Most radionuclides used in nuclear medicine are metals. In the field of diagnostic imaging, the radiolabel of choice is almost always 99mTc because of it's relatively short half-life, pure ã emission, 140keV ideal energy for imaging and allowance for daily access. One straightforward approach to radiolabel DNAs with metallic radionuclides is first to derive the antisense DNA on either of its ends with a primary amine, possibly attached by a suitable linker to minimize steric hindrance. The amine then may be conjugated with various metal bifunctional chelators, such as the anhydrides of diethylenetriamine pentaacetic acid (DTPA) or the N-hydroxysuccinimide esters of SHNH and mercaptoacetyltriglycine (MAG3). DTPA has previously been used for radiolabeling antibodies with 99mTc but was abandoned when the instability of the label was judged to be unacceptably high. As an alternative, SHNH can form stable labeling with DNAs. But Hnatowich et al. reported that DNAs labeled in this manner showed nonspecific serum protein binding, which would interfere with the biodistribution and thus affect the imaging results (3).
MAG3 seems to show better characteristic in labeling DNAs with 99mTc. In this study, a mean of 80.11%(s.d=2.96%, n=4) labeling efficiency was obtained. The radiolabeled antisense probe still kept the affinity to bind to its complement, as shown in Figure 2. After 30min incubation in fresh human serum at 37¨C, the peak radioactivity shift into two peaks. It seemed that protein binding had occurred. But since 1hr thereafter, the peak radioactivity regressed to its primary site, which most likely suggested that the labeled DNAs had dislocated from the serum proteins. This short-time serum protein binding seemed to be beneficial in protecting the labeled DNAs from being attacked by nuclease.
The extraordinary properties of DNA and RNA suggest that there is potential for the use of these oligonucleotides as radiopharmaceuticals. Despite many uncertainties concerning mechanisms, synthetic single-strand antisense deoxyribonucleic acids are now in clinical trials for the chemotherapy of several cancers, including follicular lymphoma and acute myelogenous leukemia (4-7). The question considered here is whether antisense DNAs will also be important to future nuclear medicine imaging. For successful antisense imaging, at least the following criteria must be fulfilled: 1, sufficient oncogene mRNA products in cytoplasm; 2, antisense probes of suitable base sequences; 3, efficient methods for antisense probes labeling; 4, good permeability and in vivo stability (8,9).
The c-myc oncogene works in cooperation with other oncogenes in a variety of cancers. It is an early response gene whose expression is involved in the signal transduction pathways leading to cellular proliferation (1,10-15). The concentration of the c-myc mRNA increases rapidly 30- to 50-fold during DNA synthesis, and the half-life of c-myc mRNA is sufficiently long, which makes it a suitable target for antisense imaging (16). This level and time of mRNA retention in cytoplasm may be sufficient for in vivo hybridization, retention of labeled probe and noninvasive imaging. As reported by Dewanjee and his colleagues in their imaging studies, the specific binding to isolated mRNAs was about 20-fold higher for the labeled antisense compared to the sense DNAs. The nude mice obtained about 10%-12% of the radioactivity on injected antisense DNAs and only about 1% on sense DNAs at 1hr postadministration. Preferential accumulation was revealed in tumored mice with respectable tumor/muscle ratios of 20 in antisense probes and of 0.95 in sense probes as early as 30min(17).
In contrast to single-strand DNAs, single-strand RNAs form extensive secondary structures. The secondary structures of mRNA may be required for stability and possibly for recognition by those proteins regulating translation. These structures have implications for chemotherapy, since antisense DNAs show lower affinities for duplex regions. The affinity of antisense DNA for duplex, compared to singlet, regions of mRNA has been reported to be 105-106-fold lower. Therefore, antisense strategies usually seek to target only single-strand regions of the mRNA. Since knowledge of the molecular structure of mRNA is limited, one common approach is to target either the initiation codon (AUG) and adjacent sequences or the untranslated sequences on either the 3' or 5' end (18). The c-myc mRNA antisense oligonucleotide chosen in this study was designed to target the initiation site of translation, in the hope that these regions will be accessible. This selection was proved to be act by our encouraging results similar to those of Dewanjee's. Maybe because of it's relatively high molecule or difference in radiolabeling, the tracer showed much slower pharmacokinetics in accumulation to the target and clearance from background tissues in our study than in Dewanjee et al's.
The tracer distribution reached an equilibrium value after 45 minutes post administration for both antisense and sense probes and did not change for viscera, connective tissues and tumor. The tumor could be imaged with the antisense probe as early as 45min and reached the highest tumor-to-muscle ratio of 5.9 at 4hr. This allows diagnosis of malignant tumors using short-time radionuclides as 99mTc. Besides the tumor and those highly accumulated organs as the liver and kidneys, the cellular elements of blood, mainly the nucleated cells (neutrophiles, lymphocytes and reticulocytes), retain the probe as a result of internalization and hybridization with low copy of mRNAs. Freshly formed platelets also retained a small fraction of the probes. This resulted in prolonged background clearance that the most encouraging results were not achieved until 20hr later when the background activity was diminished sufficiently. Considering the high uptake of tracers in the liver, intestinal bowels and kidneys, the 99mTc-MAG3 c-myc probe seemed unsuitable for detecting tumors in these regions. From these points of view, more investigations should be taken to improve the target/nontarget ratio and to accelerate the clearance of background activity.