Reducing circulating low density lipoprotein cholesterol (LDL-C) reduces the risk of cardiovascular disease. Current approaches to reduce LDL-C include statins, which inhibit cholesterol synthesis and up-regulate hepatic cell-surface LDL receptors (LDL-R), and ezetimibe, which blocks cholesterol absorption. Serum proprotein convertase subtilisin/kexin 9 (PCSK9) is a serine protease that binds to LDL receptors through a two step process, increasing their degradation and thereby reducing the rate at which LDL is removed from circulation [1]. Both statins and ezetimibe elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation [2]. Though PCSK9 was first identified less than a decade ago, it seems to be an important regulator of LDL metabolism. PCSK9 'gain-of-function' mutations are rare, autosomal dominant traits that cause familial hypercholesterolemia and predispose to atherosclerotic cardiovascular disease. The more common 'loss-of-function' mutations cause low LDL-cholesterol and atheroprotection [3,4].

PCSK9 inhibition

PCSK9 inhibition through gene silencing by antisense oligonucleotides or by the action of small peptides and antibodies is a novel therapeutic approach to treatment of hypercholesterolemia. Initial observations of reduction in atherosclerosis in mice, rats, and primates with PCSK-9 inhibition prompted human trials. Of the many approaches to PCSK9 inhibition, monoclonal antibodies are the furthest advance in their clinical development; small molecule oral inhibitors seem a remote prospect [5].

Gene silencing

To determine whether PCSK9 inhibition in mice could enhance LDL-C lowering effect of both statins and ezetimibe, a study utilized small interfering RNAs (siRNAs) to knock down PCSK9, together with ezetimibe, rosuvastatin, and an ezetimibe - rosuvastatin combination. Ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lowered serum cholesterol but induced the expression of PCSK9 as well as hepatic cholesterol biosynthesis. PCSK9 knockdown in combination with any treatment arm led to greater reductions in serum non-HDL-C with a near-uniform reduction of all LDL-C subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/PCSK9 siRNA exhibited a significant reduction in serum apoB and triglyceride levels, suggesting that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides [2].

Monoclonal antibodies (PCSK Mabs)

PCSK Mabs are human monoclonal antibodies that bind specifically to human PCSK9 and prevent its interaction with LDL-R, thereby decreasing LDL-C. There are 3 PCSK Mabs being actively studied in clinical trials: (i) Regeneron/Sanofi Product ALIROCUMAB/SAR236553 (Alirocumab, PraluentTM), (ii) Amgen Product EVOLOCUMAB (evolocumab, RepathaTM), and (iii) Pfizer Product RN 316 (bococizumab). Both alirocumab and evolocumab have been tested in phase I, phase II and phase III trials. Pfizer’s bococizumab has been slow to start but fast catching up with cardiovascular outcomes phase III trials of other two products.

Alirocumab (PraluentTM)

Alirocumab is a specific monoclonal antibody to PCSK9 that significantly reduced LDL-C levels in healthy volunteers and in subjects with hypercholesterolemia in phase I studies. It was subsequently tested in phase II studies in patients with primary hypercholesterolemia with impressive results [6].

Preclinical experience

In rats and monkeys, alirocumab distribution is restricted to intravascular compartment following IV administration. It is degraded into small peptides and individual amino acids with a mean half life of 98-121 hours in rats and 51-61 hours in monkeys.

Phase I studies: Three phase 1 studies of ALIROCUMAB in healthy volunteers have been reported. Of these, two were randomized, single ascending-dose studies of either intravenous (n=40) or subcutaneous (n=32) administration of alirocumab versus placebo. These studies were followed by a randomized, placebo-controlled, multiple-dose trial in adults with heterozygous familial hypercholesterolemia (HeFH) who were receiving atorvastatin (n=21) and those with non-familial hypercholesterolemia who were receiving treatment with atorvastatin (n=30) (baseline LDL-C >100 mg/dL) or a modified diet alone (n=10) (baseline LDL-C >130 mg/dL). Alirocumab doses of 50, 100, or 150 mg were administered subcutaneously on days 1, 29, and 43. Among subjects receiving alirocumab, there were no discontinuations because of adverse events (primary end point). Alirocumab significantly lowered LDL-C levels in all studies (secondary end point) [7]. In the multiple-dose study, alirocumab doses of 50, 100, and 150 mg reduced measured LDL-C levels in the combined atorvastatin-treated populations from baseline by −39.2, −53.7, and −61.0 percentage points respectively as compared with placebo (P<0.001 for all comparisons). The magnitude of these effects was similar in familial and non-familial hypercholesterolemia. There was also a favourable trend on Lp(a), particularly in patients receiving atorvastatin. There was no apparent hepatotoxicity or discontinuations for any adverse effects (Table 1) [8].

