Best Practice & Research Clinical Endocrinology & Metabolism 28 (2014) 453–461

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Best Practice & Research ClinicalEndocrinology & Metabolism

journal homepage: www.elsevier .com/locate/beem

12

Emerging therapies for raising high-densitylipoprotein cholesterol (HDL-C) and augmentingHDL particle functionality

Marcin Barylski, MD, PhD, Assistant Professor of Medicine a,Peter P. Toth, MD, PhD, Professor of Clinical Family andCommunity Medicine b,c,*, Dragana Nikolic, MD, DoctoralCandidate d, Maciej Banach, MD, Professor of Medicine e,Manfredi Rizzo, MD, PhD, Professor of Medicine d,f,Giuseppe Montalto, MD, Professor of Medicine d

aDepartment of Internal Medicine and Cardiological Rehabilitation, Medical University of Lodz, Lodz, PolandbCGH Medical Center, Sterling, IL 61081, USAcUniversity of Illinois School of Medicine, Peoria, IL, USAdBiomedical Department of Internal Medicine and Medical Specialties, University of Palermo, Palermo, ItalyeNephrology and Hypertension, Medical University of Lodz, Zeromskiego 113, 90-549 Lodz, Polandf Euro-Mediterranean Institute of Science and Technology, Palermo, Italy

Keywords:apoA-I mimeticcoronary artery diseasedelipidationendothelial lipase inhibitorfarnesoid X receptorhigh-density lipoproteinsliver X receptorreverse cholesterol transportRVX-208

* Corresponding author. University of Illinois SchoE-mail addresses: mbarylski3@wp.pl (M. Bary

Nikolic), maciejbanach@aol.co.uk (M. Banach), man

1521-690X/$ – see front matter � 2013 Elsevier Lthttp://dx.doi.org/10.1016/j.beem.2013.11.001

High-density lipoprotein (HDL) particles are highly complex pol-ymolecular aggregates capable of performing a remarkable rangeof atheroprotective functions. Considerable research is being per-formed throughout the world to develop novel pharmacologicapproaches to: (1) promote apoprotein A-I and HDL particlebiosynthesis; (2) augment capacity for reverse cholesterol trans-port so as to reduce risk for the development and progression ofatherosclerotic disease; and (3) modulate the functionality of HDLparticles in order to increase their capacity to antagonize oxida-tion, inflammation, thrombosis, endothelial dysfunction, insulinresistance, and other processes that participate in arterial wallinjury. HDL metabolism and the molecular constitution of HDLparticles are highly complex and can change in response to bothacute and chronic alterations in the metabolic milieu. To date,some of these interventions have been shown to positively impactrates of coronary artery disease progression. However, none ofthem have as yet been shown to significantly reduce risk for

ol ofMedicine, Peoria, IL, USA. Tel.:þ1 (815) 632 5093; Fax:þ1 (815) 626 5947.lski), peter.toth@cghmc.com (P.P. Toth), draggana.nikolic@gmail.com (D.fredi.rizzo@unipa.it (M. Rizzo), giuseppe.montalto@unipa.it (G. Montalto).

d. All rights reserved.

M. Barylski et al. / Best Practice & Research Clinical Endocrinology & Metabolism 28 (2014) 453–461454

cardiovascular events. In the next 3–5 years a variety of pharma-cologic interventions for modulating HDL metabolism and func-tionality will be tested in large, randomized, prospective outcomestrials. It is hoped that one or more of these therapeutic approacheswill result in the ability to further reduce risk for cardiovascularevents once low-density lipoprotein cholesterol and non-HDL-cholesterol targets have been attained.

� 2013 Elsevier Ltd. All rights reserved.

