Accepted

Manuscript

Lipid metabolism and emerging targets for

lipid-lowering therapy

Daniel Gaudet, MD, PhD, Jean-Philippe Drouin-Chartier, MSc,

Patrick Couture, MD, FRCP(C), PhD

PII: S0828-282X(17)30002-8

DOI: 10.1016/j.cjca.2016.12.019

Reference: CJCA 2343

To appear in: Canadian Journal of

Cardiology

Received Date: 3 November

2016

Revised Date: 12 December

2016

Accepted Date: 26 December

2016

Please cite this article as: Gaudet D, Drouin-Chartier J-P, Couture P,

Lipid metabolism and emerging targets for lipid-lowering therapy,
Canadian Journal of Cardiology (2017), doi: 10.1016/ j.cjca.2016.12.019.

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LIPID METABOLISM AND EMERGING TARGETS FOR LIPID-LOWERING THERAPY
Authors
Daniel Gaudet1, MD, PhD, Jean-Philippe Drouin-Chartier2, MSc, and Patrick Couture2,3,
MD, FRCP(C), PhD
From the:
1. Clinical Lipidology and Rare Lipid Disorders Unit, Department of Medicine,
Université de Montréal Community Gene Medicine Center, Chicoutimi, Canada 2. Institute of
Nutrition and Functional Foods, Laval University, Quebec City, Canada 3. Centre hospitalier
universitaire de Québec Research Center, Laval University,
Quebec City, Canada
Address for correspondence:
Patrick Couture, MD, FRCP(C), PhD Institute of Nutrition and Functional Foods (INAF) 2440,
Hochelaga Blvd. Pavillon des Services Laval University Quebec City Canada G1V 0A6 Phone: 418-
654-2106 E-mail: patrick.couture@crchul.ulaval.ca
Daniel Gaudet, MD, PhD Lipid Clinic Chicoutimi Hospital and ECOGENE-21 Clinical and
Translational Research Center 225 Saint-Vallier Université de Montréal Chicoutimi, QC Canada
G7H 7P2 Phone: 418-541-1077 E-mail: daniel.gaudet@umontreal.ca
Word count (excluding references): 4364 Word count of abstract: 244 Tables: 1 Figures: 1

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1 Brief summary 2 Dyslipidemia constitutes a major risk factor for cardiovascular disease and 3
premature atherosclerosis. However, treatment of dyslipidemia with the most widely 4
5
6
7
8 used proportion treat aiming face major lipid-related lipid-lowering HDLs of challenges. patients
 C CE
are A disorders reviewed. drugs, from i.e., cardiovascular by Despite

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reducing statins their and plasma lipid-lowering

A N
ezetimibe, risk. M Emerging levels often of effects, atherogenic targets fails

U S C RI
these to and protect lipoproteins novel novel a therapies significant

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therapies or to

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9 Abstract 10 Cardiovascular disease (CVD) is one of the leading causes of morbidity and 11
mortality worldwide, and dyslipidemia constitutes a major risk factor for CVD and 12 premature
atherosclerosis. Therapies reducing the plasma levels of atherogenic 13 lipoproteins are well-
established interventions that decrease CVD risk. However, 14 treatment of dyslipidemia with the
most widely used lipid-lowering drugs (i.e., statins and 15 ezetimibe) often fails to protect a
significant proportion of patients from cardiovascular 16 risk. The development of several novel
therapies to treat lipid-related disorders and their 17 associated risks is ongoing and includes the
 following: (a) reducing plasma levels of 18 atherogenic lipoproteins using proprotein convertase
subtilisin/kexin type 9 inhibitors, 19 antisense inhibitors of apo(a), microsomal triglyceride transfer
protein inhibitors, 20 antisense oligonucleotides of apoB for inhibiting VLDL production, and
inhibitors of 21 angiopoietin-like protein 3 or apoC-III for triglyceride-rich lipoprotein (TRL)
management 22 upstream of LDL production as well as gene replacement therapy to improve LDL
and 23 TRL clearance; (b) emerging therapies that target HDL and reverse cholesterol transport 24
using cholesteryl ester transfer protein inhibitors, HDL peptide mimetics, and autologous 25 infusion
of pre-beta HDLs. Clinical trials of several of these emerging therapies are 26 currently being
conducted. Despite the potential efficacy of these new therapies in CVD 27 prevention, their costs
may limit their use due to limited reimbursement funds. 28 Therefore, the real challenge facing the
next generation of lipid-lowering agents will 29 most likely be accessibility, reflecting a new
paradigm that applies to almost all emerging 30 therapies for any disease in the era of precision
medicine. 31
32 Keywords: Lipid metabolism, Lipid-lowering drugs