Phase 2 studies

Study 1003: This multicentre, randomised, blind, placebo-controlled phase 2 trial assessed the efficacy and safety of various doses and dosing intervals of alirocumab (150 mg, 200 mg, or 300 mg every 4 weeks, or 150 mg every 2 weeks, or placebo every 2 weeks) added to statins, to further lower LDL-C in 77 patients with HeFH with a baseline LDL-C >100 mg/dl on stable diet and statin dose with or without ezetimibe. Randomisation was stratified by concomitant use of ezetimibe at baseline. The primary endpoint of mean percent reduction in LDL-C from baseline at week 12 was 28·9% for 150 mg every 4 weeks (p=0·0113), 31·54% for 200 mg every 4 weeks (p=0·0035), 42·53% for 300 mg every 4 weeks (p<0·0001), and 67·90% for 150 mg every 2 weeks (p<0·0001), compared with 10·65% with placebo. Over 80% achieved an LDL-C <70 mg/dL. LDL-C reductions averaged nearly 100 mg/dL from baseline. No serious adverse event was reported with alirocumab. No increases of more than three times the upper limit of normal were reported for hepatic transaminases or creatinine kinase. The most common adverse event was injection-site reaction. Thus, alirocumab significantly reduced LDL-C levels in HeFH patients already on high-dose statin, with/without ezetimibe. More sustained LDL-C reducing efficacy was noted with 2 weekly regime compared to higher doses administered every 4 weeks (Table 2) [9].

Study 11565:To evaluate the LDL-C lowering efficacy of alirocumab in patients with primary hypercholesterolemia, a double-blind, randomized, parallel-group, placebo controlled trial of 12 weeks duration was conducted in 183 patients with LDL-C ≥100 mg/dl on stable-dose atorvastatin 10, 20, or 40 mg. The treatment arms were SC placebo every 2 weeks; alirocumab 50, 100, or 150 mg every 2 weeks; or 200 or 300 mg every 4 weeks, alternating with placebo. Alirocumab demonstrated a clear dose-response relationship with respect to percentage LDL-C lowering for both 2 weekly (40%, 64%, and 72%) and 4 weekly (43% and 48%) administration (p<0.0001). Alirocumab also substantially reduced non-HDL-C, apoB, and Lp(a). It improved achievement of LDL-C targets, particularly with 2 weekly regime. There were no signals for persistent or prevalent clinical or laboratory adverse events, including hepatic and muscle assessments. One patient on alirocumab had leukocytoclastic vasculitis, attributed to study drug that resolved with prednisolone [10].

Study 11566: In another phase II study in 92 patients of primary hypercholesterolemia, alirocumab plus atorvastatin 80 mg reduced LDL-C by 73%, representing a 56% additional reduction in LDL-C compared to Placebo + Atorvastatin 80 mg. Addition of alirocumab allowed significantly more subjects to attain LDL-C levels of <100 mg/dL and <70 mg/dL. A similar LDL-C magnitude of effect was seen when alirocumab was added to atorvastatin 10 mg. Co-administration of alirocumab and atorvastatin 80/10 mg was generally well tolerated and 2 weekly dosing of alirocumab appeared to maintain a steady state reduction in LDL-C [11].

Evolocumab (RepathaTM): Evolocumab, another human monoclonal IgG2 antibody against PCSK9, had been tested in 4 phase II studies.

