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Abstract

Proprotein convertase subtilisin/kexin 9 (PCSK9) is part of the proteinase K subfamily of subtilases and plays a key role in lipid metabolism. It increases degradation of the low-density lipoprotein receptor (LDL-R), modulates cholesterol metabolism and transport, and contributes to the production of apolipoprotein B (apoB) in intestinal cells. Exogenous PCSK9 modifies the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase and enhances secretion of chylomicrons by modulating production of lipids and apoB-48. Statins increase PCSK9 messenger RNA expression and attenuate the capacity to increase LDL-R levels. Therefore, the inhibition of PCSK9 in combination with statins provides a promising approach for lowering low-density lipoprotein cholesterol (LDL-C) concentrations. This review will address new therapeutic strategies targeting PCSK9, including monoclonal antibodies, antisense oligonucleotides, small interfering RNAs, and other small molecule inhibitors. Further studies are still needed to determine the efficacy and safety of the PCSK9 inhibitors not only to decrease LDL-C but also to investigate the potential underlying mechanisms involved and to test whether these compounds actually reduce cardiovascular end points and mortality.

Keywords

proprotein convertase subtilisin/kexin 9 PCSK9 statins low-density lipoprotein receptor

Introduction

Familial hypercholesterolemia (FH) is a group of genetic defects characterized by extremely increased levels of serum cholesterol that contribute to a high risk of premature coronary heart disease (CHD).¹ Familial hypercholesterolemia has an autosomal dominant inheritance, the heterozygous form having inherited a mutated gene from 1 parent and the homozygous form 1 from each parent.² The most common cause of FH is a mutation in the low-density lipoprotein receptor (LDL-R) gene that determines increased levels of low-density lipoprotein cholesterol (LDL-C) at an early age.^3,4 The prevalence of both forms of FH is estimated between 1 in 200 and 1 in 500.⁵

Hypercholesterolemia is a major risk factor for coronary artery disease (CAD).^6
-8 The classes of statins, 3-hydroxy-3-methylglutaryl-coenzyme A inhibitor drugs that lower cholesterol by inhibiting its endogenous synthesis, have been well documented to contribute to prevention and treatment of CAD, diabetes, or stroke.^9,10 Statins reduce the regulatory pool of intracellular cholesterol and increase transcription of the LDL-R gene.^11
-13 Despite the widespread availability of statins, 16% to 53% of individuals globally fail to accomplish their lipid targets. Even higher rates of failure (79%) occur in patients with FH.^14,15

In 2003, proprotein convertase subtilisin/kexin 9 (PCSK9), also referred to as neural apoptosis-regulated convertase 1, was discovered as the ninth member of the proprotein convertase family.¹⁶ Proprotein convertase subtilisin/kexin 9, expressed in the liver, gut, renal, and nervous system, is responsible for the proteolytic growth of secretory proteins such as neuropeptides, prohormones, cytokines, growth factors, receptors and serum, and cell surface proteins.^16,17 Recently, it has been revealed that PCSK9 is also expressed in human atherosclerotic plaques.¹⁸ Proprotein convertase subtilisin/kexin 9 indirectly regulates the plasma LDL-C levels by controlling the number of LDL-R molecules expressed at the plasma membrane.^19,20 The physiological importance of PCSK9 was discovered by detection of point mutations in the PCSK9 gene that cause hyper- and hypocholesterolemia as a result of gain- and loss-of-function alleles, respectively.²¹ Gain-of-function mutations in the PCSK9 gene are related to autosomal dominant hypercholesterolemia and premature cardiovascular disease, while loss-of-function mutations of the PCSK9 gene are connected to lower LDL-C levels and a significantly decreased risk of CHD.^22
-24 Beyond effects on lipid metabolism, experimental studies described an implication of PCSK9 in visceral adipogenesis, glucose homeostasis, liver regeneration, and susceptibility to hepatitis C virus infection.^25
-27

Despite the fact that PCSK9 is expressed in arterial wall, kidney, and small intestine, studies in tissue-specific PCSK9 knockout mice indicated that nearly all circulating PCSK9s are produced by the liver.²⁸ Its expression in the liver could be pivotal for the coordination between peripheral fatty acid uptake and liver lipoprotein uptake, via regulation of surface levels of the very low-density lipoprotein receptor (VLDL-R) and LDL-R.²⁷ Various molecular forms of PCSK9 circulate in the plasma, a 62-kDa band, which represents a full-length protein minus prodomain and a 55-kDa fragment, which is a cleavage product and higher molecular weight forms composed of homo- or heteromultimers.²⁹ Several methods based on enzyme-linked immunosorbent assays (ELISAs) have been developed to quantify circulating levels of PCSK9 in human plasma.^29
-31 Until now, it still has not been proved whether the levels of circulating PCSK9 affect the levels of LDL-R protein in peripheral tissues.^17,27 Experiments were carried out to establish whether an excess level of PCSK9 was sufficient to induce PCSK9/LDL-R complex formation in human hepatocyte-like C3A cells. It was demonstrated using ELISA that having excess levels of PCSK9 is not enough to stimulate complex formation between PCSK9 and the LDL-R.¹⁹ Other studies have shown that serum levels of PCSK9 assessed by ELISA appear to be directly linked with both serum LDL-C and serum total cholesterol (TC) levels³² and positively associated with fasting glucose and insulin.³³

Statins have been proven to stimulate PCSK9 expression in mice and human liver hepatocellular cell lines (hepG2) by raising the nuclear translocation of sterol regulatory element-binding protein 2 (SREBP-2).³⁴ The bile acid-regulated hepatocyte nuclear factor 1 regulates the expression of PCSK9 necessary for the full transcriptional activity of SREBP-2 at the PCSK9 promoter.^35,36 Statins can raise the plasma levels of PCSK9 by 14% to 47%, depending on the type, the dose, and the duration of treatment with statins.³⁷ Additionally, it has been demonstrated that high levels of PCSK9 are linked with cardiovascular incidents in patients with CHD having reduced statin consumption.³⁸ On the other hand, PCSK9 inhibition has potential for harm because PCSK9 plays different roles in pathways of neuronal apoptosis, regulation of sodium channels, pancreatic islet cell function, and nervous system development.³⁹ Experimental and clinical studies focused on PCSK9 as a potential therapeutic target in the treatment of conditions determined by hypercholesterolemia are reviewed.