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33 Overview of lipoprotein metabolism 34 An overview of lipoprotein metabolism and emerging targets for
lipid-lowering 35 therapy is shown in Figure 1. Lipoproteins are secreted by the liver and small intestines, 36
and their primary functions are to deliver lipids (cholesterol, triglycerides (TGs) and 37 phospholipids (PLs))
to peripheral tissues and to return lipids and cholesterol to the liver 38 for clearance or recycling. 39 In
hepatocytes and enterocytes, cholesterol, TGs and PLs are assembled with 40 apolipoprotein (apo)B under
the action of microsomal triglyceride transfer protein 41 (MTTP) to form lipoproteins.1 ApoB is responsible
for the structural integrity of 42 intestinally derived chylomicrons and hepatic lipoproteins from synthesis to
secretion 43 and clearance. Once secreted, apoB-containing lipoproteins acquire apoC-II, apoC-III 44 and
apoE in different concentrations in the circulation. Lipoprotein TGs are hydrolyzed 45 by lipoprotein lipase
(LPL), releasing free fatty acids for cellular uptake and forming 46 smaller and denser lipoprotein remnants.
LPL activity is mainly determined by the apoC- 47 II:apoC-III ratio because LPL is enhanced by apoC-II
and inhibited by apoC-III.2 48 Angiopoietin-like protein 3 (ANGPTL3) is a secretory protein that also exerts
inhibitory 49 effects on LPL and decreases TG hydrolysis.3 50 Chylomicron and very low density
lipoprotein (VLDL) remnants are cleared from 51 plasma by specific liver receptors. 4 VLDL remnants are
hydrolyzed by hepatic lipase to 52 form cholesterol-rich, low density lipoprotein (LDL) particles.5 LDLs
transport most 53 cholesterol and are cleared from plasma by binding to LDL receptors (LDLRs) mainly in
54 the liver or intestines. After endocytosis of the LDL-LDLR complex, the LDL particle is 55 degraded,
thereby releasing free cholesterol. The LDLR is subsequently recycled to the 56 cell surface. However, the
normal recycling of LDLR may be disrupted by the proprotein

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57 convertase subtilisin/kexin type 9 (PCSK9), which promotes LDLR intracellular 58 lysosomal

degradation and indirectly decreases LDL clearance.6 59 The liver also secretes free apo(a) at a genetically
defined rate.7 In the circulation, 60 apo(a) binds to LDL particles and forms lipoprotein (a) (Lp(a)). This
lipoprotein can bind 61 to the extracellular matrix via apoB- and apo(a)-related components, which confers
its 62 highly atherosclerotic properties. 63 The intestinal and hepatic secretion of lipid-poor apoA-I is
another major lipid 64 pathway. In the circulation, apoA-I is lipidated via cholesterol efflux by transporters
65 located in peripheral tissues and macrophages.8 By acquiring cholesterol, apoA-I forms 66 nascent HDL,
which matures into high-density lipoprotein (HDL). Mature HDLs interact 67 with cholesteryl ester transfer
protein (CETP), which mediates the exchange of 68 cholesteryl esters and TGs with TG-rich apoB-
containing lipoproteins and LDLs.9 This 69 exchange influences the lipid composition of HDLs. HDL-
cholesterol (C) is finally 70 selectively removed from the particle by the liver. The cholesterol is excreted in
bile, and 71 the lipid-poor HDL is hydrolyzed or returned to circulation for relipidation.8 72 Proprotein
convertase subtilisin/kexin type 9 inhibitors 73 PCSK9 plays a pivotal role in regulating LDL-C
homeostasis. By binding to 74 LDLRs at the surface of hepatocytes and promoting their lysosomal
 degradation, 75 PCSK9 reduces LDL uptake, leading to increased LDL concentrations. 10 PCSK9 has 76
emerged as an attractive target for lowering LDL-C levels to reduce the risk of coronary 77 heart disease.
Two PCSK9 monoclonal antibodies (mAbs) (alirocumab, evolocumab) 78 subcutaneously (SC) administered
either biweekly or monthly are currently being 79 studied in phase III trials with large patient populations.
By sequestering PCSK9, PCSK9 80 inhibitors block the binding of PCSK9 protein to LDLRs, preventing

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81 turn, this preserves LDLR recycling and increases receptor density on the hepatocyte 82 surface. 11 Health
Canada approved evolocumab in 2015 and alirocumab in 2016 for 83 reducing LDL-C levels in patients with
familial hypercholesterolemia (FH) or in patients 84 with very high cardiovascular risk who were not
responsive to statins. The ODYSSEY 85 clinical trial program currently comprises 14 phase III studies
ranging from 24 to 104 86 weeks in duration and involving more than 23,500 planned patients worldwide.
The 87 program was designed for further assessment of the efficacy and safety of alirocumab 88 compared
with ezetimibe in various populations, including patients with statin 89 intolerance, patients with
hypercholesterolemia with or without statin intake, patients 90 with heterozygous (HeFH) or homozygous FH
(HoFH), and patients on monotherapy.12- 91 22 Taken together, the ODYSSEY clinical trials reported a

significant reduction in LDL-C 92 by 36-61% from baseline after a minimum of 24 weeks of treatment
with alirocumab. 93 Moreover, alirocumab consistently reduced levels of total cholesterol, apoB, non-HDL-C
94 and Lp(a) independent of the dose of the background statin and the addition of 95 ezetimibe in HeFH and
non-FH populations.23 Finally, a post hoc analysis of the 96 ODYSSEY LONG TERM study showed a 52%
reduction in major cardiovascular events 97 following alirocumab treatment in patients with HeFH and high
cardiovascular risk who 98 were receiving maximally tolerated statins.24 The efficacy of evolocumab is
currently 99 being evaluated in the PROFICIO phase III clinical trial. This program consists of 14 100 clinical
trials with a planned enrollment of approximately 30,000 patients.25-29 Ranging 101 from 10-52 weeks,
patients treated with evolocumab in monotherapy and with a statin or 102 ezetimibe reported 49-65%
significant reductions in LDL-C levels with substantial 103 reductions in non-HDL-C, apoB and Lp(a), and
modest variable effects on TG and HDL- 104 C. Regression of coronary atherosclerosis following

