International Journal of Cardiology 192 (2015) 72–81

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International Journal of Cardiology

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

Review

Atherosclerosis: Recent trials, new targets and future directions

Ricardo Ladeiras-Lopes a,b, Stefan Agewall c, Ahmed Tawakol d,e, Bart Staels f, Evan Stein g, Robert J. Mentz h,
Adelino Leite-Moreira a, Faiez Zannad i, Wolfgang Koenig j,k,l,⁎
a
Department of Physiology and Cardiothoracic Surgery, Cardiovascular Research and Development Centre, Faculty of Medicine of the University of Porto, Porto, Portugal
b
Department of Cardiology, Gaia/Espinho Hospital Centre, Vila Nova de Gaia, Portugal
c
Department of Cardiology, Oslo University Hospital Ullevål and Institute of Clinical Medicine, University of Oslo, Oslo, Norway
d
Cardiology Division, Massachusetts General Hospital, Boston, MA, USA
e
Cardiology Division, Harvard Medical School, Boston, MA, USA
f
INSERM UMR1011, Univ. Lille 2, Institut Pasteur de Lille, EGID, Lille, France
g
Metabolic and Atherosclerosis Research Centre, Cincinatti, OH, USA
h
Duke Clinical Research Institute and the Division of Cardiology, Duke University Hospital, Durham, NC, USA
i
INSERM, Centre d'Investigation Clinique-9501, Université de Lorraine, Nancy, France
j
Department of Internal Medicine II — Cardiology, University of Ulm Medical Center, Ulm, Germany
k
Deutsches Herzzentrum München, Technische Universität München, Munich Germany
l
DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Mortality from cardiovascular diseases (CVD) represents the primary cause of death worldwide. Prevention or
Received 30 December 2014 treatment of atherosclerosis and its clinical sequelae is a central goal in the management of patients with
Received in revised form 9 April 2015 established vascular disease or those at high-risk for vascular events. This paper provides a review of the contem-
Accepted 6 May 2015
porary pharmacological armamentarium targeting atherosclerosis and also highlights strategies to support fu-
Available online 8 May 2015
ture clinical trial design. Powering future trials targeting LDL-cholesterol to its absolute reduction and
Keywords:
including patients with a higher LDL-C despite optimal medical therapy (or unable to tolerate statins) will in-
Atherosclerosis crease the odds of meaningful results. Mendelian randomization studies may identify new causal risk factors
Pharmacology for CVD that would help in the selection of the patients most likely to benefit from a specific new compound. Fur-
Clinical research thermore, imaging techniques integrating a morphological and functional assessment such as IVUS, OCT, PET/CT
and PET/MRI may represent in a near future robust “soft” endpoints to support successful translation of early re-
search into meaningful phase III clinical outcome trials.
© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction LDL-C after an acute coronary syndrome (ACS) suffers a subsequent

Clinical complications of atherosclerosis like myocardial infarction
(MI), stroke and peripheral arterial disease represent the leading
cause of mortality and morbidity in the world [1]. Therefore, the devel-
opment of drugs targeting the atherosclerotic process has been an im-
portant area of contemporary clinical research.
While statins are the cornerstone of prevention they are not the pan-
acea for eliminating atherosclerotic cardiovascular disease (ASCVD) [2]. A
meta-analysis published in 2004 including patients on standard statin
therapy showed a 21.7% 5-year event rate for major adverse cardiovascu-
lar event (MACE) in patients with prior ASCVD [3]. Furthermore, almost 1
in 10 high-risk patients treated with intensive statin therapy with low
⁎ Corresponding author at: Klinik für Herz-& Kreislauferkrankungen, Deutsches
Herzzentrum München, Technische Universität München, Lazarettstr. 3680636 Munich,
Germany. Tel.: +49 89 1218 3235; fax: +49 89 1218 4083.
E-mail addresses: ricardoladeiraslopes@gmail.com (R. Ladeiras-Lopes),
koenig@dhm.mhn.de (W. Koenig).
adverse event within a 2-year period [4]. In this way, there is significant
residual cardiovascular risk that persists despite low LDL-C, highlighting
the role of “atherogenic dyslipidemia” that includes low HDL-C and high
triglycerides [5].It is likely that longer duration of statin therapy combined
with early introduction of LDL-C lowering will reduce residual risk, and
with inexpensive and effective statins now available, recent guidelines
in the USA has expanded such treatment to lower risk patients [6].
The utilization of complex imaging techniques including anatomical
and functional evaluations and a better selection of high-risk patients
may foster the successful design of trials. Indeed, a large number of
lipid-associated new targets or targets reflecting other pathways of the
complex atherosclerotic process are being evaluated in mechanistic im-
aging studies as well as in large outcome trials in the endeavor of residual
vascular risk reduction (see Tables 1, 2 and 3). The latter will definitely in-
clude patient-tailored approaches in order to modulate the complex ath-
erosclerotic process, emphasizing a strategy of personalized medicine [7].
This paper results from a discussion at the 2013 Cardiovascular
Clinical Trialists' Forum held in Paris, France and aims to provide an

http://dx.doi.org/10.1016/j.ijcard.2015.05.013
0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
 R. Ladeiras-Lopes et al. / International Journal of Cardiology 192 (2015) 72–81 73

