Guest Editorial: HDL-targeted Therapies—Where Do We Go From Here?

A recent succession of negative studies— including two randomized, placebocontrolled intervention trials^{1, 2} and a large genetic association analysis3—calls for a careful re-examination of the approach to HDL-directed therapies. In May 2011 the Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides: Impact on Global Health (AIM-HIGH) trial was stopped early due to futility.4 In the setting of mildly reduced HDL-C levels (mean baseline 35 mg/dL), the addition of extended-release niacin at a daily dose of 1500-2000 mg raised HDL-C by 10 mg/dL (28%), or 5 mg/dL (13%) compared to the control group.¹ Despite the observed increase in HDL-C, niacin use was not associated with a lower incidence of cardiovascular events among patients with stable coronary artery disease (CAD) treated to aggressive LDL-C targets (mean baseline 71 mg/dL, mean on-treatment 65 mg/dL).¹ Peculiarities of study design as recently reviewed limit the generalizability of the AIM-HIGH findings.⁵ More definitive evidence regarding the effect of niacin on clinical outcomes awaits the conclusion of the Heart Protection Study 2: Treatment of HDL to Reduce the Incidence of Vascular Events (HPS2-THRIVE) trial. Dal- OUTCOMES succumbed to a similar fate as AIM-HIGH in May 2012 after interim analysis failed to demonstrate a benefit of dalcetrapib, an inhibitor of cholesteryl ester transfer protein (CETP), on hard cardiovascular endpoints in patients with acute coronary syndrome (ACS) on optimal medical therapy.² In phase II studies, dalcetrapib 600 mg daily, the dose used in the outcomes trial, increased HDL-C by an average of 25-31% and did not exhibit adverse off-target hemodynamic or hormonal effects.⁶ Details regarding the prematurely terminated study remain eagerly anticipated. As noted below, at least two more potent CETP inhibitors are still in clinical development. In addition to these two disappointing clinical trials, a large Mendelian randomization analysis published in May 2012 highlighted the potential for inconsistency between higher HDL-C levels and a lower risk of myocardial infarction (MI).³ Carriers of a single nucleotide polymorphism in the endothelial lipase gene (LIPG Asn396Ser) exhibited HDL-C levels 5.5 mg/dL higher than non-carriers with no significant difference in LDL-C or other lipids. Observational cohort studies suggest that this modest increment in HDL-C would confer a 13% reduction in MI risk. However, analysis of 20,913 MI cases and 95,407 controls demonstrated no association between LIPG Asn396Ser and MI (odds ratio 0.99, 95% CI 0.88-1.11, p=0.85). Several other variants associated with HDL-C were also found to have no clear association with MI, unless they were also associated with triglyceride or LDL- C levels.

The principal lesson from these recent findings lies in the complexity of the relationship between HDL cholesterol and cardiovascular disease. A wealth of data from traditional epidemiologic studies as well as statin trials support an inverse relationship between HDL-C and coronary events, a powerful association incorporated into most global risk equations including the Framingham risk score.⁷ However, despite the popularly ingrained concept of HDL-C as the "good" cholesterol, evidence indicates that higher levels of HDL-C are not synonymous with improved outcomes. HDL-C suffers from limitations intrinsic to its static, mass-based measurement.⁸ First, as a snapshot of the steady-state cholesterol pool, HDL-C does not directly assess the rate of centripetal cholesterol flux from peripheral foam cells to the liver, which is influenced by many factors beyond the mass of HDL-C alone. Second, circulating HDL-C values fail to convey information regarding the anti-inflammatory, antioxidant, antithrombotic, and endothelial function promoting benefits of HDL, despite evidence supporting the potential clinical significance of these pleiotropic functions.

Recent publications shed light on promising measures that assess reverse cholesterol transport, arguably the most critical anti-atherogenic function of HDL, as well as anti-inflammatory activity. One study utilized a validated ex vivo system to quantify cholesterol efflux capacity using incubation of macrophages with apo B-depleted serum.⁹ Healthy participants exhibited an inverse relationship between efflux capacity and carotid intima-media thickness before and after adjustment for HDL-C. Among subjects who underwent coronary angiography for clinically suspected CAD, efflux capacity remained a strong inverse predictor of coronary disease status after adjustment for traditional risk factors as well as HDL-C (adjusted OR for CAD per 1-SD increase in efflux capacity, 0.75; 95% CI 0.63-0.90) and apo A-I (OR 0.74; 95% CI 0.61-0.89). A second study demonstrated an association between CAD status and HDL inflammatory index (HII), the latter quantified as the ratio of in vitro LDL oxidation of a fluorescein substrate incubated with and without participant HDL.¹⁰ Among 193 symptomatic patients undergoing angiography, HII was significantly higher (less antioxidant capacity) among those with acute coronary syndrome (ACS) than those without CAD (1.57 vs 1.17, p 0.005) or with stable CAD (1.57 vs 1.11, p 0.006). Association with ACS remained significant after adjusting for traditional risk factors (OR 3.8 p 0.003).

