PCSK9 inhibition in LDL cholesterol reduction: genetics and therapeutic implications of very low plasma lipoprotein levels.

JPT-06707; No of Pages 9 Pharmacology & Therapeutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: Y.S. Chatzizisi

PCSK9 inhibition in LDL cholesterol reduction: Genetics and therapeutic implications of very low plasma lipoprotein levels A. David Marais a,⁎, Jae B. Kim b, Scott M. Wasserman b, Gilles Lambert c,d,⁎⁎ a

Chemical Pathology, University of Cape Town, National Health Laboratory Service and Medical Research Council Cape Heart Group, Cape Town, South Africa Amgen Inc., Thousand Oaks, CA, USA Université de Nantes, Faculté de Médecine, Nantes, France d Laboratoire UMR PhAN, CHU Hôtel Dieu, Nantes, France b c

a r t i c l e

i n f o

Keywords: Proprotein convertase subtilisin/kexin type 9 Low-density lipoprotein cholesterol Low-density lipoprotein receptor Genetics

a b s t r a c t Atherosclerosis is a complex process involving the build-up of arterial plaque incorporating low-density lipoprotein cholesterol (LDL-C) and an inflammatory response. Lowering plasma LDL-C confers cardiovascular benefit for patients with hypercholesterolemia resulting from genetic and/or lifestyle factors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of LDL-C metabolism. Secreted from liver cells, circulating PCSK9 binds to the LDL receptor and is subsequently internalized with the receptor, thereby promoting its cellular degradation. As a result, PCSK9 gain-of-function mutations are causatively associated with familial hypercholesterolemia, whereas PCSK9 loss-of-function mutations are associated with very low LDL-C levels and a reduced cardiovascular risk. Preventing PCSK9-mediated LDL receptor degradation with monoclonal antibodies is a novel strategy to further lower LDL-C, especially in patients with severe forms of hypercholesterolemia with elevated LDL-C despite maximal conventional treatment and/or in those intolerant to conventional therapies. Here, the safety and efficacy of these novel therapeutic agents targeting PCSK9 will be discussed with respect to recent clinical trials targeting this molecule, as well as inherited hypolipidemias and animal models that confer very low LDL-C because of PCSK9 deficiency. © 2014 Elsevier Inc. All rights reserved.

Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The low-density lipoprotein receptor: a pivotal regulator of low-density lipoprotein plasma clearance 3. Genetic contributors to dyslipidemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Proprotein convertase subtilisin/kexin type 9 . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Safety of proprotein convertase subtilisin/kexin type 9 inhibition . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: Apo, apolipoprotein; CHD, coronary heart disease; EGFA, epidermal growth factor-like repeat homology domain; ER, endoplasmic reticulum; FH, familial hypercholesterolemia; FHBL, familial hypobetalipoproteinemia; GOF, gain of function; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; IDL, intermediate-density lipoprotein; LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; LOF, loss of function; LPL, lipoprotein lipase; mAb, monoclonal antibody; MTP, microsomal triglyceride transfer protein; PCSK9, proprotein convertase subtilisin/kexin type 9; T2D, type 2 diabetes; TG, triglyceride; VLDL, very low-density lipoprotein. ⁎ Correspondence to: A. D. Marais, Chemical Pathology, University of Cape Town Health Science Faculty, Anzio Rd, Observatory 7925, South Africa. Tel.: +27 21 4066192; fax: +27 21 4488150. ⁎⁎ Correspondence to: G. Lambert, Laboratoire UMR PhAN, HNB1, CHU Hôtel Dieu, Place Alexis Ricordeau, 44093 Nantes Cedex, France. Tel.: +33 2 5348 2002; fax: +33 2 5348 2003. E-mail addresses: [email protected] (A.D. Marais), [email protected] (G. Lambert).

http://dx.doi.org/10.1016/j.pharmthera.2014.07.004 0163-7258/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Marais, A.D., et al., PCSK9 inhibition in LDL cholesterol reduction: Genetics and therapeutic implications of very low plasma lipoprotein levels, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.07.004

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A.D. Marais et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