overview of the most recent clinical data on antiatherosclerotic com- with a significant change in carotid intima-media thickness (cIMT)
pounds, emphasizing new targets and strategies for robust clinical [11]. These results challenged not only the role of ezetimibe in CVD
trial design. prevention but also pointed to the limitations of biomarkers and sur-
rogate non-invasive markers to assess the efficacy of a lipid-lowering
drug.
2. Lipoprotein metabolism However, the results of the Improved Reduction of Outcomes:
Vytorin Efficacy International (IMPROVE-IT) Trial comparing simvastat-
2.1. Cholesterol absorption inhibition in plus ezetimibe with simvastatin alone in 18,144 patients included
within 5 days post-ACS showed a modest 6.4% relative risk reduction
Ezetimibe is a selective inhibitor of cholesterol absorption at the (RRR) of the primary endpoint and thus demonstrated a beneficial
brush border of the small intestine mediated by the Niemman–Pick role of ezetimibe in vascular protection [12]. Baseline LDL-C was
C1-like 1 protein, thus impairing the uptake of intestinal lumen micelles 95 mg/dl and was reduced to 69.5 mg/dL in the simvastatin group and
into the enterocyte [8,9] (Fig. 1). Lower cholesterol absorption depletes to 53.7 mg/dL in the combination group during 7 years of follow-up
the hepatic pool of cholesterol and upregulates hepatic LDL receptors, with a good safety profile.
therefore boosting LDL-C clearance from the circulation. Ezetimibe is
able to induce a significant LDL-C reduction of 15–20% alone or by ap- 2.2. PCSK9 inhibition
proximately 50% combined with simvastatin [10].
Although the results of ezetimibe and simvastatin in Hypercholes- The proprotein convertase subtilisin/kexin (PCSK) type 9 is a serine
terolemia Enhances Atherosclerotic Regression (ENHANCE) Trial protease which modulates lipoprotein metabolism by its circulating
including patients with familial hypercholesterolemia showed a reduc- component binding with LDL to the hepatic LDL receptor promoting he-
tion in LDL-C and hsCRP when a combination therapy of simvastatin and patic internalization and intracellular degradation of the LDL receptor,
ezetimibe was compared to simvastatin alone, yet it was not associated preventing its recycling (Fig. 1) [9,13]. The latter is a fundamental player
in LDL clearance from the circulation. In addition, pre-clinical research
has also identified roles for PCSK9 in peripheral tissues. For instance,
Table 1 PCSK9 is induced by inflammation [14] and small interfering RNA
Drug targets for atherosclerosis and current research development phase. (siRNA) knockdown of PCSK9 in human macrophages attenuates the in-
flammatory response elicited by oxidized LDL (oxLDL) [15]. Lastly, stat-
Target Clinical
research in therapy increases the expression of PCSK9 [16]. The integration of
phase these data, together with overwhelming evidence that patients with
LDL lowering
loss-of-function mutations of PCSK9 have 15–30% reductions in LDL-C
PCSK9 antibodies and 47–88% reduction in the risk of coronary heart disease (CHD)
Alirocumab (SAR236553/REGN727) 3 [19], supports a role for PCSK9 as an interesting target for anti-
Evolocumab (AMG145) 3 atherosclerosis drug therapy.
Bococizimab (PF-04950615/RN316) 3
There are several ways to interfere with PCSK9: mimetic peptides and
MTTP (microsomal triglyceride transfer protein) inhibitors 3
Antisense apoB (mipomersen) 3 adnectins (with similarities to small monoclonal antibodies), small-
ATP-citrate lyase inhibitor (ETC-1002) 2 molecule inhibitors, gene silencing (e.g., antisense oligonucleotides and
TR-β agonist (MGL-3196) 1 siRNA) and monoclonal antibodies [13]. To date, the most promising
Lp(a) strategy to impair the action of PCSK9 appears to be the use of monoclo-
Antisense apo(a) 1
Triglycerides
nal antibodies. Indeed, two monoclonal antibodies targeting PCSK9,
Antisense apo C-III (ISIS 304801) 2 AMG145 (evolocumab, Amgen), SAR236553/REGN727 (alirocumab,
PPAR agonists (α:K877, α/δ:GFT505, δ:CER-002) 2 Sanofi/Regeneron) and bococizumab (Pfizer) reported encouraging re-
Omega-3 free fatty acids (eicosapentaenoic acid and 3 sults in phase 1 and 2 trials. The body of evidence accumulated since
docosahexaenoic acid)
2009 has shown that these drugs elicit a significant and sustained
Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT-1) inhibitors 2
(LCQ908) 40–70% reduction of LDL-C in patients with familial hypercholesterol-
Angiopoietin-like proteins (ANGPTLs) 3 and 4 inhibitors Pre-clinical emia (FH) [18–22] and non-FH [21,23–26]. LDL-C lowering was observed
HDL in patients with PCSK9 inhibitors, alone or in combination with a statin,
Reconstituted HDL (CSL-112) 2 and also in statin-intolerant patients [27]. Phase 3 trials are currently in
Recombinant HDL (CER-001) 2
Apo A-I inducer (RVX-208) 2
progress to assess the role of the following drugs on cardiovascular
Apo A-I milano (ETC-216/MDCO-216) 1 events: evolocumab (FOURIER; 28,000 patients with prior MI or stroke)
Apo A-I mimetics (D6F APP018, ATI-5261, …) 2 [28], alirocumab (ODYSSEY OUTCOMES; 18,000 patients with recent
ABCA1 inducer Pre-clinical ACS) [29] and bococizumab (SPIRE 1 and 2; 17,000 and 9,000 patients,
Antagomir miR-33 Pre-clinical
respectively, with high CV risk as defined by LDL-C levels) [30,31].
