Therapeutic elevation of HDL-cholesterol to prevent atherosclerosis and coronary heart disease

Pharmacology & Therapeutics 111 (2006) 893 – 908 www.elsevier.com/locate/pharmthera

Associate editor: K.E. Suckling

Therapeutic elevation of HDL-cholesterol to prevent atherosclerosis and coronary heart disease M. John Chapman ⁎ Dyslipoproteinemia and Atherosclerosis Research Unit (UMR-551), National Institute for Health and Medical Research (INSERM), France University Pierre and Marie Curie, Pavillon Benjamin Delessert, Hôpital de la Pitié, 83 Bd de l'hôpital, 75651 Paris Cedex 13, France

Abstract Innovative pharmacological approaches to raise anti-atherogenic high-density lipoprotein-cholesterol (HDL-C) are currently of considerable interest, particularly in atherogenic dyslipidemias characterized by low levels of HDL-C, such as type 2 diabetes, the metabolic syndrome, and mixed dyslipidemia, but equally among individuals with or at elevated risk for premature cardiovascular disease (CVD). Epidemiological and observational studies first demonstrated that HDL-C was a strong, independent predictor of coronary heart disease (CHD) risk, and suggested that raising HDL-C levels might afford clinical benefit. Accumulating data from clinical trials of pharmacological agents that raise HDL-C levels have supported this concept. In addition to the pivotal role that HDL-C plays in reverse cholesterol transport and cellular cholesterol efflux, HDL particles possess a spectrum of anti-inflammatory, anti-oxidative, anti-apoptotic, anti-thrombotic, vasodilatory and anti-infectious properties, all of which potentially contribute to their atheroprotective nature. Significantly, anti-atherogenic properties of HDL particles are attenuated in common metabolic diseases that are characterized by subnormal HDL-C levels, such as type 2 diabetes and metabolic syndrome. Inhibition of cholesteryl ester transfer protein (CETP), a key player in cholesterol metabolism and transport, constitutes an innovative target for HDL-C raising. In lipid efficacy trials, 2 CETP inhibitors-JTT-705 and torcetrapib-induced marked elevation in HDL-C levels, with torcetrapib displaying greater efficacy. Moreover, both agents attenuate aortic atherosclerosis in cholesterol-fed rabbits. Clinical trial data demonstrating the clinical benefits of these drugs on atherosclerosis and CHD are eagerly awaited. © 2006 Elsevier Inc. All rights reserved. Keywords: Therapeutic elevation; HDL-cholesterol; Atherosclerosis; Coronary heart disease

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Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The association between HDL-C and CHD risk . . . . . . . . . . . . . HDL-C and protection against atherosclerosis development . . . . . . . 3.1. Cholesterol homeostasis and reverse cholesterol transport . . . . 3.2. Anti-inflammatory effects . . . . . . . . . . . . . . . . . . . . . 3.3. Anti-oxidative actions . . . . . . . . . . . . . . . . . . . . . . . 3.4. Anti-infectious effects . . . . . . . . . . . . . . . . . . . . . . . Factors known to reduce HDL-C levels or alter HDL anti-atherogenicity HDL-C and guideline recommendations . . . . . . . . . . . . . . . . . Clinical management of low HDL-C levels . . . . . . . . . . . . . . .

⁎ Tel.: +33 1 42 17 78 78; fax: +33 1 45 82 81 98. E-mail address: [email protected]. 0163-7258/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2006.02.003

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6.1. 6.2.

Statins . . . . . . . . . . . . . . . . . . . . Agonists of peroxisome proliferator-activated 6.2.1. PPARα agonists: fibrates . . . . . . 6.2.2. PPARγ agonists. . . . . . . . . . . 6.2.3. PPARβ/δ agonists. . . . . . . . . . 6.2.4. Mixed PPAR agonists: PPARα/γ . . 6.3. Niacin . . . . . . . . . . . . . . . . . . . . 7. Novel therapeutic strategies . . . . . . . . . . . . . 7.1. JTT-705 . . . . . . . . . . . . . . . . . . . 7.2. Torcetrapib . . . . . . . . . . . . . . . . . . 8. Summary . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

1. Background Current international recommendations for the prevention of coronary heart disease (CHD) emphasize reductions in low-density lipoprotein-cholesterol (LDL-C) levels in individuals with or at elevated risk of premature development of cardiovascular disease (CVD) (Expert Panel on Detection Evaluation and Treatment of High Blood Cholesterol in Adults, 2001; De Backer et al., 2003). Several prospective intervention trials have established that statins are the drug of choice for efficacious reduction of LDL-C levels and have shown consistent benefit in terms of diminution in cardiovascular events in both the primary and secondary prevention of CVD (Expert Panel on Detection Evaluation and Treatment of High Blood Cholesterol in Adults, 2001). Yet, while statins have demonstrated risk reductions of 25–40% in clinical trials, a substantial proportion of treated patients retain an elevated residual CVD risk and still experience cardiovascular events. Even in patients receiving aggressive statin therapy, as exemplified by the Treating to New Targets trial, which compared atorvastatin 80 mg/day with atorvastatin 10 mg/day in patients with CHD, a relatively high event rate was observed (LaRosa et al., 2005). The socioeconomic burden attributed to cardiovascular events is considerable and, therefore, innovative, complementary therapeutic strategies for the prevention and management of CVD are required. At present, there is a great deal of interest in the clinical benefit associated with raising high-density lipoproteincholesterol (HDL-C) levels. This is partly because HDL-C has been shown to be a strong, independent, inverse risk factor for CHD but also because several new therapies that can substantially raise HDL-C are now in the later stages of clinical development. Such therapies are viewed as having major potential in clinical practice, not just for the treatment of high-risk patient populations characterized by low HDL-C levels, such as patients with type 2 diabetes or the metabolic syndrome, but equally as they provide a means to address the residual cardiovascular risk that exists in the broader group of individuals with or at risk of CHD who are receiving statin therapy. Indeed, the finding from the INTERHEART

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study across 52 countries worldwide that the apolipoprotein (apo) B/apol A-I ratio–a surrogate for the LDL-C/HDL-C ratio–was the parameter most predictive of myocardial infarction (Yusuf et al., 2004) suggests that therapeutic strategies which concomitantly target elevated LDL-C and low HDL-C levels are likely to be most effective in the treatment of CVD. 2. The association between HDL-C and CHD risk Several epidemiologic studies have demonstrated that HDLC is a strong, independent, inverse predictor of CHD risk (Fig. 1) (Gordon et al., 1977; Castelli et al., 1986; Assmann et al., 1996; Sharrett et al., 2001). In the Framingham study, which evaluated men and women aged 49–82 years who were free of CHD at baseline, HDL-C was the most potent lipid risk factor for CHD-more so than LDL-C, total cholesterol, or triglyceride levels (Gordon et al., 1977). In a 12-year follow-up of the Framingham participants, those with high HDL-C (80th percentile) were at 50% lower risk of cardiovascular events than those with low HDL-C (20th percentile) (Castelli et al., 1986). In the Prospective Cardiovascular Munster (PROCAM) study, participants with HDL-C ≥ 35 mg/dL had a 4-fold lower risk of CHD after a 6-year follow-up compared with those whose HDL-C was <35 mg/dL (Assmann et al., 1996). In a 10-year follow-up of the Atherosclerosis Risk in Communities (ARIC) Study, the strong and continuous association that was observed between progressive increase in HDL-C levels and parallel reduction in CHD risk was apparent in both men and women who were initially free of CHD (Fig. 2) (Sharrett et al., 2001). Based on the available evidence, it has been estimated that there is a 2–3% decrease in cardiovascular risk for every 1-mg increase in HDL-C (Gordon et al., 1989). Consequently, raising HDL-C is a potentially promising and innovative treatment strategy for preventing CVD not only in subjects with low HDL-C levels but also in subjects with quasi-normal HDL-C concentrations who display elevated ratios of atherogenic to non-atherogenic cholesterol, such as in hypercholesterolemia.