1. Introduction Plasma low-density lipoprotein cholesterol (LDL-C) levels strongly correlate with atherosclerotic coronary artery disease (Anderson et al., 1987; Jacobs et al., 1992; Neaton et al., 1992; Assmann et al., 1997; Sharrett et al., 2001; Menotti et al., 2008). Lowering LDL-C reduces the risk of vascular events by approximately 20% for every 1-mmol/L reduction (Baigent et al., 2005, 2010). The European guidelines for managing dyslipidemia indicate a target LDL-C of b2.5 mmol/L (97 mg/dL) for high-risk patients and b1.8 mmol/L (70 mg/dL), or a ≥50% reduction from baseline, for very high-risk patients (European Association for Cardiovascular Prevention and Rehabilitation et al., 2011). Statins, which lower LDL-C in a dose-dependent manner, are often not sufficient to achieve target levels in patients with very high LDL-C and at high risk for coronary heart disease (CHD) (Davidson et al., 2005; Jones et al., 2012). A novel class of hypocholesterolemic agents, the anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibodies, alone or in combination with statins, reduce LDL-C by N50% in patients with a wide range of dyslipidemias and cardiovascular risk. The first aim of the present report is to provide a comprehensive review of PCSK9 function as a secreted inhibitor of the LDL receptor and a synoptic overview of PCSK9 inhibitors in clinical trials. The second aim is to shed light on potential adverse effects, focusing on the consequences of very low levels of LDL that could arise from the long-term use of these new therapies.

hand, the transport of dietary lipids through the circulation is referred to as the exogenous lipoprotein pathway (Fig. 1). Ingested lipids are absorbed by enterocytes, where they are packaged into chylomicrons together with apolipoprotein (apo) B-48. Chylomicron assembly requires the action of the microsomal triglyceride transfer protein (MTP). Chylomicrons are transported along the secretory pathway and secreted into the lymph, which specifically requires the action of SARA2/SAR1B. On the other hand, the endogenous lipoprotein pathway emanates from the hepatocyte (Fig. 1). Hepatic lipids are packaged into very low-density lipoprotein (VLDL) together with apoB-100, a process also requiring the action of the MTP. In the circulation, hydrolysis of triglycerides (TG) in VLDL and chylomicrons releases fatty acids to muscle, adipose tissue, and lactating breast, a process mediated by lipoprotein lipase (LPL). Chylomicrons are hydrolyzed to smaller remnants, whereas VLDL particles are hydrolyzed to yield intermediate-density lipoproteins (IDL) and LDL (Fig. 1). The LDL receptor (LDLR) pathway is responsible for uptake and degradation of most apoB-containing lipoproteins of the exogenous and endogenous lipoprotein pathways. The LDLR clears chylomicron remnants and IDL by binding apoE on these lipoproteins and LDL by binding apoB-100. The receptor– ligand complex is internalized in clathrin-coated pits. After endocytosis, the lipoprotein undergoes lysosomal degradation, whereas the LDLR is recycled back to the cell surface (Xie et al., 2006; Goldstein and Brown, 2009). The secreted factor PCSK9 (described below in Section 4) precisely antagonizes the recycling of the receptor after endocytosis.

2. The low-density lipoprotein receptor: a pivotal regulator of low-density lipoprotein plasma clearance

3. Genetic contributors to dyslipidemia

The amount of cholesterol in the blood is carefully controlled through de novo synthesis and delivery to the liver from diet or LDL. On the one

Genetic disorders resulting in either no (abetalipoproteinemia) or very low levels (hypobetalipoproteinemia) of apoB-containing

Fig. 1. Exogenous and endogenous lipoprotein metabolic pathways. Left panel: The exogenous lipoprotein pathway transports dietary and biliary cholesterol and fatty acids from the gut through the circulation, delivering free fatty acids derived from chylomicrons through the action of lipoprotein lipase (LPL) to muscle cells and adipose tissue, while the liver takes up chylomicron remnants. Right panel: The endogenous pathway delivers triglycerides from hepatocytes in the form of very low-density lipoproteins (VLDLs). These particles are similar to chylomicrons but have apoB-100 instead of apoB-48. VLDLs release free fatty acids (FFA) into the bloodstream through the action of LPL; as they release triglycerides (TGs), they become intermediate-density lipoproteins (IDLs) and eventually low-density lipoproteins (LDLs). IDL and LDL are taken up by the liver; LDL particles are also taken up by peripheral tissues. These actions require the LDL receptor (LDLR).



lipoproteins, including LDL-C, may result from defects either in apoB or in intracellular lipoprotein assembly or trafficking (Whitfield et al., 2004). The primary causes of hypobetalipoproteinemia include autosomal dominant mutations in APOB resulting in familial hypobetalipoproteinemia (FHBL) (Burnett et al., 2003; Whitfield et al., 2004), autosomal recessive mutations in MTP resulting in abetalipoproteinemia (Wetterau et al., 1992; Schonfeld, 2003), or autosomal recessive mutations in SARA2/ SAR1B resulting in chylomicron retention disease (Dannoura et al., 1999; Jones et al., 2003; Liao and Laufs, 2005; Georges et al., 2011). Abetalipoproteinemia is characterized by a complete disruption of the apoB-containing lipoprotein assembly, severely limiting the absorption of dietary lipids and fat-soluble vitamins with consequent steatorrhea. There may be failure to thrive after birth, growth impairment, and deficiency in tocopherol, which leads to retinitis pigmentosa and neuromuscular problems at a later stage. Loss of cholesterol from red cells may result in acanthocytosis (Schonfeld, 2003). In sharp contrast, familial hypercholesterolemia (FH) is an autosomal dominantly inherited disorder with a characteristic clinical phenotype: marked elevations in plasma LDL-C, high prevalence of tendon xanthomata, and premature CHD. FH is predominantly due to mutations in the LDLR gene (Marais, 2004). Other less common causes of FH include mutations in the ligand-binding domain of apoB-100 (Soria et al., 1989) and gain-of-function (GOF) mutations in PCSK9 (Abifadel et al., 2009).

