Effects of ezetimibe on atherosclerosis in preclinical models

Atherosclerosis 215 (2011) 266–278

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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

Review

Effects of ezetimibe on atherosclerosis in preclinical models Harry R. Davis Jr. a,∗ , Robert S. Lowe b , David R. Neff c a

Merck Research Labs, In Vivo Pharmacology, RY-80Y-215, 126 East Lincoln Ave., P.O. Box 2000, Rahway, NJ 07065 USA Merck Sharp & Dohme Corp., Global Scientific and Medical Publications, Whitehouse Station, NJ, USA c Merck Sharp & Dohme Corp., Global Medical Affairs, Whitehouse Station, NJ, USA b

a r t i c l e

i n f o

Article history: Received 28 September 2010 Received in revised form 19 January 2011 Accepted 7 February 2011 Available online 17 February 2011 Keywords: Atherosclerosis ApoB Animal models Ezetimibe

a b s t r a c t Ezetimibe (Zetia® , Ezetrol® , Merck, Whitehouse Station, NJ) is a potent inhibitor of sterol absorption, which selectively blocks the uptake of biliary and dietary cholesterol in the small intestine. Clinical trials have demonstrated the beneficial effects of ezetimibe on the reduction of atherogenic lipoproteins and the attainment of guideline-recommended lipid levels. Direct evidence that these improvements translate to a reduction in atherosclerosis or cardiovascular events is limited, although reductions in major atherosclerotic events that are consistent with the LDL-C lowering achieved have recently been presented for patients with chronic kidney disease treated with ezetimibe/simvastatin 10/20 mg in the SHARP trial. Animal models of atherosclerosis have played a central role in defining the mechanisms involved in initiation and development of disease and have been used in drug development to evaluate potential therapeutic efficacy. The effect of ezetimibe on atherosclerosis has been examined in several of these animal model systems. ApoE knockout mice develop severe hypercholesterolemia and premature atherosclerosis with features similar to that seen in humans and techniques ranging from gross visualization of plaque to high-resolution MRI have demonstrated the consistent ability of ezetimibe to significantly inhibit atherosclerosis. sr-b1−/− /apoE−/− double knockout mice exhibit additional characteristics common to human coronary heart disease (CHD), and the one study of ezetimibe in sr-b1−/− /apoE−/− mice showed a significant reduction in aortic sinus plaque (57%), coronary arterial occlusion (68%), myocardial fibrosis (57%), and cardiomegaly (12%) compared with untreated controls. The effects of ezetimibe have also been evaluated in ldlr−/− /apoE−/− double knockout mice, demonstrating that functional LDL receptors were not required for ezetimibe-mediated reduction of plasma cholesterol or atherosclerosis. For the few studies that have been conducted in rabbits, ezetimibe has been shown to significantly inhibit diet and vascular-injury-induced atherosclerosis as measured by intima/media thickness, atherosclerotic lesion composition, and thrombosis. The current body of preclinical evidence consistently demonstrates that ezetimibe reduces atherosclerosis in animals, presumably due primarily to the decrease in circulating levels of atherogenic lipoproteins that the drug produces. Demonstration that ezetimibe-mediated lowering of atherogenic lipoproteins in humans has a similar effect on atherosclerosis and cardiovascular risk awaits additional results from recently completed and ongoing outcomes trials. © 2011 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The molecular target of ezetimibe and potential off-target effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of ezetimibe on atherosclerosis in animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. ApoE knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. SR-B1/ApoE knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. LDL receptor knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Rabbit models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of atherosclerosis by ezetimibe: potential mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +1 732 594 0615. E-mail address: [email protected] (H.R. Davis Jr.). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.02.010

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5.

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Atherosclerosis is a progressive disease that starts early in life and is the underlying cause of most cardiovascular disease [1]. The primary event responsible for initiation of atherosclerosis is retention of apolipoprotein (Apo) B-containing lipoproteins within the arterial intima [2]. Accumulation of lipoproteins and their subsequent modification (oxidation, aggregation) triggers an inflammatory immune response and development of atherosclerotic plaques [3]. Over time, the fibrous cap of a small percentage of plaques can deteriorate, forming a vulnerable cap that is prone to rupture, which in turn can ultimately be the cause of most acute cardiovascular events [4]. Development and progression of atherosclerosis is dependent on the level and duration of exposure to circulating ApoB lipoproteins, with higher levels and longer duration leading to more advanced disease [5]. The majority of ApoB found in blood is associated with low-density lipoprotein cholesterol (LDL-C). Historically, serum concentrations of total cholesterol and LDL-C levels have served as good surrogates for atherosclerosis, because they have correlated consistently with atherosclerotic burden and cardiovascular events in a large number of epidemiological, preclinical, and clinical studies. Numerous randomized clinical outcomes trials studying a variety of lipid-lowering therapies have provided convincing evidence of a direct relationship between reduction of LDL-C and lowering of cardiovascular disease risk. These included trials evaluating a variety of statins, niacin (alone and in combination with other medications), certain bile acid sequestrants, and intestinal by-pass surgery [6–9]. Ezetimibe is the first new drug to be approved as adjunctive therapy to diet for the treatment of dyslipidemia since the advent of statins. Ezetimibe can be used either alone or in combination with statins for patients with primary (heterozygous familial and non-familial) hyperlipidemia. It is also approved for use in combination with statins for treatment of homozygous familial hypercholesterolemia and in combination with fenofibrate for treatment of adult patients with mixed hyperlipidemia. Finally, it is approved as a monotherapy for patients with homozygous sitosterolemia [10]. Ezetimibe selectively inhibits the absorption of dietary and biliary cholesterol in the small intestine by blocking the activity of the Niemann-Pick C1 Like 1 (NPC1L1) sterol transporter, resulting in reduced incorporation of cholesterol into chylomicrons, decreased delivery of cholesterol to the liver, and upregulation of hepatic LDL receptors (LDLR), resulting in increased plasma clearance of atherogenic ApoB-containing lipoproteins [11] (Fig. 1). Reductions in ApoB and non-high-density lipoprotein cholesterol (non-HDL-C) concentrations have been demonstrated as well. In humans, co-administration of ezetimibe with statins inhibits both absorption and synthesis of cholesterol and results in significantly larger reductions in LDL-C and greater attainment of guideline-recommended LDL-C goals than with statins alone [11]. Although clinical trials have firmly established the role of statins in reducing the risk of cardiovascular events, definitive outcomes evidence beyond the benefit of statins alone does not yet exist for ezetimibe and will not be available until the conclusion of the IMProved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE-IT) in 2013 [12]. The purpose of this review is to summarize the studies that identified NPC1L1 as the molecular target of ezetimibe and to present the available preclinical data