Introduction

The high-density lipoproteins (HDLs) are functionally highly versatile and have the capacity to drivereverse cholesterol transport and exert a variety of other atheroprotective functions [1]. Elevatedserum levels of high-density lipoprotein cholesterol (HDL-C) are highly correlatedwith reduced risk forcardiovascular 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 eventsand 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 raisingHDL-C does correlate with reductions in both cardiovascular event rates as well as progression ofatherosclerotic disease [5–7]. Unfortunately, large prospective randomized outcomes trials in patientswith 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 ofstatin therapy. These studies have raised serious issues regarding the value of raising HDL-C inmodulating risk in the secondary prevention setting. Despite these setbacks, newer forms of phar-macologic interventions targeted at HDL metabolism and functionality are being developed and testedat a rapid rate. It is hoped that one or more of these novel approaches will help to reduce residual riskfor cardiovascular events once atherogenic lipoprotein burden in serum is controlled to guideline-defined 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 re-combinant HDL particles into the circulation, rather than increasing HDL indirectly by modulating HDLmetabolism. One approach uses recombinant apoA-IMilano. Individuals with the apoA-IMilano mutation(R173C)have lowHDL-C levels (10–30mg/dl), andnoapparent increasedcardiovasculardisease (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 byintravascular ultrasound with 5 once weekly treatments [13]. Procedural difficulties complicated thedevelopment of ETC-216 (clinical denomination of apoA-IMilano) and no further clinical trials with thisformulationhavebeen reported [14].More recently, itwas shownthat recombinantHDLcontainingapoA-IMilano exerts greater anti-inflammatory and plaque stabilizing properties rather than antiatheroscleroticproperties [15]. Another rHDL compound, CSL-111, consists of apoA-I purified from human plasma andcomplexedwithphosphatidylcholinederived fromsoybeans. Thefirst trial of CSL-111 examined the effectof rHDL in the Atherosclerosis Safety and Efficacy (ERASE) trial conducted in 183 patients with acutecoronary syndrome (ACS) [16]. Four weekly infusions of CSL-111 to 111 individuals randomized to the40 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 nosignificant change in atheroma volume, as measured by intravascular ultrasound (IVUS), compared withthe placebo group. Another study investigated the effect of CSL-111 on surrogate cardiovascularmarker inpatients 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 low-density lipoprotein cholesterol (LDL-C) (23%),humanrHDLdidnot improvevascular function compared toplacebo. 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 intravenousinfusionwith the use of autologous delipidated HDL [18]. Preclinical evaluation of selective delipidatedHDL in dyslipidemicmonkeys achieved a significant 6.9% reduction in aortic atheroma volume assessedby IVUS [19]. The process involves the selective removal of apoA-I HDL particles from plasma, delipi-dating them, and then reinfusing the cholesterol-depleted functional pre-b HDL. In a human trial, 28patients 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% frombaseline. [18] However, it is not yet established whether or not acute regression of atheroscleroticplaque volume is associated with decreased clinical cardiovascular events. Autologous delipidated HDLinfusions do not induce liver toxicity or hypersensitivity reactions. In the study HDL apheresis resultedin hypotension in one-third of the participants undergoing treatment. A delipidation system for humanuse is now available from Lipid Sciences Plasma Delipidation System-2 (PDS-2), which converts aHDLto preb-like HDL by selectively removing cholesterol from HDL in samples of plasma collected frompatients by apheresis. This approach remains under investigation.

HDL mimetics

ApoA-I mimetic peptides drugsApoAI 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 of18A, additional improved peptides were generated by increasing the number of phenylalanine resi-dues on the hydrophobic face (referred to as 2F, 3F, 4F, 5F, 6F, and 7F) of the polypeptide. [20] Amongthem only apoA-I mimetic peptide 4F showed promise in a number of animal models and in earlyhuman trials [21] leading to a phase I/II study in humans with high risk CVD [22]. In this study, the 4Fpeptide (synthesized from L-amino acids for L-4F) was delivered at low doses (0.042–1.43 mg/kg) byintravenous or subcutaneous administration [23]. Very high plasma peptide levels were achieved, butthere was no improvement in HDL anti-inflammatory function [23]. On the other hand, previousstudies showed that L-4F restores vascular endothelial function in murine models of hypercholes-terolemia [24]. In another clinical trial the 4F peptide synthesized from all D-amino acids (making itresistant 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 asignificantly improved HDL inflammatory index [22]. In humans with significant cardiovascular risk, asingle dose of D-4F was found to improve the inflammatory index of HDL with modest oralbioavailability [25].

Given concerns regarding possible cytotoxicity through ABCA1-independent lipid efflux, additionalpeptide mimetics have been engineered [26]. Peptides comprised of twenty-two amino acids based ondomains of apolipoprotein A-I that have a higher affinity for ABCA1 have been shown to promotecholesterol efflux without cytopathic effects [26–28]. Furthermore, such domains are conserved acrossother apolipoproteins, and similarly designed peptides from apolipoprotein E promote ABCA1-mediated reverse cholesterol transport [29]. Recently, 5A, an asymmetric bihelical peptide based on2F, 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 onapolipoprotein A-I. The 5A peptide had increased ABCA1-dependent cholesterol efflux and decreasedhemolysis compared with its parent compound [30].

