Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a hepatic enzyme that regulates the low-density lipoprotein cholesterol (LDL-c) receptor and thus circulating LDL-c levels. With overwhelming evidence now supporting the reduction in LDL-c to lower the risk of cardiovascular disease, PCSK9 inhibitors represent an important therapeutic target, particularly in high-risk populations. Here, we summarise and update the science of PCSK9, including its discovery and the development of various inhibitors, including the now approved monoclonal antibodies. In addition, we summarise the clinical applications of PCSK9 inhibitors in a range of patient populations, as well as the major randomised controlled trials investigating their use in coronary prevention.

Introduction

There is now overwhelming evidence to support reducing low-density lipoprotein cholesterol (LDL-c) to reduce atherosclerotic cardiovascular disease (ASCVD) [1]. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that regulates cell surface receptors, in particular the LDL receptor. Expressed primarily in the liver, the enzyme is responsible for regulating the degradation of the LDL receptor in response to cholesterol levels [2]. PCSK9 inhibitors have emerged as LDL-c lowering therapies and may be particularly beneficial in genetic conditions such as familial hypercholesterolaemia (FH), in patients who are statin-intolerant or patients who are already on maximum doses of statins but still require lipid reduction. We have previously reviewed the use of PCSK9 inhibitors in patients with FH [3] and now provide an overview and update on the science of PCSK9 and the clinical use of PCSK9 inhibitors in a range of populations, including recently published information on clinical outcomes.

Discovery of PCSK9

PCSK9 is a 692-amino acid glycoprotein and the ninth discovered member of the proprotein convertases, a family of secretory serine proteases [2]. It was originally identified in 2001 when elevated levels were found in studies of cerebellar neuron apoptosis and was subsequently named neural apoptosis-regulated convertase 1 [2,4,5]. In 2003, its gene was characterised when a gain-of-function mutation in PCSK9 was discovered to result in markedly elevated LDL-c. This discovery identified a third causal gene for autosomal dominant hypercholesterolaemia or FH, after mutations in the genes encoding the LDL receptor and apolipoprotein B (apoB). A second gain-of-function mutation in PCSK9 also associated with a severe hypercholesterolaemic phenotype was later identified, confirming the original finding [6,7]. In 2006, two nonsense mutations resulting in a loss-of-function were reported in an African-American population, which were associated with a 28% reduction in LDL-c levels and an 88% reduction in the 15-year risk of coronary heart disease (CHD). A third variant, found in a Caucasian population, resulted in a 15% reduction in LDL-c and a 47% reduction in CHD [8]. Three probands with very low LDL-c levels and no detectable circulating PCSK9 due to homozygous or compound heterozygous loss-of-function mutations were also reported, all of which were described as healthy and fertile ‘natural human knockouts’. The subsequent confirmation in animal studies as to the viability and cholesterol-lowering effect of PCSK9 knockouts paved the way for this protein to be developed as a new target for lipid-lowering therapies [5,7] (Figure 1).

Timeline of PCSK9 discovery

PCSK9 structure and physiology

The human 22-kb gene for PCSK9 is located on the small arm of chromosome 1p32 and contains 12 exons and 11 introns [9]. Its expression and synthesis are regulated by sterol regulatory element-binding protein 2 (SREBP-2) with intracellular cholesterol content the principal driver that controls PCSK9 gene expression [2–4,10]. The promoter region of the PCSK9 gene contains several sites, including a specificity protein 1 site, a hepatocyte nuclear factor 1α (HNF-1α) site and two sterol-responsive elements for transcription regulation [2]. The PCSK9 promoter region is also regulated by ligand-activated nuclear receptors, including farnesoid X receptor (FXR) and peroxisome proliferator-activated receptors (PPARs) [9].

PCSK9 is initially synthesised as a 75-kDa soluble zymogen known as pro-PCSK9, which contains an N-terminal signal peptide, a prodomain, a catalytic domain and a cysteine–histidine rich C-terminal domain [2,4,5]. Autocatalytic cleavage of pro-PCSK9 in the endoplasmic reticulum releases the signal peptide (amino acids 1–30), a 14-kDa prodomain and a 60-kDa mature protein, a process that is necessary for PCSK9 folding and maturation [2,4,5]. The prodomain however, remains associated with the mature protein via hydrogen bonds, facilitating its movement through the endoplasmic reticulum to the Golgi body. This association inhibits activity by blocking the catalytic site of the enzyme and chaperoning PCSK9 through the secretory pathway. As such, the only PCSK9 substrate identified so far is its own prodomain [2,4,5,11].

Once PCSK9 has been secreted, it self-associates, allowing dimeric and trimeric protein complexes to circulate alongside the monomeric form, with the levels of dimeric and trimeric PCSK9 correlating with LDL receptor degradation [2]. Mature, monomeric PCSK9 circulates either bound to LDL or as a furin-cleaved fragment that retains some residual activity [2,10,12]. Although it is not known how or where the association with LDL takes place, it does require very low-density lipoprotein (VLDL) catabolism and possibly apoB conformational change or surface exposure of unknown lipid structures during lipolysis [2]. In normolipidaemic subjects, approximately 40% of circulating PCSK9 appears to be associated with LDL [13] meaning the levels of PCSK9 in human plasma vary, existing in both the free and protein-associated forms. As a result, circulating levels measured by immunoassays may not be reliably predictive of activity, as antibodies recognise both the active and furin-cleaved inactive forms and the enzyme also exists and acts intracellularly.

Regulation of circulating PCSK9

Circulating PCSK9 levels are also influenced by nutrition and hormones, with levels reduced by fasting, glucagon and growth hormone [9,13,14]. Insulin appears to have an inhibitory effect with acute hyperinsulinaemia reducing plasma PCSK9 levels in obese postmenopausal women [15]. Marine-derived polyunsaturated fatty acids have been shown to lower plasma PCSK9 levels in premenopausal and postmenopausal women by 11.4 and 9.8% respectively [16]. Endogenous oestrogens have been demonstrated to lower plasma PCSK9 and LDL-c [17], while a Chinese study found that serum PCSK9 levels were higher in postmenopausal women compared with premenopausal women, independent of oestrogen status, with no effect of oestrogen on hepatic PCSK9 expression [18]. Inter-individual variation in plasma levels of PCSK9 in premenopausal women is further influenced by changes in endogenous oestrogen levels during the menstrual cycle [19].

Studies in healthy male volunteers and offspring of type 2 diabetic patients reveal a minimal response of plasma PCSK9 levels to a short-term, high-fat diet. In contrast, a short-term high-fructose diet increased plasma PCSK9 levels, independent of cholesterol synthesis, and was associated with insulin resistance, hepatic steatosis and triglyceride levels [20]. Other studies suggest that inflammation modulates both PCSK9 expression and release, suggestive of a role in innate immune response [11]. This is predominantly thought to be due to its effect on the clearance of lipid pathogens via the LDL receptor [21]. Inflammation induces marked changes in lipid and lipoprotein metabolism. Animal studies have demonstrated that hepatic PCSK9 levels are increased following exposure to inflammatory stimuli, which results in increased LDL receptor degradation and increased serum LDL-c levels [22]. In humans, sepsis is associated with disturbed cholesterol metabolism, with reduced cholesterol levels associated with a poor prognosis. PCSK9 expression can be up-regulated during sepsis and may contribute to organ dysfunction and tissue inflammation [21]. Finally, as statins have been shown to increase the activity and/or nuclear translocation of SREBP-2, statin therapy may increase PCSK9 mRNA expression, possibly limiting statin therapy efficacy [5]. Two previous studies have demonstrated that treatment with atorvastatin decreases LDL-c levels, while at the same time increasing serum PCSK9 protein levels [23,24].

