Best Practice & Research Clinical Endocrinology & Metabolism 28 (2014) 453–461
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Emerging therapies for raising high-density lipoprotein cholesterol (HDL-C) and augmenting HDL particle functionality Marcin Barylski, MD, PhD, Assistant Professor of Medicine a, Peter P. Toth, MD, PhD, Professor of Clinical Family and Community Medicine b, c, *, Dragana Nikolic, MD, Doctoral Candidate d, Maciej Banach, MD, Professor of Medicine e, Manfredi Rizzo, MD, PhD, Professor of Medicine d, f, Giuseppe Montalto, MD, Professor of Medicine d a
Department of Internal Medicine and Cardiological Rehabilitation, Medical University of Lodz, Lodz, Poland CGH Medical Center, Sterling, IL 61081, USA c University of Illinois School of Medicine, Peoria, IL, USA d Biomedical Department of Internal Medicine and Medical Specialties, University of Palermo, Palermo, Italy e Nephrology and Hypertension, Medical University of Lodz, Zeromskiego 113, 90-549 Lodz, Poland f Euro-Mediterranean Institute of Science and Technology, Palermo, Italy b
Keywords: apoA-I mimetic coronary artery disease delipidation endothelial lipase inhibitor farnesoid X receptor high-density lipoproteins liver X receptor reverse cholesterol transport RVX-208
High-density lipoprotein (HDL) particles are highly complex polymolecular aggregates capable of performing a remarkable range of atheroprotective functions. Considerable research is being performed throughout the world to develop novel pharmacologic approaches to: (1) promote apoprotein A-I and HDL particle biosynthesis; (2) augment capacity for reverse cholesterol transport so as to reduce risk for the development and progression of atherosclerotic disease; and (3) modulate the functionality of HDL particles in order to increase their capacity to antagonize oxidation, inflammation, thrombosis, endothelial dysfunction, insulin resistance, and other processes that participate in arterial wall injury. HDL metabolism and the molecular constitution of HDL particles are highly complex and can change in response to both acute and chronic alterations in the metabolic milieu. To date, some of these interventions have been shown to positively impact rates of coronary artery disease progression. However, none of them have as yet been shown to significantly reduce risk for
* Corresponding author. University of Illinois School of Medicine, Peoria, IL, USA. Tel.: þ1 (815) 632 5093; Fax: þ1 (815) 626 5947. E-mail addresses: [email protected] (M. Barylski), [email protected] (P.P. Toth), [email protected] (D. Nikolic), [email protected] (M. Banach), [email protected] (M. Rizzo), [email protected] (G. Montalto). 1521-690X/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.beem.2013.11.001
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cardiovascular events. In the next 3–5 years a variety of pharmacologic interventions for modulating HDL metabolism and functionality will be tested in large, randomized, prospective outcomes trials. It is hoped that one or more of these therapeutic approaches will result in the ability to further reduce risk for cardiovascular events once low-density lipoprotein cholesterol and non-HDLcholesterol targets have been attained. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction The high-density lipoproteins (HDLs) are functionally highly versatile and have the capacity to drive reverse cholesterol transport and exert a variety of other atheroprotective functions [1]. Elevated serum levels of high-density lipoprotein cholesterol (HDL-C) are highly correlated with reduced risk for cardiovascular events [2–4]. Given these data, it is quite logical to ask the question: does raising HDL-C, increasing HDL particle number, or modulating HDL functionality impact risk for cardiovascular events and can any of these changes impact the development and progression of atherosclerotic disease? A variety of post hoc results from clinical trials and a number of meta-analyses suggest that raising HDL-C does correlate with reductions in both cardiovascular event rates as well as progression of atherosclerotic disease [5–7]. Unfortunately, large prospective randomized outcomes trials in patients with established cardiovascular disease performed with cholesterol ester transfer protein inhibitors [8,9] and niacin [10,11] failed to demonstrate incremental benefit when tested against a background of statin therapy. These studies have raised serious issues regarding the value of raising HDL-C in modulating risk in the secondary prevention setting. Despite these setbacks, newer forms of pharmacologic interventions targeted at HDL metabolism and functionality are being developed and tested at a rapid rate. It is hoped that one or more of these novel approaches will help to reduce residual risk for cardiovascular events once atherogenic lipoprotein burden in serum is controlled to guidelinedefined levels. Directly augmenting apoA-I and apoA-I/phospholipid complexes Another approach to increasing serum levels of HDL is by infusing reconstituted HDL (rHDL) or recombinant HDL particles into the circulation, rather than increasing HDL indirectly by modulating HDL metabolism. One approach uses recombinant apoA-IMilano. Individuals with the apoA-IMilano mutation (R173C) have low HDL-C levels (10–30 mg/dl), and no apparent increased cardiovascular disease (CVD) risk [12]. Early studies indicated that recombinant apoA-IMilano, when delivered by intravenous infusion, promotes regression of atherosclerotic lesions to a greater extent than wild type apoA-I as measured by intravascular ultrasound with 5 once weekly treatments [13]. Procedural difficulties complicated the development of ETC-216 (clinical denomination of apoA-IMilano) and no further clinical trials with this formulation have been reported [14]. More recently, it was shown that recombinant HDL containing apoAIMilano exerts greater anti-inflammatory and plaque stabilizing properties rather than antiatherosclerotic properties [15]. Another rHDL compound, CSL-111, consists of apoA-I purified from human plasma and complexed with phosphatidylcholine derived from soybeans. The first trial of CSL-111 examined the effect of rHDL in the Atherosclerosis Safety and Efficacy (ERASE) trial conducted in 183 patients with acute coronary syndrome (ACS) [16]. Four weekly infusions of CSL-111 to 111 individuals randomized to the 40 mg/kg proved to be well tolerated and failed to meet its primary end-point. The high dose regimen (80 mg/kg) was discontinued because of abnormal liver transaminase elevations. However, there was no significant change in atheroma volume, as measured by intravascular ultrasound (IVUS), compared with the placebo group. Another study investigated the effect of CSL-111 on surrogate cardiovascular marker in patients following ACS [17]. In this trial 29 patients were randomized to a single infusion of CSL-111 (80 mg/kg over 4 h) or albumin. Following significant increases of HDL-C (64%) and reductions in lowdensity lipoprotein cholesterol (LDL-C) (23%), human rHDL did not improve vascular function compared to placebo. A modified version CSL-111 (CSL-112) is currently in phase II trials.
