In mice fed high-fat high-cholesterol diet, gene inactivation of PCSK9 significantly reduced the accumulation of cholesteryl esters in aortas, which was markedly increased by overexpression of PCSK9 resulting in accelerated development of atherosclerotic plaques [29]. Interestingly, in LDLR-deficient mice lacking or overexpressing PCSK9, no significant differences were observed in cholesteryl ester accumulation and plaque size, strongly suggesting that the process by which PCSK9 enhances atherosclerosis is primarily mediated through its action on the LDLR [29].

Cloned minipigs created by transposition of a human PCSK9 gain-of-function mutant – a model for familial hypercholesterolemia (FH) – had a significant increase in aortic atherosclerosis compared with wild-type minipigs [30]. At the opposite, inhibition of PCSK9 by a mAb, alirocumab, reduced atherosclerosis development in ApoE3Leiden. CETP mice model, and enhanced the beneficial effects of atorvastatin [31], providing an argument for statin and PCSK9 combination therapy.

The impact of PCSK9 inhibition on atherosclerotic plaques is currently evaluated by intravascular ultrasound in a large phase III trial with evolocumab, GLAGOV (GLobal Assessment of plaque reGression with a PCSK9 antibOdy as measured by intraVascular ultrasound) [32].

PCSK9 gene expression is mainly modulated by intracellular cholesterol concentrations and consequent activation of the transcription factor sterol-responsive element-binding protein 2 (SREBP2) [21] (Fig. 9.1b), similarly to other genes involved in cholesterol homeostasis, such as LDLR. The relationship between statin treatment and PCSK9 secretion has been investigated in animals and humans. In hepatic cell lines, statins upregulated the mRNA expression of LDLR and PCSK9 [33]. Statin administration to PCSK9 knockout mice enhanced LDL clearance from plasma [21]. In humans, statins induced a dose-dependent increase in the concentration of plasma PCSK9 [34, 35]. This concomitant regulation of both PCSK9 and LDLR by cholesterol via SREBP2 helps to explain the paradoxical effect of statin therapy [36], potentially limiting the pharmacological effect of statin on LDL-C concentration. This was confirmed in patients with LOF mutations in PCSK9 gene who are more responsive to statin therapy [37]. These data support PCSK9 inhibition as a very attractive target for lowering LDL-C and enhancing the efficacy of statin treatment.

Several therapeutic approaches to the inhibition of PCSK9 have been proposed [38], including inhibition of PCSK9 synthesis by gene-silencing agents, such as antisense oligonucleotides (ASOs) or small interfering RNA (siRNA); inhibition of PCSK9 binding to LDLR by mAbs, small peptides, or adnectins; and inhibition of PCSK9 autocatalytic processing by small molecule inhibitors. These strategies targeting either extracellular or intracellular PCSK9 have been extensively described in recent reviews [39–43].

Preclinical studies on inhibition of PCSK9 synthesis by ASOs were promising, but the development of two ASOs by BMS/ISIS (BMS-84421) and Santaris Pharma (SPC5001) was stopped in Phase I [43]. siRNA is another approach [44, 45]. In rats, siRNA targeting PCSK9 reduced LDL-C level by around 30 %, and in nonhuman primates, single-dose administration of 5 mg of the drug decreased LDL-C by 56–70 % [44]. In a phase I trial of ALN-PCS, an siRNA developed by Alnylam Pharmaceuticals, a dose-dependent reduction in LDL-C was observed, with a 40 % reduction with the highest dose, associated with a 70 % reduction in plasma PCSK9 concentrations [46]. Inhibition of PCSK9 binding to LDLR by small peptide inhibitors such as SX-PCK9 (Serometrix, East Syracuse, NY, USA) or adnectins such as BMS-962476 (BMS/Adnexus, Waltham, MA, USA) are in preclinical development or phase I [39–41]. On the basis of the discovery of a LOF mutation in the autocatalytic cleavage site of PCSK9 [47], inhibition of PCSK9 autocatalytic processing is the approach chosen by Cadila Healthcare and Shifa Biomedical [39] with molecules in preclinical development phase. Finally, mAbs [48] are the most studied and advanced approach in terms of clinical development with published phase I, II, and III human trials. An alternative approach for PCSK9 inhibition could be a peptide-based anti-PCSK9 active vaccination approach providing the opportunity for long-term LDL-C management [49].

Several mAbs targeting PCSK9 have been tested in preclinical studies to assess their disruption of the PCSK9-LDLR interaction or inhibition of PCSK9 internalization [39, 43]. Human data are mainly available for three mAbs: Alirocumab (SAR236553/REGN727) and evolocumab (AMG 145), two fully human mAbs developed by Sanofi/Regeneron and Amgen, respectively, and bococizumab (RN316/PF04950615), a humanized mAb developed by Pfizer/Rinat. The data obtained in phase II have been already extensively described [11, 42, 43]: globally, in combination with a statin – and also in monotherapy – mAbs induced dramatic significant decreases in all the atherogenic lipoproteins.