AIM-HIGH and dal-OUTCOMES additionally suggest that demonstrating incremental clinical benefit above and beyond current optimal medical therapy, inclusive of aggressive LDL-lowering, may be challenging for new therapies. Such a high hurdle may inadvertently weed out effective treatments for individuals unable to achieve ideal targets, including those suffering from medication intolerance (e.g., statin myopathy) or extreme phenotypes (e.g., familial dyslipidemias). This population is not insignificant in routine clinical practice. With regard to optimal management of dyslipidemia, a recent international study of 9,518 ambulatory patients on lipid-lowering therapy revealed that 34% of high-risk patients and 71% of very high-risk patients do not meet their recommended LDL-C goals.¹¹ Moreover, data from tertiary lipid clinics suggest that the majority of patients referred for complex dyslipidemia fail to achieve target LDL-C levels.^{12, 13} Broadening the scope of comparative effectiveness research to include populations such as those unable to take high-dose statin therapy addresses considerable unmet need and may also facilitate the development of useful HDLdirected therapies.

Fortunately, now more than ever, a myriad of HDL-targeted drug candidates is available to test new approaches.¹⁴ Lipidpoor apo A-I—phospholipid complexes have been studied extensively in animals and in preliminary studies in humans. Preclinical studies have demonstrated that the administration of apo A-I is associated with the inhibition or regression of atherosclerosis, enhanced macrophagespecific reverse cholesterol transport, and the inhibition of vascular inflammatory pathways, endothelial adhesion molecule expression and phospholipid oxidation.^15-17 Moreover, short exploratory clinical studies of apoA-I infusion using recombinant apoA-I Milano/phospholipid (MDCO-216),¹⁸ purified native apoA-I/ phospholipid (CSL-111),¹⁹ and autologous delipidated HDL (PDS-2)20 have yielded decreases in coronary atherosclerosis as assessed by coronary imaging.

Two novel compounds enable further testing of the CETP-inhibition strategy. Unlike dalcetrapib, which exerts modest effects on HDL-C alone, anacetrapib and evacetrapib yield significant reductions in atherogenic lipoproteins as well as more potent increases in HDL-C, at least suggesting a greater potential for clinical benefit. The phase III Determining the Efficacy and Tolerability of CETP Inhibition with Anacetrapib (DEFINE) randomized, placebo-controlled trial examined the effect of 100 mg of anacetrapib administered daily for 18 months to 1,623 patients with CAD or at high-risk for CAD events who had achieved LDL-C treatment goals with statin therapy.^{21, 22} Treatment with anacetrapib was associated with a 40% reduction in LDL-C from 81 mg/dl to 45 mg/dl (P <0.001) and a 138% increase in HDL-C from 41 mg/dl to 101 mg/dl (P <0.001) compared with placebo.²² Lp(a) decreased 36% compared with placebo from 27 nmol/l to 15 nmol/l. No increases in clinic-based blood pressure, serum aldosterone levels, or cardiovascular events were observed following anacetrapib treatment at 76 weeks. Supported by these improvements in LDL, HDL, and Lp(a), as well as an apparently benign safety profile, the Randomized Evaluation of the Effects of Anacetrapib through Lipid- Modification (REVEAL) is underway. This study will examine major coronary events, defined as coronary death, myocardial infarction and coronary revascularization procedures, in 30,000 patients with CAD, cerebrovascular atherosclerotic disease, or peripheral artery disease. The estimated study completion date is January 2017. Evacetrapib administration in daily doses of 30-500 mg decreased LDL-C from 20-51 mg/dL (14-36%) and increased HDL-C from 30-66 mg/dL (54-129%) in a 12-week randomized trial of 398 dyslipidemic patients.²³ Addition of evacetrapib 100 mg daily to statin therapy yielded further reductions in LDL-C of 16-21 mg/ dL (11-14%) and increments in HDL-C of 41-48 mg/dL (79-89%).²³ Effects on lipoprotein(a) were not reported, and no adverse events were observed in the small study. Apparently, a large phase III clinical outcomes trial is planned to elucidate the efficacy and safety of evacetrapib.