CETPi (evacetrapib, anaceptrapib) 3
rLCAT (APC-501) Pre-clinical The potential disadvantages of these agents include their subcutane-
LXR-623 Pre-clinical ous administration (although it may be performed monthly or twice
SR-B1 inhibitor (ITX5061) 1 monthly, which may be an advantage compared to a daily drug use)
Inflammation/atherosclerosis and injection site reactions (in about 6% of patients both with
Inflammation
sPLA2 (varespladib) 3
evolocumab [32] and alirocumab [32]). Furthermore, long-term im-
LpPLA2 (darapladib) 3 mune reactions even to fully human antibodies may appear, although
ox-LDL antibodies 2 available evidence from trials suggests that they are rare. The larger
5-lipoxygenase inhibitors (VIA-2291) 2 phase 3 trials underway may help to clarify the long-term tolerability
Methotrexate (CIRT) 3
of these drugs. The recently published results of a 52-week treatment
CCR-2 inhibition 2
CRP antisense 2 with evolocumab (DESCARTES; every 4 weeks) showed sustained effi-
IL1b/IL1RA/Inflammasome inhibition (canakinumab) 3 cacy in reducing LDL-C with acceptable safety and tolerability [33]. Fur-
Immunization strategies thermore, recently announced findings showed that alirocumab on top
Antibodies against modified apoB epitopes (MLDL1278A) 2 of statin therapy (administered every 2 weeks) in very-high risk pa-
ApoB vaccination strategies Pre-clinical
tients with hypercholesterolemia elicited a 48% reduction in MACE
74 R. Ladeiras-Lopes et al. / International Journal of Cardiology 192 (2015) 72–81
Fig. 1. Schematic representation of promising targets for atherosclerosis drug therapy. CETP — Cholesteryl ester transfer protein; EL — endothelial lipase; LDLr — LDL receptor; Lp-PLA2 —
lipoprotein-associated phospholipase A2; MTTP — Microsomal triglyceride transfer protein; NPC1L1 — Niemann–Pick C1-Like 1; oxLDL — oxidized LDL particles; sPLA2 — secretory
phospholipase A2.
over 78 weeks of treatment [34]. Similar results have been reported for
evolocumab from the OSLER program with evolocumab [35]. The results
of the larger, outcome-driven trials may help to clarify the role of these
promising agents in the treatment of atherosclerosis.
2.3. Microsomal triglyceride transfer protein (MTTP) inhibition
MTTP is an intracellular lipid-transfer protein with a central role in
the assembly of chylomicrons and VLDL in enterocytes and hepatocytes,
respectively [9,36]. Patients with abetalipoproteinemia have extremely
low levels of apoB-containing lipoproteins (chylomicrons, VLDL, IDL
and LDL) in circulation due to a defect in MTTP activity [37], suggesting
a potential therapeutic role for MTTP inhibition.
Lomitapide is a MTTP inhibitor approved in the US and in the EU for
the treatment of homozygous FH. Approval was based on the recently
published results of an open-label phase III trial including 29 patients
with homozygous FH which demonstrated significant reductions
of LDL-C (30–50%), triglycerides (31–45%), apoB (43–49%) and
Lp(a) (15–19%) [38]. Lomitapide had been demonstrated in a phase II
trial to elicit additional LDL-C reduction as add-on therapy to ezetimibe
in patients with non-FH [39]. However, the potential for an increase in
hepatic fat [40] and liver enzyme elevations in these trials raised con-
cerns about potential hepatotoxicity of lomitapide.
Future outcome trials with lomitapide as well as additional
studies with experimental MTTP inhibitors (implitapide [41,42] and
CP-346086 [43]) are needed. A small-molecule (SLx-4090) designed
to selectively inhibit enterocyte MTTP is also under investigation [44],
possibly overcoming the problem of hepatic liver accumulation with
previous inhibitors. However, enterocyte-specific MTTP inhibitors may
be associated with gastrointestinal adverse effects and deficiency of
fat-soluble vitamins [45], therefore further research is needed to clarify
the efficacy and tolerability of these agents.
2.4. Antisense oligonucleotides targeting apolipoproteins
Antisense oligonucleotides (ASO) are short (about 20 nucleotides)
single-stranded nucleic acid analogs that bind to complementary se-
quences in target mRNAs, preventing their translation into the protein
product [46].
Mipomersen is an ASO that blocks hepatic synthesis of apo B-100, a
central component of all proatherogenic lipoproteins. It has been shown
to decrease VLDL-C and LDL-C levels [47–49]. Its consistent efficacy led
to the FDA approval for the treatment of homozygous FH in January
2013. Mipomersen requires weekly subcutaneous administration and,
as lomitapide, is associated with hepatic fat accumulation [47].
Other ASOs targeting different apolipoproteins have also appeared
in the last several years, aiming at decreasing triglycerides and in-
creasing HDL-C and also decreasing Lp(a). ISIS-APOCIII is an ASO
targeting apo C-III that reduces triglycerides and increases HDL-C with
an acceptable safety profile [50]. Furthermore, an ASO targeting
apolipoprotein(a) is also under development, eliciting a specific, robust
and sustained dose-dependent lowering of Lp(a), an independent risk
factor for CVD [51]. Additional clinical studies assessing the clinical ben-
efit of this new generation of therapies are encouraged.
Table 2
Recently published outcome-driven phase 3 trials.

