Treatment with Volanesorsen, a 2'-O-Methoxyethyl-Modified Antisense Oligonucleotide Targeting APOC3 mRNA, Does Not Affect the QTc Interval in Healthy Volunteers.

The aim of this study was to assess the effect of volanesorsen on the corrected QT (QTc) interval. This thorough QT study enrolled 52 healthy male and female subjects who were randomized at a single site in a four-way crossover study. Subjects were randomly assigned to 1 of 12 treatment sequences and crossed over into four treatment periods over the course of which each subject was to receive a single therapeutic dose of volanesorsen as a 300 mg subcutaneous (SC) injection, a single supratherapeutic dose of volanesorsen as 300 mg intravenous (IV) infusion, a single oral (PO) dose of moxifloxacin (positive control), and placebo dose. The study demonstrated that volanesorsen 300 mg SC and 300 mg IV did not have a clinically relevant effect on ΔΔQTcF exceeding 10 ms. The largest mean effect at any postdose time point was 3.0 ms (90% confidence interval [CI]: 0.8-5.2) after SC dosing and 1.8 ms (90% CI -0.4 to 4.0) after IV dosing. Volanesorsen, at the studied therapeutic and supratherapeutic doses, does not have a clinically meaningful effect on the QTc.

From traditional pharmacological towards nucleic acid-based therapies for cardiovascular diseases.

Nucleic acid-based therapeutics are currently developed at large scale for prevention and management of cardiovascular diseases (CVDs), since: (i) genetic studies have highlighted novel therapeutic targets suggested to be causal for CVD; (ii) there is a substantial recent progress in delivery, efficacy, and safety of nucleic acid-based therapies; (iii) they enable effective modulation of therapeutic targets that cannot be sufficiently or optimally addressed using traditional small molecule drugs or antibodies. Nucleic acid-based therapeutics include (i) RNA-targeted therapeutics for gene silencing; (ii) microRNA-modulating and epigenetic therapies; (iii) gene therapies; and (iv) genome-editing approaches (e.g. CRISPR-Cas-based): (i) RNA-targeted therapeutics: several large-scale clinical development programmes, using antisense oligonucleotides (ASO) or short interfering RNA (siRNA) therapeutics for prevention and management of CVD have been initiated. These include ASO and/or siRNA molecules to lower apolipoprotein (a) [apo(a)], proprotein convertase subtilisin/kexin type 9 (PCSK9), apoCIII, ANGPTL3, or transthyretin (TTR) for prevention and treatment of patients with atherosclerotic CVD or TTR amyloidosis. (ii) MicroRNA-modulating and epigenetic therapies: novel potential therapeutic targets are continually arising from human non-coding genome and epigenetic research. First microRNA-based therapeutics or therapies targeting epigenetic regulatory pathways are in clinical studies. (iii) Gene therapies: EMA/FDA have approved gene therapies for non-cardiac monogenic diseases and LDL receptor gene therapy is currently being examined in patients with homozygous hypercholesterolaemia. In experimental studies, gene therapy has significantly improved cardiac function in heart failure animal models. (iv) Genome editing approaches: these technologies, such as using CRISPR-Cas, have proven powerful in stem cells, however, important challenges are remaining, e.g. low rates of homology-directed repair in somatic cells such as cardiomyocytes. In summary, RNA-targeted therapies (e.g. apo(a)-ASO and PCSK9-siRNA) are now in large-scale clinical outcome trials and will most likely become a novel effective and safe therapeutic option for CVD in the near future. MicroRNA-modulating, epigenetic, and gene therapies are tested in early clinical studies for CVD. CRISPR-Cas-mediated genome editing is highly effective in stem cells, but major challenges are remaining in somatic cells, however, this field is rapidly advancing.

The dedicated "Lp(a) clinic": A concept whose time has arrived?