ATI-5261 synthetic peptideNative 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 afashion similar to that of HDL and successfully enhances cholesterol efflux from macrophages andreduces aortic atherosclerosis by up to 45% after intraperitoneal injection in mice. ATI-526 increasesreverse cholesterol transport in mice [31]. The compound presently awaits early phase clinical trials inhumans.

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Endothelial lipase inhibitorsEndothelial 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 confirmedthat variation of the EL gene is an important determinant of plasma HDL-C level [32]. However, howchanges in HDL-C level attributed to EL may affect atherosclerosis is still not clear. Some human studiespropose an atherogenic role for EL, with a positive association of plasma level of EL mass and coronaryartery calcification [33]. Carriers of EL variants associated with increased HDL-C levels have been re-ported to have decreased risk of coronary artery disease [34], but this association has not beenobserved in other studies [35]. Studies in mice showed that EL overexpression reduces HDL-C andapoA-I levels [36] due to increased renal catabolism. Conversely, gene deletion of EL results inincreased HDL-C and apoA-I levels [37]. Although EL inactivation was expected to inhibit atheroscle-rosis by raising HDL-C, the effect of EL inactivation seems more complex than expected. The enthu-siasm for EL inhibitors is somewhat tempered by Mendelian randomization data showing thatvariations in the gene loci for EL and cholesterol ester transfer protein (CETP) that increase HDL-C arenot associated with protection against the development of atherosclerotic disease and its complica-tions [38].

Lecithin-cholesterol acyltransferase modulatorsSeveral drug development approaches have recently been initiated for modulating lecithin-

cholesterol acyltransferase (LCAT) activity. Early studies for the treatment of atherosclerosis byraising HDL-C through plasma LCATenzyme 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 treatmentof atherosclerosis and the dyslipidemia associated with low HDL. Intravenous infusion of human rLCATin rabbits was found to raise HDL-C, to increase fecal excretion of cholesterol, and to reduce athero-sclerosis [40]. Another potential alternative to LCAT injection for treatment of human LCAT deficiencywas recently reported in which adipocytes transfected with LCAT were transplanted into mice andwere found to raise HDL-C [41]. Only one LCATmodulator has reached early clinical development, ETC-642, but little data are available on the vascular effects of treatment with this agent [42].

Apo A-I upregulator

Reservelogix-208Reservelogix-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-Iand HDL-C [43]. Serum from human subjects treated with RVX-208 exhibited increased cholesterolefflux capacity despite a relatively modest increase in HDL-C levels [44]. In a phase II trial, modestchanges in HDL-C and apoA-I were reported in 299 statin-treated patients with stable coronary arterydisease (CAD) [45]. One Phase IIb study is ongoing and involves 172 statin-treated patients randomizedfor RVX-208 100mg or placebo twice daily for 24weeks [46]. The ASSURE trial investigated the effect ofRVX-208 on coronary atherosclerosis assessed by intravascular ultrasound [47]. The trial was negativewith no demonstrable differences in percent atheroma volume or normalized total atheroma betweenpatients treated either with placebo or RVX-208.

Synthetic liver X receptor agonistsSynthetic 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 cholesterolefflux, these compounds have been shown to raise HDL-C levels and to reduce atherosclerosis intransgenic mouse models [48]. Thus, LXR agonist activation may be a promising pharmacologic targetfor the treatment of dyslipidemia and atherosclerosis. Unfortunately, the development of first gener-ation LXR compounds has been hampered by their capacity to increase expression of lipogenic genes inthe liver, which increase levels of triglycerides and promote hepatic steatosis [49]. Various syntheti-cally engineered LXR agonists have been developed and tested in animal models. They all show highpotency for interacting with LXRa/b receptors, but none of them shows selectivity for ABCA1 andABCG1 [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 inmacrophages resulting inenriched cholesterol content in the outer layer of plasma membranes [51]. The LXR agonist LXR-623 isassociated with increased expression of ABCA1 and ABCG1 in cells [52], but adverse central nervoussystem-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 specificcellular cholesterol efflux and increased intestinal excretion of HDL-derived cholesterol [50]. Morerecently, a novel synthetic LXR agonist, ATI-111, that is more potent than T0901317, inhibited athero-sclerosis progression and prevented atheromatous plaque formation in mice [55]. Research on moreselective LXR ligands is an active area of experimental pharmacology.