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demonstrating the effects of ezetimibe on atherosclerosis in a variety of animal models. 2. The molecular target of ezetimibe and potential off-target effects Following the discovery of ezetimibe and demonstration that it was a potent inhibitor of intestinal sterol absorption [13], an intensive effort was undertaken to elucidate the molecular pathway(s) responsible for the observed biological effects. Using a genomicbioinformatics approach, Altmann et al. identified the NPC1L1 protein as the likely target of ezetimibe [14]. Additional experimentation demonstrated specific localization of NPC1L1 mRNA and protein expression to enterocytes located in the proximal jejunum, which was consistent with the known pathway for cholesterol absorption [14]. npc1l1 null (−/−) mice were generated to evaluate the role of NPC1L1 in vivo and were shown to have an 86% reduction in cholesterol absorption and a 72% inhibition of intestinal cholesterol uptake compared with wildtype (npc1l1+/+ ) mice [14]. Furthermore, npc1l1−/− mice were completely insensitive to treatment with ezetimibe, and they were resistant to diet-induced hypercholesterolemia [15]. Use of an in vitro ezetimibe binding assay with enterocyte brush border membranes from mice (wildtype and npc1l1−/− ), rats, and monkeys, as well as with membranes from human embryonic kidney cells expressing rat and human NPC1L1, provided evidence of direct interaction between ezetimibe and NPC1L1 [16]. The molecular characteristics of NPC1L1 and the role it plays in mediating cellular cholesterol transport have been recently reviewed [17]. NPC1L1 contains an extracellular sterol-binding loop, a sterol sensing domain and putative tyrosine-based sorting signals, which are all thought to be involved in clathrin-mediated NPC1L1-dependent uptake of free cholesterol and delivery to the endoplasmic reticulum for esterification and packaging into chylomicrons. In vitro binding assays have identified a 61-amino-acid site within the extracellular loop C of NPC1L1 that is required for high-affinity binding to ezetimibe [18], and cell based assays have demonstrated that ezetimibe prevents NPC1L1 internalization and cholesterol uptake [19]. The scavenger receptor type B1 (SR-B1) and NCP1L1 are both localized to the jejunal enterocytes. The authors of one in vitro binding study with brush border membrane vesicles from wildtype or NPC1L1 knockout mice suggested that SR-B1, not NPC1L1, was the molecular target of ezetimibe [20]. This specific issue was subsequently resolved by additional experiments in mice, which distinguished between cholesterol uptake into the proximal brush border membranes by SR-B1 and CD36, and cholesterol absorption across the apical brush border membrane of enterocytes, which is mediated by NPC1L1 and confirmed to be blocked by ezetimibe [21]. Additional studies using a fecal dual isotope method showed that cholesterol absorption levels were 55% for wild type mice, 50.3% for sr-b1−/− mice, 5.7% (90% reduction) for npc1l1−/− mice, and 4.2% (92% reduction) for npc1l1−/− /sr-b1−/− double knockout mice, providing further evidence that SR-B1 is not responsible for regulating cholesterol absorption [22]. Taken together, these experiments demonstrate that NPC1L1 is a major transporter of cholesterol in the intestine and the molecular target of ezetimibe. Ezetimibe is rapidly metabolized in the small intestine and liver to an active phenolic glucuronide, and both undergo enterohepatic recycling, which limits systemic exposure [23]. Because the

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Fig. 1. Mechanism of action for reduction of atherogenic ApoB-containing lipoproteins by ezetimibe, and potential effects on atherosclerotic burden. ApoB = apolipoprotein B, CE = cholesteryl ester, CETP = cholesteryl ester transfer protein, FFA = free fatty acid, HDL = high density lipoprotein, IDL = intermediate density lipoprotein, LCAT = lecithincholesterol acyltransferase, LDL = low density lipoprotein, LPL = lipoprotein lipase, NPC1L1 = Niemann-Pick C1 Like 1 sterol transporter, SR-B1 = scavenger receptor type B1, TG = triglyceride, VLDL = very low density lipoprotein.

approved 10 mg dose of ezetimibe prescribed to humans produces very low systemic plasma levels of ezetimibe, it is highly improbable that biologically relevant off-target effects of ezetimibe would occur. Ezetimibe is a minor substrate for common cytochrome P450 isoenzymes (1A2, 2D6, 2C8, 2C9, and 3A4) and does not influence their activity, and therefore, has minimal effect on the pharmacokinetics of most medications [10,24]. In order to characterize the molecular specificity of ezetimibe for NPC1L1 and identify potential off-target effects, in vitro assays were conducted for more than 100 enzymes, transporters and receptors, including all known lipid pathways. Most assays used 10 ␮M of ezetimibe, which is >400 times the level found in human plasma. No potent inhibition of biochemical or cellular processes was observed [13,25]. Ezetimibe inhibition of human acyl-coenzyme A:cholesterol acyltransferase (ACAT) 1 and 2 at >30 ␮M and rat hepatic microsomal ACAT at an IC50 of 18 ␮M was noted; however, these levels of ezetimibe are >800 times higher than the levels observed in human plasma. Non-specific biological effects of ezetimibe have also been evaluated in numerous toxicology studies in mice, rats, and dogs, and no target organ toxicity or proliferative lesions were seen in any study (Table 1) [10,25]. No evidence of mutagenicity (Ames), clastogenicity (chromosomal aberration assay), genotoxicity (in vivo mouse micronucleus test), or effect on longevity were observed [10,25]. Carcinogenesis studies in rats (20 times the human dose exposure) and mice (>150 times the human dose exposure) demonstrated no statistically significant increases in tumor incidence after 104 weeks. Dogs are similar to humans in expressing NPC1L1 in both the liver and intestine, and therefore are a particularly good model for evaluating organ toxicity. Dogs were given doses of ezetim-

ibe up to 1000 mg/kg (∼1000 times the human dose exposure) for 3 months and up to 300 mg/kg for a year, and no organ toxicity was found. These results provide further evidence of the molecular specificity of ezetimibe for a single target, namely NPC1L1. 3. Effects of ezetimibe on atherosclerosis in animal models Animal models have been instrumental in understanding the complex cellular and molecular pathways involved in initiation and progression of atherosclerotic disease and have also been important tools in evaluating the potential efficacy of drug therapy. The effect of ezetimibe on atherosclerosis has been evaluated in a number of animal models, and the results of these studies are discussed below and summarized in Table 2. 3.1. ApoE knockout mice ApoE plays an important role in facilitating transport and delivery of cholesterol and other lipids throughout the body [26]. ApoE is a component of liver-derived very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL particles, as well as intestinally synthesized chylomicrons and their remnants, and it facilitates the catabolism of these particles via LDL receptormediated clearance from the plasma. ApoE is also associated with some HDL particles, although its function in this context is poorly understood. In humans, multiple isoforms of ApoE exist. Genetic variants of these isoforms can cause diminished binding of ApoEcontaining particles to hepatic receptors, a subsequent increase in serum levels of cholesterol-rich IDL and chylomicron remnants, and