M.J. Chapman / Pharmacology & Therapeutics 111 (2006) 893–908

Fig. 1. HDL-C as a predictor of CHD risk. (A) Data from a 6-year follow up of the PROCAM study showing that the incidence of CHD decreases with higher levels of HDL-C (Assmann et al., 1996). (B) Data from the Framingham study showing that high levels of HDL-C reduce the risk of CHD at all levels of LDL-C (Gordon et al., 1977).

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2000) and minor components such as liposoluble vitamins and antioxidants (Goulinet & Chapman, 1997). The amphipathic outer layer of HDL contains the phospholipids, apolipoproteins, and free cholesterol, whereas the hydrophobic inner core consists primarily of cholesteryl ester (CE) and variable amounts of triglycerides. The relationship between HDL and its cardioprotective effects is complex due to the heterogeneity of the HDL lipoprotein class. HDL particles have been classified into various subpopulations, which differ markedly in physicochemical properties (eg, apolipoprotein/lipid composition, particle size, isoelectric point, and charge). Thus, apolipoprotein content distinguishes particles into those containing only apo A-I (Lp A-I) and those that contain both apo A-I and apo A-II (Lp A-I/A-II), while separating HDL subpopulations on the basis of size and charge by 2-dimensional non-denaturing gel electrophoresis, identifies up to 12 subpopulations classified as having either alpha or beta mobility. Despite the considerable resolution of HDL particle subpopulations by 2D electrophoretic technology, this method is primarily limited to an analytical approach. Moreover, individual electrophoretically-separated HDL subpopulations, such as α1, α2, and pre-β, may be highly heterogeneous with regard to the respective physical and chemical properties of their particle content. It is therefore relevant that isopycnic density gradient centrifugation facilitates reproducible preparative resolution of 5 HDL particle subpopulations (HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c), thereby permitting structural and functional studies (Blanche et al., 1981; Chapman et al., 1981; Kontush et al., 2003). These subpopulations are comparable to their counterparts separated and identified by

3. HDL-C and protection against atherosclerosis development HDL particles are pseudo-micellar, protein–lipid complexes that vary in density, size and chemical composition. Approximately 1/3 to 1/2 of the mass of HDL is comprised of highly specialized proteins—termed apolipoproteins (apo)predominantly apo A-I and apo A-II (Thompson et al., 2004). These apolipoproteins play a key role in maintaining the structural integrity of HDL particles but equally exert a major influence on the intravascular metabolism and function of HDL. Other apolipoproteins may also be present, such as apo A-IV, apo AV, apo C-I, apo C-II, apo C-III, apo E, and apo J. Of these, apo A-I is most intimately associated with the antiatherogenic activities of HDL. Other proteins associated with HDL include enzymes such as paraoxonase 1, plateletactivating factor acetylhydrolase (PAF-AH or Lp.PLA2), and lecithin/cholesteryl acetyl transferase (LCAT), and lipid transfer factors such as cholesteryl ester transfer protein (CETP) and PLTP (Gotto & Brinton, 2004). The remaining half of the mass of HDL is composed of phospholipids, free and esterified cholesterol, and triglycerides (Asztalos et al.,

Fig. 2. HDL-C quintiles and relative risk of CHD for women (A) and men (B) in the ARIC study (adjusted for age and race) (Sharrett et al., 2001).

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gradient gel electrophoresis (Blanche et al., 1981; Chapman et al., 1981; Kontush et al., 2003). There is evidence that some HDL subpopulations correlate more closely with CHD risk than others. In the VA-HIT trial (described in more detail in Section 6), HDL3, but not HDL2, at baseline and as a percentage change during treatment was significantly related to the development of new CHD events (Robins et al., 2001). In the Framingham Offspring Study, male CHD patients had significantly lower levels of large alpha-1 particles and significantly higher levels of alpha-3 and pre beta-1 particles compared with all or HDL-C-matched controls (Asztalos et al., 2004). The multiple mechanisms by which HDL may protect against the development of atherosclerosis are summarized in Table 1. In addition to its role in reverse cholesterol transport, HDL can attenuate inflammatory activity, promote antioxidative effects, exert vasodilatory properties and reduce endothelial dysfunction, protect arterial wall cells against apoptosis, and exert anti-thrombotic and anti-infectious actions (Calabresi et al., 2003; Barter et al., 2004; Khovidhunkit et al., 2004).

Table 1 Anti-atherogenic activities of HDL particles Activity

Documented protective effects

1. Facilitation of reverse ▪ Efflux of cholesterol from foam cells in artery cholesterol transport wall (Zhang et al., 2003) 2. Anti-inflammatory ▪ Inhibition of the synthesis of platelet-activating factor (Sugatani et al., 1996) ▪ Inhibition of leukocyte adhesion to the arterial wall via attenuation of the expression of VCAM-1 (Barter et al., 2002) and other cytokineinduced cell adhesion molecules (Cockerill et al., 2001; Nicholls et al., 2005a) ▪ Inhibition of expression of MCP-1 (Navab et al., 1991; Mackness et al., 2004) 3. Improved endothelial ▪ Stimulation of endothelial NO synthase activity function (Yuhanna et al., 2001; Mineo et al., 2003; Nofer et al., 2004) ▪ Enhanced endothelium-dependent vasodilation (Spieker et al., 2002) ▪ Prevention of endothelial cell apoptosis (Kimura et al., 2001; Nofer et al., 2001) ▪ Stimulation of prostacyclin synthesis (Fleisher et al., 1982) 4. Anti-oxidative ▪ Protection of LDL from oxidation (Kontush et al., 2003): ▪ via apo AI-mediated anti-oxidative actions (Navab et al., 2000) ▪ via paraoxonase-mediated anti-oxidative actions (Mackness et al., 2004) 5. Anti-thrombotic ▪ Protection of erythrocytes against the generation of procoagulant activity (Epand et al., 1994) ▪ Stimulation of prostacyclin synthesis (Navab et al., 1991) ▪ Inhibition of thrombin-induced endothelial tissue factor expression (Viswambharan et al., 2004) 6. Anti-infectious ▪ Reduction of the pyrogenic activity of bacterial lipopolysaccharide (Ulevitch et al., 1979) ▪ Lysis of Trypanosoma Brucei Hajduk et al. (1989)

