Synthesis and development of positron-emitting tracers, labeled with carbon-11 and radiometals (Ga-68, Zr-89, Y-90 and Lu-177), and their potential use in oncology

Study of altered metabolism as well as accurate initial diagnosis and determination of the extent of diseases continue to be a major challenge for contribution in understanding the modification that can select appropriate treatment options, monitoring the effects of therapeutic interventions, and detecting disease after recurrence. Critical for new tracers’ development is the selection of appropriate targets. Two different molecular targets have been taken into consideration: 1) COX-2 for inflammation and cancer targeting; the work foresaw the labelling of a COX-2 selective inhibitor (VA426) with carbon-11 and the subsequent evaluation in-vivo. 2) PSMA for prostate cancer targeting; the work foresaw two subprojects: a) coupling of DUPA-Pep, a PSMA-selective ligand, with two different chelating agents and labelling with different radiometals for imaging (68Ga and 89Zr) and therapy (90Y and 177Lu); b) labelling of mesoporous nanoparticles (MSNPs) for drug delivery into prostate cancer. VA426 (1-(4-fluorophenyl)-3-(2-methoxyethyl)-2-methyl-5-(4-methylsulfonyl)phenyl)-1H-pyrrole) is a COX-2-selective inhibitor provided by Anzini (University of Siena). The synthesis of [11C]VA426 was achieved by reacting [11C]methyl iodide with an excess of the corresponding alcohol precursor VA425 and NaH 60% (1:10) in DMSO. The radiochemical yield was 21 ± 1.67 % (decay-corrected, based on [11C]CH3I) (n = 3) with a specific radioactivity of 90 ± 54 GBq/µmol (n = 3). The average time of synthesis from end of bombardment including formulation was 30 min with 97.6 ± 2.6 % radiochemical purity. Preliminary investigation has been performed in vivo using BL/6 female mice. Mice treated with LPS, compared with the control, showed more intense uptake at the site of injection of the toxin. In particular, it is possible to notice a major distribution in the liver and intestine. Further studies will be performed to evaluate the potential use of this tracer in colorectal cancer cell lines expressing COX-2. DUPA-Pep, a PSMA-selective urea-based ligand, proved to be a successful imaging ligand with excellent potential for clinical studies. The current study reports the coupling of DUPA-Pep with desferrioxamine (Df) and CHX-A´´-DTPA and the subsequent labelling with 68Ga, 89Zr for imaging (Df) and 90Y, 177Lu for therapy (CHX-A´´-DTPA), as new PSMA ligands. The labelling of Df-DUPA-Pep was performed at room temperature under neutral condition in 0.25 M HEPES buffer. In both radiosyntheses, the RCYs were higher than 95 % within 10 min, making dispensable any further purification and providing the radiotracers directly applicable for further utilization. Stability of the labelled peptide, in PBS and HS at 37°C, revealed no decomposition or decreased of amount of product as confirmed by radio-HPLC. KD values and cellular uptake in PSMA-positive cell lines (LNCaP C4-2) were calculated and corresponded to 42.04 ± 4.76 and 29.84 ± 4.82 nM for the 68Ga and 89Zr labelled peptides, respectively. The inhibitor 2-PMPA completely abolished peptide binding, proving the peptide-specificity for PSMA. Study of biodistribution and tumor uptake were also performed in SCID mice implanted with PSMA-positive tumor xenografts. PET/CT images depicted a specific accumulation into the tumor. Further uptake was detected in kidney, bladder and spleen. An average tumor-to-tissue ratio of 26.75 ± 10.4 was calculated in PSMA-positive tumors. PSMA-negative xenografts showed a tumor-to-tissue ratio of 1.15 ± 0.28. Labelling of CHX-A´´-DTPA-DUPA-Pep was also performed with both 68Ga and 89Zr at room temperature under neutral pH (0.25M HEPES buffer), providing very high incorporation of 68Ga within few minutes, with a high stability and KD value at nanomolar concentration. The radiolabelling of the peptide with the therapeutic radiometals (90Y and 177Lu) was performed at room temperature in 0.5M NH4OAc buffer at pH 5.5. The incorporation of both radiometals was more than 90% within 10 minutes. Further studies of stability have to be conducted in PBS and HS at 37°C to verify that the labelled peptide is stable in order to avoid side effects caused by free radiometal. The labelling of MSNPs has been successfully performed with 89Zr . Further experiments will be conducted in order to increase the dispersibility into the suspension, through PEGylation. Tumor targeting will be evaluated by attaching DUPA-Pep to the particle surface.