Background: For pretargeting and other nuclear medicine applications, it may eventually be useful to radiolabel phosphodiamidate morpholinos (MORFs) with therapeutic radionuclides such as 188Re. However, by preparing 188Re-MORFs labelled conventionally with MAG3 as chelator, we have observed unacceptable levels of oxidation to perrhenate in vitro and in vivo in mice.
Objective: To improve upon stability, tricarbonyl labelling was considered since tricarbonyl complexes are thought to stabilize metals in low oxidation states.
Methods: An amine derivatized 25 mer MORF was conjugated with either NHS-MAG3 or NHS-Hynic. The MAG3 conjugated MORF was radiolabelled conventionally with 188Re while the Hynic conjugated MORF was radiolabelled through its tricarbonyl intermediate. Using a commercial kit modified with additional reducing agent over that required for the preparation of the 99mTc tricarbonyl complex [99mTc(CO)3(H2O)3]+, we demonstrated that the equivalent 188Re tricarbonyl, [188Re(CO)3(H2O)3]+, could be prepared. Simple incubation at elevated temperatures with the Hynic conjugated MORF then provided 188Re-(CO)3-Hynic-MORF. Confirmation was achieved by a shift assay using a complementary MORF conjugated polymer and size exclusion HPLC. To evaluate the relative stability of the tricarbonyl labelled MORF compared to the MAG3 labelled MORF in vitro, the radiolabelled MORFs were incubated in phosphate buffer and the presence of perrhenate measured periodically by strip chromatography. Stability in vivo was evaluated by biodistribution studies in normal mice.
Results: The overall yields for tricarbonyl intermediates averaged greater than 90% for 99mTc and 60-80% for 188Re. Yields following subsequent labelling to Hynic-MORF were about 60-80% for 99mTc and 15-20% for 188Re. The in vitro stability results in phosphate buffer showed that 188Re-MAG3-MORF was fully oxidized by 48 h while 188Re-(CO)3-Hynic-MORF was less than 20% oxidized at that time. Similarly, the 188Re-(CO)3-Hynic-MORF biodistribution in normal mice showed lower radioactivity level in stomach, intestines and thyroid compared with 188Re-MAG3-MORF.
Conclusion: 188Re-tricarbonyl labelling of Hynic conjugated MORFs may be considerably more stable to oxidation than the MAG3 labelled MORFs and therefore more suitable for radiotherapy trials.