Three large phase III programs have been developed with alirocumab, evolocumab, and bococizumab. The lipid-lowering phase III trials [50–73] with these three mABs are summarized in Tables 9.1, 9.2, and 9.3. Current data indicate that mAbs are very effective at lowering concentrations of atherogenic lipoproteins, with significant decreases in LDL-C, apoB, non-HDL-C, and also Lp(a) concentrations. So far, in the phase III programs, efficacy has been demonstrated

These mAbs are administered as subcutaneous (SC) injections, with various doses and strategies: in the ODYSSEY program, alirocumab SC injections are mainly realized every 2 weeks (Q2W). Two doses, 75 and 150 mg, have been tested in the majority of phase III trials, with the possibility to uptitrate alirocumab from 75 to 150 mg Q2W depending on LDL-C goal achievement. The frequency of injections every 4 weeks (Q4W) and the dose of 300 mg Q4W have been recently evaluated in CHOICE trials [60, 61]. In the PROFICIO program, two doses of evolocumab have been tested, 140 mg Q2W and 420 mg Q4W [62–68]. Finally, the SPIRE program is conducted with bococizumab 150 mg Q2W.

Globally, in combination with a statin, mAbs decrease LDL-C levels by 39–61 % in heterozygous FH and by 46–76 % in other hypercholesterolemic patients.

In the TESLA trial, the mean decrease in LDL-C is – as expected – less in patients with homozygous FH [67], with a greater response on evolocumab (−40.8 % in LDL-C) in patients defective in one or both alleles, providing a new complementary therapeutic strategy to treat these very high patients.

Moreover, treatment with mAbs induces a significant decrease in Lp(a) levels. A pooled analysis from 1359 patients in 4 phase II trials showed a dose-dependent reduction of Lp(a) levels with evolocumab (−29.5 and −24.5 % with 140 mg Q2W and 420 mg Q4W respectively) [75]. Complementary studies are needed to characterize the mechanism underlying this effect and to determine the clinical relevance of the Lp(a) reducing effect. However, especially considering that current therapies effective in reducing Lp(a) are limited to mipomersen (an anti-apoB antisense oligonucleotide) or lomitapide (an MTP inhibitor) [76], PCSK9 inhibitors might be an effective option to improve the CVD risk of patients with elevated Lp(a) plasma levels.

Overall, the mAbs tested so far have been generally safe and well tolerated, with no major safety issues and no differences in the rate of adverse events (AEs) between treatment and placebo groups from completed phase II and III studies.

In all of the phase 2 studies, alirocumab was generally well tolerated over the treatment period [8–12 weeks]. Injection-site reactions were the most common AEs in two of the phase II trials but were generally mild in severity and transient. However, in the phase II study assessing alirocumab for treatment of FH [77], one patient in the group of 300 mg dose Q4W discontinued treatment after the first dose due to injection-site reaction and generalized pruritus. In another phase II trial [78], one patient receiving atorvastatin 80 mg plus alirocumab 150 mg Q2W discontinued treatment due to a hypersensitivity reaction and rash occurring 12 days after the second injection of mAb. There was a single case of cutaneous leukocytoclastic vasculitis reported in one patient, 9 days after initiation of alirocumab 300 mg [79]. The patient responded rapidly to withdrawal of the drug and initiation of steroid therapy.

Evolocumab was also generally well tolerated throughout the phase II trials, with a similar incidence of drug-related AEs across treatment groups and no evidence of a relationship between the incidence of any AEs and evolocumab dose [42]. Small numbers of serious adverse events (SAEs) occurred, but none was considered related to the treatment. Injection-site reactions were generally infrequent and mild. Of the 1359 randomized patients in the evolocumab phase II parent studies, 1104 (81 %) elected to enroll in the OSLER study, open trial designed to evaluate mainly longer-term safety. Patients were randomized 2:1 to receive either open-label SC evolocumab 420 mg Q4W with standard of care or standard of care alone [80]. In the OSLER trial, AE occurred in 81.4 % of evolocumab-treated patients and in 73.1 % of patients in the standard of care group. SAE occurred in 7.1 % of patients in the evolocumab group and 6.3 % in the standard of care group. An injection-site reaction was reported in 5.2 % of patients in the evolocumab group. In a specific analysis of the frequency of AEs by LDL-C value on evolocumab treatment, an imbalance appeared in memory impairment in patients with LDL-C <50 mg/dL: four patients (1 %) compared to one patient (0.3 %) in evolocumab subgroup with LDL-C ≥50 mg/dL and zero in the standard of care group. This finding seems at the origin of the request from FDA to make an assessment of potential neurocognitive AEs across the phase III development program for all the mAbs against PCSK9, especially in the longer-term studies.

Indeed, more information on safety and tolerability has been obtained from phase III programs of alirocumab [50–61] and evolocumab [62–67], especially from the longer-term ODYSSEY LONG TERM [53] and DESCARTES [63] trials. In the DESCARTES trial conducted in 901 patients (599 on evolocumab 420 mg Q4W, 302 on placebo) during 52 weeks, the overall incidence of AEs was similar in the evolocumab group and the placebo group. The most common AEs in the evolocumab group were nasopharyngitis, upper respiratory tract infection, influenza, and back pain. Injection-site reactions were reported in 5.7 % of patients in the evolocumab group and 5.0 % of patients in the placebo group [63].