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Delipidated HDL infusions Another novel approach to HDL therapeutics is to raise levels of HDL particles by intravenous infusion with the use of autologous delipidated HDL [18]. Preclinical evaluation of selective delipidated HDL in dyslipidemic monkeys achieved a significant 6.9% reduction in aortic atheroma volume assessed by IVUS [19]. The process involves the selective removal of apoA-I HDL particles from plasma, delipidating them, and then reinfusing the cholesterol-depleted functional pre-b HDL. In a human trial, 28 patients with ACS received 5 weekly infusions of delipidated HDL (n ¼ 14) or placebo (n ¼ 14). Selectively increasing preb-HDL was associated with decreased total atheroma volume by 5.2% from baseline. [18] However, it is not yet established whether or not acute regression of atherosclerotic plaque volume is associated with decreased clinical cardiovascular events. Autologous delipidated HDL infusions do not induce liver toxicity or hypersensitivity reactions. In the study HDL apheresis resulted in hypotension in one-third of the participants undergoing treatment. A delipidation system for human use is now available from Lipid Sciences Plasma Delipidation System-2 (PDS-2), which converts aHDL to preb-like HDL by selectively removing cholesterol from HDL in samples of plasma collected from patients by apheresis. This approach remains under investigation. HDL mimetics ApoA-I mimetic peptides drugs ApoAI mimetics are short synthetic peptides that mimic the amphipathic a-helix of apoA-I. The first apoA-I mimetic peptide consisted of 18 amino acids (compound 18A). Based on the structure of 18A, additional improved peptides were generated by increasing the number of phenylalanine residues on the hydrophobic face (referred to as 2F, 3F, 4F, 5F, 6F, and 7F) of the polypeptide. [20] Among them only apoA-I mimetic peptide 4F showed promise in a number of animal models and in early human trials [21] leading to a phase I/II study in humans with high risk CVD [22]. In this study, the 4F peptide (synthesized from L-amino acids for L-4F) was delivered at low doses (0.042–1.43 mg/kg) by intravenous or subcutaneous administration [23]. Very high plasma peptide levels were achieved, but there was no improvement in HDL anti-inflammatory function [23]. On the other hand, previous studies showed that L-4F restores vascular endothelial function in murine models of hypercholesterolemia [24]. In another clinical trial the 4F peptide synthesized from all D-amino acids (making it resistant to hydrolysis by gastrointestinal peptidases) for D-4F was administered orally at higher doses (0.43–7.14 mg/kg). Interestingly, despite very low plasma peptide levels, it was associated with a significantly improved HDL inflammatory index [22]. In humans with significant cardiovascular risk, a single dose of D-4F was found to improve the inflammatory index of HDL with modest oral bioavailability [25]. Given concerns regarding possible cytotoxicity through ABCA1-independent lipid efflux, additional peptide mimetics have been engineered [26]. Peptides comprised of twenty-two amino acids based on domains of apolipoprotein A-I that have a higher affinity for ABCA1 have been shown to promote cholesterol efflux without cytopathic effects [26–28]. Furthermore, such domains are conserved across other apolipoproteins, and similarly designed peptides from apolipoprotein E promote ABCA1mediated reverse cholesterol transport [29]. Recently, 5A, an asymmetric bihelical peptide based on 2F, with 1 of the domains containing more alanine residues and thereby reducing its helical content, has been constructed to more closely reflect the combination of low- and high-affinity helices on apolipoprotein A-I. The 5A peptide had increased ABCA1-dependent cholesterol efflux and decreased hemolysis compared with its parent compound [30]. ATI-5261 synthetic peptide Native apoA-I is a 243 amino acid protein that contains multiple a-helical segments repeated in tandem and separated by proline residues. In vitro, ATI-5261 exerts its effects through ABCA1 in a fashion similar to that of HDL and successfully enhances cholesterol efflux from macrophages and reduces aortic atherosclerosis by up to 45% after intraperitoneal injection in mice. ATI-526 increases reverse cholesterol transport in mice [31]. The compound presently awaits early phase clinical trials in humans.
