In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells
In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cellsMartin K. Bijsterbosch*, Muthiah Manoharan1, Erik T. Rump, Remco L. A. De Vrueh, Richard van Veghel, Kathleen L. Tivel1, Erik A. L. Biessen, C. Frank Bennett1, P. Dan Cook1 and Theo J. C. van Berkel
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, PO Box 9503, 2300 RA Leiden, The Netherlands and 1ISIS Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008, USA
Received April 16, 1997; Revised and Accepted July 2, 1997
ABSTRACT
Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3 +- 3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5 +- 1.4% and 17.9 +- 1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1 +- 3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6 +- 4.5 and 4.3 +- 1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.
INTRODUCTION
The technology of cloning and sequencing of pathogenic genes enables specific therapeutic approaches at the level of nucleic acids. Oligonucleotides complementary to sequences in pathogenic genes (antisense oligonucleotides) can hybridize sequence- specifically with these genes or their messenger RNA, and can consequently highly specifically affect the expression of their target genes (1 -4 ). Such a specific inhibition of the expression of pathogenic genes affords an exciting new strategy for therapeutic intervention. An initial drawback for the therapeutic application of the antisense technology was the low stability of unmodified oligonucleotides in a biological environment, due to hydrolysis of their phosphodiester bonds by exo- and endonucleases. To overcome this problem, a wide variety of oligonucleotide analogues with modified internucleoside linkages or modified ribose moieties have been synthesized (2 ,3 ,5 ). These analogues are more resistant to nuclease activity than the unmodified phosphodiester oligonucleotides. Phosphorothioate oligodeoxynucleotides, in which a non-bridging oxygen atom is replaced by a sulfur atom, are the most extensively studied analogues. These compounds are much more stable in a biological environment than phosphodiester oligodeoxynucleotides (1 ,4 ). Numerous studies have demonstrated that phosphorothioate oligodeoxynucleotides can be effective inhibitors of the expression of a broad range of genes, including pathogenic genes such as oncogenes and viral genes (4 ). Further, it was found in animal studies that phosphorothioate oligodeoxynucleotides are well tolerated, and can be therapeutically effective in vivo (7 -11 ). Several phosphorothioate oligodeoxynucleotides have now entered clinical trials (3 ,6 ).
To fully understand the possibilities and limitations of the therapeutic application of phosphorothioate antisense oligodeoxynucleotides, it is necessary to evaluate their in vivo fate. The disposition of phosphorothioate oligodeoxynucleotides has been studied in several species, and the general conclusion from these studies was that the liver is the major site of uptake (reviewed in refs 7 ,8 ). However, the liver contains three major cell types: parenchymal cells, Kupffer cells, and endothelial cells, each equipped with specific receptor systems that are capable of internalizing extracellular ligands (12 ,13 ). The role of these different liver cell types in the clearance of phosphorothioate oligodeoxynucleotides, and the receptor systems involved, has thus far not been studied. The aim of the present study is, therefore, to address these unresolved issues, which are especially relevant for antisense phosphorothioate oligodeoxynucleotides aimed to affect gene expression in the liver. We investigated in rats the disposition of ISIS-3082, a 20mer phosphorothioate oligodeoxynucleotide specific for the murine adhesion molecule ICAM-I (9 ). The results indicate that endothelial cells are mainly responsible for the hepatic uptake of the oligonucleotide, and that uptake is mediated by `scavenger-type' receptors
MATERIALS AND METHODS
Reagents
Polyinosinic acid (5') and polyadenylic acid (5') were from Sigma (St. Louis, MO, USA). Emulsifier SafeTM or Hionic FluorTM scintillation cocktails were from Packard (Downers Grove, IL, USA). All other reagents were of analytical grade.
Oligonucleotide synthesis
ISIS-3082 phosphorothioate oligodeoxynucleotide, sequence 5'-TGCATCCCCCAGGCCACCAT-3', was synthesized as described before (9 ). The oligonucleotide was radiolabeled with 3H by heat-catalyzed exchange at C8 positions of the purine nucleotides as described earlier (14 ). The specific radioactivity of the radiolabeled oligonucleotide was 48.5 * 106 d.p.m./mg. The radiochemical purity, determined by HPLC (see below), was ~90%.