Search Strategy

All available data provided by the electronic databases PubMed/MEDLINE (1966 to April 2014), EMBASE and SCOPUS (1965 to April 2014), and DARE (1966 to April 2014) were used in the survey. The following search terms were selected for research: PCSK9, PCSK9 levels, secretion of PCSK9, PCSK9 and cardiovascular diseases, PCSK9 and lipid metabolism, PCSK9 and hypoglycemic therapy, PCSK9 and hypolipidemic therapy, PCSK9 in animal studies, PCSK9 in human studies, PCSK9 and clinical trials, PCSK9 and monoclonal antibodies, and PCSK9 and antisense oligonucleotides.

The Role of PCSK9 in Lipid Metabolism

In humans, LDL is responsible for the transport of about 60% to 70% of the TC.⁴⁰ The liver secretes and produces VLDL, a lipoprotein rich in triglyceride (TG) that contains a single copy of apolipoprotein B-100 (apoB-100) and various quantities of apolipoprotein E (apoE) and apolipoprotein C (apoC).^41,42 The LDL-C derives from the metabolism of VLDL and the major protein constituent of LDL is apoB-100.⁴³ In peripheral tissues such as adipose tissue and muscle, as a consequence of hydrolysis of TG by the action of lipoprotein lipase, VLDL particles are modified to TG-poor, apoE-rich VLDL remnants.⁴⁴ In the liver, most VLDL remnants are quickly cleared by hepatocytes, while particles that escape clearance suffer further hydrolysis to produce LDL.^42,45 The number of LDL-Rs present on the surface of hepatocytes determines the plasmatic LDL-C levels. They bind plasma LDL to the hepatocyte surface and mediate LDL uptake. Low-density lipoprotein is eliberated into the endosomal system, and the LDL-R is reused to bind new LDL particles.^46,47

Proprotein convertase subtilisin/kexin 9, a known posttranscriptional regulator of LDL-R expression, is produced in the endoplasmic reticulum (ER) as a precursor form of 692 amino acids that includes a signal peptide (residues 1-30).⁴³ Proprotein convertase subtilisin/kexin 9 is characterized by a 3-domain structure and a catalytic triad, consisting of amino acid residues asparagine, histidine, and serine, which is implicated in substrate binding and catalysis.⁴²

The role of PCSK9 in lipid metabolism is confirmed by 3 suggested hypotheses. The first one involves a heterozygous missense mutation of PCSK9 that determines autosomal dominant hypercholesterolemia through a gain-of-function mechanism.²² The second one implies that PCSK9 messenger RNA (mRNA) levels contribute to increased intracellular cholesterol.⁴⁸ The third and most important remark was that nonsense mutations in PCSK9 are associated with 15% to 30% reductions in LDL-C, with significant reductions in cardiovascular risk.^24,49 The mechanism by which PCSK9 regulates LDL-R degradation is not fully resolved, but it seems to involve different intracellular and extracellular pathways.^50,51

In the hepatocytes, PCSK9 goes through an obligatory autocatalytic cleavage in the ER before being produced, but its catalytic activity is not necessary for its capacity to regulate LDL-R surface expression.⁵² Autocatalytic activity appears to be needed for PCSK9 to leave the ER, but it is unclear whether it is needed for PCSK9-stimulated LDL-R degradation.⁵³ It is indicated that PCSK9 might cleave the LDL-R directly or might function indirectly by clipping another protein that promotes degradation of the LDL-R.⁵³ The LDL-R also appears to control the intracellular trafficking of PCSK9 from the ER to downstream sites, but the mode of interaction between the LDL-R and PCSK9 appears to vary in the ER and at the cell surface.^30,54

Extracellularly, PCSK9 binds to the LDL-R, which channels it inside to the lysosomal compartment for destruction.⁴⁷ Besides its function in regulating LDL-R levels, PCSK9 as well regulates cellular levels of 2 other LDL-R family members, the VLDL-R and the apolipoprotein E receptor 2 (apoE-R2).⁵⁵

Current Strategies Targeting PCSK9

Statins have been shown to induce an increase in PCSK9 expression, and it has been assumed that inhibiting the PCSK9-LDL-R interaction may also enhance the lipid-lowering efficacy of statins.⁵⁶ Many studies have shown that inhibition of PCSK9 together with statins offers promising therapeutic applications in the control of PCSK9-regulated pathologies.⁵⁷ At present, the strategy to inhibit PCSK9 includes (1) inhibition of PCSK9-LDL-R interaction, using monoclonal antibodies (mAbs) or mimetic peptides and adnectins, (2) inhibition of PCSK9 synthesis using gene silencing agents such as antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) oligonucleotides, and (3) inhibition of PCSK9 autocatalytic processing using small molecule inhibitors (Table 1). Mimetic peptides, adnectins, and mAbs exclusively target circulating PCSK9 and therefore its extracellular function, whereas gene silencing agents target both PCSK9 intra- and extracellular functions.

Table 1.

PCSK9-Targeted Therapies in Development.

