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

Abstract

Several studies have shown improved efficacy of cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotides. To gain insight into the mechanisms of the improved efficacy in vivo, we investigated the disposition of ISIS-9388, the 3'-cholesterol analog of the ICAM-1-specific phosphorothioate oligodeoxynucleotide ISIS-3082, in rats. Intravenously injected [(3)H]ISIS-9388 was cleared from the circulation with a half-life of 49.9 +/- 2.2 min (ISIS-3082, 23.3 +/- 3.8 min). At 3 h after injection, the liver contained 63.7 +/- 3. 3% of the dose. Compared to ISIS-3082, the hepatic uptake of ISIS-9388 is approximately 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 liver uptake of [(3)H]ISIS-9388, respectively, and intracellular concentrations of approximately 2, 75 and 50 microM, respectively, could be reached in these cells (1 mg/kg dose). Preinjection with polyinosinic acid or poly-adenylic acid reduced the hepatic uptake of [(3)H]ISIS-9388, which suggests the involvement of (multiple) scavenger receptors. Size exclusion chromatography of mixtures of the oligonucleotides and rat plasma indicated that ISIS-9388 binds to a larger extent to high molecular weight proteins than ISIS-3082. Analysis by agarose gel electrophoresis indicated that ISIS-9388 binds more tightly to plasma proteins than ISIS-3082. The different interaction of the oligonucleotides with plasma proteins possibly explains their different dispositions. We conclude that cholesterol conjugation results in high accumulation of phosphorothioate oligodeoxynucleotides in various liver cell types, which is likely to be beneficial for antisense therapy of liver-associated diseases.

Figure 1

Plasma clearance of i.v. injected [³H]ISIS-9388 and [³H]ISIS-3082. Rats were i.v. injected with [³H]ISIS-9388 (open circles) or [³H]ISIS-3082 (closed circles), both at a dose of 1 mg/kg body wt. Blood samples were taken at the indicated times and the radioactivity in the plasma was determined. Values are means ± SEM of four rats.

Figure 2

Comparison of tissue uptake of i.v. injected [³H]ISIS-9388 and [³H]ISIS-3082. Rats were i.v. injected with [³H]ISIS-9388 (open bars) or [³H]ISIS-3082 (closed bars), both at a dose of 1 mg/kg body wt. The distribution of radioactivity over all tissues was determined at 180 and 90 min after injection, respectively. Tissue uptakes are expressed as percentages of amounts of radioactivity cleared from the circulation (88.9 ± 2.3 and 97.9 ± 0.3% of the dose for ISIS-9388 and ISIS-3082, respectively). Values are means ± SEM of three rats.

Figure 3

Liver accumulation of [³H]ISIS-9388 and [³H]ISIS-3082. Rats were i.v. injected with [³H]ISIS-9388 (open circles) or [³H]ISIS-3082 (closed circles), both at a dose of 1 mg/kg body wt. At the indicated times, the amounts of radioactivity in the liver were determined. Values are means ± SEM of three or four rats.

Figure 4

Liver uptake of [³H]ISIS-9388: effects of polyanions. Rats were i.v. injected with [³H]ISIS-9388 at a dose of 1 mg/kg body wt. Shortly (1 min) prior to injection of the labeled ligand, the animals received 10 mg/kg poly(I) (closed circles), 10 mg/kg poly(A) (open circles) or an equal volume (2 ml/kg) of saline solvent (closed squares). At the indicated times, the amounts of radioactivity in the liver were determined. Values are means ± SEM of three or four rats.

Figure 5

Association of ISIS-9388 and ISIS-3082 with plasma components: analysis by size exclusion chromatography. [³H]ISIS-9388 (open circles) and [³H]ISIS-3082 (closed circles) were incubated at 37°C with rat plasma at a concentration of 20 µg/ml. After 30 min, aliquots of the incubation mixtures were subjected to size exclusion chromatography on a Superose 6 column. Fractions of 0.1 ml were collected and were assayed for radioactivity. The results are expressed as percentages of the recovered amounts (recoveries >90%). The elution volumes of LDL, HDL, serum albumin (SA) and free oligodeoxynucleotide (ODN) are indicated by arrows.

Figure 6

Association of ISIS-9388 and ISIS-3082 with LDL, HDL and serum albumin. [³H]ISIS-3082 and [³H]ISIS-9388 (20 µg/ml) were incubated at 37°C with 25 mg/ml rat [¹²⁵I]serum albumin (A and D), 0.2 mg/ml rat [¹²⁵I]LDL (B and E) or 1.0 mg/ml rat [¹²⁵I]HDL (C and F). After 30 min, aliquots of the incubation mixtures were subjected to size exclusion chromatography on a Superose 6 column. The fractions (0.1 ml) were assayed for ³H radioactivity (closed circles) and ¹²⁵I radioactivity (open circles). The results are expressed as percentages of the recovered radioactivity (recoveries >70%).

Figure 7

Interaction of ISIS-9388 with LDL-enriched plasma. [³H]ISIS-9388 (20 µg/ml) was incubated at 37°C with rat plasma supplemented with 1 mg human LDL/ml (A) or with control plasma mixed with phosphate-buffered saline instead of LDL (B). After 30 min, aliquots of the incubation mixtures were subjected to size exclusion chromatography on a Superose 6 column. The fractions were assayed for ³H radioactivity (l) and cholesterol (). Values are expressed as percentages of the recovered amounts (recoveries >75%). The elution volumes of LDL, HDL, serum albumin (SA) and free oligodeoxynucleotide (ODN) are indicated by arrows.

Figure 8

Association of ISIS-9388 and ISIS-3082 with plasma components: analysis by agarose gel electrophoresis. [³H]ISIS-9388 (A) and [³H]ISIS-3082 (B) were incubated at 37°C with rat plasma at a concentration of 20 µg/ml. After 30 min, aliquots of the incubation mixtures were subjected to electrophoresis in a 0.75% (w/v) agarose gel at pH 8.8 (75 mM Tris–hippuric acid buffer). Bromophenol blue was used as the front marker. After electrophoresis, protein in the gel was visualized by Coomassie brilliant blue staining and quantified by scanning the gel. The amounts of protein in the gel () are given in arbitrary units (AU). Slices of the gel were assayed for ³H radioactivity (l) and the amounts of radioactivity are given as percentage of the recovered amount (recoveries >60%). Migration is given relative to the migration of Bromophenol blue (5.0 cm). The migration of free oligodeoxynucleotide (ODN) is indicated by the arrow.