Hepatic PCSK9 and the LDL receptor: mechanism of action

LDL is the primary cholesterol carrying lipoprotein. Derived from VLDL in plasma, LDL particles have a cholesterol ester-rich lipid core and a single apoB at the surface [25]. The apoB acts as a ligand for the LDL receptor [12,26], which is primarily located on hepatic cell surfaces [10,27]. Once bound, the LDL/LDL receptor complex is internalised by endocytosis into clathrin-coated vesicles. Within the cytosol, LDL separates from its receptor, which is then recycled back to the cell surface [12,26]. This process is aided by the acidic pH of the endosome, reducing the binding affinity of LDL for its receptor and facilitating structural rearrangement of the receptor into a hairpin configuration that aids its recycling [4,26]. The endosomes containing the LDL then fuse with lysosomes, resulting in the degradation of LDL, hydrolysis of cholesterol esters and distribution of free cholesterol to the rest of the hepatocyte [4]. Cholesterol levels are sensed by two distinct transcription factor pathways, the SREBPs and the liver X receptors (LXR), which co-ordinate complementary gene expression, giving rise to increased LDL receptor expression and reverse cholesterol transport, respectively [28]. LXR also regulates cholesterol uptake via transcriptional induction of inducible degrader of the LDL receptor (IDOL) that triggers ubiquitination of the receptor within the cytoplasm and subsequent degradation [29].

The LDL receptor was the first PCSK9 target to be identified, highlighting the important role the protein plays in the metabolism of LDL-c [9]. The binding of PCSK9 to the LDL receptor has been demonstrated to occur within the first epidermal growth factor-like repeat A (EGF-A) domain in the extracellular portion of the LDL receptor, distinct from the apoB domain that LDL binds to [12,13]. This EGF-A domain is essential for both the PCSK9–LDL receptor complex formation and its retention in the lysosome [13]. Once formed, the PCSK9–LDL receptor complex moves to the endosomes through clathrin-mediated endocytosis, where a decrease in pH strengthens the association [9,13]. This blocks the structural rearrangement of the receptor, locking it in an open conformation and preventing its recycling back to the cell surface, instead rerouting it to the lysosomes for degradation by unknown proteases [9,10,13,26]. Additionally, PCSK9 can enhance intracellular LDL receptor degradation prior to secretion by forming a complex with the receptor within the Golgi apparatus and directing it to the lysosome for degradation instead of transport to the membrane [4,10,26,27]. Both methods of PCSK9-mediated LDL receptor degradation require the cysteine–histidine-rich domain, which appears to be essential for trafficking of the complex to the lysosomes. Although the exact mechanism for this remains unknown, it is believed to strengthen the PCSK9–LDL receptor association within the acidic endosomes [5,9]. The subsequent clearance of PCSK9 occurs mainly in the liver, via its degradation with the LDL receptor, although this process also occurs independent of LDL binding [12] (Figure 2).

Hepatic mode of action of PCSK9 (steps 1–4) and activity of statins and PCSK9 inhibitors (siRNA and monoclonal antibodies)

Other lipid receptor effects of PCSK9

In addition to the LDL receptor, animal and in vitro studies suggest PCSK9 can target other members of the LDL receptor family and transport them for lysosome degradation. These include the VLDL receptor and the apolipoprotein E (apoE) receptor 2, with binding also occurring within the EGF-A domain, as well as the epithelial cholesterol transporter [9,11]. PCSK9 can also modify CD81 on hepatocytes and CD36, a scavenger receptor, on macrophages [11] and has been suggested to affect triglyceride levels via several mechanisms. These include modulation of hepatic triglyceride metabolism through regulation of the SREBP1c pathways, transcriptional and post-transcriptional regulation of fatty acid synthase, and up-regulated synthesis of non-sterol receptor element regulated genes such as apoB, apoE, diglyceride acyltransferase and microsomal triglyceride transfer protein [2]. Recent studies demonstrate how PCSK9 inhibition accelerates the catabolism of VLDL, intermediate-density lipoprotein (IDL) and LDL particles, while also decreasing the production of IDL and LDL [30,31].

Effect of PCSK9 on lipoprotein (a)

Cell-association studies suggest that lipoprotein (a) (Lp(a)), a highly atherogenic LDL-like particle, is reduced by co-incubation with LDL-c and PCSK9, suggesting Lp(a) competes with LDL for receptor binding and internalisation [11]. Recent studies have shown that the intact form of PCSK9 is found in association with Lp(a) in the plasma of patients with elevated Lp(a) and in transgenic mice expressing human Lp(a). This association was not related to the presence of oxidised phospholipids and did not occur through apoA. It is unclear whether this association is seen in people with normal levels of Lp(a) [32]. In patients treated with PCSK9 inhibitors, reductions in plasma levels of Lp(a) have been observed, in some cases by up to 30% [33]. While the mechanisms are unknown, it has been postulated to be via several receptors, including the LDL receptor, the apoE receptor or scavenger receptor class B type 1, which may also catabolise Lp(a). Other mechanisms include targeting docking, sorting and endocytic receptors or reductions in apoB or assembly of Lp(a) on hepatocyte surface [11,34].

Statin treatment does not appear to have an effect on Lp(a) levels and carriers of the PCSK9 loss-of-function mutations generally display similar Lp(a) levels to non-carriers. In contrast, gain-of-function mutation carriers appear to have elevated Lp(a) compared with normolipidaemic controls, yet epidemiological studies reveal no association between circulating PCSK9 and Lp(a) levels [35]. In vitro studies suggest that although PCSK9 does not significantly modify Lp(a) catabolism, it does enhance the hepatic secretion, a process that is reversed with PCSK9 inhibition [36]. More recently, PCSK9 inhibition via a monoclonal antibody has been shown to reduce plasma Lp(a) by decreasing the production of Lp(a) particles. When given in combination with a statin, PCSK9 inhibition resulted in an increase in the catabolism of Lp(a) particles [37]. Thus, although the relationship between PCSK9 and Lp(a) remains unclear, the activity of PCSK9 on a range of other lipids and lipid-family receptors offers additional potential therapeutic targets.

Extrahepatic PCSK9 expression and activity

In addition to the liver, PCSK9 is also produced in the kidney, small intestine, pancreas, central nervous system and vascular system [13], modulating cellular function in a range of tissues including adipose tissue, cardiomyocytes, macrophages and cancer cells [13]. In addition to regulating genes involved in cholesterol metabolism, PCSK9 also appears to play a role in gene expression related to proliferation, apotosis and inflammation [13].

Renal expression and activity

Although expressed abundantly in the kidneys, circulating PCSK9 is unable to degrade the LDL receptor in this tissue [13]. In vitro studies demonstrate that PCSK9 reduces epithelial sodium channel expression at the cell surface via enhanced proteasomal degradation. This does not appear to require the catalytic activity of PCSK9, nor does it alter endocytosis or degradation of the channel at the cell surface, suggestive of a role in channel trafficking [2,38]. Recent mouse studies have shown that despite an increase in cleaved epithelial sodium channel in PCSK9 knockout mice, there was no subsequent effect on sodium balance or blood pressure, either basally or in hypertensive models [39].

Intestinal expression and activity

PCSK9 expression in the intestine is quantitatively second only to hepatic expression [2]. Within the small intestine, PCSK9 is thought to regulate triglyceride-rich apoB production, increase assembly and secretion of intestinal triglyceride-rich lipoproteins through both transcriptional and post-transcriptional mechanisms, as well as play a role in modulating trans-intestinal faecal cholesterol excretion [2,4,5]. In vitro studies have shown that PCSK9 stimulates intestinal apoB secretion, with subsequent increases in apoB mRNA and mRNA levels for genes involved in the biosynthesis of fatty acids and triglycerides [5].

Pancreatic expression and activity

PCSK9 appears to predominate in the endocrine pancreas, specifically δ cells, where it co-localises with somatostatin but does not appear to alter cholesterol content or glucose-stimulated insulin secretion [40]. However, animal studies have revealed that aged PCSK9 knockout mice appear to have increased LDL receptor expression and reduced insulin in their pancreas, in addition to being hypoinsulinaemic, hyperglycaemic and glucose intolerant. Furthermore, their islets showed signs of malformation, apoptosis and inflammation, suggestive of a role for PCSK9 in normal islet function [41].