Molecule	Company	Description	Phase
1D05-IgG2	Merck	Monoclonal antibody	Preclinical
1B20	Merck
J10, J16, J17	Pfizer
EGF-AB peptide fragment	Schering-Plough	Mimetic peptide	Preclinical
LDL-R (H306Y) subfragment	US National Institutes of Health
LDL-R DNA construct	US National Institutes of Health
BMS-962476	Bristol-Myers Squibb/Adnexus	Adnectin	Preclinical
SX-PCSK9	Serometrix	Small molecule inhibitor	Preclinical
TBD	Shifa Biomedical Corporation	Small molecule inhibitor	Preclinical
	Idera Pharmaceuticals	Antisense oligonucleotide	Preclinical
ISIS 394814	Isis Pharmaceuticals, Inc
SPC4061	Santaris-Pharma
SPC5011	Santaris-Pharma
SPC5001	Santaris-Pharma	Locked nucleic acid antisense oligonucleotide	Phase 1
ALN-PCS02	Alnylam Pharmaceuticals	Small interfering RNA oligonucleotide	Phase 1
LGT209	Novartis Pharmaceuticals	Monoclonal antibody	Phase 1
RN316 (PF-04950615)	Pfizer	Monoclonal antibody	Phase 1
LY3015014	Eli Lilly Pharmaceutical	Human monoclonal antibody	Phase 1
LGT209	Novartis Pharmaceuticals	Monoclonal antibody	Phase 2
Bococizumab (also known as RN316 or PF-04950615)	Pfizer		Phase 2
LY3015014	Eli Lilly Pharmaceutical		Phase 2
Alirocumab (also known as REGN727 and SAR236553)	Regeneron/Sanofi	Human monoclonal antibody	Phase 2
Evolocumab (AMG145)	Amgen	Human monoclonal antibody	Phase 2
Alirocumab (also known as REGN727 and SAR236553)	Regeneron/Sanofi	Fully human IgG1 mAb	Phase 3
Evolocumab (AMG145)	Amgen	Fully human IgG1 mAb
Bococizumab (also known as RN316)	Pfizer	Humanized IgG1 mAb
RG-7652	Roche	Monoclonal antibody

Abbreviations: LDL-R, low-density lipoprotein receptor; PCSK6, proprotein convertase subtilisin/kexin 9; IgG, immunoglobulin G.

Inhibition of PCSK9-LDL-R Interaction

Monoclonal Antibodies

Monoclonal antibodies are an emerging class of drugs developed by different pharmaceutical companies, currently undergoing experimental and clinical studies. The inhibition of PCSK9 function by therapeutic mAbs was shown to successfully lower LDL-C levels in different experimental and clinical studies.⁵⁸ Proprotein convertase subtilisin/kexin 9 antibody-based therapies require bimonthly or monthly dosing injection regimens. The antibodies change the affinity interaction between the natural gain-of-function mutant of PCSK9 and the LDL-R in both cell-free and cell-based assays.⁵⁹

Experimental studies

The first studies using antibodies that block the interaction between PCSK9 and LDL-R and inhibit the PCSK9-mediated LDL-R degradation have been performed in cell culture systems. These antibodies restored LDL uptake in HepG2 cells treated with exogenous PCSK9 and in HepG2 cells engineered to overexpress recombinant PCSK9.⁵⁹

Recently, a new PCSK9 antibody, mAb1, was identified and biophysically characterized. Using X-ray methods, it has been shown that mAb1 binds module 1 of the C-terminal domain (CTD) of PCSK9 and blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Monoclonal antibody 1 is also effective in lowering serum levels of LDL-C in cynomolgus monkeys in vivo. The study reported that blocking CTD is sufficient to partially inhibit PCSK9 function.⁵⁸ The clinical efficacy of mAbs is based on 1 of these 3 functionalities: (1) target-specific binding by the Fab domain, (2) interaction of the Fc domain with cell-surface receptors, and (3) deposition of complement on multimeric immune complexes between the mAb and the target with consequent activation of complement-dependent cytotoxicity.⁵⁶

Merck Research Laboratories (Whitehouse Station, New Jersey) identified 1D05-IgG2, a PCSK9 antibody that structurally mimics the epidermal growth factor A (EGF-A) domain of LDL-R. The 1D05-IgG2 antibody can block the inhibitory effects of wild-type PCSK9 and 2 gain-of-function human PCSK9 mutants, S127R and D374Y. In a transgenic mouse model (CETP/LDL-R-hemi), 1D05-IgG2 antibody reduced plasma LDL-C to 40% and increased hepatic LDL-R protein levels 5-fold. In healthy rhesus monkeys, despite its relatively short half-life of 3.2 days, it also effectively reduced LDL-C to 20% to 50% for over 2 weeks. The LDL-C-lowering effect was shown to be mediated by reducing the amount of PCSK9 that could bind to the LDL-R.⁶⁰ 1B20, another novel antibody developed recently by Merck & Co, binds to PCSK9 with subnanomolar affinity and antagonizes PCSK9 function in vitro. Administered for 14 days in 2 successive doses, 1B20 induced dose-dependent reductions in LDL-C (≥25%), increased PCSK9 levels bound to antibody in plasma, and reduced mRNA levels of SREBP-regulated genes PCSK9 and LDL-R in the liver.⁶¹

Pfizer-Rinat (New York, New York) developed a humanized mAb that recognizes the LDL-R binding domain of PCSK9.⁶² In vivo, J10 effectively reduces serum TC in C57BL/6 mice fed normal chow.⁶² Mouse antibody J10 was humanized and affinity matured to antibody J16 by using standard humanization and affinity maturation strategies.⁶³ The J16 antibody binds to a 3-dimensional epitope mapping to the catalytic domain of PCSK9. In Cynomolgus monkeys, J16 injected intravenously (IV; one 3 mg/kg dose, half-life of 2.3 days) decreased LDL-C levels by 70%. The effect was maintained for 10 days. When administered to monkeys fed a high-fat diet, similar results were noticed (64% decrease in LDL-C at the 3 mg/kg dose).⁶⁴ In complex with PCSK9, J16 was internalized and degraded in lysosomes by a target-mediated clearance pathway.⁶⁵ These antibodies inhibit PCSK9-LDL-R binding and PCSK9-mediated downregulation of LDL-R in vitro.⁶²

