Sintesi e valutazione di una sonda MRI a base di Gadolinio per la diagnosi del cancro alla prostata

The high spatial resolution and the great ability of differentiating soft tissues have determined the widespread success of Magnetic Resonance Imaging (MRI) in clinical diagnosis. When there is a poor contrast between healthy and pathological regions, the use of a contrast agent (CA) can be highly beneficial. The main determinants of the contrast in a MR image are the water proton relaxation times (T1 or T2) and CAs are chemicals able to alter them markedly, in the tissues where they are distributed. The most representative class of T1-positive CAs are represented by paramagnetic gadolinium complexes. The aim of this work was to synthesize and evaluate a new high relaxivity Gd(III)-based MRI positive CA for the diagnosis of prostate cancer. The agent is based on the chelating agent AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), an heptadentate ligand, whose Gd-complex is able to coordinate two molecules of water, thus nearly doubling the overall relaxivity of the probe when compared with the clinical agents that have only a single metal-coordinated water molecule. To further increase the detection sensitivity of the agent, a tetrameric version of the complex (dL-(Gd-AAZTA)4) was prepared, in which the four chelates are coupled to a small dendrimer of lysine. As cancer extracellular matrix has an abundance of proteins that can be used as biomarkers for cancer molecular imaging, including fibrin-fibronectin complexes, dL-(Gd-AAZTA)4 was then conjugated to a pentapeptide (CREKA), reported to strongly recognize the fibrin-fibronectin complex. The probe was synthesized starting from AAZTA(tBu)4C4COOH through various steps: initially a nitro-Mannich reaction was carried out to generate the seven-membered ring. Catalytic hydrogenation followed by a step of alkylation led to the methyl ester derivative that was then hydrolysed to obtain the final acid. Both CREKA and the dendrimer of lysine coupled with AAZTA were synthesized by Fmoc Solid Phase Peptide Synthesis (SPPS). The major advantage of SPPS is that no further purification between the couplings was needed because undesired intermediates were washed away from the solid support during the synthesis. Every step of the synthesis was characterized by HPLC-MS and NMR, whereas the relaxometric properties of the final product were assessed on a Stelar SpinMaster relaxometer. In vitro targeting tests were carried out on fibrin and fibrin-fibronectin clots, whereas in vivo studies were performed on male athymic nude mice bearing orthotopic PC-3 prostate cancer. Importantly, a good MRI contrast was detected in the tumour after the i.v. injection of CREKA-dL-(Gd-AAZTA)4 at a low dose (0.02 mmol Gd/kg), thus demonstrating the great potential of this agent in MR molecular imaging of prostate cancer.