Neuronal expression and activity

PCSK9 was originally discovered as a factor that is up-regulated in apoptotic neurons and expressed at high levels in the cerebellum during perinatal development [13]. More recently, it has been shown to have pro-apoptotic effects in cerebellar granule neurons that are mediated by degradation of the apoE receptor and anti-apoptotic pathways [42]. Animal studies have shown that brain PCSK9 expression is induced following ischaemic stroke, although this did not affect lesion volume or neurogenesis [43]. However, its role in both vascular dementia and Alzheimer’s disease remains controversial, with some studies showing PCSK9 to be involved in the degradation of the amyloid precursor protein-cleaving enzyme [44] and others reporting no effect [45]. Recent analysis of Phase II and III trials have suggested a higher incidence of neurocognitive adverse events in patients treated with PCSK9 inhibitors, despite the overall incidence being very low (<1%) and unrelated to the degree of LDL-c reduction. The EBBINGHAUS trial, specifically designed to evaluate cognitive effects however, observed no impairment in cognitive function in patients treated with a PCSK9 inhibitor, including those with very low LDL-c levels [46].

Cardiac and vascular expression and activity

Recent evidence suggests that PCSK9 is expressed in cells of the arterial wall including endothelial cells, smooth muscle cells and macrophages, impacting both vascular homoeostasis and atherosclerosis [2]. Within vascular smooth muscle cells, PCSK9 is metabolised and secreted similar to hepatocytes, triggering degradation of the LDL receptor on the arterial macrophage surface and uptake of lipoproteins [2,13]. In vitro studies suggest that PCSK9 secretion by smooth muscle cells plays a role in reducing macrophage LDL receptor expression, foam cell formation and atherogenesis [47]. More recently, PCSK9 has been demonstrated to have a pro-inflammatory action on macrophages, which may also contribute to smooth muscle cell proliferation and migration [2,48].

Low shear stress has been shown to up-regulate PCSK9 expression in vascular endothelial and smooth muscle cells, which appears to be coupled to reactive oxygen species production [49]. Animal studies demonstrate that PCSK9 overexpression is pro-atherogenic, while its absence is protective. These effects were only observed in wild-type and apoE knockout mice, and not LDL receptor-deficient mice, suggestive of a role for the LDL receptor in the pro-atherogenic actions of PCSK9 [50]. In addition, these pro-atherogenic effects were cholesterol-independent and increased atherosclerotic lesion inflammation [51]. In contrast, circulating cholesterol and atherosclerotic lesion development appeared to be minimally modified in the PCSK9 knockout mouse on either an LDL receptor knockout or apoE knockout background. Furthermore, the ability of anti-PCSK9 antibody to reduce atherosclerosis required a functional apoE-LDL receptor pathway and had no effect when apoE was absent [52]. PCSK9 has also been detected in human carotid atherosclerotic lesions, where it is predominantly expressed in the vascular smooth muscle cells [53].

The BIOSTAT-CHF subanalysis study investigated whether the PCSK9-LDL receptor axis could predict risk in patients with heart failure. The findings revealed a positive linear association between PCSK9 levels and risk of mortality and composite end point of death or unscheduled hospitalisation for heart failure in patients with worsening signs and/or symptoms of heart failure. A negative association was also observed for LDL receptor and mortality, suggesting a potential benefit for PCSK9 inhibitors on outcomes in patients with heart failure [54]. A prospective cohort study investigated the relationship between serum PCSK9 and incident CVD in older men and women. After 15 years follow-up, baseline serum PCSK9 concentration predicted incident CVD even after adjustment for established CVD risk factors [55]. More recently, a small study in patients receiving a PCSK9 inhibitor found an improvement in vascular function after 2 months, that was proportional to the reduction in LDL-c [56].

Adipose tissue effects

Although not expressed in adipocytes, circulating PCSK9 produced by the liver can regulate the levels of cell surface receptors within adipose tissue [9]. Animal studies have demonstrated that a lack of PCSK9 leads to visceral fat accumulation through increased expression of the VLDL receptor, resulting in triglyceride hydrolysis and free fatty acid uptake [9]. These mice have also been shown to display adipocyte hypertrophy, which appears to be independent of LDL receptor expression [2]. The Progressione della Lesione Intimale Carotidea (PLIC) study revealed that carriers of the loss-of-function PCSK9 R46L variant had lower LDL-c levels, higher body mass index and increased percentage of total and android fat mass compared with non-carriers. Although the number of carriers was low, the mutation was also associated with a two-fold increase in prevalence of hepatic steatosis and higher epicardial fat thickness, with all findings replicated in PCSK9 knockout mice. While suggestive of a role for PCSK9 in modulating lipid delivery and accumulation in peripheral tissues, it remains unclear as to how PCSK9 inhibition would affect these parameters [57].

Development of PCSK9 inhibitors as a lipid-lowering therapy

Although produced and active in a number of tissues within the body, the prominent hepatic role of PCSK9 on lipoprotein metabolism has led to extensive investigation into the development of PCSK9 inhibitors and their use as lipid-lowering agents. Much of this research was driven by the early work identifying gain-of-function and loss-of-function mutations in the PCSK9 gene [9,10] (Figure 1).

PCSK9 inhibitors function by reducing the concentration or activity of circulating and possibly intracellular PCSK9, preventing the degradation of LDL receptors and thus increasing clearance of LDL-c from the plasma [10,27]. Multiple strategies for reducing circulating PCSK9 are being investigated, including preventing PCSK9 from binding to the LDL receptor and targeting PCSK9 synthesis and processing (Figure 2). A number of approaches have been adopted including: (i) gene silencing by antisense oligonucleotides or siRNA, (ii) inhibition of PCSK9 autocatalytic sites or (iii) prevention of PCSK9 binding to LDL receptors through monoclonal antibodies, EGF-A mimetic peptides or adnectins [4,26,58,59]. Antisense oligonucleotides and siRNA target intracellular PCSK9 through gene silencing techniques to reduce its production. Antibodies, either exogenous or vaccine-derived, target extracellular PCSK9, selectively binding to specific circulating epitopes, leading to its sequestration. Small protein inhibitors (peptides/adnectins) prevent the extracellular interaction between PCSK9 and the LDL receptor [7]. Additionally, some nutraceuticals have been shown to act as inhibitors of PCSK9 expression or activity, including berberine, curcumin, polydatin, polyunsaturated fatty acids and quercetin glucoside. Despite promising benefits in vitro and in several small clinical trials, few studies have investigated their long-term safety and efficacy [60].

PCSK9 gene silencing

Antisense oligonucleotides and siRNA reduce intracellular expression by targeting PCSK9 mRNA, halting its translation and resulting in mRNA degradation [4,5]. This gene-silencing approach was thought to have the potential to affect intracellular (either nuclear or cytoplasmic) PCSK9 levels, further interfering with LDL receptor recycling and degradation [13,61]. Antisense oligonucleotides utilise a short, single strand of RNA that binds to the specific mRNA, leading to competitive inhibition of translation or degradation of the resulting complex. In contrast, siRNA uses a dsRNA complex that also recognises the target mRNA, facilitating cleavage and degradation and a halt to protein production [61].

Although reductions in PCSK9 and LDL-c levels with the antisense oligonucleotides, SPC5001 and BMS-844421 were observed in animals, further development was terminated for undisclosed reasons [4]. A Phase I clinical trial of the anti-PCSK9 siRNA, ALN-PCS reduced free PCSK9 levels by 70% and LDL-c levels by 40% [62]. More recently, the Phase I trial of inclisiran (ALN-PCSsc) demonstrated that single or multiple doses of 300 mg or more significantly reduced levels of PCSK9 (up to 83.8%) and LDL-c (up to 59.7%) for at least 6 months with no treatment discontinuation or serious adverse events reported. These effects were also observed in patients taking stable statin therapy. While the present study was randomised and placebo controlled, it was limited by a small sample size and single-blind design [63]. Further evaluation of the safety and potential for 3- or 6-month administration of inclisiran was investigated in a Phase II trial, which revealed significant reductions in both PCSK9 and LDL-c. Serious adverse events occurred in 11% of the inclisiran patients and 8% of placebo patients [64,65]. ORION-1, a Phase II trial demonstrated significant and prolonged reductions in atherogenic lipoproteins following a bi-annual dosing regimen [66]. Five large Phase III trials assessing LDL-c lowering and cardiovascular outcomes with inclisiran are currently being planned [61]. Advantages of the siRNA methods include the long duration of cholesterol reduction, reductions in total (rather than just free) PCSK9 levels, reduction in treatment administration and increased target specificity improving drug potency and reducing side effects [61]. Recent developments in gene-editing technology through CRISPR-Cas9 have also paved the way for changes in DNA to be introduced via targeting of specific sequences. Animal studies have revealed reductions in PCSK9 expression using a lipid-like nanoparticle CRISPER-Cas9 delivery system [67]. Although still in the proof-of-concept safety and efficacy stages, PCSK9-targeted CRISPR-Cas9 is currently in preclinical development [58].