4. Proprotein convertase subtilisin/kexin type 9 PCSK9, formerly known as neural apoptosis-related convertase 1, was identified in a patented database by searching for previously unidentified proprotein convertases that could cleave nonbasic residues (Seidah et al., 2003). In adults, it is predominantly expressed in the liver and small intestine (Seidah et al., 2003). In 2003, GOF mutations in PCSK9 were identified as the third cause of FH (once mutations in LDLR or APOB had been excluded) in two French families whose total

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cholesterol level was in the 90th percentile (Abifadel et al., 2003). Other PCSK9 GOF mutations were later described in FH patients from Utah, Norway, and the United Kingdom (Leren, 2004; Timms et al., 2004; Naoumova et al., 2005).

4.1. Structure, mode of action, and gene expression PCSK9 is synthesized as a 72-kDa zymogen in the endoplasmic reticulum (ER) (Seidah et al., 2003). It comprises a signal peptide (residues 1–30), a prodomain (residues 31–152) preceding a catalytic domain (residues 153–451) that contains the canonical N–H–S catalytic triad, followed by a C-terminal domain (residues 452–692) (Benjannet et al., 2004). In the ER, the mature 62-kDa enzyme is formed through autocatalytic intramolecular cleavage between residues Q152 and S153 of pro-PCSK9. After cleavage, the prodomain remains within the catalytic site of the enzyme through noncovalent interactions, hindering access of potential substrates to the catalytic pocket. This PCSK9/prodomain complex moves to the Golgi apparatus and is secreted (Lambert et al., 2012). PCSK9 reduces hepatic LDLR primarily as a secreted factor. Specifically, PCSK9 interacts with the extracellular domain of the LDLR at the first epidermal growth factor-like repeat homology domain (EGFA). Binding occurs with an affinity of 170 to 750 nM at the neutral pH of plasma but is much greater (affinity of 1–8 nM) within the acidic endosomes (Lambert et al., 2012). A series of tight molecular interactions between PCSK9 and the LDLR lock the receptor in an extended (or open) conformation, precluding normal cycling of the LDLR back to the plasma membrane, and reroute it for lysosomal degradation (Fig. 2). The exact molecular mechanism by which the PCSK9-LDLR complex is routed to the lysosome after endocytosis has yet to be fully elucidated and appears to be cell-type dependent. First, the presence of the autosomal recessive hypercholesterolemia adaptor protein is required in most (but not all) cell types to allow endocytosis of the PCSK9-LDLR complex into clathrin-coated pits (Wang et al., 2012).

Fig. 2. The proprotein convertase subtilisin/kexin type 9 (PCSK9) pathway. Left panel: In the absence of bound PCSK9, the acidic environment of the endosome releases the low-density lipoprotein receptor (LDLR) from the low-density lipoprotein (LDL) particle, and the receptor is recycled to the cell surface while the LDL particle is delivered to the lysosome. Loss-offunction (LOF) mutations in PCSK9 result in increased availability of LDLR on the cell surface. Right panel: When bound to PCSK9, the LDLR traffics to the lysosomal compartment and is degraded along with the LDL particle. Gain-of-function (GOF) mutations in PCSK9 lead to decreased availability of LDLR on the hepatocyte.