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Table 1 Pre-clinical safety studies with ezetimibe. Species

Duration; route of administration

Ezetimibe dose (mg/kg)

Findings

Mouse

3-Month dose range finding

0, 100, 500, and 2000

Decreased mean body weight of males and females (6.9 and 6.3%, respectively, relative to controls) at 2000 mg/kg

2-Year carcinogenicity; diet admix

0, 25, 100, 500 female 0, 25, 100, 500 male

No effect on tumors or mortality No toxicological findings

3-Month with 1-month post-dose; diet admix

0, 20, 100, 500, and 1500

No test article-related findings in females Serum cholesterol decreased in males at all doses, and decreased triglycerides at 1500 mg/kg No test article-related findings in post-dose period

6-Month; diet admix

0, 150, 750, 1500 males 0, 50, 250, 500 females

No test article-related findings in males Mean serum AST increased (1.7× control value) in females at 500 mg/kg with no histopathologic correlate in liver or muscle

2-Year carcinogenicity; diet admix

0, 50, 250, 500 female 0, 150, 750, 1000 male

No effect on tumors or mortality Slight decrease in body weight gain of males at ≥750 mg/kg

3-Month with 1-month post dose; oral (capsule)

0, 10, 30, 100, and 300

Slight to moderate dose-related increased incidence of soft/loose stool At all doses, non dose-dependent decreases in serum cholesterol (56–73% of pretest values) and triglycerides, which approached or returned to pretest during post-dose period

3-Month; oral (gavage)

0, 300, 600, and 1000

6-Month; oral (gavage)

0, 30, 100, and 300

1-Year; oral (gavage)

0, 30, 100, and 300

Dose-related increase in incidence of soft stool in all dose groups Non dose-dependent decreases in mean serum cholesterol (62–73%) at all doses Non dose-dependent decreases in mean serum cholesterol (58–70%) in males at >30 mg/kg and females at all doses Decreases in mean serum cholesterol (61–75%) at all doses

Rat

Dog

Modified with permission from Toxicology [25], Copyright ©Elsevier 2009.

the premature development of atherosclerosis. In the absence of ApoE, mice develop severe hypercholesterolemia and atherosclerosis similar to that seen in humans, due to the accumulation of very high plasma levels of VLDL and chylomicron remnants [27]. ApoE knockout mice (−/−) have been established as an animal model for studying the development and progression of atherosclerosis and have been used to evaluate the effects of ezetimibe on this disease process. One of the first studies to assess the effects of ezetimibe on atherosclerosis was conducted by Davis et al., using apoE−/− mice receiving a high-fat western diet (40 kcal% butterfat, 0.15% cholesterol), low-fat diet (10 kcal% corn oil, 0.15% cholesterol), or semi-synthetic, cholesterol-free diet (10 kcal% corn oil) with or without ezetimibe (0.005%) for six months [28]. Compared with untreated controls, ezetimibe was shown to inhibit cholesterol absorption by >90% and significantly lower plasma cholesterol, VLDL/chylomicron remnants, and LDL-C. Ezetimibe produced significant reductions in the atherosclerotic lesion surface area of the entire aorta, from 20% to 4% in mice fed the western diet and from 24% to 7% in the low-fat cholesterol diet group (p < 0.05) (Fig. 2). Ezetimibe also significantly reduced atherosclerosis in the aortic arch (81–87%), thoracic aorta (68–74%), and abdominal aorta (47–71%) (p < 0.05 for all). Atherosclerosis of the proximal right carotid artery develops rapidly in apoE−/− mice, and was significantly inhibited by ezetimibe (p < 0.05), with the intimal lesion cross-sectional area reduced by 97% for mice receiving the western diet (0.003 mm2 vs. 0.098 mm2 for controls) or low-fat diet (0.004 mm2 vs. 0.142 mm2 for controls), and by 91% in the cholesterol-free group (0.004 mm2 vs. 0.044 mm2 for controls). A second study introduced a knockout mutation of NPC1L1 in apoE−/− mice, which produced a result similar to treatment of apoE−/− mice with ezetimibe [29]. Cholesterol absorption was reduced by 77–83% compared with controls, mice were completely resistant to diet-induced hypercholesterolemia, and atherosclerosis was dramatically reduced by >90% (99% in aortic lesion surface area, 94–97% in innominate artery lesion area, and >90% in aor-

tic root lesion area) relative to apoE−/− mice without the NPC1L1 mutation. These observations demonstrate that both pharmacologic blockade of NPC1L1 with ezetimibe as well as the genetic knockout of NPC1L1 can reduce cholesterol absorption, modulate cholesterol metabolism, and impede the formation of atherosclerosis in the apoE−/− mouse model. Nakagami et al. also used apoE−/− mice to evaluate the vascular protective effects of ezetimibe. Mice were fed either normal chow or a western diet (10% coconut oil, 0.5% cholesterol) with or without ezetimibe (5 mg/kg/day) for three months, and markers associated with atherosclerosis were assessed [30]. Ezetimibe significantly reduced plasma cholesterol (76%) and LDL-C (78%), and Oil red O staining showed significant inhibition of lipid-rich plaques in the aorta (Fig. 3). In addition, ezetimibe improved endothelial dysfunction of the aorta as demonstrated by an increased vasodilator response to acetylcholine, up-regulation of endothelial nitric oxide synthase (both mRNA and protein), and inhibition of IL-6 mRNA expression, a marker of inflammation. In a six-month study evaluating the effect of plant sterol ester-supplements (PSE) and ezetimibe (0.005%) in apoE−/− mice, PSE and ezetimibe were shown to decrease serum cholesterol in mice fed a western diet (40 kcal% butterfat, 0.15% cholesterol) from 1190 mg/dL (untreated controls) to 454 mg/dL (PSE) and 388 mg/dL (ezetimibe) [31]. Co-administration of ezetimibe with PSE reduced serum cholesterol levels significantly more than seen with ezetimibe alone (251 mg/dL, p < 0.05). Ezetimibe reduced the atherosclerotic plaque area in the aortic sinus from 44% (control mice) to 10% (p < 0.05), which was significantly greater than the effects seen with PSE alone (20%, p <0.05), and comparable to the effects of ezetimibe + PSE (13.2%). Similar results were seen in mice on a normal chow diet (14 kcal% vegetable oil), with ezetimibe and PSE both reducing plasma cholesterol from 460 mg/dL (control) to 206 mg/dL, PSE plus ezetimibe reducing cholesterol levels to 190 mg/dL, and ezetimibe treatment decreasing atherosclerosis plaque area from 24.5% (control) to 3.5%, to 6.6% with ezetimibe + PSE, and to 13.7% with PSE alone (p < 0.05 compared with