3.1. Cholesterol homeostasis and reverse cholesterol transport Cholesterol homeostasis in the whole organism involves 2 opposing processes: (1) the transfer of cholesterol of both exogenous and endogenous origin from the liver to peripheral tissues via apo B-containing lipoproteins (very low-density lipoprotein [VLDL], intermediate-density lipoprotein [ILDL], and low-density lipoprotein [LDL]) for cellular uptake by the LDL receptor and (2) the transfer of cholesterol from peripheral tissues and the arterial wall back to the liver for removal from the body by biliary secretion of both bile acids and cholesterol. When considered as a concerted biological pathway, this second process is known as reverse cholesterol transport. The central role that HDL plays in reverse cholesterol transport is pivotal to cholesterol homeostasis in the whole organism, and is believed to constitute the principal mechanism by which HDL exerts an atheroprotective effect on the vasculature (Toth, 2003; Wang & Briggs, 2004). The key pathways of reverse cholesterol transport are depicted in Fig. 3. The process can be considered to begin with the efflux of excess intracellular free cholesterol from peripheral cells by 1 of several routes. Most commonly cited is cholesterol efflux mediated via the ATP-binding cassette transporter A1 (ABCA1) (Brewer et al., 2004). In this case, lipid-poor apo A-I secreted by the intestine and liver or liberated during intravascular remodeling of HDL particles interacts with ABCA1 and acquires free cholesterol and phospholipid to form discoidal pre-beta HDL (also known as nascent HDL). The free cholesterol in pre-beta HDL is esterified by LCAT to CE, generating a neutral lipid core and resulting in the formation of mature, spherical, alpha-migrating HDL. Alternative routes of cholesterol efflux from peripheral cells include the scavenger receptor class B, type 1 (SR-B1) receptor (Williams et al., 1999) the recently identified ABCG1 receptor (Wang et al., 2004) and passive diffusion (Yancey et al., 2003). Unlike ABCA1mediated cholesterol efflux, the major acceptor for cholesterol effluxed via these latter routes appears primarily to be mature, alpha-HDL (Asztalos et al., 2005). Cholesterol associated with mature alpha-HDL can be returned to the liver via 1 of at least 3 pathways. Firstly, HDL particles can deliver cholesterol to the liver, mostly as free cholesterol but also as CE, by direct interaction with hepatic receptors—primarily via the SR-B1 receptor, which facilitates delivery as free cholesterol and as CE by a selective uptake process, and to a lesser degree, via the LDL receptor, when HDL particles contain apo E (Ji et al., 1999; Williams et al., 1999). Through this process, both lipid-poor apo A-I particles and spherical HDL particles depleted in free cholesterol and CE are regenerated. The apo A-I particles can be recycled to stimulate further ABCA1-mediated efflux from peripheral cells but are also susceptible to glomerular filtration and endocytosis by the cubulin/megalin receptors in the kidney (Kozyraki, 2001). Secondly, holo-HDL particles may potentially be endocytosed by hepatocytes, although the precise identity of the specific receptor(s) involved remains controversial. Thirdly, the CE content of HDL particles can be transferred to atherogenic apo B-containing lipoproteins with ultimate uptake

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Peripheral Cells; Macrophages

FC

FC

ABCA1

ABCG1 LCAT

Lipid-Poor Apo AI

FC

LCAT

HDL3 CE:FC

SRB1

HDL2 CE:FC

CE

Pre-ß-HDL CETP

HL LCAT PLTP EL

SRB1 Hepatocyte

FC, CE

CE

VLDL VLDL-R IDL LDL

HDL2 Apo E CE:FC

HDL.R

LDL.R

Holo-HDL

FC, CE

Fig. 3. Intravascular apo A-I and HDL particle remodelling, reverse cholesterol transport, and the role of CETP. Refer to manuscript text for further explanation.

by LDL receptors in the liver. Pivotal to this mechanism is the action of CETP, which is secreted primarily by the liver and circulates in plasma associated mostly with HDL (Tall, 1993). CETP promotes net mass transfer of CEs from HDL to apo B-containing lipoproteins in heteroexchange for triglycerides, which move in the opposite direction. Recent evidence suggests the CETP-mediated transfer of CE to apo B-containing lipoproteins may account for an elevated proportion of the total cholesterol returned to the liver in humans (Schwartz et al., 2004). Equally, the CE in apo B-containing lipoproteins may be returned to peripheral cells via LDL receptor-mediated uptake. CETP therefore possesses the potential to simultaneously exert both pro-atherogenic and anti-atherogenic effects. The potential of CETP as a therapeutic target is discussed in greater detail in Section 7. In addition to CETP-mediated heterotransfer of CE and TG between HDL and apo B-containing lipoproteins, several other key component processes of HDL metabolism, of HDL particle recycling, and of reverse cholesterol transport represent potential therapeutic targets for efficacious raising of circulating HDL-C level (Fig. 3). These are (i) stimulation of hepatic and/or intestinal apo A-I production, (ii) stimulation of cellular cholesterol efflux to primary acceptors, preferentially in peripheral tissue and arterial macrophages via stimulation of ABCA1 transporter activity, (iii) stimulation of cholesterol efflux via ABCG1 and/or ABCG4 transporters and/or the SRB1 receptor in peripheral macrophages to mature spherical HDL particles, (iv) attenuation of the activity of the hepatic and/or endothelial lipases, (v) reduction in the uptake of mature HDL particles by putative holo-HDL particle receptors in the liver, and (vi) enhanced lipolysis of triglyceride-rich lipoproteins by upregulating lipoprotein lipase activity, and thereby liberating surface fragments in increased amounts which may sequester to the HDL pool. It is noteworthy that strategies targeting

mechanisms (ii)–(iv) all involve enhanced lipidation of apo A-I, thereby increasing its residence time in circulating HDL particles. As discussed below, present knowledge of the mechanisms of action of currently available agents for HDL-C raising (statins, fibrates as PPARα agonists, niacin and CETP inhibitors) indicates that they act by regulating 1 or more of these component processes of HDL metabolism. 3.2. Anti-inflammatory effects Atherosclerosis is now known to be a chronic inflammatory disorder characterized by activation of endothelial cells and by accumulation of macrophages and T lymphocytes in the arterial intima (Libby et al., 2002). Indeed, endothelial dysfunction–a direct deleterious effect of the integrated action of diverse risk factors at the endothelial surface (for example, dyslipidemia, elevated blood pressure, hyperglycemia, and smoking)–is frequently manifested during the early stages of atherosclerosis and is characterized by reduced nitric oxide (NO) bioavailability. Reduced NO bioavailability is associated with increased leukocyte affinity, impaired vascular tone, and increased levels of oxygen free radicals (Weissberg, 2000). HDL particles exert an array of effects that impact the atherosclerotic inflammatory process. Critically, during early atheroma development, HDL inhibit leukocyte adhesion to the arterial wall by attenuating the expression of vascular cell adhesion molecule (VCAM-1) and monocyte chemotactic protein 1 (MCP-1), and inhibit the oxidative modification of LDL (see below) (Barter et al., 2002). These atheroprotective actions of HDL have been demonstrated in a porcine model, in which the injection of reconstituted discoidal HDL inhibited endothelial cell activation (Cockerill et al., 2001) and in a rabbit model, in which infusion of reconstituted HDL, apo A-I, and

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phospholipids inhibited early pro-oxidant and pro-inflammatory changes induced by a carotid periarterial collar (Nicholls et al., 2005a). HDL have been shown to produce an additional anti-adhesive effect by inhibiting platelet-activating factor (PAF)-induced adhesion of leukocytes to the activated endothelium, as observed in a study using human endothelial cells (Sugatani et al., 1996). HDL can also increase NO bioavailability by stimulating endothelial NO synthase activity. This may be brought about by HDL binding to SR-B1 (Yuhanna et al., 2001; Mineo et al., 2003) or to the lysophospholipid receptor S1P3 (Nofer et al., 2004). In the latter case, the endothelial protective action of HDL is believed to be mediated by bioactive lysophospholipids that are present in small quantities in HDL (Nofer et al., 2004). Of note, it has been demonstrated that endothelium-dependent vasodilation was improved and NO bioavailability increased in hypercholesterolemic patients infused intravenously with reconstituted HDL (Spieker et al., 2002). In addition to the beneficial effects that HDL may exert on endothelial cell adhesive function and NO bioavailability, in vitro studies have also demonstrated that HDL can prevent endothelial cell apoptosis (Kimura et al., 2001; Nofer et al., 2001). Recently, it has become clear that dysfunctional HDL particles in patients with CVD lack atheroprotective properties and may actually be pro- rather than anti-inflammatory (Ansell et al., 2005). Myeloperoxidase (MPO)-generated oxidants appear to be key participants in generating this dysfunctional HDL (Nicholls et al., 2005b). MPO is known to bind to HDL via apo A-I and this has been shown to facilitate the selective targeting of apo A-I for site-specific chlorination and nitration by MPO-generated reactive oxidants in vivo. One consequence of MPO-catalyzed apo A-I oxidation is the reduced capacity of HDL to promote cellular cholesterol efflux via ABCA1. Thus, MPO-induced loss of the atheroprotective properties of HDL provides a good example of the interplay between inflammation and oxidative stress in the development of atherosclerotic lesions. 3.3. Anti-oxidative actions Oxidative stress is a well documented risk factor for CVD and, as a component of inflammation, is a key contributor to atherosclerotic plaque initiation and progression. Diminution of NO bioavailability and a local increase in oxygen free radical production in the arterial wall promotes oxidative stress and, subsequently, LDL oxidation. Oxidized LDL particles exert a spectrum of potent pro-inflammatory activities, including stimulation of production of oxygen-free radicals, notably in monocyte-derived macrophages, thereby enhancing oxidative modification of native LDL particles (Ohara et al., 1993; Young & Parthasarathy, 1994; Rader & Dugi, 2000). Oxidized LDL are recognized and internalized via several distinct receptor families on the macrophage, leading to intracellular cholesterol accumulation and transformation to proinflammatory foam cells (Ross, 1999). Accumulating evidence shows that HDL protects LDL from oxidation (Assmann & Gotto, 2004; Chapman et al., 2004).

