PCSK9 inhibition and LDL cholesterol lowering: the biology of an attractive therapeutic target and critical review of the latest clinical trials

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1 Clinical Lipidology ISSN: (Print) (Online) Journal homepage: PCSK9 inhibition and LDL cholesterol lowering: the biology of an attractive therapeutic target and critical review of the latest clinical trials David Rhainds, Benoit J Arsenault & Jean Claude Tardif To cite this article: David Rhainds, Benoit J Arsenault & Jean Claude Tardif (2012) PCSK9 inhibition and LDL cholesterol lowering: the biology of an attractive therapeutic target and critical review of the latest clinical trials, Clinical Lipidology, 7:6, To link to this article: Copyright 2012 Future Medicine Ltd Published online: 18 Jan Submit your article to this journal Article views: 146 View related articles Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 18 November 2017, At: 00:17

2 review PCSK9 inhibition and LDL cholesterol lowering: the biology of an attractive therapeutic target and critical review of the latest clinical trials PCSK9 is a serine protease expressed in the liver and the intestine. Gain-of-function mutations in PCSK9 are associated with autosomal dominant hypercholesterolemia, independently of mutations in apob-100 or the LDL receptor (LDLR). Wild-type PCSK9 binds to the extracellular domain of the LDLR at the surface of hepatocytes, is internalized with the receptor and redirects it to lysosomes where the complex is degraded. Conversely, lossof-function mutations in PCSK9 are associated with lower levels of LDL cholesterol (LDL-C) and reduced cardiovascular risk. Inhibition of PCSK9 binding to the LDLR with human monoclonal antibodies or reduction of PCSK9 expression with sirna are the two main strategies to reduce LDL-C, which are actively pursued in the clinical development phases. In this article, the current evidence for PCSK9 as a target for reducing LDL-C and results of Phase I and clinical trials with anti-pcsk9 therapeutic strategies are reviewed. keywords: familial hypercholesterolemia n PCSK9 n sirna n statin LDL cholesterol as a cardiovascular disease risk factor LDL cholesterol (LDL C) is a well established risk factor for cardiovascular disease (CVD). The evidence supporting this belief arises from the well accepted notions that: n High levels of LDL C have consistently been associated with increased risk of CVD in numerous population based prospective studies [1] ; n n Both familial (dominant) hypercholester olemia (FH) and primary (polygenic) hyper cholesterolemia, characterized by elevated LDL C levels, are associated with premature atherosclerosis [2] ; Cholesterol has been shown to trigger the earliest steps of atherogenesis in animal models [3] ; n LDL receptor n monoclonal antibody which block cholesterol synthesis and increase LDL receptor (LDLR) levels in the liver. In this article, we review in vitro and in vivo evidence for inhibition of the PCSK9 mediated degradation of LDLR in the liver as a new therapeutic strategy to increase LDL uptake in the liver and reduce LDL C levels by a mechanism that is different from statins and provides further LDL C reduction. A comprehensive assessment of the different strategies under development to inhibit PCSK9 in humans is presented and the clinical efficacy, tolerability and safety of PCSK9 inhibitors in published Phase I and Phase clinical studies is discussed. and planned Phase I studies of PCSK9 inhibitors are also listed. In the context of clinical management of elevated LDL C levels, increasingly lower LDL C treatment goals have been adopted by international panels of experts [5]. To reach these goals a relatively small number of drug classes have been developed and are currently in use, including the widely prescribed inhibitors of HMG CoA reductase also known as statins, Therapeutic arsenal for reduction of LDL C levels HMG CoA reductase inhibitors or statins were introduced into the clinic 25 years ago. With clinical trials suggesting that statins usually reduce LDL C levels by approximately 4050%, statin therapy has rapidly become the cornerstone of lipid lowering therapy [5]. The evidence from large scale clinical trials suggests that statins reduce CVD risk by 2540% depending on statin dose and patient characteristics, and that achieving lower LDL C levels with increasing statin dose further decreases cardiovascular risk /CLP Future Medicine Ltd Clin. Lipidol. (2012) 7(6), n Drugs that target blood LDL C levels delay and may prevent the onset of CVD [4]. David Rhainds*1, Benoit J Arsenault1, Jean Claude Tardif*1,2 1 Atherosclerosis Research Group, Montreal Heart Institute, 5000 Belanger Street, Montreal, Quebec, H1T 1C8, Canada 2 Faculty of Medicine, Université de Montréal, Montreal, Quebec, Canada *Authors for correspondence: Tel.: ext Fax: david.rhainds@icm-mhi.org jean-claude.tardif@icm-mhi.org part of ISSN

3 review Rhainds, Arsenault & Tardif in primary and secondary prevention studies [4]. A recently published report has also provided evidence suggesting that statin therapy decreases cardiovascular risk similarly across all risk factor categories, lending support for the widespread use of this class of drugs [6]. Although the efficacy and safety profile of statin therapy is acceptable, it must be considered that more than half of cardiovascular events are not being prevented by statins. This may be attributable to the fact that such individuals have elevated residual risk, which encompasses the spectrum of other unrelated risk factors for CVD, such as the hightriglyceride, low HDL cholesterol (HDL C) dyslipidemia, elevated blood pressure, insulin resistance or Type 2 diabetes, among others [7]. Other individuals may be less compliant to statin therapy because of muscle pain or other forms of statin intolerance. There is an apparent unmet medical need for these individuals who are not compliant to statin therapy in which lifestyle modification therapy is often not sufficient to reduce LDL C levels and who could benefit from additional pharmacological treatments. Several classes of drugs aiming at reducing blood LDL C, such as cholesterol absorption inhibitors (ezetimibe), niacin and bile acid sequestrants are currently available in the clinic and have been used in the past years for the treatment of hypercholesterolemia (HC) without considerable influence on CVD risk. On the other hand, investigational agents targeting a handful of molecular targets may soon become available for the treatment of FH (1:500 frequency for heterozygotes) and other forms of primary HC. Mipomersen is an antisense oligonucleotide delivered by subcutaneous (sc.) injection that inhibits the hepatic synthesis of apob-100 and represents one of these new therapeutic agents that has been tested in a Phase I trial in patients with homozygous FH on maximally tolerated lipid-lowering therapy [8]. In addition to reducing LDL C levels by 25% after 26 weeks, mipomersen also provides significant decreases in apob and Lp(a) levels, without changing apob-48 or chylomicron levels. This drug is currently being evaluated by regulatory authorities as a potential adjunct treatment to currently available drugs for patients with FH [301]. Inhibition of microsomal triglyceride transfer protein (MTP) with an oral compound called lomitapide is another strategy that is currently being pursued to reduce LDL C levels in patients with FH. MTP primarily acts in the liver and the intestine to assemble 622 Clin. Lipidol. (2012) 7(6) triglyceride-rich apob-containing VLDLs and chylomicrons. Studies have shown that this compound could decrease LDL C and apob levels by approximately 50% [9]. Close follow-up of patients treated with these investigational drugs will be important as administration of mipomersen has been associated with adverse injection site reaction in most study participants (76%) [8]. In addition, by reducing the secretion of apob-containing lipoproteins, these agents have been shown to promote hepatic fat accumulation [10]. Carefully monitoring liver transaminases and directly evaluating hepatic fat content, or slowly titrating up to identify doselimiting side effects in the case of lomitapide may be warranted. Rationale for PCSK9 as a target for LDL C lowering Genetic basis The PCSK9 gene spans a 25 kb region on the short arm of chromosome 1 (1p32.3) and encodes a 692 amino acid serine protease. To date, more than 50 nonsynonymous variants in PCSK9 have been shown to affect plasma LDL C levels in humans as reported in the PCSK9 mutation database [302]. A selection of variants corresponding to gain-of-function (higher LDL C levels) and loss-of-function (lower LDL C levels) PCSK9 mutations is shown in Figure 1A. In 2003, the first evidence supporting a role of PCSK9 in altering lipoproteincholesterol metabolism in humans came from the identification of two gain-offunction mutations in PCSK9, S127R and F216L, in two French families with an elevated proportion of autosomal dominant HC (ADH) cases unrelated to LDLR or apob mutations [11]. The S127R mutation may interfere with cellular trafficking and secretion of PCSK9 [12], while the F216L mutation could alter a potential cleavage site in PCSK9, thereby increasing its half-life in the blood [13]. This breakthrough led to the identification of additional families and additional mutations in PCSK9 that are present in approximately 2% of ADH patients. In the general population, gain-of-function variants in PCSK9, such as the E670G, are associated with an increased risk of ischemic stroke, as suggested by the Belgian Stroke Study [14], and with the presence of atherosclerosis in participants of the LCAS [15] and of the PLIC study [16]. The genetic evidence suggesting a potential role of PCSK9 inhibition in reducing blood LDL C levels came from the identification

4 PCSK9 inhibition & LDL cholesterol lowering S P 30 SP H VFAQ D 152 Prodomain LDL-C L6 L5 C-ter D374Y L4 L3 L2 R469W A443T L253F EGF-B Pro 688 P 692 R357H N425S R496W H553R R218S LDL-C R46L G106R Q152H R97 Y142X Cat EGF-A 533 G 425 F216L D129G L7 386 S Catalytic domain S127R C-ter 317 N review L1 Q554E C679X NH2 β LDLR type A repeats EGF-C EGF-like repeats YWTD repeats of the β-propeller domain Serine/threonine-rich region (O-glycosylated) C Transmembrane domain Clin. Lipidol. Future Science Group (2012) Figure 1. Schematic of PCSK9 and LDL receptor structure and interaction. (A) Major structural features of PCSK9 (upper part) and location of selected human PCSK9 mutations (lower part). The locations are aspartate (D), histidine (H) and serine (S) residues that comprise the catalytic triad, as well as the asparagine (N) forming the oxyanion hole, are indicated in black letters. Location of known post-translational modifications including sulfation (S) at Y38, phosphorylation (P) at S47 and S688 and N-glycosylation (G) at N533 is indicated with color bullets. Key amino acid residues defining the autocatalytic cleavage site location (VFAQ ) between the prodomain and catalytic domain are also indicated. Location of gain-of-function mutations leading to increased LDL cholesterol levels (shown in red) and loss-of-function mutations leading to decreased LDL cholesterol (shown in green) are indicated. See [302] for an updated database of PCSK9 mutations. (B) Major structural domains of the LDLR and its interaction with PCSK9. The LDLR is a single-pass transmembrane protein with a short cytoplasmic tail (C-ter) and a large extracellular domain, which is involved in binding of PCSK9 and LDL particles. Seven LDLR type A repeats form the ligand-binding domain for attachment to LDL particles in a calcium-dependent manner. Two EGF-like repeats (EGF-A and EGF-B), a six-bladed b-propeller domain and a third EGF-like repeat (EGF-C) altogether form the EGF-precursor homology domain involved in PCSK9 binding at the cell surface. The LDLR extracellular domain is also comprised of a serine- and threonine-rich domain that undergoes extensive O-glycosylation in the terminal secretory pathway. As indicated, the PCSK9 catalytic domain binds to the LDLR EGF-A module, while the PCSK9 prodomain contacts the LDLR b-propeller. C-ter: C-terminal; LDLR: LDL receptor; SP: Signal peptide. of loss-of-function mutations and common polymorphisms associated with lower LDL C levels (Figure 1A). One of the most striking examples is the daughter of a black woman enrolled