Poster P170 4/28/11
ANTISENSE OLIGONUCLEOTIDES TARGETING SR-BI AND CD36: CONCENTRATION, DISTRIBUTION, AND ACTIVITY WITHIN ATHEROSCLEROTIC TISSUE
Aaron J Donner, Wuxia Fu, Mark J Graham, Rosanne M Crooke, Adam E Mullick, ISIS Pharmaceuticals, Carlsbad, CA
In order to quantify the accumulation, distribution and activity of 2’-O-methoxyethyl (MOE) 20mer gapmer antisense oligonucleotides (ASOs) in atherosclerotic aortic tissue, ASOs targeting the class B scavenger receptors, CD36 and SR-BI, were evaluated in mouse models of atherosclerosis. The ASOs were administrated for 3 weeks at 25, 50 or 100 mg/kg/wk. RT-PCR analysis of target gene inhibition revealed potent dose-dependent reductions of CD36 and SR-BI (see table below). ASO tissue concentrations were measured by LC/MS and demonstrated dose-dependent increases in both the liver and atherosclerotic aortic tissue. For example, liver ASO concentrations were 228±28, 270±18 and 346±30 and aortic ASO concentrations were 31±6, 51±2 and 88±7 ug/g for the 25, 50 and 100 mg/kg/wk doses, respectively. Thus, at each higher dose, there was an enrichment of atherosclerotic aortic tissue ASO accumulation relative to the liver, and at the highest dose, the liver:aortic ASO tissue concentrations were approximately 4:1. Immunohistochemistry of atherosclerotic tissue from micetreated with 50 mg/kg/wk of an ASO demonstrated drug localization and accumulation within endothelial cells and macrophages in early plaque deposits and the adventitia. CD36 ASO treatment demonstrated anti-atherosclerotic activity in two murine models of atherosclerosis. In summary, these data demonstrate robust ASO distribution, activity and pharmacology within atherosclerotic tissue suggesting that these regions may represent another opportunity for antisense therapeutic intervention.
Poster P138 4/28/11
A PHASE 1 STUDY IN HEALTHY VOLUNTEERS TO EVALUATE THE PHARMACOKINETICS, SAFETY, AND TOLERABILITY OF MIPOMERSEN IN 3 DOSING REGIMENS
JoAnn D Flaim, Isis Pharmaceuticals, Inc, Carlsbad, CA; Marjie Hard, Genzyme Corp, Cambridge, MA; John Grundy, John Su, Isis Pharmaceuticals, Inc, Carlsbad, CA; Joanne Donovan, Genzyme Corp, Cambridge, MA
Mipomersen (ISIS 301012) is an investigational apolipoprotein (apo) B synthesis inhibitor, which has demonstrated significant reductions in LDL-C, non-HDL-C, apo B and Lp(a) in four Phase 3 studies at a dose of 200 mg subcutaneous (SC) once weekly when added to stable lipid-lowering therapy. With a goal to provide prescribers and patients dosing alternatives as needed in the future, the current study investigated 3 SC dosing regimens for PK, safety and tolerability, including assessment of biomarkers for a number of inflammatory pathways: IL-1β (activation of inflammasomes), IL-6 (Toll-like receptor stimulation), hsCRP (acute phase response), IL-13 (mast cell activation), IFN-α and IFN-β (type 1 interferon signaling), MCP-1 and MIP-1α (chemokine release), and C5a and Bb (complement activation). Healthy volunteers were randomized to 1 of 3 cohorts (n=28/cohort) – 30 mg daily (QD), 70 mg 3x/wk (TIW) or 200 mg once weekly (QW) – with each cohort further randomized to receive either mipomersen or volume-matched placebo (saline) in a 3:1 ratio for 3 weeks. The primary PK objective was to evaluate the relative bioavailability of the two test regimens (30 mg QD and 70mg TIW) to the reference regimen (200 mg QW) by comparison of the post-distribution phase plasma concentrations. Comparable post-distribution phase concentrations were observed 7 days after the last dose across the 3 dose regimens (geometric mean test:reference ratios [90% CI] were 0.92 [0.75, 1.13] for 30 mg QD and 1.02 [0.83, 1.25] for 70 mg TIW) suggesting the two test regimens resulted in similar tissue exposure to the 200 mg QW regimen. Injection site reactions were frequently reported, but were mild in severity and did not lead to treatment discontinuation. In general, there were no differences in inflammatory biomarker signals between treatment groups and placebo with the exception of transient post-dose elevations of hsCRP in the mipomersen 200 mg QW group (week 1, median +3.8 mg/L) that tended to decrease with continued dosing (week 3, median +2.3 mg/L). These results support assessment of these dose regimens in longer-term studies.
