Kexin Type 9 (PCSK9) Inhibition and the Future of Lipid Lowering Therapy

    Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibition and the Future of Lipid Lowering Therapy Lee Joseph, Jennifer G. Robinson PII: DOI: Reference:

S0033-0620(15)00029-8 doi: 10.1016/j.pcad.2015.04.004 YPCAD 655

To appear in:

Progress in Cardiovascular Diseases

Please cite this article as: Joseph Lee, Robinson Jennifer G., Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibition and the Future of Lipid Lowering Therapy, Progress in Cardiovascular Diseases (2015), doi: 10.1016/j.pcad.2015.04.004

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ACCEPTED MANUSCRIPT Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibition and

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Lee Joseph, MBBS

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the Future of Lipid Lowering Therapy

Jennifer G Robinson, MD, MPH

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University of Iowa

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Corresponding Author Jennifer G. Robinson, MD, MPH

Director, Prevention Intervention Center Department of Epidemiology

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College of Public Health

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Professor, Departments of Epidemiology & Medicine

University of Iowa

Office Assistant Fax

319.384.1563

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USA

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Iowa City, IA 52242-2007

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145 N Riverside Drive S455 CPHB

319.384.1540 319.384.4155

Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Low-density lipoprotein cholesterol (LDL-C) reduction with statins is the cornerstone of

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atherosclerotic cardiovascular disease (CVD) prevention. The LDL-C lowering non-statin therapy ezetimibe also modestly reduces CVD risk when added to statin therapy. There remains

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a clinical need for additional LDL-C lowering agents to reduce CVD risk in patients with genetic

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hypercholesterolemia, statin intolerance, or who are at high risk due to clinical CVD or diabetes. In clinical trials proprotein convertase subtilisin/kexin type 9,( PCSK9) inhibition using

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monoclonal antibodies has demonstrated robust LDL-C lowering efficacy of 50% to 65% and a favorable safety profile. These agents are a promising therapeutic strategy for addressing the

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unmet needs for additional CVD risk reduction. Regulatory approval for PCSK9 monoclonal

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antibodies may occur in the near future, and additional agents for PCSK9 inhibition are under development. This review focuses on the mechanism of LDL-C reduction using PCSK9

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inhibition, as well as the Phase I to III clinical trials of PCSK9 inhibitors. Results of the ongoing

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Phase III CVD outcomes trials are eagerly awaited.

Keywords: Familial hypercholesterolemia, Heterozygous FH, Monoclonal antibodies, Proprotein convertase subtilisin/kexin type 9

Abbreviations: ACC/AHA-American College of Cardiology/American Heart Association AEs – Adverse events 2

ACCEPTED MANUSCRIPT Apo A- Apolipoprotein A

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Apo B –Apolipoprotein B

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CVD – Cardiovascular disease

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FH-Familial hypercholesterolemia HDL-High-density lipoprotein

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HDL-C- HDL-cholesterol

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HeFH-Heterozygous FH

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LDL- Low –density lipoprotein

LDL R-LDL receptors

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LDL-C- LDL cholesterol

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mAbs-Monoclonal antibodies

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MI- Myocardial infarction

PCSK9- Proprotein convertase subtilisin/kexin type 9 SREBP-2- Sterol regulatory element-binding protein 2 TEAEs-Treatment emergent Aes TGs- Triglycerides

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ACCEPTED MANUSCRIPT Background

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Cardiovascular disease (CVD) remains the leading cause of mortality worldwide, accounting for

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30% of global mortality.1 Low-density lipoprotein (LDL) cholesterol (LDL-C) reduction with statin therapy is the cornerstone of CVD prevention.2 Currently, statins are the most effective

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drugs available for lowering LDL-C. High intensity statins result in 50 to 60% LDL-C reductions

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on average.3 A large meta-analysis of 174,149 subjects from 27 randomized trials by Cholesterol Treatment Trialists' Collaborators reported 21% reduction in the risk of major CVD events for

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every 39 mg/dL LDL-C level lowering with statin therapy, independent of the baseline LDL-C levels.4

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Based on a rigorous systematic review of randomized trial evidence, the 2013 American College

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of Cardiology/American Heart Association (ACC/AHA) blood cholesterol treatment guideline recommends statin therapy for patient groups most likely to experience a net benefit.2 High-

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intensity statins are recommended for high-risk patients unless safety concerns are present, and

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moderate-intensity statins for patients with safety concerns for high intensity statin, and in primary prevention. Use of non-statins may be considered in high risk patients who may benefit from additional LDL-C lowering, preferably with a non-statin shown to reduce CVD events in randomized trials. To date, ezetimibe is the only non-statin found to reduce CVD events when added to statin therapy in high risk patients.5 The American Diabetes Association recently released similar recommendations focused on statin-intensity and level of risk, as has the United Kingdom National Institute for Health and Care Excellence.6,7 A number of other guidelines based on expert consensus continue to recommend treatment targeting LDL-C levels or percentage reduction in LDL-C level, with the level determined by the 4

ACCEPTED MANUSCRIPT degree of CVD risk.8,9 However, accumulating data suggests that the risk-based approach recommended in the 2013 ACC/AHA cholesterol guideline would more appropriately treat high

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risk patients and prevent more CVD events than earlier approaches based on LDL-C level. 10,11,12

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Indeed, there appears to be no lower LDL-C limit to the reduction in CVD events or atherosclerotic regression with statin therapy, suggesting more aggressive LDL-C lowering

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beyond LDL-C thresholds <100 or <70 mg/dl could further reduce CVD risk. 13,14

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The currently available LDL-C lowering agents for management of blood cholesterol to reduce

