Design and evaluation of potential bioreductive radiopharmaceuticals based on the 3+1 mixed ligand approach

Aim: A family of potential bioreductive oxorhenium and oxotechnetium mixed ligand complexes (general formula MOLC, M = Tc or Re, L R-N(CH2CH2SH)2, C= p-NO2-C4H5-R'SH) bearing a nitro aromatic group as pharmacophore in the structure of the monodentate coligand has been synthesised and characterised both 'in vitro' and 'in vivo'. Materials and Methods: Oxorhenium complexes were synthesised at carrier level using ReOCl3(PPh3)2 and characterised by UV-vis and IR spectra, and elemental analysis. Oxotechnetium complexes were prepared by using 99mTc-glucoheptonate as precursor. The lipophilic species formed were extracted by dichloromethane and analysed by HPLC to assess the radiochemical purity. Cytotoxicity to V-79 cells (Chinese hamster lung fibroblasts) 'in vitro' was studied at a single dose (20-0.1 μM) in air or hypoxia for 2 hours. Negative and positive controls were used to validate the assay. Biodistribution studies in normal mice (CD1, female, 25-30 g) and in mice bearing induced sarcoma were performed at 30 minutes, 2 and 24 hours post-injection. Images in rats and mice bearing spontaneous adenocarcinoma were acquired between 1 and 24 hours after injection. Results: The oxorhenium complexes were isolated as crystalline products in high yield (40-70%). All analytical results were consistent with the proposed ''3+1'' structure. High cytotoxicity was found for compounds bearing the N',N'-dialkyl-ethylenediamine [(R)2 -N-(CH2)2-N) moiety in the structure of the tridentate ligand, however this toxicity was not selective in hypoxia. Labelling with 99mTc was achieved with high yield (> 85%) and radiochemical purity (> 90%). Biodistribution studies in normal mice showed high initial blood, lungs and liver uptake and excretion through the hepatobiliary and urinary tracts (total excretion in 24 hours > 70%). Tumour uptake was moderate (about 1.2 %/g at 30 min.) but the tumour/muscle ratio was 2-3 at 2 hours post-injection. Centellographic images in animals bearing spontaneous adenocarcinoma showed similar results. Uptake in an animal bearing an abscess instead of a tumour was negligible. Conclusions: A family of 99mTc complexes with potential application in oncological Nuclear Medicine was designed using the '3+1' mixed ligand approach and a nitro aromatic moiety as pharmacophore. Labelling was successfully performed and chemical analysis confirmed the proposed structure. Cytotoxicity was found to be highly dependent of the structure of the tridentate ligand. In vivo evaluation corroborated selective affinity of these compounds for tumoral tissue. These results are encouraging and demonstrate the potentiality of our design approach