Radiolabelling morpholinos with 188Re tricarbonyl provides improved in vitro and in vivo stability to re-oxidation

BACKGROUND: For pretargeting and other nuclear medicine applications, it may eventually be useful to radiolabel phosphodiamidate morpholinos (MORFs) with therapeutic radionuclides such as Re. However, by preparing Re-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 Re 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 Tc tricarbonyl complex [Tc(CO)3(H2O)3], we demonstrated that the equivalent Re tricarbonyl, [Re(CO)3(H2O)3], could be prepared. Simple incubation at elevated temperatures with the Hynic conjugated MORF then provided Re-(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 Tc and 60-80% for Re. Yields following subsequent labelling to Hynic-MORF were about 60-80% for Tc and 15-20% for Re. The in vitro stability results in phosphate buffer showed that Re-MAG3-MORF was fully oxidized by 48 h while Re-(CO)3-Hynic-MORF was less than 20% oxidized at that time. Similarly, the Re-(CO)3-Hynic-MORF biodistribution in normal mice showed lower radioactivity level in stomach, intestines and thyroid compared with Re-MAG3-MORF. CONCLUSION: Re-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.