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105 been reported.30 Finally, bococizumab has been investigated in the SPIRE 1 and SPIRE 106 2
clinical trials to assess its efficacy and safety in patients at high risk for cardiovascular 107 events. 31
However, on November 1st, 2016, Pfizer announced the discontinuation of the 108 global clinical
development program for bococizumab because of an unanticipated 109 attenuation of LDL-C
lowering over time, as well as a higher level of immunogenicity and 110 a higher rate of injection-
site reactions than shown with the other agents in this class. 111 Overall, meta-analyses indicated that
PCSK9 inhibitors had a strong effect in 112 lowering LDL-C and other lipid levels with satisfactory
safety and tolerability in patients 113 with hypercholesterolemia.32 Moreover, a recent meta-analysis
of placebo-controlled, 114 randomized clinical trials of alirocumab and evolocumab demonstrated
significant 115 reductions in all-cause mortality and myocardial infarctions.33 Additionally, these two
116 agents showed limited side effects and high tolerability in patients with statin 117 intolerance.28,
34
Furthermore, the phase I trial of inclisiran, a long-acting RNAi agent 118 targeting the synthesis of
PCSK9, showed no major adverse events as well as 119 reductions of 75% and 51% in PCSK9 and
LDL-C concentrations, respectively, with 120 inclisiran doses ≥300 mg.35, 36 RNAi is another gene-
silencing approach that suppresses 121 the translation of specific mRNAs and the production of
corresponding gene products. 122 Further investigation of the safety and efficacy of inclisiran is
currently underway in a 123 phase II trial. Thus, PCSK9 inhibitors are a promising therapeutic
strategy to address 124 additional reductions in LDL-C concentrations to reduce CVD in high-risk
populations or 125 in patients with statin intolerance.37, 38 126 Cholesteryl ester transfer protein
inhibitors 127 Well-documented evidence has shown that HDL-C concentration is an 128
independent inverse predictor of the risk for developing a cardiovascular event. 39 CETP
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129 is a plasma glycoprotein that mediates the net exchange of cholesteryl ester from HDL 130 for TG in
apoB-containing lipoproteins, including chylomicrons, VLDL and LDL.40 The 131 inhibition of CETP
increases HDL-C levels and reduces levels of cholesterol in 132 atherogenic apoB-containing lipoproteins.41
The first CETP inhibitor tested in humans 133 was torcetrapib, and its effects on cardiovascular events were
investigated in the 134 ILLUMINATE trial, which included 15,067 high-risk statin-treated subjects.42
Although 135 torcetrapib increased HDL-C levels by 72% and decreased LDL-C levels by 25%, it also 136
increased blood pressure,42 synthesis and secretion of aldosterone, and endothelin-1 137 levels in the artery
wall. Torcetrapib failed in phase III clinical development due to 138 increased risks of major cardiovascular
events and mortality.43 The cardio-protective 139 effects of two other CETP inhibitors, dalcetrapib and
evacetrapib, were investigated in 140 phase III clinical trials that included more than 10,000 subjects (dal-
OUTCOMES and 141 ACCELERATE trials).44-46 Despite favorable modulation of lipid parameters, the 142
development of these two CETP inhibitors was halted because no differences in 143 cardiovascular event
rates were found between the treated and placebo groups. The 144 DEFINE study was an 18-month trial
designed to assess the lipid efficacy and safety of 145 anacetrapib in high-risk, statin-treated patients
(n=1623).47 Anacetrapib increased 146 concentrations of HDL-C (+138%) and apoA-I (+45%) and reduced
levels of LDL-C (- 147 30%) and Lp(a) (-39%). Anacetrapib therapy did not result in any adverse effects. A
148 large phase III clinical trial (REVEAL) is under way and aims to enroll 30,000 149 participants older
than 50 years. This trial is predicted to have a follow-up of 150 approximately 4 to 5 years and will ultimately
determine the efficacy of treatment with 151 anacetrapib for secondary prevention of CVD. Finally, TA-8995
is a new promising 152 CETP inhibitor. In the phase II TULIP trial, 364 patients with mild dyslipidemia were
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153 enrolled in a randomized, double-blind, placebo-controlled, parallel-group study to 154 assess the
tolerability and efficacy of TA-8995.48 LDL-C and apoB levels were 155 significantly reduced by 47% and
34%, respectively, whereas HDL-C and apoA-I levels 156 were significantly increased by >160% and
approximately 60%, respectively after TA- 157 8995 treatment. Moreover, TA-8995 was well-tolerated
without any safety concerns or 158 apparent adverse effects on the liver and muscle. 159 In summary, CETP
inhibitors exert beneficial effects on lipid parameters in 160 patients with dyslipidemia. Because HDL-C
levels are inversely associated with CVD- 161 related mortality, CETP inhibitors could be a novel therapeutic
option for CVD. 162 Antisense inhibitor of apolipoprotein (a) 163 Lp(a) is an LDL-like lipoprotein particle
with one apo(a) molecule, a large 164 hydrophilic glycoprotein covalently bound to apoB-100 by a disulfide
bond.49 Plasma 165 levels of Lp(a) are primarily genetically determined by variations in the LPA gene coding
166 for apo(a).50 High Lp(a) levels have been shown to explain residual CVD risk in FH 167 patients and in
the general population.51 However, Lp(a) is relatively refractory to both 168 lifestyle and drug intervention, 52
with current therapeutic options limited to nicotinic acid, 169 which shows consistent reductions of 15% to
25%53, 54 and recently, PCSK9 inhibitors.55, 170 56 However, these studies examined short durations with