270

Table 2 Characteristics and results of studies evaluating the effects of ezetimibe in animal models. Reference

Technique

Ezetimibe dose

Time

Animal model −/−

Gamori trichrome stain

0.005% (w/w)

6 months

apoE

Davis et al. [29]

Morphometric analysis Oil red O stain Oil red O stain

None

24 weeks

5 mg/kg/day (oral) 0.005% (w/w)

3 months 6 months

apoE−/− /npc1l1−/− mice apoE−/− mice apoE−/− mice

MRI MRI H&E, Oil red O, CD68 staining

±5 mg/kg/day 0.005% prophylactic treatment

15 months 6, 12, 20 weeks

apoE−/− mice

0.005% early treatment

8, 12 weeks

0.005% adult treatment

5, 12, 16 weeks

0.005% aged treatment

5, 12 weeks

Nakagami et al. [30] Weingartner et al. [31] Dietrich et al. [32] Tang et al. [33]

mice

apoE−/− mice

5 mg/kg/day

4, 6, 8 months

apoE−/− mice

Braun et al. [36] Davis et al. [37]

Near-Infrared Fluorescence Imaging Sudan III stain Oil red O stain NR

0.005% 5 mg/kg/day

∼3 months 6 months

sr-b1−/− /apoE−/− mice ldlr−/− /apoE−/− mice

Basso et al. [39]

Oil red O staining

15 mg/kg/day

24 weeks

Davis et al. [41]

Cholesterol ester content (mg/g wet wt)

Lovastatin (1.2 mg/kg/day) Ezetimibe (0.6 mg/kg/day) L + E combined E (0.6 mg/kg/day) Simvastatin (5 mg/kg/day) E+S 1 mg/kg/day

4 weeks

ldlr−/− vs. ABCG5/G8 × ldlr−/− mice Rabbits

Graf et al. [34]

Gomez-Garre et al. [42]

Orcein stain

Patel et al. [43]

Aortic wall cholesterol, microscopy

Plasma cholesterol

Atherosclerosis

WTD Low-fat cholesterol Cholesterol free diet NC WTD WTD WTD NC WTD NC to WTD at 6 weeks, ±E at week 6 NC to WTD at 4 weeks, ±E at week 16 NC to WTD at 4 weeks, ±E at week 24 NC to WTD at 4 weeks, ±E at week 40 WTD

↓↓↓ ↓↓↓ ↓↓↓

↓↓↓↓ ↓↓↓↓ →

↓↓ ↓↓↓ ↓↓↓↓ ↓↓↓ ↓↓↓ ↓↓↓ ↓↓

↓↓↓↓ ↓↓↓↓ ↓↓ ↓↓↓↓ ↓↓↓↓ ↓↓ ↓↓↓↓

↓↓

↓↓

↓↓

↓↓

↓↓

↓

↓↓

↓↓

NC Semi-synthetic cholesterol free WTD

→ ↓ ↓

↓↓↓

1% cholesterol diet

→

→

↓↓↓

↓↓↓

↓↓↓ → →

↓↓↓ ↓ ↓↓

→ NR

↓↓ ↓↓

6 weeks

Rabbits (New Zealand)

2% cholesterol, 6% peanut oil

6 months

Rabbits

High cholesterol diet

↓↓ IDL/LDL

E = ezetimibe, L = lovastatin, NC = normal chow, NR = not reported, S = simvastatin, WTD = western type diet; Percent reduction: → (no change), ↓ (>0–25%), ↓↓ (>25–50%), ↓↓↓ (>50–75%), ↓↓↓↓ (>75–100%).

↓↓↓ ↓↓

H.R. Davis Jr. et al. / Atherosclerosis 215 (2011) 266–278

Davis et al. [28]

Diet

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Fig. 2. Representative aortic arch images from male apoE−/− mice following 6 months of diet ± ezetimibe (Davis et al. [28] – personal communication).

ezetimibe). When evaluating the composition of atherosclerotic plaques in mice on a western diet, ezetimibe was shown to reduce the accumulation of lipids and infiltration of macrophages as well as increase the levels of interstitial collagen when compared with matched controls, consistent with the observed reduction of atherosclerosis. Recently, high-resolution magnetic resonance imaging (MRI) was used to evaluate the long-term effects of ezetimibe on atherosclerotic lesion formation in the thoracic aorta of apoE−/− mice in vivo [32]. apoE−/− mice were fed a high-fat western type diet (19.4% lard fat, 1.5% cholesterol) with or without ezetimibe (5 mg/kg/day) for 15 months and compared with each other and age-matched wild type mice receiving normal chow. MRI demonstrated significant reductions in the vessel wall thickness of the aortic root (29.5%) and aortic arch (29.9%) in ezetimibe-treated animals, with concomitant reductions in vessel wall area of the aortic root (43.4%) and aortic arch (42.9%) when compared with untreated apoE−/− mice (p < 0.01). When compared with wild type mice, aortic root vessel wall thickness increased by 189.1% in apoE−/− mice and 117.9% in apoE−/− mice receiving ezetimibe, while aortic arch vessel wall thickening was increased by 147.2% without ezetimibe and completely prevented during ezetimibe treatment. Histological staining with Sudan III confirmed the effects observed by MRI. Tang et al. also used in vivo high-resolution MRI and ApoE knockout mice to demonstrate the effectiveness of ezetimibe in reducing atherosclerosis [33]. Two models were utilized. In the prophylactic model, mice were treated with ezetimibe prior to development of atherosclerosis. These mice were switched from normal chow to a western diet (21% fat, 0.15% cholesterol) containing ezetimibe (0.005%) or placebo at six weeks of age. In the therapeutic model, mice were allowed to develop atherosclerosis prior to treatment by switching from a normal chow to a western diet at four weeks of age followed by administration of ezetimibe or placebo starting at 16 weeks (early pre-adult), 24 weeks (adult), or 40 weeks (aged). Baseline MRIs were established prior to treatment and evaluated again at defined intervals within a 3–6 month treatment period. Ezetimibe produced significant reductions in plasma cholesterol for all groups compared with the untreated controls (p < 0.01). Evaluation of MRI data demonstrated nearly complete inhibition of atherosclerosis after 6 months of treatment with ezetimibe in the

