ABSTRACT:The pharmacokinetics of a 2-O-(2-methoxyethyl)-modified oligonucleotide, ISIS 301012 [targeting human apolipoprotein B-100 (apoB-100)], was characterized in mouse, rat, monkey, and human. Plasma pharmacokinetics following parental administration was similar across species, exhibiting a rapid distribution phase with t 1/2␣ of several hours and a prolonged elimination phase with t 1/2␤ of days. The prolonged elimination phase represents equilibrium between tissues and circulating drug due to slow elimination from tissues. Absorption was nearly complete following s.c. injection, with bioavailability ranging from 80 to 100% in monkeys. Plasma clearance scaled well across species as a function of body weight alone, and this correlation was improved when corrected for plasma protein binding. In all of the animal models studied, the highest tissue concentrations of ISIS 301012 were observed in kidney and liver. Urinary excretion was less than 3% in monkeys and human in the first 24 h. ISIS 301012 is highly bound to plasma proteins, probably preventing rapid removal by renal filtration. However, following 25 mg/kg s.c. administration in mouse and 5-mg/kg i.v. bolus administration in rat, plasma concentrations of ISIS 301012 exceeded their respective protein binding capacity. Thus, urinary excretion increased to 16% or greater within the first 24 h. Albeit slow, urinary excretion of ISIS 301012 and its shortened metabolites is the ultimate elimination pathway of this compound, as demonstrated by 32% of dose recovered in total excreta by 14 days in a rat mass balance study. The pharmacokinetics of ISIS 301012 in human is predictable from the pharmacokinetics measured in animals. The pharmacokinetic properties of ISIS 301012 provide guidance for clinical development and support infrequent dose administration.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:The pharmacokinetics of a 2-O-(2-methoxyethyl)-ribose modified phosphorothioate oligonucleotide, ISIS 104838 (human tumor necrosis factor-␣ antisense), have been characterized in mouse, rat, dog, monkey, and human. Plasma pharmacokinetics after i.v. administration exhibited relatively rapid distribution from plasma to tissues with a distribution half-life estimated from approximately 15 to 45 min in all species. Absorption after s.c. injection was high (80-100%), and absorption after intrajejunal administration in proprietary formulations was as high as 10% bioavailability compared with i.v. administration. Urinary excretion of the parent drug was low, with less than 1% of the administered dose excreted in urine after i.v. infusion in monkeys at clinically relevant doses (<5 mg/ kg). ISIS 104838 is highly bound to plasma proteins, likely preventing renal filtration. However, shortened oligonucleotide metabolites of ISIS 104838 lose their affinity to bind plasma proteins. Thus, excretion of radiolabel (mostly as metabolites) in urine (75%) and feces (5-10%) was nearly complete by 90 days. Elimination of ISIS 104838 from tissue was slow (multiple days) for all species, depending on the tissue or organ. The highest concentrations of ISIS 104838 in tissues were seen in kidney, liver, lymph nodes, bone marrow, and spleen. In general, concentrations of ISIS 104838 were higher in monkey tissues than in rodents at body weight-equivalent doses. Plasma pharmacokinetics scale well across species as a function of body weight alone. This favorable pharmacokinetic profile for ISIS 104838 provides guidance for clinical development and appears to support infrequent and convenient dose administration.

In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.

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Improving cellular uptake and biodistribution remains one of the major obstacles for a successful and broad application of peptide nucleic acids (PNAs) as antisense therapeutics. Recently, we reported the identification and functional characterization of an antisense PNA, which redirects splicing of murine CD40 pre-mRNA. In this context, it was discovered that a simple octa(l-lysine) peptide covalently linked to the PNA is capable of promoting free uptake of the conjugate into BCL1 cells as well as primary murine macrophages. On the basis of this peptide motif, the present study aimed at identifying the structural features, which define effective peptide carriers for cellular delivery of PNA. While the structure-activity relationship study revealed some clear correlations, only a few modifications actually led to an overall improvement as compared to the parent octa(l-lysine) conjugate. In a preliminary PK/tissue distribution study in healthy mice, the parent conjugate exhibited relatively broad tissue distribution and only modest elimination via excretion within the time frame of the study.