Eli Lilly (Indianapolis, IN) developed a mAb against the PCSK9 catalytic domain that inhibits PCSK9 binding to LDL-R and its degradation in HepG2 cells. After a single intravenous dose of 5 mg/kg administered to healthy Cynomolgus monkeys, a 60% reduction in LDL-C was observed. The LDL-C reduction was maintained below baseline levels for approximately 8 weeks. Subcutaneous administration was as effective in lowering LDL-C levels as intravenous injection.⁶⁶

Amgen Inc (Thousand Oaks, California) developed a humanized anti-PCSK9 mAb1 that blocks the PCSK9:LDL-R interaction and prevents LDL-R degradation in HepG2 cells and in vivo in mice and nonhuman primates.⁶⁷ New mAbs against PCSK9 currently under investigation for potential use in humans are PF-04950615/RN-316 (Pfizer; phase 2), LGT 209 (Novartis, Basel, Switzerland; phase 2), and MPSK3169A (Roche-Genentech, phase 2, South San Francisco, California).⁵⁶

Clinical studies

Phase 1 studies

Regeneron Pharmaceuticals, Inc (Tarrytown, New York)/Sanofi SA (Paris, France) conducted the first phase I studies in humans with REGN727/SAR236553, a mAb against PCSK9, now known as alirocumab. REGN727 decreased LDL-C levels in 3 randomized double-blind placebo-controlled phase I studies.⁶⁸ The first 2 single-dose studies were done in healthy volunteers, one by intravenous and the other by subcutaneous administration. A dose-dependent decrease in LDL-C levels was observed (28%-65% and 32%-46%, respectively). The third study was multiple dose, performed in patients with FH and non-FH on atorvastatin. The control group of 10 patients with non-FH was on a modified diet alone. The drug was administered subcutaneously (SC) on days 1, 29, and 43. In statin-treated patients with FH and non-FH, subcutaneous REGN727 reduced LDL-C levels by an additional 40% to 60%, depending on dosage. REGN727 decreased LDL-C to comparable levels in patients with non-FH with or without statin therapy.

Amgen Inc developed AMG145 (Evolocumab), an investigational human mAb that inhibits PCSK9. AMG145 was studied in 2 randomized, double-blind, placebo-controlled, ascending-dose phase 1 studies in healthy volunteers and patients with hypercholesterolemia on statins. Healthy adults (phase 1a) were randomized to 1 dose of AMG145: 7, 21, 70, 210, or 420 mg SC; 21 or 420 mg IV; or matching placebo. In a phase 1b dose-escalation study, repeated doses of AMG145 were administered SC as add-on therapy to statins to patients with hypercholesterolemia (FH or non-FH; low to moderate dose or high dose). Overall incidence of treatment-emergent adverse effects was similar in AMG145 versus placebo groups: 69% versus 71% (phase 1a); 65% versus 64% (phase 1b). Phase 1 studies revealed that AMG145 significantly reduced serum LDL-C in healthy volunteers and in patients with hypercholesterolemia on statin, including those with heterozygous FH or taking the highest doses of atorvastatin or rosuvastatin, with an overall adverse effects profile similar to placebo.⁶⁹

Novartis International AG/KaloBios Pharmaceuticals, Inc (South San Francisco, California) developed LGT209, a humanized mAb derived from a mouse precursor that binds to the C-terminal residues 680 to 692 of PCSK9.⁷⁰ LGT209 poorly disrupts the PCSK9 and LDL-R interaction but inhibits LDL-R degradation and restores LDL-C uptake in HepG2 cells. LGT209 was tested in a phase I study by subcutaneous injections in healthy volunteers with increased cholesterol and in patients with hypercholesterolemia on statins.⁷¹

Phase 2 studies

AMG145 has been tested in different 12-week phase 2 studies. In Goal Achievement After Utilizing an anti-PCSK9 Antibody in Statin-Intolerant Subjects (GAUSS), 236 patients intolerant to ≥1 statins due to side effects were treated for 12 weeks with evolocumab at varying doses. The study demonstrated a 40% to 60% LDL-C reduction and only minimal side effects.⁷²

Trial evaluating PCSK9 antibody in Subjects with LDL Receptor Abnormalities (TESLA), a phase 2 open-label, single-arm, multicenter 12-week trial, evaluated 8 patients with homozygous FH on previous stable drug therapy for 4 weeks or more. Patients were given 420 mg subcutaneous evolocumab once monthly for a minimum of 12 weeks, followed by every 2 weeks for another 12 weeks. The primary end point was the percentage of reduction from baseline in LDL-C at week 12. The study demonstrated significant and dose-related LDL-C reduction in patients with homozygous FH having defective LDL-R activity but no reduction in those who were receptor negative.⁷³

The Monoclonal Antibody against PCSK9 to Reduce Elevated LDL-C in Patients Currently Not Receiving Drug Therapy for Easing Lipid Levels (MENDEL) trial is a randomized, double-blind, placebo-controlled, phase 2 study. The study tested AMG145 as monotherapy in 406 patients with hypercholesterolemia from 52 centers in Europe, the United States, Canada, and Australia.⁷⁴ The patients received AMG145 70, 105, or 140 mg every 2 weeks or 280, 350, and 420 mg every 4 weeks versus placebo or ezetimibe 10 mg/d. After 12 weeks, the modification from baseline in LDL-C varied from −39% to −51% with AMG145 compared with −3.7% and +4.5% for the 2 placebo regimens (every 2 weeks and every 4 weeks, respectively) and +15% with ezetimibe (P < .0001 for all doses vs placebo or ezetimibe).⁷⁴