in the Dallas Heart Study who carries two mutations in PCSK9 (Y142X and DR97) [17]. She has virtually no detectable PCSK9 in the blood and remarkably low LDL C levels (14 mg/dl) [17]. Interestingly, this aerobics instructor is an apparently healthy, fertile, normotensive and well-educated woman with normal liver and renal function. Seeking mutations in PCSK9 in a population from Zimbabwe, Hooper and colleagues identified a subject homozygous for the C679X mutation with very low LDL C levels (16 mg/dl) [18]. Unfortunately, no information is available regarding the health status of this individual. Long-term follow-up of these individuals with very low LDL C levels secondary to loss-of-function mutations in PCSK9 will be important to identify potential adverse effects of PCSK9 inhibition. Nonetheless, investigators of the Dallas Heart Study also documented that a common variant in PCSK9, R46L, was associated with lower LDL C levels in white 623

5 review Rhainds, Arsenault & Tardif subjects [19]. To document the impact of lossof-function mutations in PCSK9 on CVD risk in the general population, investigators of the ARIC study genotyped the Y142X and C679X variants in 3363 black participants and the R46L variant in 9524 white participants [20]. In blacks, 85 participants carried one of the two sequence variants. These participants had on average 29% lower LDL C levels and a lower carotid intimamedia thickness. During the 15 year follow-up in initially CVD-free black participants, only one individual with a sequence variant in PCSK9 had a cardiovascular event, which conferred an 88% reduction in CVD risk compared with individuals without sequence variants in PCSK9. In whites, the 301 carriers of the R46L polymorphism had 15% lower LDL C levels and a reduction of 44% in the incidence of CVD compared with noncarriers [20]. The CARDIA study also suggested that genetic variants in PCSK9 contributed to lower LDL C levels in AfricanAmericans and in whites, and documented that these differences could be tracked over time [21]. This study was also one of the first to document that such mutations exerted their effect additively to apoe genotypes as across the spectrum of the studied PCSK9 genetic variants, participants with either the e2/e2 or e3/e2 isoforms of apoe had the lowest LDL C levels. One of the most comprehensive population-based assessment of PCSK9 genotypes and risk of CVD has been performed by Danish investigators in three large-scale studies (Copenhagen City Heart Study, Copenhagen General Population Study and Copenhagen Ischemic Heart Disease Study) that included more than 45,000 participants from prospective and casecontrol studies [22]. In the three studies combined, carriers of the R46L polymorphism had 13% lower LDL C levels and a 30% lower risk of ischemic heart disease. In the subsequent meta-ana lysis of seven studies, the mean reduction of LDL C levels associated with the R46L polymorphism was 12% and the reduction in ischemic heart disease risk was 30%. The fact that several of the above studies have reported that CVD risk reduction considerably exceeds what could be expected with similar LDL C reductions in other circumstances, such as in statin trials, and the observation that mutations in PCSK9 are associated with low LDL C starting in childhood [23], lend great support to the concept that lifetime exposure to low LDL C levels entails lowering of CVD risk. In line with this concept, it was suggested 624 Clin. Lipidol. (2012) 7(6) that statin treatment initiated at a younger age in patients at high risk of CVD could reduce LDL C levels before severe atherosclerosis has been established [24]. Although genetic variations in the region harboring PCSK9 have a significant impact on plasma LDL C, little is known about the contribution of common genetic variants in predicting plasma PCSK9 levels. In normal subjects, mean plasma PCSK9 has been reported between 6590 ng/ml (12 nm), although the levels can vary almost 100-fold from 30 to 3000 ng/ml [25,26]. In an attempt to identify genetic variants associated with plasma PCSK9 levels, Chernogubova and colleagues performed a genome-wide association study in two Swedish cohorts (n = 1215) [27]. Although no unsuspected regions showed association with plasma PCSK9 levels, they reported, in a candidate gene approach, that the R46L polymorphism explained less than 1% of the variation in PCSK9 levels, which may be attributable to the low frequency of this protective allele (1.9%). Additional work investigating the contribution of clinical and genetic factors associated with plasma PCSK9 levels is needed to better understand the molecular basis of high PCSK9 levels and their effect on LDL C levels. Molecular basis of regulation of LDLR levels by PCSK9 PCSK9 is a serine protease predominantly expressed in the liver, intestine and, to a lesser extent, in the kidneys [28]. Its sequence shows homology with bacterial subtilase in its prosegment and catalytic region, while its C-terminal histidine- and cysteine-rich domain adopts a new fold with homology to homotrimeric resistin, not found in other proprotein convertases [29]. The 692 amino acid prepro-pcsk9 has an apparent molecular weight of 74 kda. The protein is N-glycosylated on one site, phosphorylated on two other sites and sulfated on one clearly identified residue, as shown in Figure 1A [13,28,30]. After cotranslational removal of the signal peptide, pro-pcsk9 undergoes an autocatalytic cleavage at Q152 in the endoplasmic reticulum into two products of 14 kda (prodomain) and 60 kda (mature PCSK9), respectively (Figure 1A). The prodomain or prosegment remains associated with the mature protein and acts as a chaperone in the secretory pathway. Contrary to other PCs, it is secreted with PCSK9 as an inactive complex. Absence of the autocatalytic cleavage

