Pharmacokinetics Of Oligonucleotides

A. Cellular Pharmacokinetics

Cellular uptake of phosphorothioate oligonucleotides has been documented to occur in most mammalian cells (96-103). Cellular uptake of oligonucleotide is time and temperature dependent. It is also influenced by cell type, cell culture conditions and media, and the length/sequence of the oligonucleotide itself (96). No obvious correlation between the lineage of cell, whether the cells are transformed or virally infected, and uptake has been identified. Cellular uptake appears to be an active process (i.e., oligonucleotide) will accumulate in greater concentration intracellular than in the medium and is energy dependent. Despite the fact that mammalian cells in culture will readily accumulate oligonucleotides, it has been necessary to further facilitate cytosolic delivery for many, but not all, cells with transfection agents such as cationic lipids, dendrimers, fu-sogenic peptides, electroporation, etc., (38,46,104-108). In the absence of these facilitators, it has been difficult to demonstrate true antisense effects in cultured cells., although there are some exceptions. However, in vivo, this is not the case. It has become apparent that in vitro cell uptake studies do not predict in vivo cell uptake and pharmacokinetics of oligonucleotides (96,109-113). Our understanding of cellular and subcellular uptake has evolved as superior analytical tools have been developed. These advances include development of immunohisto-chemical techniques use oligonucleotide-specific antibodies (114), and in situ perfusion of whole organs followed by cell sorting and subcellular separation techniques coupled with capillary gel electrophoresis (110).

Our understanding of cellular and subcellular distribution and pharmacokinetics of oligonucleotides in whole animals is emerging. In our laboratories, we use more specific tools for qualification and even quantification of intact oligonucleotide (110,114-116). Phosphorothioate oligonucleotides rapidly distribute to whole tissue with distribution half-lives range from 30 to 60 min in vivo. Approximately half of the oligonu-cleotide associated with the liver (as an example) is intracellular in both parenchymal and nonparenchymal cells by 4 h after intravenous administration (110,117). The other half of the organ-associated oligonucleotide appears to be associated with extracellular matrix or interstitium, or loosely bound to the cell membrane. Consistent with this observation, others have shown that phosphorothioates have been localized to connective tissue and can bind to various proteins within these matrices, such as laminin and fibronectin (114,118,119). Some of this matrix- associated oligonucleotide will diffuse to cells over time or be lost to efflux from the organ (114). It is likely that both of these processes are functioning up to 24 h after administration of oligonucleotide. By 24 h after injection of phosphorothioate oligonucleotide, little is seen to be associated with extracellular matrix (114). Thus, it is likely that whole organ pharmacokinetic evaluation after 24 h will parallel cellular clearance kinetics.

Although the in vitro studies fail to predict well which cell types will take up oligonucleotide in vivo, the general trend of variability from cell type to cell type continues to be observed in vivo (114). Based on these results, one would not expect to uniformly inhibit expression of a targeted gene product within a tissue or whole organism, resulting in differential sensitivity of different tissues and cells within tissue to the antisense effect. Subcellular distribution has been shown to be broad, and the extent of cytosolic and nuclear distribution differs between cells (110). In general, the total number of oligonucleotide molecules is greatest in the cytosol. However, because of the much smaller volume of the nucleus, the nucleus may often contain a higher concentration of oligonucleo-tide than the cytosol.

Nuclease metabolism has been shown to account for the clearance of phosphorothioate oligonucleotide from organs of distribution. Within the cells, the pattern of metabolites appears to be quite similar between cell types and the subcellular compartments (membrane associated, cytosolic, and nuclear). Increasing doses from 5 to 50 mg/kg only moderately decreased metabolism intracellularly, consistent with whole organ data (110).

Several studies have suggested that active uptake processes, including receptor-mediated endocytosis and pino-cytosis, are involved in uptake of oligonucleotides in vivo. At very low doses (less than 1 mg/kg), competition of binding for scavenger receptors in vivo altered the whole organ distribution of oligonucleotides in liver but not in kidney (120-122). However, distribution studies conducted in scavenger receptor knockout mice did not show significantly altered intracellular and whole organ distribution of phosphorothioate oligonucleotides (123).