The Study of LDL-Cholesterol Reduction Using a Monoclonal PCSK9 Antibody in Japanese Patients with Advanced Cardiovascular Risk (YUKAWA) is a 12-week, randomized, double-blind, placebo-controlled, phase 2 study that evaluated the efficacy and safety of evolocumab in 310 statin-treated Japanese patients at high cardiovascular risk and hypercholesterolemia. The study showed that evolocumab significantly reduced LDL-C and was well tolerated by Japanese patients at high cardiovascular risk with hypercholesterolemia on stable statin therapy.⁷⁵

Reduction of LDL-C with PCSK9 Inhibition in Heterozygous Familial Hypercholesterolemia Disorder (RUTHERFORD) is a multicenter phase 2, double-blind, randomized, placebo-controlled trial, evaluating the efficacy and safety of AMG145 in 168 patients with heterozygous FH. AMG145 given every 4 weeks generated fast and significant decrease in LDL-C in patients with heterozygous FH on intensive statin use, with or without ezetimibe. The treatment was well tolerated, with few adverse events.⁷⁶

The Open-Label Study of Long-Term Evaluation against LDL-C (OSLER) randomized trial evaluated the efficacy and safety of 52 weeks’ administration of evolocumab (AMG145) in 1104 patients with hypercholesterolemia from 4 separate phase 2 dose-ranging studies that lasted 12 weeks: patients with statin intolerance from GAUSS, patients on statins from LDL-C Assessment with PCSK9 Monoclonal Antibody Inhibition Combined with Statin ThErapy (LAPLACE)-thrombolysis in myocardial infarction 57,⁷⁷ patients with FH from RUTHERFORD, and patients with low-risk hypercholesterolemia not on statins from MENDEL trials.⁷⁸ Adverse events and serious adverse events took place in 81.4% and 7.1% of the evolocumab-plus-standard-of-care group patients and in 73.1% and 6.3% of the standard-of-care-group patients, respectively. Musculoskeletal and connective tissue disorders were observed in 33.0% and 34.7% of patients when LDL decreased below 50 or 25 mg/dL, respectively, compared with ∼25% in those with levels of ≥50 mg/dL. Injection site reactions occurred in ∼5% of patients with evolocumab. Evolocumab dosed every 4 weeks showed continued efficacy, safety, and tolerability over 1 year of treatment.^78,79

A pooled analysis of 1359 patients in 4 phase 2 trials studied the efficacy and safety of evolocumab in patients with hyperlipidemia on various background lipid therapies. The trials randomized 1359 patients to various doses of subcutaneous evolocumab every 2 weeks (Q2W) or 4 weeks (Q4W), placebo, or ezetimibe for 12 weeks. Evolocumab, dosed either Q2W or Q4W, showed significant changes in other atherogenic and antiatherogenic lipoproteins and reduced LDL-C levels and was well tolerated over the 12-week treatment period.⁸⁰

In a 12-week phase 2 study of patients with LDL-C levels ≥100 mg/dL on a stable dose of atorvastatin (10, 20, or 40 mg/d), SAR236553/REGN727 (doses 50-150 mg) as add-on given SC every 2 weeks resulted in a 40% to 72% reductions in LDL-C.⁶⁸

Roche/Genentech created RG7652 (MPSK3169A), an antibody against the catalytic domain of PCSK9. It was tested in a phase 2 randomized, placebo-controlled, double-blind trial in patients with CAD or high risk of CAD. MPSK3169A was given SC every 4 weeks over a 24-week period and demonstrated a good profile of safety and remarkable cholesterol-lowering effects.⁸¹ The humanized antibody LGT209, created by Novartis International AG/KaloBios Pharmaceuticals, Inc, was recently tested.

Phase 3 studies

MENDEL-2, a phase 3 trial, evaluated safety, tolerability, and efficacy of evolocumab in 614 patients with high cholesterol (LDL-C ≥ 100 mg/dL and < 190 mg/dL) who were not receiving lipid-lowering therapy. The patients were randomized to 1 of the 6 treatment groups to compare 2 dosing regimens of evolocumab (140 mg every 2 weeks or 420 mg monthly) with placebo and ezetimibe (10 mg daily). The study demonstrated the safety across treatment groups. The most common adverse events (>2% in the evolocumab combined group) were headache, diarrhea, nausea, and urinary tract infection.⁸²

The Durable Effect of PCSK9 Antibody Compared with Placebo Study (DESCARTES) is a phase 3, randomized, multicenter, double-blind trial that evaluated safety, tolerability, and efficacy in 901 patients with cardiovascular risk and high LDL-C. Evolocumab significantly reduced LDL-C, from baseline at week 52 compared to placebo. At week 12, LDL-C reduction was comparable with the efficacy at week 52.⁸³ The mean percentage of reduction in LDL-C was consistent with the results observed in the 52-week analysis of the phase 2 OSLER study.

The RUTHERFORD-2 was a phase 3 trial done in 329 patients with heterozygous FH on a stable dose of statin and other lipid-lowering therapies, which showed that subcutaneous evolocumab significantly reduced mean LDL-C by 59% to 66% from baseline compared to placebo (P < .001). Some adverse events were more often seen with evolocumab (≥2% in the evolocumab combined group and ≥2% compared to placebo), namely nasopharyngitis, contusion, back pain, nausea, influenza, and myalgia. These data suggest that evolocumab may therefore offer a new treatment option as an add-on therapy to existing lipid-lowering medication, in patients with heterozygous FH. Recently, the positive results from these phase 3 separate studies, MENDEL-2, DESCARTES, and RUTHERFORD-2 showed that evolocumab significantly reduces LDL-C by 55% to 66% in patients with high cholesterol levels.