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CVD risk have several limitations, including statin intolerance, the need for additional LDL-C lowering in patients with genetic hypercholesterolemia, the inability to achieve the anticipated

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reduction in LDL-C cholesterol or lipid goals in some patients, and safety and tolerability concerns with non-statins other than ezetimibe. Statin intolerance is commonly encountered in

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lipid management. Muscle symptoms are commonly reported by patients taking statins, with up

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to 10 to 30% patients reporting muscle symptoms in observational studies.15 However, rates are much lower in double-blind placebo-controlled trials and many patients can tolerate a statin on

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re-challenge.16,17

For high-risk patients who have suboptimal LDL-C control, patients with genetic hypercholesterolemia, and statin intolerant patients who have an indication for statin therapy, non-statin lipid lowering therapy is reasonable per the 2013 AHA/ACC guidelines. With the exception of ezetimibe in the recently reported IMPROVE-IT trial, adequately powered trials have not overall found incremental CVD risk reduction from adding niacin or fenofibrate to statin therapy, and have found evidence of excess harm with niacin.18-20 Notably, despite the optimal treatment with currently available therapies, 70 to 80% remain at high CVD risk and may benefit from additional LDL-C lowering.21-23 LDL-C reduction by means of proprotein 5

ACCEPTED MANUSCRIPT convertase subtilisin/kexin type 9 (PCSK9) inhibition is a promising novel therapeutic strategy. The PCSK9 monoclonal antibodies ( mAbs) under development have generated tremendous

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interest due to their remarkable LDL-C lowering efficacy and favorable safety profile in clinical

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trials to date. This review focuses on the mechanism of LDL-C reduction from PCSK9

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inhibition, as well as the Phase I to III clinical trials of PCSK9 inhibitors.

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PCSK9

In 2003, genetic studies identified two gain-of-function mutations in the PCSK9 gene in a French

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family as a cause for autosomal dominant familial hypercholesterolemia (FH), a condition associated with premature CVD and death.24 This was the third autosomal dominant mutation for

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FH, in addition to mutations in the LDL receptor (LDL-R) and apolipoprotein B (ApoB) genes.

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The precise role of PCSK9 (initially known as NARC-1) in cholesterol regulation was

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subsequently elucidated in animal studies.25 STRUCTURE. PCSK9 is a secreted enzymatic protein of the subtilisin family of serine

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proteases.26 It is primarily synthesized in the liver, but is also found in the intestine and kidney.27 Pro-PCSK9, the 692-aminoacid and 75 kDa precursor of PCSK9, consists of a pro-domain, catalytic domain, C-terminal domain and a signal sequence. It is produced in the endoplasmic reticulum, modified in the Golgi apparatus, where it undergoes autocatalytic cleavage to enter the secretory pathway, and is then released into the circulation. ROLE OF PCSK9 IN CHOLESTEROL HOMEOSTASIS REGULATION. The majority of LDL particles are removed from the circulation via the hepatic transmembrane LDL-R [Figure 1].28,29 LDL-R binds to a single LDL particle and the complex is then internalized via endocytosis. Due to a drop in pH in the vesicle, the complex of LDL-R and the LDL particle 6

ACCEPTED MANUSCRIPT dissociates, freeing the LDL-R to be recycled to the cell membrane to repeat the cycle. Each LDL-R is recycled up to 150 times. The LDL particle is degraded within the lysosome to release

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cholesterol for either storage or other cellular activities. Regulation of the hepatic LDL-R activity is performed by sterol regulatory element-binding

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protein 2 (SREBP-2) during transcription and by PCSK9 post transcriptionally [Figure 1].30 PCSK9 down-regulates the LDL-R expression on the hepatocyte surface by directly and

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irreversibly binding with the LDL-R/ LDL complex.31 The larger complex is internalized and

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degraded by the lysosome, resulting in subsequent degradation of the LDL-R. As a result, LDLC clearance is decreased, leading to increased plasma LDL levels.

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PCSK9 REGULATION. At the transcriptional level, both PCSK9 and LDL-R are regulated mainly by intracellular cholesterol levels via SREBP-2. By inhibiting cholesterol synthesis by

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HMGCoA reductase, statins induce expression of SREBP-2. Increased SREBP-2 levels, in turn,

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induce LDL-R and PCSK9 gene expression in a dose dependent fashion, the former gene being more robustly upregulated [Figure 1].25,31 Fibrates also increase PCSK-9 levels via SREBP-2.32

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The up-regulation of PCSK9 levels by both statins and fibrates suggest PCSK9 inhibition may enhance the lipid-lowering efficacy of these drugs. PCSK9 MUTATIONS & CVD risk. Gain-of-function PCSK9 mutations are associated with FH and premature CVD.33 In contrast, loss-of-function mutations in PCSK9 can cause hypobetalipoproteinemia which is associated with reduced CVD risk due to the decreased ability of PCSK9 to bind LDL-R resulting in decreased LDL-R degradation, increased LDL-R recycling and decreased plasma LDL-C levels. Carriers of two nonsense mutations in PCSK9 (Y142X and C679X) among African Americans had 40% lower LDL-C level in the Dallas Heart Study (DHS) cohort.34 PCSK9 was subsequently sequenced in the Atherosclerosis Risk in 7

ACCEPTED MANUSCRIPT Communities (ARIC) study participants. This study found that 2.6% of 3363 black subjects had similar PCSK9 nonsense mutations (Y142X and C679X).35 These mutant carriers had a 28%

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LDL-C reduction and 88% reduction in coronary events over a 15 year observation period.