HPLC analysis
The integrity of [3H]ISIS-3082 in stock solutions, plasma samples, and urine was assessed by ion-exchange HPLC, using a Partisil SAX-10 column (0.46 * 25.0 cm; Alltech, Deerfield, IL, USA). The column was eluted at a flow rate of 1 ml/min, using the following mobile phases: buffer A, 0.1 M Tris-HCl (pH 7.4) + 25% (v/v) acetonitrile; buffer B, 0.1 M Tris-HCl (pH 7.4) + 25% (v/v) acetonitrile + 2.5 M KCl. After injection of the samples (0.5 ml), the column was washed for 5 min with buffer A, followed by gradient of 0-100% buffer B (20 min). Subsequently, the column was washed for 50 min with buffer B. The retention time of ISIS-3082 under these conditions was ~57 min. A series of 3'-truncated ISIS-3082 oligodeoxynucelotides were used as standards. It was found that 12-, 14-, 16-, and 18mer oligonucleotides eluted at ~27, 30, 34, and 42 min, respectively. Plasma or urine samples were processed before separation as follows. Aliquots of 0.2 ml were diluted with 0.4 ml H2O, and spiked with 40 [mu]g of authentic ISIS-3082, which served both as carrier to minimize non-specific adsorption and as internal standard. To eliminate non-specific interactions with serum proteins, plasma samples were heated for 2 min at 95oC and then rapidly chilled on ice, immediately before separation (15 ).
Determination of plasma clearance and tissue distribution
Male Wistar rats weighing between 200 and 350 g were used. The animals were anaesthesized by intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight), and the abdomen was opened. Radiolabeled oligonucleotide, dissolved in phosphate- buffered saline (10 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl), was injected via the vena penis (2 ml/kg body weight). At the indicated times, blood samples of 0.2-0.3 ml were taken from the inferior vena cava and collected in heparinized tubes. The samples were centrifuged for 2 min at 16 000 g, and the plasma assayed for radioactivity. The total amount of radioactivity in plasma was calculated using the equation: plasma volume (ml) = [0.0219 * body weight (g)] + 2.66 (16 ). At the indicated times, liver lobules were tied off and excised, and at the end of the experiment the remainder of the liver was removed. The amount of liver tissue tied off successively did not exceed 15% of the total liver mass. The amount of radioactivity in the liver at each time point was calculated from the radioactivities and weights of the liver samples. Uptake by extrahepatic tissues was determined by removing the tissues at the end of the experiment, and counting of radioactivity. Uptake by bone marrow was determined by sampling from femurs and tibiae, and assuming that bone marrow constitutes 1.32 +- 0.14% of the body weight (calculated from data in refs 17 ,18 ). Radioactivity in the tissues was corrected for radioactivity in plasma present in the tissue at the time of sampling (19 ).
Determination of the distribution over liver cell types
Rats were anaesthesized and injected with radiolabeled ISIS-3082 as described above. The liver was perfused at 60 min after injection, and parenchymal, Kupffer and endothelial cells were isolated from the liver as described in detail earlier (20 ). The cell fractions were assayed for radioactivity and protein. Shortly before separation of the cells, a liver lobule was tied off and excised to determine the total liver uptake. The contributions of the various cell types to the total liver uptake was calculated as described previously (20 ). As found with other ligands (16 ,20 ), no significant amounts of radioactivity were lost from the cells during the isolation procedure. This was checked in each experiment by comparing the calculated liver uptake (i.e. the summation of the contributions of the various cell types) with the value actually measured in the liver lobule.
Subcellular fractionation
Rats were anaesthesized and injected with radiolabeled ISIS-3082 as described above. After 60 min, the liver was perfused with ice-cold 0.25 M sucrose + 10 mM Tris-HCl (pH 7.4), and subsequently divided into subcellular fractions as described previously (21 ). In brief, the liver was homogenized in sucrose/Tris-HCl (see above) using a homogenizer of the Potter-Elvehjem type. Fractions enriched in nuclei, mitochondria, lysosomes and microsomes were obtained by collecting pellets obtained after consecutive centrifugation steps of 5 min at 1200 g, 5 min at 9000 g, 15 min at 22 000 g and 30 min at 210 000 g, respectively (g forces in middle of tubes). The final supernatant was the cytosol fraction. The fractions were assayed for the activity of marker enzymes as described earlier (21 ). Radioactivity and protein were determined as described below.
Determination of proteins
Protein concentrations in cell suspensions and subcellular fractions were determined by the method of Lowry et al. (22 ), with a bovine serum albumin standard.
Determination of radioactivity
Liquid samples were counted without further processing by liquid scintillation spectroscopy, using Emulsifier SafeTM or Hionic FluorTM scintillation cocktails. Tissue samples were processed using a Packard 306 Sample Oxidizer. Some tissues (e.g. bone) were dissolved in 10 M NaOH at 95oC before counting. All radioactivity measurements were performed in a Packard Tri-Carb 1500 liquid scintillation counter.
