Development of Antisense Drugs for Dyslipidemia

J Atheroscler Thromb, 2016; 23: 1011-1025. doi: 10.5551/jat.RV16001 Review Development of Antisense Drugs for Dyslipidemia Tsuyoshi Yamamoto1, 2, Fumito Wada1, 2 and Mariko Harada-Shiba2 1Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan 2Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Suita, Japan Abnormal elevation of low-density lipoprotein (LDL) and triglyceride-rich lipoproteins in plasma as well as dysfunction of anti-atherogenic high-density lipoprotein (HDL) have both been recognized as essential components of the pathogenesis of atherosclerosis and are classified as dyslipidemia. This review describes the arc of development of antisense oligonucleotides for the treatment of dyslipidemia. Chemically-armed antisense candidates can act on various kinds of transcripts, including mRNA and miRNA, via several different endogenous antisense mechanisms, and have exhibited potent systemic anti-dyslipidemic effects. Here, we present specific cutting-edge technologies have recently been brought into antisense strategies, and describe how they have improved the potency of antisense drugs in regard to pharmacokinetics and pharmacodynamics. In addition, we discuss per- spectives for the use of armed antisense oligonucleotides as new clinical options for dyslipidemia, in the light of outcomes of recent clinical trials and safety concerns indicated by several clinical and pre- clinical studies. Key words: Antisense drug, Chemical modification, Lipid lowering drug, Molecular targeting, Dyslipidemia Copyright©2016 Japan Atherosclerosis Society This article is distributed under the terms of the latest version of CC BY-NC-SA defined by the Creative Commons Attribution License. without this genetic background4-6). FH is an autoso- 1. Introduction mal dominant-type genetic disorder caused by specific Consecutive dysregulation of lipoprotein metab- gene mutations relevant to LDL metabolism. FH olism is the greatest contributor to the development shows severe hyper-LDL cholesterolemia and prema- and progression of atherosclerosis, which leads to cor- ture CAD. Although the stronger class of statins has onary artery disease (CAD). Abnormal elevation of largely helped to attenuate severe blood LDL choles- plasma low-density lipoprotein (LDL) and triglyceride terol, statins are not always effective and may not pro- (TG)-rich lipoproteins as well as the dysfunction of vide sufficient LDL reduction particularly for homo- anti-atherogenic high-density lipoprotein (HDL) are zygous FH (HoFH) patients and severe heterozygous both recognized as essential components of the patho- FH patients (HeFH). Therefore, alternative or addi- genesis of atherosclerosis and are classified as dyslipid- tional drugs are required for these patients. emia. In this regard, the significant quantitative bene- There is extensive evidence that elevated TG and fits of modifying blood LDL cholesterol concentra- low HDL cholesterol levels are both independent risk tions for both primary and secondary prevention have factors for CAD7). In addition, an extremely high been demonstrated in a number of large-scale clinical blood TG level increases the risk of pancreatitis. Fur- trials, as well as meta-analyses, using statins1-3). thermore, current lipid-lowering drug interventions For patients with familial hypercholesterolemia do not achieve sufficient efficacy in patients with (FH), the necessity of earlier identification of their severe hypertriglyceridemia accompanied by low HDL disease and life-long intense LDL cholesterol manage- cholesterolemia having such diseases as familial com- ment is greater than in hypercholesterolemic patients bined hyperlipidemia (FCHL), familial chylomicronemia syndrome (FCS) and familial partial lipodystro- Address for correspondence: Tsuyoshi Yamamoto, Graduate phy (FPL). School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 561-0884, Japan Recently developed chemically-armed antisense E-mail: [email protected] oligonucleotides (AONs) are potent enough to pro- Received: April 26, 2016 vide a therapeutic option even for patients with severe Accepted for publication: May 11, 2016 inherited dyslipidemia. In fact, numerous molecular 1011 Yamamoto et al. Table 1. Antisense drugs that are under clinical development or have been approved. Target Type Company Disease Pre-clinical Phase Ⅰ Phase Ⅱ Phase Ⅲ Approved KYNAMRO® 2’-MOE modified APOB Ionis Hypercholesteremia (Mipomersen) Gapmer IONIS-APOCⅢRX 2’-MOE modified APOC3 Ionis/Akcea Chylomicronemia (volanesorsen) Gapmer 2’-MOE modified IONIS-APO(a)-LRX APO(a) Ionis/Akcea Very high Lp(a) Gapmer 2’-MOE modified Mixed IONIS-ANGPTL3-LRX ANGPTL3 Ionis/Akcea Gapmer dyslipidemias 2’F/MOE-modified anti-miR-33 mir-33a/b Regulus