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Among 9524 white subjects, a 15% LDL-C reduction and 47% coronary event reduction [hazard ratio (95% CI), 0.5 (0.3 to 0.8)] was found in the 3.2% subjects with PCSK9 sequence variation

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(R46L allele). Reduced LDL-C levels has also been reported in homozygous and heterozygous

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carriers of the A443T substitution and L253F mutant carrriers among African Americans, and the R46L mutant carriers among Europeans.36 A meta-analysis of 66,698 individual subjects

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confirmed that the carriers of PCSK9 R46L genotype was associated with 12% LDL-C reduction and 28% reduction in the risk of ischemic heart disease [HR (95% CI): 0.7 (0.6 to 0.8)].37 The

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remarkable CVD risk reduction noted in these studies of up to 88% well exceeds the relative risk

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reductions observed in randomized trials, and is attributed to the life-long lower LDL-C levels in

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these patients.

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The Evolution of PCSK9 Inhibition-Directed Therapies The genetic and epidemiologic observations were rapidly translated into research to determine whether PCSK9 inhibition could be utilized as a LDL-C lowering therapeutic strategy. Potential approaches for PCSK9 inhibition include inhibiting PCSK9 synthesis, antagonizing PCSK9 interaction with LDL-R, and increasing clearance of PCSK9.38 Tremendous progress in developing PCSK9 inhibiting therapies has been made since the initial 2003 report [Figure 2]. Drugs currently under investigation are based on the first two strategies and fall into 2 categories: biologic therapy and small molecule pharmacotherapy. Biologic therapies inhibiting PCSK9/ LDL-R interactions via mAbs are farthest along in development. Development of 8

ACCEPTED MANUSCRIPT small-molecule pharmacotherapy for PCSK9 inhibition has been difficult due to the structure of the PCSK9 molecule, which does not have suitable clefts for small molecule binding that reduce

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its activity. Large molecule biologic therapy has been more successful since the mAbs

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effectively binds the flat portion of the PCSK9 molecule resulting in reduced function.

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Several PCSK9 mAbs are well into Phase III development. These mAbs bind to circulating PCSK9 and prevent it from binding with the LDL-R/LDL-C complex, thus restoring normal

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LDL-R recycling [Figure 1]. Gene-specific silencing by means of antisense oligonucleotides or

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small interference RNA and or mimetic peptides are also under development. In contrast to small-molecule pharmacotherapy, biologic therapeutics are large, complex proteins requiring

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parenteral administration. They are highly specific to their target, potentially limiting off-target adverse effects. They also have a long serum half-life and do not penetrate the blood-brain

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barrier; their elimination is via the reticuloendothelial system.

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Monoclonal Antibodies

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The mAbs are specific antibodies produced by a single clone of cells or cell lines. The first generation therapeutic mAbs were mouse-derived, often leading to the development of human anti-mouse antibodies and resulting in reduced efficacy as well as increased risk for hypersensitivity.39 Subsequently, chimeric, humanized and fully human mAbs were produced with a human constant region, with each type having increasing proportions of human sequence in the variable region and decreasing immunogenicity. The mAbs are delivered intravenously or subcutaneously, generally tolerated well and have limited potential for drug-drug interactions due to their target specificity. They neither interact with cytochrome P450 or other transport proteins in the body nor affect the QT interval changes due to absence of cardiac potassium 9

ACCEPTED MANUSCRIPT ion/human ether-à-go-go related gene channel inhibition.39 The major adverse effects of mAbs are limited to target-specific toxicity, immunogenicity related hypersensitivity reactions and off-

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target non-specific adverse reactions including injection site reactions and infusion reactions.

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PCSK9 Monoclonal Antibodies in Development Three PCSK9 mAbs, alirocumab (REGN727/SAR236553; Regeneron/Sanofi), evolocumab

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(AMG-145; Amgen) and bococizumab (RN316/PF-04950615; Pfizer) are currently being studied in Phase III trials with larger patient populations. The PCSK9 mAbs, LGT209 (Novartis) and

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LY3015014 (Eli Lilly), are undergoing Phase II trials. RG7652 (Roche/Genentech), another PCSK9 mAb, was discontinued in 2014. The role of these drugs is under investigation as add-on

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to statins or other lipid-lowering therapy and as monotherapy in a variety of patient populations,

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including patients at high CVD risk, statin intolerant patients, and those with FH.

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PCSK9 mAbs result in substantial reductions in total cholesterol, LDL-C, ApoB and lipoprotein (a) in a dose dependent fashion when used as add-on or monotherapy. When added to maximal

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statin therapy, PCSK9 can lower LDL-C to <25 mg/dl, a level that has not been regularly observed in clinical trials to date.40 Theoretical concerns have arisen regarding the long-term exposure to very low levels of LDL-C with respect to hemorrhagic stroke, cancer, hypertension, reproductive function and neurocognitive dysfunction risks.41,42 The healthy individuals with homozygous PCSK9 loss-of-function mutations who have had lifetime LDL-C levels as low as 14-16 mg/dL have remained healthy in all aspects including neurologic and reproductive functions based on epidemiological studies, suggesting that long-term inhibition of PCSK9 itself is unlikely to have significant adverse effects.43-45 Another concern is development of immunogenicity due to anti-drug antibody formation and loss of treatment efficacy. Notably, 10

ACCEPTED MANUSCRIPT fully human mAbs may carry the least potential for immunogenicity. Adverse effects and antidrug antibodies are being closely monitored in the ongoing long term Phase III trials of

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PCSK9 mAbs. Whether the 50 to 65% reductions in LDL-C from PCSK-9 mAbs will translate into a reduction

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in CVD events is under investigation in several ongoing CVD outcomes trials. These trials are evaluating the incremental CVD event reduction when PSCK9 mAbs are added to maximal

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background statin therapy in high risk patients. It is not clear whether there is a linear or curvilinear relationship between LDL-C lowering and CVD event reduction in the clinical trials