small numbers of subjects. 171 Furthermore, relationships to dose, gender, and background lipid therapy
have not been 172 fully established. Due to expanding evidence indicating that Lp(a) contributes to CVD, 173
antisense oligonucleotide (ASO) drugs have emerged as a promising approach to 174 reduce Lp(a) levels in
the clinical setting. An animal study57 showed that ISIS 144367, a 175 second generation apo(a)-specific
ASO, significantly reduced hepatic apo(a) mRNA 176 expression in mouse models after 4 weeks of treatment.
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177 significantly decreased apo(a) levels by 19.2% and 86% in three different transgenic 178 mouse models
and reduced Lp(a) levels by 24.8% in one of these models. A more 179 optimal ASO (ISIS-APO (a)Rx) was

also studied in LPA transgenic mice and cynomolgus 180 monkeys.58 ISIS-APO (a)Rx produced dose-

dependent reductions in hepatic apo(a) 181 mRNA and apo(a) in plasma after two weeks of ASO
administration in transgenic mice. 182 In chow-fed cynomolgus monkeys, ISIS-APO (a)Rx significantly

reduced hepatic apo(a) 183 mRNA function by 70% to 97% of the dose administered during a 13-week
period. As 184 anticipated, plasma Lp(a) levels were concomitantly reduced by 23% to 90% relative to 185
baseline levels. A double-blinded, placebo-controlled, dose-escalation phase I trial in 186 healthy volunteers
with Lp(a) levels of >25 nmol/L (>10 mg/dL) assessed the efficacy, 187 safety, and pharmacokinetics of

ISIS-APO(a)Rx.59 ISIS-APO(a)Rx (100-300 mg) showed 188 dose-dependent mean percentage reductions
in plasma Lp(a) levels of 39.6% from 189 baseline in the 100 mg group, 59.0% in the 200 mg group, and
77.8% in the 300 mg 190 group. 191 In summary, ISIS-APO(a)Rx showed great potential to reduce plasma

