Lipid metabolism and the targeting of angiopoietin-like 3: Experimental drugs under development

KEYWORDS:

• angiopoietin-like 3
• evinacumab
• vupanorsen
• ARO-ANG3
• dyslipidemia
• cardiovascular disease

1. Introduction

Angiopoietin-like protein 3 (ANGPTL3) is an important regulator of lipid metabolism [1]. ANGPTL3 inhibits lipoprotein lipase (LPL), resulting in an increase in serum triglyceride levels [2]. In addition, ANGPTL3 inhibits endothelial lipase, which in turn induces a reduction in high-density lipoprotein cholesterol (HDL-C) levels [3]. Near-complete suppression of ANGPTL3 activity is also associated with a reduction in low-density lipoprotein cholesterol (LDL-C) levels through a yet unclear mechanism [4,5]. In mendelian randomization studies, subjects with heterozygous loss-of-function variants in ANGPTL3 had lower levels of triglycerides, HDL-C and LDL-C than participants without these variants and also had 34–41% lower risk for coronary heart disease [4,5].

Several strategies that inhibit ANGPTL3 are currently under evaluation including monoclonal antibodies, antisense oligonucleotides, small interfering RNAs, vaccines and in vivo gene editing approaches to permanently knock out ANGPTL3 using clustered regularly interspaced palindromic repeats-associated protein 9 (CRISPR/Cas9). Monoclonal antibodies selectively inhibit circulating ANGPTL3. Antisense oligonucleotides are single-stranded deoxyribonucleotides, complementary to the ANGPTL3 mRNA and bind to the ANGPTL3 mRNA within the nucleus to form a DNA-RNA heteroduplex, which is then cleaved by the RNAse H endonuclease resulting in reduced mRNA translation [6]. Small interfering RNAs are double-stranded RNAs that enter into the cytoplasm to form the RNA-induced silencing complex (RISC) and then induce cleavage of the ANGPTL3 mRNA, reducing mRNA translation [6]. Vaccines aim to trigger the generation of host antibodies against ANGPTL3. The CRISPR/Cas9 system introduces double-strand breaks at desired sites within the ANGPTL3 gene, which are then repaired resulting in either frameshift mutations or in-frame insertions/deletions and therefore ANGPTL3 gene knock out [7]. Notably, statins reduce ANGPTL3 levels and therefore a blunting of the effect of agents that inhibit ANGPTL3 is expected in statin-treated patients [8]. The effects of other lipid-lowering agents, including ezetimibe and proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors, on ANGPTL3 levels is unknown.

2. Evinacumab

Evinacumab is a fully human monoclonal antibody against ANGPTL3 and has been evaluated in several studies. A phase-2, double-blind, randomized, placebo-controlled study enrolled 272 patients with or without heterozygous familial hypercholesterolemia who had refractory hypercholesterolemia (screening LDL-C ≥ 70 mg/dl with atherosclerosis or ≥ 100 mg/dl without atherosclerosis despite treatment with a PCSK9 inhibitor with or without a statin and ezetimibe). Evinacumab, at a dose of 300 or 450 mg weekly or 300 mg every 2 weeks administered subcutaneously, reduced LDL-C levels by 53, 56 and 38%, respectively, at 16 weeks [9]. When infused intravenously (5 or 15 mg/kg of body weight every 4 weeks), evinacumab lowered LDL-C levels by 24 and 50%, respectively, and reduced triglyceride levels by 25 and 61%, respectively, at 16 weeks [9]. On the other hand, a 15–31% reduction in HDL-C levels was also observed in patients treated with evinacumab 5 or 15 mg/kg of body weight every 4 weeks, respectively [9]. Urinary tract infections, injection-site erythema, arthralgias and myalgias occurred more frequently in the evinacumab than in the placebo group but were generally mild [9]. In a phase 3, double-blind, randomized, placebo-controlled study in 65 patients with homozygous familial hypercholesterolemia, intravenous infucsion of evinacumab (15 mg/kg of body weight every 4 weeks) reduced LDL-C levels by 49% and triglyceride levels by 50% at 24 weeks [10]. Reductions in LDL-C levels were similar in patients with null-null variants and in those with non-null variants [10]. A 30% reduction in HDL-C levels was also observed in patients treated with evinacumab [10]. Adverse event rates did not differ between the evinacumab and placebo groups [10]. Based on these findings, the US Food and Drug Administration and the European Medicines Agency approved evinacumab for the management of patients ≥ 12 years-old with homozygous familial hypercholesterolemia as an adjunct to diet and other lipid-lowering therapies [11,12]. In a more recent study in 56 patients with serum triglyceride levels ≥ 150 mg/dl and LDL-C levels ≥ 100 mg/dl, evinacumab (administered subcutaneously at a dose of 150, 300 or 450 mg once weekly or 300 or 450 mg every 2 weeks or infused intravenously at a dose of 20 mg/kg of body weight every 4 weeks) reduced triglyceride levels by up to 88%, LDL-C levels by up to 25% and was well tolerated [13]. A decrease in HDL-C levels by up to 24% was also observed in patients treated with evinacumab [13]. An ongoing single-arm, open-label study (NCT04233918) is evaluating the safety, efficacy and pharmacokinetics of evinacumab in pediatric (5–11 years-old) patients with homozygous familial hypercholesterolemia [14]. The primary objective is to demonstrate a reduction in LDL-C levels by evinacumab [14].

