Elsevier

The Lancet

Volume 386, Issue 10002, 10–16 October 2015, Pages 1472-1483
The Lancet

Articles
Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study

https://doi.org/10.1016/S0140-6736(15)61252-1Get rights and content

Summary

Background

Lipoprotein(a) (Lp[a]) is a risk factor for cardiovascular disease and calcific aortic valve stenosis. No effective therapies to lower plasma Lp(a) concentrations exist. We have assessed the safety, pharmacokinetics, and pharmacodynamics of ISIS-APO(a)Rx, a second-generation antisense drug designed to reduce the synthesis of apolipoprotein(a) (apo[a]) in the liver.

Methods

In this randomised, double-blind, placebo-controlled, phase 1 study at the PAREXEL Clinical Pharmacology Research Unit (Harrow, Middlesex, UK), we screened for healthy adults aged 18–65 years, with a body-mass index less than 32·0 kg/m2, and Lp(a) concentration of 25 nmol/L (100 mg/L) or more. Via a randomisation technique, we randomly assigned participants to receive a single subcutaneous injection of ISIS-APO(a)Rx (50 mg, 100 mg, 200 mg, or 400 mg) or placebo (3:1) in the single-dose part of the study or to receive six subcutaneous injections of ISIS-APO(a)Rx (100 mg, 200 mg, or 300 mg, for a total dose exposure of 600 mg, 1200 mg, or 1800 mg) or placebo (4:1) during a 4 week period in the multi-dose part of the study. Participants, investigators, and study staff were masked to the treatment assignment, except for the pharmacist who prepared the ISIS-APO(a)Rx or placebo. The primary efficacy endpoint was the percentage change from baseline in Lp(a) concentration at 30 days in the single-dose cohorts and at 36 days for the multi-dose cohorts. Safety and tolerability was assessed 1 week after last dose and included determination of the incidence, severity, and dose relation of adverse events and changes in laboratory variables, including lipid panel, routine haematology, blood chemistry, urinalysis, coagulation, and complement variables. Other assessments included vital signs, a physical examination, and 12-lead electrocardiograph. This trial is registered with European Clinical Trials Database, number 2012-004909-27.

Findings

Between Feb 27, 2013, and July 15, 2013, 47 (23%) of 206 screened volunteers were randomly assigned to receive ISIS-APO(a)Rx as a single-dose or multi-dose of ascending concentrations or placebo. In the single-dose study, we assigned three participants to receive 50 mg ISIS-APO(a)Rx, three participants to receive 100 mg ISIS-APO(a)Rx, three participants to receive 200 mg ISIS-APO(a)Rx, three participants to receive 400 mg ISIS-APO(a)Rx, and four participants to receive placebo. All 16 participants completed treatment and follow-up and were included in the pharmacodynamics, pharmacokinetics, and safety analyses. For the multi-dose study, we assigned eight participants to receive six doses of 100 mg ISIS-APO(a)Rx, nine participants to receive six doses of 200 mg ISIS-APO(a)Rx, eight participants to receive six doses of 300 mg ISIS-APO(a)Rx, and six participants to receive six doses of placebo. Whereas single doses of ISIS-APO(a)Rx (50–400 mg) did not decrease Lp(a) concentrations at day 30, six doses of ISIS-APO(a)Rx (100–300 mg) resulted in dose-dependent, mean percentage decreases in plasma Lp(a) concentration of 39·6% from baseline in the 100 mg group (p=0·005), 59·0% in the 200 mg group (p=0·001), and 77·8% in the 300 mg group (p=0·001). Similar reductions were observed in the amount of oxidized phospholipids associated with apolipoprotein B-100 and apolipoprotein(a). Mild injection site reactions were the most common adverse events.

Interpretation

ISIS-APO(a)Rx results in potent, dose-dependent, selective reductions of plasma Lp(a). The safety and tolerability support continued clinical development of ISIS-APO(a)Rx as a potential therapeutic drug to reduce the risk of cardiovascular disease and calcific aortic valve stenosis in patients with elevated Lp(a) concentration.