6 PCSK9 inhibition & LDL cholesterol lowering critically impacts PCSK9 secretion as shown by mutations such as H226A in the catalytic triad and Q152H in the autocatalytic cleavage site [28,31]. Crystallographic studies have shown that the prodomain prevents access to the catalytic site in the complex by potential substrates [29,32]. Accordingly, the successful production in vitro of a catalytically inactive PCSK9 protein by coexpressing the wild-type prodomain as a chaperone for the mature protein inactivated by mutation of the catalytic triad serine (S386A) confirmed that PCSK9 activity is not required for its action on the LDLR in vitro and in mice [3335]. As a secreted protein, PCSK9 circulates in human plasma at a mean concentration of 6590 ng/ml [3638]. As shown by many groups, extracellular PCSK9 binds to the cell surface of LDLR and is internalized with the receptor. The complex is then dragged into lysosomes where both proteins are degraded [25,3942]. It has been suggested that LDL particles may interfere with PCSK9 binding to the LDLR, but this has not been defined at the molecular level as it relates to the ill-described conformation of the LDLR when bound to LDL [43]. The LDLR is the prototypical member of a family of modular proteins involved in clearance of lipoproteins and proteaseantiprotease complexes and intracellular signaling. This 839 amino acid protein has a large N-terminal extracellular domain (ECD), one transmembrane domain and a short C-terminal cytoplasmic tail, which harbours the internalization signal. The LDLR structural domains are shown in Figure 1B. The LDLR ECD is comprised of three types of modules, which are present in all LDLR family members in various numbers: LDLR type A (LA) repeats, EGF repeats and a b-propeller (or YWTD) module, plus a serine/threonine-rich region close to the plasma membrane, which is highly O-glycosylated [44]. In the LDLR ECD, the ligand-binding domain is formed by seven LA repeats, while the EGF-precursor homology domain is formed by two EGF repeats (EGF-A and EGF-B), the b-propeller and another EGF repeat. Binding of PCSK9 to the LDLR is calcium dependent (as is binding of LDL to the receptor) and requires the presence of EGF-A to allow binding and degradation of the receptor [45]. A minimal array of three LA repeats is also required to cause degradation of the complex [46]. Absence of the entire LDLR ligand-binding domain or b-propeller allows internalization but not degradation of the LDLR [46]. The binding review affinity for the LDLR increases at acidic ph in various assays, which suggests that stronger interactions between PCSK9 and LDLR are formed at endosomal ph [45,47,48]. Direct binding between EGF-A and fulllength PCSK9 has been demonstrated from co-crystals formed at both acidic [47] and neutral ph [49,50]. The PCSK9 catalytic domain forms a small (~500 Å 2 ) and relatively flat contact surface with the EGF-A repeat [47,51]. This interaction involves distant amino acid patches of the N-terminal catalytic domain (amino acids ) and close to the catalytic site (amino acids ) of PCSK9 [47]. Recently, an additional contact patch of 70 Å 2 was identified in co-crystals of PCSK9 and full-length LDLR ECD at neutral ph, which is formed by the prosegment of PCSK9 and the b-propeller of the LDLR [51]. This dual binding site of PCSK9 on the LDLR is shown in the PCSK9LDLR complex of Figure 1B. Comparison of LDLR ECD at neutral versus acidic ph suggests that PCSK9 holds the LDLR EGF-precursor homology domain in an extended conformation and may hamper a conformational change that is required to promote its recycling (as well as LDL release) [51]. In all models of the PCSK9LDLR interaction, the PCSK9 C-terminal domain is solvent exposed and allows binding to modulators such as annexin A2 [52]. The C-terminal could also recruit coreceptors implicated in internalization and/or trafficking to lysosomes of PCSK9LDLR complexes [29,51,53]. Recently, the PCSK9 C-terminal domain has been suggested to interact with the LDLR-binding domain at endosomal ph [54,55]. Absence of either domain causes rapid release of PCSK9 at acidic endosomal ph, in agreement with the established requirement of the C-terminal domain for LDLR degradation [40,46,56]. A schematic view of PCSK9 trafficking, sites of complex formation with the LDLR and resulting degradation is shown in Figure 2. While extracellular PCSK9 was shown to induce degradation of the LDLR in numerous publications, PCSK9 binding to LDLR could also occur early in the secretory pathway compartments while both proteins are trafficking towards the cell surface. Intracellular PCSK9 could interact with the LDLR precursor, that is, its nonterminally glycosylated form, and direct it to an acidic/lysosomal compartment for degradation [39,40]. The concept of an intracellular route for PCSK9-mediated LDLR degradation is supported by studies showing 625

7 review Rhainds, Arsenault & Tardif Secreted PCSK9 LDL LDL binding Coated pit LDLR recycling Endocytosis Golgi Coreceptor? Endosome? PCSK9 LDLR pro-pcsk9 ER Lysosome Gene mrnas Nucleus Clin. Lipidol. Future Science Group (2012) Figure 2. Cellular trafficking of PCSK9, mechanism of PCSK9-induced LDL receptor degradation and strategies for PCSK9 inhibition. Soon after its synthesis in the rough ER and removal of the signal peptide (not shown), the pro PCSK9 undergoes an autocatalytic cleavage (blue arrow) of its prodomain (red), which remains complexed to mature PCSK9 even after its secretion in the extracellular compartment. It has been suggested that PCSK9 and LDLR could bind in post-er secretory compartments (Golgi and/or trans-golgi network) and lead to lysosomal degradation of the complex by an uncharacterized route (dashed arrow). The PCSK9 is ultimately secreted and binds to the LDLR at the extracellular neutral ph. After binding to an LDL particle and clustering in clathrincoated pits, the LDLR normally undergoes autosomal recessive hypercholesterolemia protein-dependent endocytosis into early endosomes, where it undergoes a conformational change due to the lower ph. The ligand-binding domain folds into the b-propeller domain in a closed conformation, which releases LDL particles and allows protected LDLR to recycle towards the plasma membrane. When PCSK9 binds to the cell surface LDLR, the complex is endocytosed in an autosomal recessive hypercholesterolemia proteindependent manner, but the interaction is strengthened by the lower ph. Moreover, the LDLR ligand binding domain interacts with the C-terminal domain of PCSK9 (dashed arrow) and adoption of the closed