Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation

Several studies have shown improved efficacy of cholesteryl-conjugated phos phorothioate antisense oligodeoxynucleotides. To gain insight into the mech anisms of the improved efficacy in vivo, we investigated the disposition of ISIS-9388, the 3'-cholesterol analog of the ICAM-1-specific phosphorothioa te oligodeoxynucleotide ISIS-3082, in rats. Intravenously injected [H-3]ISI S-9388 was cleared from the circulation with a half-life of 49.9 +/- 2.2 mi n (ISIS-3082, 23.3 +/- 3.8 min). At 3 h after injection, the liver containe d 63.7 +/- 3.3% of the dose. Compared to ISIS-3082, the hepatic uptake of I SIS-9388 is similar to 2-fold higher. Endothelial, Kupffer and parenchymal cells accounted for 45.7 +/- 5.7, 33.0 +/- 5.9 and 21.3 +/- 2.6% of the liv er uptake of [H-3]ISIS-9388, respectively, and intracellular concentrations of similar to 2, 75 and 50 mu M, respectively, could be reached in these c ells(1 mg/kg dose). Preinjection with polyinosinic acid or polyadenylic aci d reduced the hepatic uptake of [H-3]ISIS-9388, which suggests the involvem ent of (multiple) scavenger receptors. Size exclusion chromatography of mix tures of the oligonucleotides and rat plasma indicated that ISIS-9388 binds to a larger extent to high molecular weight proteins than ISIS-3082. Analy sis by agarose gel electrophoresis indicated that ISIS-9388 binds more tigh tly to plasma proteins than ISIS-3082, The different interaction of the oli gonucleotides with plasma proteins possibly explains their different dispos itions. We conclude that cholesterol conjugation results in high accumulati on of phosphorothioate oligodeoxynucleotides in various liver cell types, w hich is likely to be beneficial for antisense therapy or liver-associated d iseases.