In statin-intolerant patients with hypercholesterolemia, GAUSS-2, a phase 3, randomized, double-blind, ezetimibe-controlled trial, evaluated the effects of 12 weeks of evolocumab 140 mg every 2 weeks or 420 mg every month.⁸⁴ A statistically substantial decline in LDL-C between 37% and 39%, compared to ezetimibe, was noted. GAUSS-2 involved patients with intolerance to ≥2 statins.

The LAPLACE-2, a phase 3, randomized, double-blind, placebo-, and ezetimibe-controlled trial, evaluated evolocumab in combination with statin therapy as compared to placebo and ezetimibe in 1896 patients with primary hypercholesterolemia and mixed dyslipidemia. It was observed that safety was similar between the different treatment groups. The study demonstrated a statistically significant reduction in LDL-C of 55% to 76% compared to placebo when used in combination with statin therapy in patients with high cholesterol. No adverse events occurred in more than 2% of the evolocumab combined group.⁸⁵

The TESLA, a phase 3, double-blind, randomized, placebo-controlled, multicenter 12-week trial, evaluated the safety, tolerability, and efficacy of evolocumab compared to placebo in 49 patients with homozygous FH. The patients were on a stable dose of statin or other lipid-lowering medication. Subcutaneous evolocumab 420 mg was administered monthly versus placebo. The most common observed adverse events in the evolocumab group were upper respiratory tract infection, influenza, gastroenteritis, and nasopharyngitis.⁸⁶

Program to Reduce LDL-C and Cardiovascular Outcomes Following Inhibition of PCSK9 in Different Populations (PROFICIO) is a large clinical trial program that will evaluate evolocumab in 20 phase 3 clinical trials, with a total planned enrollment of approximately 30 000 patients. Evolocumab will be administered every 2 weeks and monthly in different patient populations with hyperlipidemia as stand-alone treatment (THOMAS-1, THOMAS-2, and MENDEL-2), in combination with statins (LAPLACE-2 and YUKAWA-2), or statin intolerance (GAUSS-2 and GAUSS-3), and in patients with heterozygous (RUTHERFORD-2 and TAUSSIG) and homozygous (TESLA and TAUSSIG) FH.⁸⁴

The Further Cardiovascular Outcomes Research with PCSK9 inhibitors in subjects with elevated risk (FOURIER) phase 3 trial will compare evolocumab in combination with statin therapy with placebo and statin therapy. The primary end point of the study will be reduction of recurrent cardiovascular event. The study will enroll approximately 22 500 patients with cardiovascular disease at increased risk of cardiovascular events over 5 years. The purpose of this study is to verify whether additional LDL reduction with PCSK9 antibodies will contribute to reduce major cardiovascular events better than statin therapy alone.⁸⁷

Global Assessment of Plaque Regression with a PCSK9 Antibody as Measured by Intravascular Ultrasound (GLAGOV), a phase 3, randomized, double-blind, multicenter, placebo-controlled, parallel-group trial, will evaluate the effect of evolocumab on atherosclerotic disease burden as measured by intravascular ultrasound in approximately 950 patients with CAD undergoing cardiac catheterization taking lipid-lowering therapy, in week 78.⁸⁸

Evaluation of Cardiovascular Outcomes after an Acute Coronary Syndrome during Treatment with alirocumab SAR236553 (REGN727; ODYSSEY) is a randomized, double-blind, placebo-controlled, parallel-group trial. The ODYSSEY currently comprises a number of clinical trials undergoing in 2000 study centers globally and enrolling more than 23 500 patients with hypercholesterolemia who had an acute coronary syndrome. The ODYSSEY is designed to compare the effect of alirocumab with placebo on the occurrence of cardiovascular events over a 5-year period. Patients will be on lipid-lowering therapy and dietary management but not having reached their LDL-C target.⁸⁹ The first results showed that alirocumab was much more effective in LDL-C reduction in patients with hypercholesterolemia when compared with ezetimibe (ODYSSEY MONO trial).⁹⁰

The ODYSSEY MONO is a 24-week, phase 3 study that included 103 patients on 75 mg of alirocumab SC every 2 weeks. The dose was increased at week 12 to 150 mg if the measurement of LDL-C at week 8 was >70 mg/dL. In patients treated with alirocumab, the mean reduction in LDL-C was 47.2% versus 15.6% reduction for patients treated with ezetimibe 10 mg. Adverse events were observed in 78.4% of the ezetimibe group and 69.2% of the alirocumab group, the most common reported with alirocumab being nasopharyngitis, influenza, and upper respiratory tract infections. The companies are currently conducting 11 other phase 3 studies (ODDYSSEY OUTCOMES) with alirocumab in different patient populations, drug combinations, and dosing regimens.⁹⁰

Bococizumab, the proposed generic name for RN316, significantly reduced LDL-C in a phase 2b, 24-week, randomized, placebo-controlled, dose-ranging study in 354 statin-treated patients. The study evaluated 2 dosing regimens twice monthly (bococizumab 50, 100, or 150 mg) and once monthly (bococizumab 200 or 300 mg). Bococizumab twice and once monthly dosing regimens were associated with significant placebo-adjusted reductions in LDL-C at week 12, with the greatest reductions seen with 150 mg for the twice monthly regimen and 300 mg for the once monthly regimen.⁹⁰

To date, clinical trials conducted with mAbs against PCSK9 have shown that these antibodies are efficacious, with an excellent safety profile, but their long-term impact on cardiovascular events and potential side effects (including neurocognitive adverse events as was suggested by FDA in March this year) is currently under investigation.