Inhibition of PCSK9 autocatalytic sites

Because autocatalytic processing of PCSK9 is a necessary step for the secretion of the mature protein, intracellular inhibition of this process with small molecules offers another therapeutic target [5]. Despite being cheaper to produce and available as an orally administered treatment, these carry the risk of increased side effects due to reduced selectivity [5]. Use of these molecules to inhibit the PCSK9 autocatalytic site is still in the preclinical investigation phase and is made difficult because the site of interaction between the catalytic subunit of PCSK9 and the EGF-A domain of the LDL receptor is flat, lacking pockets necessary for small molecules to bind [5,26].

Preventing PCSK9 binding to the LDL receptor

Utilisation of monoclonal antibodies that bind to PCSK9 and prevent its subsequent binding to and degradation of the LDL receptor have been the most effective approach thus far [10,26]. These function by binding to the catalytic domain and prodomain of PCSK9, blocking interaction with the LDL receptor and neutralising PCSK9 activity selectively in the plasma [61]. Studies have shown maximal suppression of circulating unbound PCSK9 within 4–8 h of antibody administration, with subsequent LDL-c reductions in ∼65% in healthy and ∼60–80% in hypercholesterolaemic patients [4]. To date, there are six monoclonal antibodies that have been or are currently being developed and tested. Two of these, alirocumab (formerly SAR236553/REGN727) and evolocumab (formerly AMG145), have been approved for use by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) in patients with FH and those with clinical ASCVD who require additional lipid lowering despite maximal statin dose and diet control [7,26]. These two antibodies are fully human IgG1 and IgG2 respectively, and work by preventing PCSK9 from binding to the LDL receptor, promoting increased recycling and cell surface density of hepatic LDL receptors and plasma LDL clearance [12]. A third monoclonal antibody, bococizumab, is a humanised monoclonal antibody that retains ∼3% murine sequences, which resulted in the production of high titres of antidrug antibodies in some patients, directly attenuating the LDL-c lowering response and duration. As a result, development of this antibody was halted in November 2016 [4]. A fourth antibody, LY3015014, was shown to bind to intact but not truncated forms of PCSK9, blocking its interaction with the LDL receptor, but allowing normal proteolytic cleavage of PCSK9, thus limiting its accumulation. Despite dose-dependent and durable LDL-c reductions in hypercholesterolaemic patients when used as add-on therapy, development was discontinued due to lower reductions compared with other monoclonal antibodies [65]. RG7652 (MPSK3169A), a fully human monoclonal antibody, was trialled in Phase I and II studies, with and without statin therapy to establish safety and efficacy. The antibody was found to have dose-dependent lowering effect on LDL-c, with additional reductions in apoB and Lp(a). Adverse events were generally mild and there were no unexpected safety issues [68,69].

Targeted screening of virus-like particle peptide vaccines against PCSK9 has revealed that vaccinated mice, particularly those vaccinated with the PCSK9Qβ-003 vaccine, developed high-titre IgG antibodies against PCSK9. This resulted in reductions in total cholesterol and plasma PCSK9 levels in both wild-type and LDL receptor heterozygous knockouts, as well as increased LDL receptor expression and up-regulation of SREBP-2, HNF-1α and HMG-CoA reductase [70]. More recently a peptide vaccine consisting of short peptides conjugated to a carrier protein was developed. When given to apoE knockout mice, the vaccine elicited a significant antibody response against PCSK9, which was maintained for 24 weeks. Decreased plasma levels of total cholesterol, VLDL and chylomicrons were also observed, along with a significant increase in hepatocyte LDL receptors [71]. Vaccination has also been shown to produce an even greater LDL-c lowering response in animals treated with statins, with additional reductions in inflammatory markers and aortic inflammation [65]. In humans, the AT04A vaccine is an active immunisation against PCSK9. Animal studies have shown that AT04A was able to induce a high immune response against PCSK9 leading to significant reductions in plasma lipids without side effects. This was associated with reductions in systemic and vascular inflammation and aortic atherosclerotic lesions [72]. Phase I trials are currently underway and with the reduced cost and simple administration, vaccines against PCSK9 offer the potential for improved compliance [73].

Small mimetic peptides targeting the EGF-A domain, the catalytic domain, the prodomain and the C-terminal domain of PCSK9 have been developed. Smaller, highly selective and cheaper than the monoclonal antibodies, these mimetic peptides are designed to modify the interaction of PCSK9 with the LDL receptor through competitive inhibition. Although their effectiveness has been demonstrated in in vitro studies, there are currently no clinical trials testing their use [5]. Adnectins, also known as monobodies, bind to their target protein with high specificity and affinity by exchanging amino acids in β-sheet loops while preserving structural stability of PCSK9. Preclinical studies of BMS-962476 have demonstrated their effectiveness in cynomolgus monkeys [5]; however despite being well tolerated in humans and showing reductions in LDL-c and PCSK9 levels, development has been discontinued [65]. More recently, DS-9001a, a novel small biologic alternative was found to significantly reduce LDL-c in cynomolgus monkeys up to 21 days after a single administration, with additional benefits observed when co-administered with statins in transgenic mice [74].

Clinical trials involving PCSK9 monoclonal antibodies

Despite widespread use of lipid-lowering therapies such as statins and ezetimibe, there remains a significant portion of patients who cannot meet lipid targets. With advances in the development of PCSK9 inhibitors, particularly the monoclonal antibodies, a number of studies have looked at their effectiveness in a range of populations (Table 1). The PROFICIO and ODYSSEY programmes investigating evolocumab and alirocumab respectively represented a new approach to clinical trials by involving significant numbers of patients through extensive collaboration of many countries. A recent meta-analysis of 35 randomised controlled trials (n=45539) indicated that treatment with a PCSK9 monoclonal antibody (alirocumab or evolocumab) is well tolerated, reduces LDL-c and improves cardiovascular clinical outcomes (myocardial infarction (MI), stroke, coronary revascularisation), despite no benefit observed for all-cause or cardiovascular mortality [75]. Below, we summarise the major PCSK9 monoclonal antibody trials in various populations carried out to date.

