Preclinical Evaluation of DO3P-AME-DO3P: A Polyazamacrocyclic Methylene Phosphonate for Diagnosis and Therapy of Skeletal Metastases

A phosphonate derivative 10′-bis(acetamido)-ethane-bis[1,4,7-tri(methylene phosphonic acid)-1,4,7,10-tetraazacyclododecane] (DO3P-AME-DO3P), was synthesized with 90% yield in high purity. It was labeled with <sup>99m</sup>Tc in 97.5% efficiency and specific activity of 112−250 MBq/μmol. The binding affinity of <sup>99m</sup>Tc-DO3P-AME-DO3P towards bone minerals was tested in vitro by using hydroxy apatite as a bone model with absorption of 93% during the first hour of the experiment. Receptor binding assay on human bone cell line SAOS-2 demonstrated <i>K</i><sub>d</sub> value of 1.07 nM. Cell binding studies of DO3P-AME-DO3P on osteoblasts and osteoclasts cells performed in vitro displayed preferential affinity of the compound towards osteoclast (167.95 ± 3.56% dose/mg protein). The serum stability of <sup>99m</sup>Tc complex was found to be 96.8% after 24 h. Blood kinetics of <sup>99m</sup>Tc-DO3P-AME-DO3P performed on normal rabbits showed fast clearance with <i>t</i><sub>1/2</sub>(<i>F</i>) = 15 min ± 0.014 min and <i>t</i><sub>1/2</sub>(<i>S</i>) = 4 h 3 min ± 0.09 min. Biodistribution studies carried out in normal BALB/c mice showed bone-to-blood ratio of 20 and bone-to-muscle ratio of 33. The bone tissue demonstrated highest concentration of bound radioactivity with 10.73% ID/g at 1 h post injection. The protonation and stability constants were determined by pH-potentiometry titrations. The stability constants of DO3P-AME-DO3P with Lu(III), Sm(III), and Ho(III) were 19.7, 21.8, and 20.2 determined by “out of cell” method. The excellent bone seeking properties of DO3P-AME-DO3P make it a candidate of choice for SPECT imaging and preferential uptake of the compound in osteoclasts in comparison to osteoblasts; BMM and BMC can be used to understand the pathway of pathogenesis of osteoporosis and skeletal metastases.