Vol. 302, Issue 2, 619-626, August 2002

bis-Cholesteryl-Conjugated Phosphorothioate Oligodeoxynucleotides Are Highly Selectively Taken Up by the Liver

Martin K. Bijsterbosch, Muthiah Manoharan, Rick Dorland, Richard van Veghel, Erik A. L. Biessen and Theo J. C. van Berkel

Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands (M.K.B., R.D., R.V, E.A.L.B., T.J.C.B.); and ISIS Pharmaceuticals, Carlsbad, California (M.M.)

We previously modulated, by conjugating a single cholesterol, plasma protein binding and liver cell uptake of a phosphorothioate oligodeoxynucleotide (PS-ODN). In this study, we investigated the biological fate of a PS-ODN, denoted ISIS-9389 (3',5'-bis-cholesteryl-conjugated ISIS 3082), provided with two cholesteryl moieties. After intravenous injection of into rats, [³H]ISIS-9389 was cleared from plasma with a half-life of 23.6 ± 0.3 min. After 90 min (approximately 95% cleared), the liver contained 83.0 ± 0.8% of the dose. Spleen and bone (marrow), which constitute with the liver the reticuloendothelial system, contained 3.1 ± 0.3 and 4.3 ± 0.2%, respectively. All other tissues accumulated together <5% of the dose. The hepatic uptake of [³H]ISIS-9389 occurred mainly by endothelial cells (51.9 ± 6.4% of the liver uptake). Parenchymal and Kupffer cells were responsible for 24.9 ± 7.7 and 23.3 ± 2.5%, respectively. Preinjected polyinosinic acid and polyadenylic acid reduced hepatic uptake, albeit the latter was less effective. This finding suggests implication of (multiple) scavenger receptors in liver uptake of ISIS-9389. The interaction of ISIS-9389 with plasma proteins, analyzed by size exclusion chromatography, differs from that of unconjugated PS-ODN and PS-ODN with a single cholesterol. Plasma-incubated ISIS-9389 was mainly recovered as a high molecular weight complex. In conclusion, conjugation of PS-ODNs with two cholesteryl moieties results in almost quantitative uptake by the liver. The liver targeting exceeds the already impressive gain in liver uptake achieved by conjugation of a single cholesterol, and is expected to increase the therapeutic activity against liver-associated targets and reduce side effects in nonhepatic tissues.