Lp(a) levels 192 in a dose-dependent manner and is a promising therapeutic drug to reduce CVD risk 193
and calcific aortic valve stenosis in patients with high Lp(a) concentrations. With potent 194 ASO-lowering
Lp(a) levels, the hypothesis that lowering Lp(a) levels will lead to 195 reductions in CVD events can be
tested. 196 Lomitapide, mipomersen and inhibition of VLDL production 197 The development of lomitapide
(an inhibitor of MTTP) and mipomersen (an apoB 198 ASO) was a result of the decryption of the
mechanisms of VLDL assembly and 199 production in hepatocytes. Both drugs target HoFH patients who
have very few 200 functional LDLR available to clear LDL particles. By hindering VLDL production and
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201 secretion (i.e., upstream of LDL formation), pro-atherogenic LDL particle accumulation is 202
prevented. This finding was underscored by the identification of null MTTP gene variants 203 as a
cause of abetalipoproteinemia,60 a disease characterized by low cholesterol 204 concentrations and
very small amounts of circulating LDL. A similar rationale was 205 applied to the development of
mipomersen.61 ApoB is the backbone of all lipoproteins of 206 the VLDL pathway. Interference with
the transcription of the APOB gene was considered 207 an attractive approach to decrease VLDL
production and reduce the number of LDL 208 particles in HoFH. Mipomersen is an antisense agent
that inhibits the synthesis of apoB. 209 Both lomitapide and mipomersen have demonstrated
significant (up to >50%) reductions 210 in LDL-C.61, 62 Although very effective, these agents are
also associated with side effects 211 and increased risk of liver steatosis in a significant proportion of
treated patients.63, 64 212 Because of the risk of liver toxicity, mipomersen is only available in the US
as an adjunct 213 therapy for HoFH at a weekly dose of 200 mg SC, through a restricted program
termed 214 Risk Evaluation and Mitigation Strategy.61 Lomitapide is approved in Canada for HoFH
215 patients. 216 ApoC-III inhibitors and LPL-dependent or LPL-independent management of
TRL 217 ApoC-III is a 79-amino acid glycoprotein primarily synthesized in the liver and in 218 the
small intestine to a lesser extent. It binds to apoB- or apoA-containing lipoproteins, 219 including
chylomicrons and remnants of VLDL, IDL, LDL and HDL particles.65 Novel 220 sensitive assays
have recently detected Lp(a)-bound apoC-III.66 The clinical relevance 221 of Lp(a)-apoC-III has not
been documented. ApoC-III has previously been reported as 222 an independent CVD risk factor.67
However, the risk associated with circulating plasma 223 apoC-III is dependent on its association
with specific lipoproteins. In multivariate 224
analyses, LDL or HDL containing apoC-III was shown to be associated with high risk of
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225 CVD events, even after adjusting for TG concentrations. In contrast, LDL or HDL lacking 226
apoC-III did not predict CVD incidence.67 The development of apoC-III inhibitors has 227 been
accelerated by these observations and by the identification of a rare R19X null 228 APOC3 gene
variant in the Amish founder population.68 This variant confers a favorable 229 plasma lipid profile
and apparent cardio-protection. The observation that APOC3 loss-of- 230 function (LoF) variants
were associated with lower plasma TG concentrations and 231 possibly lower CVD risk has been
replicated in larger cohorts. 69 ApoC-III is a key 232 regulator of plasma TG and TG-rich lipoprotein
(TRL) metabolism by combining both 233 lipoprotein lipase (LPL)-dependent and LPL-independent
mechanisms.70, 71 ApoC-III is a 234 known potent inhibitor of apoC-II-mediated activation of LPL
that inhibits hepatic lipase 235 activity, promotes intrahepatic VLDL assembly and secretion, and
interferes with the 236 turnover of TRL remnants through a hepatic clearance mechanism mediated
by LDL 237 family receptors (LRP1) and others65, 71 and possibly through non-canonical clearance
238 mechanisms. The mechanisms of action and regulation of LPL-independent pathways 239 have
yet to be elucidated, but their importance has recently been highlighted in a phase 240 II proof-of-
concept study with an apoC-III ASO (volanesorsen) in patients lacking LPL 241 activity.70 In this
study, three patients with <5% normal LPL activity and fasting plasma 242 TG values between 15.9-
23.5 mmol/L were treated with volanesorsen at a weekly dose 243 of 300 mg SC. After 13 weeks of
study-drug administration, plasma apoC-III levels were 244 reduced by 71% to 90% and TG levels
were reduced by 56% to 86%. However, LPL 245 activity remained below 5%. Ongoing phase III
clinical trials with volanesorsen target 246 several subtypes of severe dyslipidemia: familial and
multifactorial chylomicronemia and 247 different types of severe hypertriglyceridemia and
lipodystrophies (Table 1). 70, 72 248
Volanesorsen has recently been shown to lower apoC-III levels in atherogenic TRL and
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249 apoB-containing lipoproteins, including Lp(a). 66 In addition to volanesorsen, a small 250 RNAi
therapeutic agent (ALN-AC3) is currently in clinical development. 251 ANGPTL3 inhibitors and
TRL-mediated management of upstream LDL production 252 ANGPTL3, 4 and 8 are key members
of the angiopoietin-like family and are potent 253 regulators of lipid metabolism. ANGPTL3 has
been a target for drug development since 254 the identification of LoF mutations in the ANGPTL3
gene, which have been associated 255 with combined hypolipoproteinemia.73, 74 Affected subjects
have reduced plasma 256 cholesterol and TG concentrations.74, 75 This suggests that therapies
inhibiting 257 ANGPTL3 could reduce levels of TG or LDL-C and provide benefits to patients with
258 severe hypertriglyceridemia, severe hypercholesterolemia (FH) or combined 259 hyperlipidemia.
ANGPTL3 is secreted and expressed primarily in the liver and acts to 260 increase the plasma levels
of TG, LDL-C and HDL-C by affecting the content and 261 characteristics of secreted VLDL,
inhibiting the activity of LPL and endothelial lipase 262 (EL) and by modulating the clearance of
TG-rich lipoproteins upstream of LDL 263 production.74, 76 ANGPTL3 deficiency has also been
associated with decreased serum 264 levels of nonesterified fatty acids (NEFA) and increased insulin
sensitivity in patients 265 who are homozygous for LoF mutations in ANGPTL3.73 Humans carrying
these variants 266 were otherwise healthy and present reduced risks of CVDs. Improved insulin
sensitivity 267 and reductions in hepatic steatosis have been observed in murine models with 268
ANGPTL3-ASO.77 ANGPTL3 deficiency significantly affects post-prandial lipid 269 metabolism in
humans. Patients homozygous for LoF ANGPTL3 variants and without 270 detectable protein
activity have reduced postprandial lipemia (total TG, TRL and apoB- 271 48), expanded postprandial
NEFA pools and decreased influx of dietary-derived fatty 272
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273 Two forms of ANGPTL3 inhibitors are in clinical development: an ANGPTL3 ASO 274 and a fully
human ANGPTL3-mAb, evinacumab.76, 77 Although both inhibitors are 275 specific inhibitors of ANGPTL3,
the mechanisms and sites of action of these two agents 276 differ. The ANGPTL3-ASO mainly acts in liver
cells, whereas evinacumab remains in the 277 circulation. The clinical relevance of these differences has yet
to be determined. A 278 recent study was conducted to determine whether evinacumab reduced LDL-C levels
in 279 patients (with genetically and phenotypically confirmed HoFH) on stable lipid-lowering 280 regimens.
The findings of this study showed dramatic incremental reductions of 35% to 281 90% of LDL-C after four
weeks of treatment. LDL-C reduction was sustained up to 24 282 weeks after the last dose of the study drug.79
283 Emerging therapies targeting HDL and reversing cholesterol transport 284 The biology of HDL
particles is complex, and their anti-atherogenic properties go 285 far beyond increasing HDL-C
concentrations.80 Several emerging therapeutic agents are 286 designed to target HDL particles, not solely to