LDLR conformation is prevented, which results in the complex trafficking to late endosomal/lysosomal compartment where it is degraded in a manner similar to LDL particles. The C-terminal domain of PCSK9, while not essential for PCSK9 binding, is required for endocytosis and degradation of the LDLR, and binding to modulators such as annexin A2. Thus, the participation of a coreceptor (green stick) for PCSK9 at the cell surface or in endosomes has been suggested. Strategies to inhibit PCSK9 activity to prevent formation of the PCSK9LDLR complex in the liver are indicated: (A) inhibition of PCSK9 synthesis by antisense oligonucleotide- or sirna-based delivery to hepatic cells, or reduction of PCSK9 gene transcription; (B) inhibition of PCSK9 autocatalytic processing by small-cell-permeant molecules; (C) extracellular capture of mature PCSK9 by antibodies against LDLR-binding domains of PCSK9; (D) inhibition of PCSK9 binding to LDLR by small molecules; (E) putative inhibition of PCSK9 binding to a coreceptor involved in its endocytosis or trafficking to lysosomes. ER: Endoplasmic reticulum; LDLR: LDL receptor. that wild-type PCSK9 or gain-of-function mutants expressed in some cells do not reduce LDLR in neighboring untransfected cells [57] or may even degrade LDLR despite being retained intracellularly [12]. Also, the effect of 626 Clin. Lipidol. (2012) 7(6) extracellular PCSK9 on LDLR levels has often been shown at supraphysiological levels of PCSK9 (>100 nm), such as in human PCSK9 liver-specific transgenic mice [25,58], in mice injected with recombinant PCSK9 [35] or with

8 PCSK9 inhibition & LDL cholesterol lowering cultured hepatocytes [57]. Taking into account the relatively low affinity of PCSK9 for the LDLR at neutral ph (~170 nm) [59], it seems logical that a high local concentration of PCSK9 is required to induce paracrine effects on the LDLR in the microenvironment of hepatic cells. This might be compensated with gainof-function mutants that show higher affinity for the LDLR, such as the D374Y mutant [49,50,59]. Despite being secreted at similar levels (1012 nm) by transfected HepG2 hepatocytes, D374Y, but not the wild-type PCSK9, acts on neighboring cells to decrease LDLR in a paracrine fashion [57]. Finally, secreted PCSK9 can be cleaved in its catalytic domain by hepatic furin, a basic amino acid PC, in vitro and in vivo. Inactivation of PCSK9 activity by furin causes a significant fraction of PCSK9 to circulate in the bloodstream as an inactive fragment [13,60]. Thus, the action of PCSK9 on the LDLR may take place in the secretory pathway, at the cell surface or after PCSK9 has circulated for some time before returning to the liver. This has led to the development of various inhibition strategies aiming at interrupting one or more of these steps. Inhibition of PCSK9 as a strategy to increase LDLRs & lower LDL C levels The identification of PCSK9 as a gene responsible for ADH in French families, in which mutations in APOB or LDLR were excluded, was rapidly followed by studies where its impact at the wholebody level was demonstrated in mice, confirming its natural role as a modulator of liver LDLR and plasma LDL C levels. Indeed, PCSK9 knock-out mice exhibit a 4050% decrease in total cholesterol [58,61], which results from the almost complete absence of LDL C (-80%) and from a 30% decrease of HDL C likely caused by an increased clearance of apoe-containing HDL in mice [61]. There was no other obvious phenotype in the PCSK9 knockout mouse aside from their limited capacity for liver regeneration after partial hepatectomy [58]. Conversely, liverand kidney-specific PCSK9 transgenic mice had marked elevations of LDL C [25,58,62]. These studies have fostered the efforts to develop therapeutic agents aiming at blocking PCSK9 activity towards the LDLR. Proof-of-concept studies have been performed with hepatic cell models and animal species including mice and nonhuman primates (NHP), and are described in the following section. Therapeutic strategies against PCSK9 can be broadly classified by the review mechanism of inhibition: inhibition of binding to the LDLR, inhibition of synthesis via RNAi or reduction of translation of the PCSK9 gene, and inhibition of autocatalytic processing as shown in Figure 2AE ; they can be further classified by the nature of the therapeutic agent, that is, monoclonal antibody (mab), small molecule and sirna, among others, as summarized in Table 1. Preclinical studies In vitro studies When expressed in hepatocytes, the LDLR ECD behaves as a secreted, soluble protein. Accordingly, it was shown to block degradation of the LDLR by exogenous purified PCSK9, thus acting as a decoy receptor [41]. This initial report was confirmed with LDLR ECD fragments directly involved in PCSK9 binding: either full-length EGF-A or the EGF-A/B domain peptides could inhibit binding to the LDLR and prevent its degradation in human hepatic cell lines [50,63]. A seemingly straightforward strategy is to identify small mimetic peptides that could interfere with the PCSK9LDLR interaction, despite the rather f lat-binding surface between the PCSK9 catalytic domain and the LDLR EGF-A repeat [64]. Nonetheless, a mimetic peptide (SX-PCK9) has been designed by Serometrix (NY, USA) and is undergoing structureactivity relationship studies to strengthen its allosteric interference with the PCSK9LDLR-binding step [303]. Another approach has been taken by Genentech (CA, USA) researchers who identified modified EGF-A sequences by phage display with increased affinity for PCSK9 in the absence of calcium [65]. A fusion protein of the Fc region of immunoglobulins and modified EGF-A could rescue liver LDLR in mice injected with purified PCSK9 [65]. mabs represent a clinically validated therapeutic approach to target secreted proteins [66]. As expected from the implication of PCSK9 catalytic and C-terminal domains in LDLR degradation (see Figure 1B for a schematic view of the PCSK9LDLR complex), antibodies against the PCSK9 catalytic domain could block binding to LDLR in HepG2 cells in the presence of overexpressed or exogenous purified PCSK9 [67]. On the other hand, a Fab against the C-terminal domain was inefficient in preventing binding, but blocked internalization of the complex and partially (~50%) restored LDL uptake in HepG2 cells [53]. Similarly to 627