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Endothelial lipase inhibitors Endothelial lipase (EL) inhibition may represent potential future therapies to reduce apoA-I catabolism and to increase plasma apoA-I and HDL-C levels. Human genetic studies have confirmed that variation of the EL gene is an important determinant of plasma HDL-C level [32]. However, how changes in HDL-C level attributed to EL may affect atherosclerosis is still not clear. Some human studies propose an atherogenic role for EL, with a positive association of plasma level of EL mass and coronary artery calcification [33]. Carriers of EL variants associated with increased HDL-C levels have been reported to have decreased risk of coronary artery disease [34], but this association has not been observed in other studies [35]. Studies in mice showed that EL overexpression reduces HDL-C and apoA-I levels [36] due to increased renal catabolism. Conversely, gene deletion of EL results in increased HDL-C and apoA-I levels [37]. Although EL inactivation was expected to inhibit atherosclerosis by raising HDL-C, the effect of EL inactivation seems more complex than expected. The enthusiasm for EL inhibitors is somewhat tempered by Mendelian randomization data showing that variations in the gene loci for EL and cholesterol ester transfer protein (CETP) that increase HDL-C are not associated with protection against the development of atherosclerotic disease and its complications [38]. Lecithin-cholesterol acyltransferase modulators Several drug development approaches have recently been initiated for modulating lecithincholesterol acyltransferase (LCAT) activity. Early studies for the treatment of atherosclerosis by raising HDL-C through plasma LCAT enzyme activity were initiated by Zhou et al. in a rabbit model [39]. It was shown that recombinant LCAT administration may represent a novel approach for the treatment of atherosclerosis and the dyslipidemia associated with low HDL. Intravenous infusion of human rLCAT in rabbits was found to raise HDL-C, to increase fecal excretion of cholesterol, and to reduce atherosclerosis [40]. Another potential alternative to LCAT injection for treatment of human LCAT deficiency was recently reported in which adipocytes transfected with LCAT were transplanted into mice and were found to raise HDL-C [41]. Only one LCAT modulator has reached early clinical development, ETC642, but little data are available on the vascular effects of treatment with this agent [42]. Apo A-I upregulator Reservelogix-208 Reservelogix-208 (RVX-208) is a small molecule that increases endogenous synthesis of apoA-I. In African green monkeys, oral administration of RVX-208 resulted in increased levels of plasma apoA-I and HDL-C [43]. Serum from human subjects treated with RVX-208 exhibited increased cholesterol efflux capacity despite a relatively modest increase in HDL-C levels [44]. In a phase II trial, modest changes in HDL-C and apoA-I were reported in 299 statin-treated patients with stable coronary artery disease (CAD) [45]. One Phase IIb study is ongoing and involves 172 statin-treated patients randomized for RVX-208 100 mg or placebo twice daily for 24 weeks [46]. The ASSURE trial investigated the effect of RVX-208 on coronary atherosclerosis assessed by intravascular ultrasound [47]. The trial was negative with no demonstrable differences in percent atheroma volume or normalized total atheroma between patients treated either with placebo or RVX-208. Synthetic liver X receptor agonists Synthetic liver X receptor (LXR) agonists including LXRa/b are known to induce the transcription of ABCA1 and ATP-binding cassette transporter G1 (ABCG1). As potent activators of cellular cholesterol efflux, these compounds have been shown to raise HDL-C levels and to reduce atherosclerosis in transgenic mouse models [48]. Thus, LXR agonist activation may be a promising pharmacologic target for the treatment of dyslipidemia and atherosclerosis. Unfortunately, the development of first generation LXR compounds has been hampered by their capacity to increase expression of lipogenic genes in the liver, which increase levels of triglycerides and promote hepatic steatosis [49]. Various synthetically engineered LXR agonists have been developed and tested in animal models. They all show high potency for interacting with LXRa/b receptors, but none of them shows selectivity for ABCA1 and ABCG1 [49,50]. T091317 is a LXR activator, which consistently decreases atherosclerosis in mouse
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models and induces gene expression of Niemann-Pick C1 (NPC1) and NPC2 in macrophages resulting in enriched cholesterol content in the outer layer of plasma membranes [51]. The LXR agonist LXR-623 is associated with increased expression of ABCA1 and ABCG1 in cells [52], but adverse central nervous system-related effects were noted in more than half of patients, leading to termination of the study [53]. Other agonists (AZ876 and GW3965) were shown to reduce the number of atherosclerotic lesions [54]. The LXR agonist GW6340, an intestine specific LXRa/b agonist, promoted macrophage specific cellular cholesterol efflux and increased intestinal excretion of HDL-derived cholesterol [50]. More recently, a novel synthetic LXR agonist, ATI-111, that is more potent than T0901317, inhibited atherosclerosis progression and prevented atheromatous plaque formation in mice [55]. Research on more selective LXR ligands is an active area of experimental pharmacology. Synthetic farnesoid X receptor agonists The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that plays an important role in the regulation of cholesterol and, more specifically, HDL homeostasis [56]. Preclinical studies showed that activation of FXR leads to both pro- and antiatherosclerotic effects and a major metabolic effect of FXR agonists in animal models is a reduction of plasma HDL [56,57]. Hambruch et al. showed that FXR agonists promote HDL-derived cholesterol excretion into feces in mice and monkeys [57]. For these reasons, FXR agonists have received attention as a potential therapeutic target [58], and different agonists have been generated as a strategy for HDL-C raising therapies. These include GW4064, 6ECDCA, FXR-450, and PX20606 [57]. GW4064 has potential cell toxicity and uncertain bioavailability prevents its development for clinical studies [58]. In normolipidemic monkeys treated with PX20606, HDL2 is decreased without changing apoA-I levels. In these studies, the basic mechanisms of FXR mediating HDLC clearance are conserved in mice and monkeys. These observations will support further studies to investigate the potential roles of FXR activation on HDL metabolism and speciation. Gene therapy Animal experiments with apoA-I transgenes have yielded beneficial results for the prevention of atherosclerosis [59,60]. To date, this approach has little application in man. Animal data supports novel Table 1 Summary of selected strategies to increase HDL/apoA-I and potential compound under development. Pharmacotherapeutic strategy
Drug
Aim
Recombinant apoA-I Milano/phospholipids Purified native apoA-I/phospholipids Upregulators of endogenous apoA-I production ApoA-I mimetic peptides
ETC-216
Directly augmenting apoA-I/HDL pool