The emergence of pathophysiological, epidemiologic, and genetic data strongly supports the causality for lipoprotein(a) [Lp(a)] in cardiovascular disease (CVD) and calcific aortic valve disease (CAVD). In parallel, novel Lp(a) lowering approaches have been developed that have re-invigorated clinical interest in Lp(a). Because Lp(a) is the most prevalent monogenetic lipid disorder globally, with prevalence of Lp(a) > 50 mg/dL estimated at >1.4 billion people, the rationale for diagnosing and managing Lp(a)-mediated risk is now stronger than ever. Patients with elevated Lp(a) are significantly under-diagnosed and the diagnosis is frequently made ad hoc rather than systematically. Elevated Lp(a) levels are associated with atherothrombotic risk and patients present with varied clinical phenotypes, ranging from stroke in pediatric age groups, to ST-segment elevation myocardial infarction in young males, to CAVD in elderly individuals. A new clinical care paradigm of a dedicated "Lp(a) Clinic" would serve to evaluate and manage such patients who have elevated Lp(a) as the pathophysiological etiology. Such a clinic would include multidisciplinary expertise in lipid metabolism, clinical cardiology, vascular medicine, valvular disease, thrombosis, and pediatric aspects of clinical care. This viewpoint argues for the rationale of an Lp(a) outpatient clinic where patients with elevated Lp(a) and their affected relatives can be referred, evaluated, managed and followed, to ultimately reduce Lp(a)-mediated CVD and CAVD risk.

Oxidized Phospholipids on Apolipoprotein B-100 and Recurrent Ischemic Events Following Stroke or Transient Ischemic Attack.

BACKGROUND: Biomarkers to predict recurrent stroke and targets of therapy to prevent stroke are lacking. OBJECTIVES: This study evaluated whether patients with prior cerebrovascular events and elevated levels of oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB), but without prior coronary artery disease (CAD), are at risk for recurrent stroke and CAD events following high-dose statin therapy. METHODS: In the SPARCL (Stroke Prevention by Aggressive Reduction in Cholesterol Levels) trial, OxPL-apoB levels were measured in 4,385 patients with stroke or transient ischemic attack at baseline and in 3,106 patients at 5 years following randomization to placebo or 80 mg atorvastatin. The primary endpoint was the time from randomization to a second nonfatal or fatal stroke. Secondary endpoints included first major coronary events and any cardiovascular event. RESULTS: Patients with recurrent stroke had higher baseline median OxPL-apoB levels than patients without (15.5 nmol/l vs. 11.6 nmol/l; p < 0.0001). After multivariable adjustment, elevated baseline OxPL-apoB predicted recurrent stroke (hazard ratio [HR]: 4.3; p < 0.0001), first major coronary events (HR: 4.0; p < 0.0001), and any cardiovascular event (HR: 4.4; p < 0.0001). These comparisons for any endpoint did not differ by treatment, shown as a nonsignificant interaction test. The net reclassification improvement, integrated discrimination improvement, and area under the receiver-operating characteristic curve (AUC) were all significantly improved by adding OxPL-apoB to the models, with ΔAUC +0.0505 (p < 0.0001) for recurrent stroke, ΔAUC +0.0409 (p < 0.0001) for first major coronary event, and ΔAUC +0.0791 (p < 0.0001) for any cardiovascular event. CONCLUSIONS: Elevated OxPL-apoB levels predicted recurrent stroke and first major coronary events in patients with prior stroke or transient ischemic attack. The lack of statin-OxPL-apoB treatment interaction suggested that OxPLs might be statin-independent therapeutic targets to reduce risk of cardiovascular events. (Lipitor in the Prevention of Stroke, for Patients Who Have Had a Previous Stroke [SPARCL]; NCT00147602).

Effect of an oral astaxanthin prodrug (CDX-085) on lipoprotein levels and progression of atherosclerosis in LDLR(-/-) and ApoE(-/-) mice.