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Second, hepatic, but not adrenal, expression of the LDLR appears highly sensitive to elevated circulating PCSK9 levels in mice (Tavori et al., 2013a). Third, PCSK9 readily dissociates from the LDLR within the early endosome of SV-589 fibroblasts, thereby limiting PCSK9mediated LDLR degradation in this cell type (Nguyen et al., 2014). Additionally, PCSK9 binds to the EGFA domain of other LDLR family members, such as the very low-density lipoprotein receptor (Poirier et al., 2008; Roubtsova et al., 2011), the apolipoprotein receptor 2 (Poirier et al., 2008; Kysenius et al., 2012), and the LDLR-related protein-1 receptor (Canuel et al., 2013). As a result, there is a positive correlation between circulating PCSK9 and LDL-C levels in the population (Lambert et al., 2008; Lakoski et al., 2009; Welder et al., 2010). The LDLR appears to be the major route for PCSK9 removal from the circulation, as shown in LDLR-knockout mice (Tavori et al., 2013a) and in homozygous FH patients (Raal et al., 2013). This reciprocal regulation between the LDLR and PCSK9 appears extremely complex because (i) distinct domains of the LDLR are binding to the LDL particle and to PCSK9 and (ii) a significant proportion (30%–40%) of circulating PCSK9 is carried onto LDL particles (Kosenko et al., 2013; Tavori et al., 2013b). For instance, in severe FH, lipoprotein apheresis reduces plasma LDL levels by 77% and circulating (mostly LDL-bound) PCSK9 levels by 52% (Tavori et al., 2013b). It has been proposed that LDL particles inhibit PCSK9 binding to the LDLR at the cell surface and thereby lower the ability of PCSK9 to induce LDLR degradation in vitro (Kosenko et al., 2013). Besides the well-characterized extracellular mode of action of PCSK9 described above, PCSK9 could, to some extent, act on the LDLR intracellularly (i.e., before secretion, by enhancing LDLR degradation endogenously) (Poirier et al., 2009) and/or by chaperoning the LDLR from the ER to the plasma membrane (Strom et al., 2014). The most striking cooperative phenomenon occurring between PCSK9 and the LDLR, however, is their transcriptional coregulation by the sterol regulatory element binding protein-2 pathway, most notably through sterol deprivation or treatment with statins (Dubuc et al., 2004; Mayne et al., 2008). Thus, statin treatment is associated with an increase in circulating PCSK9 levels as well as LDLR, a phenomenon that explains, at least in part, why the power of LDL-C reduction by statins is plateaued. Noteworthy, statins also show benefits outside of their LDL-C-lowering abilities (i.e., pleiotropic effects), which include improving endothelial function, increasing stability of atherosclerotic plaques, reducing oxidative stress and inflammation, and suppressing the thrombogenic response (Liao and Laufs, 2005). Many of these benefits are mediated through further downstream 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) suppression, mainly through decreased isoprenylation of signaling molecules such as Rac, Rho, and Ras, which modulates signaling pathways (Liao and Laufs, 2005). 4.2. Loss-of-function proprotein convertase subtilisin/kexin type 9 mutations Discovery of the PCSK9 GOF mutations causal for FH led to the hypothesis that loss-of-function (LOF) mutations in this gene could have the opposite effect (Cohen et al., 2006) (Fig. 2). Several LOF mutations in PCSK9 are associated with low plasma LDL-C in adults and children (Hallman et al., 2007; Huang et al., 2009). Such mutations in PCSK9 were found in 2% of African Americans, who mainly carried the C142X or C679X nonsense mutations (Cohen et al., 2005). Heterozygous carriers exhibit LDL-C levels of 2.6 mmol/L, compared with 3.6 mmol/L for noncarriers, accompanied by an 88% reduction in global CHD risk (Cohen et al., 2006). The C142X mutation likely promotes mRNA decay, whereas the PCSK9-C679X protein is retained within the ER and is not secreted from cells (Zhao et al., 2006). PCSK9-R46L is a missense mutation found in approximately 2% to 3% of the white population (Cohen et al., 2006; Benn et al., 2010). Compared with noncarriers, heterozygous carriers have a 13% reduction in LDL-C levels, a 15% to 25% reduction in plasma PCSK9, a 47% reduction

