Max N. Moore

Comparison of pharmacokinetics and tissue disposition of an antisense phosphorothioate oligonucleotide targeting human Ha-ras mRNA in mouse and monkey

The plasma pharmacokinetics and tissue disposition of ISIS 2503 were studied in mice following single and multiple bolus intravenous (iv) injections of 1-50 mg/kg, and in monkeys following single and multiple 2-h iv infusions of 1-10 mg/kg and bolus iv injections of 1 mg/kg of ISIS 2503. ISIS 2503 and its metabolites were measured in plasma, urine, and tissues using solid-phase extraction followed by capillary gel electrophoresis (CGE). In both species, the plasma clearance of ISIS 2503 was characterized by rapid distribution to tissues, and to a lesser extent, metabolism. The plasma clearance in mice was at least two-fold more rapid than in monkeys at equivalent doses. The plasma disposition (t1/2) increased with dose. The highest concentrations of oligonucleotide were consistently observed in the kidney and liver in both species. At equivalent doses, tissue concentrations in monkeys were much higher than tissue concentrations in mice. Urinary excretion of total oligonucleotide was a minor elimination pathway in both species at doses < 10 mg/kg. However, urinary excretion of total oligonucleotide in mice was increased to 12-29% as dose increased from 20 to 50 mg/kg.

Synthesis of antisense oligonucleotides. using environmentally friendly and safe deprotection procedures

It is demonstrated that mixed-sequence phosphorothioate oligodeoxyribonucleotides can be synthesized on scales up to 80 mmol without using chlorinated solvents like dichloromethane, while preserving both high yield and purity of the product. A solution of dichloroacetic acid in toluene cleanly and efficiently removes 4,4′-dimethoxytrityl groups from the 5′-terminus of the growing oligonucleotide chain during synthesis on solid support. Ammonium hydroxide treatment at room temperature at atmospheric pressure furnishes deprotected oligonucleotides reducing the risk that pressurized reaction glass vessels pose. To ensure facile separation of polymer beads (Primer HL 30) and oligonucleotide solution, minimum agitation of the reaction mixture is applied.