Hayes M. Dansky

Safety of Anacetrapib in Patients with or at High Risk for Coronary Heart Disease

BACKGROUND Anacetrapib is a cholesteryl ester transfer protein inhibitor that raises high-density lipoprotein (HDL) cholesterol and reduces low-density lipoprotein (LDL) cholesterol. METHODS We conducted a randomized, double-blind, placebo-controlled trial to assess the efficacy and safety profile of anacetrapib in patients with coronary heart disease or at high risk for coronary heart disease. Eligible patients who were taking a statin and who had an LDL cholesterol level that was consistent with that recommended in guidelines were assigned to receive 100 mg of anacetrapib or placebo daily for 18 months. The primary end points were the percent change from baseline in LDL cholesterol at 24 weeks (HDL cholesterol level was a secondary end point) and the safety and side-effect profile of anacetrapib through 76 weeks. Cardiovascular events and deaths were prospectively adjudicated. RESULTS A total of 1623 patients underwent randomization. By 24 weeks, the LDL cholesterol level had been reduced from 81 mg per deciliter (2.1 mmol per liter) to 45 mg per deciliter (1.2 mmol per liter) in the anacetrapib group, as compared with a reduction from 82 mg per deciliter (2.1 mmol per liter) to 77 mg per deciliter (2.0 mmol per liter) in the placebo group (P<0.001)--a 39.8% reduction with anacetrapib beyond that seen with placebo. In addition, the HDL cholesterol level increased from 41 mg per deciliter (1.0 mmol per liter) to 101 mg per deciliter (2.6 mmol per liter) in the anacetrapib group, as compared with an increase from 40 mg per deciliter (1.0 mmol per liter) to 46 mg per deciliter (1.2 mmol per liter) in the placebo group (P<0.001)--a 138.1% increase with anacetrapib beyond that seen with placebo. Through 76 weeks, no changes were noted in blood pressure or electrolyte or aldosterone levels with anacetrapib as compared with placebo. Prespecified adjudicated cardiovascular events occurred in 16 patients treated with anacetrapib (2.0%) and 21 patients receiving placebo (2.6%) (P = 0.40). The prespecified Bayesian analysis indicated that this event distribution provided a predictive probability (confidence) of 94% that anacetrapib would not be associated with a 25% increase in cardiovascular events, as seen with torcetrapib. CONCLUSIONS Treatment with anacetrapib had robust effects on LDL and HDL cholesterol, had an acceptable side-effect profile, and, within the limits of the power of this study, did not result in the adverse cardiovascular effects observed with torcetrapib. (Funded by Merck Research Laboratories; ClinicalTrials.gov number, NCT00685776.).

Noninvasive In Vivo High-Resolution Magnetic Resonance Imaging of Atherosclerotic Lesions in Genetically Engineered Mice

BACKGROUND The pathogenesis of atherosclerosis is currently being investigated in genetically engineered small animals. Methods to follow the time course of the developing pathology and/or the responses to therapy in vivo are limited. METHODS AND RESULTS To address this problem, we developed a noninvasive MR microscopy technique to study in vivo atherosclerotic lesions (without a priori knowledge of the lesion location or lesion type) in live apolipoprotein E knockout (apoE-KO) mice. The spatial resolution was 0.0012 to 0.005 mm3. The lumen and wall of the abdominal aorta and iliac arteries were identified on all images in apoE-KO (n=8) and wild-type (n=5) mice on chow diet. Images obtained with MR were compared with corresponding cross-sectional histopathology (n=58). MR accurately determined wall area in comparison to histopathology (slope=1.0, r=0.86). In addition, atherosclerotic lesions were characterized in terms of lesion shape and type. Lesion type was graded by MR according to morphological appearance/severity and by histopathology according to the AHA classification. There was excellent agreement between MR and histopathology in grading of lesion shape and type (slope=0.97, r=0.91 for lesion shape; slope=0. 64, r=0.90 for lesion type). CONCLUSIONS The combination of high-resolution MR microscopy and genetically engineered animals is a powerful tool to investigate serially and noninvasively the progression and regression of atherosclerotic lesions in an intact animal model and should greatly enhance basic studies of atherosclerotic disease.

