Introduction

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Active antisense oligonucleotide therapeutic clinical programs now exist for numerous disease states (Crooke, 1996; Dorr and Kisner, 1998; Yacyshyn et al., 1998). Indeed, the first antisense drug targeting viral replication of cytomegalovirus was approved by the Food and Drug Administration in 1998 (Stix, 1998). In all clinical studies reported to date, the routes of administration for antisense oligonucleotides are either local or by intravenous or subcutaneous injection. With few exceptions, the systemically administered oligonucleotides being studied today in the clinic are phosphorothioate oligodeoxynucleotides. Like DNA, these compounds are multiply charged molecules at physiological pH and exhibit high aqueous solubility. Pharmacokinetic studies of this class of compounds indicate that they distribute rapidly and broadly to tissues but do not pass the blood-brain barrier (Cossum et al., 1993, 1994; Agrawal et al., 1995a; Geary et al., 1997; Phillips et al., 1997) and exhibit little to no oral bioavailability (Agrawal and Zhang, 1998; Nicklin et al., 1998).

The methods used for predicting oral bioavailability of organic compounds have been extensively studied and include in vitro (for review, see Artursson et al., 1996), ex vivo (for review, see Barthe et al., 1999), and in situ (Doluisio et al., 1969) approaches. In vitro methods include Caco-2 and intestinal epithelial cell culture permeability assays. These in vitro models of the gastrointestinal barrier have shown great potential for providing rapid throughput screening of multiple compounds. Very limited work has been reported, however, for compounds with molecular weights in excess of 1000 g/mol. Antisense oligonucleotides are polar, charged hydrophilic compounds and as such will not passively diffuse across intact lipid bilayer membranes. It is, therefore, likely that oligonucleotides cross cellular barriers by passing through paracellular junctions and water channels. Tight junctions of cellular monolayers such as provided by the Caco-2 cell monolayer in vitro method are, therefore, not likely to be the optimal model for studying permeability of this chemical class of compounds (Barthe et al., 1999). Furthermore, it is known that cells in culture vary greatly in their ability to take up oligonucleotides and, in fact, do not predict cell uptake in vivo (Crooke et al., 1995). We have, therefore, used in situ whole animal models for initial screening of oligonucleotides and for characterization of relative permeabilities in the rat intestine (Khatsenko et al., 2000). In this study, we report additional characterization of the intestinal bioavailability of oligonucleotides of differing chemistries in more traditional whole animal absolute bioavailability studies compared directly to intravenous injection.

It has been hypothesized that the presystemic instability of the first-generation phosphorothioate oligodeoxynucleotides may contribute to their poor oral bioavailability (Agrawal and Zhang, 1998; Khatsenko et al., 2000). To test this hypothesis, whole animal oral bioavailability studies were used. In this study, we characterized the contribution of permeability, presystemic metabolism and the choice of radiolabel on the ultimate interpretation of in vivo oral bioavailability results for a number of antisense oligonucleotides of differing chemistry.

	Materials and Methods

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Antisense Oligonucleotides. Oligonucleotides were synthesized at Isis Pharmaceuticals, Inc. Radiolabel was incorporated using ³⁵S for ISIS 2302, ISIS 14725, and ISIS 11159 (all contain phosphorothioate backbone). The ³⁵S was incorporated at five nucleotide linkages in from the 5' end to ensure prolonged stability. For ISIS 16952 (PO MOE), a nonexchangeable tritium label was incorporated in the 5' methyl of the tritium base located three bases in from the 5' end. The oligonucleotide sequence and chemistry are detailed in Table 1.