9 review Rhainds, Arsenault & Tardif Table 1. Anti-PCSK9 therapeutic agents currently in development. Company Name Type Clinical development status Phase I reported Phase ongoing Phase reported Phase I planned Phase I completed Phase planned Phase Preclinical Phase ongoing Phase I planned Preclinical Ref. Inhibition of PCSK9 binding to LDLR Amgen AMG 145 Fully human mab Regeneron/Sanofi-Aventis REGN727/SAR Fully human mab Pfizer/Rinat Neuroscience PF /RN316 mab Novartis Merck Roche/Genentech BMS/Adnexus Serometrix LGT209 BMS SX-PCK9 mab mab mab Adnectin Small peptide mimetic [69] [85] [308] [112] [304] [303] Inhibition of PCSK9 synthesis (gene silencing) Alnylam ISIS/BMS Santaris ALN-PCS sirna in LNP (SNALP delivery) PCSK9Rx/BMS Phosphorothioate 2 -MOE RNase H antisense oligonucleotide SPC5001 Phosphorothioate LNA RNase H antisense oligonucleotide [75] Phase I Terminated in Phase I [74] Terminated in Phase I [76] Preclinical Preclinical [82] Inhibition of PCSK9 autocatalytic processing Cadila Healthcare Shifa Biomedical Small molecule Small molecule [83] Agents are sorted according to the mechanism of PCSK9 inhibition. 2 -MOE: 2 -O-monoxyethyl modified; Adnectin: 12-kDa fragment of the tenth repeat of fibronectin type I extracellular domain exposing a PCSK9-binding loop; LDLR: LDL receptor; LNA: Locked nucleic acid antisense; LNP: Lipidoid nanoparticle; mab: Monoclonal antibody; SNALP: Stable nucleic acid lipid particle. achieve blockade with a PCSK9 mab, Adnexus Therapeutics (MA, USA) and Bristol-MyersSquibb (NY, USA) are codeveloping a PCSK9 adnectin, which consists of the tenth repeat of the fibronectin type I ECD exposing a PCSK9-binding loop [304]. This type of biological agent will likely require modification (e.g., PEGylation) to increase its stability in circulation [68], but it has the advantage of being small (12 kda), thus reducing its manufacturing cost. Animal studies In animal models such as mice and NHP (rhesus or cynomolgus monkeys), results of currently available studies provide strong evidence of the clinical efficacy of therapeutic mabs directed against the catalytic domain of PCSK9 [6973] and gene-silencing strategies in reducing LDL C levels [7477]. Immunization of mice with human PCSK9 also reduces plasma LDL C due to the development of cross-reacting antimouse PCSK9 antibodies [78]. Subcutaneous, intravenous (iv.) and intraperitoneal routes of administration are all effective for reducing total cholesterol or LDL C in animal models, but the sc. route remains the preferred route for long-term 628 Clin. Lipidol. (2012) 7(6) administration in humans. Antibody-mediated PCSK9 capture was clearly effective in normal mouse models [69,72,73] and also in mice with a humanized lipoprotein profile, such as human cholesteryl ester transfer protein transgenic, LDLR hemizygous mice, which have lower HDL C and higher LDL C on a chow diet compared with normal mice [70,71]. As expected from the PCSK9 knockout mice phenotype, sustained reduction of PCSK9 activity resulted in lower HDL C levels, which impacted total cholesterol levels after mab or phosphorothioate antisense oligonucleotide injection [69,72,74]. Injected mabs were also effective in reducing total cholesterol in human PCSK9 transgenic mice, despite very high levels of circulating PCSK9 protein [69]. Significant and sustained reductions of LDL C levels in NHP have been obtained with gene-silencing and mab-based strategies. A seminal report in NHP came from Alnylam (MA, USA) using sirna encapsulated in a lipidoid nanoparticle (LNP) [79], a technology developed by Tekmira (BC, Canada) under the name SNALP (stable nucleic acid lipid particle) [80]. Infusion of a single dose of LNP resulted in a maximal 53 and 75% reduction of LDL C and

10 PCSK9 inhibition & LDL cholesterol lowering plasma PCSK9 levels after 3 days, respectively [75]. A similar LDL C reduction (-50%) was reached when a 13-mer locked nucleotide antisense was injected weekly for 4 weeks after an initial loading dose. This reduction was sustained for the treatment period and was stable up to day 56 [77]. There was no effect on HDL C levels in gene-silencing studies, in agreement with most mab injection studies in chow-fed NHP [70,72]. However, injection of Amgen s (CA, USA) mabs to NHP resulted in a 26% reduction in HDL C levels, which could be explained by the profound (~80%) reduction of LDL C levels [69]. Nonetheless, in hypercholesterolemic NHP, which show a moderate increase in LDL C (120 vs 50 mg/dl in chow-fed animals), HDL C levels remained stable [72]. While all therapeutic mabs show high-affinity binding to PCSK9, their terminal half-life is short ( days) and also dose dependent, a finding that suggests antigen-dependent clearance of the complex [73]. This is in agreement with the observed LDLR-independent clearance of PCSK9 in LDLR knockout mice and in homozygous FH subjects [81]. To circumvent PCSK9-mediated degradation of mabantigen complexes, mab J17, a refined version of mab J16 that has ph-sensitive binding to PCSK9, was engineered [72,73]. At acidic ph in the endosomal pathway, mab J17 detaches from PCSK9 and is rescued from degradation by the FcRn receptor. In mice and NHP, J17 had a maximal effect on total cholesterol equal to its parent mab J16, but the time for recovery to baseline cholesterol levels was two- to three-times longer [73]. Recycling of J17 via ph-dependent binding to PCSK9 resulted in an eightfold longer half-life in NHP. [73] Thus, antigen-dependent clearance of PCSK9mAb complexes can be counterbalanced by antibody engineering, which will increase the duration of LDL C reduction. As further reduction of LDL C in humans will often be attempted in patients who are already treated with the maximal tolerable dose of statins, demonstration of the efficacy of PCSK9 inhibitors is required in statin-treated animal models. Moreover, statins elevate plasma PCSK9 levels, an effect that could limit their LDL C-lowering effect (see section Modulation of PCSK9 expression by lipid-modulating drugs ). In NHP, two different mabs from Merck (NJ, USA) provided additional