Mackness et al. (2000) first demonstrated that HDL particles possess the capability to protect atherogenic LDL from structural modification as a result of oxidative stress while Bowry et al. (1992) showed that HDL may act as a sink for oxidized lipids and, notably, lipid hydroperoxides. Most recently, Kontush et al. (2003) have shown that among the HDL particle subpopulations in normolipidemic subjects, it is the small dense HDL particles which possess the most potent anti-oxidative activity. Such activity appears to represent the sum of the activities of several HDL components, including apo A-I, phospholipids, and anti-oxidative enzymes, such as paraoxonase 1. Apo A-I has been shown to reduce peroxides of phospholipids and CEs and to remove products of 12lipoxygenase from native LDL (Navab et al., 2000, 2001). Paraoxonase 1 and lipoprotein-associated phospholipase A2 (i.e. PAF acetylhydrolase) can inactivate LDL-derived oxidized phospholipids (Assmann & Gotto, 2004; Chapman et al., 2004); indeed, animals deficient in paraoxonase 1 were observed to be more susceptible to diet-induced atherosclerosis (Shih et al., 1996, 1998). Supportive cohort study data from the Caerphilly Prospective Study has shown that low paraoxonase 1 activity was a significant risk factor for coronary events in men at high risk for CHD (Mackness et al., 2003). The neutral lipid core content (CE and triglycerides) in HDL also appears to exert major influence on HDL anti-oxidative activity. Indeed, when the triglyceride/CE ratio in the HDL core is elevated, as in metabolic diseases such as type 2 diabetes and the metabolic syndrome, there is significant attenuation (up to 50%) of the anti-oxidative activity of dense HDL-3, as measured on a perparticle basis. The influence of neutral lipid content on the antioxidative activity of HDL appears to be expressed, at least in part, via its impact on apo A-I conformation (Hansel et al., 2004; Nobecourt et al., 2005). 3.4. Anti-infectious effects There is some evidence that HDL particles can exert antiinfectious effects. This property of HDL was first demonstrated by Ulevitch et al. (1979), who showed that the ability of plasma to inhibit the pyrogenic activity of bacterial lipopolysaccharide, lost after delipidation, was restored after the addition of HDL. Equally of note is the capacity of HDL particles to induce lysis of the parasite Trypanosoma brucei (Hajduk et al., 1989). 4. Factors known to reduce HDL-C levels or alter HDL anti-atherogenicity Low levels of HDL-C are common in certain populations, such as smokers, patients with type 2 diabetes or the metabolic syndrome, the obese, the physically inactive, individuals on very high carbohydrate diets (>60% of energy intake), persons with mixed dyslipidemia, patients with hypertriglyceridemia of any cause, and patients taking certain medicines (e.g. betablockers, anabolic steroids, progestational agents) (Expert Panel on Detection Evaluation and Treatment of High Blood Cholesterol in Adults, 2001). In many individuals there may be a primary genetic cause for low HDL-C (Genest et al., 1992)

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including mutations or deficiency of the apo A-I, ABCA1, LCAT and CETP genes (Assmann et al., 1993; Lawn et al., 1999). One recent study has estimated that nearly 40% of the variation in HDL-C levels between individuals is genetically determined, of which 25% may be attributed to functional polymorphisms in the CETP gene (Knoblauch et al., 2004) such as promoter polymorphism −629 C (Dachet et al., 2000). In some dyslipidemic individuals, HDL particles may be transformed to dysfunctional, lipid-poor, small HDL with diminished residence time in the plasma compartment. For instance, in moderate hypertriglyceridemia, high CETP activity preferentially targets elevated levels of triglyceride-rich VLDL-1 leading to their enrichment in CEs but equally to enhanced triglyceride content in HDL (Guerin et al., 2001). Hepatic lipase hydrolyzes triglyceride-enriched HDL to form small dense HDL. Such small dense HDL of abnormal composition (high triglyceride/CE ratio) has a shorter half-life in plasma than large HDL and, as a result, HDL levels decrease (Lewis & Rader, 2005). Small dense HDL particles in hypertriglyceridemic states also exhibit impaired anti-oxidative activity versus larger HDL particles, as demonstrated recently in a study that compared plasma and serum from patients with the metabolic syndrome with that from matched, normolipidemic, healthy controls (Hansel et al., 2004). In the metabolic syndrome patients, systemic oxidative stress, as measured by plasma 8isoprostane levels, was elevated 4-fold and small, dense HDL subfractions displayed altered chemical composition (notably a high triglyceride/CE core lipid ratio) with impaired antioxidative activity. Because patients with the metabolic syndrome exhibit moderately elevated levels of glucose in plasma, there may be glycation of apo A-I. This abnormality of apo A-I, along with the potential for elevated oxidative stress, is known to impair the anti-atherogenic properties of HDL, including the ability to act as lipid acceptors and to promote cellular cholesterol efflux (Decossin et al., 1995; Therond et al., 1999). Similarly, abnormal intrinsic physicochemical properties of small, dense HDL particles in patients with type 2 diabetes has recently been shown to result in reduced anti-oxidative activity (Nobecourt et al., 2005). Thus, patients with low HDL-C levels may therefore be at additional risk as a result of both quantitative and qualitative anomalies in HDL particles. 5. HDL-C and guideline recommendations Guidelines from the National Cholesterol Education Program (NCEP) reflect the importance of HDL-C as a major CV risk factor. In the NCEP ATP II guidelines, an HDL-C level of > 60mg/dL was classified as a negative risk factor for CVD (Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II), 1993). In 2003, the revised NCEP guidelines (ATP III) modified the definition of low HDL-C from ≤ 35mg/dL to ≤ 40mg/dL and introduced the concept of 10-year absolute CHD risk (Expert Panel on Detection Evaluation and Treatment of High Blood Cholesterol in Adults, 2001). The introduction of a CHD risk score indirectly