Trial, year Drug therapy Population Number Mean baseline Length of Effect on lipid Primary outcome Results (primary Comments
published of lipids (mg/dL) follow-up concentration outcome)
(reference) patients (months)
enrolled

ILLUMINATE, Torcetrapib 60 mg daily + 45–75 years, 15,067 LDL 80; HDL 49; 12 ↓ LDL 22 mg/dL; ↑HDL Death from CAD, non-fatal 6.2% event-rate in the Torcetrapib increased cardiovascular
2007 [78] atorvastatin versus atorvastatin history of CVD apoA1 128; (prematurely 6 mg/dL; ↓ TG 10 mg/dL; MI, stroke, hospitalization for intervention group; 5.0% and non-cardiovascular death; 5.4

R. Ladeiras-Lopes et al. / International Journal of Cardiology 192 (2015) 72–81

only or type 2 apoB 73 terminated) ↑ apoA1 25% (at 3 months); unstable angina in placebo; HR = 1.25 mm Hg increase in SAP
diabetes ↓ apoB 13% (at 3 months) (1.09–1.44)
AIM-HIGH, ER Niacin (1500–2000 mg) + ≥45 years, 3,414 LDL 74; HDL 35; 36 ↓ LDL 14%; ↑HDL 25%; Death from CAD, non-fatal 16.4% event-rate in the No incremental benefit of niacin in
2011 [72] simvastatin 40–80 mg+/− history of CVD TG 168; apoA-1 ↓ TG 31% MI, ischemic stroke, intervention group; reducing CV events despite
ezetimibe 10 mg daily versus 122; apoB 83 hospitalization for ACS or 16.2% in placebo; HR = significant increase in HDL-C and
simvastatin 40–80 mg+/− symptom-driven coronary or 1.02 (0.87–1.21) decrease in TG
ezetimibe 10 mg daily cerebral revascularization
Dal-OUTCOMES, Dalcetrapib 600 mg daily + ≥45 years; 15,871 LDL 76; HDL 42; 31 Minimal effect on LDL; Major coronary event 8.3% event rate with Substantial increase in HDL with
2012 [60] OMT versus OMT recent ACS apoA1 137; ↑HDL 31–40%; ↑ TG 4–10%; (including death from CAD) intervention; 8.0% with no effect on CV outcomes; 0.6 mm
apoB 81 ↑ apoA1 9%; minimal effect and ischemic stroke place; HR 1.04 g increase in SAP; ↑18% CRP levels
on apoB (0.93–1.16)
HPS-2 THRIVE, ER niacin (2 g) + laropiprant 50–80 years, 25,673 LDL 63; HDL 44; 48 ↓ LDL 10 mg/dL; ↑HDL Major coronary event 13.2% event-rate at 4 Significant excess of new-onset
2013 [73] (40 mg) daily versus simvastatin history of CVD TG 125 34 mg/dL; ↓ TG 33 mg/dL (including death from CAD), years in the intervention diabetes, diabetic complications,
40 mg (+/− ezetimibe) stroke (including death or group; 13.7% in placebo; bleeding, infection in the
hemorrhagic), HR = 0.96 (0.90–1.03) intervention group
revascularization
VISTA-16, 2014 Varespladib 500 mg daily + ≥40 years; 5,145 LDL 105; HDL 4 ↓ LDL 29%; ↑HDL 5.1%; Death from CAD, non fatal 6.1% event rate with Significant increased risk of MI and
[102] OMT versus OMT recent ACS 43; TG 154 (prematurely ↓ TG 22% MI, stroke, hospitalization for intervention; 5.1% with combined cardiovascular mortality,
terminated) unstable angina place; HR 1.25 MI and stroke in varespladib-treated
(0.97–1.61) patients; 6-month survival data
collected in only 31% of patients;
↓85% CRP levels
STABILITY, Darapladib 160 mg daily + ≥18 years; 15,828 LDL 80 (median) 44 N/A Cardiovascular death, MI or 9.7% event rate in No benefit of darapladib in stable
2014 [97] OMT versus OMT established CVD stroke intervention group; CAD
or T2DM with 10.4% in placebo; HR
documented 0.94 (0.85–1.03)
CAD
SOLID-TIMI 52, Darapladib 160 mg daily + ≥18 years; 13,026 LDL 75; HDL 42; 30 N/A Death from CAD, non-fatal 16.3% event rate in the Darapladib was associated with
2014 [98] OMT versus OMT recent ACS TG 135 MI, or urgent coronary intervention group; increased odor-related concerns
( 30 days) revascularization 15.6% with placebo; HR and diarrhea (10.6% vs 5.6%)
1.00 (0.91–1.09)

75
76 R. Ladeiras-Lopes et al. / International Journal of Cardiology 192 (2015) 72–81

Table 3
Ongoing outcome-driven phase 3 trials.