in global CHD risk, and a 30% lower incidence of ischemic heart disease (Humphries et al., 2009; Benn et al., 2010). Additional studies suggest that the PCSK9-R46L mutation may also protect carriers from myocardial infarction (Kathiresan and Myocardial Infarction Genetics Consortium, 2008; Kostrzewa et al., 2008). The PCSK9-R46L mutation was investigated in heterozygous FH patients with defined LDLR defects. These patients had lower-than-expected LDL-C levels while on statin therapy, potentially because of an increased sensitivity to statins conferred by these PCSK9 mutations (Berge et al., 2006). PCSK9-R46L was shown to be more susceptible to proteolytic degradation (Dewpura et al., 2008) and to have a reduced affinity for the LDLR compared with wild-type PCSK9 in Biacore studies (Fisher et al., 2007). Other reported PCSK9-inactivating mutations include truncating (W428X and L82X) (Fasano et al., 2007; Miyake et al., 2008) and insertion (c.43_44insCTG) mutations (Yue et al., 2006). PCSK9 missense mutations segregating with hypocholesterolemia include R93C, G106R, Q152H, R237W, L253F, G236S, N354I, A443T, S462P, and the monoallelic double-mutant R104C/V114A, all of which preclude normal processing and secretion of the PCSK9 protein (Berge et al., 2006; Cameron et al., 2006; Kotowski et al., 2006; Cameron et al., 2008; Miyake et al., 2008; Cameron et al., 2009; Cariou et al., 2009; Huang et al., 2009; Mayne et al., 2011). Interestingly, the case of a compound heterozygous woman who inherited the Y142X mutation from her mother and the c.290_ 292delGCC mutation from her father was reported (Zhao et al., 2006). The latter mutation deletes R97 (Δ97) and prevents autocatalytic cleavage and secretion of PCSK9. Her plasma PCSK9 was undetectable and her LDL-C level was 0.36 mmol/L (Zhao et al., 2006). She was healthy, fertile, normotensive, and college educated, with normal liver and renal function, all of which suggest that absence of PCSK9 is compatible with normal health. Two homozygous cases of PCSK9-R46L were found in an elderly population. Compared with wild-type and PCSK9-R46L heterozygous women, who displayed LDL-C levels of 3.8 mmol/L and 3.3 mmol/L, respectively, homozygous women had mean LDL-C levels of 3.1 mmol/L (Polisecki et al., 2008). One woman received pravastatin, which reduced LDL-C by 63% compared with an average reduction of 36% in women with both PCSK9 wild-type alleles and R46L heterozygotes (Polisecki et al., 2008). A Zimbabwean woman attending an antenatal clinic was found to be homozygous for the PCSK9-C679X mutation. She had an LDL-C level of 0.4 mmol/L postpartum (Hooper et al., 2007). The wild-type individuals and the 23 PCSK9-C679X heterozygous women had mean ± SD LDL-C levels of 2.2 ± 0.7 mmol/L and 1.6 ± 0.3 mmol/L, respectively (Hooper et al., 2007). These cases suggest that a complete or nearcomplete absence of PCSK9 resulting in very low levels of LDL-C does not appear deleterious in humans and is compatible with a normal pregnancy.

4.3. Animal models of proprotein convertase subtilisin/kexin type 9 deficiency PCSK9-knockout mice have approximately 2.8-fold higher hepatic LDLR expression and 42% to 48% lower total plasma cholesterol and lower apoB levels than wild-type animals (Rashid et al., 2005; Zaid et al., 2008). These mice are particularly sensitive to statins, and their LDL-C fractional catabolic rate is markedly increased compared with controls (Rashid et al., 2005). Similar features are observed in hepatocyte-specific conditional PCSK9-knockout mice, which have 27% lower plasma cholesterol than controls (Zaid et al., 2008). LDL-C is reduced by 80% and 60% in the plasma of complete PCSK9-knockout and hepatocyte-specific PCSK9-knockout mice, respectively. The lipoprotein profiles of LDLR-knockout mice and of PCSK9/LDLR doubleknockout mice are nearly identical, indicating that PCSK9 modulates circulating cholesterol levels exclusively via the LDLR (Zaid et al., 2008).


AMG145 (Evolocumab)

REGN727/SAR236553 (Alirocumab)

Monotherapy (MENDEL) (Koren et al., 2012)

Combination therapy (LAPLACE) (Giugliano et al., 2012)

HeFH (RUTHERFORD) (Raal et al., 2012)

Statin-intolerant (GAUSS) (Sullivan et al., 2012)

Open-label 52-week trial (OSLER) (Koren et al., 2014)

Combination therapy (Roth et al., 2012)

Combination therapy (McKenney et al., 2012)

HeFH (Stein et al., 2012)

Patient population

LDL-C ≥2.6 and b4.9 mmol/L (≥100 and b190 mg/dL)

LDL-C N2.2 mmol/L (N85 mg/dL)

HeFH LDL-C ≥2.6 mmol/L (≥100 mg/dL)

Statin intolerance, LDL-C not at NCEP target

LDL-C ≥2.6 mmol/L (≥100 mg/dL)

LDL-C ≥2.6 mmol/L (≥100 mg/dL)

HeFH or non-FH, LDL-C ≥2.6 mmol/L (≥100 mg/dL)

Background therapy

None

Statin ± EZE

Statin ± EZE

ATV

ATV

Statin ± EZE

Patients, N

406

629

167

No or low-dose statin ± EZE 157

Open-label extension of MENDEL, LAPLACE, RUTHERFORD, and GAUSS None, statin ± EZE 1104

92

183

77

Mean LDL-C change vs baseline at 12 wks Treatment −39% to −51% Placebo/control −4% to +5%

−42% to −66%a NA

−43% to −55% +1%

−41% to −63% −15%

Mean at 52 wks −52% −3%

Mean LDL-C change vs baseline at 12 wks −66% to −73%b −40% to −72% −5% −17%b

−29% to −68% −11%

Mean HDL-C change vs baseline at 12 wks Treatment +5% to +12% Placebo/control +1% to +6%