Laplace Timi 57 study: Laplace-Timi 57 study assessed the efficacy, safety, and tolerability of EVOLOCUMAB in stable patients with hypercholesterolemia on a statin. In this dose-ranging, multinational, blinded study in the USA, Canada, and Europe, 631 patients (18-80 years) with LDL-C >85 mg/dL on a stable dose of statin, with or without ezetimibe, were randomly assigned equally, to SC injections of EVOLOCUMAB 70 mg, 105 mg, or 140 mg, or matching placebo every 2 weeks; or subcutaneous injections of EVOLOCUMAB 280 mg, 350 mg, or 420 mg, or matching placebo every 4 weeks. At the end of the dosing interval at week 12, the primary end point of mean LDL-C reduction was 41·8% to 66·1% in 2 weekly regime; p<0·0001 for each dose and 41·8% to 50·3%; in 4 weekly regime; p<0·0001, without any treatment-related serious adverse events (Table 3) [12].

Mendel study: This multinational phase II study assessed the effects of EVOLOCUMAB in 406 patients (18-75 years) with hypercholesterolaemia (serum LDL-C 100-190 mg/dL) in the absence of concurrent lipid-lowering treatment. Subjects were randomly assigned equally to SC EVOLOCUMAB 70 mg, 105 mg, or 140 mg, or placebo every 2 weeks; SC EVOLOCUMAB 280 mg, 350 mg, or 420 mg or placebo every 4 weeks; or open label ezetimibe 10 mg/day. The primary endpoint was percentage change from baseline in LDL-C concentration at week 12. EVOLOCUMAB significantly reduced LDL-C in all dose groups (mean baseline LDL-C 142 mg/dl; changes from baseline with every 2 weeks EVOLOCUMAB 70 mg: −41·0% 105 mg −43·9%; 140 mg −50·9%; every 4 weeks EVOLOCUMAB 280 mg −39·0%; 350 mg −43·2%; 420 mg −48·0%; placebo every 2 weeks −3·7%; placebo every 4 weeks 4·5%; and ezetimibe −14·7% (p<0·0001 for all doses vs placebo or ezetimibe). Treatment-emergent adverse events occurred in 136 (50%) of 271 patients in the EVOLOCUMAB groups, 41 (46%) of 90 patients in the placebo groups, and 26 (58%) of 45 patients in the ezetimibe group; no deaths or serious treatment-related adverse events were reported (Table 4) [13].

Rutherford study: Despite statin treatment, many patients with HeFH do not reach desired LDL-C targets. This phase 2, multicenter, double-blind, randomized, placebo-controlled, dose-ranging study evaluated the efficacy and safety of EVOLOCUMAB in HeFH patients diagnosed by Simon Broome criteria with LDL-C >100 mg/dL despite statin therapy with or without ezetimibe. The subjects (n=167) were randomized 1:1:1 to EVOLOCUMAB 350 mg, 420 mg, or placebo-administered SC every 4 weeks. At week 12, the primary end point of mean percentage change from baseline in LDL-C was 43% and 55% with EVOLOCUMAB 350 mg and 420 mg, respectively, compared with 1% increase with placebo (P<0.001 for both dose groups). Serious adverse events (not considered treatment-related) occurred in 2 patients on EVOLOCUMAB. Thus, EVOLOCUMAB administered every 4 weeks yielded rapid and substantial reductions in LDL-C in HeFH patients despite intensive statin use, with or without ezetimibe, with minimal adverse events and good tolerability (Table 5) [14].

Gauss Study: In this study of 160 statin intolerant patients with a mean LDL-C of 193 mg/dl, EVOLOCUMAB 280, 350, 420 mg once in 4 w or EVOLOCUMAB 420 + ezetemibe, or ezetemibe alone, the magnitude of LDL-C reduction at 12 weeks was EVOLOCUMAB alone: 41% to 51%, EVOLOCUMAB + ezetemibe: 63%, and ezetemibe alone: 15%. The proportion of subjects achieving LDL-C <100 mg/dl was EVOLOCUMAB alone: 47% to 61%, EVOLOCUMAB + ezetemibe: 90%, and ezetemibe alone: 7%. Myalgia was reported by 3% on monotherapy, CK elevation >10 times was found in 2 patients and none developed significant elevation of hepatic transaminases (Table 6) [15].