Distribution in the kidney has been more thoroughly studied, and drug has been shown to be present in Bowman's capsule, the proximal convoluted tubule, the brush border membrane, and the renal tubular epithelial cells (114,124). These data suggested that the oligonucleotides are filtered by the glomerulus and then reabsorbed by the proximal convoluted tubule epithelial cells. Moreover, the authors identified a specific protein in the brush border that may mediate uptake. In subsequent studies, the authors have purified the 45-kDa protein, reconstituted it in phospholipid vesicles and demonstrated that it served as a channel allowing nucleic acid to pass through phospholipid bilayers (125). In separate studies, other investigators have shown that, although some oligonu-cleotide is taken up from the tubular lumen brush border, the distribution to the tubule epithelial cells is predominantly from the capillary serosal side (126). The uptake from capillary circulation may not be receptor mediated. In summary, it is likely that there are multiple processes involved in the uptake of oligonucleotides into cells in vivo. Additional research will be required to further elucidate these mechanisms.

B. Whole Animal Oligonucleotide Pharmacokinetics

1. Phosphorothioate Oligodeoxynucleotides

The plasma pharmacokinetics of phosphorothioate oligodeoxynucleotides are characterized by rapid and dose-dependent clearance (30-60 min half-life) driven primarily by distribution to tissue and secondarily by metabolism. Urinary and fecal excretion are minor pathways for elimination of phos-phorothioate oligonucleotides. Dose-dependent clearance from plasma is predominantly a function of saturable tissue distribution (127,128). Metabolism has been shown to be unchanged in plasma over a large dose range (1-50 mg/kg) and after repeated administration up to 1 month, suggesting that metabolism is neither inhibited or induced by repeat administration (129).

The plasma pharmacokinetics are quite similar between animals and man, and they scale from one species to the next on the basis of body weight, not surface area (129-133). For example, it is possible to show that, when dosed on the basis of body weight, the concentrations of oligonucleotides in plasma administered by a 2-h constant intravenous infusion are similar between humans and monkeys. Thus, it has been possible to predict plasma concentrations in humans from nonclinical pharmacokinetic data.

Phosphorothioate oligonucleotides bind to circulating plasma proteins such as albumin and a-2 macroglobulin (134). The apparent affinity for human serum albumin is low (10-30 ^M). Therefore, plasma protein binding provides a repository for these drugs preventing rapid renal excretion. Because serum protein binding is saturable at high concentrations, intact oligonucleotide may be found in urine in increasing amounts as dose rate and/or amount is increased (129, 135,136).

Phosphorothioate oligonucleotides are rapidly and extensively absorbed after intradermal, subcutaneous, intramuscular, or intraperitoneal administration (109,127,137,138). Non-parenteral absorption has been characterized for pulmonary and oral routes of administration. Estimates of bioavailability range from 3% to 20% following intranasal dosing and < 1% by the oral route (139,140). Although it is likely that permeability in the intestine is low, stability of these compounds in the intestine (prior to absorption) may be a rate-limiting factor to oral absorption (141,142). As discussed below, somechem-ical modifications to the oligonucleotide enhance oral absorption. The metabolic half-life of a 20-mer phosphorothioate oligonucleotide in the rat intestine (in vivo) is less than 1 h (data shown in Section VI).

Phosphorothioates are broadly distributed to all peripheral tissues. Highest concentrations of oligonucleotides are found in the liver, kidney, spleen, lymph nodes, and bone marrow with no measurable distribution to the brain (109,127,129, 135,141). Many other tissues take up smaller amounts of oligonucleotide, resulting in lower tissue concentrations. Phos-phorothioate oligonucleotides are primarily cleared from tissues by nuclease metabolism. Rate of clearance differs between tissues with the spleen, lymph nodes, and liver, generally clearing more rapidly than kidney, for example. In general, the clearance rates result in half-lives of elimination ranging from 2 to 5 days in rodents and primates (128,133).