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raises the prominence of HDL-C, since a high level of HDL-C reduces the score. The NCEP ATP III guidelines also identify low HDL-C as a risk determinant for the metabolic syndrome and recognize low HDL-C as a potential secondary target of therapy in (i) those with isolated low HDL-C (if triglycerides are <200 mg/dL) and (ii) when present as a component of the metabolic syndrome. Consistent with the NCEP ATP III recommendations for HDL-C in metabolic syndrome, the International Diabetes Federation recently announced a new definition for this syndrome, in which low HDL-C is now identified as a key criterion (International Diabetes Federation, 2005). Although the NCEP guidelines emphasize that low HDL-C is an important risk factor for CVD, no specific targets for its elevation are stipulated. Nevertheless, other influential bodies in both Europe (the European Consensus Panel on HDL-C) (Chapman et al., 2004) and the US (the Expert Group on HDL-C) (Sacks, 2002) have published papers in which targets for HDL-C are suggested. With specific regard to CVD prevention in women, the American Heart Association's recent evidence-based guidelines state that the optimal level of HDL-C is >50 mg/dL (Mosca et al., 2004). Reduced levels of HDL-C are of particular concern in patients with Type 2 diabetes. Over the years, HDL-C has risen in prominence in the guidelines provided by the American Diabetes Association. In 2003, optimal HDL-C levels were defined as > 40mg/dL, with low risk defined as an HDL-C ≥ 60 mg/dL and high risk defined as an HDL-C < 40mg/dL (these HDL values should be increased by 10 mg/dL in women) (American Diabetes Association, 2003). In 2004, an HDL-C target of > 40mg/dL was recommended (Haffner, 2004); a goal of >50 mg/dL can be considered in women. These targets were sustained in the 2005 recommendations (Standards of Medical Care in Diabetes, 2005). 6. Clinical management of low HDL-C levels The available options for elevating low HDL-C levels have been recently reviewed by Ashen and Blumenthal (2005). While HDL-C levels may be increased by 10% or more by implementing therapeutic lifestyle changes, including weight reduction (Wilsgaard & Arnesen, 2004), exercise (SpateDouglas & Keyser, 1999), smoking cessation (Maeda et al., 2003) and moderate alcohol consumption (Hannuksela et al., 1992), many patients will also require pharmacologic intervention. Current therapeutic options are summarized in Table 2 and are described in more detail below. 6.1. Statins Statins produce modest elevations in HDL-C (up to 16%) (Brewer, 2003). The mechanisms implicated in such elevation are incompletely understood. Nonetheless statins attenuate CETP-mediated CE transfer to apo B-containing particles partially by lowering plasma CETP concentration and by reducing the number of apo B-containing lipoprotein particles that are available to accept CE (Le Goff et al., 2004). Statins

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Table 2 Lipid modulating drugs and effects on HDL-C: present and future Drug class Existing therapies Statins

Mechanism of action for HDL elevation Effect on HDL

Lower plasma CETP concentration; reduce CETP-mediated transfer of CE to acceptor apo B-containing lipoproteins via reductions in particle number (Le Goff et al., 2004); enhance hepatic apo A-I production (Schaefer et al., 1999) Fibrates Upregulate expression of apo A-I, apo A-II, SR-B1, and ABCA1 through the binding and activation of PPAR-alpha; increase lipolysis of VLDL, with enhanced release of surface fragments to HDL, via upregulation of LPL expression (Chapman, 2003) Niacin Suppresses lipolysis of triacylglycerol in adipose tissue via inhibition of the hormone-sensitive triglyceride lipase (Karpe & Frayn, 2004; Carlson & Oro, 1962), thereby decreasing triglyceride levels; stimulates cholesterol efflux by activating ABCA1 (Rubic et al., 2004) Thiazolidinediones Unclear, although probably related to the observed reduction in triglyceride levels (Raskin et al., 2001)

↑ up to 16% (Brewer, 2003)

↑ up to 10% (Birjmohun et al., 2005)

↑ up to 35% (Birjmohun et al., 2005)

↑ up to 20%

Novel therapies in development Apo A-I Milano Elevated efficiency as a cellular Not applicable cholesterol acceptor through an arginine to cysteine substitution at position 173 (Calabresi et al., 1994) JTT-705 Inhibits CETP, probably by binding ↑ up to 34% a to Cys-13 Torcetrapib Inhibits CETP by enhancing the ↑ up to 106% b association of CETP with its lipoprotein substrates (Clark et al., 2004) a b

At a dose of JTT-705 900mg/day in dyslipidemic patients. At a dose of torcetrapib 120mg twice daily in patients with low HDL-C.

also appear to enhance hepatic apo A-I production (Schaefer et al., 1999). Because of the significant effects of statins on the entire cascade of apo B-containing lipoproteins, it is difficult to assess the extent to which the smaller effect on HDL-C levels contributes to observed reductions in cardiovascular risk. However, lowering LDL-C with statins in persons with low HDL-C at baseline does significantly reduce CHD risk. For instance, in the 4S (The Scandinavian Simvastatin Survival Study Group, 1995), CARE (Sacks et al., 1996) and LIPID (The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group, 1998) trials, the reduction in risk with statin therapy in patients with low baseline levels of HDL-C was similar to that in those with high baseline levels of HDL-C. Moreover, patients in the 4S trial with elevated LDL-C, low HDL-C, and elevated triglyceride levels had an increased risk for CHD events versus placebo and experienced greater benefit with simvastatin than patients with LDL-C elevations alone (Ballantyne et al., 2001). The AFCAPS/TexCAPS study specifically recruited patients with low HDL-C and/or elevated

LDL-C. In this trial, lovastatin increased HDL-C by 6%. Patients whose baseline HDL-C was < 40mg/dL experienced a 3-fold reduction (45–15%) in their risk for first-time CHDrelated events compared with patients with HDL-C ≥ 40mg/dL (Downs et al., 1998). In an angiographic trial of fluvastatin therapy, reductions in the progression of atherosclerosis were also observed in patients with low HDL-C (Ballantyne et al., 1999). 6.2. Agonists of peroxisome proliferator-activated receptors Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors which are implicated in the transactivation or transrepression of a wide spectrum of genes which possess specific response elements for PPARs in their promoter regions. To date, 3 distinct isoforms of PPARs have been identified: α, β/δ, and γ. The relative distribution of PPAR subtypes and their transcriptional responses to activation vary in a tissue and ligand-specific manner. PPARα is mainly expressed in liver, skeletal muscle and heart, while PPARγ is abundant in adipose tissue. By contrast, PPAR β/δ exhibits wide tissue distribution. As ligand-activated nuclear receptors, PPARs modulate the expression of genes involved in lipid transport and metabolism, in fatty acid and glucose metabolism, in inflammation, and in hemostasis. 6.2.1. PPARα agonists: fibrates Fibrates are PPARα ligands of moderate binding affinity which typically increase HDL-C levels by up to 10% (Birjmohun et al., 2005) via several mechanisms (Fruchart et al., 2001; Chapman, 2003). Firstly, they upregulate expression of apo A-I and apo A-II. Secondly, they enhance macrophage cholesterol efflux via upregulation of ABCA1 and SR-B1. Thirdly, they induce expression of lipoprotein lipase (LPL), which increases lipolysis of VLDL, releasing surface fragments to HDL-a process that may also be enhanced by fibrate-induced attenuation of the hepatic production of apo C-III. The benefits of fibrate therapy are primarily illustrated by 4 clinical trials. In the Diabetes Atherosclerosis Intervention Study (DAIS), 418 patients with type 2 diabetes, mild lipid abnormalities, and at least 1 visible coronary lesion were randomized to micronized fenofibrate or placebo (Effect of fenofibrate on progression of coronary-artery disease in type 2 diabetes: the Diabetes Atherosclerosis Intervention Study, a randomised study, 2001). Patients randomized to fenofibrate experienced significant changes in lipid parameters compared with placebo (all p's < .001). The fenofibrate group also showed a significantly smaller increase in percentage diameter stenosis (p = .02), a significantly smaller decrease in minimum lumen diameter (p = .029), and a non-significantly smaller decrease in mean segment diameter (p = .17) compared with placebo, indicating that fenofibrate reduced the angiographic progression of CHD in patients with type 2 diabetes. In DAIS, the shift in LDL particle size that was observed with fenofibrate, from small dense LDL to more buoyant particles, appeared to make a significant contribution to the clinical benefits (Vakkilainen et al., 2003). In the Helsinki Heart Study of 4081 men with non-

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the pioglitazone group and 572 of 2633 patients in the placebo group having at least 1 event (HR 0.90, 95% CI 0.80–1.02, p = .095). However, in terms of the main secondary endpoint (a composite of all-cause mortality, non-fatal MI, and stroke), pioglitazone was significantly more effective than placebo with 301 patients in the pioglitazone group and 358 patients in the placebo group having an event (HR 0.84, 95% CI 0.72–0.98, p = .027).