Activation of liver X receptors (LXRs) has been demonstrated to promote mobilization of intracellular cholesterol, increase macrophage cholesterol efflux via macrophage ABCA1 and ABCG1, and augment intestinal HDL generation.^24-26 Two LXR isoforms have been identified— LXRα and LXRβ. Therapeutic development of LXR agonists has been hindered by hepatic steatosis and increased plasma triglyceride concentrations reported in preclinical studies of these drugs.²⁷ Fortunately, dissociating LXR efficacy and toxicity might be possible owing to the differential effects of LXR agonism by receptor isoform and by tissue-specific effects. Administration of a nonselective LXR agonist to LXRα-deficient mice stimulated macrophage ABCA1 expression and cholesterol efflux without inducing fatty liver and with minimal upregulation of hepatic triglyceride synthesis.²⁸ A second approach to safer LXR development might be to selectively activate intestinal LXR. Fatty liver arises from activation of hepatic LXR, which, through upregulation of SREBP1c, stimulates lipogenesis.²⁹ Elevation of triglyceride levels occurs via SREBP1c and the subsequent suppression of apo A-V, which inhibits VLDL synthesis and stimulates VLDL hydrolysis. On the other hand, LXR expression on both macrophages and the small intestine contributes to the regulation of reverse cholesterol transport. An intestine-specific LXRα/β agonist, GW6340, promoted macrophage-specific reverse cholesterol transport, augmenting the fecal excretion of radiolabeled sterol by 52% via increased intestinal HDL production and intestinal excretion of HDL-derived cholesterol.³⁰ Elucidation of posttranscriptional pathways of HDL metabolism has identified additional targets for pharmacotherapeutic intervention. Short non-coding sequences of RNA, termed microRNAs (miRNAs), inhibit gene expression by binding to complementary 3’ untranslated regions of messenger RNAs (mRNAs) and causing translational repression and/or mRNA destabilization.³¹ Genome-wide screening identified miR-33, encoded within intron 16 of sterol regulatory element binding transcription factor 2 (SREBF2), from a subset of differentially expressed miRNAs modulated by cellular cholesterol content.³² In vitro and in vivo murine studies demonstrated, in the setting of miR-33 overexpression, suppressed macrophage and hepatocyte expression of ABCA1, reduced circulating HDL-C levels, and attenuated efflux to apoA-I. Conversely, silencing of miR-33 was associated with greater macrophage and hepatocyte expression of ABCA1 and increased HDL-C levels. In a mouse model of atherosclerosis, administration of an antisense oligonucleotide (ASO) to miR-33 significantly increased HDL-C by 35% and promoted macrophage-specific reverse cholesterol transport, augmenting hepatic and fecal delivery of radiolabeled tracer by 42% and 82%, respectively.³³ Importantly, these favorable changes in HDL parameters were accompanied by atheroma regression (35% reduction in aortic sinus lesion area compared to baseline and controls) as well as histologic evidence of remodeling toward a more stable plaque phenotype (28% decrease in lipid accumulation, 35% reduction in macrophage content, and 2-fold increase in collagen content).³³ In a non-human primate model of dyslipidemia, subcutaneous delivery of anti-miR-33 ASO over a 12-week period increased HDL-C up to 50%.³⁴ Greater macrophage cholesterol efflux was observed following incubation of foam cells with serum obtained from treated monkeys compared to equivalent volumes of serum isolated from control monkeys, correlating with the HDL-C levels in the two groups. Monkeys administered the anti-miR-33 also exhibited attenuated expression of genes involved in fatty acid synthesis, enhanced expression of genes involved in fatty acid oxidation, and a decrement in VLDL triglycerides of up to 50%, suggesting therapeutic potential for additional metabolic derangements associated with insulin resistance.³³ It will be interesting to see if anti-miR-33 approaches are taken into clinical development.

In conclusion, recent negative studies suggest the need for a revised approach to the evaluation of novel HDL-directed therapies. Therapeutic elevation of HDL-C does not necessarily mitigate atherothrombotic risk. Assessment of HDL functionality, particularly reverse cholesterol transport, is important to characterize the potential anti-atherogenic activity of new compounds; however, validation of emerging HDL assays remains critical prior to their use as surrogate measures. Finally, apoA-I mimetics, CETP inhibitors, LXR agonists, and miR-33 antagonists comprise several HDL-targeted candidate drug classes under current investigation.

Disclosure statement: Dr. deGoma has no disclosures to report. Dr. Rader has received consulting fees from AstraZeneca, Bristol-Myers Squibb, Daiichi Sankyo Inc., Eli Lilly & Co., GlaxoSmithKline, Johnson and Johnson, Merck & Co., Novartis Pharmaceuticals, Pfizer Inc., Regeneron, Roche, Sanofi, Alnylam, Catabasis, and Omthera.