UNLABELLED: Oxidative stress and inflammation are key promoters of atherosclerosis and myocardial damage. When orally administered, the novel astaxanthin prodrug CDX-085 delivers high levels of the xanthophyll antioxidant astaxanthin that protects LDL from oxidation and reduces primary thrombosis. In this study, we analyzed whether delivery of astaxanthin from administration of the CDX-085 prodrug reduces plasma lipoprotein levels and the progression of atherosclerosis in low-density lipoprotein receptor negative (LDLR(-/-)) and apolipoprotein E deficient (ApoE(-/-)) mice. METHODS: Relative circulating levels of astaxanthin derived from CDX-085 administration compared to administration of pure astaxanthin was initially evaluated in a canine model. In mouse Study #1, 16 wild-type and 16 LDLR(-/-) mice on 0.5% cholesterol diet supplemented with either 0.0%, 0.08%, 0.2% and 0.4% CDX-085 were used to assess plasma levels and lipoprotein biodistribution measured by FPLC after 4 weeks treatment. In Study #2, 36 male LDLR(-/-) mice were randomized to a 0.5% cholesterol chow diet (CHOW group, n=12) or 0.5% cholesterol chow fortified with 0.08% CDX-085 (n=12) or 0.5% cholesterol chow with 0.4% CDX-085 (n=12) for 12 weeks. In Study #3, 34 male ApoE(-/-) mice were randomized in the same fashion as the Study #2 and fed similar diets for 9 weeks. RESULTS: CDX-085 administration was shown to result in significantly higher levels of circulating astaxanthin (p<0.001 ANOVA) over a 72 h period compared to pure, non-esterified astaxanthin in a single-dose pharmacokinetic study in beagles. In Study #1, plasma astaxanthin levels were 5-9-fold higher in LDLR(-/-) mice compared to wild-type mice. Astaxanthin was highly distributed among all lipoprotein fractions, generally reflecting cholesterol content of lipoproteins. In Study #2, administration of CDX-085 resulted in significantly lower total cholesterol levels (528±68 mg/dL vs. 550±67 mg/dL vs. 602±80 mg/dL, p=0.047) and aortic arch atherosclerosis (9.0±4.2% vs. 9.8±3.5% vs. 13.2±3.6%, p=0.023) in the 0.4% CDX-085 group compared to the 0.08% CDX-085 and CHOW groups, respectively. In ApoE(-/-) mice, a 72% reduction in triglycerides in the 0.4% CDX-085 group and 50% reduction in the 0.08% CDX-085 groups was noted compared to CHOW group (final levels 17±11 mg/dL vs. 30±15 mg/dL vs. 60±32 mg/dL, respectively, p=0.001). CONCLUSION: Oral administration of the novel astaxanthin prodrug CDX-085 shows that it distributes among lipoproteins. CDX-085 lowers total cholesterol and aortic arch atherosclerosis in LDLR(-/-) mice and triglyceride levels in ApoE(-/-) mice and shows promise for further evaluation in human studies.

Phospholipase A2 enzymes, high-dose atorvastatin, and prediction of ischemic events after acute coronary syndromes.

BACKGROUND: Secretory phospholipase A2 (sPLA(2)) and lipoprotein-associated phospholipase A2 (Lp-PLA(2)) are enzyme biomarkers of increased cardiovascular risk and targets of emerging therapeutic agents. Their relationship to cardiovascular events in the setting of high-dose statin therapy compared with placebo in patients with acute coronary syndrome is not known. METHODS AND RESULTS: sPLA(2) and Lp-PLA(2) mass and activity were measured in 2587 patients in the Myocardial Ischemia Reduction With Acute Cholesterol Lowering (MIRACL) trial at baseline and after 16 weeks of treatment with atorvastatin 80 mg/d or placebo. Baseline levels of sPLA(2) and Lp-PLA(2) mass and activity were not associated with the primary efficacy measure of the trial of death, myocardial infarction, or unstable angina. However, in the overall cohort, baseline sPLA(2) mass predicted risk of death after multivariable adjustment (hazard ratio for 2-fold increase, 1.30; 95% confidence interval, 1.09-1.56; P=0.004). This association remained significant when examined separately in the placebo group but not in the atorvastatin group. Compared with placebo, atorvastatin reduced median sPLA(2) mass (-32.1% versus -23.1%), sPLA(2) activity (-29.5% versus -19.2%), Lp-PLA(2) mass (-35.8% versus -6.2%), and Lp-PLA(2) activity (-24.3% versus 5.4%; P<0.001 for all). Atorvastatin reduced the hazard of death associated with elevated sPLA(2) mass and activity by ≈50%. CONCLUSIONS: sPLA(2) mass independently predicts death during a 16-week period after acute coronary syndrome. High-dose atorvastatin significantly reduces sPLA(2) and Lp-PLA(2) mass and activity after acute coronary syndrome and mitigates the risk of death associated with sPLA(2) mass. Atorvastatin may exert antiinflammatory effects on phospholipases that contribute to its therapeutic benefit after acute coronary syndrome.