In Vitro Metabolism in Gastrointestinal Tissues. Homogenates were prepared from pancreas, stomach, small intestine, and large intestine of fed and fasted rats using modifications of a procedure described previously for rat liver (Crooke et al., 2000). Briefly, rats were anesthetized with a 50 mg/kg i.p. injection of sodium pentobarbital. Sections of the gastrointestinal tract were removed from animals, rinsed in ice-cold 1× phosphate-buffered saline without calcium and magnesium and placed in a 50-ml polycarbonate centrifuge tube containing an ice-cold buffer consisting of 100 mM Tris-HCl and 1 mM magnesium acetate, pH 8.0 (nuclease buffer). Either pancreas, stomach, small or large intestines were transferred to a large plastic weigh boat on ice with 5 ml of fresh nuclease buffer and minced into 1- to 2-mm pieces. Minced tissue was transferred into 2-ml Fastprep tubes (Bio 101, Inc., Vista, CA) containing 1 ml of cold nuclease buffer and 100 µl of Matrix Green lysing beads (Bio 101, Inc) and homogenized with a Bio 101 Fastprep Savant Tissue/Cell disruptor for 10 to 25 s at an energy setting of 4.5. After homogenization, the tubes were placed in ice, individual homogenates were pooled, and the protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) based on the method of Bradford (1976). The homogenates were diluted to a final total protein concentration of 50 µg/ml and distributed to 2.0-ml microfuges to perform nuclease assays as described below.

In Vivo Stability and Bioavailability in Rats. Sprague-Dawley male rats weighing between 200 and 220 g were surgically modified by implanting an intraduodenal catheter. Animals were maintained under isoflurane gas anesthesia throughout the surgical procedure. To access the stomach, an incision was made on the abdominal midline through the skin at approximately 1 cm immediately caudal to the xyphoid appendix. A medical grade silastic catheter was inserted through a pinhole on the fundus of the stomach. After having routed the catheter through the pylorus to the duodenum, one purse string suture and one mattress suture were used to secure the catheter in place. After exteriorization of the dosing end of the catheter and closure of the abdominal incision, the animal was placed in a jacket to secure the catheter.

Bioanalytical Methods Used for in Vivo Studies. Total radioactivity was measured using liquid scintillation techniques. Plasma and urine were mixed directly with liquid scintillation fluid. Tissues were digested in concentrated (35%) tetraethylammonium hydroxide aqueous solution (tetraethylammonium hydroxide digestion media).

Calculations and Statistics. Absolute bioavailability was calculated by the ratio of the area under the plasma concentration-time curve following intraduodenal administration divided by the intravenous area under the plasma concentration-time curve and corrected for intact oligonucleotide as measured by HPLC and capillary gel electrophoresis as well as dose. Ratio of specific tissue concentrations comparing intraduodenal to intravenous was also used to further clarify the bioavailability of the oligonucleotides from the intestinal administrations. Finally, urine excretion of radiolabel was compared. Descriptive statistics were applied for presentation of the data, including calculation of averages and standard deviation.

	Results

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In Vitro Stability of Oligonucleotides. The most aggressive tissues for degradation of unmodified and modified phosphorothioate oligodeoxynucleotide were pancreas and small intestine (Fig. 1). The stomach and large intestinal tissues were somewhat less aggressive in their ability to degrade PS ODN in vitro. Greater stability in these in vitro matrices was seen for 2'-modified oligonucleotides (Fig. 2). PS ODN 20-mer was approximately 50% degraded by 2 h of incubation with small intestinal tissue, whereas the two 2'-O-MOE modification motifs were still greater than 80% intact at 8 h.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Stability of an unmodified phosphorothioate oligodeoxynucleotide (1 µM) in various fasted digestive tissues homogenates (50 µg of protein/ml) over an 8-h period at 37°C. Pancreas, ; stomach, ; small intestine, ; large intestine, . Values represent the mean ± standard deviation of six to eight replicates from three separate experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Stability of oligonucleotides (1 µM) of varying chemistry and degree of modification in fasted rat small intestine homogenate (50 µg of protein/ml) over an 8-h period at 37°C. PS ODN, ; 3',5' capped PS 2' MOE, ; 3' capped PS 2' MOE (14725 eg.), . Values represent the mean ± standard deviation of six to eight replicates from three separate experiments.