Mimetic peptides and adnectins

Proprotein convertase subtilisin/kexin 9 function can be inhibited by peptides mimicking the EGF-A domain of the LDL-R that interacts with PCSK9 at the plasma membrane.^52,91,92 A synthetic EGF-A peptide that binds the LDL-R inhibits PCSK9-mediated degradation of LDL-R in a dose-dependent manner in HepG2 cells.⁹¹

It has been shown that an anti-PCSK9 antigen binding fragment can interrupt the connection between PCSK9 and the LDL-R by restoring cellular LDL uptake.^44,52 The lack of the CTD may damage the potential of PCSK9 to internalize into cells and inhibit LDL uptake.⁴⁴ A substitute is to use PCSK9 peptide sequences that are too short to promote LDL-R degradation but long enough to compete with full-length PCSK9.⁹²

An anti-PCSK9 Fab, 1G08, with subnanomolar affinity for PCSK9, was observed using a phage display library.⁴⁴ The PCSK9-dependent inhibitory effects on LDL uptake were evaluated by measuring LDL uptake in HEK293 and HepG2 cells.⁴⁴ 1G08 did not modify the PCSK9-LDL-R connection but inhibited the internalization of PCSK9 in these cells. Mutagenesis studies have reported that 1G08 Fab binds to a region of β-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 CTD.⁴⁴ The PCSK9 CTD inhibits LDL-R function mostly through cellular uptake of PCSK9 and LDL-R complex. 1G08 Fab is a useful new tool for delineating the mechanism of PCSK9 uptake and LDL-R degradation.⁴⁴

Adnectins are a new family of therapeutic-engineered target-binding proteins created to bind with high affinity and specificity to targets as high as those of antibodies, but more easily manipulated genetically and compatible with bacterial expression systems. Adnectins differ from antibodies in the primary sequence and have a simpler, single-domain structure without disulfide bonds.⁹³ BMS-962476 (Bristol-Myers Squibb/Adnexus, Waltham, Massachusetts) is an adnectin currently being tested in a phase 1 trial.

Inhibition of PCSK9 Synthesis by Gene Silencing

Another method to inhibit PCSK9 synthesis is by using gene silencing agents such as ASO and siRNA oligonucleotides. RNA interference (RNAi)-based gene silencing agents have been used in human studies. Ongoing clinical trials will provide alternatives to traditional small molecule therapies.⁹⁴

Antisense oligonucleotides

Antisense oligonucleotides are short single strands of DNA comprising 12 to 20 nucleotides. The term antisense refers to the relationship between an oligonucleotide and its complementary target nucleic acid. The oligonucleotides bind directly to mRNA modulating the amount of mRNA made from the gene, or to a selected DNA portion forming a triple-stranded structure.⁹⁵ The binding prevents translation of mRNA, thereby preventing translation and the production of the respective proteins.⁹⁶ Antisense DNA and siRNA produce PCSK9 mRNA degradation.

Locked nucleic acid (LNA) are oligonucleotides consisting of 1 or more nucleotide building blocks in which the ribose moieties in the C3′-endo (β-d-LNA) or the C2′-endo (α-l-LNA) conformation are fixed by an extra methylene bridge.⁹⁷ Locked nucleic acid technology, based on nucleic acid analogs with a steady, unchanged conformational status, can be used to produce ASOs for PCSK9.⁹⁸ The benefits of LNA-based drugs involve higher specificity and affinity to targets providing enhanced potency and favorable pharmacokinetic and tissue-penetrating qualities that allow systemic delivery of these drugs.

The first evidence for the efficacy of an LNA ASO that targets both human and mouse PCSK9 was described by Gupta et al. The investigators employed human hepatocyte-derived cell lines HepG2 and HuH7 and a pancreatic mouse β-TC3 cell line known to express high endogenous levels of PCSK9.⁹⁹

Isis Pharmaceuticals developed ISIS 394814, a second generation ASO inhibitor targeting murine PCSK9, to determine its potential as a lipid-lowering agent. After administration of a PCSK9 ASO to high fat-fed mice for 6 weeks, TC was reduced by 53%, and LDL reduced by 38%.¹⁰⁰ This was the first demonstration that injectable ASO inhibitors could be used to reduce PCSK9 hepatic levels.¹⁰¹

Another 2 LNA ASOs targeting PCSK9 produced a 50% reduction in circulating LDL-C in nonhuman primates after a loading dose (20 mg/kg) and 4 weekly maintenance doses (5 mg/kg). A reduction by 85% in PCSK9 mRNA and serum PCSK9 protein was observed. The compounds were well tolerated with no observed toxic effects.¹⁰² Therefore, LNA ASOs targeting PCSK9 are probable complements to statins in controlling increased LDL-C levels.¹⁰²

Different LNA ASOs, such as SPC4061, SPC4955, and SPC5001, were developed by Santaris-Pharma for patients as multiple approaches are needed to lower high cholesterol levels.¹⁰³ Among the nucleic acid-based therapies, SPC5001 (Santaris Pharma A/S), a LNA-based inhibitor, and BMS-844421 (Bristol-Myers Squibb), an antisense RNA therapy, have completed clinical trials.

Small Interfering RNA Oligonucleotides

Alnylam Pharmaceuticals developed a new technology of gene silencing using siRNA, capable of targeting murine, rat, nonhuman primate, and human PCSK9. The siRNA was incorporated into injectable lipidoid nanoparticles (LNPs) to minimize toxicity and IV infused in rats, mice, and monkeys. The effects of PCSK9 silencing lasted for 3 weeks after a single intravenous administration and determined a 50% to 70% reduction in PCSK9 mRNA in mouse and rat liver. In Cynomolgus monkeys, LDL-C levels were decreased by more than 50% and the effects lasted for nearly 21 days.¹⁰⁴ The siRNAs silenced the human PCSK9 transcript by >70% and substantially lowered PCSK9 plasma protein levels in transgenic mice expressing human PCSK9.⁷⁰