+2% to +8%a NA

+9% to +10% +2%

+6% to +12% −1%

Mean at 52 wks +9% +4%

Mean HDL-C change vs baseline at 12 wks +3% to +6% +4% to +9% −4% −1%

−7% to +12% +2%

Mean Lp(a) change vs baseline at 12 wks Treatment −9% to −27% Placebo/control +2% to +9%

NR NR

−19% to −27% +4%

−20% to −29% −8%

Median at 52 wks −30% −9%

Mean Lp(a) change vs baseline at 12 wks −31% to −35% −8% to −29% −3% 0%

−7% to −23%c −4%c

Adverse events Treatment Placebo/control

42% to 58% 42% to 49%

49% to 66% 42%

58% to 66% 59%

48% to 69% 59%

81% 73%

45% to 60% 61%

50% to 65% 45%

75% to 87% 60%

Serious adverse events Treatment Placebo/control

0% to 2%d 0%

1% to 5% 5%

0% to 4%d 0%

0% to 6%d 0%

7% 6%

0% to 3% 0%

0% to 4% 3%

0% 7%



Table 1 Key results of PCSK9 monoclonal antibodies in phase 2 clinical trials.

ATV = atorvastatin; EZE = ezetimibe; GAUSS = Goal Achievement After Utilizing an anti-PCSK9 antibody in Statin-intolerant Subjects; HDL-C = high-density lipoprotein cholesterol; HeFH = heterozygous familial hypercholesterolemia; LAPLACE = LDL-C Assessment With PCSK9 Monoclonal Antibody Inhibition Combined With Statin Therapy; LDL-C = low-density lipoprotein cholesterol; Lp(a) = lipoprotein(a); MENDEL = Monoclonal Antibody Against PCSK9 to Reduce Elevated LDL-C in Patients Currently Not Receiving Drug Therapy for Easing Lipid Levels; NA = not available; NCEP = National Cholesterol Education Program; NR = not reported; OSLER = Open-label Study of Long-term Evaluation Against LDL-C; PCSK9 = proprotein convertase subtilisin/kexin type 9; RUTHERFORD = Reduction of LDL-C With PCSK9 Inhibition in Heterozygous Familial Hypercholesterolemia Disorder. a Mean change versus placebo. b Results at 8 wks. c Median values. d None of the serious adverse events were considered to be related to treatment.

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Potential adverse effects of the PCSK9-knockout approach include an increased level of amyloid-β peptide, which could increase the risk of Alzheimer disease (Jonas et al., 2008), but PCSK9 single-nucleotide polymorphisms associated with lower LDL-C have not been associated with cognitive impairment in humans (Postmus et al., 2013). PCSK9 deficiency in mice has also been associated with increased hepatic CD81 expression, a major receptor for the hepatitis C virus (Labonte et al., 2009). The accumulation of the very low-density lipoprotein receptor in the visceral adipose tissue of PCSK9-knockout mice has resulted in marked adipocyte hypertrophy and triglyceride accumulation in this tissue, particularly in female mice (Roubtsova et al., 2011). But PCSK9-knockout mice appear to be protected from postprandial hyperlipemia following oil gavage (Le May et al., 2009), which could be a consequence of increased intracellular degradation of apoB before secretion (Sun et al., 2012a) and/or of increased transintestinal cholesterol excretion (Le May et al., 2013). Most importantly, PCSK9 deletion in mice reduces the incidence of atherosclerosis in an LDLR-dependent manner. PCSK9-knockout mice fed a Western diet were protected from cholesterol ester deposition in the aorta on the C57BL/6 (74% reduction) as well as on the apoEknockout (39% reduction) backgrounds, but not on the LDLR-knockout background (Denis et al., 2012). Conversely, overexpression of PCSK9 resulted in the development of severe atherosclerotic lesions in mice (Denis et al., 2012) and in pigs (Al-Mashhadi et al., 2013). 4.4. Clinical studies targeting proprotein convertase subtilisin/kexin type 9 Several therapies targeting PCSK9, which are in preclinical or clinical evaluation, have shown promising results. Two antisense nucleotides directed at reducing PCSK9 expression, BMS-84442/ISIS-405879 and SPC5001, have discontinued development (Garber, 2012). Toxic acute tubular injury with a transient reduction in renal function was reported in a healthy volunteer following SPC5001 treatment (van Poelgeest et al., 2013). ALN-PCS, a small interfering RNA targeting PCSK9 synthesis, has recently been shown in a phase 1 clinical trial to reduce LDL-C by 40% relative to placebo at the highest dose tested 3 days following intravenous infusion in healthy volunteers (Fitzgerald et al., 2014). To date, the most advanced PCSK9 inhibitors in terms of clinical development are the fully human monoclonal antibodies (mAbs) alirocumab (formerly REGN727/SAR236553) and evolocumab (formerly AMG 145), as well as the humanized mAb bococizumab (formerly RN316/PF-04950615). Data from published phase 2 clinical trials of PCSK9 inhibitors are summarized in Table 1. In phase 2 clinical studies examining alirocumab (8 or 12 weeks' duration), mean reductions in LDL-C ranged from 29% to 73% relative to baseline when used in combination with statins in patients with or without FH (McKenney et al., 2012; Roth et al., 2012; Stein et al., 2012). In phase 2 trials of evolocumab, mean reductions in LDL-C at 12 weeks ranged from 39% to 63% relative to baseline when used as monotherapy or in combination with statins ± ezetimibe (Koren et al., 2012; Raal et al., 2012;