CSL-111 and CSL 112 RVX-208
Directly augmenting apoA-I/HDL pool Directly augmenting apoA-I/HDL pool
D-4F L-4F ATI-5261 Selective HDL delipidated miR-33
Mimicking apoA-I functionality
Autologous delipidated HDL Gene therapy Liver X receptor agonists
Niacin receptor agonists Farnesoid X receptor Cholesteryl ester transfer inhibitors Endothelial lipase inhibition LCAT activators
LXRa/b agonists LxR-623 T0901317, GW3965 ATI-111 ARI-3037MO FxR-450 Anacetrapib MK-0859 Evacetrapib LY248595 Boronic acid inhibitors Selective sulfonylfuran urea rLCAT ETC-642
Directly augmenting apoA-I/HDL pool Modulating HDL levels and cholesterol efflux expression Enhancing RCT & Macrophage cholesterol efflux
Indirectly augmenting apoA-I and HDL-cholesterol Modulate HDL levels Indirectly augmenting apoA-I and HDL-cholesterol Increasing HDL-cholesterol Enhancing RCT
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gene-based approaches to increase HDL-C. A potential mechanism to increase of HDL-C is increasing ABCA1 and ABCG1 expression through the therapeutic manipulation of microRNA metabolism (inhibition of miR-33 being the most promising candidate to date). Overexpression of antisense miR-33 using a lentivirus vector in mice showed a 50% increase in hepatic ABCA1 protein levels and a concomitant 25% increase in plasma HDL levels after 6 days [61]. Marquart et al. showed that injection of an anti-miR-33 oligonucleotide in mice resulted in a substantial increase in ABCA1 expression and HDL levels [62]. Furthermore, it was shown in mice that miR-33 decreases cholesterol efflux [63]. These data suggest that miR-33 might be a possible target for the treatment of cardiovascular and metabolic disorders. Table 1 summarizes selected strategies to increase HDL/apoA-I and describes potential compounds under development. Conclusion Unlike the path taken to therapeutically modulate LDL-C levels, that for HDL-C has turned out to be much more difficult and complicated. Two CETP inhibitors, RVX-208, and multiple other agents have failed in clinical trials. Is the apparent protectiveness of HDL-C suggested by epidemiology simply an epiphenomenon? Is it possible that the exceptional complexity of HDL’s proteome and lipidome really have little bearing on the structural and functional integrity of arterial walls? It is hoped that these and many other questions concerning HDL therapeutics will be answered in the years ahead with the innovative approaches summarized herein. In the meantime, we will await the results of clinical trials testing the ability of these various agents to impact rates of atherosclerotic disease progression and risk for cardiovascular events.
Practice points At the present time, HDL-C is not a target of therapy. The “HDL hypothesis” (i.e., that raising HDL-C, HDL particles, or modulating HDL functionality impacts risk for CV events) is a matter of intensive investigation. A variety of new pharmacologic interventions are being developed that: 1. Increase serum HDL particle concentration by infusing autologous delipidated HDL, human native apoA-I and apoA-I(Milano) incorporated into liposomes, and apoA-I mimetics. 2. The regulation of RCT is being studied by agonizing nuclear transcription factors (LXR-alpha and FXR) and modulating the activity of enzymes responsible for HDL metabolism in serum (LCAT, CETP, endothelial lipase). Gene therapy is being tested in a murine model to evaluate the safety and efficacy of an antisense molecule to miR-33. Antisense technology is already being used in humans to treat familial hypercholesterolemia with mipomersen, an antisense oligonucleotide directed against the mRNA for apoprotein B.
Research agenda HDL particles are highly heterogeneous with diverse lipid and protein cargos. It will be important to establish: 1. How specific HDL interventions impact both the serum concentration of HDL particles and their functionality. 2. If functionality is greatly increased, is it even necessary to robustly elevate serum levels of HDL?
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3. If specific functions (e.g., RCT, anti-oxidative capacity) are augmented or even adversely affected despite elevations in serum levels of HDL? 4. Long-term safety of these agents in humans. 5. Whether or not they can safely be used in combination with other lipid modifying drugs such as the statins or fibrates 6. The impact of specific agents on rates of atherosclerosis disease progression and cardiovascular events (myocardial infarction, ischemic stroke, death, need for coronary and peripheral revascularization) will have to be assessed in large prospective outcomes and imaging trials.
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Intestinal specific LXR activation stimulates reverse cholesterol transport and protects from atherosclerosis. Cell Metab 2010;12:187–93. [49] Oosterveer MH, Grefhorst A, Groen AK, et al. The liver X receptor: control of cellular lipid homeostasis and beyond: implications for drug design. Prog Lipid Res 2010;49:343–52. *[50] Yasuda T, Grillot D, Billheimer JT, et al. Tissue-specific liver X receptor activation promotes macrophage reverse cholesterol transport in vivo. Arterioscler Thromb Vasc Biol 2010;30:781–6. [51] Rigamonti E, Helin L, Lestavel S, et al. Liver X receptor activation controls intracellular cholesterol trafficking and esterification in human macrophages. Circ Res 2005;97:682–9. [52] Quinet EM, Basso MD, Halpern AR, et al. LXR ligand lowers LDL cholesterol in primates, is lipid neutral in hamster, and reduces atherosclerosis in mouse. J Lipid Res 2009;50:2358–70. *[53] Katz A, Udata C, Ott E, et al. Safety, pharmacokinetics, and pharmacodynamics of single doses of lxr-623, a novel liver Xreceptor agonist, in healthy participants. J Clin Pharmacol 2009;49:643–9. [54] Bradley MN, Hong C, Chen M, et al. Ligand activation of LXR beta reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR alpha and apoE. J Clin Invest 2007;117:2337–46. [55] Peng D, Hiipakka RA, Xie JT, et al. Anovel potent synthetic steroidal liver X receptor agonist lowers plasma cholesterol and triglycerides and reduces atherosclerosis in LDLR-/- mice. Br J Pharmacol 2011;162:1792–804. [56] Mencarelli A, Fiorucci S. FXR an emerging therapeutic target for the treatment of atherosclerosis. J Cell Mol Med 2010;14: 79–92. *[57] Hambruch E, Miyazaki-Anzai S, Hahn U, et al. Synthetic farnesoid X receptor agonists induce high-density lipoproteinmediated transhepatic cholesterol efflux in mice and monkeys and prevent atherosclerosis in cholesteryl ester transfer protein transgenic low-density lipoprotein receptor (/) mice. J Pharmacol Exp Ther 2012;343:556–67. [58] Fiorucci S, Cipriani S, Baldelli F, et al. Bile acid-activated receptors in the treatment of dyslipidemia and related disorders. Prog Lipid Res 2010;49:171–85. [59] Plump AS, Scott CJ, Breslow JL. Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse. Proc Natl Acad Sci U S A 1994;91:9607–11. *[60] Maeda N, Li H, Lee D, et al. Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridemia and protection from postprandial hypertriglyceridemia. J Biol Chem 1994;269:23610–6. *[61] Rayner KJ, Sheedy FJ, Esauet CC, et al. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J Clin Invest 2011;121:2921–31.