reduction of LDL C over simvastatin, that is, -20% with 1B20 and -40% with J16 [71,72]. Remarkably, addition of mab J16 to simvastatin resulted review in a total 80% decrease in LDL C levels in hypercholesterolemic monkeys [72]. Both mabbased and sirna based therapies are being investigated in clinical development phases (see section Clinical studies of PCSK9 inhibitors ). In NHP, single-dose mab-based studies revealed higher maximal effects (7080%) on LDL C levels compared with gene-silencing strategies (5055%). In the absence of any modifications to enhance antibody half-life, frequency of administration and absence of liver toxicity will be critical to the success of both mab-based and sirna-based strategies. Notably, one monkey injected with PCSK9 sirna in LNP showed aspartate aminotransferase and alanine aminostransferase levels that were more than threefold the upper limit of normal [75]. An alternative strategy for PCSK9 inhibition is based on the absolute requirement of autocatalytic processing to allow for proper folding and secretion of PCSK9. Recently, patients carrying the Q152H mutation in the autocatalytic cleavage site were identified in FrenchCanadian kindred. In vitro, the mutant protein did not undergo processing, was not secreted and, in fact, acted as a dominant negative protein over wild-type PCSK9 [31]. These findings support the concept that the development of an orally active, cell-permeable small molecule inhibiting the autocatalytic processing of PCSK9 in the endoplasmic reticulum can be envisioned [82]. Notably, two companies have undertaken the identification of such molecules, Shifa Biomedical (PA, USA) [83] and Cadila Healthcare (Ahmedabad, India) [201]. These molecules are still in their preclinical development phase. Clinical studies of PCSK9 inhibitors Taking into consideration the evidence provided by studies investigating human loss-of-function mutations, in vitro studies and animal models, therapeutic strategies to inhibit PCSK9 activity in humans have rapidly progressed through clinical development phases. A summary of Phase I and clinical studies performed as of November 2012 is presented in Tables 2 & 3. Studies which have been published in peerreviewed journals or in an abstract form will be discussed below, including molecules from Alnylam (ALN PCS02), Amgen (AMG 145) and Regeneron (NY, USA)/Sanofi (Paris, France; REGN727), with particular emphasis on their efficacy, safety and tolerability. Because very limited information is available on the clinical 629

11 review Rhainds, Arsenault & Tardif Table 2. Clinical studies of anti-pcsk9 therapeutic agents, including completed Phase I studies. Therapeutic Number of patients; BL LDL C Duration; agent mean age (years); cohort (mg/dl) dosage Plasma PCSK9 (% change) LDL C (% change from BL) ALN PCS02 Mean -68% (peak -84%) Mean -41% (peak -50%) NCT Change in LDL C related to degree and duration of reduction in free PCSK9 Max -2865% iv. until day 64; max -3346% sc. until day 22 NCT Mean in combined Atorva (n = 51) at day 57: -39% 50 mg -54% 100 mg -61% 150 mg -81% Q1W -75% Q2W -66% Q4W NCT % Q2W NCT REGN727 n = 32; 46; volunteers, no statins 146 n = 40; n = 32; 35; volunteers 133 n = 21; 40; hefh on low/moderate dose Atorva n = 30; 52; HC on low/moderate dose Atorva n = 10; HC on diet; no CAD AMG 145 RN316/ PF weeks; single-dose, iv. ( mg/kg) Single-dose (0.312 mg/kg iv., mg sc.) 6 weeks; multiple doses, sc. (50, 100 and 150 mg on days 1, 29 and 43) n = 51; 58; HC on moderate dose statin (Rosuva <40, Atorva <80 or Simva 2080 mg) HC on high-dose statin (Rosuva 40 mg or Atorva 80 mg) n = 48; volunteers >130 n = 25; HC on Atorva 40 mg SPC5001 Terminated in 2011 ISIS/ Terminated in 2010 BMS weeks; multiple doses, sc. (Q1W 6, Q2W 3, Q4W 2 ) 6 weeks; multiple doses, sc. (Q2W 3 ) Single escalating doses, iv. (0.318 mg/kg) Single-dose, iv. (0.5 or 4 mg/kg) Ref. [84,305] NCT [85] [85] NCT [86] [86] Completed NCT Completed NCT NCT NCT Full details of ongoing trials can be found at ClinicalTrials.gov [309]. Atorva: Atorvastatin; BL: Baseline; CAD: Coronary artery disease; LDL C: LDL cholesterol; HC: Hypercholesterolemia; HeFH: Heterozygous familial hypercholesterolemia; iv.: Intravenous; Q1W: Every week; Q2W: Once every 2 weeks; Q4W: Once every 4 weeks; Rosuva: Rosuvastatin; sc.: Subcutaneous; Simva: Simvastatin. development of mabs by other companies such as Roche/Genentech, Merck, Novartis (Basel, Switzerland) and Pfizer (NY, USA), they will not be described further. Efficacy & regimen of PCSK9 inhibitors In Phase I studies, the three compounds were tested in statin-naive patients with high LDL C and no evidence of coronary artery disease (CAD) [8486]. Alnylam s results with ALN PCS02, a LNP-delivered sirna, have shown a mean maximal 41% reduction in LDL C levels after a single dose of 0.4 mg/kg iv. after 4 weeks [84,305]. This was accompanied by a 68% decrease in plasma PCSK9, independently of baseline levels. It is expected that higher doses of ALN PCS02 will be tested to match LDL C reductions obtained with mabs, but its efficacy will likely have to be documented further: after sc. injection rather than iv. injection, in hypercholesterolemic subjects (with or without 630 Clin. Lipidol. (2012) 7(6) CAD) and in statin-treated patients with higher PCSK9 levels (see section Modulation of PCSK9 expression by lipid-modulating drugs ). Regeneron/Sanofi s REGN727 was tested by Stein and colleagues in volunteers with high baseline LDL C (mean: 133 mg/dl) by iv. or sc. injection with single ascending doses [85]. Mean maximal LDL C level reductions were 65% (for 64 days) and 46% (for 22 days) after iv. or sc. injection, respectively. The higher maximal dose of REGN727 tested in the iv. group (i.e., 12 mg/kg [~800 mg] compared with 250 mg sc.) could be responsible for the longer duration and maximal effect on LDL C [85]. In a multiple-dose protocol in a young population of hypercholesterolemic patients taking low/moderate statin doses, of which 21 were heterozygous FH, sc. administration of REGN727 decreased LDL C levels by up to 61%, but a rebound in LDL C level was observed 4 weeks after the injection [85]. These