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to date.46-48 However, recent data have shown that there appears to be no lower LDL-C limit to

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the reduction in CVD events from statin therapy.14

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Alirocumab

Alirocumab, a fully human mAb to PCSK9, is administered subcutaneously and if approved, will

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be marketed for administration every 2 weeks, and possibly every 4 weeks, using an auto-

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injector pen. It has a dose dependent mean terminal half-life of 5 to 9 days.38 Phase I and II clinical studies of alirocumab have found substantial dose-dependent reductions in LDL-C of 50% as a monotherapy and up to 65% as an add-on therapy to statins. The greater efficacy in statin treated patients is attributed to the up-regulation of PCSK9 levels by statins. Alirocumab consistently reduces total cholesterol, ApoB, non-high density lipoprotein cholesterol (non-HDL-C) and lipoprotein (a) irrespective of the dose of the background statin and addition of ezetimibe in both heterozygous FH (HeFH) and non-HeFH populations 49-52 Modest increases in HDL-C and apolipoprotein A1 (ApoA1) and decreases in triglycerides have also been observed in some trials. The precise mechanism of decrease in lipoprotein (a) is yet to 11

ACCEPTED MANUSCRIPT be established. A rebound in LDL-C level towards the baseline was noted 2 weeks after monthly alirocumab doses, whereas biweekly alirocumab doses demonstrated a consistent LDL-C

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reduction pattern.49,51 Combining the safety data of Phase I and II trials, the most common

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treatment emergent adverse events (TEAE) were mild injection-site reactions. There have been no persistent or prevalent liver or skeletal muscle adverse events (AEs). Neurocognitive

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dysfunction was not formally assessed. Though low levels of anti-alirocumab antibodies were

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reported in the Phase II trials, there was no evidence of AEs or a reduction in alirocumab

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efficacy related to these antibodies.

ODYSSEY Phase III Clinical Trial Program

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The ODYSSEY clinical trial program currently comprises 14 Phase III studies ranging 24 to 104 weeks duration and involving more than 23,500 planned patients worldwide, to further assess the

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efficacy and safety of alirocumab [Figure 2]. These trials use a dose-up-titration approach based

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on the LDL-C level and are enrolling patients receiving the current standard of care. With the exception of the ODYSSEY HIGH FH & LONG-TERM trials, the initial dose of alirocumab is

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75 mg biweekly. If LDL-C remained >70 mg/dl in high risk patients (or >100 mg/dl in lower risk patients) after 8 weeks, alirocumab was up-titrated in a blinded fashion to 150 mg biweekly at week 12. In the ODYSSEY OUTCOMES trial, alirocumab is up-titrated from 75 mg to 150 mg every 2 weeks if LDL-C is >50 mg/dl. The trials include patients with high CVD risk, HeFH, or well-defined statin intolerance and assess different treatment options including alirocumab monotherapy, combination therapy with statins and other lipid lowering therapies and flexible dosing options. A. Monotherapy

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ACCEPTED MANUSCRIPT ODYSSEY MONO. In this trial, patients did not receive statins. Alirocumab monotherapy resulted in an LDL-C reduction of 47% in comparison to 16% in the ezetimibe arm over a 24

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week period, with similar rates of injection-site reactions and muscle-related AEs in both groups

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(each rate below 4% in both groups).53

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B. HeFH Population

ODYSSEY FH trials. In HeFH patients with poorly controlled hypercholesterolemia on

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maximally tolerated doses of statins, the ODYSSEY FH I and II trials showed that alirocumab

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resulted in significantly greater reduction in LDL-C from baseline to 24 weeks compared with placebo in both FH I (49% vs. 9% respectively) and FH II (49% vs. 3% respectively) trials,

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which was maintained at 52 weeks.54 Alirocumab was up-titrated to 150 mg biweekly at week 12 in 43% and 39% of the FH I and FH II trial subjects, respectively. On safety analysis, TEAEs

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leading to drug discontinuation were similar (3.1% vs. 3.7%). The HIGH FH study showed

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alirocumab 150 mg every 2 weeks significantly reduced LDL-C compared with placebo (46% vs. 7%) at 24 weeks.55 Alirocumab arm had numerically higher TEAEs requiring treatment

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discontinuation (4.2% vs. 2.9%), adjudicated CVD events (8.3% vs. 0%) and injection-site reactions (8.3% vs. 2.9%). C. Combination Therapy ODYSSEY COMBO I & II. The COMBO studies investigated the efficacy and safety of alirocumab as add-on therapy to maximally tolerated statin in high CVD risk patients with LDLC levels >70 or >100 mg/dL (depending on the level of risk), with or without other lipidlowering therapy versus placebo (COMBO I) or ezetimibe (COMBO II). Alirocumab dose was increased to 150 mg every 2 weeks in 17% of the COMBO I and 18% of the COMBO II trial

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ACCEPTED MANUSCRIPT patients at week 12.There was 48% reduction in LDL-C at 24 weeks from baseline in the alirocumab arm compared with 2% reduction in the placebo arm in the COMBO I study.56 In the

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COMBO II study, alirocumab resulted in 51% reduction in LDL-C compared with 21% for

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ezetimibe at 24 weeks that was maintained during a 52 week follow up period.57 There were no

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significant differences in AEs.