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[62] Marquart TJ, Allen RM, Ory DS, et al. miR-33 links SREBP-2 induction to repression of sterol transporters. Proc Natl Acad Sci U S A 2010;107:12228–32. [63] Horie T, Ono K, Horiguchi M, et al. MicroRNA-33 encoded by an intron of sterol regulatory element-binding protein 2 (Srebp2) regulates HDL in vivo. Proc Natl Acad Sci U S A 2010;107:17321–6.
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Emerging therapies for raising high-density lipoprotein cholesterol (HDL-C) and augmenting HDL particle functionality Marcin Barylski, MD, PhD, Assistant Professor of Medicine a, Peter P. Toth, MD, PhD, Professor of Clinical Family and Community Medicine b, c, *, Dragana Nikolic, MD, Doctoral Candidate d, Maciej Banach, MD, Professor of Medicine e, Manfredi Rizzo, MD, PhD, Professor of Medicine d, f, Giuseppe Montalto, MD, Professor of Medicine d a
Department of Internal Medicine and Cardiological Rehabilitation, Medical University of Lodz, Lodz, Poland CGH Medical Center, Sterling, IL 61081, USA c University of Illinois School of Medicine, Peoria, IL, USA d Biomedical Department of Internal Medicine and Medical Specialties, University of Palermo, Palermo, Italy e Nephrology and Hypertension, Medical University of Lodz, Zeromskiego 113, 90-549 Lodz, Poland f Euro-Mediterranean Institute of Science and Technology, Palermo, Italy b
Keywords: apoA-I mimetic coronary artery disease delipidation endothelial lipase inhibitor farnesoid X receptor high-density lipoproteins liver X receptor reverse cholesterol transport RVX-208
High-density lipoprotein (HDL) particles are highly complex polymolecular aggregates capable of performing a remarkable range of atheroprotective functions. Considerable research is being performed throughout the world to develop novel pharmacologic approaches to: (1) promote apoprotein A-I and HDL particle biosynthesis; (2) augment capacity for reverse cholesterol transport so as to reduce risk for the development and progression of atherosclerotic disease; and (3) modulate the functionality of HDL particles in order to increase their capacity to antagonize oxidation, inflammation, thrombosis, endothelial dysfunction, insulin resistance, and other processes that participate in arterial wall injury. HDL metabolism and the molecular constitution of HDL particles are highly complex and can change in response to both acute and chronic alterations in the metabolic milieu. To date, some of these interventions have been shown to positively impact rates of coronary artery disease progression. However, none of them have as yet been shown to significantly reduce risk for
* Corresponding author. University of Illinois School of Medicine, Peoria, IL, USA. Tel.: þ1 (815) 632 5093; Fax: þ1 (815) 626 5947. E-mail addresses: [email protected] (M. Barylski), [email protected] (P.P. Toth), [email protected] (D. Nikolic), [email protected] (M. Banach), [email protected] (M. Rizzo), [email protected] (G. Montalto). 1521-690X/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.beem.2013.11.001
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cardiovascular events. In the next 3–5 years a variety of pharmacologic interventions for modulating HDL metabolism and functionality will be tested in large, randomized, prospective outcomes trials. It is hoped that one or more of these therapeutic approaches will result in the ability to further reduce risk for cardiovascular events once low-density lipoprotein cholesterol and non-HDLcholesterol targets have been attained. Ó 2013 Elsevier Ltd. All rights reserved.
