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Stability of polycationic complexes of an antisense oligonucleotide in rat small intestine homogenates
María González Ferreiro1, Rosanne M Crooke, Lloyd Tillman
1College of Pharmacy, Freie Universität Berlin, Berlin, Germany.
Abstract:
Presystemic degradation in the gastrointestinal tract is one of the major problems contributing to the poor oral absorption of antisense oligonucleotides. Complexes between the antisense phosphorothioate oligodeoxynucleotide ISIS 2302 and the polycationic carriers protamine sulfate grade X, protamine chloride grade V, protamine phosphate grade X, poly-L-lysine hydrobromide (PLL), spermidine phosphate salt, spermine diphosphate salt, and Protasan G113 and CL113 were formulated in order to increase stability against intestinal nucleolytic degradation. Specific conductivity measurements were carried out to determine the charge ratio of the complex systems. Nuclease stability assays were performed in a rat small intestine homogenate model, which displayed significant exo- and endonuclease activity. Full-length oligonucleotide and metabolites were analyzed by capillary gel electrophoresis with UV detection at 260 nm. Most of the complexes of ISIS 2302 and the polycationic materials, except PLL-based systems, showed a better protection against enzymatic metabolism than free oligonucleotide. Protamine sulfate and protamine chloride considerably enhanced the nuclease stability of the phosphorothioate antisense oligonucleotide. The association of oligonucleotides with several polycationic substances proved to be an alternative to chemical modification in order to stabilize oligonucleotides in the gastrointestinal tract against nucleolytic degradation.
Insights
Formulating antisense oligonucleotides with polycationic carriers like protamine sulfate enhances their stability against degradation in the gastrointestinal tract. This approach improves oral absorption by protecting the therapeutic oligonucleotides from nucleolytic enzymes.
Area of Science:
- Biochemistry
- Pharmaceutical Sciences
- Drug Delivery
Background:
- Antisense oligonucleotides (ASOs) face significant presystemic degradation in the gastrointestinal (GI) tract, leading to poor oral absorption.
- Nucleolytic degradation by enzymes in the GI tract is a primary challenge for oral delivery of ASOs.
Purpose of the Study:
- To formulate complexes of the antisense phosphorothioate oligodeoxynucleotide ISIS 2302 with various polycationic carriers.
- To enhance the stability of ISIS 2302 against intestinal nucleolytic degradation.
- To evaluate polycationic substances as an alternative to chemical modification for stabilizing oligonucleotides.
Main Methods:
- Complex formation between ISIS 2302 and polycationic carriers (protamine sulfate, protamine chloride, poly-L-lysine hydrobromide, etc.).
- Determination of charge ratios using specific conductivity measurements.
- Nuclease stability assays in a rat small intestine homogenate model.
- Analysis of oligonucleotide integrity using capillary gel electrophoresis with UV detection.
Main Results:
- Most complexes, excluding poly-L-lysine hydrobromide (PLL)-based systems, demonstrated improved protection against enzymatic degradation compared to free ISIS 2302.
- Protamine sulfate and protamine chloride significantly enhanced the nuclease stability of the phosphorothioate antisense oligonucleotide.
- The study identified specific polycationic carriers that effectively stabilize oligonucleotides in a simulated GI environment.
Conclusions:
- Association of oligonucleotides with certain polycationic substances offers a viable strategy to improve their stability in the GI tract.
- Polycationic complexation represents a promising alternative to chemical modifications for enhancing the oral bioavailability of antisense oligonucleotides.