Long-term safety and efficacy of anacetrapib in patients with atherosclerotic vascular disease.

AIMS: REVEAL was the first randomized controlled trial to demonstrate that adding cholesteryl ester transfer protein inhibitor therapy to intensive statin therapy reduced the risk of major coronary events. We now report results from extended follow-up beyond the scheduled study treatment period. METHODS AND RESULTS: A total of 30 449 adults with prior atherosclerotic vascular disease were randomly allocated to anacetrapib 100 mg daily or matching placebo, in addition to open-label atorvastatin therapy. After stopping the randomly allocated treatment, 26 129 survivors entered a post-trial follow-up period, blind to their original treatment allocation. The primary outcome was first post-randomization major coronary event (i.e. coronary death, myocardial infarction, or coronary revascularization) during the in-trial and post-trial treatment periods, with analysis by intention-to-treat. Allocation to anacetrapib conferred a 9% [95% confidence interval (CI) 3-15%; P = 0.004] proportional reduction in the incidence of major coronary events during the study treatment period (median 4.1 years). During extended follow-up (median 2.2 years), there was a further 20% (95% CI 10-29%; P < 0.001) reduction. Overall, there was a 12% (95% CI 7-17%, P < 0.001) proportional reduction in major coronary events during the overall follow-up period (median 6.3 years), corresponding to a 1.8% (95% CI 1.0-2.6%) absolute reduction. There were no significant effects on non-vascular mortality, site-specific cancer, or other serious adverse events. Morbidity follow-up was obtained for 25 784 (99%) participants. CONCLUSION: The beneficial effects of anacetrapib on major coronary events increased with longer follow-up, and no adverse effects emerged on non-vascular mortality or morbidity. These findings illustrate the importance of sufficiently long treatment and follow-up duration in randomized trials of lipid-modifying agents to assess their full benefits and potential harms. TRIAL REGISTRATION: International Standard Randomized Controlled Trial Number (ISRCTN) 48678192; ClinicalTrials.gov No. NCT01252953; EudraCT No. 2010-023467-18.

Blood transfusion and ischaemic outcomes according to anemia and bleeding in patients with non-ST-segment elevation acute coronary syndromes: Insights from the TAO randomized clinical trial.

BACKGROUND: The benefits and risks of blood transfusion in patients with acute myocardial infarction who are anemic or who experience bleeding are debated. We sought to study the association between blood transfusion and ischemic outcomes according to haemoglobin nadir and bleeding status in patients with NST-elevation myocardial infarction (NSTEMI). METHODS: The TAO trial randomized patients with NSTEMI and coronary angiogram scheduled within 72h to heparin plus eptifibatide versus otamixaban. After exclusion of patients who underwent coronary artery bypass surgery, patients were categorized according to transfusion status considering transfusion as a time-varying covariate. The primary ischemic outcome was the composite of all-cause death or MI within 180 days of randomization. Subgroup analyses were performed according to pre-transfusion hemoglobin nadir and bleeding status. RESULTS: 12,547 patients were enrolled. Among these, blood transfusion was used in 489 (3.9%) patients. Patients who received transfusion had a higher rate of death or MI (29.9% vs. 8.1%, p<0.01). This excess risk persisted after adjustment on GRACE score and nadir of hemoglobin (HR 3.36 95%CI 2.63-4.29 p<0.01). Subgroup analyses showed that blood transfusion was associated with a higher risk in patients without overt bleeding (adjusted HR 6.25 vs. 2.85; p-interaction 0.001) as well as in those with hemoglobin nadir > 9.0 g/dl (HR 4.01; p-interaction<0.0001). CONCLUSION: In patients with NSTEMI, blood transfusion was associated with an overall increased risk of ischaemic events. However, this was mainly driven by patients without overt bleeding and those hemoglobin nadir > 9.0g/dl. This suggests possible harm of transfusion in those groups.

Randomized Evaluation of the Effects of Anacetrapib through Lipid-modification (REVEAL)-A large-scale, randomized, placebo-controlled trial of the clinical effects of anacetrapib among people with established vascular disease: Trial design, recruitment, and baseline characteristics.