In summary, pharmacokinetic studies of phosphorothioate oligonucleotides demonstrate that they are well absorbed from parenteral sites, distribute broadly to all peripheral tissues, do not cross the blood-brain barrier, and are eliminated primarily by slow metabolism. In short, once-a-day or every-other-day systemic dosing should be feasible. In general, the pharmaco-kinetic properties of this class of compounds appear to be largely driven by chemistry rather than sequence. Additional studies are required to determine whether there are subtle sequence-specific effects on the pharmacokinetic profile of this class of drugs.

2. Second-generation Oligonucleotides

The plasma pharmacokinetics of 2'-O-methyl-, 2'-O-propyl-, or 2'-O-methoxyethyl-modified oligonucleotides do not differ significantly from their oligodeoxynucleotide congeners (79,80,143,144). Because metabolism plays only a minor role in the plasma distribution kinetics, this modification is expected to do little to alter the distribution and excretion kinetics. Early studies in our laboratory indicate that the binding affinity to serum albumin may be somewhat lessened by 2 -ribose sugar modifications, but the overall capacity of the plasma proteins to bind these oligonucleotides is not significantly changed (Table 3). Therefore, urinary excretion remains a minor route of elimination, and these compounds are broadly distributed to peripheral tissues.

Several of the 2'-ribose sugar modification produces enough of an increase in nuclease resistance that it is possible

Table 3 Serum Albumin Affinity, Whole Plasma Fraction Bound to Proteins (Fb), and Fraction of Dose Excreted in Urine (/excreted, 0-24 h) Following Intravenous Administration at 3 mg/kg—Comparison of First- and Second-Generation Chemistries

Compound no. Chemistry Kd (^M) Fb m /excreted

ISIS 2302 PS ODNa 17.7 99.2 0.003

ISIS 11159 PS 2'-MOEb 29.3 95.5 0.032

aPS ODN, phosphorothioate oligodeoxynucleotide.

bPS 2'-MOE, 2'-O-methoxyethyl ribose modified phosphorothioate (all nucleotides were modified). cPO 2'-MOE, 2'-O-methoxyethyl ribose modified phosphodiester (all nucleotides were modified).

to produce relatively stable oligonucleotides with phos-phodiester linkages (Table 2). Thus, this modification allows for elimination or reduction in the number of sulfurs contained in the internucleotide bridge, but these compounds are less stable than their 2'-modified phosphorothioate congeners (145). In addition, as sulfur is removed, plasma protein binding is greatly decreased and rapid removal from plasma by filtration in the kidney increases significantly. This pharmaco-kinetic characteristic may limit the use of phosphodiester second-generation modified oligonucleotides intended for treatment of systemic disease (79). Alternatively, this pharma-cokinetic profile may be ideal for locally administered oligo-nucleotides because it limits the accumulation of systemically absorbed drug.

Absorption for parenterally administered modified oligo-nucleotides is consistently rapid and nearly complete. Some of the second-generation modified oligonucleotides have exhibited improved intestinal permeability (141) as well as significantly improved stability in the intestine (142). It is likely this combination of improved biochemical characteristics have led to the observation of improved oral bioavailability (141) for this class of oligonucleotide compounds.

The distribution pattern of the 2'-ribose-modified phospho-rothioate oligonucleotides are similar to first-generation phos-phorothioates and similarly not altered by changes in sequence. Kidney, liver, spleen, bone marrow, and lymph nodes are the major sites of distribution. The most exciting difference in pharmacokinetics is, not surprisingly, manifested in prolonged terminal elimination half-lives from tissues of distribution. The elimination half-lives appear to be increased nearly 5 to 10-fold, suggesting that once-weekly systemic dosing may be feasible (Table 4).

In summary, pharmacokinetic studies of 2'-modified ribose phosphorothioate oligonucleotides demonstrate that they are well absorbed from parenteral sites, may have improved oral absorption attributes, and distribute broadly to all peripheral tissues. Although stability has been greatly enhanced, nuclease metabolism is likely the primary mechanism for ultimate elimination of these modified oligonucleotides. In short, once-a-week systemic dosing should be feasible and oral administration may be possible in the near future. Additional studies are

Table 4 Summary of Observed Organ Clearance Half-lives (in days) Comparing Second- and First-Generation Chemistries
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