Table 1
Summary of clinical trials investigating effects of PCSK9 monoclonal antibodies on lipid levels
Study Population Treatment Design Outcome 
LAPLACE-TIMI 57 [76High-risk CVD 631 Evolocumab Phase II, dose-ranging RCT Significant reductions in LDL-c, non-HDL cholesterol and apoB 
    12 weeks  
MENDEL-2 [77Hypercholesterolaemia 614 Evolocumab RCT Significant reductions in LDL-c, non-HDL cholesterol and apoB 
    12 weeks  
GLAGOV [78, 79Angiographic coronary disease 968 Evolocumab RCT Significant reductions in % atheroma, total atheroma volume and LDL-c 
    76 weeks Significant increase in plaque regression 
OSLER [80Patients from OSLER-1 and OSLER-2 4465 Evolocumab Pooled data Significant reductions in LDL-c and rate of CVD events 
    1 year  
FOURIER [81–83ASCVD and LDL-c > 1.8 mmol/l 27564 Evolocumab RCT Significant reduction in LDL-c 
    48 weeks Significant reduction in composite of CV death, MI, stroke, UA or revascularisation 
SPIRE-2 [84High-risk CVD 10621 Bococizumab RCT Significant reduction in in CV death, MI, stroke or revascularisation in high-risk patients 
    12 months-terminated early  
ODYSSEY Outcomes [85Acute coronary syndrome, LDL-c > 1.8 mmol/l and statin therapy 18924 Alirocumab RCT Significant reduction in LDL-c 
    2.8 years Significant reduction in composite of CHD death, non-fatal MI, ischaemic stroke, UA requiring hospitalisation 
GAUSS-2 [86Statin intolerant hypercholesterolaemic patients 307 Evolocumab RCT Significant reductions in LDL-c 
    12 weeks  
GAUSS-3 [87Statin-intolerant hypercholesterolaemic patients 218 Evolocumab Two-stage RCT Significant reductions in LDL-c 
    24 weeks  
ODYSSEY Alternative [88Statin intolerance and moderate-high risk CVD 361 Alirocumab Two-stage RCT Significant reductions in LDL-c 
    24 weeks  
Multicentre [89HetFH and LDL-c > 2.6 mmol/l 77 Alirocumab Phase II RCT Significant reductions in LDL-c, non-HDL cholesterol and apoB 
    12 weeks  
ODYSSEY FH I and FH II [90HetFH and elevated LDL-c 735 Alirocumab 2 RCTS Significant reductions in LDL-c 
    24 weeks  
RUTHERFORD-2 [91HetFH and LDL-c > 2.6 mmol/l 331 Evolocumab RCT Significant reductions in LDL-c 
    12 weeks  
ODYSSEY ESCAPE [92,93HetFH on apheresis 62 Alirocumab RCT Significant reductions in LDL-c and rate of apheresis 
    18 weeks  
SPIRE Program [94Phenotypically defined FH patients 1578 Bococizumab Pooled SPIRE program trials 11.2 months – terminated early Significant reductions in LDL-c, total cholesterol, apoB, non-HDL cholesterol, Lp(a) and triglycerides 
TESLA [95HomFH 50 Evolocumab RCT Significant reductions in LDL-c 
    12 weeks  
TAUSSIG [96HomFH with and without apheresis 106 Evolocumab Open-label Significant reductions in LDL-c and Lp(a) 
    Interim subset analysis  
ODYSSEY DM-INSULIN [97Type 1 and 2 diabetes and elevated LDL-c 4983 Alirocumab Pooled data Significant reductions in LDL-c 
    24–104 weeks  
Study Population Treatment Design Outcome 
LAPLACE-TIMI 57 [76High-risk CVD 631 Evolocumab Phase II, dose-ranging RCT Significant reductions in LDL-c, non-HDL cholesterol and apoB 
    12 weeks  
MENDEL-2 [77Hypercholesterolaemia 614 Evolocumab RCT Significant reductions in LDL-c, non-HDL cholesterol and apoB 
    12 weeks  
GLAGOV [78, 79Angiographic coronary disease 968 Evolocumab RCT Significant reductions in % atheroma, total atheroma volume and LDL-c 
    76 weeks Significant increase in plaque regression 
OSLER [80Patients from OSLER-1 and OSLER-2 4465 Evolocumab Pooled data Significant reductions in LDL-c and rate of CVD events 
    1 year  
FOURIER [81–83ASCVD and LDL-c > 1.8 mmol/l 27564 Evolocumab RCT Significant reduction in LDL-c 
    48 weeks Significant reduction in composite of CV death, MI, stroke, UA or revascularisation 
SPIRE-2 [84High-risk CVD 10621 Bococizumab RCT Significant reduction in in CV death, MI, stroke or revascularisation in high-risk patients 
    12 months-terminated early  
ODYSSEY Outcomes [85Acute coronary syndrome, LDL-c > 1.8 mmol/l and statin therapy 18924 Alirocumab RCT Significant reduction in LDL-c 
    2.8 years Significant reduction in composite of CHD death, non-fatal MI, ischaemic stroke, UA requiring hospitalisation 
GAUSS-2 [86Statin intolerant hypercholesterolaemic patients 307 Evolocumab RCT Significant reductions in LDL-c 
    12 weeks  
GAUSS-3 [87Statin-intolerant hypercholesterolaemic patients 218 Evolocumab Two-stage RCT Significant reductions in LDL-c 
    24 weeks  
ODYSSEY Alternative [88Statin intolerance and moderate-high risk CVD 361 Alirocumab Two-stage RCT Significant reductions in LDL-c 
    24 weeks  
Multicentre [89HetFH and LDL-c > 2.6 mmol/l 77 Alirocumab Phase II RCT Significant reductions in LDL-c, non-HDL cholesterol and apoB 
    12 weeks  
ODYSSEY FH I and FH II [90HetFH and elevated LDL-c 735 Alirocumab 2 RCTS Significant reductions in LDL-c 
    24 weeks  
RUTHERFORD-2 [91HetFH and LDL-c > 2.6 mmol/l 331 Evolocumab RCT Significant reductions in LDL-c 
    12 weeks  
ODYSSEY ESCAPE [92,93HetFH on apheresis 62 Alirocumab RCT Significant reductions in LDL-c and rate of apheresis 
    18 weeks  
SPIRE Program [94Phenotypically defined FH patients 1578 Bococizumab Pooled SPIRE program trials 11.2 months – terminated early Significant reductions in LDL-c, total cholesterol, apoB, non-HDL cholesterol, Lp(a) and triglycerides 
TESLA [95HomFH 50 Evolocumab RCT Significant reductions in LDL-c 
    12 weeks  
TAUSSIG [96HomFH with and without apheresis 106 Evolocumab Open-label Significant reductions in LDL-c and Lp(a) 
    Interim subset analysis  
ODYSSEY DM-INSULIN [97Type 1 and 2 diabetes and elevated LDL-c 4983 Alirocumab Pooled data Significant reductions in LDL-c 
    24–104 weeks  

Abbreviations: CVD, cardiovascular disease; HDL, high-density lipoprotein; HetFH, heterozygous FH; HomFH, homozygous FH; RCT, randomised controlled trial; UA, unstable angina.

Dyslipidaemia

The LAPLACE-TIMI 57 trial examined the efficacy of evolocumab in patients at high risk of major CVD events who were unable to meet lipid targets on stable statin therapy with or without ezetimibe. Evolocumab significantly reduced LDL-c levels with additional reductions in non-HDL cholesterol and apoB. There were no differences in the rate of serious adverse events, treatment-related adverse events, liver or kidney function [76]. The MENDEL-2 trial compared the effect of evolocumab with placebo and ezetimibe in patients with hypercholesterolaemia. Evolocumab significantly reduced LDL-c and favourably affected other lipid parameters (reductions in apoB and non-HDL cholesterol). There were no differences in treatment-related or muscle-related adverse events or biochemical anomalies [77].

ASCVD

Elevated LDL-c is causally associated with the development and progression of ASCVD [98]. Earlier optimism of a nearly 50% reduction in ASCVD event rates, demonstrated by open label extension data and post hoc analysis of Phase III studies, has been tempered somewhat by the results of large randomised controlled trials.

The GLAGOV study investigated the effect of evolocumab on progression of coronary atherosclerosis in statin-treated patients. Evolocumab significantly decreased LDL-c, nominal change in percent atheroma and total atheroma volume in addition to inducing more plaque regression. There were no differences in serious adverse events, treatment-related adverse events or rates of laboratory anomalies. Although not powered to assess effects on cardiovascular events, there were fewer adverse outcomes, non-fatal MIs and coronary revascularisation procedures in the evolocumab-treated patients [78,79]. Interestingly, recent analysis of the GLAGOV study has demonstrated that addition of evolocumab to a statin did not produce differential changes in plaque composition compared with statin alone. This suggests that assessment of plaque burden through virtual histology may not provide additional information on the beneficial effects of PCSK9 inhibitors on atherosclerotic plaque [99]. THE OSLER trials included patients who had completed evolocumab Phase II and III trials (OSLER-1 and OSLER-2 respectively) and whose primary goal was to gather long-term safety data and pre-specified exploratory analysis on cardiovascular outcomes. In addition to reductions in LDL-c, the rate of cardiovascular events at 1 year was reduced in the evolocumab group [80].