ODYSSEY OPTIONS I & II. The ODYSSEY OPTIONS studies found that alirocumab as add-

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on therapy to statin (atorvastatin in OPTIONS I and rosuvastatin in OPTIONS II) was superior to

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other lipid-lowering strategies. The LDL-C reduction from baseline to 24 weeks for alirocumab compared with ezetimibe, double-dose statin and rosuvastatin 40 mg/day was 36-54% vs. 11-

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23% vs. 5-16% vs. 21%, respectively.58 AE rates were similar among the different treatment arms. In contrast to the other ODYSSEY studies that showed significant LDL-C reduction with

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alirocumab in comparison with placebo or ezetimibe on background of all rosuvastatin doses,

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there was no statistically significant difference in LDL-C reduction between alirocumab and doubling rosuvastatin 20 mg daily.

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D. Statin Intolerant Population ODYSSEY ALTERNATIVE. In this active-control trial of patients with a history of statin intolerance and moderate to very high CVD risk, alirocumab was superior to ezetimibe for LDLC reduction (45% vs. 15%) with a lower rate of muscular side effects (33% vs. 41% in the ezetimibe arm vs. 46% in the atorvastatin 20 mg arm).59 E. Long Term Results ODYSSEY LONG TERM. In this trial, biweekly alirocumab 150 mg resulted in a consistent reduction in LDL-C compared with placebo (-61% vs. 1%) (Figure 3), which was maintained up 14

ACCEPTED MANUSCRIPT to 52 weeks in patients with HeFH or high CVD risk on maximum tolerated statin therapy regardless of the baseline LDL-C level, PCSK9 level, HeFH status and sex differences.60

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Alirocumab also substantially reduced total cholesterol, ApoB, lipoprotein (a) and triglycerides

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(TGs), and modestly increased HDL-C and ApoA1. In a post hoc analysis, adjudicated CVD events [coronary death, acute myocardial infarction (MI), hospitalization for unstable angina, or

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ischemic stroke] were lower in the alirocumab group compared to placebo (1.4% vs. 3.0%;

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hazard ratio 0.46 (95% CI 0.26-0.82; p<0.01). TEAEs were comparable in alirocumab arm,

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placebo arm, and in 562 patients with 2 consecutive LDL-C level <25 mg/dL. F. CVD Outcomes

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ODYSSEY OUTCOMES. This trial [ClinicalTrials.gov Identifier: NCT01663402] is an ongoing large CVD outcomes study of 18,000 patients who suffered acute coronary syndrome event

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within a year to determine the long-term impact of alirocumab and low LDL-C levels on the

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CVD event rates over a treatment period of 64 months.61,62 Patients are randomized to receive biweekly subcutaneous alirocumab (75 mg up-titrated to 150 mg if LDL-C >50 mg/dl at week 8)

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or matching placebo on a background of maximum tolerated statin. The primary efficacy outcome is time to first occurrence of coronary death, MI, hospitalization for unstable angina, or ischemic stroke.

Evolocumab Evolocumab, a fully human mAb to PCSK9, has a non-linear pharmacokinetics, an elimination half-life of 20 to 21 days and a subcutaneous route of administration. In Phase I and II clinical trials, evolocumab emerged as an efficacious and safe option for LDLC reduction as monotherapy or as add on therapy to statin, in statin intolerant patients, in high 15

ACCEPTED MANUSCRIPT CVD risk patients as well as the HeFH population [Figure 2].63-67 As an add-on therapy to statins, evolocumab led to substantial dose-dependent reductions in LDL-C of up to 51% as a

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monotherapy, up to 66% for 140 mg every 2 week and up to 50% as 420 mg once a month.

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Evolocumab substantially reduced total cholesterol, ApoB, non-HDL-C and lipoprotein (a). Evolocumab 420 mg administered every 4 weeks has similar efficacy to 140 mg every 2 weeks

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on average, but there is more variability in LDL-C levels due to consumption of the PCSK9 mAb

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toward the end of the 4-week dosing period. Combining safety analyses of Phase I to III studies,

with treatment periods of 65 weeks.

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there appear to be no imbalances in adverse effects in the evolocumab treated groups to date,

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The PROFICIO Phase III Clinical Trial Program The umbrella Phase III program for evolocumab, named PROFICIO consists of 22 clinical trials

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with a planned enrollment of approximately 30,000 patients [Figure 2]. It includes studies

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A. Monotherapy

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evaluating the effects of evolocumab in a wide range of patients.

MENDEL-2. In this trial of patients who did not receive a statin, evolocumab monotherapy significantly reduced LDL-C by 55% to 57% more than placebo and by 38% to 40% more than ezetimibe in patients with hypercholesterolemia.68 No imbalance in AEs was observed. B. HeFH Population RUTHERFORD 2. In this study, evolocumab administered either 140 mg biweekly or 420 mg monthly in patients with HeFH on stable lipid lowering therapy reduced LDL-C by 59% to 61% at week 12 compared with placebo without significant increase in the rates of AEs.69

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ACCEPTED MANUSCRIPT C. Combination Therapy LAPLACE-2. In patients with primary hypercholesterolemia and mixed dyslipidemia

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randomized to either a moderate or a high intensity statin, biweekly evolocumab 140 mg reduced LDL-C levels by 66% to 75%, monthly evolocumab 420 mg by 63% to 75%, and ezetimibe by

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19% to 32% compared with placebo at the mean of weeks 10 and 12; biweekly and monthly dosing regimens of evolocumab were equivalent.70 The efficacy of evolocumab in LDL-C

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reductions was similar irrespective of the background statin type, dose or intensity, possibly due

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to the greater up-regulation of PCSK9 levels by high intensity statin. Evolocumab resulted in achieved LDL-C levels of 39-49 mg/dl in the moderate intensity statin groups, and 35-38 mg/dl

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in the high intensity statin groups (Figure 4). Evolocumab substantially reduced non-HDL-C, ApoB and lipoprotein (a), with variable modest effects on TGs and HDL-C.

