Editorial

10.1517/13543780802170048 © 2008 Informa UK Ltd ISSN 1354-3784 969

Antisense technology for the prevention or the treatment of cardiovascular disease: The next blockbuster? Vasilios G Athyros , Anna I Kakafi ka , Konstantinos Tziomalos , Asterios Karagiannis & Dimitri P Mikhailidis † † University College London, Department of Clinical Biochemistry (Vascular Prevention Clinic), Royal Free Hospital, London, UK

Antisense technology might be a gateway to the treatment of diseases by targeting the expression of genes rather than permanently altering them. Thus, there will be fewer ethical concerns. Antisense oligonucleotides (ASO) can alter target gene expression by binding to RNA. Once bound, the ASO either disables or induces the degradation of the target RNA. This technology may be used to treat various conditions (including cancer, diabetes, and hypertension, as well as autoimmune and cardiovascular diseases). ASOs are potentially potent, selective and well-tolerated drugs. Mipomersen (ISIS 301012) inhibits human apolipoprotein (apo)B-100 synthesis and lowers circulating apoB and low-density lipoprotein cholesterol levels. ASO technology may provide a spectrum of agents targeting other vascular risk factors or mediators of atherosclerosis.

Keywords: antisense oligonucleotide , cardiovascular disease , gene expression , low-density lipoprotein cholesterol , mipomersen , RNA

Expert Opin. Investig. Drugs (2008) 17(7):969-972

1. Introduction

Further developments in gene technology are required before its full therapeutic potential can be realized for the treatment of dyslipidaemia and cardiovascular disease (CVD) [1] . There is also a need to define widely accepted ethical issues in this field.

Antisense technology may represent a new approach to the treatment of diseases by targeting the expression of genes, rather than permanently altering them. Thus, there will be fewer ethical concerns. An antisense oligonucleotide (ASO) can alter target gene expression by binding to RNA [2] . Once bound, the ASO either disables or induces the degradation of the target RNA [3,4] . This technology may be used to treat various conditions (including CVD, cancer, diabetes, hypertension and autoimmune diseases). There are more than 20 antisense drugs in clinical development [5] .

Regarding CVD, ASO inhibitors can target apolipoprotein B-100 (apoB-100) mRNA [6-10] , proprotein convertase (PC) SK9, degradation of the low-density lipoprotein (LDL) receptor [11] , intercellular adhesion molecule-1 (ICAM-1) mRNA [12] , MyD88 splicing, as well as pro-inflammatory signalling through the interleukin (IL)-1R [13] , tumour necrosis factor-alpha (TNF- α ) [14] and acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) mRNA [15] . Moreover, reduction of acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) expression with ASO in obese animals can reduce hepatic lipogenesis and hepatic steatosis as well as attenuate hyperlipidaemia [16] . Acetyl-CoA carboxylases 1 and 2 (Acc1 and Acc2) regulate both mitochondrial fatty acid oxidation and fat synthesis [17] .

1. Introduction

2. Clinical and experimental

evidence of antisense

oligonucleotide administration

3. Expert opinion

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Antisense technology for the prevention or the treatment of cardiovascular disease: The next blockbuster?

970 Expert Opin. Investig. Drugs (2008) 17(7)

Suppression of both enzymes with a single ASO promoted fat oxidation in animal models [17] .

Familial hypobetalipoproteinaemia (FHBL) is caused by mutations in the apoB gene, resulting in inability to translate full-length apoB-100. These patients are at a low risk for CVD (longevity syndrome) and have plasma levels of apoB and LDL-cholesterol (LDL-C) that are often 25% of normal [18] . This observation resulted in interest in the pharmacological inhibition of apoB-100 [19] . The apoB-100 antisense compound, ISIS 147764, reduced apoB-100 mRNA levels in the liver and in serum. Total cholesterol and LDL-C decreased by 25 – 55% and 40 – 88%, respectively [6] . Unlike small-molecule inhibitors of micro-somal triglyceride transfer protein, ISIS 147764 did not produce hepatic or intestinal steatosis, nor did it affect dietary fat absorption or elevate plasma transaminase activity [6] .