Sullivan et al., 2012). Relative to placebo, reductions in LDL-C at 12 weeks were 42% to 66% (Giugliano et al., 2012). Preliminary results from phase 2 trials of the humanized anti-PCSK9 mAb RN316 (bococizumab) in patients with hypercholesterolemia showed LDL-C reductions of up to 84% relative to baseline (single dose) with or without atorvastatin (Gumbiner et al., 2012b) and reductions of up to 66% at 6 weeks relative to baseline following multiple doses (4 weekly doses) (Gumbiner et al., 2012a). Thus, mAb inhibition of PCSK9 alone or in combination with existing lipid-lowering treatments has proven effective at reducing LDL-C in all the population groups evaluated to date, including those with FH, non-FH hypercholesterolemia, and statin intolerance (Stein and Raal, 2014). In that respect, we recently published that elevated PCSK9 levels are equally detrimental in non-FH and FH patients alike, as a 100-ng/mL increase in PCSK9 will lead to an increase in LDL-C of 0.20 to 0.25 mmol/L in non-FH and heterozygous FH patients, irrespective of their LDLR mutation (Lambert et al., 2014). In the largest and longest anti-PCSK9 mAb trial published to date, long-term administration of evolocumab 420 mg every 4 weeks showed a 52% reduction in LDL-C versus baseline at 1 year (Koren et al., 2014). Taken together, these studies suggest that PCSK9 inhibition is effective and well-tolerated over a 12-week period in a diverse patient population. This therapeutic approach may confer substantial benefits for patients who cannot tolerate doses of statins required to achieve LDL-C treatment targets. Whether anti-PCSK9 antibodies will reduce the rates of myocardial infarction, stroke, and unstable angina is currently being tested in three large phase 3 clinical trials. Initial results for outcome studies for Amgen's evolocumab (22,500 patients included) and SanofiRegeneron's alirocumab (18,000 patients included) are anticipated in 2018, and results for Pfizer's bococizumab (18,300 patients included) are anticipated in 2017 (Table 2).

5. Safety of proprotein convertase subtilisin/kexin type 9 inhibition The phase 1 and 2 trials examining the use of alirocumab and evolocumab as monotherapy or in combination with statins showed rates of adverse events comparable to placebo (Table 1). Binding antibodies were observed in one patient each in the evolocumab and placebo groups (Koren et al., 2012). No neutralizing antibodies were detected in any of the evolocumab trials (Giugliano et al., 2012; Koren et al., 2012; Raal et al., 2012; Sullivan et al., 2012). In alirocumab trials, antidrug antibodies were observed in a total of eight patients at minimally detectable levels (McKenney et al., 2012; Roth et al., 2012; Stein et al., 2012). The rates of muscle-related adverse events, elevated creatine kinase or aminotransferases greater than 5 or 10 times the upper limit of normal, were not significantly different in evolocumab- or alirocumabtreated patients compared with controls (Giugliano et al., 2012; Koren et al., 2012; McKenney et al., 2012; Raal et al., 2012; Roth et al., 2012; Stein et al., 2012; Sullivan et al., 2012).