Intensification of Statin Therapy Results in a Rapid Reduction in Atherosclerotic Inflammation Results of a Multicenter Fluorodeoxyglucose-Positron Emission Tomography/Computed Tomography Feasibility Study

OBJECTIVES The study sought to test whether high-dose statin treatment would result in greater reductions in plaque inflammation than low-dose statins, using fluorodeoxyglucose-positron emission tomography/computed tomographic imaging (FDG-PET/CT). BACKGROUND Intensification of statin therapy reduces major cardiovascular events. METHODS Adults with risk factors or with established atherosclerosis, who were not taking high-dose statins (n = 83), were randomized to atorvastatin 10 versus 80 mg in a double-blind, multicenter trial. FDG-PET/CT imaging of the ascending thoracic aorta and carotid arteries was performed at baseline, 4, and 12 weeks after randomization and target-to-background ratio (TBR) of FDG uptake within the artery wall was assessed while blinded to time points and treatment. RESULTS Sixty-seven subjects completed the study, providing imaging data for analysis. At 12 weeks, inflammation (TBR) in the index vessel was significantly reduced from baseline with atorvastatin 80 mg (% reduction [95% confidence interval]: 14.42% [8.7% to 19.8%]; p < 0.001), but not atorvastatin 10 mg (% reduction: 4.2% [-2.3% to 10.4%]; p > 0.1). Atorvastatin 80 mg resulted in significant additional relative reductions in TBR versus atorvastatin 10 mg (10.6% [2.2% to 18.3%]; p = 0.01) at week 12. Reductions from baseline in TBR were seen as early as 4 weeks after randomization with atorvastatin 10 mg (6.4% reduction, p < 0.05) and 80 mg (12.5% reduction, p < 0.001). Changes in TBR did not correlate with lipid profile changes. CONCLUSIONS Statin therapy produced significant rapid dose-dependent reductions in FDG uptake that may represent changes in atherosclerotic plaque inflammation. FDG-PET imaging may be useful in detecting early treatment effects in patients at risk or with established atherosclerosis.

Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice

Macrophage foam cells are integral in the development of atherosclerotic lesions. Gene expression analysis of lesional macrophage foam cells is complicated by the cellular heterogeneity of atherosclerotic plaque and the presence of lesions of various degrees of severity. To overcome these limitations, we tested the ability of laser capture microdissection (LCM) and real-time quantitative reverse transcription PCR to selectively analyze RNA from lesional macrophages of apolipoprotein E (apoE)-deficient mice. Proximal aortic tissue sections were immunostained for macrophagespecific CD68/macrosialin by a rapid (≈15-min) protocol. Alternating sections from each animal were used to isolate RNA either from entire sections (analogous to isolation from whole tissue) or by LCM selection of CD68-positive cells. We measured the mRNA levels of CD68, a macrophage-specific marker, α-actin, a smooth muscle cell marker, and cyclophilin A, a control gene. Compared with whole sections, CD68 mRNA levels were greatly enriched (33.6-fold) in the laser-captured lesional macrophages. In contrast to whole sections, LCM-derived RNA had undetectable levels of α-actin. To illustrate the ability of this method to measure changes in lesional macrophage gene expression, we injected 100 μg of lipopolysaccharide i.p. into apoE-deficient mice and detected in laser-captured lesional macrophages increased mRNA expression for vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, and monocyte chemoattractant protein-1 (11.9-, 32.5-, and 31.0-fold, respectively). By selectively enriching foam cell RNA, LCM provides a powerful approach to study the in situ expression and regulation of atherosclerosis-related genes. This approach will allow the study of macrophage gene expression under various conditions of plaque formation, regression, and response to genetic and environmental perturbations.

Diabetes induces endothelial dysfunction but does not increase neointimal formation in high-fat diet fed C57BL/6J mice.

Studies of diabetic vascular disease have traditionally used murine models of type 1 diabetes and genetic models of type 2 diabetes. Because the majority of patients with type 2 diabetes have diet induced obesity, we sought to study the effect of diabetes on arterial disease in a mouse model of diet induced obesity/diabetes. C57Bl/6 mice fed a high-fat diet for 9 weeks developed type 2 diabetes characterized by elevated body weight, hyperglycemia, and hyperinsulinemia. Arteries from diabetic mice exhibited a marked decrease in endothelium-dependent vasodilation, a modest decrease in endothelium independent vasodilation, and an increase in sensitivity to adrenergic vasoconstricting agents. Insulin stimulated protein kinase B (akt) and endothelial nitric oxide synthase (eNOS) phosphorylation were preserved in arteries from diabetic mice; however, eNOS protein dimers were markedly diminished. Arterial nitrotyrosine staining indicated that increased levels of peroxynitrite contributed to eNOS dimer disruption in the diabetic mice. The abnormal vasomotion was not an acute response to the high-fat diet, as short term high-fat diet feeding had no effect on endothelium dependent dilation. A trend toward smaller neointimal lesions was noted in high-fat diet fed mice after femoral artery wire denudation injury. In summary, disrupted eNOS dimer formation rather than impaired insulin mediated eNOS phosphorylation contributed to the endothelial dysfunction in diet induced obese/diabetic mice. The lack of an increase in neointimal formation indicates that additional diabetes associated parameters (such as hyperlipidemia and atherosclerotic vascular disease) may need to be present to increase neointimal formation in this model.