prophylactic model (Fig. 4A), while plaque progression was clearly visible in the three ascending arterial branches in the untreated adult therapeutic group (Fig. 4B). The combination of innominate, left carotid, and left subclavian artery plaque volume was used to evaluate the development of atherosclerosis over time. Ezetimibe produced almost complete inhibition of plaque progression in the prophylactic group while partial inhibition of progression was seen in the therapeutic models, with earlier treatment producing a greater beneficial effect (Fig. 4C). In addition, ezetimibe appeared to produce a rapid effect on blocking progression of atherosclerosis once it was administered, whether at 16, 24, or 40 weeks of age. MRI results were in excellent agreement with subsequent histological examination. Graf et al. utilized Sudan III staining and a novel near-infrared fluorescence imaging (NIRF) method specific for atherosclerotic lesions to monitor the effect of ezetimibe on atherosclerosis formation in ApoE knockout mice [34]. Mice were fed a high-fat diet (21% fat) with or without ezetimibe (5 mg/kg/day) for 4, 6, and 8 months followed by NIRF imaging and Sudan staining of the thoracic aorta. Serum cholesterol levels were significantly reduced by ezetimibe at 6 and 8 months compared with controls (6 months, 203 vs. 784 mg/dL; 8 months, 215 vs. 589 mg/dL; p < 0.01). At 4 months of age, Sudan III staining detected the presence of atherosclerotic lesions in both treated and untreated mice. Atherosclerosis significantly progressed at each subsequent time interval in the untreated group, with up to 40% of the total aortic area positively stained by 8 months. Ezetimibe-treated mice, however, showed minimal Sudan III staining and NIRF signal intensity at 6 and 8 months. Evaluation of lesion formation in brachiocephalic arteries produced similar results; control animals revealed a significant increase in plaque size between 4 and 8 months (from 7.2% to 32.0%) while ezetimibe produced a significant reduction (from 6.9% to 2.1%, p < 0.05). These results demonstrated an ezetimibe-mediated regression of atherosclerosis over this time period. 3.2. SR-B1/ApoE knockout mice SR-B1 is the receptor that typically clears ApoA1-containing HDL-C from the circulation. Braun et al. developed an

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Fig. 3. Atherosclerosis in ApoE-deficient mice after 3 months on a normal or high fat ± ezetimibe diet. (A) Representative photomicrographs of hematoxylin and eosin staining and Oil red O staining in cross-sections of the descending aorta (100× magnification). (B) Representative photomicrographs of Oil red O staining in longitudinally opened aorta. (C) Percent of intimal surface stained with Oil red O (pink color). *p < 0.01 compared with “normal diet”; † p < 0.01 compared with “high-fat diet” (Reprinted with permission from Atherosclerosis [30]. Copyright © 2008 Elsevier Ireland Ltd.).

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Fig. 4. (A) Ezetimibe treatment in the prophylactic group. MRI, (left) and histological (right) images after 26 weeks of treatment with 0.005% ezetimibe. IA = innominate artery, LC = left carotid artery, LS = left subclavian artery. (B) Untreated adult therapeutic group. Baseline MRI (left), 12-week MRI (middle) and 12-week histological (right) images. (C) Prophylactic and therapeutic effects of ezetimibe on plaque progression in three index arteries (combination of innominate, left carotid, and left subclavian arteries). Arrows indicate the initiation of ezetimibe therapy for prophylactic (6 weeks), early pre-adult (16 weeks), adult (24 weeks), or aged (40 weeks) models (Reprinted with permission from the International Society of Magnetic Resonance in Medicine [33]. Copyright © 2009).

sr-b1−/− /apoE−/− double knockout mouse model that significantly limits hepatic cholesterol clearance of both circulating ApoB- and ApoA1-containing lipoproteins. This model exhibits several characteristics not found in apoE−/− mice that are common in human coronary heart disease (CHD), including occlusive coronary arterial atherosclerosis, cardiac hypertrophy, myocardial infarction, cardiac dysfunction, and premature death at about 6 weeks of age [35]. These mice have been used to evaluate the effects of cholesterol absorption inhibitors on disease progression. In one study, mice received ∼12 mg/kg/day ezetimibe (0.005%, w/w) along with a standard chow diet at weaning (21 days old), and after three weeks of treatment they were compared with control animals receiving no drug [36]. Ezetimibe did not modify plasma levels of total cholesterol, phospholipids, triglycerides, HDL, or VLDL; however a 35% decrease of cholesterol in the IDL/LDL size range was observed (140 mg/dL for ezetimibe vs. 217 mg/dL for controls, p = 0.043). Ezetimibe significantly reduced atherosclerosis, with a 57% decrease in aortic sinus plaque area (Fig. 5A) and a 68% decrease in the percentage of severely occluded coronary arteries (Fig. 5B). Ezetimibe also significantly improved cardiac pathology, with an overall 57% reduction in myocardial fibrosis and a 12% decrease in cardiomegaly compared with controls (p = 0.015 and 0.04, respectively). In addition, ezetimibe increased the mean survival time by 27% (44 days for control animals vs. 56 days for ezetimibe, p < 0.0001).

Fig. 5. Effects of ezetimibe on atherosclerosis. sr-b1 (−/− )/apoE (−/− ) double knockout mice were fed standard chow with or without ezetimibe from weaning. Hearts were harvested at 39–44 days of age. (A) Oil red O-stained lesions in the aortic root. Average lesion areas (mm2 , horizontal lines) were no drug, 0.109 ± 0.032 (n = 4) and ezetimibe 0.047 ± 0.037 (n = 6). (B) Coronary arterial atherosclerosis. Left: representative Oil red O-stained sections of coronary arteries (neutral lipid stains red) from mice receiving no drug or ezetimibe. Bar: 50 ␮m. Right: extent of coronary arterial occlusions (average percent of coronary vessels with 0–10%, 10–50% or 50–100% occlusions). *p ≤ 0.01 for no drug vs. ezetimibe-treated (Reprinted with permission from Atherosclerosis [36]. Copyright © 2008 Elsevier Ireland Ltd.).