increase HDL-C levels. These include 287 intravenous infusions of HDL peptide mimetics and autologous
infusions of pre-beta 288 HDL. Three fully human HDL mimetic compounds and one autologous pre-beta
HDL 289 plasmapheresis technique are currently being evaluated in proof-of-concept clinical 290 studies.
Upon infusion, these drugs are expected to increase cholesterol efflux 291 capacity. 81 The efficacy and safety
of HDL peptide mimetics have recently been 292 evaluated in HoFH82 and are currently being assessed in
acute coronary artery disease 293 (CAD)83 or in severe hypoalphalipoproteinemia due to mutations in ATP-
binding 294 cassette 1 (ABCA1) or APOA1 genes. These bio-drugs mimic the structural and 295 functional
biological properties of natural, nascent HDL (pre-beta HDL) and stimulate 296 reverse lipid transport (RLT)
capacity. A highly specific plasmapheresis technique has
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297 also been developed to assess the efficacy of serial (weekly) autologous infusions of 298 delipidated
HDL on arterial walls and plasma LDL-C concentrations in acute coronary 299 syndrome (ACS). This
approach uses a patient's plasma to transform the majority of 300 circulating HDL into pre-beta HDL. Once
re-infused, the pre-beta HDLs are expected to 301 stimulate the removal of excess cholesterol and other lipids
from the arterial wall and 302 other tissues and become mature HDL and thereby increase cholesterol efflux
capacity 303 through RLT. The autologous infusion of pre-beta HDL is currently being evaluated in 304
severe FH. 305 Other HDL targets for drug development include lecithin-cholesterol 306 acyltransferase
(LCAT) recombinants.84 Increased LCAT levels may be beneficial for 307 CVD prevention and familial
LCAT deficiency (FLD), a rare HDL disorder characterized 308 by low HDL-cholesterol. 309 Gene
replacement therapy to improve LDL clearance or TRL hydrolysis 310 Gene replacement therapy introduces
functional genes into cells to treat 311 disease. The first gene replacement therapy trial in lipidology was
conducted more than 312 25 years ago to treat HoFH by transplanting autologous hepatic tissue transfected ex
313 vivo with competent copies of LDLR gene.85 The results were mitigated. The potential of 314 more
sophisticated in vivo techniques is currently under investigation for HoFH and 315 other lipid disorders. Pre-
clinical studies involving different gene-based approaches to 316 upregulate LDLR have recently been
completed, including stem cell therapy and 3- 317 hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase or
PCSK9 RNAi.35, 86, 87 318 The most advanced gene replacement for HoFH in humans is the in vivo 319
transfection of an adeno-associated viral vector serotype 8 (AAV8) vector encoding 320 human LDLR cDNA.
A first-in-human phase I/IIa study using recombinant LDLR-
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321 expressing AAV8 to treat 12 patients with HoFH is ongoing.88 HoFH is not the only 322 target
for gene replacement therapy. Recently, the development of in vivo LPL gene 323 therapy for LPL
deficiency (LPLD) led to the conditional approval of alipogene tiparvovec 324 in Europe. The gene
replacement therapy agent alipogene tiparvovec (AAV1-LPLS447X) 325 uses a non-replicating
serotype 1 AAV (AAV1) to deliver and express the human gain- 326 of-function S447X variant LPL
transgene.89 Alipogene tiparvovec specifically targets 327 LPLD, a rare autosomal recessive disease
caused by homozygosity for null LPL gene 328 mutations. It is not suitable for patients with other
forms of severe hypertriglyceridemia.89 329 After up to 6 years, treatments with alipogene tiparvovec
were associated with lower 330 incidences of pancreatitis (or severe abdominal pain events
consistent with pancreatitis) 331 in a significant proportion of patients. However, important inter-
individual differences 332 were noticed.90 Unfortunately, the lengths of available post-treatment
observational 333 period studies are not sufficient to definitively determine the outcome.
Furthermore, not 334 all participants showed clear-cut, long-term (> 5 years) benefits. However, the
treatment 335 appears to be safe in the long-term, with low rates of nuclear integration (a few issues
336 were observed in mitochondrial DNA and on chromosomes 1 and 5)91 and favorable 337
immune response profiles.92 Conditionally available in Europe (and not in Canada), 338 alipogene
tiparvovec is the first gene replacement therapy to be authorized in the 339 western world. Low HDL
(hypoalphalipoproteinemia) and other phenotypes are also 340 promising targets for gene-based
therapy.85 341 Other emerging therapies acting on TRL hydrolysis, VLDL production, cell 342
signaling, LDL clearance or peripheral tissues 343 Additional agents targeting unmet medical
needs in patients intolerant to statins 344 and subgroups of patients with severe
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345 hypertriglyceridemia, fatty liver and steatohepatitis (NASH), lipodystrophies or lipid 346 storage diseases
are currently being developed. Novel therapies in advanced clinical 347 development for severe
hypercholesterolemia include bempedoic acid, gemcabene, and 348 peroxisome proliferator-activated receptor
(PPAR)-delta agonists.93-96 Bempedoic acid is 349 an LDL-C-lowering oral agent currently in phase II trials.
It inhibits ATP citrate lyase 350 (ACL), an enzyme involved in fatty acid and cholesterol synthesis.
Bempedoic acid 351 enters the liver through cell surface receptors different from those that take up statins. 93
352 Gemcabene is a dialkyl ether (dicarboxylic acid) that blocks the production of hepatic TG 353 and
cholesterol synthesis (possibly via inhibition of acetyl-coenzyme A carboxylase at 354 steps before HMGCoA
reductase) and enhances the clearance of VLDL and VLDL 355 remnants upstream of LDL production. Based
on phase II clinical trials, the combined 356 effects of these mechanisms reduce plasma VLDL-C, LDL-C,
TG, apoC-III and high- 357 sensitivity C-reactive protein (hsCRP) levels and increase HDL-C levels
compared with 358 placebo.94 Novel plasma TG-lowering or peripheral fat-mobilizing agents include 359
selective PPARα-modulators (SPPARMα), apoC-II peptide mimetics, sterol regulatory 360 element-binding
protein 1 (SREBP-1) agonists, diacylglycerol acyltransferase 1 (DGAT- 361 1) inhibitor (pradigastat), and
DGAT-2 antisense.97-99 Pemafibrate is a novel selective 362 PPARα modulator (SPPARMalpha). All PPARα
agonists are ligand-activated 363 transcription factors that modulate lipid metabolism and gene expression of
key 364 apoproteins that affect HDL and TRL hydrolysis (e.g., apoA-1, apoC-II, and LPL), control 365
cellular cholesterol trafficking in macrophages and influence inflammation. Compared 366 with fenofibrate,
pemafibrate has significantly more potent PPARα-activating efficacy in 367 vitro and in vivo.100 In a recent
randomized, double-blind, active- and placebo-controlled 368 phase II trial, the effect of pemafibrate was
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369 and HDL-C levels. Furthermore, the occurrence of adverse effects was lower than with 370
fenofibrate.100 371 Conclusion 372 Several novel therapies that treat lipid-related disorders and
associated risks are 373 in clinical development. To be clinically useful, these agents must cover an
unmet need 374 and be safe, effective, affordable and available. Each of these emerging therapies is
375 being developed around specific targets to address unmet medical needs and residual 376
risks.101 These novel agents specifically target well-defined subgroups of patients in 377 specific
niches (a characteristic of precision medicine). Several of these novel 378 treatments are bio-drugs or
depend on mechanisms, principles of action or delivery 379 systems that are technically sophisticated
and very expensive to produce. Thus, even if 380 these agents are established to be safe, effective,
and available, their production costs 381 may limit their use due to limited financial resources. The
real challenge of the next 382 generation of lipid-lowering agents will likely be accessibility. This is
a new paradigm 383 that applies to almost all emerging therapies involving precision medicine.