Major findings in Phase II trials: Overall, subcutaneous (sc) administration of PCSK Mabs has been able to achieve 40% to 60% LDL-C reduction beyond what can be achieved by statins. In general, 2-weekly regimes have been more effective than 4-weekly dosing intervals. The most common side effects have been injection site reactions and gastrointestinal symptoms. Myalgia was reported in 5% to 10%. Liver or renal dysfunction has not been reported with PCSK Mabs.

PCSK9 and Lp(a): In a pooled analysis of 1359 patients on background statin therapy in 4 phase II trials of 12 weeks duration, evolocumab resulted in ~25% to ~30% reductions in Lp(a) compared to control (p < 0.001). While the mean percentage of reduction was significantly greater in those patients with baseline Lp(a) of ≤125 nmol/l, the absolute reduction was substantially larger in those with levels >125 nmol/l. Mean percentage reductions did not differ based on age or sex but the trend was greater in those patients taking statins [16].

Phase III Studies with Alirocumab

Odyssey combo I: The ODYSSEY COMBO I study evaluated the efficacy and safety of alirocumab as add-on therapy to stable maximally tolerated daily statin with or without other lipid-lowering therapy in high cardiovascular risk patients with suboptimally controlled hypercholesterolemia. This multicenter, phase 3, randomized, double-blind, 52-week trial enrolled 316 patients with established coronary heart disease or coronary heart disease risk equivalents and hypercholesterolemia. The primary efficacy end point of percent change in LDL-C from baseline to week 24 was -48.2% and -2.3% for alirocumab and placebo, respectively (P < .0001). Treatment-emergent adverse events were comparable between groups (Table 7) [17,18].

Odyssey combo II: The 720-patient, double-blind ODYSSEY COMBO II trial evaluated the long-term safety and efficacy of alirocumab vs. ezetimibe in combination with a maximally tolerated statin dose. Results showed greater decrease in LDL-C with alirocumab versus ezetimibe at week 24 (51% vs. 21%, P<0.0001). In addition, 77 percent of alirocumab patients achieved LDL-C <70 mg/dL at week 24 [19].

Odyssey long-term: Odyssey Long-term was a randomized trial involving 2341 patients at high risk for cardiovascular events who had LDL cholesterol levels of >70 mg/dL and were receiving treatment with statins at the maximum tolerated dose with or without other lipid-lowering therapy. Patients were randomly assigned in a 2:1 ratio to receive alirocumab (150 mg) or placebo every 2 weeks for 78 weeks. At week 24, the difference between the alirocumab and placebo groups in the mean percentage change from baseline in calculated LDL-C was −62% points (P<0.001); the treatment effect remained consistent over a period of 78 weeks. In a post-hoc analysis, the rate of major adverse cardiovascular events was lower with alirocumab than with placebo (1.7% vs. 3.3%; hazard ratio, 0.52, nominal P = 0.02) [20].

Odyssey mono: In this study, the efficacy and safety of alirocumab were compared with ezetimibe in hypercholesterolemic patients at moderate cardiovascular risk not receiving statins or other lipid-lowering therapy. The primary endpoint of mean LDL-C percentage change from baseline to 24 weeks was 47% with alirocumab versus 16% with ezetimibe [21].

Odyssey alternative: Odyssey alternative compared alirocumab with ezetimibe in patients at moderate to high cardiovascular risk with statin intolerance. Patients (n=361) received single-blind (sc) and oral placebo for 4 weeks during placebo run-in. Patients reporting muscle-related symptoms during the run-in were to be withdrawn. Continuing patients were randomized to double-blind alirocumab, ezetimibe, or atorvastatin 20 mg/d for 24 weeks. Primary end point was percent change in LDL-C from baseline to week 24 for alirocumab vs ezetimibe. Alirocumab reduced mean LDL-C by 45.0% vs 14.6% with ezetimibe (P=0.0001). Skeletal muscle-related events were less frequent with alirocumab vs atorvastatin (hazard ratio 0.6, P=0.042) [22].