Funding

Isis Pharmaceuticals.

Introduction

Lipoprotein(a) (Lp[a]) consists of apolipoprotein(a) (apo[a]) covalently linked to apolipoprotein B-100 (apoB). Apo(a) is synthesised by hepatocytes and binds covalently to newly synthesised apoB to form Lp(a).1, 2 The primary determinant of plasma Lp(a) concentration is the LPA gene, which evolved through duplication and remodelling of the kringle (K) domains of the plasminogen gene. The plasminogen gene has five kringle domains (K), but the LPA gene is composed only of KIV, KV, and a catalytically inactive protease domain. Ten KIV domain subtypes have arisen through duplication, of which KIV2 is present in multiple and variable copies that define the heterogeneity of Lp(a) molecular size and plasma concentration.3

Lp(a) concentrations in the atherogenic range (>250–300 mg/L or 62·5–75 nmol/L) are highly prevalent, affecting 20–30% of the population worldwide. Lp(a) concentration is inversely associated with the number of KIV2 repeats and positively associated with single nucleotide polymorphisms (SNPs) rs10455872 and rs3788220.4, 5 Ordinarily, environmental and dietary factors have minimal effects on Lp(a) concentrations. A physiological function of Lp(a) has not been identified. However, epidemiological studies and meta-analysis in primary and secondary prevention settings, genome-wide association studies, and Mendelian randomisation studies strongly support its causal, independent role in myocardial infarction, stroke, peripheral arterial disease, and calcific aortic valve stenosis.5, 6, 7, 8 Findings from these studies consistently suggest that risk of cardiovascular disease and calcific aortic valve stenosis is driven by increased plasma Lp(a) concentration rather than independent effects of LPA genotypes. Clinical trials to lower Lp(a) concentrations and reduce the risk of cardiovascular disease in patients with elevated Lp(a) concentrations have not been done because of the lack of effective drugs to specifically lower Lp(a) without affecting other lipid parameters. For the same reason, most clinicians do not routinely measure Lp(a) concentrations to ascertain its contribution to risk of cardiovascular disease, not even in high-risk patients with established cardiovascular disease or early family history of cardiovascular disease or in patients who are undergoing revascularisation procedures.

Research in context

Evidence before this study

We searched PubMed and ClinicalTrials.gov without language restrictions for trials published between Jan 1, 1990, and Feb 1, 2015, using the search terms “Lp(a)”, “lipoprotein(a)”, and “lipoprotein (a)”. No randomised controlled trials could be identified that randomly assigned a specific medical therapy that lowers only lipoprotein(a) (Lp[a]) versus placebo in patients with increased Lp(a) concentrations. Findings from the Specific Lp(a) Apheresis for Regression of Coronary and Carotid Atherosclerosis (LaRCA) study (NCT02133807) showed potent reductions of Lp(a) with an Lp(a)-specific apheresis column and angiographic evidence of atheroma regression. The Lipoprotein Apheresis in Refractory Angina study (NCT01796912) recruited patients with refractory angina and Lp(a) concentrations greater than 50 mg/dL (125 nmol/L) and LDL-C concentrations less than 80 mg/dL (2·072 nmol/L). Drugs with multiple effects on lipoproteins are being tested in trials with patients who have increased plasma Lp(a) concentrations. For example, the Early Aortic Valve Lipoprotein(a) Lowering Trial (EAVaLL, NCT02109614) is assessing niacin (nicotinic acid) versus placebo in patients with aortic sclerosis, and the study on the effects of TA-8995 on Lp(a) in patients with increased Lp(a) (NCT02241772) is assessing a cholesteryl ester transfer protein inhibitor.