The Frank-Kamenetsky study utilized a single dose of 5 mg/kg of LNP-PCS-A2 or LNP-PCS-B2 in nonhuman primates, which conducted to a considerable decrease in LDL-C in monkeys. The effect had appeared on day 3 after the single dose and LDL-C levels returned to baseline over ∼14 days (for LNP-PCS-A2) and ∼21 days (LNP-PCS-B2). A ∼60% decrease in LDL-C was observed that lasted for 2 to 3 weeks after a single IV injection. Furthermore, they showed that the lowering of LDL-C in the treated animals correlated with reduced circulating apoB levels.^57,104

Single IV doses of siRNA successfully decreased serum TC levels in rats or mice transgenic for human PCSK9, without affecting liver TG content.^57,104 The above-mentioned results confirm that PCSK9 targeting with RNAi can specifically lower LDL-C, confirming the need for development of PCSK9-lowering agents.¹⁰⁴

A randomized, single-blind, placebo-controlled, phase 1 trial in healthy volunteers studied the effect of ALN-PCS02 (an RNAi drug by Alnylam Pharmaceuticals) on both PCSK9 synthesis and serum LDL-C levels.^56,105 The patients received 1 dose of intravenous ALN-PCS (with doses ranging from 0.015 to 0.400 mg/kg) or placebo using a 3:1 ratio computer algorithm. The safety and tolerability of ALN-PCS02 were the primary end point and pharmacokinetic characteristics of ALN-PCS, and its pharmacodynamic effects on PCSK9 and LDL cholesterol were the secondary end points of the study. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (79% vs 88%). This study is the first to show an RNAi drug being used to affect LDL cholesterol in human beings.¹⁰⁵

Inhibition of PCSK9 Autocatalytic Processing by Small Molecule Inhibitors

Intracellular inhibitors of PCSK9 catalytic activity by small molecule inhibitors seem to be another promising strategy since autocatalytic processing of PCSK9 is required for secretion of the protein from the ER. Now being tested are synthetic mimetics to enhance activity and understanding at these induced fit interfaces. It seems that the oral administration of a small inhibitory molecule is difficult since PCSK9 undergoes autocatalytic cleavage activity in the hepatocyte.

Proprotein convertase subtilisin/kexin 9 is unique because the mature enzyme exhibits a cleaved prosegment complexed with the catalytic subunit and has no protease activity toward other substrates. A recent study suggested a novel strategy for the design of PCSK9 inhibitors. Since the in trans presence of the PCSK9 prosegment was shown to interfere with the action of PCSK9 on the LDL-R and the prosegment cannot be secreted alone, a chimeric protein using the Fc-region of human IgG1 fused to the PCSK9 prosegment was conceived. The expression of this Fcpro-fusion protein in HEK293 and HepG2 cells determined the secretion of a protein that binds PCSK9 and inhibits its action on the LDL-R. Fcpro interacts with the prosegment and/or catalytic subunit of the prosegment-PCSK9 complex and allosterically modulates its function.¹⁰⁶

Serometrix (East Syracuse, New York) developed a novel family of small molecule inhibitors, SX-PCK9, for the PCSK9 interaction with LDL-R for the potential treatment of FH. Initial in vitro assays have shown specific antagonist activity against interaction sites.⁶⁶ Shifa Biomedical Corporation created TBD, another small molecule PCSK9 modulator, which is currently undergoing preclinical tests.

Conclusions

Proprotein convertase subtilisin/kexin 9 inhibitors are currently under evaluation in different clinical outcome studies and are anticipated to find wide application either as monotherapy or as an adjunct to statins. Recent studies indicated that PCSK9 inhibition by mAbs, gene silencing, gene-repair techniques, silencing RNA, genetic knockdown of PCSK9, or mimetic peptides could be effective and seems to be safe as treatment modalities in patients with hypercholesterolemia intolerant to statins and associated cardiovascular diseases. Currently, the most advanced pharmacological PCSK9 inhibitors are mAbs, defining the direction for therapeutic strategy. In case of positive outcomes, we should expect to have them in use in 2016.

However, further studies are still needed on the efficacy and safety of the PCSK9 inhibitors not only to decrease LDL-C but also to investigate the potential underlying mechanisms involved and to test whether these compounds actually reduce cardiovascular end points and mortality. These therapies might be of major benefit to patients with FH and high-risk patients (eg, with metabolic syndrome and after acute coronary syndrome) who do not have a good lipid-lowering response to statins.^107,108 They might be crucial lipid-lowering agents in patients with statin intolerance. It seems that PCSK9 inhibitors might be the main and the most important component of the combined therapy of lipid disorders with statins. However, we do not think they might take over the place of statins in the future. It is connected both with their mechanism of action (strong lipid-lowering properties and probably lack of other—cholesterol-independent effects) and large estimated cost of these medications once they get approved.

Footnotes

Author Contributions

S. Dragan contributed to conception and design of the study, analysis and interpretation of the data, and drafting of the article; C. Serban contributed to conception and design of the study, analysis and interpretation of the data, drafting of the article, with substantial contribution to data acquisition; and M. Banach contributed to conception and design of the study, analysis and interpretation of the data, drafting of the article, and critical revision of the article for intellectual content.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Proprotein Convertase Subtilisin/Kexin 9 Inhibitors

Abstract

Keywords

Introduction

Search Strategy

The Role of PCSK9 in Lipid Metabolism

Current Strategies Targeting PCSK9

Inhibition of PCSK9-LDL-R Interaction

Monoclonal Antibodies

Experimental studies

Clinical studies

Phase 1 studies

Phase 2 studies

Phase 3 studies

Mimetic peptides and adnectins

Inhibition of PCSK9 Synthesis by Gene Silencing

Antisense oligonucleotides

Small Interfering RNA Oligonucleotides

Inhibition of PCSK9 Autocatalytic Processing by Small Molecule Inhibitors

Conclusions

Footnotes

Author Contributions

Declaration of Conflicting Interests

Funding

References