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3.3. LDL receptor knockout mice The LDL receptor (LDLR) is instrumental in the hepatic clearance of cholesterol from circulation and maintenance of cholesterol homeostasis, especially through clearance of circulating ApoB lipoproteins synthesized by the liver (ApoB-100) and intestine (ApoB-48). Studies were therefore conducted to assess the potential role of the LDLR in ezetimibe-mediated lowering of plasma cholesterol and reduction of atherosclerosis in ApoE knockout mice [37]. ldlr −/− /apoE−/− double knockout mice were fed a semi-synthetic, cholesterol-free diet with or without ezetimibe (5 mg/kg/day) and evaluated after six months. Ezetimibe reduced total cholesterol from 1205 mg/dL to 934 mg/dL and atherosclerotic lesion cross sectional area by 48% in the aorta and 20% in the carotid artery in the absence of any functional LDLR, demonstrating that the LDLR was not required for the reduction of cholesterol and atherosclerosis. Other studies in ldlr−/− mice suggest that inhibition of sterol absorption by ezetimibe and subsequent reduction of cholesterol delivery to the liver leads to a reduced rate of hepatic cholesterol secretion in the plasma [38]. The ATP binding cassette (ABC) G5 and G8 half transporters have been shown to play a key role in cholesterol and non-cholesterol sterol excretion from the body. Basso et al. were interested in evaluating the effect of liver-specific over-expression of the ABCG5/G8 transporter on reduction of atherosclerosis in LDLR knockout mice [39]. ldlr−/− crossed with ABCG5/G8 transgenic mice, which overexpress ABCG5/G8 only in the liver, and ldlr−/− control mice were fed a western diet (21.2% fat, 0.2% cholesterol) and evaluated over 24 weeks for levels of cholesterol absorption, biliary secretion, and atherosclerosis. In previous experiments, ldlr−/− × ABCG5/G8 mice demonstrated an increased secretion of biliary cholesterol compared with ldlr−/− control mice; however, levels of plasma cholesterol and atherosclerotic progression were similar for both (cholesterol levels between 250 and 300 mg/dL; mean proximal aortic lesion area of approximately 100–150 × 103 ␮m2 at 10 weeks and 250–300 × 103 ␮m2 at 13 weeks) due to the intestinal reabsorption and recirculation of cholesterol back to the liver [40]. In the current study, ldlr−/− × ABCG5/G8 mice that were fed a western diet containing ezetimibe (15 mg/kg/day) had their cholesterol reabsorption inhibited down to 5% and had a significant reduction in the mean aortic lesion area of 52–59% compared with ldlr−/− controls, demonstrating that inhibition of cholesterol absorption was required for reduction of atherosclerosis in the ldlr−/− × ABCG5/G8 mouse model. 3.4. Rabbit models Rabbit models have been used to elucidate the molecular mechanisms responsible for many aspects of atherosclerosis, including plaque formation, plaque composition, inflammation, and thrombosis. Rabbits have also been used to evaluate the effect of ezetimibe on atherosclerosis. Rabbits with pre-established, diet-induced hypercholesterolemia were treated with ezetimibe (0.6 mg/kg/day), lovastatin (1.2 mg/kg/day), or a combination of both for four weeks, and then assessed for levels of plasma cholesterol as well as atherosclerosis of the aortic arch and thoracic aorta, using cholesterol ester content as a marker for atherosclerotic progression [41]. Although lovastatin had no effect on plasma cholesterol levels or atherosclerosis, ezetimibe significantly reduced plasma cholesterol by 67% (primarily in the chylomicron and VLDL + LDL fractions, p < 0.05) and inhibited aortic atherosclerosis by 64% compared with control animals (∼1.8 mg cholesterol ester/g wet wt for ezetimibe vs. 5.0 mg/g for controls, p < 0.05). Co-administration of ezetimibe with lovastatin further enhanced the reduction of plasma and lipoprotein levels and caused significant reductions in atheroscle-

rosis compared with either lovastatin-treated animals or controls (p < 0.05). Recently, New Zealand rabbits were used to evaluate the effects of ezetimibe alone or in combination with simvastatin on intimamedia thickness (IMT) and atherosclerotic plaque composition [42]. Femoral atherosclerosis was induced by an atherogenic diet (containing 2% cholesterol and 6% peanut oil) and vascular injury, and animals were treated with ezetimibe (0.6 mg/kg/day), simvastatin (5 mg/kg/day), ezetimibe plus simvastatin, or no treatment for 6 weeks. Results showed a reduction in the intima/media ratio by ezetimibe (13%), simvastatin (27%), and ezetimibe plus simvastatin (28%) treatment compared with untreated animals (Fig. 6). In addition, ezetimibe and simvastatin monotherapy significantly reduced lipid-rich areas within the atherosclerotic lesions, and a further reduction was observed during combined treatment. Assessment of fibrosis within the femoral lesions showed no significant differences between any of the treatment groups. Evaluation of lesions for monocytes/macrophage infiltration showed significant reduction with ezetimibe and simvastatin alone, and significantly greater reduction with both drugs together (p < 0.01). Interestingly, ezetimibe treatment alone had no effect on hyperlipidemia, while ezetimibe plus simvastatin showed incremental but non-significant reductions in total cholesterol, VLDL, and LDL compared with placebo or ezetimibe-treated animals. In another study, Abela et al. used a rabbit model to assess the effect of ezetimibe on plaque progression and thrombosis [43]. Atherosclerosis was induced by balloon de-endothelialization followed by a high-cholesterol diet, either with or without ezetimibe (1 mg/kg/day). After six months, rabbits were triggered with Russell viper venom and histamine to induce plaque disruption [44], euthanized, and evaluated for levels of serum cholesterol and aortic wall cholesterol. A strong correlation was observed when comparing the thrombus area (mm2 ) to serum cholesterol levels (mg/dL) and arterial wall cholesterol levels (mg/g) (r2 = 0.5, p < 0.002). Lowering cholesterol levels with ezetimibe reduced thrombosis and cholesterol crystal formation.

