A number of 2'- O-modified antisense oligonucleotides have been reported for their potential use in oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2'-O-MOE (2'-O-methoxyethyl)- and 2'-O-Me (2'-O-methyl)-modified ASOs (antisense oligonucleotides). We now report the synthesis and biological activity of another 2'-O-modification, namely 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA). This modification resulted in an increase in the affinity of antisense oligonucleotides to complementary RNA similar to 2'-O-MOE-modified ASOs as compared to first-generation antisense oligodeoxynucleotides. The ASO modified with 2'-O-NMA reduced expression of PTEN mRNA in vitro and in vivo in a dose-dependent manner similar to 2'-O-MOE modified ASO. Importantly, toxicity parameters such as AST, ALT, organ weights, and body weights were found to be normal similar to 2'-O-MOE ASO-treated animal models. The data generated in these experiments suggest that 2'-O-NMA is a useful modification for potential application in both antisense and other oligonucleotide-based drug discovery efforts.

Abstract-Cholesterol oxidation products (ChOx) have been reported to cause acute vascular injury in vivo; however, the pharmacokinetics of ChOx after administration and the mechanisms by which they cause chronic vascular injury are not well understood. To further study the pharmacokinetics and atherogenic properties of ChOx, New Zealand White rabbits were injected intravenously (70 mg per injection, 20 injections per animal) with a ChOx mixture having a composition similar to that found in vivo during a 70-day period. Total ChOx concentrations in plasma peaked almost immediately after a single injection, declined rapidly, and returned to preinjection levels in 2 hours. After multiple injections, the ChOx concentrations rose gradually to levels 2-to 3-fold above baseline levels, increasing mostly in the cholesteryl ester fraction of LDL and VLDL. Rabbit serum and the isolated LDL/VLDL fraction containing elevated ChOx concentrations were cytotoxic to V79 fibroblasts and rabbit aortic endothelial cells. At the time of killing, cholesterol levels in the aortas from ChOx-injected rabbits were significantly elevated despite the fact that plasma cholesterol levels remained in the normal range. In addition, aortas from the ChOx-injected rabbits retained more 125 I-labeled horseradish peroxidase, measured 20 minutes after intravenous injection. Transmural concentration profiles across the arterial wall also showed increased horseradish peroxidase accumulation in the inner half of the media from the thoracic aorta in ChOx-injected rabbits. In conclusion, ChOx injection resulted in accumulation of circulating ChOx and induced increased vascular permeability and accumulation of lipids and macromolecules. This study reveals that even under normocholesterolemic conditions, ChOx can cause endothelial dysfunction, increased macromolecular permeability, and increased cholesterol accumulation, parameters believed to be involved in the development of early atherosclerotic lesions.

Differences in sensitivity of monkeys and humans to antisense oligonucleotide (ASO)-induced complement alternative pathway (AP) activation were evaluated in monkeys, humans, and in serum using biochemical assays. Transient AP activation was evident in monkeys at higher doses of two 29-O-methoxyethyl (29-MOE) ASOs (ISIS 426115 and ISIS 183750). No evidence of AP activation was observed in humans for either ASO, even with plasma ASO concentrations that reached the threshold for activation in monkeys. The absence of complement activation in humans is consistent with a query of the Isis Clinical Safety Database containing 767 subjects. The in vivo difference in sensitivity was confirmed in vitro, as monkey and human serum exposed to increasing concentrations of ASO indicated that monkeys were more sensitive to AP activation with this class of compounds. The mechanistic basis for the greater sensitivity of monkeys to AP activation by 29-MOE ASO was evaluated using purified human or monkey factor H protein. The binding affinities between a representative 29-MOE ASO and either purified protein are similar. However, the IC 50 of fluid-phase complement inhibition for monkey factor H is about 3-fold greater than that for human protein using either monkey serum or factor H-depleted human serum. Interestingly, there is a sequence variant in the monkey complement factor H gene similar to a single nucleotide polymorphism in humans that is correlated with decreased factor H protein function. These findings show that monkeys are more sensitive to 29-MOE ASO-mediated complement activation than humans likely because of differences in factor H inhibitory capacity.