RESULTS
Plasma clearance and tissue uptake of ISIS-3082
The disposition of ISIS-3082 was studied after a bolus injection of the radiolabeled oligonucleotide into rats. The administered amount, 1 mg/kg body weight, was in the range of phosphorothioate oligodeoxynucleotide doses that have been found to be effective in vivo (9 -11 ). Figure 1 shows that after an initial rapid distribution phase (distribution volume 63.9 +- 9.3 ml/kg body weight), radioactivity was cleared from the circulation with a half-life of 23.3 +- 3.8 min (means +- S.E.M. of four rats). HPLC-analysis of plasma samples indicated that at 60 and 90 min after injection >85% and >75% of the radioactivity, respectively, represented oligonucleotide that was at least 70% full length. At 90 min after injection, when >90% of the injected dose had been cleared from the circulation, the distribution of the radioactivity over the body was determined (Table 1 ). A small amount of the administered radioactivity (2.5 +- 0.4% of the dose) was recovered in the urine. HPLC analysis indicated that the radioactivity in urine represented degradation products. The liver, which contained 40.5 +- 1.4% of the dose, was found to be mainly responsible for the clearance of the oligonucleotide. Spleen and bone marrow, which constitute with the liver the reticuloendothelial system (RES), contained smaller amounts of radioactivity (0.7 +- 0.1 and 6.7 +- 0.7% of the dose, respectively). The kidneys contained the second highest amount of radioactivity (17.9 +- 1.3% of the dose), and the remainder of the radioactivity was distributed over a large number of tissues. Table 1 only lists tissues containing >1% of the dose. Bulky tissues, like muscle and skin contained 5.5 +- 0.8 and 8.4 +- 1.7% of the dose, respectively. The carcass contained a substantial amount of radioactivity (10.4 +- 0.5% of the dose), which is mainly present in bone marrow (see above). The specific uptake, expressed as relative specific radioactivity, was highest in the kidneys and in the RES organs liver, spleen, and bone marrow.
. Tissue distribution of intravenously injected [3H]ISIS-3082
Tissue
Radioactivity (% of recovered)
Relative specific radioactivity
Blood plasma
2.1 +- 0.3
Urine
2.5 +- 0.4
Liver
40.5 +- 1.4
8.1 +- 0.2
Spleen
0.7 +- 0.1
3.3 +- 0.4
Bone marrow
6.7 +- 0.7
4.7 +- 0.5
Kidneys
17.9 +- 1.3
22.4 +- 2.0
Intestines
6.4 +- 0.6
1.0 +- 0.1
Pancreas
2.2 +- 4.4
2.2 +- 0.3
Muscles
5.5 +- 0.8
0.2 +- 0.0
Skin
8.4 +- 1.7
0.5 +- 0.1
Carcass (including marrow)
10.4 +- 0.5
0.5 +- 0.0
All other tissues
3.3 +- 0.1
0.3 +- 0.0
Rats were intravenously injected with [3H]ISIS-3082 at a dose of 1 mg/kg body weight. At 90 min after injection, the distribution of radioactivity over all tissues was determined. The results are expressed as percent of the recovered amount of radioactivity and as relative specific activity (percent recovered radioactivity divided by percent recovered weight). Recoveries of radioactivity and tissues were 84.6 +- 4.2% and 94.1 +- 0.7%, respectively. Only tissues containing >1.0% of the recovered dose and/or a relative specific radioactivity >2.0 are listed. Values are means +- S.E.M. of three rats.
Dose-dependence of plasma clearance and tissue uptake of ISIS-3082
The high specific uptake of ISIS-3082 by the RES organs and kidneys points to a selective uptake mechanism, possibly receptor-mediated endocytosis. As receptor-mediated clearance mechanisms are saturable, we investigated the effect of the injection of different doses of ISIS-3082. Figure 2 compares the plasma clearance of radioactivity after injection of [3H]ISIS-3082 at a dose of 0.05 mg/kg or 1.0 mg/kg. After injection of 0.05 mg of ISIS-3082/kg body weight, radioactivity was cleared from the circulation with a half-life of 13.1 +- 2.0 min, which is significantly (P <0.05) faster than observed after injection of the oligonucleotide at a dose of 1.0 mg/kg (half-life 23.3 +- 3.8 min). The distribution volume was not significantly different (83.2 +- 13.0 ml/kg versus 63.9 +- 9.3 ml/kg). The rate of uptake by the liver, being the most important organ for disposition, was also investigated. Figure 3 shows that the hepatic uptake of [3H]ISIS-3082 proceeded more rapidly at a dose of 0.05 mg/kg than at 1.0 mg/kg. The dose-dependence of extrahepatic uptake of [3H]ISIS-3082 was also investigated (Fig. 4 ). As the sampling times and rates of plasma clearance are different for the two different doses, the uptake of the oligonucleotide is expressed as uptake index, which allows the most accurate comparison. The uptake indexes of liver, spleen, bone marrow, and kidneys were significantly lower at a dose of 1.0 mg/kg than at 0.05 mg/kg, which indicates that the uptake systems in these organs are saturable. The uptake indexes of muscle, skin and intestine were dose-independent and thus uptake in these organs is, at least in the range of 0.05-1.0 mg/kg, not saturable.
ACKNOWLEDGEMENT
Part of this work was supported by a grant from the European Commission (Biomed 2 programme; grant PL 951439).
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