RADIOLABELED ANTISENSE OLIGODEOXYNUCLEOTIDES - IN-VITRO AND IN-VIVO APPLICATIONS

Radiopharmaceuticals labeled with nonmetallic (I-123, F-18) and metall ic radionuclides (Tc-99m, Ga-67, In-111, Y-90, Sm-153) are used routin ely for diagnosis and therapy. Described is a new type of radioligand based on antisense oligodeoxy-nucleotide analogs labeled with I-123, T c-99m and In-111 and which are called ''antisense radiopharmaceuticals '' or ''radiolabeled antisense oligonucleotide (RASON)'' probes. The o ligodeoxynucleotides provide us with a new avenue of intracellular del ivery of radionuclides, drugs and toxins post-intravenous administrati on. These probes have been developed and tested in both in vitro (cult ured cells and tissues) and in vivo systems (mice, dogs and pigs). Qua lity assurance of RASON probes have been carried out by thin-layer, ge l electrophoresis, size-exclusion and ion-exchange chromatography tech niques for chemical, radionuclidic and radiochemical impurities. Size- exclusion techniques were used for the quantification of hybridization kinetics of RASON probes with specific mRNA of interest. In vitro upt ake was dependent on nucleotide-length, time and temperature. Since pr obes are internalized efficiently in target cancer cells of interest a fter IV injection, high level of radiation could be delivered for cell -killing. For therapy, RASON probes could be labeled with I-131 (beta, gamma) Sm-153 (beta,gamma), Re-186 (beta,gamma), Re-188 (beta), Y-90 ( beta), At-211 (alpha) etc. In vitro studies of RASON probes with cultu red cells and fixed tissues, identified the parameters of stability, s pecificity and hybridization kinetics. The pharmacokinetics of several In-111 labeled deoxynucleotides (oxo and thio derivatives, sense and antisense) of different nucleotide length and sequence, specific for b inding mRNAs of beta-actin, histone4, c-myc, c-myb, c-fos and c-erbB2 were studied in Balb/c and nude mice, Beagle dogs and Yorkshire pigs. The paradigm of maximal probe uptake was tested in mammary-tumor beari ng Balb/c mice and human tumor xenograft (MCF-7) innude mice. For neut rophil labeling, the histone4 mRNA in the neutrophils was targeted for direct labeling in whole blood. Using the P388 cell-line (murine mono cytic leukemia) and neutrophils from canine, porcine and human blood a s model cells, several RASON probes have been screened for optimum nuc leotide length (15, 20, 25-mer). In vivo studies characterized the par ameters of specific activity on biodistribution, in vivo metabolism, r adiation dosimetry and optimum imaging time with these new probes.