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D. Statin Intolerant Population

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GAUSS-2. Among hypercholesterolemic patients with statin intolerance who were not receiving

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a statin, evolocumab reduced LDL-C levels by 53% to 56% compared to 37% to 39% reductions with oral ezetimibe. The evolocumab group had a lower incidence of muscle related AEs than the ezetimibe group (12% vs. 23%).71 E. Long Term Trials DESCARTES. Evolocumab 420 mg monthly resulted in 49-57% reductions in LDL-C from baseline to week 52 compared with placebo in patients treated with diet alone, atorvastatin 10 mg, atorvastatin 80 mg, or atorvastatin 80 mg plus ezetimibe 10 mg.72 There were more serious AEs in the evolocumab arm than placebo arm, however, there were no clear indications of any specific risks. 17

ACCEPTED MANUSCRIPT OSLER. OSLER was an open-label extension trial that enrolled participants from the 12-week Phase 2 and 3 trials. Eligible patients were re-randomized (2:1) to open-label evolocumab 140

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mg every 2 weeks or 420 mg every 4 weeks or standard of care. Over 52 weeks, patients who

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received evolocumab in OSLER had a mean 52% reduction in LDL-C compared to standard of care.73 LDL-C levels returned to near baseline levels in patients who discontinued evolocumab

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on entry to OSLER. Monthly evolocumab demonstrated continued efficacy and was well-

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tolerated over 1 year of treatment.

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F. CVD Outcomes Trial

FOURIER TRIAL. FOURIER study [ClinicalTrials.gov Identifier: NCT01764633] is an ongoing

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large scale trial for assessing the safety and efficacy of biweekly or monthly evolocumab compared to moderate or high intensity statin therapy in 27,500 patients with clinical

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atherosclerotic CVD.74 The primary end point of this study is a composite of CVD death, MI,

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Bococizumab

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hospitalization for unstable angina, stroke, or coronary revascularization.

Bococizumab, a humanized mAb, is in Phase III trial stage of development (Figure 2), however, the results of the Phase I and II studies have not been reported in peer reviewed publications yet. In a Phase II trial, subcutaneous bococizumab at different doses (biweekly 50 mg, 100 mg, and 150 mg, monthly 200 mg or 300 mg) was compared with placebo for 24 weeks.75 Doses were reduced in 35% of subjects as two consecutive LDL-C levels were ≤ 25 mg/dL. Bococizumab 150 mg biweekly dose showed the most robust reduction in LDL-C of 53 mg/dL from baseline compared with placebo at week 12. There was a dose-dependent increase in treatment-related

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ACCEPTED MANUSCRIPT AEs. Though there were no hypersensitivity reactions, anti-drug antibodies were detected in 7.2% of patients, resulting in diminution of LDL-C reduction only in 1 patient.

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Currently, five Phase III clinical trials testing the safety and efficacy of bococizumab are underway, three 12 week studies investigating its effect on LDL-C levels compared to placebo in

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high to very high CVD risk patients on background statin therapy [SPIRE-HR and SPIRE-LDL studies], and HeFH patients and high to very high CVD risk subjects [SPIRE-HF trial] and two

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CVD outcome trials studying its effect on 5 year CVD event reduction in high CVD risk

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population compared to placebo [SPIRE-1 and SPIRE-2 trials, ClinicalTrials.gov Identifiers: NCT01975376 and NCT01975389, respectively].76,77

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Clinical Considerations and Future Perspectives

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PCSK9 mAb inhibitors can reduce LDL-C by up to 50% when used as monotherapy and up to

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65% when added to background statin therapy with an excellent margin of safety. PCSK9 mAbs have been shown to be efficacious as monotherapy or as add on therapy to other lipid lowering

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agents in a variety of patient populations including patients with high CVD risk, statin intolerance, HeFH, high lipoprotein (a) and renal insufficiency. Alirocumab and evolocumab have comparable efficacy and safety when used at comparable doses (150 or 140 mg, respectively) every 2 weeks Bococizumab and other PSCK-9 mAbs are not as far along in development. Preliminary analyses suggest that the addition of PCSK9 inhibitors may further reduce CVD risk in statin-treated patients. The ongoing long term CVD outcomes trials will provide more evidence for the CVD risk reduction potential and long term safety concerns with PCSK9 inhibitors and low LDL-C levels.

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ACCEPTED MANUSCRIPT PCSK9 inhibitors have some drawbacks. Firstly, the parenteral administration and the unfamiliarity of patients with injectable agents may be a reason for patient discomfort. Patient

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education programs similar to the programs for diabetes management or biologics used in other

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disease areas will need to be designed to address this concern. Secondly, the cost-effectiveness of

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these agents will need to be established.

If approved by regulatory bodies, PCSK9 inhibitors may be used clinically in special populations

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such as statin intolerant patients, HeFH patients and patients with CVD who need additional

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LDL-C lowering before outcome trial results are available. The 2013 ACC/AHA cholesterol guideline emphasizes maximization of statin therapy, but there may be patient populations for

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whom additional cholesterol-lowering non-statin therapy may be considered. Although ezetimibe has been shown to reduce CVD events in clinical trials, PCSK9 mAbs may still have a role in

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high risk patients due to their greater LDL-C lowering efficacy and potential for considerably

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greater CVD event reduction .