Patients with prior vascular disease remain at high risk for cardiovascular events despite intensive statin-based treatment. Inhibition of cholesteryl ester transfer protein by anacetrapib reduces low-density lipoprotein (LDL) cholesterol by around 25% to 40% and more than doubles high-density lipoprotein (HDL) cholesterol. However, it is not known if these apparently favorable lipid changes translate into reductions in cardiovascular events. METHODS: The REVEAL study is a randomized, double-blind, placebo-controlled clinical trial that is assessing the efficacy and safety of adding anacetrapib to effective LDL-lowering treatment with atorvastatin for an average of at least 4years among patients with preexisting atherosclerotic vascular disease. The primary assessment is an intention-to-treat comparison among all randomized participants of the effects of allocation to anacetrapib on major coronary events (defined as the occurrence of coronary death, myocardial infarction, or coronary revascularization). RESULTS: Between August 2011 and October 2013, 30,449 individuals in Europe, North America, and China were randomized to receive anacetrapib 100mg daily or matching placebo. Mean (SD) age was 67 (8) years, 84% were male, 88% had a history of coronary heart disease, 22% had cerebrovascular disease, and 37% had diabetes mellitus. At the randomization visit (after at least 8weeks on a protocol-defined atorvastatin regimen), mean plasma LDL cholesterol was 61 (15) mg/dL and HDL cholesterol was 40 (10) mg/dL. INTERPRETATION: The REVEAL trial will provide a robust evaluation of the clinical efficacy and safety of adding anacetrapib to an effective statin regimen. Results are anticipated in 2017.

Global risk assessment for lipid therapy to prevent coronary heart disease.

Randomized clinical trials have established that lipid- lowering pharmacologic therapy can substantially reduce morbidity and mortality in patients with known coronary artery disease (CAD). Researchers are now working to define the role of lipid-lowering agents in the primary prevention of CAD to extend their benefit to patients at increased risk for future coronary events. The risk assessment models presently used for secondary prevention are not sufficient to identify high-risk, asymptomatic patients. Building on the accumulated data about the physiologic mechanisms and metabolic factors that contribute to CAD, novel serum markers and diagnostic tests are being critically studied to gauge their utility for the assessment of high-risk patients and occult vascular disease. New risk prediction models that combine traditional risk factors for CAD with the prudent use of new screening methods will allow clinicians to target proven risk reduction therapies at high-risk patients before they experience a cardiac event.

Inducible polymorphic ventricular tachyarrhythmias in a transgenic mouse model with a long Q-T phenotype.

We created a mouse model with a prolonged Q-T interval and spontaneous arrhythmias by overexpressing the NH(2) terminus and first transmembrane segment (Kv1.1N206Tag) of a delayed rectifier potassium channel (LQT(+/-) mouse). Analyses were performed using whole cell recordings of cardiac myocytes, surface electrocardiography, and programmed electrical stimulation. Action potential duration (APD) was prolonged to the same extent and was more highly variable in myocytes derived from LQT(+/-) and LQT(+/+) mice than in myocytes derived from wild-type (WT) FVB mice. Under ketamine anesthesia, the Q-T interval of both LQT(+/+) and LQT(+/-) mice was comparably prolonged versus that of WT mice. Stimulation of the right ventricle using an intracardiac catheter induced polymorphic ventricular tachyarrhythmias in 50% of the LQT(+/-) mice and 36% of the LQT(+/+) mice, whereas polymorphic ventricular tachyarrhythmias were not inducible in WT mice. The analyses of LQT(+/-) and LQT(+/+) mice indicate that prolongation of the Q-T interval in LQT mice is associated with prolonged APD, increased dispersion of APD among cardiocytes, and inducibility of polymorphic ventricular tachycardia, providing the substrate for spontaneous arrhythmias in these animals.

Activation of nitric oxide synthase (NOS3) by mechanical activity alters contractile activity in a Ca2+-independent manner in cardiac myocytes: role of troponin I phosphorylation.