Trial Drug therapy Population Estimated Time frame Start Primary outcome Estimated
(reference) enrollment (years) date completion
date

IMPROVE-IT [12] Ezetimibe 10 mg +
simvastatin 40 mg
(single tablet) versus
simvastatin 40 mg
REVEAL [79] Anacetrapib 100 mg daily
ACCELERATE [80] Evacetrapib 130 mg daily
FOURIER [28] Evolocumab (AMG145)
every 2 weeks or monthly
ODYSSEY Alirocumab (SAR236553)
OUTCOMES [29] 75 mg every 2 weeks
SPIRE 1 [30] Bococizumab
(PF-04950615) 150 mg
every 2 weeks
SPIRE 2 [31] Bococizumab
(PF-04950615) 150 mg
every 2 weeks
CANTOS [104] Canakinumab 50/150/300
mg every 3 months
CIRT [105] Methotrexate 15–20 mg
weekly plus 1 mg folic acid
6 days/week
≥18 years; recent ACS; LDL-C 18,141 Minimum October All-cause death, MI, stroke September 2014
125 mg/dL (not on statin) or (completed) follow-up of 2005
LDL-C 100 mg/dL (on statin 2.5 years for
therapy) each patient
≥50 years; established ASCVD 30,000 4 June 2011 Major coronary event January 2017
(MI, ischemic stroke, carotid (coronary death, myocardial
revascularization, PAD) or infarction or coronary
T2DM and symptomatic CAD revascularization)
≥18 years; established ASCVD 12,000 4 October CV death, MI, stroke, January 2016
(ACS, cerebrovascular, PAD) or 2012 coronary revascularization or
T2DM with documented CAD hospitalization for unstable
angina
40–85 years; established ASCVD 22,500 5 January Cardiovascular death, MI, February 2018
(ACS, cerebrovascular, PAD); 2013 coronary revascularization,
LDL-C ≥ 70 mg/dL OR non- hospitalization for unstable
HDL-C ≥ 100 mg/dL; fasting angina, stroke
triglycerides ≤ 400 mg/dL
≥40 years; recent ACS 18,000 Up to 6 years October Cardiovascular death, MI or January 2018
(b52 weeks) 2012 stroke, hospitalization for
unstable angina
≥18 years; on background 12,000 5 October Cardiovascular death, MI or August 2017
lipid lowering treatment; 2013 stroke, hospitalization for
LDL-C ≥ 70 and 100 mg/dL unstable angina needing
OR non-HDL-C ≥ 100 urgent revascularization
and 130 mg/dL
≥18 years; on background 6300 5 October Cardiovascular death, MI or August 2017
lipid lowering treatment; 2013 stroke, hospitalization for
LDL-C ≥ 100 mg/dL OR unstable angina needing
non-HDL-C ≥ 130 mg/dL urgent revascularization
≥18 years; MI in the last 17,200 3 April 2011 Cardiovascular death, MI or April 2017
30 days; hs-CRP N 2 mg/L stroke
≥18 years; MI in the last 7000 Up to 6 years April 2013 Cardiovascular death, MI or December 2018
5 years; T2DM or metabolic stroke
syndrome