2. Clinical and experimental evidence of antisense oligonucleotide administration

Mipomersen (ISIS 301012) is the first ASO used in clinical trials to decrease apoB-100 production in the liver [7] . In a clinical trial, 50 – 400 mg of ISIS 301012 administered weekly via subcutaneous injection for 4 weeks reduced apoB-100 by 14.3 – 47.4% and LDL-C by 5.9 – 40% at 55 days [7] . The concentration of apoB-100 closely mirrors the total number of very low-density lipoprotein, intermediate-density lipoprotein and LDL particles in plasma because each hepatic apoB-containing lipoprotein secreted by the liver carries one molecule of apoB-100 [20] . In cases with reductions in apoB-100 greater than those of LDL-C (as above), there will be an increase in LDL particle size, which became larger and more buoyant [21] . These LDL subfractions are less atherogenic [22] . Thus, there is a beneficial change in LDL particles, both quantitatively and qualitatively [23,24] . The most frequent adverse event of mipomersen was injection-site erythema that resolved spontaneously [7] .

The pharmacokinetics of mipomersen, were assessed in mice, rats, monkeys and humans [19] . Plasma pharmacokinetics following parenteral administration was similar across species, with a rapid distribution phase (t ½ of several hours and a prolonged elimination phase with t ½β of days) [19] . The prolonged elimination phase represents equilibrium between tissues and circulating drug due to slow elimination from tissues. Absorption was nearly complete following subcutaneous injection, with bioavailability of 80 – 100% in monkeys. In all the animal models, the highest tissue concentrations of mipomersen were observed in kidney and liver [19] . Urinary excretion was < 3% in monkeys and humans in the first 24 h. Mipomersen is highly bound to plasma proteins, probably preventing rapid removal by renal filtration. Though slow, urinary excretion of mipomersen (ISIS 301012) and its shortened metabolites is the ultimate elimination pathway. The pharmacokinetics

of mipomersen in humans, predicted from animal models, support infrequent administration [19] .

A subcutaneous injectable formulation of mipomersen (ISIS 301012) is undergoing Phase II clinical trials, while Phase I trials are underway with an oral formulation [9] . In a double-blind, randomized, placebo-controlled, dose-escalation study, conducted in 36 volunteers with mild dyslipidaemia [10] , apoB-100 was reduced by a maximum of 50% (p = 0.002) from baseline. This decrease in apoB-100 concurred with a maximum of 35% reduction of LDL-C (p = 0.001). Both LDL-C and apoB remained significantly below baseline (p < 0.05) up to 3 months after the last dose [10] . Of note, besides reducing apoB-100 and LDL-C levels, this compound also significantly lowers plasma triglycerides (TGs) without significantly affecting high-density lipoprotein cholesterol (HDL-C) levels [8] .

An ASO inhibitor targeting PCSK9 has also been developed [9] . PCSK9 is a member of a family of proteases that is thought to promote the degradation of the LDL receptor (LDLR) through an undefined mechanism. Administration of a PCSK9 ASO to high fat-fed mice led to the increase in the number and activity of the LDLR, which reduced total cholesterol and LDL-C by 53 and 38%, respectively [11] . Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolaemia in patients with dysfunctional LDLR.

Besides influencing one CVD risk factor, such as dyslipidaemia, ASO technology may target unfavourable molecules against oxidation and/or inflammation [25] . Other targets involved in atherosclerosis include lipoprotein-associated phospholipase A 2 , 5-lipoxygenase-activating protein, acyl-CoA:cholesterol acyltransferase, chemokine receptors, and protein kinases [25] .

ISIS 104838 is a second-generation ASO that binds tumour TNF- α mRNA [13] . It is generally well tolerated intravenously and subcutaneously, and the pharmacokinetics support an infrequent dosing interval. This and ISIS 2302, an ASO targeting ICAM-1 mRNA that is currently in clinical trials [12] , could be used to ‘halt’ atherosclerosis.