Cardiac myocytes express the calcium-responsive nitric oxide synthase (eNOS or NOS3). Activation of NOS3 by increased intracellular Ca2+ concentration, [Ca2+]i, has been demonstrated to decrease myocyte contractile responsiveness, although this appears to occur in a Ca2+-independent manner. Therefore, the aim of this study was to examine the possibility that contractile activity could be modulated by an NO-mediated alteration in the phosphorylation status of troponin I, which is known to alter myofilament sensitivity to Ca2+. During pacing at 3 Hz, 32P-labeled myocytes exhibited a 59 +/- 9% increase in TnI phosphorylation compared to quiescent cells (p < 0.05), an effect that was significantly attenuated by either methylene blue or l-nitroarginine (l-NA). While exposure to methylene blue significantly increased the contractile amplitude of paced myocytes, this was not accompanied by an alteration in intracellular Ca2+. These data indicate that the NO-mediated effects on myocyte contraction may be elicited through an alteration in myofilament Ca2+ sensitivity that results from an alteration in the phosphorylation status of troponin I.

S-nitrosothiols inhibit neuronal norepinephrine transport.

Although it has been recently shown that nitric oxide (NO) and its congeners (NO(x)), including nitrosothiols, may modify catecholamine turnover in the brain, it is not known whether NO(x) affect norepinephrine (NE) uptake by sympathetic neurons. The nitrosothiol NO donor S-nitroso-acetylpenicillamine (SNAP, 100 microM for 1 h) elicited a concentration-dependent reduction in desipramine-sensitive [3H]NE uptake into PC-12 cells (66 +/- 3%; P < 0.01) or cultured rat superior cervical ganglia (74 +/- 5%; P < 0.001), whereas desipramine-insensitive [3H]NE uptake was unaffected, indicating a selective effect on uptake-1-mediated transport. Short-term coculture of PC-12 cells with microvascular endothelial cells expressing the cytokine-inducible NO synthase (NOS2) also exhibited a reduction in [3H]NE uptake (33 +/- 3%, P < 0.001) that could be prevented by the addition of the NOS inhibitor N-monomethyl-L-arginine (L-NMMA, 1 mM). Endogenous production of NO(x) by nerve growth factor-pretreated PC-12 cells also exhibited an L-NMMA-inhibitable reduction in [3H]NE uptake. Whereas SNAP resulted in a 10-fold elevation of PC-12 guanosine 3',5'-cyclic monophosphate (cGMP) content (P < 0.01), its effect on [3H]NE uptake was not mimicked by exposure to 8-bromo-cGMP. However, the inhibitory effect of SNAP on uptake-1-mediated [3H]NE transport could be attenuated by 1 mM cysteine, a sulfhydryl compound that could act as a sink for NO(x)-mediated nitrosation reactions, although cysteine did not affect the increase in intracellular cGMP with SNAP. These data suggest that an endogenous NO(x) source(s) modifies the activity of the uptake-1 catecholamine transporter in postganglionic sympathetic neurons, which, as we demonstrate, express both NOS1 and NOS3 isoforms, possibly by S-nitrosothiol-mediated nitrosation of regulatory sites on the transporter.

The role of the NO pathway in the control of cardiac function.

Nitric oxide (NO) acts as an autocrine- and paracrine-acting signaling autacoid that, among other functions, has been shown to regulate cardiac contractile responsiveness to beta-adrenergic and muscarinic cholinergic agonists. Nitric oxide (NO) is formed by the oxidation of one of two equivalent guanidino nitrogens in L-arginine by O2 to form NO and L-citrulline. This reaction is catalyzed by a family of enzymes termed NO synthases. Three distinct isoforms of NOS have been identified, each the product of a separate gene. Cellular constituents of cardiac muscle, including ventricular myocytes as well as microvascular endothelial cells, have been shown to express the "endothelial constitutive" isoform of NO synthase (ecNOS or NOS3) in vivo, and both cell types also express the NO synthase isoform induced by specific inflammatory cytokines (iNOS or NOS2) in vivo and in vitro. While NO-dependent intracellular signalling in cardiac myocytes clearly involves the activation of guanylate cyclase and downstream signalling by cGMP, there is accumulating evidence that non-cGMP-dependent regulatory signalling events are also initiated by NO. In addition, decreased contractile responsiveness of cardiac myocytes to beta-adrenergic agonists, following induction of NOS2 by inflammatory cytokines, requires the presence of insulin and the co-induction of enzymes responsible for production of tetrahydrobiopterin, a NOS co-factor. Inappropriate or excessive production of NO by cardiac myocytes and by microvascular endothelial cells likely contributes to the cardiac contractile dysfunction characteristic of the systemic inflammatory response syndrome and cardiac allograft rejection.