Localization of atherosclerosis susceptibility loci to chromosomes 4 and 6 using the Ldlr knockout mouse model

Atherosclerosis is a complex disease resulting from the interaction of multiple genes. We have used the Ldlr knockout mouse model in an interspecific genetic cross to map atherosclerosis susceptibility loci. A total of 174 (MOLF/Ei × B6.129S7-Ldlrtm1Her) × C57BL/6J-Ldlrtm1Her backcross mice, homozygous for the Ldlr null allele, were fed a Western-type diet for 3 months and then killed for quantification of aortic lesions. A genome scan was carried out by using DNA pools and microsatellite markers spaced at ≈18-centimorgan intervals. Quantitative trait locus analysis of individual backcross mice confirmed linkages to chromosomes 4 (Athsq1, logarithm of odds = 6.2) and 6 (Athsq2, logarithm of odds = 6.7). Athsq1 affected lesions in females only whereas Athsq2 affected both sexes. Among females, the loci accounted for ≈50% of the total variance of lesion area. The susceptible allele at Athsq1 was derived from the MOLF/Ei genome whereas the susceptible allele at Athsq2 was derived from C57BL/6J. Inheritance of susceptible alleles at both loci conferred a 2-fold difference in lesion area, suggesting an additive effect of Athsq1 and Athsq2. No associations were observed between the quantitative trait loci and levels of plasma total cholesterol, high density lipoprotein cholesterol, non-high density lipoprotein cholesterol, insulin, or body weight. We provide strong evidence for complex inheritance of atherosclerosis in mice with elevated plasma low density lipoprotein cholesterol and show a major influence of nonlipoprotein-related factors on disease susceptibility. Athsq1 and Athsq2 represent candidate susceptibility loci for human atherosclerosis, most likely residing on chromosomes 1p36–32 and 12p13–12, respectively.

High-Density Lipoproteins Retard the Progression of Atherosclerosis and Favorably Remodel Lesions Without Suppressing Indices of Inflammation or Oxidation

Objective—Protective properties of high-density lipoproteins (HDL) may include reverse cholesterol transport and suppression of oxidation and inflammation. These were investigated in vivo, as were the effects of HDL on the characteristics of atherosclerotic lesions. Methods and Results—Male apolipoprotein E knockout (apoE−/−) and apoE−/− mice expressing human apolipoprotein AI (hAI/apoE−/−) were studied up to 20 weeks after commencing a high-fat diet. Plasma HDL cholesterol was twice as high in hAI/apoE−/− mice. Over time, aortic root lesion area remained less in hAI/apoE−/− mice, although plaques became complex. In advanced lesions, plaque lipid was higher in apoE−/− mice, whereas plaque collagen and alpha actin were increased in hAI/apoE−/− mice. In nonlesional aorta, mRNA abundance for pro-inflammatory proteins (vascular cell adhesion molecule [VCAM]-1, intercellular adhesion molecule-1 [ICAM-1], monocyte chemoattractant protein-1 [MCP-1]) increased between 4 and 16 weeks in apoE−/− (but not wild-type) mice, and were not reduced by elevated HDL. Autoantibodies to malondialdehyde low-density lipoprotein (LDL) increased progressively in apoE−/− mice, with similar results in hAI/apoE−/− mice. Conclusions—HDL retarded plaque size progression despite greatly elevated plasma cholesterol. This effect was over a wide range of lesion severity. Expression of hAI reduced plaque lipid and increased the proportion of plaque occupied by collagen and smooth muscle cells, but did not affect indicators of inflammation or LDL oxidation.

