Pharmacokinetics and protein binding of cholesterol-conjugated heteroduplex oligonucleotide

Heteroduplex oligonucleotide (HDO) is a novel oligonucleotide therapeutic consisting of an antisense oligonucleotide (ASO) and its complementary RNA. A recent report showed that cholesterol-conjugated HDO (Chol-HDO) exhibited antisense activity in various tissues, including the brain; however, little information is available on the pharmacokinetic and plasma protein-binding properties of HDO and Chol-HDO. In the present study, we investigated the tissue distributions of an ASO, HDO, and Chol-HDO in mice and rats after intravenous injection. Tissue distribution was evaluated by measuring the concentration of ASO in tissue samples using liquid chromatography and tandem mass spectroscopy. ASO and HDO disappeared rapidly from the plasma, whereas Chol-HDO showed prolonged retention in the systemic circulation. The amount of ASO in the brain tissue was highest after injection of Chol-HDO, confirming its efficient delivery to the brain. The tissue distribution of oligonucleotides differed less in rats than in mice. Hepatic uptake of ASO and HDO, but not of Chol-HDO, was significantly inhibited by co-administration with the scavenger receptor inhibitor dextran sulfate sodium. The binding to plasma proteins was evaluated. Compared to ASO, HDO showed lower protein binding, but Chol-HDO showed much higher binding, with remarkable differences in binding to high-density and low-density lipoproteins. The binding of Chol-HDO to these proteins was also confirmed in mouse plasma after injection. These results indicate that the binding of Chol-HDO to plasma proteins, especially lipoproteins, is critical for determining tissue distribution and brain delivery after intravenous injection.