Poster P405 4/29/11
ANTISENSE INHIBITION OF APOLIPOPROTEIN C-III REDUCES PLASMA LIPIDS AND INCREASES HDL FUNCTIONALITY IN CETP TRANSGENIC, LDL RECEPTOR NULL MICE
Thomas A Bell, Rosanne Crooke, Mark Graham, Richard Lee, Adam Mullick, Wuxia Fu, ISIS Pharmaceuticals, Carlsbad, CA
Inhibition of apoC-III with an antisense oligonucleotide significantly reduced plasma cholesterol and triglyceride while increasing HDL, apoA1 protein and PON1 activity Mice given the apoC-III ASO also displayed reductions in CETP protein and activity. Treatment with apoC-III ASO enhanced HDL cholesterol clearance. The results from these studies suggest that in a process facilitated by CETP, the apoC-III ASO mediated lowering of plasma lipids can have positive effects on HDL metabolism and function. Numerous epidemiological studies have associated elevated plasma triglycerides (TG) with an increase in cardiovascular disease risk. Apolipoprotein C-III (apoC-III) is a key regulator of plasma TG levels via modulation of lipoprotein lipase. In humans, plasma TG is primarily associated with VLDL and LDL and can readily exchange with cholesteryl ester (CE) from HDL in a process mediated by cholesteryl ester transfer protein (CETP). The primary focus of these studies was to examine the effects of an apoC-III antisense inhibitor (ASO) on plasma lipids and lipoprotein metabolism in hyperlipidemic LDLr-deficient mice that express human CETP. These mice were fed a western diet (42% calories from fat, 0.2% cholesterol) and administered either saline, a control ASO, or anapoC-III ASO at 12.5 mg/kg/wk for 4 weeks. At the end of treatment, mice that were administered the apoC-III ASO displayed dramatic reductions in plasma cholesterol and TG when compared to either saline or control ASO groups. Mice in the apoC-III ASO group also had an increase in apoA1 protein, paraoxonase-1 activity, and HDL cholesterol levels as well as significant reductions in CETP protein and activity relative to the control groups. To directly evaluate the effects of apoC-III inhibition on HDL metabolism, CETP tg, LDLr-deficient mice were also treated with the apoC-III ASO for 6 weeks, then injected with radiolabeled HDL, and clearance of labeled HDL was monitored over 24 hours. When compared to the control groups, mice treated with the apoC-III ASO displayed an enhanced rate of radiolabeled HDL clearance of from plasma that was associated with a greater accumulation of label in the liver, suggesting that inhibition of apoC-III in these mice improves reverse cholesterol transport. These preliminary studies suggest that the apoC-III ASO-mediated lowering of plasma lipids can exert downstream changes in HDL metabolism and function. These effects on HDL appear to result from lowering of CETP protein and activity and additional studies will be required to delineate the interplay between plasma lipid, apoC-III, CETP and atherosclerosis.