2.5. Targeting HDL
The epidemiological association between low HDL-C and increased
risk of CVD in the general population is strong and consistent [52]. How-
ever, Mendelian randomization studies using variants in genes control-
ling HDL-C levels, such as the adenosine triphosphate binding cassette
A1 (ABCA1) [53] and lecithin-cholesterol acyl transferase (LCAT) [54]
genes, do not support a causal role for HDL-C in determining CV risk.
Furthermore, gene polymorphisms at the CETP and endothelial lipase
loci associated with increased HDL-C do not confer cardioprotection
[55]. Therefore, raising HDL may not uniformly lower CV risk and cur-
rent studies focus on identifying determinants of HDL functionality,
rather than on merely HDL-C raising.
Furthermore, there is conflicting data from studies assessing the as-
sociation between low HDL-C and residual CV risk of patients with
established atherosclerotic disease on intensive statin therapy. A recent
post-hoc analysis of the COURAGE trial showed an increased risk of
MACE in patients with low HDL-C, despite optimal medical therapy in-
cluding intensive statin treatment [56]. On the other hand, several stud-
ies found no association between low HDL-C and MACEs in patients
using intensive lipid lowering medication [57–60].
At present, strategies shift from approaches solely aiming to increase
HDL-C levels to ones that involve a better understanding of HDL func-
tion [61,62], through analysis of its functionality and composition in-
cluding several lipids and proteins [63]. There is a need for robust
biomarkers of HDL function that could be addressed in future clinical tri-
als. For example, in patients receiving high-dose statin therapy, the
number of HDL particles may be a better marker of residual risk than
HDL-C or apoA-I levels [64]. Furthermore, cholesterol efflux capacity
from macrophages, an index of HDL function [65], is impaired in
humans with ASCVD and dysfunctional oxidized HDL particles [66].
Myeloperoxidase (MPO) has a definite role on the immune phago-
cytic response but this enzyme also represents a potential link between
inflammation and atherosclerosis. Its circulating levels are increased in
stable coronary artery disease and ACS [67,68] and are associated with
cardiovascular mortality [69]. Oxidative modification of apoA-I leading
to impaired HDL function may partially account for the deleterious car-
diovascular effect of MPO [70]. Again, fully functional HDL particles
might be more important than merely high HDL-C levels.
The clinical impact of pharmacological interventions to increase
plasma HDL-C at present is questionable. Indeed, the available evidence
suggests that this therapeutic strategy is either neutral or harmful to our
patients. Therefore, we would like to see a better characterization of
HDL function in future studies, in order to translate such new evidence
to improved therapeutic approaches.
2.5.1. Niacin
Niacin decreases plasma triglycerides, LDL-C, Lp(a) and increases
HDL-C by several mechanisms, including the inhibition of adipose tissue
lipolysis and hepatic triglyceride synthesis [71]. While the neutral re-
sults of the AIM-HIGH trial are still in our mind (no decrease in cardio-
vascular death, non-fatal MI, hospitalization for ACS or coronary
revascularization, despite significant improvements in HDL-C and tri-
glyceride levels) [72], the role of niacin in the treatment of hypercholes-
terolemia and CVD prevention suffered another major drawback with
the results of the Heart Protection Study 2-Treatment of HDL to Reduce
the Incidence of Vascular Events (HPS2-THRIVE) trial. This large phase
III trial demonstrated no benefit of adding niacin and laropiprant to
 R. Ladeiras-Lopes et al. / International Journal of Cardiology 192 (2015) 72–81
simvastatin (with or without ezetimibe) in reducing MACEs [73], with a
significant excess of serious adverse events (hemorrhagic stroke, infec-
tions, diabetes and myopathy) in the niacin/laropiprant group [74].
Interestingly, despite a 20% reduction in LDL-C and 17% increase
in HDL-C, no impact on hard outcomes was demonstrated in 25,673 pa-
tients with a baseline LDL-C of 64 mg/dL. This low baseline LDL-C
might have potentially hampered the trial (see below “Pathway for suc-
cessful trial design — Power to absolute reduction”). The evidence ob-
tained from this trial questions results from previous trials which
suggested a potential beneficial role of niacin in CVD prevention [75].
2.5.2. Cholesteryl ester transfer protein (CETP) inhibitors
CETP is a glycoprotein secreted by the liver that mediates lipid trans-
fer between lipoproteins in plasma [9,76]. CETP participates in the
indirect pathway of reverse cholesterol transport (RCT) by transferring
cholesteryl esters from HDL to VLDL, IDL and LDL, in this way
reintroducing cholesterol in the endogenous lipid pathway. Therefore,
blocking CETP has the biological rationale to become a promising ther-
apeutic target.
Preliminary studies demonstrated that CETP inhibitors substantially
increase HDL-C and decrease LDL-C [77], depending on their potency.
However, the initial large trials did not demonstrate outcome benefits
with these agents (ILLUMINATE trial with torcetrapib [78] and dal-
OUTCOMES trial with dalcetrapib [60]). The results of several phase III
outcome-driven trials with two new, more potent agents are pending:
the Randomized Evaluation of the Effects of Anacetrapib through
Lipid-modification (REVEAL) [79] and A Study of Evacetrapib in High-
risk Vascular Disease (ACCELERATE) [80].
2.5.3. Apolipoprotein A1 inducers
The upregulation of endogenous synthesis of apolipoprotein A-I
(apoA-I), the major protein carried on HDL particles, represents another
potential target to increase HDL-C by generating nascent HDL particles
[9,81]. RVX-208 is an orally administered small molecule that
upregulates apoA-I synthesis through an epigenetic mechanism (RVX-
208 is an inhibitor of BET transcriptional regulators [82]). Patients
with coronary artery disease treated for 12 weeks with RVX-208 on
top of statin therapy showed a dose-dependent increase in apoA-I and
HDL-C levels [83]. Furthermore, RVX-208 is also associated with an in-
crease in the number and size of HDL particles [81]. Yet, results of the re-
cently reported ApoA-I Synthesis Stimulation and Intravascular
Ultrasound for Coronary Atheroma Regression Evaluation (ASSURE)
trial (323 patients followed for 24 weeks) failed to demonstrate plaque
regression as assessed by intravascular ultrasound imaging (IVUS) [84];
however, increases in ApoA1 by RVX-208 were very modest. Since this
is not a validated surrogate endpoint in atherosclerosis trials, future
studies are needed to identify whether there is a role for this class of
agents. Consequently, there is still room for potent apoA-I inducers to
be tested for efficacy against ASCVD.
2.5.4. HDL mimetics
An intravenous recombinant form of apoA-I Milano (a naturally oc-