A comparison and summary of the three major outcomes studies, FOURIER, SPIRE-2 and ODYSSEY OUTCOMES, is presented in Table 2. The FOURIER trial observed significant reductions in LDL-c as well as the risk of the primary end point (composite of cardiovascular death, MI, stroke, hospitalisation for unstable angina or coronary revascularisation) and the secondary end point (composite of cardiovascular death, MI or stroke) with evolocumab. There were no significant differences in outcomes when patients were stratified for baseline LDL-c levels, nor were there any differences in adverse events. [81]. Pre-specified secondary analysis revealed that patients who achieved lower LDL-c levels had progressively fewer cardiovascular events with no evidence for a plateau and no increase in adverse events, suggestive of no safety concerns for very low LDL-c levels [82]. Further analysis revealed that evolocumab significantly reduced the risk of cardiovascular events in patients with peripheral arterial disease, with a reduced risk of major adverse limb events that was consistently associated with lower LDL-c levels [83]. Reductions in risk were also seen in patients closer to their most recent MI, with multiple prior MIs or with residual multivessel coronary artery disease with evolocumab [100]. Despite early termination, SPIRE-2 revealed no beneficial effect on major adverse cardiovascular events in lower risk patients, but a significant reduction in the primary end point (cardiovascular death, MI, stroke, urgent revascularisation) in high-risk patients treated with bococizumab [84].

Table 2
Summary of cardiovascular outcomes in clinical trials of PCSK9 monoclonal antibodies
Design characteristics SPIRE-2 [84FOURIER [81ODYSSEY OUTCOMES [85
Patient population High risk CVD Stable ASCVD Recent ACS and high-intensity statin 
n 10621 27564 18924 
Age (median) 63 years 63 years 58 years 
LDL-c (mmol/l)    
  Entry 2.6 1.8 1.8 
  Baseline 3.3 2.4 2.4 
Lipid-lowering drugs    
  Statins 73% 69% 89% 
  Ezetimibe 13% 5% 3% 
PCSK9 inhibitor and dose Bococizumab Evolocumab Alirocumab 
 150 mg biweekly and titrated 140 mg biweekly or 420 mg monthly 75 or 150 mg biweekly and titrated 
Follow-up duration 1 year 2.2 years 2.8 years 
 Terminated early   
Primary end point CV death, MI, stroke, urgent revascularisation CV death, MI, stroke, hospitalisation for UA, coronary revascularisation CHD death, MI, ischaemic stroke, hospitalisation for UA 
Change in LDL-c 52% reduction 59% reduction 61% reduction 
Primary end point 21% reduction 15% reduction 15% reduction 
MI 24% reduction 27% reduction 14% reduction (NPR) 
Stroke 21% reduction 21% reduction 27% reduction (NPR) 
Unstable angina 5% reduction 1% reduction 39% reduction (NPR) 
CVD death 18% reduction (NS) 5% increase (NS) 8% reduction (NS) 
All-cause death 9% reduction (NS) 4% increase (NS) 15% reduction (NS) 
Design characteristics SPIRE-2 [84FOURIER [81ODYSSEY OUTCOMES [85
Patient population High risk CVD Stable ASCVD Recent ACS and high-intensity statin 
n 10621 27564 18924 
Age (median) 63 years 63 years 58 years 
LDL-c (mmol/l)    
  Entry 2.6 1.8 1.8 
  Baseline 3.3 2.4 2.4 
Lipid-lowering drugs    
  Statins 73% 69% 89% 
  Ezetimibe 13% 5% 3% 
PCSK9 inhibitor and dose Bococizumab Evolocumab Alirocumab 
 150 mg biweekly and titrated 140 mg biweekly or 420 mg monthly 75 or 150 mg biweekly and titrated 
Follow-up duration 1 year 2.2 years 2.8 years 
 Terminated early   
Primary end point CV death, MI, stroke, urgent revascularisation CV death, MI, stroke, hospitalisation for UA, coronary revascularisation CHD death, MI, ischaemic stroke, hospitalisation for UA 
Change in LDL-c 52% reduction 59% reduction 61% reduction 
Primary end point 21% reduction 15% reduction 15% reduction 
MI 24% reduction 27% reduction 14% reduction (NPR) 
Stroke 21% reduction 21% reduction 27% reduction (NPR) 
Unstable angina 5% reduction 1% reduction 39% reduction (NPR) 
CVD death 18% reduction (NS) 5% increase (NS) 8% reduction (NS) 
All-cause death 9% reduction (NS) 4% increase (NS) 15% reduction (NS) 

Abbreviations: ACS, acute coronary syndrome; CVD, cardiovascular disease; NS, not significant; NPR, no P-value reported; UA, unstable angina.

More recently, ODYSSEY OUTCOMES observed significant reductions in LDL-c and the primary composite end point (death from CHD, non-fatal MI, fatal and non-fatal ischaemic stroke and unstable angina requiring hospitalisation) with alirocumab. Risk reduction was of a very similar magnitude to that seen with evolocumab in FOURIER. These effects were greatest in patients with higher baseline LDL-c levels (>2.6 mmol/l or 100 mg/dl) and consistent with that seen in the SPIRE-2 trials. Although a composite of death from any cause, non-fatal MI or non-fatal ischaemic stroke was significantly reduced, death from CHD, cardiovascular causes and death from any cause were not analysed due to the hierarchical testing plan [85]. Interestingly, pre-specified analysis to determine the effect of alirocumab on total (first and subsequent) non-fatal cardiovascular events and all-cause deaths revealed reductions in total non-fatal cardiovascular events and death in the presence of a strong association between fatal and non-fatal event risk. The total number of non-fatal cardiovascular events and deaths prevented with alirocumab was twice the number of first events prevented, suggesting that total event reduction is a more comprehensive metric when considering the clinical efficacy of alirocumab [101]; this approach has also been adopted in certain statin trials, such as PROVE IT-TIMI 22, IDEAL, TNT and IMPROVE-IT [102–106]. Within ODYSSEY OUTCOMES, there was a similar number of adverse events reported between groups, with the exception of injection site reactions. Interestingly, an upward drift in LDL-c levels in the alirocumab group over the 48-month treatment follow-up period was observed. A number of reasons were proposed for this including premature treatment discontinuation, dose reduction or blinded substitution to placebo as specified by the protocol, and reduction in the intensity of statin treatment [85]. Although the OUTCOMES trial was not included, pooled analysis of eight controlled Phase III ODYSSEY trials has revealed that alirocumab is well tolerated and effective with a similar safety profile in high-risk ASCVD patients with or without prior revascularisation [107].

Of note is the impact of the PCSK9 monoclonal antibodies on Lp(a) levels in these major clinical outcome trials. While previous studies suggest that PCSK9 inhibitors can lower Lp(a) levels by approximately 30% [33], a recent review suggests that even modest lowering of Lp(a) may have some clinical benefit [108]. Within the FOURIER trial, no interaction was observed between effect of evolocumab and baseline Lp(a); however, the greatest clinical benefit was seen in patients who achieved the greatest reductions in both LDL-c and Lp(a). In ODYSSEY OUTCOMES, the extent of reduction in Lp(a), but not baseline values, predicted the degree of benefit seen with alirocumab, which appeared to be independent of the extent of LDL-c reduction, suggestive of an additional benefit from Lp(a) reduction alone. While neither of these two outcomes have been formally published, the results presented suggest (but do not prove) an additional mechanism for the benefit of PCSK9 inhibition on ASCVD [108].

Statin intolerance

Statin intolerance, predominantly in the form of muscle-related side effects, is thought to occur in as many as 20–30% of statin users [109,110] and is the most common cause of statin discontinuation. PCSK9 inhibitors, which act via a mechanism distinct to that of statins, provide an alternate therapeutic option for these patients to achieve lipid targets.