Frequency-dependent activation of a constitutive nitric oxide synthase and regulation of contractile function in adult rat ventricular myocytes.

Cardiac myocytes have recently been shown to express a constitutive Ca(2+)-sensitive isoform of NO synthase (NOS3), although the mechanism(s) responsible for activation of NOS3 and its physiological function remain to be determined. Since the activity of NOS3 is known to be regulated in part by the intracellular Ca2+ activity ([Ca2+]i) in endothelial cells, we determined whether increasing myocyte [Ca2+]i by uniform electric field pacing was accompanied by an increase in NOS3 activity, detected as nitrite accumulation in the medium. A higher [Ca2+]i with increasing pacing frequencies was shown to be accompanied by a time-dependent accumulation of nitrite in medium that bathed adult rat ventricular myocytes stimulated at 3 Hz. Nitrite release by paced cells was significantly attenuated by treatment with either the NO synthase inhibitor nitro-L-arginine (L-NA, 1 mmol/L) or the intracellular Ca2+ chelator BAPTA-AM (20 mumol/L). Paced myocytes also exhibited a frequency- and time-dependent increase in intracellular cGMP content that could be inhibited significantly by either L-NA or the soluble guanylate cyclase inhibitor LY83583 (5 mumol/L). To determine whether the increase in NOS3 activity with pacing affected contractile function, myocytes were sequentially paced at frequencies from 0.5 to 3 Hz. Methylene blue, L-NA, and LY83583 all increased the amplitude of shortening of myocytes paced at 3 Hz. Furthermore, a significantly greater positive inotropic response to high extracellular Ca2+ (3 mmol/L) was demonstrated by myocytes pretreated with L-NA compared with control cells. These data indicate that myocyte NOS3 activity is regulated in part by [Ca2+]i, whether induced by changes in pacing frequency or [Ca2+]o, and depresses myocyte contractile responsiveness to higher stimulation frequencies.

Pyridostigmine-mediated growth hormone release: evidence for somatostatin involvement.

Pyridostigmine (PD), a cholinesterase inhibitor, has been shown to elicit GH release when given alone and to potentiate the GH response to GH-releasing hormone (GHRH) in man. Numerous experiments have indirectly indicated that somatostatin (SS) inhibition is its likely mechanism of action. This study sought to establish the ability of PD to induce GH release in the rat, determine the dose-response relationship, and test the hypothesis that SS inhibition is the method of action. Three experiments were performed to monitor the GH response to PD. I) Five groups of male rats were food deprived for 72 h. The groups were then treated iv with saline, SS antibody (SS-ab), and 10, 100, and 1000 micrograms/kg PD, respectively. Blood samples were drawn before and after treatment. II) Two groups of male rats were pretreated iv with GHRH antibody (GHRH-ab) and either SS-ab or normal sheep serum (NSS). Blood samples were drawn every 30 min for 8.5 h, during which time each animal was injected with PD (10 micrograms/kg) in the third hour and again in the sixth hour. III) Male rats received a PD injection (10 micrograms/kg, iv) during a spontaneous GH trough period and a second PD injection during a spontaneous GH peak period. Blood samples were drawn at regular intervals preceding and following treatments. In Exp I, PD induced a clear 4- to 5-fold increase in GH concentrations in food-deprived rats. The maximal GH responses occurred after the 10 and 100 micrograms/kg doses, although the pattern and duration were different with these two doses. In Exp II, PD induced an approximately 2-fold increase in GH values in animals pretreated with GHRH-ab and NSS, but failed to induce a change in GH in the animals treated with GHRH-ab and SS-ab. In Exp III, PD failed to induce any change in GH concentration when administered during spontaneous GH peaks or troughs. The first two experiments suggest that PD increases GH secretion in the rat via inhibition of SS. The failure of PD to alter GH during a spontaneous peak is consistent with the current hypothesis that the level of SS is low at this time. Its failure to alter GH during trough periods may be related to very high SS tone. In conclusion, our results support the hypothesis that PD acts via inhibition of SS secretion.