HDL-C >200 mg/dL but without CHD, gemfibrozil 1200 mg/ day increased HDL-C by 11%, decreased triglyceride by 35%, and decreased LDL-C levels by 11% compared with placebo after 5years (Frick et al., 1987). Major coronary events were reduced by 34% and estimated to be partly due to elevations in HDL-C. In the VA-HIT trial of 2531 men with known CHD, low HDL-C (mean 32mg/dL), modestly elevated triglycerides (mean 161 mg/dL), and average LDL-C (mean 111 mg/dL), gemfibrozil 1200 mg/day increased HDL-C by 6%, decreased triglycerides by 31%, and had no observable effect on LDL-C. Major coronary events were significantly reduced by 22% compared with placebo (Rubins et al., 1999). Changes in HDL-C and, in particular, HDL3 were significantly associated with reduction in coronary events, whereas changes in triglycerides or LDL-C were not (Robins et al., 2001). Providing further support for the benefits of fibrate therapy, data from a recent meta-analysis of 53 studies showed that fibrates reduced the risk for major coronary events by 25% (Birjmohun et al., 2005). Finally, in the recent Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial, the impact of fenofibrate (200 mg/day) on cardiovascular events in 2131 patients with previous CV disease and in 7664 without was evaluated over a 5-year period (Keech et al., 2005). Fenofibrate did not significantly diminish the risk of the primary outcome of CHD death and non-fatal MI; nonetheless, an 11% relative reduction in the primary endpoint was observed on fenofibrate treatment, which involved a significant 24% reduction in nonfatal MI (p = 0.01). In addition, total cardiovascular events were significantly reduced by 11% (p = 0.035). Higher rates of starting statin therapy in patients in the placebo group may have masked larger treatment benefit in the active arm.

6.2.3. PPARβ/δ agonists The potential interest of PPARβ/δ agonists as modulators of fatty acid oxidation and lipid homeostasis is currently emerging (Oliver et al., 2001; Sprecher et al., 2004; Wallace et al., 2005). Thus the high affinity, selective PPARβ/δ agonist GW 501516 was found to reduce plasma triglyceride levels and concomitantly to increase those of HDL-C in a non human primate model of obesity and type 2 diabetes (Oliver et al., 2001). More recently, this lead compound at a maximal dose of 3 mg/kg was demonstrated to induce a maximal elevation of 43% in HDL-C levels in the normotriglyceridemic St. Kitts vervet, a non human primate model of atherosclerosis; such elevation equally involved increase in plasma apo AI and apo AII concentrations concomitantly with increased HDL particle size (Wallace et al., 2005). The efficacy of GW 501516 in raising HDL-C levels in man was revealed in phase I studies at doses up to 10 mg/day over a 2-week period (Sprecher et al., 2004). Thus, HDL-C concentrations rose by 19% (p < 0.01) at the highest dose. Triglyceride levels decreased in parallel (− 20%), thereby suggesting that PPARβ/δ agonists may enhance lipolysis of triglyceride-rich lipoproteins with increased release of surface remnants to the HDL pool, although alternative mechanisms cannot be excluded.

6.2.2. PPARγ agonists Glitazones (thiazolidinediones) are a new class of antihyperglycemic agent and are currently indicated for type 2 diabetes. Two compounds are currently available, rosiglitazone and pioglitazone. Glitazones are synthetic ligands of PPARγ and thus increase fatty acid clearance (Miyazaki et al., 2001) probably by enhancing adipogenesis and/or lowering rates of lipolysis in adipocytes (Spiegelman, 1998). Such action diminishes the amount of substrate available for triglyceride production in the liver. In general, glitazones increase HDL levels by up to 20%, most likely due to their effect on triglyceride levels, although the LDL to HDL ratio may remain unaltered due to increased levels of LDL or total cholesterol (Raskin et al., 2001). The effect of pioglitazone versus placebo on morbidity and mortality in high-risk patients with type 2 diabetes was evaluated in a prospective, randomized controlled trial of 5238 patients with type 2 diabetes who had evidence of macrovascular disease (the PROACTIVE study) (Dormandy et al., 2005). In terms of the primary endpoint (a composite of allcause mortality, non-fatal MI, stroke, acute coronary syndrome, endovascular or surgical intervention in the coronary or leg arteries, and amputation above the ankle), pioglitazone was not significantly superior to placebo with 514 of 2605 patients in

6.2.4. Mixed PPAR agonists: PPARα/γ The considerable evolution in the occurrence of metabolic diseases involving insulin resistance together with perturbed glucose and lipid and lipoprotein metabolism underlies the growing need for efficacious pharmacological agents in this area, and particularly as such disease states involve elevated cardiovascular risk. The spectrum of genes whose expression is modified by PPARα and γ represents an optimal therapeutic target as PPARα and γ agonists modulate fatty acid, lipid and glucose metabolism (Ljung et al., 2002; Fagerberg et al., 2005). Initial studies of a dual PPARα/γ agonists (AZ 242) in ob/ob mice and Zucker rats suggested that such an agent might exert beneficial action not only on insulin resistance and insulin sensitivity, but also on carbohydrate and lipid metabolism (Ljung et al., 2002). Such promise was upheld in recent studies of AZ 242 (Tesaglitazar) in hypertriglyceridemic, non diabetic subjects with abdominal obesity and insulin resistance, in which a maximal dose-dependent triglyceride-lowering effect of 37% was accompanied by a 40% reduction in plasma non-esterified fatty acid levels and 16% increase in HDL-C (Fagerberg et al., 2005). This effect on HDL-C levels is significant as subjects presenting the criteria of the metabolic syndrome are frequently at elevated CV risk due in part to low HDL-C concentrations (Hansel et al., 2004).

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6.3. Niacin Of the options currently available for elevating HDL-C, niacin (nicotinic acid) is the most effective, raising levels by approximately 20% (Birjmohun et al., 2005). Tolerability can be an issue with niacin, with serious side effects including loss of glucose control and liver toxicity: such effects are intimately related to the use of the immediate-release formulation of niacin. Flushing, another side effect, may occur frequently (in up to 70–80% of patients), but is attenuated by use of the prolonged-release formulation of niacin (Chapman et al., 2004). As a result of this side effect profile, analogues of niacin with high affinity for the nicotinic acid receptor (see below) and with an attenuated vasodilatory profile are in development. We cannot however exclude the possibility that niacin-induced flushing and associated vasodilatory effects may be beneficial in certain vascular pathologies, such as coronary spasm and angina. Three main mechanisms contribute to the HDL-C-elevating action of niacin. The first mechanism is dependent upon the ability of niacin to suppress lipolysis of triacylglycerol in adipose tissue via inhibition of the hormone-sensitive triglyceride lipase (Karpe & Frayn, 2004). This anti-lipolytic action of niacin involves reduction of intracellular cyclic adenosine monophosphate (cyclic AMP) levels in adipose tissue via a G-protein-coupled receptor that mediates inhibition of adenylyl cyclase (Karpe & Frayn, 2004). In man, the orphan G-proteincoupled receptor (HM74) has been identified as the nicotinic acid receptor in adipose tissue. HM74 functions as a lowaffinity receptor for niacin, whereas the shorter homologous form (HM74A) seems to be a high-affinity receptor. Modulation of the intracellular signaling cascade upon niacin-receptor binding results in diminished lipolysis and hence reduced circulating levels of non-esterified fatty acids, the main substrates for hepatic triglyceride synthesis. Since hepatic triglyceride is integrated into nascent VLDL particles and secreted into the circulation, niacin lowers plasma VLDLtriglyceride levels and, as a consequence, attenuates CETPmediated depletion of HDL-CE. Thus, the triglyceride-lowering action of niacin favors retention of CE in HDL with normalization of HDL neutral lipid content, increases HDL particle size, and prolongs the plasma HDL/apo A-I residence time in vivo, effectively increasing HDL-C levels (Shepherd et al., 1979). The second mechanism by which niacin elevates HDL-C levels is by facilitating cholesterol efflux from macrophages to HDL acceptors via the ABCA1 membrane transporter, thereby increasing reverse cholesterol transport (Rubic et al., 2004). Whether this action causes local, intraplaque depletion of cholesterol remains speculative. The third mechanism appears to relate to niacin's ability to reduce uptake of HDL particles by the liver. While the precise mechanism remains to be elucidated, the effect is to again increase the plasma residence time of HDL and apo A-I (Shepherd et al., 1979; Jin et al., 1997). Clinical trials have demonstrated the benefits of niacin for reducing cardiovascular risk. For example, the Coronary Drug Project evaluated the effect of 5 different lipid-modifying