4. Inhibition of atherosclerosis by ezetimibe: potential mechanisms of action Current understanding of the mechanisms believed to contribute to the development of atherosclerosis have been well described [2,3], and some degree of hyperlipidemia appears to be required. Long-term exposure of the arterial intima to elevated levels of LDL can promote continued lipoprotein retention, which leads to chronic inflammation and progression of disease. Ezetimibe is a potent inhibitor of intestinal cholesterol absorption and can reduce circulating levels of atherogenic ApoBcontaining lipoproteins. Cholesterol derived from the diet or biliary cholesterol from the liver enters the enterocyte by NPC1L1mediated transport [19] and is assembled with triglyceride and ApoB-48 into chylomicrons [45]. After hydrolysis and metabolism in plasma, the residual cholesterol-rich chylomicron remnant lipoproteins are removed from the circulation by the liver, which in turn adds to the hepatic cholesterol pool. In monkeys, ezetimibe prevents the increase in plasma cholesterol seen with a high-fat, high-cholesterol diet and has been shown to reduce the postprandial cholesterol content of chylomicrons, thereby decreasing the amount of cholesterol delivered by chylomicron remnants to the liver [46]. A significant reduction in plasma levels of ApoB-100containing particles (VLDL, IDL, LDL) is also observed, suggesting that reduced delivery of cholesterol to the liver results in compensatory up-regulation of hepatic LDL receptors and enhanced particle clearance. Studies of ezetimibe in mice [38] and miniature

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Fig. 6. Morphological lesions of femoral arteries of healthy control rabbits (A), normolipidemic diet (ND, B), untreated (C), ezetimibe-treated (Eze, D), simvastatin-treated (Simva, E) and ezetimibe + simvastatin-treated (Eze + Simva, F) rabbits. Photographs show a representative orcein-stained femoral section from each group. No lesions were observed in arteries from healthy control rabbits (A). Magnification 4× for all photographs. (G) Quantitative analysis of neointimal lesion size. Data are mean ± SEM, n = 6–18 arteries in each group from 4 to 10 animals per group. *p < 0.05 vs. untreated rabbits (Reprinted with permission from British Journal of Pharmacology [42]. Copyright © 2009 The British Pharmacological Society).

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pigs [47] provide direct evidence that reduced cholesterol absorption by ezetimibe increases LDL receptor expression and the rate of plasma LDL clearance. Similar effects have been seen in men with hypercholesterolemia, where ezetimibe significantly increased the fractional catabolic rate of LDL ApoB-100 by 24%, resulting in a decreased LDL ApoB-100 pool size of 23% [48]. The anti-atherosclerotic effects of reducing LDL-C and non-HDLC are firmly established, and this is presumed to represent the key mechanism responsible for inhibition of atherosclerosis by ezetimibe. It is possible, however, that additional mechanisms may also play a role. Efflux of cholesterol from macrophage foam cells by HDL-mediated reverse cholesterol transport (RCT) is thought to play a role in the stabilization and regression of atherosclerotic plaques [49]. Several in vivo studies have explored the effects of ezetimibe on RCT. In one study, ezetimibe suppressed dietary cholesterol absorption by 85% in mice, and promoted a six-fold increase in cholesterol efflux from macrophages to feces compared with untreated controls [50]. A similar study by Briand et al. showed that inhibition of NPC1L1 by ezetimibe increased macrophageto-feces transport by 87% compared with control mice, and the authors suggest that inhibition of intestinal cholesterol absorption, which promotes fecal excretion of HDL-derived cholesterol, was responsible for the effect [51]. Using a novel 13 C2 -cholesterol tracer methodology to assess global reverse cholesterol transport in rats, Turner et al. demonstrated that ezetimibe significantly increased the flux of cholesterol from plasma to feces (as neutral sterols), and produced an upward trend in calculated cholesterol efflux from tissues to plasma [52]. Similar results were seen in a randomized, double-blind, placebo-controlled crossover study of 31 hyperlipidemic patients, where ezetimibe was shown to significantly increase the flux of plasma-derived cholesterol into fecal neutral sterols by 52% (p = 0.035) [53]. Taken together, these findings suggest that inhibition of intestinal cholesterol absorption by ezetimibe has the potential to promote RCT; however, the clinical relevance of these findings is unknown.

5. Discussion Development of ezetimibe as a potent inhibitor of cholesterol absorption and subsequent discovery of the NPC1L1 cholesterol transporter has elucidated a key pathway involved in maintenance of cholesterol homeostasis. Evaluation of ezetimibe in a number of preclinical studies found no issues with safety or off-target biological effects, which is consistent with the safety and tolerability profiles observed in the clinic. Several animal models and a variety of detection methods have demonstrated the ability of ezetimibe to inhibit cholesterol absorption, lower plasma levels of cholesterolcontaining atherogenic lipoproteins, and impede development of atherosclerosis. A limited number of studies suggest that ezetimibe may also influence other processes associated with atherosclerosis; however, the clinical significance of these observations are currently hypothetical. While the beneficial effect of ezetimibe on atherosclerosis in animal models is unambiguous, similar effects in humans have not yet been clearly demonstrated. Several clinical trials have used carotid intimal-media thickness (cIMT) as a surrogate marker to evaluate the effects of ezetimibe combined with statins on atherosclerosis. The Ezetimibe and Simvastatin in Hypercholesterolemia Enhances Atherosclerosis Regression (ENHANCE) trial compared the effects of simvastatin 80 mg combined with either placebo or ezetimibe 10 mg in 720 patients with heterozygous familial hypercholesterolemia (HeFH) [54]. After 24 months, changes in mean cIMT were similar for the simvastatin alone and simvastatin plus ezetimibe arms (0.0058 ± 0.0037 mm and 0.0111 ± 0.0038 mm, respectively, p = 0.29). Some suggest that the normal baseline cIMT values