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Disclosures
DG received funding during the last five years from the Canadian Institutes for
Health Cymabay, received Research, the Canada, Institute, Innovations. Dairy Research, funding and

Merck Farmers Gemphire, Agriculture Dow A Frosst, during Genome Agrosciences), of

C C E
Canada Ionis, and Pfizer, the Agri-Food Quebec, last Novartis, (DFC), Amgen,

P T E D
five Dairy years Aegerion, Canola Canada Pfizer, Sanofi, Research

A
from Regeneron, Council (Growing Kaneka M Amgen, the Institute, Canadian of

N U S
Corporation, Catabasis, Forward Canada, Sanofi, Dairy Institutes program and

C R I
Flax Australia, Cerenis, and Uniqure. Council for Atrium supported Health Chiesi,
 P
Danone of PC

T
by
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Treatment of Gaudet lipidology. apolipoprotein monkeys. the Med. Statins: in October Abstract 2016.

in MBX-8025 triglycerides A in Prevention. and the C. hepatic II of hypercholesterolemic randomized,

U
patients a of 2015;372:1500-1509. Efficacy clinical to 2015;14:8. PCSK9 genomic novel anti-PCSK9

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safety D. promises atherosclerosis: Effect intramuscular 2016). EAS16-1024 2016;249:36-43. with

Atherosclerosis. Effect 2016;10:556-567.

C
transcriptional therapeutics. selective in trial with of and ETC-1002 C-II Can with in of the era: 2016.