curring variant of apoA-I associated with protection from vascular dis-
ease despite very low levels of HDL-C [85]), ETC-216, showed a
significant reduction in coronary atheroma volume using IVUS [86].
Event-driven trials are needed to clarify the role of this therapy.
The recently published CHI-SQUARE (Can HDL Infusions Significant-
ly Quicken Atherosclerosis Regression) trial was the largest randomized
clinical trial to date testing the efficacy of serial HDL infusions in patients
with recent ACS [87]. The HDL mimetic consisted of a recombinant
apoA-I combined with two phospholipids. This study missed its primary
efficacy endpoint of a reduction in total coronary atheroma volume by
IVUS. Therefore, despite the mechanistic potential of HDL mimetics
[88], further research is needed to clarify the role of these agents in ath-
erosclerosis treatment.
77
2.6. MicroRNAs
MicroRNAs (miRNAs) are short non-coding RNAs that modulate
messenger RNA stability, therefore exerting a post-transcriptional regu-
lator role that may represent a novel and promising way of targeting the
atherosclerotic process [89]. Indeed, miRNAs seem to play an important
but still vaguely characterized role on early atherosclerosis plaque for-
mation, as well as on plaque rupture and erosion [90]. Current evidence
suggests that a miRNA profile including the combination of several cir-
culating miRNAs will probably represent a better approach to identify
vulnerable patients and disease states. For instance, a specific miRNA
profile in patients with ACS has already been described [91].
Targeting miRNAs may represent a new pharmacological tool to
modulate the atherosclerotic process. For example, miRNA-33 a/b re-
presses the expression of the cholesterol transporter ABCA1, which is
a pivotal regulator of HDL genesis. Inhibition of this mi-RNA in non-
human primates raised plasma HDL and lowered VLDL triglycerides, a
promising finding for this new type of pharmacological intervention
[92]. At the moment we still have more questions than answers:
which miRNAs should be targeted? Should we target site-specific
miRNAs or will it be possible to administer a systemic inhibitor? Clearly,
further studies are strongly encouraged to help us understand the role
of miRNAs in atherosclerosis treatment.
3. Inflammation and the atherosclerotic process
Atherosclerosis is a chronic local inflammatory disease characterized
by a systemic low-grade inflammatory response that is associated with
MACE including MI and stroke [93,94]. The opportunity to modulate the
inflammatory pathway that plays a central role in the transition from a
stable to a vulnerable plaque, platelet and coagulation activation and
thus increasing risk for clinical events is both attractive and feasible.
The phospholipase A2 (PLA2) family includes several enzymes in-
volved in the generation of precursors of proinflammatory mediators
(e.g., leukotrienes, eicosanoids, platelet-activating factors) and is also
involved in lipoprotein remodeling yielding highly atherogenic oxidized
LDL particles [95].
Darapladib is an orally active inhibitor of lipoprotein-associated
PLA2 (Lp-PLA2) that is currently in phase III testing despite an earlier
neutral study assessing plaque deformability and CRP levels in the Inte-
grated Biomarkers and Imaging Study (IBIS)-2 trial (darapladib
prevented necrotic core expansion, though) [96]. The recently pub-
lished results of the Stabilization of Atherosclerotic Plaque by Initiation
of Darapladib Therapy (STABILITY) trial, including patients with stable
CAD, did not reduce time to first CV event [97], but reduced several sec-
ondary endpoints. Darapladib also did not significantly reduce CV
events in patients with recent ACS in the SOLID-TIMI 52 (Stabilization
of Plaques using Darapladib — Thrombolysis in Myocardial Infarction)
trial [98]. Still, analyses focusing on treatment response depending on
baseline levels of Lp-PLA2 activity are pending.
Varespladib targets isoforms IIa, V and X of secretory PLA2 (sPLA2).
In phase II programs it showed efficacy reducing sPLA2 activity, LDL-C
and CRP [99–101]. However, this drug not only failed to reduce the
risk of MACE in the Vascular Inflammation Suppression to Treat Acute
Coronary Syndrome for 16 Weeks (VISTA-16) phase III trial, but actually
increased the risk of MI [102]. In line, a previous Mendelian randomiza-
tion study showed that that sPLA2 is not causally related to ASCVD
[103].
The CANTOS trial is assessing whether inhibition of inflammation by
blocking the inflammasome product and inflammatory cytokine IL-1β
with canakinumab (subcutaneously administered fully human mono-
clonal antibody that selectively neutralizes IL-1β) will reduce cardiovas-
cular events in 10,000 patients with prior MI [104]. Furthermore, the
CIRT trial will assess the role of low-dose methotrexate in the secondary
cardiovascular prevention of patients with prior MI and either metabol-
ic syndrome or type 2 diabetes mellitus [105]. These eagerly awaited
78 R. Ladeiras-Lopes et al. / International Journal of Cardiology 192 (2015) 72–81
results may shed light on the role of targeting inflammation to prevent
cardiovascular events.
The intriguing idea of developing immunity to atherosclerosis
derives from the growing body of evidence targeting several
atherosclerosis-related antigens using active immunization or antibody
infusion [106]. Epitopes of apoB, the only protein permanently associat-
ed with LDL particles, are targeted in the development of pharmacolog-
ical tools to induce atheroprotective immune responses. Indeed,
elevated circulating levels of anti-apoB IgG were found to be inversely
related to the severity of coronary atherosclerosis and associated with
lower risk of MI [107]. Further research is needed to refine basic re-
search data and pave the way for future studies.
4. New targets for selected populations
4.1. Genetics and Mendelian randomization studies
Genome-wide association studies (GWAS) assess the association be-
tween genetic variants distributed throughout the genome and a phe-
notypic trait [108]. In recent years, these techniques have identified
gene variants associated with altered lipoprotein metabolism and the
risk of CAD [109]. The aim of integrating the large body of genetic data
to better understand the causes of CVD and find new targets for drug
therapy has resulted in novel study designs including Mendelian ran-
domization studies aimed at demonstrating causality between the
trait and ASCVD [110].
To establish causality in CV disease, the viewpoint from Mendelian
randomization begins with epidemiological association and suggests
that if a biomarker is causally associated with CVD risk, then a genetic
variant that affects its plasma concentration or activity should itself be
associated with CV risk [110]. A growing body of evidence exploring
this concept confirmed LDL-C (using PCSK9 nonsense mutations) [17]