AN INTRAVITAL MICROSCOPIC ANALYSIS OF ANTISENSE OLIGONUCLEOTIDE DEPLETION OF FXI, PLAVIX® TREATMENT, AND THEIR COMBINATION ON PLATELET AGGREGATION AND FIBRIN FORMATION
Dacao Gao, Jeff Crosby, Alexey Revenko, Gourab Bhattacharjee, Chenguang Zhao, Chris May, Robert Macleod, Brett Monia, Isis Pharmaceutical, Carlsbad, CA
During pathogenic intravascular thrombosis, platelet aggregation and fibrin formation occur simultaneously. Platelets detect injury through the collagen receptor, aggregate in response to activation, and contribute to thrombus propagation via the secretion of soluble agonists. Fibrin formation creates a meshwork to stabilize the propagating thrombus. We have previously demonstrated that Antisense Oligonucleotides (ASOs) targeting coagulation factor XI (fXI) demonstrate potent antithrombotic effects in several mouse models with no bleeding risk. Plavix® is a widely used anti-platelet treatment targeting the P2Y12 receptor. In this study we address how a combination of our anticoagulant antisense drug and an existing anti-platelet therapy can impact thrombus formation in live mice using intravital microscopy. Independently, fXI ASO and Plavix® both demonstrate inhibition of platelet aggregation and fibrin formation in a dose dependent manner. At low doses, Plavix® shows only inhibition of platelet aggregation with no effect on fibrin formation. Interestingly, the opposite effect is observed with fXI ASO. Combination of low doses of both fXI ASO and Plavix® effects potent inhibition of both platelet aggregation and fibrin deposition. Our results support the combination of fXI ASO and Plavix® as a potent antithrombotic strategy in the treatment of cardiovascular pathologies.
ADMINISTRATION OF AN APOLIPOPROTEIN C-III ANTISENSE OLIGONUCLEOTIDE LOWERS PLASMA TRIGLYCERIDE AND POSTPRANDIAL HYPERTRIGLYCERIDEMIA IN C57BL/6 BUT NOT APOLIPOPROTEIN C-III -/- MICE
Richard Lee, Wuxia Fu, Adam Mullick, Alex Bell, Mark Graham, Rosanne Crooke, Isis Pharmaceuticals, Carlsbad, CA
Hypertriglyceridemia, one of the components of the metabolic syndrome, has been positively associated with coronary heart disease and insulin resistance. Apolipoprotein C-III (apoC-III), a 79 aa protein secreted from the liver and small intestine, modulates plasma triglyceride(TG) levels by inhibiting the actions of lipoprotein lipase. Our study aim was to compare/contrast the effects of apoC-III antisense oligonucleotide (ASO) mediated inhibition to systemic apoC-III knockout using apoC-III deficient (-/-) mice. Chow fed C57BL/6 mouse and apoC-III -/- mice were treated for six weeks with either a control ASO (12.5 mg/kg/wk), which has no sequence homology to any annotated gene in the mouse genome, or a mousespecific apoC-III ASO (12.5 mg/kg/wk). After five weeks, mice were fasted overnight, gavaged with a TG bolus, and plasma TG levels were quantified in 1 hour increments over a 4 hour period. Relative to control ASO treated mice, hepatic apoC-III mRNA expression was significantly reduced in apoC-III ASO treated mice as well as apoC-III -/- mice treated with either control or apoC-III ASO. In the proximal third of the small intestine (SI), apoC-III mRNA reduction in apoC-III ASO treated wildtype mice was less than in apoC-III -/- mice treated with either control ASO or apoC-III ASO. Plasma TG concentrations were also significantly reduced in apoC-III treated C57BL/6 mice, control ASO treated apoC-III -/- mice, and apoC-III ASO treated apoC-III -/- mice compared to the control ASO C57BL/6 mice. Finally, plasma TG area under the curve after administration of a TG bolus was significantly reduced in apoC-III ASO treated wildtype mice, relative to the control groups. Therefore, apoC-III ASO treatment effectively replicated the phenotype observed in apoC-III -/- mice. Additionally, apoC-III ASO treatment had no effect on apoC-III -/- mice, demonstrating that the effects of the apoC-III ASO are target specific. These data suggest that pharmacologic inhibition of apoC-III may be an important therapeutic intervention for hypertriglyceridemia in man.