R
evolocumab of apolipoprotein IMPROVE-IT and of double familial safety mimetic patients. the in
 I
treatment vaccines J lipoprotein a Cardiol. vivo challenges. new PPARalpha administration

P
DGAT1 LB43 current blind, of alone patients

landscape
CRISPR-

T
paradigm. peptide

Journal
- 43. in of
an
lipase
on
and
E-
approach for long-term LDLc management. PLoS One. 2014;9:e114469. 108. REGENXBIO.
http://www.regenxbio.com/pages/programs/index.htm?panel=4
(Accessed October 2016). 2016. 109. IONIS Pharmaceuticals. http://www.ionispharma.com/pipeline
(Accessed January 2016)
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110. Viney NJ, van Capelleveen JC, Geary RS, et al. Antisense oligonucleotides targeting
apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-
controlled, dose-ranging trials. Lancet. 2016.
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Table
Sites VLDL and production mechanisms and secretion
of action of novel or emerging Mechanism MTTP ApoB ACL inhibition therapies inhibition inhibition for

AN U S
M lipid disorders Emerging Bempedoic (ApoB Mipomersen(ETC-

C R I P T
1002)Lomitapide and ASO) Therapy disease acid 62

102 93 61
prevention
Targeted Diseases
HoFH
HoFH

CCEPTED
A
HoFH

Dicarboxylic acid derivative

Acetyl-coA carboxylase inhibition
HoFH
(gemcabene)94
VLDL secretion ANGPTL3 inhibition
Evinacumab (ANGPTL3 mAb)76 HoFH ANGPTL3-GalNAc-ASO Severe hyperTG (IONIS-ANGPTL3-Lrx)77
VLDL and TG-rich lipoprotein hydrolysis
Volanosersen (ApoC-III ASO)71 FCS, including LPLD ApoC-III inhibition
ApoC-III RNAi
Severe hyperTG (ALN-AC3)103
Primary lipodystrophies
Evinacumab76
ANGPTL3 inhibition ANGPTL3-GalNAc-ASO77
HoFH Severe hyperTG PPARα agonism SPPARMα (K-877)100 Severe HyperTG
Alipogene tiparvovec90
LPL replacement
LPLD ANGPTL3 inhibition Evinacumab76
AAV1-LPLS447X gene therapy TG-rich lipoprotein clearance
ANGPTL3-GalNAc-ASO77
HoFH Severe hyperTG ApoC-III inhibition Volanesorsen70 FCS, including LPLD
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ALN-AC3103 Severe hyperTG
Exchange between HDL and TG-rich lipoproteins HDL metabolism

CC EPT ED
A CETP LCAT upregulation Inhibition

AN U S C R
M Recombinant
protein
CETP
(TA-8995)
inhibitor
(ACP-501)

human
IP T 48

LCAT
84

Hypercholesterolemia
CVD, LCAT deficiency with severe hypoalphalipoproteinemia
ApoA-I-mimetic peptide
ApoA-1 upregulation ApoA1 Milano transgene
ApoA-I DNA plasmid80
Tangier Disease, APOA-1 deficiency HoFH, ACS HDL mimetic infusion HDL mimetic peptides104
 Infusions of autologous
HDL autotransfusion
delipidated HDL/preβ-
HoFH Severe FH enriched plasma81
ACS
LDL clearance
PCSK9 mAb (Alirocumab, Evolocumab) 24, 31, 105 PCSK9 GalNAc-RNAi agent
PCSK9 inhibition
conjugate (ALN- PCSsc/inclisiran)35, 36
FH and CAD
AAV8-
Genome editing (PCSK9 CRISPR/Cas9)106 Peptide-based Anti-PCSK9 vaccine107 LDLR replacement
LDLR gene therapy

HoFH
(RGX-501)108
DGAT1 or DGAT2
Peripheral fat metabolism
inhibition
DGAT1 inhibitor
(pradigastat)97 Severe hyperTG
Apo(a) ASO
DGAT2-ASO NASH (IONIS-DGAT2Rx)109 Lp(a) production Apo(a) inhibition

(IONIS-APO(a)-LRx)110 Elevated lipoprotein (a)

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ACL: Adenosine triphosphate (ATP) -citrate lyase; ACS: Acute coronary syndrome; ANGPTL3: Angiopoietin-like
protein 3; APO: Apolipoprotein;
ASO: Antisense oligonucleotides; CAD: Coronary artery disease; CETP: cholesteryl ester transfer protein; CRISPR/Cas9:
Clustered Regularly
Interspaced FCS: hypercholesterolemia; density Microsomal siRNA: Familial Small lipoprotein triglyceride Short
interfering chylomicronemia Palindromic receptor; HyperTG: transfer RNAs; Lp(a): Repeats syndrome; SPPARMα:

protein; Hypertriglyceridemia; Lipoprotein (CRISPR)/CRISPR-associated PCSK9: FH: selective A (a); Familial

C C E P
Proprotein LPL: PPARα LCAT: hypercholesterolemia; Lipoprotein convertase modulator;

TE D
Lecithin–cholesterol lipase;
 A
9; subtilisin/kexin TG: CVD: LPLD: M Triglyceride; HDL: Cardiovascular Lipoprotein acyltransferase;

N U S C R
High-density type and 9; lipase PPAR: VLDL: disease; lipoprotein; LDL: deficiency;

IP T
Very Peroxisome DGAT: Low-density

low-density HoFH: Mab: Diacylglycerol proliferator-activated Homozygous monoclonal lipoprotein; lipoprotein

acyltransferase;
LDLR: antibody; familial
receptor;
Low-
MTTP:
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Figure Legend

Overview of lipoprotein metabolism and emerging targets for lipid-lowering therapies.

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