Initial investigations of 99mTc-labeled morpholinos for radiopharmaceutical applications.

This laboratory is evaluating phosphorothioate deoxyribonucleic acids (DNAs) and peptide nucleic acids (PNAs) for a variety of nuclear medicine applications. Morpholinos (MORFs) are a new class of oligomers with a nuclease-resistant, nonionic and water-soluble phosphorodiamidate backbone. We now report on the in vitro and in vivo properties of MORFs labeled with technetium-99m. Both 15-mer and 18-mer MORFs were obtained, each with a primary amine attached to the 3' equivalent end via a three-carbon beta-alanine linker. The amine was used to conjugate with NHS-MAG3 for 99mTc radiolabeling. By surface plasmon resonance at room temperature, the association rate constant for hybridization of the 18-mer MORF to its complementary oligomer (cMORF) was equivalent to that of DNAs and PNAs of comparable length. Hybridization of 99mTc-MORF in vitro to free cMORF, to a cMORF polymer and to cMORF beads was nearly quantitative under a variety of conditions. Kinetic studies in vitro at room temperature showed rapid (2-5 min) and nearly quantitative (90%) binding to cMORF beads. Using size-exclusion high-performance liquid chromatography, the stability of the 99mTc-MORF was found to be greater than 85% over 24 h in 37 degrees C serum with minimal protein binding. In normal mice, the 99mTc-MORF showed rapid pharmacokinetics, with only 21% and 8% remaining in the whole body at 3 and 24 h post administration, respectively. In vivo targeting with 99mTc-MORF of cMORF beads in one thigh of normal mice compared to control beads in the other thigh showed target/control thigh ratios of 2-10 between 3 and 24 h. These results demonstrate that MORF oligomers are capable of in vivo hybridization. Their properties of hybridization affinity and kinetics and their in vivo stability and pharmacokinetics make them suitable subjects for in vivo studies.

Influence of a 99mTcN core on the biological and physicochemical behavior of 99mTc complexes of L,L-EC and L,L-ECD.

99mTc-nitrido complexes of L,L-ethylene dicysteine (99mTcN-L,L-EC) and 99mTcN-L,L-ethylene dicysteine diethylester (99mTcN-L,L-ECD) were prepared and their characteristics compared to those of the respective 99mTc-oxo complexes. 99mTcN-L,L-EC and 99mTcO-L,L-EC migrate to similar extents during electrophoresis at pH 12, but, at pH6, 99mTcN-L,L-EC migrates further than 99mTcO-L,L-EC. Renal excretion of 99mTcN-L,L-EC is inferior to that of 99mTcO-L,L-EC, indicating that the TcN-glycine sequence has lower affinity for the renal tubular system. Both 99mTcO-L,L-ECD and 99mTcN-L,L-ECD are neutral, but 99mTcN-L,L-ECD is hydrophilic and shows minimal brain uptake in both mice and the baboon.

Comparison of reversed phase and reversed phase ion pair high performance liquid chromatography for analysis of TcO and TcN complexes of L,L-ethylene dicysteine di-ethylester and its acid analogues.

99mTc(V)-oxo complexes of L,L-ethylene dicysteine (L,L-EC) and its di-ester derivative L,L-ethyl cysteinate dimer (L,L-ECD) are useful tracer agents for evaluation of renal function and cerebral blood flow respectively. Labelling of these molecules with a 99mTc(V)-nitrido core instead of the 99mTc(V)-oxo core alters their biological and physiochemical behavior. In the reversed phase high performance liquid chromatography (HPLC) method presently used to analyse 99mTc(V)-oxo preparations of L,L-EC and L,L-ECD, the 99mTc(V)-nitrido-L,L-EC complex and the possible impurities of a 99mTc(V)-nitrido-L,L-ECD preparation were found to elute with the void volume. In this study, a reversed phase ion pair HPLC method has been developed that is useful for the analysis of both 99mTc(V)-oxo and 99mTc(V)-nitrido preparations of L,L-EC and L,L-ECD. Tetrabutylammonium hydroxide is used as a cationic ion pairing agent.

Stabilisation of high-activity 99mTc-d,l-HMPAO preparations with cobalt chloride and their biological behaviour.

It has been reported that the stability of a 1.11-GBq (30 mCi) technetium-99m d,l-hexamethyl-propylene amine oxime (HMPAO) preparation can be improved to up to 5 h by the addition of 200 micrograms CoCl2.6H2O within 2 min after reconstitution. However, it is not clear whether this method is also efficient for high-activity preparations (5.55 GBq) and whether this modified 99mTc-d,l-HMPAO has the same biological properties and can safely be used. We have now studied CoCl2-stabilised 99mTc-d,l-HMPAO preparations containing different amounts of "in-house" HMPAO ligand and SnCl2 and reconstituted with activities from 1.11 GBq to 5.55 GBq 99mTc. The characteristics of the generator eluates were also divergent, ranging from fresh eluates from a generator eluted less than 2 h previously to 4-h-old eluates from a generator not eluted during the preceding 72 h. Preparations containing up to 5.55 GBq 99mTc and as low as 2 micrograms SnCl2.2H2O can be efficiently stabilised for at least 6 h by the addition of CoCl2 shortly after reconstitution. Interestingly, it was found that the stabilisation method is not efficient if the cobalt ions are added prior to reconstitution of the preparation. This implies that the cobalt chloride cannot be incorporated in the labelling kit. Also, preparations with amounts of the ligand lower or higher than 0.5 mg formed the 99mTc-d,l-HMPAO complex with low radiochemical yield or showed rapid degradation. Therefore, combination of a subdivision and storage of Ceretec kits in fractions (as reported in the literature) is contra-indicated with this CoCl2 stabilisation method. CoCl2-stabilised Ceretec kits reconstituted with 5550 MBq 99mTcO4- and used 4-5 h after preparation retain the diagnostic usefulness of the fresh 1110-MBq preparation with regard to leucocyte labelling and brain imaging. Although baboon brain uptake of the stabilised preparation was 6%-9% lower, this small difference could not be distinguished in the tomographic images. The data obtained with both inhouse prepared d,l-HMPAO and Ceretec kits suggest that the eluate restrictions recommended by the Ceretec manufacturer can be neglected if the preparation is stabilised with Co2+ ions. Studies with 57Co-spiked CoCl2 added to d,l-HMPAO preparations demonstrated that the Co2+ ions clearly interact with the d,l-HMPAO ligand, probably to form one or more complexes. From biodistribution studies in mice it became evident that the toxicological profile of the Co2+ ions in the presence of d,l-HMPAO should be more favourable than that of cobaltous ions. For these reasons, it seems justifiable that CoCl2-stabilised 99mTc-d,l-HMPAO preparations should undergo rigorous studies to elucidate their clinical usefulness and pharmacological safety.