A number of pro-inflammatory cytokines, including IL-1 β , signal through the adaptor protein MyD88. The ASO (ISIS 337846) binds to MyD88 pre-mRNA [14] . By manipulating levels of MyD88 splicing, pro-inflammatory signalling through the IL-1R has been shown to be diminished, in both cell cultures and mouse liver. This mechanism may be useful as a novel modulator of inflammatory stimuli [14] .

Furthermore, there is evidence that specific pharmaco-logical inhibition of ACAT2 (by ACAT2-specific ASOs), without affecting ACAT1, is atheroprotective [15] . Hepatic ACAT2 plays a role in driving the production of atherogenic lipoproteins [13,15] .

Another enzyme, DGAT2, may play a significant role in glucose and lipid metabolism [16] . High-fat diet-induced

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Athyros, Kakafi ka, Tziomalos, Karagiannis & Mikhailidis

Expert Opin. Investig. Drugs (2008) 17(7) 971

obese C57BL/6J mice and ob/ob mice were treated with DGAT2 ASO, control ASO, or saline. DGAT2 ASO treatment reduced DGAT2 mRNA levels by > 75% in both liver and fat, without changing DGAT1 mRNA levels in either of these tissues. This resulted in decreased DGAT activity in liver, but not in fat [16] . DGAT2 ASO treatment did not cause significant changes in body weight, adiposity, metabolic rate, insulin sensitivity, or skin microstructure. However, DGAT2 ASO treatment caused a marked reduction in hepatic TG content and improved hepatic steatosis, which was consistent with a decrease in TG synthesis and an increase in fatty acid oxidation observed in primary mouse hepatocytes treated with DGAT2 ASO. Non-alcoholic fatty liver disease (NAFLD) is associated with CVD [26-28] and is common in people with metabolic syndrome, diabetes or obesity [27] . A study in rats with diet-induced NAFLD showed that knocking down DGAT2 with an ASO protects against fat-induced hepatic insulin resistance and increases hepatic fatty acid oxidation [29] .

Malonyl-CoA, generated by Acc1 and Acc2, is a key regulator of both mitochondrial fatty acid oxidation and fat synthesis [17] . Suppression of both enzymes with a single ASO was significantly more effective in promoting fat oxidation. Suppression of Acc1 also inhibited lipogenesis, on which Acc2 reduction had no effect. In rats with NAFLD,

suppression of both enzymes with a single ASO was required to significantly reduce hepatic malonyl-CoA levels in vivo , lower hepatic lipids (long-chain acyl-CoAs, diacylglycerol, and TGs) and improve hepatic insulin sensitivity [17] .

3. Expert opinion

ASOs are potentially potent, selective and well-tolerated drugs. Mipomersen (ISIS 301012) inhibits human apoB-100 synthesis and lowers circulating apoB and LDL-C levels during short-term treatment. This compound also reduces TG levels and does not seem to affect HDL-C. This apoB mRNA inhibitor (initially designed for familial homozygous or heterozygous hypercholesterolaemia) might be used as mono-therapy or in addition to statin therapy. Longer-term studies are currently ongoing. Besides mipomersen (ISIS 301012), ASO technology may provide a spectrum of agents targeting other CVD risk factors or mediators of atherosclerosis.

Declaration of interest

The present editorial was written independently; no company or institution supported it financially. Some of the authors have attended conferences and participated in advisory boards and other trials sponsored by various pharmaceutical companies.

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Affi liation Vasilios G Athyros 1 , Anna I Kakafi ka 1 , Konstantinos Tziomalos 2 , Asterios Karagiannis 1 & Dimitri P Mikhailidis † 2 † Author for correspondence 1 Aristotle University of Thessaloniki, Second Propedeutic Department of Internal Medicine, Medical School, Hippokration Hospital, Thessaloniki, Greece 2 Royal Free and University College Medical School (University of London), Department of Clinical Biochemistry (Vascular Prevention Clinic), Royal Free Hospital, Pond Street, London NW3 2QG, UK Tel: +44 20 7830 2258 ; Fax: +44 20 7830 2235 ; E-mail: MIKHAILIDIS@aol.com

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