12 PCSK9 inhibition & LDL cholesterol lowering review Table 3. Clinical studies of anti-pcsk9 therapeutic agents, including planned and ongoing studies in Phase and Phase I. NCT number (study name) Number of patients; cohort BL LDL-C (mg/dl) Duration; dosage LDL-C reduction (% change from BL) NCT (GAUSS) n = 160; statin-intolerant HC patients 193 NCT (RUTHERFORD) n = 167; hefh on statin (not 156 at goal) NCT (LAPLACE-TIMI57) n = 631; HC on low-dose statin 125 NCT (MENDEL) n = 406; low-risk patients (no lipid-lowering drug) 144 NCT (DESCARTES) NCT (TESLA) NCT (TAUSSIG) n = 900; HC >75 n = 67; hofh > weeks; multiple doses -15% Eze 10 mg qd Q4W sc. -41% 280 mg -43% 350 mg -51% 420 mg -63% 420 mg + Eze 10 mg qd 12 weeks; multiple doses +1% placebo Q4W sc. -43% 350 mg -55% 420 mg 12 weeks; multiple doses -42% 70 mg Q2W Q2W or Q4W sc. -60% 105 mg Q2W -66% 140 mg Q2W -42% 280 mg Q4W -50% 350 mg Q4W -50% 420 mg Q4W 12 weeks; multiple doses -15% Eze 10 mg qd Q2W or Q4W sc. -4% placebo Q2W -41% 70 mg Q2W -44% 105 mg Q2W -51% 140 mg Q2W +4.5% placebo Q4W -39% 280 mg Q4W -43% 350 mg Q4W -48% 420 mg Q4W 52 weeks; multiple doses Q4W sc. 12 weeks; multiple doses, sc. 5 years; multiple doses Q4W or Q2W sc. Phase Ref. [87] [88] [89,90] [91] /I /I [92] [93] [95] AMG 145 n = 105; hofh or ADH (LDLR >130 or PCSK9 mutations) REGN727 NCT n = 77; hefh on high-dose statin NCT n = 183; primary HC (polygenic) on Atorva 10 mg NCT n = 92; primary HC 123 (polygenic) on Atorva 10 mg NCT n = 12; he or ho PCSK9 GOF >70 mutations % placebo -68% Q2W 150 mg -29% Q4W 150 mg -32% Q4W 200 mg -43% Q4W 300 mg 12 weeks, 20 weeks for -5% placebo -40% 50 mg Q2W safety; multiple doses, -64% 100 mg Q2W sc. (Q2W 50, 100, -72% 150 mg Q2W 150 mg, Q4W 200 or -43% 200 mg Q4W 300 mg) -48% 300 mg Q4W -18% Atorva 80 mg qd 8 weeks, 16 weeks for -66% Atorva 10 mg safety; multiple doses qd + REGN Q2W sc. (switch to Atorva 80 mg or Atorva -73% Atorva 80 mg qd 10 mg + REGN or Atorva +REGN 80 mg + REGN) 2 weeks, 20 weeks for safety; sc. 12 weeks, 20 weeks for safety; multiple doses, sc. (Q2W 150 mg, Q4W 150, 200, 300 mg) Full details of ongoing trials can be found at ClinicalTrials.gov [309]. ACS: Acute coronary syndrome; ADH: Autosomal dominant hypercholesterolemia; Atorva: Atorvastatin; BL: Baseline; Eze: Ezetimibe; FH: Familial hypercholesterolemia; GOF: Gain-of-function; HC: Hypercholesterolemia; he: Heterozygous; ho: Homozygous; iv.: Intravenous; LDL-C: LDL cholesterol; LDLR: LDL receptor; Q2W: Once every 2 weeks; Q4W: Once every 4 weeks; qd: Once daily; Rosuva: Rosuvastatin; sc.: Subcutaneous

13 review Rhainds, Arsenault & Tardif Table 3. Clinical studies of anti-pcsk9 therapeutic agents, including planned and ongoing studies in Phase and Phase I (cont.). NCT number (study name) Number of subjects, cohort BL LDL-C (mg/dl) Duration; dosage LDL-C reduction (% change from BL) Phase Ref. n = 2100; HC not at goal (high-risk) or hefh > weeks, 20 months for safety, sc. I [306] n = 471; hefh not at goal (high-risk) n = 105; hefh > weeks, 20 months for safety, sc. 24 weeks, 20 months for safety, sc. 24 weeks, 20 months for safety, sc. 24 weeks, 20 months for safety, sc. 24 weeks, 20 months for safety, sc. I _ I _ n = 100; HC >100 I _ n = 660; on maximally tolerated statin dose n = 250; primary HC, statin intolerant >100 I _ I _ n = 306; on maximally tolerated statin dose n = 250; hefh on statin > weeks, 20 months for safety, sc. 24 weeks, 20 months for safety, sc. 64 months, 66 months for safety I _ Planned I _ n = 18,000; 416 weeks post-acs patients >70 I [307] n = 46; on high-dose statin (Atorva 80 mg or Rosuva 40 mg) n = 111; on high-dose statin >80 12 weeks, 20 for safety; multiple doses Q4W iv. Completed > weeks, 20 for safety; multiple doses Q4W iv. Completed REGN727 ODYSSEY program NCT (ODYSSEY Long term) NCT (ODYSSEY FH I) NCT (ODYSSEY High FH) NCT (ODYSSEY Mono) NCT (ODYSSEY Combo ) NCT (ODYSSEY Alternative) NCT (ODYSSEY Combo I) NCT (ODYSSEY FH ) NCT (ODYSSEY Outcomes) >160 >100 >70 RN316/PF NCT NCT Full details of ongoing trials can be found at ClinicalTrials.gov [309]. ACS: Acute coronary syndrome; ADH: Autosomal dominant hypercholesterolemia; Atorva: Atorvastatin; BL: Baseline; Eze: Ezetimibe; FH: Familial hypercholesterolemia; GOF: Gain-of-function; HC: Hypercholesterolemia; he: Heterozygous; ho: Homozygous; iv.: Intravenous; LDL-C: LDL cholesterol; LDLR: LDL receptor; Q2W: Once every 2 weeks; Q4W: Once every 4 weeks; qd: Once daily; Rosuva: Rosuvastatin; sc.: Subcutaneous. results suggest that a once every 2 weeks (Q2W) regimen should be considered with REGN727 in patients treated with statins. Amgen has reported Phase I data with AMG 145 in abstract form [86]. This study randomized older (mean age: 58 years) hypercholesterolemic patients in subgroups with either low/moderate or high doses (rosuvastatin 40 mg or atorvastatin 80 mg) of statins in one of three regimens: once weekly (Q1W) or once every 2 weeks (Q2W) for 6 weeks, or once every 4 weeks (Q4W) for 8 weeks. After 6 weeks on the Q2W regimen (three injections), AMG 145 was somewhat better in the low-dose statin group (-75% LDL C vs placebo), but was nonetheless beneficial to the high-statin group (-63% LDL C). The Q4W group (two injections) had a 64% decrease in LDL C after 8 weeks, but whether or not a rebound of LDL C levels was observed after 2 weeks is 632 Clin. Lipidol. (2012) 7(6) unknown. At the American Heart Association scientific sessions in November 2012, Amgen presented four Phase studies in very dissimilar cohorts totalling >1300 subjects, the results of which were simultaneously published in three journals. The GAUSS [87] and RUTHERFORD [88] trials recruited 160 statin-intolerant patients (due to muscle-related effects) and 167 on-statin FH heterozygotes with baseline LDL-C levels of 193 and 156 mg/dl, respectively. In both settings, the usual Q4W regimen with AMG 145 reduced LDL-C levels by up to 51% in statinintolerant patients and 55% in FH heterozygotes after 12 weeks. Daily ezetimibe (10 mg) could reduce LDL-C by 15% in statin-intolerant patients, but the combination of AMG 145 and ezetimibe resulted in an almost additive 63% reduction of LDL-C [87]. Two larger studies were also presented in November 2012, LAPLACE-TIMI 57 [89,90] and MENDEL [91],