and Lp(a) [111] as causal risk factors for CVD but refuted the role of
CRP [112], HDL-C (using the endothelial-lipase gene LIPG, as well as
other single nucleotide polymorphisms) [55] and homocysteine [113]
as causal factors. Furthermore, Mendelian randomization studies em-
phasize the importance of some (e.g., IL-6), but not all (e.g., CRP) inflam-
matory markers as causal factors for CVD [114,115] (Fig. 2). Clearly, the
availability of huge cohorts necessary for the complex Mendelian ran-
domization studies will help identify markers of increased CV risk and
foster the development of new drug therapies to reduce CVD burden.
5. Pathways for successful clinical trial design
5.1. Surrogate endpoints
In the era of dual antiplatelet therapy and intensive statin therapy in
patients with recent ACS, phase III clinical trials with “hard” endpoints
(i.e., death or CV events) require large numbers of patients followed
for several years to be adequately powered, being prohibitively expen-
sive to perform. Numerous hurdles exist considering enrolment and
funding of these trials. To overcome these limitations, the substitution
Fig. 2. Results from Mendelian randomization studies addressing the role of several mol-
ecules as risk factors or biomarkers of cardiovascular disease.
of “hard” endpoints for surrogate markers to demonstrate potential
clinical benefit has emerged as a promising trial perspective.
As reviewed in this paper, the most recent drugs in development for
atherosclerosis target functional (e.g., inflammation) and cellular
(e.g., cholesterol efflux) processes (Fig. 1). In this way, imaging modali-
ties that allow the direct visualization of atherosclerotic plaque
progression, remodeling and inflammation are an attractive way to
mechanistically strengthen the clinical development process of a novel
therapeutic strategy [116].
The most common invasive imaging techniques to evaluate coro-
nary atherosclerotic lesions are IVUS and optical coherence tomography
(OCT), both permitting post-procedural plaque characterization [117,
118]. The comprehensive assessment of plaque characteristics fostered
the development of plaque discrimination scores to identify patients
with high-risk plaques, in this way being more prone to rupture and
to suffer from a clinical event [119]. Yet the concept of vulnerable plaque
is still discussed controversially. Furthermore, during recent years a
growing body of evidence demonstrated the huge potential of non-
invasive imaging techniques to characterize the complex atherosclerot-
ic lesion both anatomically and functionally [120]. The most promising
methods combine an integrated assessment of morphological and func-
tional plaque characteristics and include PET/CT and PET/MRI. These hy-
brid techniques may allow the non-invasive detection of high-risk
plaques and assist in the selection of patients at increased risk of MI.
We believe they are the most promising techniques to successfully
serve as surrogate endpoints, yet their value has not been firmly
established.
There is a potential role for these imaging methods to facilitate the
selection of high-risk patients and also serve as prospectively validated
surrogate markers. However, additional research is needed to strength-
en the available evidence in the complex task translating successful re-
sults with imaging endpoints to net clinical benefit. As an example, the
validity of cIMT as a surrogate in clinical trials was recently questioned
by several meta-analyses showing that cIMT progression was not asso-
ciated with MACEs in the general population [121]. On the other hand,
coronary atheroma volume as assessed by IVUS predicts MACEs in pa-
tients receiving high-intensity statin therapy [122]. Moreover, robust
data comparing OCT information on plaque characteristics to “hard”
outcomes are lacking [123]. Although there is a great potential for
plaque characterization, no clear and validated criteria have been de-
fined yet [124].
5.2. Power to absolute LDL-C reduction
The absolute event rate in cardiovascular outcomes trials for
LDL-reduction is related to baseline LDL-C, i.e., the higher the baseline
LDL-C the greater the event rate in the “control” group [125]. Further-
more, the relative risk reduction (RRR) is related to absolute LDL-C re-
duction and not to the percentage of decrease [126], suggesting that
including patients with a higher baseline LDL-C will allow a greater
reduction in CV risk. Therefore, trials enrolling patients with a low base-
line LDL-C are stalled by (i) lower absolute event rates, demanding
more patients and/or longer treatment duration, (ii) lower absolute
LDL-C reduction between groups translating into less risk reduction,
(iii) down-titration or cessation of drug therapy due to “too low”
LDL-C. In this way it is our opinion that future outcome studies
should include patients with a higher LDL-C than 1.8 mmol/L
(70 mg/dL) despite optimal medical therapy (or unable to tolerate
statins) and be powered considering the absolute reduction of LDL-C
in order to avoid missing the primary endpoint.
6. Conclusion
The interest in reducing the residual cardiovascular risk of patients
on currently considered optimal medical therapy is driving the search
for new targets and drugs to modulate the atherosclerotic process. The
 R. Ladeiras-Lopes et al. / International Journal of Cardiology 192 (2015) 72–81
latest evidence available confirms that atherosclerosis is dependent on
the crucial interplay between lipoprotein metabolism and inflammation
and represents a complex multifactorial system. Despite the successful
completion of many phase I and II programs in atherosclerosis, several
drugs have recently failed to reduce cardiovascular events in a baseline
population with low LDL-C and a robust background treatment. More
evidence is needed to bring forward solid biomarkers and imaging tech-
niques to smooth the path for successful clinical translation.
Conflict of interest
R.L.L., B.S. and A.L.M. have no conflicts of interest to declare. S.A.,
honoraria and consultancy, advisory board fees, from Astra Zeneca and
Thermo Fisher. A.T., grant support from Takeda and Genentech; consult-
ing Takeda, Actelion, Amgen. E.S., consultant fees in the last 3 years from
Amgen, BMS, Regeneron/Sanofi, Roche/Genetech, AstraZeneca,
Gemphire, Catabasis and Cymabay. R.J.M., research support from AZ.
F.Z., grants to institution: Roche Diagnostics; steering committee fees:
Bayer, Boston Scientific, Janssen, Novartis, Pfizer, Resmed and Takeda;
consultant/scientific advisory board: Air Liquide, Amgen, CVRx, Servier,
St Jude, Stealth peptide. W.K., consulting fees from Roche, diaDexus,
Cerenis, Novartis, Amgen, The Medicines Company, Genzyme,
AstraZeneca and Pfizer, honoraria for lectures from AstraZeneca,
Amgen, Novartis and MSD, and research support from Roche Diagnos-
tics, Abbott, Beckmann and Singulex.
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