BIODISTRIBUTION AND METABOLISM OF INTERNALLY H-3 LABELED OLIGONUCLEOTIDES .2. 3',5'-BLOCKED OLIGONUCLEOTIDES

The pharmacokinetics and metabolism of four radiolabeled phosphodieste r oligonucleotides with 3'- and 5'-blocked ends were studied in mice a nd compared with previously studied, unblocked, all-phosphodiester and all-phosphorothioate oligonucleotides. The radiolabel was a tritiated methyl group enzymatically attached at an internal cytidine. The ends of the blocked phosphodiester oligonucleotides were protected by cycl ization or by incorporation of either phosphorothioate or methylphosph onate linkages. Although these modifications protected the blocked oli gonucleotides from degradation by exonucleases present in mouse serum, degradation initiated by endonucleases was 50% complete in 0.5-5 hr. After intravenous injection, the blocked oligonucleotides were much le ss stable than the all-phosphorothioate oligonucleotide and only margi nally more stable than the previously studied, unblocked phosphodieste r oligonucleotide. Even a ''chimeric'' blocked oligonucleotide with 16 phosphorothioate linkages and eight contiguous phosphodiester linkage s was rapidly degraded. Despite the favorable serum binding, tissue ac cumulation, and stability observed with phosphorothioate oligonucleoti des, these properties did not provide the chimeric oligonucleotide acc ess to a compartment where its phosphodiester linkages were stable. In other respects, the blocked and chimeric phosphodiester oligonucleoti des also resembled the unblocked phosphodiester oligonucleotide; radio label was cleared rapidly from the blood, there was little evidence of tissue accumulation, high performance liquid chromatographic analysis of tissue extracts showed extremely rapid degradation to mononucleoti des, and only mononucleotide metabolites were present in urine. In sum mary, blocked phosphodiester oligonucleotides are rapidly attacked by endonucleases present in mice. Unless this problem is less serious in primates, such blocked oligonucleotides will be relatively unattractiv e candidates for drug development.