3. Vupanorsen

Vupanorsen (previously known as IONIS-ANGPTL3-L_Rx and AKCEA-ANGPTL3-L_Rx), is a N-acetylgalactosamine (GalNAc)-conjugated antisense oligonucleotide targeting ANGPTL3 mRNA [15]. GalNAc exhibits high affinity to hepatocyte-specific asialoglycoprotein receptors [16]. Given that ANGPTL3 is produced primarily by hepatocytes, the use of GalNAc increases the potency of vupanorsen [15]. Administration of vupanorsen to 44 healthy adults (a 10, 20, 40, or 60 mg subcutaneous injection once weekly for a total of 6 doses) lowered LDL-C levels by up to 33%, triglyceride levels by up to 63% and was well-tolerated [15]. However, patients treated with evinacumab also experienced a reduction in HDL-C levels by up to 27% [15]. In a more recent double-blind, placebo-controlled, dose-ranging study in 286 patients with triglyceride levels > 150 mg/dl despite treatment with statins, vupanorsen (80, 120 or 160 mg subcutaneously every 4 weeks, or 60, 80, 120 or 160 mg subcutaneously every 2 weeks) reduced triglyceride levels by 41–57% [17]. However, LDL-C levels decreased modestly (8–16%) and patients treated with vupanorsen experienced more frequently injection-site reactions, increase in aminotransferase levels and in hepatic steatosis compared to patients randomized to placebo [17]. In another recent double-blind, placebo-controlled, dose-ranging, phase 2 study in 105 patients with triglyceride levels >150 mg/dl, type 2 diabetes mellitus and hepatic steatosis, vupanorsen given subcutaneously 40 or 80 mg every 4 weeks or 20 mg every week reduced triglyceride levels by 36, 53 and 47%, respectively [18]. However, LDL-C decreased minimally (by up to 12%) and a reduction in HDL-C levels by up to 18% was observed in patients treated with vupanorsen [18]. Injection site reactions were the most frequent adverse event and were generally mild [18]. Following these results [17], the clinical development program of vupanorsen was halted due to the moderate reduction in LDL-C levels and the unfavorable safety profile [17].

4. ARO-ANG3

ARO-ANG3 is small interfering RNA targeting hepatic ANGPTL3 and is also conjugated to GalNAc [19,20]. In a pilot study in 12 healthy volunteers, ARO-ANG3 administered subcutaneously at a dose of 100, 200 or 300 mg on days 1 and 29 reduced triglyceride levels by up to 65%, LDL-C levels by up to 54% [19]. These reductions lasted for at least 12 weeks and the agent was well-tolerated [19]. On the other hand, a reduction in HDL-C levels by up to 37% was also recorded [19]. In another study in 17 patients with heterozygous familial hypercholesterolemia, the same dosing regimen of ARO-ANG3 reduced LDL-C levels by 23–37% and triglyceride levels by 25–43% and was well-tolerated [20]. In the same study, patients at high cardiovascular risk but without familial hypercholesterolemia exhibited similar reduction in LDL-C and triglyceride levels [20]. An ongoing double-blind, placebo-controlled, phase 2b study (NCT04832971) is evaluating the safety and efficacy of ARO-ANG3 in 204 patients with mixed dyslipidemia [21]. The primary outcome is the change in TG levels at week 24 [21]. Another ongoing open-label study (NCT05217667) is evaluating the safety and efficacy of ARO-ANG3 in patients with homozygous familial hypercholesterolemia [22]. The primary outcome is the change in LDL-C levels up to week 24 [22].

5. Other experimental approaches targeting ANGPTL3

In preclinical studies, a virus-like particle-based vaccine targeting the LPL-binding domain of ANGPTL3 reduced triglyceride levels in mice [23]. A peptide vaccine against ANGPTL3 reduced not only triglyceride levels in mice but also reduced LDL-C levels, hepatic steatosis and delayed the development of atherosclerosis in mice [24].