Odyssey FH I and FH II: Odyssey FH I (n=486) and FH II (n=249) assessed the long-term (78 weeks) effects of alirocumab treatment in patients with HeFH, inadequately controlled on maximally tolerated lipid-lowering therapy. Mean LDL-C levels decreased by 57.9% vs. placebo at week 24 in FH I and by 51.4% vs. placebo in FH II (P < 0.0001). These reductions were maintained through Week 78. LDL-C <70 mg/dl was achieved at week 24 by 59.8% and 68.2% of alirocumab-treated patients in FH I and FH II, respectively [23].

Odyssey option I: To compare the LDL-C-lowering efficacy of adding alirocumab with other lipid-lowering strategies, 355 patients with very high CVD risk and LDL-C >70 or high CVD risk and LDL-C >100 mg/dL on baseline atorvastatin 20 or 40 mg were randomized to: 1) add-on alirocumab, 2) add-on ezetimibe, 3) double atorvastatin dose, or 4) for atorvastatin 40 mg regimen only, switch to rosuvastatin 40 mg. The primary end point was percentage change in calculated LDL-C from baseline to 24 weeks. Among atorvastatin 20 and 40 mg regimens, respectively, add-on alirocumab reduced LDL-C levels by 44.1% and 54.0% (P=0.001 vs all comparators); add-on ezetimibe, 20.5% and 22.6%; doubling of atorvastatin dose, 5.0% and 4.8%; and switching atorvastatin 40 mg to rosuvastatin 40 mg, 21.4%. Most alirocumab-treated patients (87.2% and 84.6%) achieved their LDL-C goals. Treatment-emergent adverse events occurred in 65.4% of alirocumab patients vs 64.4% ezetimibe and 63.8% double atorvastatin/switch to rosuvastatin [24,25].

Phase III Trials with Evolocumab

Mendel-2: Mendel-2 compared evolocumab with placebo and oral ezetimibe in 614 adult patients with fasting LDL-C between 100 m/dl and <190 mg/dl and Framingham risk scores ≤10%. Evolocumab reduced LDL-C from baseline by ~55% more than placebo and 38% more than ezetimibe (p < 0.001 for both). Adverse events and muscle-related symptoms were comparable across treatment groups (Table 8) [26].

Gauss-3: GAUSS-3 trial evaluated evolocumab in patients with high cholesterol who cannot tolerate statins. It met its co-primary endpoints of mean percent reductions from baseline in LDL-C at weeks 22 and 24, and the percent reduction from baseline in LDL-C at week [27].

Descartes: Descartes was a phase 3 placebo-controlled trial to evaluate the safety and efficacy of 52 weeks of treatment with evolocumab 420 mg every 4 weeks or placebo in 901 patients with an LDL-C of >75 mg/dL despite a background diet or diet plus atorvastatin (10/80 mg daily), or atorvastatin 80 + ezetimibe 10 mg. The primary end point of the change from baseline in LDL-C at week 52 was 57% in evolocumab group as compared to placebo (P<0.001) [28].

Laplace-2: To evaluate the efficacy and tolerability of evolocumab when used in combination with a moderate- vs high-intensity statin, this Phase 3, 12-week, randomized, double-blind, placebo- and ezetimibe-controlled study in patients with primary hypercholesterolemia and mixed dyslipidemia (n = 2067) were randomized to 1 of 24 treatment groups in 2 steps. After a 4-week lipid-stabilization period, patients (n = 1899) were randomized to compare evolocumab (140mg every 2 weeks or 420mg monthly) with placebo or ezetimibe or placebo. 63% to 75%. Adverse events 0were reported in 36%, 40%, and 39%of evolocumab, ezetimibe-, and placebo-treated patients, respectively. The most common adverse events in evolocumab-treated patients were back pain, arthralgia, headache, muscle spasms, and pain in extremity (all <2%) [29].

Rutherford-2: Rutherford-2 investigated the effect of evolocumab on LDL-C in patients with heterozygous familial hypercholesterolaemia (HeFH). This multicentre, randomised, double-blind, placebo-controlled trial was undertaken with 331 adult patients with HeFH on stable lipid-lowering therapy with a fasting LDL-C of 2·6 mmol/L or higher, were randomly allocated to receive sc evolocumab every 2 weeks (n=111), 4 weeks (n=110), or placebo (n=110) for 12 weeks. Compared with placebo, evolocumab at both dosing schedules led to a significant ~60% reduction in mean LDL-C at week 12 (both p<0·0001). The most common adverse events were nasopharyngitis (9%) and muscle-related adverse events (5%) [30].

