection of Alloc/Allylprotecting groups of Lys 4 andGlu 10 , respectively [3]. AfterHPLC purification, peptideswere obtained in good yieldswith a purity >98%.The [ 99m Tc(N)(peptide)(PNPn)] + complexes were prepared using two different methods.The first method is a two-step procedure: 1) the 99m Tc–nitrido precursors [ 99m Tc≡N] 2+ wasprepared by reaction of Na 99m TcO 4 (50.0 MBq-3.0 GBq) with succinic dihydrazide (SDH),SnCl 2 and ethanol in 30 minutes at room temperature; 2) the PNPn ligand and NAP-NS1/2were simultaneously added to [ 99m Tc≡N] 2+ , and the reaction mixture was heated at 80°C for518