Table 2 PCSK9 monoclonal antibodies in phase 3 cardiovascular outcomes clinical trials.⁎ Compound

Phase 3 trial primary endpoint

Inclusion age, y

Completion date

Number of patients

Alirocumab REGN727 SAR236553 Bococizumab RN316 PF-04950615

ODYSSEY-OUTCOMES (NCT01663402) Time from randomization to first occurrence of one of the following clinical events: CHD death, any nonfatal MI, fatal and nonfatal ischemic stroke, unstable angina requiring hospitalization SPIRE 1 & 2 (NCT01975376 & NCT01975389) The time from randomization to the first adjudicated and confirmed occurrence of a primary endpoint major cardiovascular event; a composite endpoint that includes CV death, nonfatal MI, nonfatal stroke, and hospitalization for unstable angina needing urgent revascularization FOURIER (NCT01764633) Time to CV death, MI, hospitalization for unstable angina, stroke, or coronary revascularization

≥40

Oct 2012–Jan 2018 (5 years)

18,000

≥18

Oct 2013–Aug 2017 (3 − 4 years)

12,000 and 6300

40–85

Jan 2013–Feb 2018 (5 years)

22,500

Evolocumab AMG145

CHD = coronary heart disease; CV = cardiovascular; MI = myocardial infarction; PCSK9 = proprotein convertase subtilisin/kexin type 9. ⁎ As listed on www.clinicaltrials.gov.



5.1. Potential adverse effects of very low low-density lipoprotein cholesterol levels PCSK9 inhibition, especially when used in combination with other lipid-lowering therapies, has yielded LDL-C reductions down to very low levels (b0.65 mmol/L or b25 mg/dL). Some studies have noted a relationship between low LDL-C levels and risk of hemorrhagic stroke (Amarenco et al., 2006), cancer (Law and Thompson, 1991), and newonset type 2 diabetes (T2D) (Preiss et al., 2011; Ridker et al., 2012). The rate of hemorrhagic stroke was observed to be numerically higher in patients who achieved low LDL-C in one study (Amarenco et al., 2006); however, a subsequent analysis found no association between low LDL-C and hemorrhagic stroke (Huisa et al., 2010). Further large analyses found no significant association (Baigent et al., 2010; Baigent et al., 2011). Although a link was initially observed between low LDL-C and incidence of cancer in a retrospective analysis (Law and Thompson, 1991), subsequent meta-analyses found no association (Cholesterol Treatment Trialists et al., 2012). In addition, PCSK9 deficiency has recently been shown to be protective against melanoma metastasis in mice livers (Sun et al., 2012b). 5.2. Potential adverse effects of low-density lipoprotein receptor upregulation Cholesterol accumulation in β-cells in pancreatic islets may be linked to the development of T2D (Brunham et al., 2007). Statin treatment combined with a PCSK9 inhibitor may induce LDL-C uptake via the LDLR in those cells, potentially interfering with cholesterol homeostasis and increasing the risk of T2D. Indeed, an increased risk of T2D has been reported in a meta-analysis of statin trials (Sattar et al., 2010; Preiss et al., 2011; Ridker et al., 2012); however, only one new case of T2D was observed for every 500 (Preiss et al., 2011) or 1000 (Sattar et al., 2010) patients treated each year in these meta-analyses. Therefore, any potential risk of T2D development should be outweighed by the benefit of LDL-C lowering (Sattar et al., 2010; Preiss et al., 2011). Still, new onset T2D will need to be monitored carefully in anti-PCSK9 phase 3 trials. Because the LDLR may act as a virus entry route, upregulation of the LDLR with statin and/or PCSK9 inhibitor treatment (Labonte et al., 2009) may facilitate viral infections in some instances. Despite these potential safety concerns, phase 2 results of PCSK9 inhibitors have shown that the rates of adverse and serious adverse events were mostly balanced between treatment groups (Table 1). The results of phase 3 trials (Table 2) will provide further information on the longterm safety of these agents and any potential concerns associated with achieving very low levels of LDL-C. 6. Conclusions The clinical features of PCSK9 LOF mutation carriers, the phenotype of PCSK9-knockout mice, and results from clinical trials examining fully human and humanized mAbs suggest a novel therapeutic option for lowering LDL-C. The marked LDL-C-lowering effects with PCSK9 inhibitors in early trials, alone or in combination with statins or other lipid-lowering therapies, are compelling, and the results from phase 3 studies will further clarify the use of PCSK9 inhibitory modalities in defined dyslipidemias (e.g., homozygous and severe heterozygous FH). Cardiovascular outcomes studies will provide additional data on the effects of PCSK9 inhibition on the reduction of cardiovascular events as well as useful information for integration of these agents into clinical practice. Conflict of interest David Marais—none. Jae Kim—employee of AMGEN.

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Scott Wasserman—employee of AMGEN. Gilles Lambert—has received honoraria and research funding from AMGEN, SANOFI-REGENERON, and PFIZER.

Acknowledgments We thank Miranda Tradewell, PhD, of Complete Healthcare Communications, Inc. (whose work was funded by Amgen) and Meera Kodukulla, PhD, of Amgen Inc. for the editorial assistance.

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Alirocumab: PCSK9 inhibitor for LDL cholesterol reduction.

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An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo.

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How Do PCSK9 Inhibitors Stack Up to Statins for Low-Density Lipoprotein Cholesterol Control?

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