Introduction The high-density lipoproteins (HDLs) are functionally highly versatile and have the capacity to drive reverse cholesterol transport and exert a variety of other atheroprotective functions [1]. Elevated serum levels of high-density lipoprotein cholesterol (HDL-C) are highly correlated with reduced risk for cardiovascular events [2–4]. Given these data, it is quite logical to ask the question: does raising HDL-C, increasing HDL particle number, or modulating HDL functionality impact risk for cardiovascular events and can any of these changes impact the development and progression of atherosclerotic disease? A variety of post hoc results from clinical trials and a number of meta-analyses suggest that raising HDL-C does correlate with reductions in both cardiovascular event rates as well as progression of atherosclerotic disease [5–7]. Unfortunately, large prospective randomized outcomes trials in patients with established cardiovascular disease performed with cholesterol ester transfer protein inhibitors [8,9] and niacin [10,11] failed to demonstrate incremental benefit when tested against a background of statin therapy. These studies have raised serious issues regarding the value of raising HDL-C in modulating risk in the secondary prevention setting. Despite these setbacks, newer forms of pharmacologic interventions targeted at HDL metabolism and functionality are being developed and tested at a rapid rate. It is hoped that one or more of these novel approaches will help to reduce residual risk for cardiovascular events once atherogenic lipoprotein burden in serum is controlled to guidelinedefined levels. Directly augmenting apoA-I and apoA-I/phospholipid complexes Another approach to increasing serum levels of HDL is by infusing reconstituted HDL (rHDL) or recombinant HDL particles into the circulation, rather than increasing HDL indirectly by modulating HDL metabolism. One approach uses recombinant apoA-IMilano. Individuals with the apoA-IMilano mutation (R173C) have low HDL-C levels (10–30 mg/dl), and no apparent increased cardiovascular disease (CVD) risk [12]. Early studies indicated that recombinant apoA-IMilano, when delivered by intravenous infusion, promotes regression of atherosclerotic lesions to a greater extent than wild type apoA-I as measured by intravascular ultrasound with 5 once weekly treatments [13]. Procedural difficulties complicated the development of ETC-216 (clinical denomination of apoA-IMilano) and no further clinical trials with this formulation have been reported [14]. More recently, it was shown that recombinant HDL containing apoAIMilano exerts greater anti-inflammatory and plaque stabilizing properties rather than antiatherosclerotic properties [15]. Another rHDL compound, CSL-111, consists of apoA-I purified from human plasma and complexed with phosphatidylcholine derived from soybeans. The first trial of CSL-111 examined the effect of rHDL in the Atherosclerosis Safety and Efficacy (ERASE) trial conducted in 183 patients with acute coronary syndrome (ACS) [16]. Four weekly infusions of CSL-111 to 111 individuals randomized to the 40 mg/kg proved to be well tolerated and failed to meet its primary end-point. The high dose regimen (80 mg/kg) was discontinued because of abnormal liver transaminase elevations. However, there was no significant change in atheroma volume, as measured by intravascular ultrasound (IVUS), compared with the placebo group. Another study investigated the effect of CSL-111 on surrogate cardiovascular marker in patients following ACS [17]. In this trial 29 patients were randomized to a single infusion of CSL-111 (80 mg/kg over 4 h) or albumin. Following significant increases of HDL-C (64%) and reductions in lowdensity lipoprotein cholesterol (LDL-C) (23%), human rHDL did not improve vascular function compared to placebo. A modified version CSL-111 (CSL-112) is currently in phase II trials.
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Delipidated HDL infusions Another novel approach to HDL therapeutics is to raise levels of HDL particles by intravenous infusion with the use of autologous delipidated HDL [18]. Preclinical evaluation of selective delipidated HDL in dyslipidemic monkeys achieved a significant 6.9% reduction in aortic atheroma volume assessed by IVUS [19]. The process involves the selective removal of apoA-I HDL particles from plasma, delipidating them, and then reinfusing the cholesterol-depleted functional pre-b HDL. In a human trial, 28 patients with ACS received 5 weekly infusions of delipidated HDL (n ¼ 14) or placebo (n ¼ 14). Selectively increasing preb-HDL was associated with decreased total atheroma volume by 5.2% from baseline. [18] However, it is not yet established whether or not acute regression of atherosclerotic plaque volume is associated with decreased clinical cardiovascular events. Autologous delipidated HDL infusions do not induce liver toxicity or hypersensitivity reactions. In the study HDL apheresis resulted in hypotension in one-third of the participants undergoing treatment. A delipidation system for human use is now available from Lipid Sciences Plasma Delipidation System-2 (PDS-2), which converts aHDL to preb-like HDL by selectively removing cholesterol from HDL in samples of plasma collected from patients by apheresis. This approach remains under investigation. HDL mimetics ApoA-I mimetic peptides drugs ApoAI mimetics are short synthetic peptides that mimic the amphipathic a-helix of apoA-I. The first apoA-I mimetic peptide consisted of 18 amino acids (compound 18A). Based on the structure of 18A, additional improved peptides were generated by increasing the number of phenylalanine residues on the hydrophobic face (referred to as 2F, 3F, 4F, 5F, 6F, and 7F) of the polypeptide. [20] Among them only apoA-I mimetic peptide 4F showed promise in a number of animal models and in early human trials [21] leading to a phase I/II study in humans with high risk CVD [22]. In this study, the 4F peptide (synthesized from L-amino acids for L-4F) was delivered at low doses (0.042–1.43 mg/kg) by intravenous or subcutaneous administration [23]. Very high plasma peptide levels were achieved, but there was no improvement in HDL anti-inflammatory function [23]. On the other hand, previous studies showed that L-4F restores vascular endothelial function in murine models of hypercholesterolemia [24]. In another clinical trial the 4F peptide synthesized from all D-amino acids (making it resistant to hydrolysis by gastrointestinal peptidases) for D-4F was administered orally at higher doses (0.43–7.14 mg/kg). Interestingly, despite very low plasma peptide levels, it was associated with a significantly improved HDL inflammatory index [22]. In humans with significant cardiovascular risk, a single dose of D-4F was found to improve the inflammatory index of HDL with modest oral bioavailability [25]. Given concerns regarding possible cytotoxicity through ABCA1-independent lipid efflux, additional peptide mimetics have been engineered [26]. Peptides comprised of twenty-two amino acids based on domains of apolipoprotein A-I that have a higher affinity for ABCA1 have been shown to promote cholesterol efflux without cytopathic effects [26–28]. Furthermore, such domains are conserved across other apolipoproteins, and similarly designed peptides from apolipoprotein E promote ABCA1mediated reverse cholesterol transport [29]. Recently, 5A, an asymmetric bihelical peptide based on 2F, with 1 of the domains containing more alanine residues and thereby reducing its helical content, has been constructed to more closely reflect the combination of low- and high-affinity helices on apolipoprotein A-I. The 5A peptide had increased ABCA1-dependent cholesterol efflux and decreased hemolysis compared with its parent compound [30]. ATI-5261 synthetic peptide Native apoA-I is a 243 amino acid protein that contains multiple a-helical segments repeated in tandem and separated by proline residues. In vitro, ATI-5261 exerts its effects through ABCA1 in a fashion similar to that of HDL and successfully enhances cholesterol efflux from macrophages and reduces aortic atherosclerosis by up to 45% after intraperitoneal injection in mice. ATI-526 increases reverse cholesterol transport in mice [31]. The compound presently awaits early phase clinical trials in humans.