Synthetic farnesoid X receptor agonistsThe 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 studiesshowed that activation of FXR leads to both pro- and antiatherosclerotic effects and a major metaboliceffect of FXR agonists in animal models is a reduction of plasma HDL [56,57]. Hambruch et al. showedthat FXR agonists promote HDL-derived cholesterol excretion into feces in mice and monkeys [57]. Forthese reasons, FXR agonists have received attention as a potential therapeutic target [58], and differentagonists have been generated as a strategy for HDL-C raising therapies. These include GW4064, 6-ECDCA, FXR-450, and PX20606 [57]. GW4064 has potential cell toxicity and uncertain bioavailabilityprevents 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 FXRmediating HDLC clearance are conserved inmice andmonkeys. These observationswill support furtherstudies 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 ofatherosclerosis [59,60]. To date, this approach has little application in man. Animal data supports novel

Table 1Summary of selected strategies to increase HDL/apoA-I and potential compound under development.

Pharmacotherapeutic strategy Drug Aim

Recombinant apoA-IMilano/phospholipids

ETC-216 Directly augmenting apoA-I/HDL pool

Purified native apoA-I/phospholipids CSL-111 and CSL 112 Directly augmenting apoA-I/HDL poolUpregulators of endogenous apoA-I

productionRVX-208 Directly augmenting apoA-I/HDL pool

ApoA-I mimetic peptides D-4FL-4FATI-5261

Mimicking apoA-I functionality

Autologous delipidated HDL Selective HDL delipidated Directly augmenting apoA-I/HDL poolGene therapy miR-33 Modulating HDL levels and

cholesterol efflux expressionLiver X receptor agonists LXRa/b agonists

LxR-623T0901317, GW3965ATI-111

Enhancing RCT & Macrophagecholesterol efflux

Niacin receptor agonists ARI-3037MO Indirectly augmenting apoA-I andHDL-cholesterol

Farnesoid X receptor FxR-450 Modulate HDL levelsCholesteryl ester transfer inhibitors Anacetrapib MK-0859

Evacetrapib LY248595Indirectly augmenting apoA-I andHDL-cholesterol

Endothelial lipase inhibition Boronic acid inhibitorsSelective sulfonylfuran urea

Increasing HDL-cholesterol

LCAT activators rLCATETC-642

Enhancing RCT

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gene-based approaches to increase HDL-C. A potential mechanism to increase of HDL-C is increasingABCA1 and ABCG1 expression through the therapeutic manipulation of microRNA metabolism (inhi-bition of miR-33 being the most promising candidate to date). Overexpression of antisense miR-33using a lentivirus vector in mice showed a 50% increase in hepatic ABCA1 protein levels and aconcomitant 25% increase in plasma HDL levels after 6 days [61]. Marquart et al. showed that injectionof an anti-miR-33 oligonucleotide in mice resulted in a substantial increase in ABCA1 expression andHDL levels [62]. Furthermore, it was shown inmice that miR-33 decreases cholesterol efflux [63]. Thesedata suggest that miR-33 might be a possible target for the treatment of cardiovascular and metabolicdisorders. Table 1 summarizes selected strategies to increase HDL/apoA-I and describes potentialcompounds under development.

Conclusion

Unlike the path taken to therapeutically modulate LDL-C levels, that for HDL-C has turned out to bemuch more difficult and complicated. Two CETP inhibitors, RVX-208, and multiple other agents havefailed in clinical trials. Is the apparent protectiveness of HDL-C suggested by epidemiology simply anepiphenomenon? Is it possible that the exceptional complexity of HDL’s proteome and lipidome reallyhave little bearing on the structural and functional integrity of arterial walls? It is hoped that these andmany other questions concerning HDL therapeutics will be answered in the years ahead with theinnovative approaches summarized herein. In the meantime, we will await the results of clinical trialstesting the ability of these various agents to impact rates of atherosclerotic disease progression and riskfor 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, humannative 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-alphaand 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 anantisense molecule to miR-33. Antisense technology is already being used in humans to treatfamilial hypercholesterolemia with mipomersen, an antisense oligonucleotide directed againstthe mRNA for apoprotein B.

Research agenda

HDL particles are highly heterogeneous with diverse lipid and protein cargos. It will beimportant to establish:

1. How specific HDL interventions impact both the serum concentration of HDL particles andtheir functionality.

2. If functionality is greatly increased, is it even necessary to robustly elevate serum levels ofHDL?

3. If specific functions (e.g., RCT, anti-oxidative capacity) are augmented or even adverselyaffected 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 combinationwith other lipid modifying drugs such

as the statins or fibrates6. The impact of specific agents on rates of atherosclerosis disease progression and cardiovas-

cular events (myocardial infarction, ischemic stroke, death, need for coronary and peripheralrevascularization) will have to be assessed in large prospective outcomes and imaging trials.

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