regimens on the primary outcome of all-cause mortality in 8341 men with previous MI. Long-term follow-up showed that monotherapy with niacin significantly reduced all-cause mortality by 11% compared with placebo (Canner et al., 1986). Other studies have assessed the benefits of niacin when combined with other cardiovascular therapies (Guyton et al., 1998; Brown et al., 2001; Wolfe et al., 2001; Kashyap et al., 2002; Capuzzi et al., 2003; Taylor et al., 2004;Whitney et al., 2005). The benefits of combining niacin with a statin are best demonstrated by the results of the HDL-Atherosclerosis Treatment Study (HATS) (Brown et al., 2001) and the Arterial Biology for the Investigation of the Treatment Effects of Reducing Cholesterol (ARBITER) 2 trial (Taylor et al., 2004). In HATS, the frequency of a first cardiovascular event among patients with CHD following 3 years of treatment was 3% with niacin plus simvastatin versus 24% with placebo (Brown et al., 2001). In ARBITER 2, the addition of niacin to background statin therapy in subjects with known CHD and moderately low HDL-C slowed the progression of atherosclerosis, as measured by changes in carotid intima-media thickness, compared with placebo (Taylor et al., 2004). Combining niacin with fibrates has also proved beneficial, as shown by a randomized trial conducted by Whitney et al. (2005) (the Armed Forces Regression Study [AFREGS]), in which subjects with CHD and low HDL-C levels received combination therapy with gemfibrozil, niacin, and cholestyramine or corresponding placebos. After 30 months, the composite cardiovascular event endpoint was reached in 26% of subjects in the placebo group and 13% of those in the combination therapy group (difference, 13.7% [95% CI, 0.9–26.5%]). 7. Novel therapeutic strategies While the epidemiological evidence that HDL particles are atheroprotective is persuasive, data from clinical trials is limited. Such a paucity of clinical data most likely reflects the current lack of well-tolerated drugs that both specifically target and substantially raise HDL-C and highlights a need for novel therapies. As indicated earlier, it is clear that inflammation plays a major role in the pathogenesis of atherosclerosis. Functional HDL has numerous anti-inflammatory properties while HDL with defective function may exert an attenuated anti-inflammatory impact. Apo A-I is intimately involved in HDL antiatherogenic activity. Several apo A-I mimetic peptides have now been identified that can interact with lipids and provide anti-inflammatory and other atheroprotective activity (Navab et al., 2005; Reddy et al., 2006). For example, in vitro when added to human plasma at nanomolar concentrations, D-4F stimulated the formation of pre-beta HDL, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity, and converted HDL from pro-inflammatory to antiinflammatory. While these amphipathic peptides appear to have therapeutic potential as oral agents, clinical trials have yet to be initiated. By contrast, a related therapy, infusion of recombinant apo A-1-Milano, is more clinically advanced with encouraging preliminary results (see Table 2).

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Apo A-I-Milano is a variant form of apo A-I that differs from the wild type by an arginine to cysteine substitution at position 173. This difference alters the functionality of apo A-I-Milano making it a more efficient cholesterol acceptor (Calabresi et al., 1994; Franceschini et al., 1999). In a phase 2 study (Nissen et al., 2003), weekly intravenous infusions of recombinant apo AI-Milano/phospholipid complexes initiated within 2 weeks of an acute coronary event and administered for a duration of 5 weeks were shown to decrease the primary endpoint of mean percent atheroma volume as measured by intravascular ultrasound by − 1.06% (standard deviation, 3.17%; median, − 0.81%; 95% CI, − 1.53% to − 0.34%; p = 0.02 compared with baseline). In the placebo group, mean percent atheroma volume increased by 0.14% (standard deviation, 3.09%; median, 0.03%; 95% CI, − 1.11–1.43%; p = 0.97 compared with baseline). These results clearly suggest that acute HDL therapy, via infusion of apo A-I or apo A-I mimetic peptides, has the potential to attenuate plaque progress, favor plaque stability, and decrease cardiovascular events. Nevertheless, they do not allow us to equate these beneficial effects with either a single or multiple antiatherogenic action(s) of HDL. It may however be hypothesized that both cholesterol efflux and anti-inflammatory actions of the infused recombinant particles may have contributed significantly to the overall effect. Large-scale trials of this novel approach with appropriate clinical endpoints are now required. Strategies aimed at decreasing cardiovascular risk by infusion of HDL-like products are reviewed in further detail by Thompson et al. (2004). The discovery that genetic deficiency in CETP is associated with elevated levels of HDL-C has led to the concept that CETP inhibition might be a viable therapeutic strategy for raising HDL-C levels (Barter et al., 2003) especially as there is a lack of definitive evidence for any excess CV disease in individuals presenting genetic CETP deficiency. Indeed, data in a cohort of Ashkenazi Jews showed that CETP gene polymorphisms associated with lower CETP levels were associated with longevity (Barzilai et al., 2003). However, as already described, the role of CETP in cholesterol metabolism is complex, and CETP-mediated transfer of CE from HDL to the apo Bcontaining lipoproteins, VLDL and LDL, may potentially result in both pro- and anti-atherogenic effects (Barter & Rye, 2001). On the one hand, CETP activity decreases direct reverse cholesterol transport via HDL and hepatic SR-B1 receptors (a potentially pro-atherogenic effect). On the other, it increases the cholesterol load in apo B-containing lipoproteins, which increases reverse cholesterol transport via LDL and hepatic LDL receptors (an anti-atherogenic effect) but also increases the mass of cholesterol transported by potentially atherogenic VLDL, IDL, and LDL, which may subsequently be returned to peripheral tissues and the arterial wall (a potentially proatherogenic effect). To date, there is considerable evidence to support the concept of CETP inhibition as a therapeutically efficacious approach to raising HDL-C (Barter et al., 2003; Le Goff et al., 2004). However, large-scale, randomized, clinical trials are ultimately required to assess the impact of CETP inhibitors on atherosclerosis and cardiovascular events. Currently, 2 small-