Atherosclerosis etc. mixmer LNA-modified miRagen/ Hypertrophic anti-miR-208 miR-208a mixmer Servier cardiomyopathy targets responsible for severe dyslipidemia have been identified and some AONs targeting these molecules have shown great therapeutic potential against dyslipidemia in animal model studies. In addition, some ongoing clinical trials are evaluating AONs in patients with severe inherited dyslipidemia and interim reports on the lipid-controlling effects of AONs have just been published (Table 1). In this review, we provide general and extensive detailed information on recent advances in antisense drug development platforms as well as individual clinical candidates for the treatment Fig.1. Possible modification sites of a nucleotide unit. of dyslipidemia. 2. Chemical Modifications for AONs the past four decades. The first innovation was phosphorothioate inter- AONs are synthetic short single-stranded nucleic nucleotide modification technology, which drastically acid oligomers (typically 5-25 nucleotides-long) avoids unintended nuclease digestion of AONs under designed to form hybrids with target transcripts that biological conditions and improves their pharmacoki- have complementary sequences. The recognition of netics9). Ionis Pharmaceuticals, a leading company target RNAs by AONs is highly accurate and binding developing antisense drugs, produced the first FDA- is tight due to their specific Watson-Crick-type base- approved clinical antisense drug, Vitravene®, based on pairing interaction. It was only recently that therapeu- this technology in 1998. The second generation of tic AONs exhibited perceptible systemic activity with- AONs was also developed by Ionis Pharmaceuticals, out delivery vehicles and achieved excellent outcomes achieved by introducing an affinity-enhancing modifi- in clinical trials when furnished with chemically- cation into a nucleic acid building block called MOE armed nucleic acid building blocks. The key to success (2’-O-methoxyethyl RNA)10). They demonstrated that in improvement of the in vivo potency of AONs was the complementary characteristics of MOE on a ribose the introduction of chemical modifications into the moiety and phosphorothioate backbone modification AON structure that makes AONs more stable in a further strengthened the potency of AONs, enabling biological context and give them higher binding affin- systemic application. The second generation technol- ity to target RNAs. There are three motifs comprising ogy eventually led to the development of Kynamro®, a the AON architecture: phosphate backbone, ribose FDA-approved anti-apolipoprotein B (ApoB) AON and nucleobase (Fig.1)8), all of which are potentially for homozygous FH, in 2013 (discussed below). chemically modifiable, and numerous chemical modi- Our group first succeeded in developing a novel fications have been introduced into the motifs over ribose modification, 2’,4’-bridged nucleic acid (2’,4’- 1012 Development of Antisense Drugs for Dyslipidemia Cytosol Figure. 2 Nucleus RNase H1 AON pre-mRNA or RNase H1 non-coding RNA mRNA Cleavage 3’ 1 5’ XRN2 2 XRN2 Exosome, Dis3, ExoSC10 3 RNase H1 mRNA 4 3’ Cleavage 5 XRN1 6 5’ XRN1 Exosome, Dis3L1 7 Fig.2. RNase H mediated functional mechanism of ASO and degradative path- way of cleavage products. BNA) (also known as locked nucleic acid, LNA), in idemia come under this mechanistic class. RNase H is 1997, which exhibits very strong target RNA binding a ubiquitously expressed endoribonuclease that prefer- and biological stability11-13). The impact of this next entially binds to the DNA-RNA hetero-duplex over generation antisense scaffold was so devastating that a RNA-RNA and DNA-DNA homo-duplexes. After an number of researchers, including us, and pharmaceu- AON binds to the target RNA, RNase H selectively tical firms started to use 2’,4’-BNA/LNA-modified hydrolizes the RNA strand of the AON-RNA AONs as research tools and develop them as clinical duplex23, 24), and RNase H1 is more likely to be respon- antisense drugs (http://www.exiqon.com/lna-technol- sible for this mechanism than RNase H225). The AON ogy). In addition, a wide variety of 2’,4’-BNA/LNA is expected to be recycled after the target RNA is cleaved analogues have been designed and chemically synthe- by RNase H1 for the next catalytic reaction26, 27). As sized in order to find the best one for antisense thera- RNase H1 is found in both the nucleus and cyto- peutics in this specific class of chemical modifica- plasm, both organelles are potential sites of action of tions14-21 etc). an AON that utilizes the RNase H1 mechanism. Puta- tive molecular targets for an AON are therefore regarded as not only cytosolic mature mRNA, but also 3. Antisense Mechanism

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