In LDL receptor-knockout mice, a model for homozygous familial hypercholesterolemia, a variation of the CRISPR/Cas9 genome editing that does not require DNA double-strand breaks was delivered using an adenoviral vector. Mice showed a reduction in cholesterol and TG levels by 51 and 56%, respectively [25]. In wild-type C57BL/6 mice, a lipid nanoparticle delivery platform carrying Cas9 mRNA and guide RNA for CRISPR-Cas9-based genome editing of ANGPTL3, resulted in a reduction in LDL-C and TG levels by 57 and 29%, respectively [26].

6. Conclusions

Targeting ANGPTL3 appears to represent a promising approach for reaching treatment targets in both patients with elevated LDL-C levels and in those with hypertiglyceridemia mentioned in Table 1. However, whether the beneficial effects on the lipid profile will translate into a reduction in cardiovascular morbidity should be evaluated in appropriately designed large clinical trials.

Table 1. Summary of safety, efficacy and current status of strategies targeting angiopoietin-like 3.

Strategy	Agent	Efficacy	Safety	Current status
Monoclonal antibodies	Evinacumab	↓ LDL-C by up to 56% ↓ TG by up to 88%	↓ HDL-C by up to 31% No other differences compared with placebo	Approved by the US Food and Drug Administration and the European Medicines Agency
Antisense oligonucleotides	Vupanorsen	↓ LDL-C by up to 33% ↓ TG by up to 63%	↓ HDL-C by up to 27% Increase in aminotransferase levels and in hepatic steatosis	Clinical development program discontinued
Small interfering RNAs	ARO-ANG3	↓ LDL-C by up to 54% ↓ TG by up to 65%	↓ HDL-C by up to 37% No other differences compared with placebo	Phase II studies ongoing
Vaccines, gene editing	(-)	(-)	(-)	Animal models

LDL-C: low-density lipoprotein cholesterol; TG: triglycerides; HDL-C: high-density lipoprotein cholesterol

7. Expert opinion

The development of therapeutic strategies that target ANGPTL3 provides a new tool for the management of patients with dyslipidemia. Evinacumab has already been approved for the management of patients with homozygous familial hypercholesterolemia but might also be useful in patients with heterozygous familial hypercholesterolemia, in patients who are intolerant to statins and in patients with very high cardiovascular risk who cannot achieve LDL-C targets despite treatment with statins and ezetimibe. However, in these groups of patients, PCSK9 inhibitors have already shown a reduction in cardiovascular events in addition to their LDL-C lowering efficacy. Therefore, the effects of evinacumab on cardiovascular events should be evaluated before it is considered in the place of PCSK9 inhibitors in patients who cannot achieve LDL-C targets despite treatment with potent statins and ezetimibe. In this context, all ANGPTL3 reduce HDL-C levels and it is unclear whether this effect will blunt the cardiovascular benefit associated with the reduction in LDL-C levels. Indeed, it is unknown whether ANGPTL3 inhibitors affect HDL functionality, which appears more strongly related to cardiovascular risk than HDL quantity. It is also unclear whether the reduction in triglyceride levels by ANGPTL3 inhibitors will contribute to the prevention of cardiovascular events. Indeed, fibrates have failed to show a reduction in cardiovascular morbidity despite their efficacy in lowering triglyceride levels. Cost is another consideration, since evinacumab is more expensive than PCSK9 inhibitors. Another disadvantage of evinacumab is the need for monthly infusion whereas antisense oligonucleotides and particularly small interfering RNAs appear to induce more long-lasting reductions in LDL-C and TG levels. On the other hand, vupanorsen induced smaller reductions in LDL-C and TG levels than evinacumab. It is therefore unclear whether the therapeutic strategy (monoclonal antibodies, antisense oligonucleotides or small interfering RNAs) or the specific agents used are more important determinants of LDL-C- and TG-lowering. It is also unclear whether the site of ANGPTL3 inhibition (intrahepatic or circulating) might increase the risk for hepatic steatosis as was seen with vupanorsen. On the other hand, monoclonal antibodies have been used in several diseases for a long time and patients might be more eager to receive a monoclonal antibody than a treatment targeting RNA. Hopefully, the efficacy and safety of RNA-based vaccines against COVID19 might alleviate potential fears against RNA-based therapies. In this context, vaccination against ANGPTL3 might offer an even longer-lasting inhibition of this pathway and further improve adherence to treatment. However, studies in humans are needed to evaluate the safety of this approach, which carries a theoretical risk for T-cell activation and off-target adverse effects. Finally, gene editing might represent the future in the inhibition of ANGPTL3 since it will have to be administered even less frequently. However, longer-term follow-up of the safety of other strategies that inhibit ANGPTL3 is needed before testing this approach in patients.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

A reviewer on this manuscript has disclosed they are parttime employed by Novo Nordisk, Denmark. Peer reviewers on this manuscript have no other relevant financial relationships or otherwise to disclose.

Additional information

Funding

This paper was not funded.