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molecule CETP inhibitors are in clinical development (see Table 2). 7.1. JTT-705 JTT-705 is a chemical CETP inhibitor, which is thought to inactivate CETP by binding to Cys-13. A number of preliminary studies have assessed the efficacy of this new compound. In 1 study, oral administration of JTT-705 to cholesterol-fed rabbits raised HDL-C by 90% and significantly reduced the progression of aortic atherosclerosis (Okamoto et al., 2000). Yet, in a further study of JTT-705 in rabbits, the difference in the deposition of aortic cholesterol was not significantly different from the control group, despite the elevation in HDL-C being similar to that in the original study (Huang et al., 2002). Nevertheless, in the second study, the diet contained a higher concentration of cholesterol and treatment was for a shorter period (3 months vs. 6 months) than in the original study, suggesting that HDL elevation alone may be inadequate when levels of atherogenic lipoproteins are markedly elevated. Trials of JTT-705 have also been conducted in humans. In a 4-week, randomized, double-blind, placebo-controlled study in 198 healthy individuals with mild dyslipidemia, JTT-705 was administered at doses of 300, 600 and 900mg/day (de Grooth et al., 2002). After 1 week, a dose-dependent decrease in CETP activity was measured, reaching a maximum of − 37% after 4 weeks of treatment with 900 mg/day. At this highest dose, HDL-C levels were increased by 34%. In another study, JTT-705 was assessed in combination with pravastatin in a trial conducted in 152 individuals with LDL-C > 160mg/dL (Kuivenhoven et al., 2004). After 4 weeks, JTT-705 600mg plus pravastatin 40 mg led to a 30% decrease from baseline in CETP activity and a 28% increase from baseline in levels of HDL-C (p < 0.001 for both parameters vs. placebo), while LDL-C decreased by 5% from baseline. JTT-705 300mg plus pravastatin 40 mg was observed to be about half as effective as the higher JTT-705 dose, decreasing CETP activity by approximately 16% and increasing HDL-C by approximately 14%. This study raises the possibility of a combined therapeutic approach involving the association of LDL-lowering and HDL-raising agents, which would potentially attenuate influx and accumulation of atherogenic cholesterol-rich particles in the arterial intima, while concomitantly enhancing cholesterol efflux to HDL. Overall reduction in plaque burden should then predictably result. 7.2. Torcetrapib Torcetrapib is a lipophilic, small-molecule inhibitor of CETP, which acts by binding to CETP with 1:1 stoichiometry and blocking both neutral lipid and phospholipid transfer activities by inducing a nonproductive complex with HDL (Clark et al., 2004; Clark et al., 2006). It has also been demonstrated that the degree to which plasma CETP shifts from a free to an HDL-bound state in the presence of torcetrapib is tightly correlated to the percent inhibition of CE-transfer activity (Clark et al., 2006).

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Torcetrapib was initially evaluated in 2 small placebocontrolled studies and was shown to produce substantial increases in HDL-C of up to 100% at the highest doses. The first study was a phase 1, multidose study (10, 30, 60, 120 mg/day, and 120 mg/twice daily) that investigated the effects of torcetrapib on plasma lipoproteins and apolipoproteins in 40 healthy normolipidemic subjects (Clark et al., 2004). Mean inhibition of CETP activity was 12 ± 17, 35 ± 17, 53 ± 8 and 80 ± 6 for the 30, 60, 120 mg/day and 120 mg/twice daily groups, respectively. HDL-C levels relative to baseline increased from 16% to 91% (10 mg/day to 120 mg/twice daily). In addition, LDL-C levels decreased from −7% to −42% (60 mg/day to 120 mg/twice daily). The second study evaluated the effects of torcetrapib alone or in combination with atorvastatin on plasma lipoprotein levels in 19 subjects with low levels of HDL-C (< 40 mg/dL) (Brousseau et al., 2004). Torcetrapib was administered for 4 weeks at a dose of 120 mg/ day either as monotherapy or with concomitant atorvastatin 20 mg/day. A subset of study participants received an additional 4 weeks of torcetrapib alone at 120 mg/twice daily. HDL-C levels increased by 46%, 61% and 106%, while LDL-C levels decreased by −8%, −17% and −17%, with torcetrapib 120mg/day, 120mg/day plus atorvastatin 20 mg/ day, and 120mg/twice daily, respectively. More recently, the results of 2 larger phase 2 trials of torcetrapib have been presented (Davidson et al., 2005). These trials provide further evidence of the lipid-modifying benefits of torcetrapib when administered with or without background atorvastatin to patients with low HDL-C levels, demonstrating substantial increases in HDL-C (up to 55%), modest decreases in LDL-C (up to − 19%), and increases in both LDL and HDL particle size. Also of interest are recent data from a cholesterol-fed rabbit model, in which elevation of HDL-C via CETP inhibition with torcetrapib was shown to significantly inhibit aortic atherosclerosis (Morehouse et al., 2004). The clinical benefit of torcetrapib in combination with atorvastatin is the subject of several ongoing phase 3 trials. The RADIANCE (Rating Atherosclerotic Disease Change by Imaging With a New CETP inhibitor) 1 and 2 studies are evaluating the benefits of torcetrapib/atorvastatin versus atorvastatin alone using changes in CIMT as a surrogate endpoint (Bots et al., 2005; Kastelein et al., 2005). RADIANCE 1 has randomized 907 patients with heterozygous familial hypercholesterolemia, while RADIANCE 2 has randomized 758 patients with mixed dyslipidemia. Results of these trials are anticipated in 2007. The ILLUSTRATE (Investigation of Lipid Level Management Using Coronary Ultrasound to Assess Reduction of Atherosclerosis by CETP Inhibition and HDL Elevation) study is evaluating changes in atheroma volume in coronary arteries using IVUS (Nissen et al., 2005). A total of 1191 patients with CHD have been randomized to treatment, with results also expected in 2007. Finally, ILLUMINATE (Investigation of Lipid Level Management to Understand Its Impact in Atherosclerotic Events) is a large clinical endpoint trial that is being conducted at 250 sites in 7 countries. Approximately 13,000 subjects with CHD or CHD risk equivalents will be randomized to either torcetrapib/atorvastatin

or atorvastatin alone. The primary endpoint is a composite of CHD death, nonfatal MI, or stroke. This trial will provide us with critical evaluation of the therapeutic validity of CETP as a target for HDL-C elevation and concomitant clinical benefit across a wide range of lipid phenotypes. 8. Summary Convincing epidemiological data suggest that HDL-C is a strong, independent, inverse predictor of CHD risk and there is much evidence that HDL particles exert a spectrum of antiatherogenic effects. Greater understanding of HDL metabolism in recent years has enabled the identification of potential new targets for therapeutic intervention and has led to the development of novel drugs for the efficacious elevation of HDL-C levels. Nevertheless, the clinical benefits of novel therapies for elevating HDL-C remain largely unproven. Animal studies in rabbits and initial clinical trials have provided promising results, but large-scale cardiovascular endpoint trials are still required to fully evaluate the clinical benefits of these innovative strategies. Acknowledgments Referenced studies that were conducted in the author's laboratory were supported by the National Institute for Health and Medical Research (INSERM). I am indebted to Dr Matthew Smith for literature analysis and editorial development of this manuscript, and to Mme Françoise Berneau for assistance in its finalisation. References American Diabetes Association. (2003). Management of dyslipidemia in adults with diabetes. Diabetes Care 26, S83–S86. Ansell, B. J., Watson, K. E., Fogelman, A. M., Navab, M., & Fonarow, G. C. (2005). High-density lipoprotein function recent advances. J Am Coll Cardiol 46, 1792–1798. Ashen, M. D., & Blumenthal, R. S. (2005). Clinical practice. Low HDL cholesterol levels. N Engl J Med 353, 1252–1260. Assmann, G., von Eckardstein, A., & Funke, H. (1993). High density lipoproteins, reverse transport of cholesterol, and coronary artery disease. Insights from mutations. Circulation 87, III28–III34. Assmann, G., Schulte, H., von Eckardstein, A., & Huang, Y. (1996). Highdensity lipoprotein cholesterol as a predictor of coronary heart disease risk. The PROCAM experience and pathophysiological implications for reverse cholesterol transport. Atherosclerosis 124, S11–S20 (Suppl.). Assmann, G., & Gotto Jr., A. M. (2004). HDL cholesterol and protective factors in atherosclerosis. Circulation 109, III8–III14. Asztalos, B. F., Roheim, P. S., Milani, R. L., Lefevre, M., McNamara, J. R., Horvath, K. V., et al. (2000). Distribution of ApoA-I-containing HDL subpopulations in patients with coronary heart disease. Arterioscler Thromb Vasc Biol 20, 2670–2676. Asztalos, B. F., Cupples, L. A., Demissie, S., Horvath, K. V., Cox, C. E., Batista, M. C., et al. (2004). High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants of the Framingham Offspring Study. Arterioscler Thromb Vasc Biol 24, 2181–2187. Asztalos, B. F., de la Llera-Moya, M., Dallal, G. E., Horvath, K. V., Schaefer, E. J., & Rothblat, G. H. (2005). Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux. J Lipid Res 46, 2246–2253.

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