Radiolabeling Morpholino With ～(111)In, ～(90)Y, ～(188)Re, ～(99m)Tc And Its Biodistribution In Normal Mice

Over the past years, techniques have been developed to label biomolecules(McAb, peptides, oligos) with Indium-111(111In) and Technetium-99m(99mTc) for radionuclide imaging. However, an increased effort has been made to label these biomolecules with B-emitters such as Ytrium-90(90Y) and Rhenium-188(188Re) for radionuclide therapy.It is well known that McAb acting as carrier has been used widely for Radioimmuno imaging(RII) and therapy(RIT). Oligonucleotide, a new labeled ligands that specifically hybridize to target sequence through complementary base pairing has been investigating for antisense imaging and therapy.A fairly large number of oligomers have been investigated for in vivo use, e.g. phosphodiester DNA, phosphorothioate DNA(sDNA) and peptide nucleic acids (PNA), but no one can be used exclusively due to their instabilities to nucleases, high protein binding affinities and poor aqueous solubilities.Morpholinos(MORFs), novel synthetic oligomers, are potential clinical therapeutic agents for targeted radionuclide therapy because of their resistance to degradative nucleases, low protein binding affinities and, in particular, good aqueous solubility.90Y and 188Re are both clinically acceptable B-emitting radionuclide, useful for therapy due to their hig energetic beta emissions. Since 90Y is a pure beta emitter, this therapeutic radionuclide is often matched with 111In for similar chemistries and 111In decays via two prominent gamma rays suitable for imaging. l88Re also decays with a 155 KeV photon in 15% abundance, which permits in situ monitoring of tumor uptake and dosimetry.Numerous chelating agents have been investigated for imaging and radionuclide therapy. The macrocyclic chelating agent l,4,7,10-tetraazacyclododecane-N,N',NN-tetraacetic acid (DOTA) and Mercaptoacetyltriglycine(MAG3), have become two subjects of widespread interest because they forms extremely stable complexes with a variety of metals and are attractive candidates for conjugation of radiometals such as 111In, 90Y and 99mTc, 188Re to antibody.This study describes the conjugation of a 25-mer MORF with DOTA and NHS-MAG3, and radiolabeling with 111In. 90Y, 188Re and Tc. The in vitro stability of labeled MORFs were investigated and biodistribution was carried out in normal mice.Materal and MethodA 25 mer MORF with a primary amine on the 3' equivalent end attached via a 6 member linker was conjugated with an isothiocyanate backbone derivative of DOTA (for labeling with 111In and 90Y) and with NHS-MAG3 (for labeling with 188Re and 99mTc). The conjugate products were radiolabeled in various buffer over a range of concentrations and pH, as well as stannouschloride dihydrate on Re labeling. The effect of temperature and reaction time were examined and optimal radiolabeling conditions were identified. The in vitro stability and hybridization ability of four labeled MORFs were investigated in saline at room temperature and in fresh serum at 37 C, the biodistribution and kinetics at different time points were carried out in normalmice.Results1. As evident by size exclusion HPLC, ITLC and Sep-Pak analysis, Morpholino oligos were successfully radiolabeled by four radionuclides via DOTA and MAGS as chelator.2. In each case, the labeled MORFs showed one sharp peak in HPLC that shifted completely to earlier retention times following addition of a polymer conjugated with the complementary MORF.3. In saline at room temperature and in 37 C serum, the radioactivity profile of 111In and 99mTc was unchanged over 48h4. The 90Y profile showed a pronounced lower molecular weight peak which did not shifted and was shown due to 90Y-DOTA resulting from radiolysis.5. In addition, the recovery of 188Re on HPLC decreased as samples aged probably due to oxidation to perrhenate.6. There are no influence on hybridization of conjugation with chelators and radiolabeling with gamma or bata emitting radionuclides.7. The biodistributions in normal mice showed major amount...