Osler-2: To examine long-term efficacy of evolocumab, 1104 (81%) of 1359 randomized patients in the 4 evolocumab phase 2 parent studies who elected to enroll in OSLER study were included in this open-label study. Regardless of their treatment assignment in the parent study, patients were randomized 2:1 to receive either open-label sc evolocumab every 4 weeks with standard of care (SOC) (n=736) or SOC alone (n=368). Around 90% of patients in both groups completed 52 weeks of follow-up. Patients who had first received evolocumab in OSLER experienced a mean 52.3% reduction in LDL-C at week 52 (P<0.0001). Patients who received 1 of 6 dosing regimens of evolocumab in the parent studies and received evolocumab+SOC in OSLER had persistent mean LDL-C reduction of 50% (P=0.31). In patients who discontinued evolocumab on entry, LDL-C levels returned to near baseline levels [31].

Tesla part B: Tesla Part B was a randomised, double-blind, placebo-controlled phase 3 trial involving 49 patients above 12 years with homozygous familial hypercholesterolaemia (HoFH) on stable lipid therapy and not receiving lipoprotein apheresis. The subjects were randomly allocated to receive sc evolocumab or placebo every 4 weeks for 12 weeks. The primary endpoint was percentage change in LDL-C. Compared with placebo, evolocumab significantly reduced LDL-C at 12 weeks by 31% (p<0·0001). No serious clinical or laboratory adverse events occurred, and no anti-evolocumab antibody development was detected during the study [32].

Taussig:Taussigstudy presented recently in the European Atherosclerosis Society 2016 Congress showed a 23% reduction in LDL-C over 48 weeks with open-label treatment with evolocumab in 106 patients >12 years with HoFH, ~a third of whom were also on lipoprotein apheresis. Though statistically underpowered to assess clinical events, investigators also estimated an annualized cardiac event rate of 2.14% for patients taking evolocumab, which compares favourably with an annualized mortality of 3.5% and a cardiovascular event rate of about 11% per year in other studies of HoFH patients on a statin plus ezetimibe combination [33,34].

PCSK9 inhibition and cardiovascular outcomes

The ultimate utility of PCSK9 inhibitors shall be determined by their effect on clinical outcomes. Preliminary evidence of cardiovascular benefit with evolocumab was suggested in OSLER-1 and OSLER-2 studies and in post hoc analysis of the ODYSSEY LONG TERM trial conducetd with alirocumab. Statistically powered cardiovascular outcome trials are underway with evolocumab (FOURIER; NCT01764633), alirocumab (ODYSSEY OUTCOMES; NCT01663402), and bococizumab (SPIRE-1; NCT01975376 and SPIRE-2; NCT01975389).

Approved clinical indications of PCSK9 inhibitors

Based on the results of RUTHERFORD-2 and TESLA Part B, evolocumab has recently been approved in several countries including US and EU. In the US, it is indicated in adults as an adjunct to other therapies for treatment of adults with HeFH, HoFH or clinical atherosclerotic cardiovascular disease (ASCVD). Alirocumab is also approved as adjunct to diet and maximally tolerated statin therapy for the treatment of adults with HeFH ASCVD, who require additional lowering of LDL-C. It is noteworthy that at the time of writing, the effect of both evolocumab and alirocumab on cardiovascular morbidity and mortality has not been determined.

Monoclonal antibodies have demonstrated LDL-C lowering of up to 60% as monotherapy and up to 70% when added to statins. Lipoprotein (a) levels may also be significantly lowered. LDL-C lowering is the primary target for ASCVD risk reduction as well as mortality reduction. However, 70% of events occur even on statin therapy. This residual risk may be attenuated with additional LDL-C reduction, though this is not yet proved. Statin intolerance is common, occurring in about 10%. There is also a significant inter-individual variation LDL-C response to statins. Treating to targets is also often an elusive goal in familial hypercholesterolemia. PCSK9 inhibition holds promise in management of all of these conditions.