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Endothelial lipase inhibitors Endothelial lipase (EL) inhibition may represent potential future therapies to reduce apoA-I catabolism and to increase plasma apoA-I and HDL-C levels. Human genetic studies have confirmed that variation of the EL gene is an important determinant of plasma HDL-C level [32]. However, how changes in HDL-C level attributed to EL may affect atherosclerosis is still not clear. Some human studies propose an atherogenic role for EL, with a positive association of plasma level of EL mass and coronary artery calcification [33]. Carriers of EL variants associated with increased HDL-C levels have been reported to have decreased risk of coronary artery disease [34], but this association has not been observed in other studies [35]. Studies in mice showed that EL overexpression reduces HDL-C and apoA-I levels [36] due to increased renal catabolism. Conversely, gene deletion of EL results in increased HDL-C and apoA-I levels [37]. Although EL inactivation was expected to inhibit atherosclerosis by raising HDL-C, the effect of EL inactivation seems more complex than expected. The enthusiasm for EL inhibitors is somewhat tempered by Mendelian randomization data showing that variations in the gene loci for EL and cholesterol ester transfer protein (CETP) that increase HDL-C are not associated with protection against the development of atherosclerotic disease and its complications [38]. Lecithin-cholesterol acyltransferase modulators Several drug development approaches have recently been initiated for modulating lecithincholesterol acyltransferase (LCAT) activity. Early studies for the treatment of atherosclerosis by raising HDL-C through plasma LCAT enzyme activity were initiated by Zhou et al. in a rabbit model [39]. It was shown that recombinant LCAT administration may represent a novel approach for the treatment of atherosclerosis and the dyslipidemia associated with low HDL. Intravenous infusion of human rLCAT in rabbits was found to raise HDL-C, to increase fecal excretion of cholesterol, and to reduce atherosclerosis [40]. Another potential alternative to LCAT injection for treatment of human LCAT deficiency was recently reported in which adipocytes transfected with LCAT were transplanted into mice and were found to raise HDL-C [41]. Only one LCAT modulator has reached early clinical development, ETC642, but little data are available on the vascular effects of treatment with this agent [42]. Apo A-I upregulator Reservelogix-208 Reservelogix-208 (RVX-208) is a small molecule that increases endogenous synthesis of apoA-I. In African green monkeys, oral administration of RVX-208 resulted in increased levels of plasma apoA-I and HDL-C [43]. Serum from human subjects treated with RVX-208 exhibited increased cholesterol efflux capacity despite a relatively modest increase in HDL-C levels [44]. In a phase II trial, modest changes in HDL-C and apoA-I were reported in 299 statin-treated patients with stable coronary artery disease (CAD) [45]. One Phase IIb study is ongoing and involves 172 statin-treated patients randomized for RVX-208 100 mg or placebo twice daily for 24 weeks [46]. The ASSURE trial investigated the effect of RVX-208 on coronary atherosclerosis assessed by intravascular ultrasound [47]. The trial was negative with no demonstrable differences in percent atheroma volume or normalized total atheroma between patients treated either with placebo or RVX-208. Synthetic liver X receptor agonists Synthetic liver X receptor (LXR) agonists including LXRa/b are known to induce the transcription of ABCA1 and ATP-binding cassette transporter G1 (ABCG1). As potent activators of cellular cholesterol efflux, these compounds have been shown to raise HDL-C levels and to reduce atherosclerosis in transgenic mouse models [48]. Thus, LXR agonist activation may be a promising pharmacologic target for the treatment of dyslipidemia and atherosclerosis. Unfortunately, the development of first generation LXR compounds has been hampered by their capacity to increase expression of lipogenic genes in the liver, which increase levels of triglycerides and promote hepatic steatosis [49]. Various synthetically engineered LXR agonists have been developed and tested in animal models. They all show high potency for interacting with LXRa/b receptors, but none of them shows selectivity for ABCA1 and ABCG1 [49,50]. T091317 is a LXR activator, which consistently decreases atherosclerosis in mouse
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models and induces gene expression of Niemann-Pick C1 (NPC1) and NPC2 in macrophages resulting in enriched cholesterol content in the outer layer of plasma membranes [51]. The LXR agonist LXR-623 is associated with increased expression of ABCA1 and ABCG1 in cells [52], but adverse central nervous system-related effects were noted in more than half of patients, leading to termination of the study [53]. Other agonists (AZ876 and GW3965) were shown to reduce the number of atherosclerotic lesions [54]. The LXR agonist GW6340, an intestine specific LXRa/b agonist, promoted macrophage specific cellular cholesterol efflux and increased intestinal excretion of HDL-derived cholesterol [50]. More recently, a novel synthetic LXR agonist, ATI-111, that is more potent than T0901317, inhibited atherosclerosis progression and prevented atheromatous plaque formation in mice [55]. Research on more selective LXR ligands is an active area of experimental pharmacology. Synthetic farnesoid X receptor agonists The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that plays an important role in the regulation of cholesterol and, more specifically, HDL homeostasis [56]. Preclinical studies showed that activation of FXR leads to both pro- and antiatherosclerotic effects and a major metabolic effect of FXR agonists in animal models is a reduction of plasma HDL [56,57]. Hambruch et al. showed that FXR agonists promote HDL-derived cholesterol