The GAUSS-2 trial compared evolocumab with ezetimibe in statin-intolerant hypercholesterolaemic patients. Evolocumab reduced LDL-c to a greater extent than ezetimibe, with treatment-related adverse events and laboratory anomalies comparable across the groups [86]. An extension trial, GAUSS-3 observed greater reductions in LDL-c levels with evolocumab compared with ezetimibe and no difference in muscle-related side effects [87]. Three hundred and eighty-two statin-intolerant patients who had completed GAUSS-1 and GAUSS-2 were further enrolled in the OSLER studies, where evolocumab plus standard of care was found to be persistently safe, tolerable and efficacious for up to 2 years [111]. The ODYSSEY ALTERNATIVE trial compared alirocumab with ezetimibe and low-dose statin in patients with moderate to high CVD risk and statin intolerance. Alirocumab reduced LDL-c compared with ezetimibe, with muscle-related events less frequent in the alirocumab group compared with the atorvastatin arm [88].

FH

FH is the most common monogenic disorder of lipid metabolism and gives rise to elevated LDL-c levels from birth, resulting in premature ASCVD [3]. It is most frequently caused by mutations in the LDLR gene, which encodes the LDL receptor. Less common causes include a defective APOB gene encoding an apoB that does not bind normally to the LDL receptor or a gain-of-function mutation in PCSK9 [3]. Mutation of one allele causes heterozygous FH, which occurs in approximately 1 in 200 people and causes ASCVD by middle age. Compound heterozygosity or true homozygosity causes homozygous FH, which is much less common but can cause ASCVD, disability and death in childhood if untreated. FH can be further characterised as either LDL receptor-defective (2–25% normal activity) or LDL receptor negative (<2% activity). Response to PCSK9 inhibitors varies depending on the functionality of the LDL receptor [3].

In patients with heterozygous FH and elevated LDL-c, on a stable diet and statin dose with or without ezetimibe, significant reductions in LDL-c, total cholesterol, non-HDL cholesterol and apoB were observed with alirocumab treatment. No serious adverse events were reported [89]. ODYSSEY FH I and FH II assessed long-term alirocumab treatment in patients with heterozygous FH not achieving lipid targets on maximal lipid therapy. Alirocumab was well tolerated and resulted in significant reductions in LDL-c and greater achievement of LDL-c targets, regardless of CVD risk [90]. RUTHERFORD-2 investigated patients with heterozygous FH on stable lipid-lowering therapy and with elevated LDL-c. Compared with placebo, evolocumab significantly reduced mean LDL-c levels and was well tolerated with adverse event rates similar to placebo [91]. Open-label extension trials of both RUTHERFORD and RUTHERFORD-2 demonstrated that continued use of evolocumab with standard of care treatment in heterozygous FH patients resulted in persistent and significant LDL-c reductions, with no serious adverse events reported [112]. Secondary analysis of phenotypically defined FH patients in the SPIRE clinical trials program revealed significant reductions in LDL-c, total cholesterol, triglycerides, Lp(a), non-HDL cholesterol levels and major adverse cardiovascular events with bococizumab compared with placebo, although these were not different when compared with non-FH patients [94].

The ODYSSEY ESCAPE trial in heterozygous FH patients undergoing regular apheresis investigated the effect of alirocumab on the frequency of apheresis treatments. Alirocumab treated patients had a 75% reduction in the standardised rate of apheresis compared with the placebo group. There was no difference in adverse event rates between groups [92]. Although the ODYSSEY ESCAPE study demonstrates the benefits of alirocumab reducing the need for apheresis, which is both demanding and expensive, further studies, particularly in homozygous FH patients and patients with elevated Lp(a), are still required [93].

The TESLA Part B study of patients with homozygous FH and on stable lipid therapy (but not apheresis), examined the effect of evolocumab and observed significant reductions in LDL-c. Despite this significant reduction, a patient with two negative mutations did not respond to the PCSK9 inhibitor at all, while patients with one negative mutation and one receptor-defective mutation (some functional LDL receptor activity), had limited response to treatment. In patients with two receptor-defective mutations and relatively more functional LDL receptor activity, response to treatment was more pronounced [95]. More recently, interim subset analysis of the TAUSSIG study revealed significant reductions in LDL-c and Lp(a) in homozygous FH patients, with or without apheresis, following treatment with evolocumab [96].

A recent review [113] has summarised the effect of evolocumab in different FH populations. In heterozygous patients, the inhibitor appears to be as effective as in non-FH patients. In homozygous patients however, the response is dependent on the type of mutation [113], with residual LDL receptor expression being a major determinant of LDL-c levels that may drive the response to evolocumab. These individual responses appeared variable even among carriers with identical LDL receptor genetic defects [114]. The efficacy of alirocumab has also been investigated in patients with double heterozygous, compound heterozygous or homozygous FH. Patients from six trials were investigated and clinically significant reductions in LDL-c were observed in all patient groups [115].

Obesity and glucose metabolism

ODYSSEY DM-INSULIN investigated the efficacy and safety of alirocumab in patients with type 2 or insulin-treated type 1 diabetes with elevated LDL-c despite maximally tolerated statin therapy. In addition to reductions in LDL-c, glycated haemoglobin and fasting plasma glucose levels remained stable for the study duration [97]. Pooled analysis of the ODYSSEY Phase III trials also revealed beneficial reductions on LDL-c in patients with pre-diabetes at baseline, with no effect on glycaemia and no significant difference on adverse event rates compared with normoglycaemic patients [116]. Prespecified analysis of the FOURIER trial demonstrated a significant reduction in cardiovascular risk in patients with and without diabetes following treatment with evolocumab, with a greater absolute risk reduction in patients with diabetes. Furthermore, there was no increased risk of newly onset diabetes even in prediabetic patients, nor a worsening of glycaemia [117].

Safety and tolerability

Current long-term data on alirocumab and evolocumab suggest they are well tolerated and generally safe. Pooled data does suggest an increase in injection site reactions and upper respiratory tract symptoms with both monoclonal antibodies [12]. Recent pooled safety analysis of Phase II and III randomised and placebo or comparator-controlled trials, as well as open-label extension trials support a favourable benefit-risk profile for evolocumab [118] and alirocumab [119]. To date, neutralising antibodies to either treatment have not been found to be clinically relevant, as they are generally transient and have limited to no impact on the drug’s lipid-lowering effect [12]. There has been no association with the use of PCSK9 inhibitors and either glycaemic control or the development of type 2 diabetes. Similarly, no effects on neurocognition have been observed [12,120], despite an increase in the frequency of neurocognitive events reported in the evolocumab group in the OSLER-1 and OSLER-2 trials [80]. Contention still remains as to the long-term safety of very low LDL-c levels, although secondary analysis of the FOURIER trial suggests this is not an issue [82]. Furthermore, recent analysis from the Cholesterol Treatment Trialists Collaboration (CTTC) affirmed a continuous relative risk reduction in major vascular events per change in LDL-c levels, suggesting that additional lowering of LDL-c beyond the lowest current targets would further reduce risk [121].

Guidelines on the use of PCSK9 inhibitors

PCSK9 monoclonal antibodies are currently only approved for use in specific patient groups. Furthermore, guidelines are complex and require consideration of the population being treated, a confirmed diagnosis of FH, the presence or absence of additional risk factors or clinical evidence of ASCVD, target LDL-c levels, as well as evidence of statin intolerance. Additional considerations of patient treatment preference and cost also need to be addressed. The American Heart Association and European Society of Cardiology/European Atherosclerosis Society Task Force all recommend the use of PCSK9 inhibitors as third-line pharmacotherapy after high-dose statin and ezetimibe in patients with FH. This includes patients without clinically diagnosed ASCVD, at high or very high CVD risk [122]. The European Society of Cardiology/European Atherosclerosis Society Task Force recommends the use of PCSK9 inhibitors in non-FH patients with ASCVD who are already on maximally tolerated lipid-lowering therapy (statin ± ezetimibe) but are not achieving lipid targets [123,124]. These recommendations are also endorsed by the American College of Cardiology, the American Society for Preventative Cardiology, the American Association of Clinical Endocrinologists, National Lipid Association and the FH Foundation [127].