Added value of this study

Antisense therapy is based on a unique pharmacological mechanism to mediate therapeutic effects, and joins small molecules and monoclonal antibodies as a third platform to modulate targets for therapeutic purposes. Its unique mechanism is to lower plasma Lp(a) concentrations by directly binding to apolipoprotein(a) (apo[a]) mRNA in the nucleus of hepatocytes, which then leads to destruction of the antisense:mRNA complex by RNase H1 and the specific inhibition of apo(a) synthesis. Lp(a) has no known enzymatic activity, and how it becomes degraded or causes atherosclerosis is unknown. Thus, it would be very difficult to develop a small molecule to either affect Lp(a) plasma concentrations or affect its atherogenic properties. Furthermore, Lp(a) cannot be easily targeted with biological drugs, such as monoclonal antibodies, because of the wide variability in plasma concentrations between individuals and its overall high plasma concentrations. Thus, the use of a second-generation antisense drug to inhibit apo(a) synthesis is theoretically an ideal mechanistic approach to reduce Lp(a) concentrations.

Implications of all the available evidence

The specialty of RNA therapeutics, and antisense drugs in particular, is a rapidly emerging field with about 50 companies developing drugs that target more than 150 disease-associated proteins. Only one RNA therapeutic (a second-generation antisense drug), Kynamro, which targets the production of apoB-100, is approved in the USA for the treatment of homozygous familial hypercholesterolaemia. However, there are multiple ongoing phase 1–3 trials of drugs that target RNA. ISIS-APO(a)Rx will allow the testing of the hypothesis that the reduction of Lp(a), without affecting other lipoproteins, will lead to therapeutic benefit.

Antisense oligonucleotides are a class of RNA therapeutic drugs designed to selectively bind mRNA coding for specific proteins and are often designed to cause degradation of mRNA by the ribonuclease RNase H1, ultimately reducing plasma concentrations of the encoded protein.9 ISIS-APO(a)Rx is a second-generation 2′-O-(2-methoxyethyl) (2′-MOE) modified antisense oligonucleotide drug with the sequence 5′-TGCTCCGTTGGTGCTTGTTC-3′. ISIS-APO(a)Rx has been modified to improve potency, duration of action, and tolerability and contains five 2′-MOE modified ribonucleosides at the 5′ and 3′ ends and ten 2-O-deoxyribonucleosides within the central portion of the molecule. All the internucleotide phosphates are chemically modified with a phosphorothioate substitution, in which one of the non-bridging oxygen atoms is substituted with sulphur. ISIS-APO(a)Rx was designed to reduce the synthesis of apo(a) in the liver and consequently reduce the level of Lp(a) in plasma (figure 1).

We assessed the safety, pharmacokinetics, and pharmacological effects of ISIS-APO(a)Rx in healthy volunteers.

Section snippets

Study design and participants

This randomised, placebo-controlled, double-blind, phase 1 study was done at the PAREXEL Clinical Pharmacology Research Unit (Harrow, Middlesex, UK). We tested ascending doses of ISIS-APO(a)Rx in healthy adults, aged 18–65 years, with body-mass index (BMI) less than 32·0 kg/m2 and Lp(a) concentration of 25 nmol/L (100 mg/L) or more. To be included, women had to not be pregnant and not lactating and either surgically sterile (eg, tubal occlusion, hysterectomy, bilateral salpingectomy, bilateral

Results

Between Feb 27, 2013, and July 15, 2013, we screened 206 volunteers (figure 2). 47 (23%) volunteers were eligible for participation in the study. We randomly assigned four groups of four participants to receive 50 mg of ISIS-APO(a)Rx (n=3), 100 mg of ISIS-APO(a)Rx (n=3), 200 mg of ISIS-APO(a)Rx (n=3), 400 mg of ISIS-APO(a)Rx (n=3), or placebo (n=4). All 16 participants allocated to receive the single-dose regimen completed treatment and follow-up and were included in the pharmacodynamic,

Discussion

This study demonstrates the first selective and potent reduction of plasma Lp(a) concentrations by ISIS-APO(a)Rx, a second-generation antisense oligonucleotide targeting hepatic apo(a) mRNA. The mechanism by which ISIS-APO(a)Rx suppresses apo(a) protein translation and lowers Lp(a) concentration in the circulation is depicted in figure 1 and described in the appendix. Dose-dependent reductions in plasma concentrations of Lp(a) as well as OxPL-apoB and OxPL-apo(a), which mediate the

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