observed in this study (0.69–0.70 mm) and the prior aggressive lipid-lowering treatment already received by most of these patients may not have allowed for any additional reduction in cIMT [55]. This would be consistent with the fact that high dose atorvastatin treatment has also been seen to fail to reduce cIMT in similar HeFH populations that had also received prior aggressive treatment [56,57]. The Stop Atherosclerosis in Native Diabetics Study (SANDS) evaluated the effects of treatment to standard vs. aggressive lipid and blood pressure targets in 499 American Indians with Type II diabetes (standard targets – LDL-C < 100 mg/dL with statin alone and systolic blood pressure [SBP] < 130 mm Hg; aggressive targets – LDL-C < 70 mg/dL with statin alone or statin plus ezetimibe and SBP < 115 mm Hg). After three years of therapy, progression of cIMT was seen with standard treatment compared with a regression of cIMT during aggressive therapy. Betweentreatment differences were significant (p < 0.001) [58]. Post hoc analysis of patients receiving aggressive therapy showed that addition of ezetimibe to statins was as effective as higher-dose statin monotherapy in reducing cIMT (−0.025 mm and −0.012 mm, respectively, p = 0.999) [59]. Recently, the Arterial Biology for the Investigation of the Treatment Effects of Reducing Cholesterol 6 – HDL and LDL Treatment Strategies (ARBITER-6 HALTS) trial evaluated patients with CHD or CHD risk equivalents who were taking statins and had LDL cholesterol < 100 mg/dL and low HDL cholesterol. This study showed that the mean cIMT was unchanged after 14 months of treatment with ezetimibe 10 mg/day added to ongoing statin therapy (−0.0007 ± 0.0035 mm, p = 0.48) and significantly reduced with extended-release niacin 2000 mg/day added to statins (−0.0142 ± 0.0041 mm, p < 0.001) [60]. While no progression of intimal thickening was observed in the ezetimibe group, the potential effect of ezetimibe on preventing cIMT progression could not be evaluated since this study did not contain a placebo control arm. Development and progression of sub-clinical atherosclerosis ultimately results in cardiovascular disease, and three large clinical trials were designed to assess the effects of ezetimibe on clinical outcomes. The Simvastatin and Ezetimibe in Aortic Stenosis (SEAS) study evaluated the effect of simvastatin 40 mg plus ezetimibe 10 mg compared with placebo in 1873 patients with asymptomatic aortic stenosis who were not otherwise considered to require lipid lowering therapy. After a median follow-up of 52 months, therapy did not reduce the primary composite outcome of aortic valve-related events and ischemic cardiovascular events or a key secondary endpoint of aortic-valve-related events [61]. However, a significant 22% reduction in the other key secondary endpoint of ischemic cardiovascular events was observed (p = 0.02). Patients with less severe aortic stenosis showed the greatest reduction of ischemic events (47% reduction for patients in the lowest tertile, 36% reduction for patients in the middle tertile, no significant effect in the tertile of patients with the most severe aortic stenosis). The level of ischemic event reduction in the two tertiles of less severe aortic stenosis (where the authors proposed there was less confounding of “ischemic” events by aortic-stenosis-related events) was consistent with that expected for the magnitude of LDL-C lowering [62]. In the second trial, Study of Heart and Renal Protection (SHARP), over 9000 patients with advanced or endstage chronic kidney disease and no known history of myocardial infarction or coronary revascularization were treated with ezetimibe/simvastatin 10/20 mg vs. placebo for a median follow-up of 4.9 years [63]. Based on a comparison of ezetimibe/simvastatin 10/20 mg, simvastatin 20 mg alone and placebo during the first year of the study, the ezetimibe component of the combination accounted for a little more than 30% of the observed LDL-C reduction. At study end, ezetimibe/simvastatin had significantly reduced the incidence of first major atherosclerotic events (nonfatal MI or coronary death, non-hemorrhagic stroke, revascularization) by 17% [64]. This result is particularly noteworthy since one third of the

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SHARP population at baseline was already on dialysis (∼50% by study end), and two prior studies evaluating atorvastatin 20 mg and rosuvastatin 10 mg therapy in hemodialysis patients failed to demonstrate a significant benefit for their primary endpoints [65,66]. Major vascular events (the original SHARP primary endpoint, consisting of the same composite but including all strokes and all cardiac deaths analyzed in the sub-population of patients originally randomized to ezetimibe 10/20 mg or placebo) were significantly reduced by 16%. The SHARP investigators noted that the reduction in major atherosclerotic events in this trial was consistent with that predicted by the degree of LDL-C reduction, based on the largest meta-analysis of statin studies to date [67]. A third ongoing study, IMProved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE-IT), is directly comparing the efficacy of ezetimibe/simvastatin 10/40 mg vs. simvastatin 40 mg monotherapy in reducing major cardiovascular events in approximately 18,000 patients enrolled within 10 days of stabilized acute coronary syndrome and is estimated to complete in mid-2013 [12]. Based on the well-established link between elevated levels of atherogenic lipoproteins and cardiovascular disease, and the demonstration that reduction of LDL-C with statins and several other lipid-lowering therapies leads to reductions in atherosclerosis and cardiovascular events, the expectation is that ezetimibe will have similar favorable effect. The two clinical outcomes trials that have completed to date have shown that ezetimibe in combination with simvastatin can improve the clinical outcome of patients with asymptomatic aortic stenosis or advanced chronic kidney disease. The definitive demonstration of the clinical benefit of ezetimibe beyond that seen with statin monotherapy awaits the results of IMPROVE-IT. Conflict of interest HRD Jr., RSL, and DRN are employees of Merck. Acknowledgements The authors gratefully acknowledge the editorial assistance of Martha Vollmer, MA, of Merck. References [1] Lusis AJ. Atherosclerosis. Nature 2000;407:233–41. [2] Tabas I, Williams KJ, Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis – update and therapeutic implications. Circulation 2007;116:1832–44. [3] Tabas I. Macrophage death and defective inflammation resolution in atherosclerosis. Nat Rev Immunol 2010;10:36–46. [4] Kolodgie FD, Burke AP, Farb A, et al. The thin-cap fibroatheroma: a type of vulnerable plaque: the major precursor lesion to acute coronary syndromes. Curr Opin Cardiol 2001;16:285–92. [5] Steinberg D, Glass CK, Witztum JL. Evidence mandating earlier and more aggressive treatment of hypercholesterolemia. Circulation 2008;118:672–7. [6] Heart Protection Study Collaborative Group. MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet 2002;360:7–22. [7] Brown BG, Zhao XQ, Chait A, et al. Simvastatin and niacin, antioxidant vitamins, or the combination for the prevention of coronary disease. N Engl J Med 2001;345:1583–92. [8] The Lipid Research Clinics Coronary Primary Prevention Trial Results. I. Reduction in incidence of coronary heart disease. JAMA 1984;251:351–64. [9] Buchwald H, Williams SE, Matts JP, Nguyen PA, Boen JR. Overall mortality in the program on the surgical control of the hyperlipidemias. J Am Coll Surg 2002;195:327–31. [10] Zetia (ezetimibe) tablets [package insert]. North Wales, PA, USA: Merck/Schering-Plough Pharmaceuticals; 2009. [11] Bays HE, Neff D, Tomassini JE, Tershakovec AM. Ezetimibe: cholesterol lowering and beyond. Expert Rev Cardiovasc Ther 2008;6:447–70. [12] Cannon CP, Giugliano RP, Blazing MA, et al. Rationale and design of IMPROVEIT (IMProved Reduction of Outcomes: Vytorin Efficacy International Trial): comparison of ezetimbe/simvastatin versus simvastatin monotherapy on cardiovascular outcomes in patients with acute coronary syndromes. Am Heart J 2008;156:826–32.

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