Genetic and Pharmacologic Inactivation of ANGPTL3 and Cardiovascular Disease

BACKGROUND Loss‐of‐function variants in the angiopoietin‐like 3 gene (ANGPTL3) have been associated with decreased plasma levels of triglycerides, low‐density lipoprotein (LDL) cholesterol, and high‐density lipoprotein (HDL) cholesterol. It is not known whether such variants or therapeutic antagonism of ANGPTL3 are associated with a reduced risk of atherosclerotic cardiovascular disease. METHODS We sequenced the exons of ANGPTL3 in 58,335 participants in the DiscovEHR human genetics study. We performed tests of association for loss‐of‐function variants in ANGPTL3 with lipid levels and with coronary artery disease in 13,102 case patients and 40,430 controls from the DiscovEHR study, with follow‐up studies involving 23,317 case patients and 107,166 controls from four population studies. We also tested the effects of a human monoclonal antibody, evinacumab, against Angptl3 in dyslipidemic mice and against ANGPTL3 in healthy human volunteers with elevated levels of triglycerides or LDL cholesterol. RESULTS In the DiscovEHR study, participants with heterozygous loss‐of‐function variants in ANGPTL3 had significantly lower serum levels of triglycerides, HDL cholesterol, and LDL cholesterol than participants without these variants. Loss‐of‐function variants were found in 0.33% of case patients with coronary artery disease and in 0.45% of controls (adjusted odds ratio, 0.59; 95% confidence interval, 0.41 to 0.85; P=0.004). These results were confirmed in the follow‐up studies. In dyslipidemic mice, inhibition of Angptl3 with evinacumab resulted in a greater decrease in atherosclerotic lesion area and necrotic content than a control antibody. In humans, evinacumab caused a dose‐dependent placebo‐adjusted reduction in fasting triglyceride levels of up to 76% and LDL cholesterol levels of up to 23%. CONCLUSIONS Genetic and therapeutic antagonism of ANGPTL3 in humans and of Angptl3 in mice was associated with decreased levels of all three major lipid fractions and decreased odds of atherosclerotic cardiovascular disease. (Funded by Regeneron Pharmaceuticals and others; ClinicalTrials.gov number, NCT01749878.)

Design of the DEFINE trial: Determining the EFficacy and Tolerability of CETP INhibition with AnacEtrapib

BACKGROUND Residual cardiovascular (CV) risk often remains high despite statin therapy to lower low-density lipoprotein cholesterol (LDL-C). New therapies to raise high-density lipoprotein cholesterol (HDL-C) are currently being investigated. Anacetrapib is a cholesteryl ester transfer protein (CETP) inhibitor that raises HDL-C and reduces LDL-C when administered alone or with a statin. Adverse effects on blood pressure, electrolytes, and aldosterone levels, seen with another drug in this class, have not been noted in studies of anacetrapib to date. METHODS Determining the EFficacy and Tolerability of CETP INhibition with AnacEtrapib (DEFINE) is a randomized, double-blind, placebo-controlled trial to assess the efficacy and safety profile of anacetrapib in patients with coronary heart disease (CHD) or CHD risk equivalents (clinical trials.gov NCT00685776). Eligible patients at National Cholesterol Education Program-Adult Treatment Panel III LDL-C treatment goal on a statin, with or without other lipid-modifying medications, are treated with anacetrapib, 100 mg, or placebo for 18 months, followed by a 3-month, poststudy follow-up. The primary end points are percent change from baseline in LDL-C and the safety and tolerability of anacetrapib. Comprehensive preplanned interim safety analyses will be performed at the 6- and 12-month time points to examine treatment effects on key safety end points, including blood pressure and electrolytes. A preplanned Bayesian analysis will be performed to interpret the CV event distribution, given the limited number of events expected in this study. RESULTS A total of 2,757 patients were screened at 153 centers in 20 countries, and 1,623 patients were randomized into the trial. Lipid results, clinical CV events, and safety outcomes from this trial are anticipated in 2010.

Changes in lipoprotein subfraction concentration and composition in healthy individuals treated with the CETP inhibitor anacetrapib.

We investigated the effects of the cholesteryl ester (CE) transfer protein inhibitor anacetrapib (ANA) on plasma lipids, lipoprotein subfraction concentrations, and lipoprotein composition in 30 healthy individuals. Participants (n = 30) were randomized to ANA 20 mg/day, 150 mg/day, or placebo for 2 weeks. Changes in concentration of lipoprotein subfractions were assessed using ion mobility, and compositional analyses were performed on fractions separated by density gradient ultracentrifugation. ANA 150 mg/day versus placebo resulted in significant decreases in LDL-cholesterol (26%) and apo B (29%) and increases in HDL-cholesterol (82%). Concentrations of medium and small VLDL, large intermediate density lipoprotein (IDL), and medium and small LDL (LDL2a, 2b, and 3a) decreased whereas levels of very small and dense LDL4b were increased. There was enrichment of triglycerides and reduction of CE in VLDL, IDL, and the densest LDL fraction. Levels of large buoyant HDL particles were substantially increased, and there was enrichment of CE, apo AI, and apoCIII, but not apoAII or apoE, in the mid-HDL density range. Changes in lipoprotein subfraction concentrations and composition with ANA 20 mg/day were similar to those for ANA 150 mg/day but were generally smaller in magnitude. The impact of these changes on cardiovascular risk remains to be determined.