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Biodistribution and metabolism of internally 3H-labeled oligonucleotides. II. 3',5'-blocked oligonucleotides
H Sands1, L J Gorey-Feret, S P Ho
1DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880.
Abstract:
The pharmacokinetics and metabolism of four radiolabeled phosphodiester oligonucleotides with 3'- and 5'-blocked ends were studied in mice and 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 cyclization or by incorporation of either phosphorothioate or methylphosphonate linkages. Although these modifications protected the blocked oligonucleotides 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 less stable than the all-phosphorothioate oligonucleotide and only marginally more stable than the previously studied, unblocked phosphodiester oligonucleotide. Even a "chimeric" blocked oligonucleotide with 16 phosphorothioate linkages and eight contiguous phosphodiester linkages was rapidly degraded. Despite the favorable serum binding, tissue accumulation, and stability observed with phosphorothioate oligonucleotides, these properties did not provide the chimeric oligonucleotide access to a compartment where its phosphodiester linkages were stable. In other respects, the blocked and chimeric phosphodiester oligonucleotides also resembled the unblocked phosphodiester oligonucleotide; radiolabel 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 mononucleotides, and only mononucleotide metabolites were present in urine. In summary, 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 unattractive candidates for drug development.
Insights
Blocked phosphodiester oligonucleotides show poor stability in mice due to rapid endonuclease degradation. These findings suggest limited potential for blocked phosphodiester oligonucleotides as drug candidates in humans.
Area of Science:
- Pharmacokinetics and Drug Metabolism
- Oligonucleotide Chemistry
Background:
- Phosphodiester oligonucleotides are a promising class of therapeutics.
- Modifications to enhance stability, such as end-blocking, are crucial for drug development.
Purpose of the Study:
- To evaluate the pharmacokinetics and metabolism of end-blocked phosphodiester oligonucleotides in mice.
- To compare their stability and degradation profiles with unblocked and all-phosphorothioate oligonucleotides.
Main Methods:
- Four radiolabeled phosphodiester oligonucleotides with 3'- and 5'-blocked ends were synthesized.
- These were administered intravenously to mice, and their degradation was analyzed using high-performance liquid chromatography.
- Metabolites in serum, tissues, and urine were characterized.
Main Results:
- End-blocking protected against exonuclease degradation but not endonuclease degradation, with 50% degradation occurring in 0.5-5 hours.
- Blocked phosphodiester oligonucleotides exhibited limited stability in vivo, comparable to unblocked versions.
- Rapid clearance from blood, minimal tissue accumulation, and extensive degradation to mononucleotides were observed.
Conclusions:
- Endonuclease susceptibility significantly limits the in vivo stability of blocked phosphodiester oligonucleotides in mice.
- Current modifications do not confer sufficient stability for therapeutic applications in this model.
- Further investigation is needed to determine if primates exhibit reduced endonuclease activity for potential therapeutic use.