Underpinned by new results of major outcomes studies [81,85], the American Heart Association Task Force on Clinical Practice Guidelines, together with the American College of Cardiology released an updated cholesterol guideline [125]. The recommendations regarding the use of PCSK9 inhibitors remain for high risk ASCVD patients with LDL-c ≥ 1.8 mmol/l (70 mg/dl) who are already on maximal statin therapy and ezetimibe. In addition, patients with severe hypercholesterolaemia (LDL-c ≥ 4.9 mmol/l or 190 mg/dl) whose LDL-c levels remain ≥ 2.6 mmol/l (100 mg/dl) despite treatment with statin and ezetimibe, a PCSK9 inhibitor should be considered if multiple ASCVD risk factors are present, with the caveat that long-term safety (>3 years) is uncertain and economic value is low at mid-2018 U.S. prices. For the heterozygous FH patients whose LDL-c remains ≥ 2.6 mmol/l (100 mg/dl) on maximal statin and ezetimibe therapy, a PCSK9 inhibitor should be considered. However, in patients with FH without evidence of clinical ASCVD and taking statin and ezetimibe therapy, PCSK9 inhibitors were considered to provide uncertain value at mid-2018 U.S. prices [125].

Guidelines are as yet unable to fully address the use of PCSK9 inhibitors in children, adolescents and pregnant women, which are particularly important in FH populations.

Cost-effectiveness of PCSK9 inhibitors

The cost of prescribing PCSK9 monoclonal antibodies remains a significant impediment to their widespread use. This is further compounded by all published economic evaluations being based on simulation models as none of the aforementioned trials have included prospective economic evaluation [126]. At present, PCSK9 monoclonal antibodies in the U.S. are 100-times more expensive than generic statins, with only a small fraction of this cost likely to be recovered by prevention of cardiovascular events. These high costs can impede patient access to the drug and impact long-term adherence [126]. It is unclear what the impact of recent announcements regarding reductions in price of both alirocumab and evolocumab from Rengeron/Sanofi and Amgen respectively, will have on their use. The Cardiovascular Disease Policy Model determined that use of PCSK9 monoclonal antibodies in patients with heterozygous FH or ASCVD did not meet generally acceptable cost-effectiveness thresholds and would substantially increase U.S. healthcare costs [127], with similar results seen in Australia [128]. Other economic models had variable conclusions on the economics of PCSK9 monoclonal antibodies, with studies sponsored by drug manufacturers reporting more favourable results. All models did agree that PCSK9 monoclonal antibodies will increase lifetime medical costs and any cost savings from preventing cardiovascular events would only offset a small fraction of the added cost of the drug. Furthermore, while all models projected improved survival from the use of these inhibitors, they differed in their assumptions of underlying risk of the treated population studied and therefore the number of quality of life years added by the therapy [126]. Two competing risk models developed from the Treating to New Targets trial suggest that individual estimated lifetime benefit from PCSK9 inhibition in patients with stable CAD on high-dose statins varied from <6 to ≥12 months free from stroke or MI. The greatest benefit was expected in younger patients with higher risk burden and relatively high LDL-c [129].

Insights from the FOURIER trial suggest that a statin plus PCSK9 monoclonal antibody had a low probability (<1%) of being cost effective at the commonly accepted $100000 per quality-adjusted life-year societal threshold. Furthermore, PCSK9 monoclonal antibodies produced a negative return on investment for 86% of private payers. Threshold analysis suggested the price of the drug would need to drop by 62% to meet conventional cost-effectiveness standards [130]. Recent budget impact analysis of PCSK9 monoclonal antibodies in patients with heterozygous FH or ASCVD suggest that the budget impact of these inhibitors as add-ons to statin therapy is relatively low compared with published estimates of other specialty biologics, with drug cost rebates and discounts likely to further reduce the impact [131]. A more recent analysis of the ODYSSEY OUTCOMES trial presented at the 2018 American Heart Association meeting suggested that at high LDL-c levels, alirocumab is cost effective at ≤$6319 per year, with the accepted willingness to pay threshold being $100000 per quality-adjusted life-year. It should be noted however, that this was based on non-significant mortality data, U.S. healthcare costs and rebates, and with no follow-up visits factored in. Further investigation regarding this cost-effectiveness was also investigated using data gathered from the Department of Veterans Affairs [132].

In general, there is little threat from ‘biosimilars’ after patent expiration, meaning the market exclusivity period for PCSK9 monoclonal antibodies is likely to be longer than the initial patent. When biosimilars are developed, they typically cost only 15–20% less than the original product, in contrast with generic small-molecule drugs (generally 85–90% cheaper). Additionally, the population who may benefit from PCSK9 monoclonal antibodies is large, with nearly 10 million U.S. adults potentially eligible. Their use as a preventative treatment for atherosclerosis, rather than as a cure, also requires their lifelong use, thus extending the cost [126].

Conclusions and future perspectives

The science of PCSK9 inhibitors continues to evolve, with a greater understanding of the role of this protein in hepatic lipid control facilitating new and improved therapeutic treatment options. Although the monoclonal antibodies remain the most successful PCSK9 inhibitors to date, alternative strategies are actively pursued. Of these, the most promising are the siRNA therapies and vaccines against PCSK9. The benefits of siRNA include the potential for sustained cholesterol reductions via reduced levels of total PCSK9. In addition, treatment administration is less frequent compared with the monoclonal antibodies, with increased target selectivity of siRNA therapy increasing drug potency and reducing the potential for side effects. Vaccines may offer a more affordable treatment option, which combined with a simple administration route would facilitate greater compliance and widespread use in the patient populations that require them. However, studies into the safety and efficacy of both of these inhibitors is still required, in addition to studies on their use in conjunction with other lipid-lowering therapy, their effect on other atherogenic particles such as Lp(a) and triglycerides, as well as comparisons of effectiveness with approved monoclonal antibodies, alirocumab and evolocumab.

While alirocumab and evolocumab offer additional therapeutic options for patients with severe hypercholesterolaemia associated with FH, as well as high-risk ASCVD patients unable to meet lipid targets, several issues still remain. Despite recent clinical trial evidence investigating their impact on clinical outcomes and mortality, their long-term (>5 years) use as well as the long-term implication of very low levels of LDL-c, particularly with respect to neurocognition, still needs to be confirmed. Additionally, patient restrictions and the large cost currently associated with using PCSK9 monoclonal antibodies makes their widespread use limited. In addition, the use of these monoclonal antibodies in conjunction with other treatments, including background high intensity statins and ezetimibe still requires investigation. To date, none of the large clinical outcomes trials have answered questions regarding PCSK9 monoclonal antibody use and mortality, as well as inter-individual variability and effects on other lipid targets, including Lp(a) and triglycerides and their contribution to reduced ASCVD risk. In high-risk patients on statins with residual elevation in triglycerides, eicosapentanoic acid (EPA) treatment was recently shown to have beneficial effects on ischaemic events, including cardiovascular death, that was accompanied by reductions in triglyceride levels [133]. The clinical trials of anti-inflammatory agents have not been universally consistent [134,135], suggesting that the combination of a PCSK9 inhibitor and high-dose EPA could be the winning new combination on top of standard therapies in the secondary prevention of ASCVD. Furthermore, the use of all lipid-lowering therapies, including PCSK9 inhibitors, in children, adolescents, the very elderly and pregnancy still requires investigation.

Funding

The authors declare that there are no sources of funding to be acknowledged.

Competing interests

GFW acknowledges research support and honoraria for advisory panels from Regeneron, Sanofi, Amgen, Gemphire and Kowa. NCW and MMP have no competing interests.

Abbreviations

     
  • apoB

    apolipoprotein B

  •  
  • apoE

    apolipoprotein E

  •  
  • ASCVD

    atherosclerotic cardiovascular disease

  •  
  • CHD

    coronary heart disease

  •  
  • EGF-A

    epidermal growth factor-like repeat A

  •  
  • EPA

    eicosapentanoic acid

  •  
  • FH

    familial hypercholesterolaemia

  •  
  • HNF-1α

    hepatocyte nuclear factor 1α

  •  
  • IDL

    intermediate-density lipoprotein

  •  
  • LDL-c

    low-density lipoprotein cholesterol

  •  
  • Lp(a)

    lipoprotein (a)

  •  
  • MI

    myocardial infarction

  •  
  • PCSK9

    proprotein convertase subtilisin/kexin type 9

  •  
  • SREBP-2

    sterol regulatory element-binding protein 2

  •  
  • VLDL

    very low-density lipoprotein

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