In Vivo Stability of Oligonucleotides. The in vivo rank order of stability for the respective oligonucleotide chemistries was similar to that predicted by the in vitro tissue assays (Fig. 3). Also the in vivo kinetics of degradation in the intestine was similar with approximately 50% of ISIS 2302 (unmodified PS ODN) degraded between 1 and 3 h after administration. By 8 h after administration in the intestine there was no measurable intact ISIS 2302 (PS ODN) remaining. This observation was at a time (8 h) when less than 1% of ISIS 2302 had absorbed into the systemic circulation. The partially modified (3' capped) 2'-O-MOE oligonucleotide (ISIS 14725) was more stable than its unmodified PS ODN analog in vivo with approximately 50% remaining intact at 8 h. Fully modified oligonucleotides were unaffected by the digestive tract with essentially 100% of the compound remaining intact in the intestine as late as 24 h after instillation.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   In vivo stability of varying oligonucleotide chemistry and degree of modification in the rat gastrointestine. Each point is represented as the mean of three separate rats (n = 3). At each time point, rats were sacrificed and the gastrointestinal contents were pooled for analysis by capillary gel electrophoresis.

In Vivo Bioavailability. The total absorption of the ³⁵S radiolabel from the phosphorothioate oligonucleotides was substantially greater than that measured for intact oligonucleotide. Indeed, plasma and urine radiolabel concentrations overestimated the actual bioavailability of intact oligonucleotide for all oligonucleotides studied. Nevertheless, intact oligonucleotide was detected for all chemistries tested both in plasma and in systemic tissue by capillary gel electrophoresis. Plasma concentration of the intact oligonucleotide and radiolabel was observed soon after administration. In general the plasma profile suggested slow and continued absorption of the radiolabel (Fig. 4, all panels). Radiolabel bioavailability was estimated to be 26% for ISIS 2302 (unmodified PS ODN) and 39% for ISIS 14725 (partially modified 2'-O-MOE phosphorothioate oligonucleotide), respectively (Table 2). Total radioactivity represents both intact (parent) oligonucleotide together with any metabolites formed. When bioavailability was corrected for intact oligonucleotide, the absolute fraction absorbed was estimated to be approximately 1% for the phosphorothioate oligodeoxynucleotide (ISIS 2302) and approximately 5% for the partially modified phosphorothioate 2'-O-MOE (ISIS 14725) (Table 3).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Intravenous (3 mg/kg) and intraduodenal (30 mg/kg) plasma concentration radiolabel equivalents of oligonucleotide in plasma of the rat. Each time point represents three to six separate samples (n = 3).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Comparison of intact oligonucleotide plasma concentrations () and radiolabel concentrations (equivalent, ). Intestinal segments were collected from rats 24 h after administration. Values represent the mean ± standard deviation of three separate rats (n = 3).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Local intestine concentrations of oligonucleotide following intravenous or intraduodenal administrations to rats.

	Discussion

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We have shown in this study that although preabsorption metabolism in the intestine likely plays a role in preventing absorption of intact oligonucleotide, it is not the only significant barrier to absorption. Oligonucleotides that have been chemically modified to significantly increase their stability also exhibited low bioavailabilities similar to their less stable analog, a phosphorothioate oligodeoxynucleotide. A partially modified analog sequence that provided improved but not complete protection from nuclease degradation in the intestine exhibited superior bioavailability. These data may suggest that other variables such as oligonucleotide sequence, degree of modification, physical chemistry of the modified oligonucleotide, and secondary structure associated with chemistry and sequence may also play a role in determining optimal absorption for this class of compound. Studies that control for these variables are needed to confirm any dependence of absorption from the intestinal tract on chemical modification design or sequence criteria.