The potential to achieve very low LDL-C levels may allow, for the first time, exploration of the

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possibility of extensive regression of atherosclerosis. Robinson and Gidding have proposed a new paradigm focused on “curing” atherosclerosis in its early stages.78 This paradigm emphasizes very aggressive LDL-C lowering early in the course of atherosclerosis, with the potential for complete regression, as was observed in animal models. LDL-C lowering treatments can then be repeated at decade or more intervals to maintain the atherosclerosis in an arrested or regressed phase. The potential for avoidance of long term drug therapy, long term drug adherence and adverse effects, and potential reduction in the costs related to the burden of clinical atherosclerotic CVD could make this model a favorable option. Future studies will be needed to determine the appropriate age and burden of atherosclerosis to initiate this approach, 20

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Conclusion

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In conclusion, PCSK9 inhibition using mAbs reduced LDL-C levels by 50% to 65% with a favorable safety profile in several Phase I to III clinical trials. These agents are a promising

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therapeutic strategy to address the unmet needs for additional LDL-C lowering to reduce CVD risk in high risk populations, particularly, those with genetic hypercholesterolemia, clinical

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CVD, or statin intolerance.

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55. Henry N Ginsberg DJR, Frederick J Raal, John R Guyton, Christelle Lorenzato, Robert Pordy, Marie T Baccara-Dinet, Eric S Stroes. ODYSSEY HIGH FH: Efficacy and Safety of Alirocumab in Patients With Severe Heterozygous Familial Hypercholesterolemia under Late-Breaking Clinical Trial Abstracts. Circulation 2014;130:2119. 56. Dean J Kereiakes JGR, Christopher P Cannon, Christelle Lorenzato, Robert Pordy, Umesh Chaudhari, Helen M Colhoun. Efficacy and Safety of Alirocumab in High Cardiovascular Risk Patients With Suboptimally Controlled Hypercholesterolemia on Maximally Tolerated Doses of Statins: The ODYSSEY COMBO I Study under Late-Breaking Clinical Trial Abstracts. Circulation 2014;130:2105-26. 57. Cannon C CB, Blom D, McKenney JM, Lorenzato C, Pordy R, Chaudhari U, Colhoun HM. . Efficacy and safety of alirocumab in high cardiovascular risk patients with inadequately controlled hypercholesterolemia on maximally tolerated daily statin: results from the ODYSSEY COMBO II study. Late-breaking clinical trial presented at the European Society of Cardiology Congress, Barcelona , Spain. 2014. 58. Harold Bays MF, Daniel Gaudet, Robert Weiss, Juan Lima Ruiz, Gerald F Watts, Ioanna GouniBerthold, Jennifer G Robinson, Peter H Jones, Randall Severance, Maurizio Averna, Elisabeth SteinhagenThiessen, Helen M Colhoun, Jian Zhao, Yunling Du, Corinne Hanotin, Stephen Donahue. Efficacy and Safety of Combining Alirocumab With Atorvastatin or Rosuvastatin versus Statin Intensification or Adding Ezetimibe in High Cardiovascular Risk Patients: ODYSSEY OPTIONS I and II under Late-Breaking Clinical Trial Abstracts. Circulation 2014;130:2118. 59. Patrick M Moriarty PDT, Christopher P Cannon, John R Guyton, Jean Bergeron, Franklin J Zieve, Eric Bruckert, Terry A Jacobson, Marie T Baccara-Dinet, Jian Zhao, Robert Pordy, Daniel Gipe. ODYSSEY ALTERNATIVE: Efficacy And Safety of the Proprotein Convertase Subtilisin/kexin Type 9 Monoclonal Antibody, Alirocumab, versus Ezetimibe, in Patients With Statin Intolerance as Defined by a Placebo Run-in and Statin Rechallenge Arm under Late-Breaking Clinical Trial Abstracts. Circulation 2014;130:2108. 60. Robinson JG FM, Krempf M, Bergeron J, Luc G, Averna M, Stroes E, Langlet G, Raal FJ, Shahawy ME, Koren MJ, Lepor N, Lorenzato C, Pordy R, Chaudhari U, Kastelein JJP. . Long-term safety, tolerability and efficacy of alirocumab versus placebo in high cardiovascular risk patients: first results from the ODYSSEY LONG TERM study in 2,341 patients. Late-breaking clinical trial presented at the European Society of Cardiology Congress, Barcelona , Spain. In; 2014 December 2, 2014; 2014. p. 2120. 61. Schwartz GG, Bessac L, Berdan LG, et al. Effect of alirocumab, a monoclonal antibody to PCSK9, on long-term cardiovascular outcomes following acute coronary syndromes: Rationale and design of the ODYSSEY Outcomes trial. American heart journal 2014;168:682-9. e1. 62. ODYSSEY Outcomes: Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab SAR236553 (REGN727). (Accessed February 15, 2015, at https://clinicaltrials.gov/ct2/show/NCT01663402.) 63. Dias CS, Shaywitz AJ, Wasserman SM, et al. Effects of AMG 145 on Low-Density Lipoprotein Cholesterol LevelsResults From 2 Randomized, Double-Blind, Placebo-Controlled, Ascending-Dose Phase 1 Studies in Healthy Volunteers and Hypercholesterolemic Subjects on Statins. Journal of the American College of Cardiology 2012;60:1888-98. 64. Giugliano RP, Desai NR, Kohli P, et al. Efficacy, safety, and tolerability of a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 in combination with a statin in patients with hypercholesterolaemia (LAPLACE-TIMI 57): a randomised, placebo-controlled, dose-ranging, phase 2 study. The Lancet 2012;380:2007-17. 65. Raal F, Scott R, Somaratne R, et al. Low-density lipoprotein cholesterol-lowering effects of AMG 145, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 serine protease in patients with heterozygous familial hypercholesterolemia: the Reduction of LDL-C with PCSK9 Inhibition in

