The in vivo pharmacokinetics/pharmacodynamics of 2'-O-(2-methoxyethyl) (2'-MOE) modified antisense oligonucleotides (ASOs), targeting apolipoprotein B-100 (apoB-100), were characterized in multiple species. The species-specific apoB antisense inhibitors demonstrated target apoB mRNA reduction in a drug concentration and time-dependent fashion in mice, monkeys, and humans. Consistent with the concentration-dependent decreases in liver apoB mRNA, reductions in serum apoB, and LDL-C, and total cholesterol were concurrently observed in animal models and humans. Additionally, the long duration of effect after cessation of dosing correlated well with the elimination half-life of 2'-MOE modified apoB ASOs studied in mice (t(1/2) congruent to 20 days) and humans (t(1/2) congruent to 30 days) following parental administrations. The plasma concentrations of ISIS 301012, observed in the terminal elimination phase of both mice and monkeys were in equilibrium with liver. The partition ratios between liver and plasma were similar, approximately 6000:1, across species, and thus provide a surrogate for tissue exposure in humans. Using an inhibitory E-max model, the ASO liver EC50s were 101 +/- 32, 119 +/- 15, and 300 +/- 191 mu g/g of ASO in high-fat-fed (HF) mice, transgenic mice containing the human apoB transgene, and monkeys, respectively. The estimated liver EC50 in man, extrapolated from trough plasma exposure, was 81 +/- 122 mu g/g. Therefore, extraordinary consistency of the exposure-response relationship for the apoB antisense inhibitor was observed across species, including human. The cross-species PK/PD relationships provide confidence in the use of pharmacology animal models to predict human dosing for second-generation ASOs targeting the liver. (C) 2008 Elsevier Inc. All rights reserved.

Due to its ability to esterify cholesterol in hepatocytes and enterocytes, ACAT2 is believed to promote atherosclerosis by driving both cholesterol absorption in the intestine and promoting packaging of cholesterol into VLDL in the liver. The purpose of these studies was to determine how liver-specific depletion of ACAT2 via in vivo antisense oligonucleotide (ASO)-mediated inhibition alters hepatic cholesterol and lipoprotein metabolism in a hyperlipidemic mouse model. Liver-specific depletion (>85%) of ACAT2 resulted in a dramatic reduction in the packaging of cholesterol into apoB-containing lipoproteins (VLDL, IDL, and LDL), while HDL cholesterol levels were not affected. In the liver of ACAT2-ASO treated mice, total (TC), esterified (CE), free (FC) cholesterol and triglyceride (TG) concentrations all were decreased when compared to control mice. Furthermore, cholesterol, phospholipid, and bile acid concentrations in gall bladder bile were not altered in ACAT2-ASO treated mice. Interestingly, during isolated liver perfusion, ACAT2-ASO treated livers had augmented perfusate accumulation rates of FC and phospholipid (PL). Collectively, these studies provide the first insight into the hepatic itinerary of cholesterol, when cholesterol esterification is specifically inhibited within the liver.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human.

A number of proinflammatory cytokines, including IL-1β, signal through the adaptor protein MyD88. This signaling leads to phosphorylation of IL-1R-associated kinase-1 (IRAK-1) and, ultimately, activation of the NF-κB transcription factor. A splice variant of MyD88 (MyD88S), which lacks the ability to couple IRAK-1 to NF-κB, has been described. A chemically modified antisense oligonucleotide (ASO) that alters the splicing ratio of MyD88 to MyD88S in both cell culture and in animals has been identified. The ASO (ISIS 337846) binds to exon II donor sites in the MyD88 pre-mRNA. By manipulating levels of MyD88 splicing, proinflammatory signaling through the IL-1R has been shown to be diminished, both in cell culture and in mouse liver. To our knowledge, this represents the first example of modulation of RNA splicing of an endogenous gene target in animals after systemic ASO dosing and suggests that this mechanism may be useful as a novel modulator of inflammatory stimuli.

s presented at the 7th Annual Conference on Arteriosclerosis, Thrombosis and Vascular Biology

The pharmacokinetics of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, were characterized within rodent whole liver, and cellular and subcellular compartments. Cross-species comparisons were performed using Sprague-Dawley rat and CD-1 mouse strains. Although whole liver oligonucleotide deposition and the proportion of drug found within parenchymal and nonparenchymal cells were similar between the two rodent species as a function of both time and dose, dramatic differences in subcellular pharmacokinetics were observed. Specifically, within murine hepatocyte nuclei, drug was observed at the 10 mg/kg dose, whereas in the rat nuclear-associated levels required the administration of 25 mg/kg. Under all experimental regimens, murine hepatic nuclear-associated drug concentrations were at least 2-fold higher than those found in rat liver cells. More detailed metabolic analysis was also performed using high performance liquid chromatography/electrospray-mass spectrometry (HPLC/ES-MS) and demonstrated that although the extent of metabolism was similar for rat and mouse, the pattern of n-1 metabolites varied as a function of both species and cell type. While rat and mouse hepatocytes and rat nonparenchymal cellular metabolites were predominantly products of 3′-exonuclease degradation, mouse nonparenchymal cells contained a majority of n-1 metabolites produced by 5′-exonucleolytic activity. Based upon these data, it would appear that subcellular oligonucleotide disposition and metabolism among rodent species are more divergent than whole organ pharmacokinetics might predict.