Michael V. Templin

Correlation of Toxicity and Pharmacokinetic Properties of a Phosphorothioate Oligonucleotide Designed to Inhibit ICAM-1

ISIS 2302 is a phosphorothioate oligodeoxynucleotide with a sequence complementary to the mRNA of human intercellular adhesion molecule 1 (ICAM-1). Hybridization of ISIS 2302 to the mRNA inhibits expression of the ICAM-1 protein in response to inflammatory stimuli. A murine active antisense oligonucleotide, ISIS 3082, has been used for in vivo pharmacology studies and has anti-inflammatory activity in models of organ transplant rejection, ulcerative colitis, and collagen-induced arthritis at doses ranging from 0.03 to 5 mg/kg. The safety assessment for ISIS 2302 includes general toxicity studies up to 6 mo in duration in mice and monkeys, genetic toxicity studies, and reproductive/fertility studies. ISIS 3082 was examined in parallel with ISIS 2302 in mouse toxicity and reproductive studies. The toxicities observed following systemic administration of ISIS 2302 and ISIS 3082 were similar and consistent with those observed for other compounds in this chemical class and, therefore, are independent of the suppression of ICAM-1 expression. Toxicokinetic evaluation demonstrated that toxicities occurred in organs containing the highest concentrations of ISIS 2302. Evidence of immune stimulation, including dose-dependent splenomegaly, lymphoid hyperplasia, and multiorgan mixed mononuclear cell infiltrates, was the most common finding in rodent studies. Monkeys were much less sensitive than mice to immune stimulation. Kidney contained the highest concentrations of ISIS 2302. Morphologic changes observed in kidney included atrophic and regenerative changes in proximal tubular epithelium; however, there was no evidence of functional abnormalities. Additional histologic changes noted in proximal tubular epithelium included basophilic granules, which were reflective of oligonucleotide distribution and uptake in these cells. Liver also contained high concentrations of oligonucleotide, which were associated with Kupffer cell hypertrophy in mice. Changes in serum transaminases, cholesterol, and triglycerides were reflective of hepatic alterations. In monkeys, high concentrations of oligonucleotide caused a transient increase in clotting times and activation of the alternative complement pathway. All toxicities associated with ISIS 2302 were reversible and occurred at doses well above those required for pharmacologic activity or currently used in clinical trials. In addition, there has been no evidence of genetic toxicity associated with ISIS 2302, and no changes in reproductive performance, fertility, or fetal development have been noted in animals treated with ISIS 2302 or ISIS 3082.

Comparison of pharmacokinetics and tissue disposition of an antisense phosphorothioate oligonucleotide targeting human Ha-ras mRNA in mouse and monkey

The plasma pharmacokinetics and tissue disposition of ISIS 2503 were studied in mice following single and multiple bolus intravenous (iv) injections of 1-50 mg/kg, and in monkeys following single and multiple 2-h iv infusions of 1-10 mg/kg and bolus iv injections of 1 mg/kg of ISIS 2503. ISIS 2503 and its metabolites were measured in plasma, urine, and tissues using solid-phase extraction followed by capillary gel electrophoresis (CGE). In both species, the plasma clearance of ISIS 2503 was characterized by rapid distribution to tissues, and to a lesser extent, metabolism. The plasma clearance in mice was at least two-fold more rapid than in monkeys at equivalent doses. The plasma disposition (t1/2) increased with dose. The highest concentrations of oligonucleotide were consistently observed in the kidney and liver in both species. At equivalent doses, tissue concentrations in monkeys were much higher than tissue concentrations in mice. Urinary excretion of total oligonucleotide was a minor elimination pathway in both species at doses < 10 mg/kg. However, urinary excretion of total oligonucleotide in mice was increased to 12-29% as dose increased from 20 to 50 mg/kg.

Pharmacokinetics and Tissue Disposition in Monkeys of an Antisense Oligonucleotide Inhibitor of Ha-Ras Encapsulated in Stealth Liposomes

AbstractPurpose. This study examined the pharmacokinetics and tissue distribution of an antisense oligonucleotide ISIS 2503, formulated in stealth (pegylated) liposomes (encapsulated) or in phosphate-buffered saline (unencapsulated). Methods. Encapsulated or unencapsulated ISIS 2503 was administered to rhesus monkeys by intravenous infusion. The concentrations of ISIS 2503 and metabolites in blood, plasma, and tissue samples were determined by capillary gel electrophoresis. Results. Plasma concentrations of encapsulated ISIS 2503 decreased mono-exponentially after infusion with a mean half-life of 57.8 hours. In contrast, the concentration of unencapsulated ISIS 2503 in plasma decreased rapidly with a mean half-life of 1.07 hours. Both encapsulated and unencapsulated ISIS 2503 distributed widely into tissues. Encapsulated ISIS 2503 distributed primarily to the reticulo-endothelial system and there were few metabolites observed. In contrast, unencapsulated ISIS 2503 distributed rapidly to tissue with highest concentration seen in kidney and liver. Nuclease-mediated metabolism was extensive for unencapsulated oligonucleotide in plasma and tissues. Conclusions. The data suggest that stealth liposomes protect ISIS 2503 from nucleases in blood and tissues, slow tissue uptake, and slow the rate of clearance from the systemic circulation. These attributes may make these formulations attractive for delivering oligonucleotides to sites with increased vasculature permeability such as tumors or sites of inflammation.

Local and systemic tolerability of a 2′O-methoxyethyl antisense oligonucleotide targeting interleukin-4 receptor-α delivery by inhalation in mouse and monkey

Abstract Antisense oligonucleotides (ASOs) bind and facilitate degradation of RNA and inhibit protein expression in pathways not easily targeted with small molecules or antibodies. Interleukin (IL)-4 and IL-13 potentiate signaling through the shared IL-4 receptor-α (IL-4Rα) subunit of their receptors. ASO targeting of IL-4Rα mRNA in a mouse model of asthma led to attenuation of airway hyperactivity, demonstrating potential benefit in asthma patients. This study focused on tolerability of inhaled IL-4Rα-targeting ASOs. Toxicity studies were performed with mouse- (ISIS 23189) and human-specific (ISIS 369645) sequences administered by inhalation. Four week (monkey) or 13 week (mouse) repeat doses at levels of up to 15 mg/kg/exposure (exp) and 50 mg/kg/exp, respectively, demonstrated dose-dependent effects limited to increases in macrophage size and number in lung and tracheobronchial lymph nodes. The changes were largely non-specific, reflecting adaptive responses that occur during active exposure and deposition of ASO and other material in the lung. Reversibility was observed at a rate consistent with the kinetics of tissue clearance of ASO. Systemic bioavailability was minimal, and no systemic toxicity was observed at exposure levels appreciably above pharmacological doses and doses proposed for clinical trials.