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Heterozygous Familial Hypercholesterolemia Disorder (RUTHERFORD) randomized trial. Circulation 2012;126:2408-17. 66. Sullivan D, Olsson AG, Scott R, et al. Effect of a monoclonal antibody to PCSK9 on low-density lipoprotein cholesterol levels in statin-intolerant patients: the GAUSS randomized trial. JAMA 2012;308:2497-506. 67. Koren MJ, Scott R, Kim JB, et al. Efficacy, safety, and tolerability of a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 as monotherapy in patients with hypercholesterolaemia (MENDEL): a randomised, double-blind, placebo-controlled, phase 2 study. The Lancet 2012;380:19952006. 68. Koren MJ, Lundqvist P, Bolognese M, et al. Anti-PCSK9 monotherapy for hypercholesterolemia: the MENDEL-2 randomized, controlled phase III clinical trial of evolocumab. J Am Coll Cardiol 2014;63:2531-40. 69. Raal FJ, Stein EA, Dufour R, et al. PCSK9 inhibition with evolocumab (AMG 145) in heterozygous familial hypercholesterolaemia (RUTHERFORD-2): a randomised, double-blind, placebo-controlled trial. The Lancet 2014;pii: S0140-6736(14)61399-4. . 70. Robinson JG, Nedergaard BS, Rogers WJ, et al. Effect of evolocumab or ezetimibe added to moderate- or high-intensity statin therapy on LDL-C lowering in patients with hypercholesterolemia: the LAPLACE-2 randomized clinical trial. JAMA 2014;311:1870-82. 71. Stroes E, Colquhoun D, Sullivan D, et al. Anti-PCSK9 antibody effectively lowers cholesterol in patients with statin intolerance: the GAUSS-2 randomized, placebo-controlled phase 3 clinical trial of evolocumab. Journal of the American College of Cardiology 2014;63:2541-8. 72. Blom DJ, Hala T, Bolognese M, et al. A 52-Week Placebo-Controlled Trial of Evolocumab in Hyperlipidemia. New England Journal of Medicine 2014;370:1809-19. 73. Koren MJ, Giugliano RP, Raal FJ, et al. Efficacy and safety of longer-term administration of evolocumab (AMG 145) in patients with hypercholesterolemia: 52-week results from the Open-Label Study of Long-Term Evaluation Against LDL-C (OSLER) randomized trial. Circulation 2014;129:234-43. 74. Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Subjects With Elevated Risk (FOURIER). (Accessed February 14, 2015, at https://clinicaltrials.gov/ct2/show/NCT01764633.) 75. Ballantyne CM, Neutel J, Cropp A, et al. Efficacy and Safety of Bococizumab (RN316/PF04950615), a monoclonal antibody against proprotein convertase subtilisin/kexin type 9 in statintreated hypercholesterolemic subjects: Results from a randomized, placebo-controlled, dose-ranging study. Journal of the American College of Cardiology 2014;63. 76. The Evaluation of Bococizumab (PF-04950615; RN316) in Reducing the Occurrence of Major Cardiovascular Events in High Risk Subjects (SPIRE-2). (Accessed February 15, 2015, at https://clinicaltrials.gov/ct2/show/NCT01975389.) 77. The Evaluation of Bococizumab (PF-04950615;RN316) in Reducing the Occurrence of Major Cardiovascular Events in High Risk Subjects (SPIRE-1). (Accessed February 15, 2015, at https://clinicaltrials.gov/ct2/show/NCT01975376.) 78. Robinson JG, Gidding SS. Curing Atherosclerosis Should Be the Next Major Cardiovascular Prevention Goal. Journal of the American College of Cardiology 2014;63:2779-85.

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Regulation of cholesterol homeostasis (A), role of PCSK9 in cholesterol homeostasis (B), impact

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of statin therapy on PCSK9 (C) and PCSK9 inhibition using monoclonal antibodies (D).

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Abbreviations: LDLR, LDL receptor; mAb, monoclonal antibodies

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Figure 2

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The evolution of PCSK9 inhibition directed therapies Figure 3

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ODYSSEY LONG TERM. Calculated LDL cholesterol levels over time for alirocumab 150 mg every 2 weeks vs placebo in high risk patients treated with statins with/without other lipid-

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lowering therapy (A), percent change in calculated LDL cholesterol from baseline to week 24

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according to HeFH status (B), and baseline LDL cholesterol subgroup (C) (ITT analysis)

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Abbreviations: HeFH, heterozygous familial hypercholesterolemia; ITT, intent-to-treat; LDL, low-density lipoprotein; LLT, lipid-lowering therapy; LS, least-squares; Q2W, every 2 weeks; SE, standard error.

In panel A, values above data points indicate LS mean absolute LDL cholesterol levels, values below data points indicate LS mean % change from baseline. Values below chart indicate number of patients with LDL cholesterol values available for ITT analysis at each time point, ie, LDL cholesterol measurements taken both on-treatment and post-treatment (for patients who had discontinued study treatment but had returned to the clinic for assessments). Missing data were accounted for using a mixed effects model with repeated measures.

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LAPLACE-2. Evolocumab efficacy in patients randomized to a moderate or high intensity statin.

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In panel A, observed achieved LDL- cholesterol levels at baseline and mean of weeks 10 & 12 are shown. The horizontal dashed lines represent LDL-cholesterol goals/levels of interest.

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treatment group. Abbreviations: LDL-C, low-density lipoprotein cholesterol; wk, week. Panel B indicates mean percent change from baseline in key lipid parameters at the mean of

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weeks 10 and 12. Error bars indicate 95% confidence intervals. Baseline was measured post lipid-stabilization period and prior to administration of first dose of study drug. Abbreviations:

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ApoB, apolipoprotein B; EvoMab Q2W, evolocumab 140 mg every 2 weeks; EvoMab QM,

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evolocumab 420 mg monthly; HDL-C, high-density lipoprotein cholesterol; Q2W, every 2

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weeks; QD, daily; QM, every month.

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