excretion into feces in mice and monkeys [57]. For these reasons, FXR agonists have received attention as a potential therapeutic target [58], and different agonists have been generated as a strategy for HDL-C raising therapies. These include GW4064, 6ECDCA, FXR-450, and PX20606 [57]. GW4064 has potential cell toxicity and uncertain bioavailability prevents its development for clinical studies [58]. In normolipidemic monkeys treated with PX20606, HDL2 is decreased without changing apoA-I levels. In these studies, the basic mechanisms of FXR mediating HDLC clearance are conserved in mice and monkeys. These observations will support further studies to investigate the potential roles of FXR activation on HDL metabolism and speciation. Gene therapy Animal experiments with apoA-I transgenes have yielded beneficial results for the prevention of atherosclerosis [59,60]. To date, this approach has little application in man. Animal data supports novel Table 1 Summary of selected strategies to increase HDL/apoA-I and potential compound under development. Pharmacotherapeutic strategy
Drug
Aim
Recombinant apoA-I Milano/phospholipids Purified native apoA-I/phospholipids Upregulators of endogenous apoA-I production ApoA-I mimetic peptides
ETC-216
Directly augmenting apoA-I/HDL pool
CSL-111 and CSL 112 RVX-208
Directly augmenting apoA-I/HDL pool Directly augmenting apoA-I/HDL pool
D-4F L-4F ATI-5261 Selective HDL delipidated miR-33
Mimicking apoA-I functionality
Autologous delipidated HDL Gene therapy Liver X receptor agonists
Niacin receptor agonists Farnesoid X receptor Cholesteryl ester transfer inhibitors Endothelial lipase inhibition LCAT activators
LXRa/b agonists LxR-623 T0901317, GW3965 ATI-111 ARI-3037MO FxR-450 Anacetrapib MK-0859 Evacetrapib LY248595 Boronic acid inhibitors Selective sulfonylfuran urea rLCAT ETC-642
Directly augmenting apoA-I/HDL pool Modulating HDL levels and cholesterol efflux expression Enhancing RCT & Macrophage cholesterol efflux
Indirectly augmenting apoA-I and HDL-cholesterol Modulate HDL levels Indirectly augmenting apoA-I and HDL-cholesterol Increasing HDL-cholesterol Enhancing RCT
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gene-based approaches to increase HDL-C. A potential mechanism to increase of HDL-C is increasing ABCA1 and ABCG1 expression through the therapeutic manipulation of microRNA metabolism (inhibition of miR-33 being the most promising candidate to date). Overexpression of antisense miR-33 using a lentivirus vector in mice showed a 50% increase in hepatic ABCA1 protein levels and a concomitant 25% increase in plasma HDL levels after 6 days [61]. Marquart et al. showed that injection of an anti-miR-33 oligonucleotide in mice resulted in a substantial increase in ABCA1 expression and HDL levels [62]. Furthermore, it was shown in mice that miR-33 decreases cholesterol efflux [63]. These data suggest that miR-33 might be a possible target for the treatment of cardiovascular and metabolic disorders. Table 1 summarizes selected strategies to increase HDL/apoA-I and describes potential compounds under development. Conclusion Unlike the path taken to therapeutically modulate LDL-C levels, that for HDL-C has turned out to be much more difficult and complicated. Two CETP inhibitors, RVX-208, and multiple other agents have failed in clinical trials. Is the apparent protectiveness of HDL-C suggested by epidemiology simply an epiphenomenon? Is it possible that the exceptional complexity of HDL’s proteome and lipidome really have little bearing on the structural and functional integrity of arterial walls? It is hoped that these and many other questions concerning HDL therapeutics will be answered in the years ahead with the innovative approaches summarized herein. In the meantime, we will await the results of clinical trials testing the ability of these various agents to impact rates of atherosclerotic disease progression and risk for cardiovascular events.
Practice points At the present time, HDL-C is not a target of therapy. The “HDL hypothesis” (i.e., that raising HDL-C, HDL particles, or modulating HDL functionality impacts risk for CV events) is a matter of intensive investigation. A variety of new pharmacologic interventions are being developed that: 1. Increase serum HDL particle concentration by infusing autologous delipidated HDL, human native apoA-I and apoA-I(Milano) incorporated into liposomes, and apoA-I mimetics. 2. The regulation of RCT is being studied by agonizing nuclear transcription factors (LXR-alpha and FXR) and modulating the activity of enzymes responsible for HDL metabolism in serum (LCAT, CETP, endothelial lipase). Gene therapy is being tested in a murine model to evaluate the safety and efficacy of an antisense molecule to miR-33. Antisense technology is already being used in humans to treat familial hypercholesterolemia with mipomersen, an antisense oligonucleotide directed against the mRNA for apoprotein B.
Research agenda HDL particles are highly heterogeneous with diverse lipid and protein cargos. It will be important to establish: 1. How specific HDL interventions impact both the serum concentration of HDL particles and their functionality. 2. If functionality is greatly increased, is it even necessary to robustly elevate serum levels of HDL?
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3. If specific functions (e.g., RCT, anti-oxidative capacity) are augmented or even adversely affected despite elevations in serum levels of HDL? 4. Long-term safety of these agents in humans. 5. Whether or not they can safely be used in combination with other lipid modifying drugs such as the statins or fibrates 6. The impact of specific agents on rates of atherosclerosis disease progression and cardiovascular events (myocardial infarction, ischemic stroke, death, need for coronary and peripheral revascularization) will have to be assessed in large prospective outcomes and imaging trials.
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