In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish.

Oxidative modification of LDL is an early pathological event in the development of atherosclerosis. Oxidation events such as malondialdehyde (MDA) formation may produce specific, immunogenic epitopes. Indeed, antibodies to MDA-derived epitopes are widely used in atherosclerosis research and have been demonstrated to enable cardiovascular imaging. In this study, we engineered a transgenic zebrafish with temperature-inducible expression of an EGFP-labeled single-chain human monoclonal antibody, IK17, which binds to MDA-LDL, and used optically transparent zebrafish larvae for imaging studies. Feeding a high-cholesterol diet (HCD) supplemented with a red fluorescent lipid marker to the transgenic zebrafish resulted in vascular lipid accumulation, quantified in live animals using confocal microscopy. After heat shock-induced expression of IK17-EGFP, we measured the time course of vascular accumulation of IK17-specific MDA epitopes. Treatment with either an antioxidant or a regression diet resulted in reduced IK17 binding to vascular lesions. Interestingly, homogenates of IK17-EGFP-expressing larvae bound to MDA-LDL and inhibited MDA-LDL binding to macrophages. Moreover, sustained expression of IK17-EGFP effectively prevented HCD-induced lipid accumulation in the vascular wall, suggesting that the antibody itself may have therapeutic effects. Thus, we conclude that HCD-fed zebrafish larvae with conditional expression of EGFP-labeled oxidation-specific antibodies afford an efficient method of testing dietary and/or other therapeutic antioxidant strategies that may ultimately be applied to humans.

Relation of oxidative biomarkers, vascular dysfunction, and progression of coronary artery calcium.

The relation between oxidative stress and coronary artery calcium (CAC) progression is currently not well described. The present study evaluated the relation among the biomarkers of oxidative stress, vascular dysfunction, and CAC. Sixty asymptomatic subjects participated in a randomized trial evaluating the effect of aged garlic extract plus supplement versus placebo and underwent measurement of CAC. The postcuff deflation temperature-rebound index of vascular function was assessed using a reactive hyperemia procedure. The content of oxidized phospholipids (OxPL) on apolipoprotein B-100 (apoB) particles detected by antibody E06 (OxPL/apoB), lipoprotein(a), IgG and IgM autoantibodies to malondialdehyde-low-density lipoprotein and apoB-immune complexes were measured at baseline and after 12 months of treatment. CAC progression was defined as an annual increase in CAC >15%. Vascular dysfunction was defined according to the tertiles of temperature-rebound at 1 year of follow-up. From baseline to 12 months, a strong inverse correlation was noted between an increase in CAC scores and increases in temperature-rebound (r(2) = -0.90), OxPL/apoB (r(2) = -0.85), and lipoprotein(a) (r(2) = -0.81) levels (p <0.0001 for all). The improvement in temperature-rebound correlated positively with the increases in OxPL/apoB (r(2) = 0.81, p = 0.0008) and lipoprotein(a) (r(2) = 0.79, p = 0.0001) but inversely with autoantibodies to malondialdehyde-low-density lipoprotein and apoB-immune complexes. The greatest CAC progression was noted with the lowest tertiles of increases in temperature-rebound, OxPL/apoB and lipoprotein(a) and the highest tertiles of increases in IgG and IgM malondialdehyde-low-density lipoprotein. In conclusion, the present results have documented a strong relation among markers of oxidative stress, vascular dysfunction, and progression of coronary atherosclerosis. Increases in OxPL/apoB and lipoprotein(a) correlated strongly with increases in vascular function and predicted a lack of progression of CAC.