Passive diffusion of small molecules has been extensively studied and the general consensus is clear that there exists a molecular weight cut-off for passive absorption of compounds from the intestine. This cut-off varies somewhat depending on the lipophilicity of the compound, but in general ranges from 550 to 800 g/mol. Antisense oligonucleotides range in length from approximately 15 to 25 nucleotides. The molecular weight range is in excess of 5000 g/mol. In addition, these compounds contain multiple negative charges associated with their phosphorothioate or phosphodiester backbone (pK_a < 3). Although mechanistic reports confirming absorption mechanisms for oligonucleotides have not been forthcoming, it is likely that oral absorption of these compounds occurs via the paracellular pathways. Previous reports have shown that hydrodynamic radius of the cross section of long linear molecules, such as polymers, may play a role in the ultimate uptake from the gastrointestinal tract via water channels and the paracellular pathway (Lane et al., 1996). From these observations one can speculate that controlling for sequence and chemistry modifications that may alter three-dimensional structure of oligonucleotides could play a role in their optimal design for absorption via the paracellular pathway.

We have recently reported additional evidence that oligonucleotides are also taken up into the epithelial cells lining the intestine (Khatsenko et al., 2000). These data suggest that oligonucleotides are able to cross the epithelial cell lipid bilayer, and thus the transcellular component of absorption of these large polar molecules cannot be ruled out. Although it is unclear at this time how much each of these mechanisms of absorption may play a role in their systemic absorption, it is clear from this study that absorption across the gastrointestinal epithelium does occur, but that the rate and extent of absorption are low.

Because this class of compounds is rapidly cleared from plasma and absorption from the intestine is slow and inefficient, it follows that plasma concentrations will be low (Nicklin et al., 1998; Khatsenko et al., 2000). Rapid clearance from plasma is largely attributed to rapid distribution to organs (Cossum et al., 1993, 1994; Agrawal et al., 1995a; Geary et al., 1997; Phillips et al., 1997). However, tissue clearance is relatively slow for phosphorothioate oligodeoxynucleotides with half-lives in excess of 24 h (Cossum et al., 1993; Geary et al., 1997; Levin et al., 1998). Elimination half-lives from tissue are further prolonged (4-10-fold longer) by 2'-ribose-modified oligonucleotides (Bennett et al., 2000). To fully assess the bioavailability of these compounds, concentration of the oligonucleotide in "sentinel" or target tissues is likely to yield a more accurate assessment of their pharmacological bioavailability. It will be important to characterize the relationship between plasma bioavailability and ultimate end-organ bioavailability in nonclinical animal models to allow for rational interpretation of plasma bioavailability in the clinic.

Because measuring total radiolabel in plasma and urine includes inactive metabolites not related to true absorption of the full-length compound, use of radiolabel will overestimate bioavailability of oligonucleotides. For oligonucleotide absorption studies it is, therefore, critical to give careful thought to the placement and type of radiolabel. Furthermore, it is imperative that cold (nonradiolabel) assay methods be incorporated into the accurate assessment of oral bioavailability of this class of compound. Previous reports of high bioavailability for 2'-ribose modified compounds based upon ³⁵S radioactivity are, therefore, likely overestimate the actual bioavailability of the oligonucleotide itself (Agrawal et al., 1995b; Wang et al., 1999).

The concentration of radiolabeled oligonucleotide was found to be manyfold higher in the local intestinal tissue for all oligonucleotide chemistries compared with concentrations in intestine following the intravenous route of administration. These data suggest that local treatment of local gastrointestinal disease may be facilitated by oral administration of more stable, modified oligonucleotides. Alternatively, formulations that deliver unmodified phosphorothioates protected from nuclease digestion and released local to bowel disease may allow for local treatment of the intestine. The concept of treating local disease locally has been further validated by recent reports of successful local antisense treatment directed against the p65 subunit of nuclear factor-B in an experimental colitis model (Neurath et al., 1996).

Improved permeability and stability of these antisense compounds can be gained by chemical alteration. Improved stability alone may not result in improved bioavailability. It is likely that the combination of improved stability and improved permeability is required to impart significantly better absorption characteristics to these compounds. These improvements, combined with formulations that impart permeation enhancement and targeted release to the small intestine, thus bypassing the acidity of the stomach, may provide the best opportunity for clinically useful oral antisense therapeutics.