Aged garlic extract supplemented with B vitamins, folic acid and L-arginine retards the progression of subclinical atherosclerosis: a randomized clinical trial.

OBJECTIVES: Previous studies demonstrated that aged garlic extract reduces multiple cardiovascular risk factors. This study was designed to assess whether aged garlic extract therapy with supplements (AGE+S) favorably affects inflammatory and oxidation biomarkers, vascular function and progression of atherosclerosis as compared to placebo. METHODS: In this placebo-controlled, double-blind, randomized trial (conducted 2005-2007), 65 intermediate risk patients (age 60+/-9 years, 79% male) were treated with a placebo capsule or a capsule containing aged garlic extract (250 mg) plus Vitamin B12 (100 microg), folic acid (300 microg), Vitamin B6 (12.5 mg) and l-arginine (100 mg) given daily for a 1 year. All patients underwent coronary artery calcium scanning (CAC), temperature rebound (TR) as an index of vascular reactivity using Digital Thermal Monitoring (DTM), and measurement of lipid profile, autoantibodies to malondialdehyde (MDA)-LDL, apoB-immune complexes, oxidized phospholipids (OxPL) on apolipoprotein B-100 (OxPL/apoB), lipoprotein (a) [Lp (a)], C-reactive protein (CRP), homocysteine were measured at baseline and 12 months. CAC progression was defined as an increase in CAC>15% per year and an increase in TR above baseline was considered a favorable response. RESULTS: At 1 year, CAC progression was significantly lower and TR significantly higher in the AGE+S compared to the placebo group after adjustment of cardiovascular risk factors (p<0.05). Total cholesterol, LDL-C, homocysteine, IgG and IgM autoantibodies to MDA-LDL and apoB-immune complexes were decreased, whereas HDL, OxPL/apoB, and Lp (a) were significantly increased in AGE+S to placebo. CONCLUSION: AGE+S is associated with a favorable improvement in oxidative biomarkers, vascular function, and reduced progression of atherosclerosis.

High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial.

BACKGROUND: Oxidized phospholipids (OxPL) are present within atherosclerotic plaques and bound by lipoprotein (a) [Lp(a)] in plasma. This study evaluated the impact of atorvastatin on oxidized LDL (OxLDL) in patients with acute coronary syndromes (ACS). METHODS AND RESULTS: OxLDL-E06 (OxPL content on apolipoprotein B-100 [apoB] detected by antibody E06), apoB-100 immune complexes (apoB-IC), OxLDL autoantibodies, and Lp(a) levels were measured in 2341 patients at baseline and after 16 weeks of treatment with atorvastatin 80 mg/d or placebo. The OxLDL-E06 and apoB-IC data are reported per apoB-100 particle (OxPL/apoB, IC/apoB) and as total levels on all apoB-100 particles (total apoB-OxPL and total apoB-IC [eg, OxPL/apoB or IC/apoBxapoB-100 levels]). Compared with baseline values, atorvastatin reduced apoB-100 (-33%), total apoB-OxPL (-29.7%), total apoB-IC IgG (-29.5%), and IgM (-25.7%) (P<0.0001 for all), whereas no change or an increase was observed with placebo. When normalized per apoB-100, compared with placebo, atorvastatin increased OxPL/apoB (9.5% versus -3.9%, P<0.0001) and Lp(a) (8.8% versus -0.7%, (P<0.0001). A strong correlation was noted between OxPL/apoB and Lp(a) (R=0.85, P<0.0001), consistent with previous data that Lp(a) binds OxPL. CONCLUSIONS: After atorvastatin treatment, total OxPL on all apoB-100 particles was decreased. However, there was enrichment of OxPL on a smaller pool of apoB-100 particles, in parallel with similar increases in Lp(a), suggesting binding by Lp(a). These data support the hypothesis that atorvastatin promotes mobilization and clearance of proinflammatory OxPL, which may contribute to a reduction in ischemic events after ACS.

Lysine-phosphatidylcholine adducts in kringle V impart unique immunological and potential pro-inflammatory properties to human apolipoprotein(a).

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.