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An abstract of the study "The Effect of Darbepoetin Alfa Treatment on Clinical Outcomes in Anemic Patients With Symptomatic Heart Failure: A Preplanned Pooled Analysis of Two Randomized, Double-Blind, Placebo-Controlled Trials," by W. T. Abraham and colleagues is presented.

Importance: Lipoprotein(a) (Lp[a]) is thought to be the major carrier of oxidized phospholipids (OxPL). OxPL are believed to be a potent driver of inflammation and atherosclerosis. Olpasiran, a small interfering RNA, blocks Lp(a) production by inducing degradation of apolipoprotein(a) messenger RNA. Olpasiran's effects on OxPL and systemic markers of inflammation are not well described., Objective: To assess the effects of olpasiran on OxPL, high-sensitivity interleukin 6 (hs-IL-6), and hs-C-reactive protein (hs-CRP) in the OCEAN(a)-DOSE randomized clinical trial., Design, Setting, and Participants: OCEAN(a)-DOSE was an international, multicenter, placebo-controlled, phase 2, dose-finding randomized clinical trial conducted between July 2020 and November 2022. A total of 281 patients with atherosclerotic cardiovascular disease and Lp(a) levels greater than 150 nmol/L were included., Intervention: Participants were randomized to receive 1 of 4 active subcutaneous doses of olpasiran vs placebo: (1) 10 mg, administered every 12 weeks (Q12W); (2) 75 mg, Q12W; (3) 225 mg, Q12W; or (4) 225 mg, administered every 24 weeks (Q24W). OxPL on apolipoprotein B (OxPL-apoB), hs-CRP, and hs-IL-6 were assessed at baseline, week 36, and week 48 in 272 patients., Main Outcomes and Measures: The primary outcome was placebo-adjusted change in OxPL-apoB from baseline to week 36., Results: Among 272 participants, median (IQR) age was 62 years (56-69), and 86 participants (31.6%) were female. Baseline median (IQR) Lp(a) concentration was 260.3 nmol/L (198.1-352.4) and median (IQR) OxPL-apoB concentration was 26.5 nmol/L (19.7-33.9). The placebo-adjusted mean percentage change in OxPL-apoB from baseline to week 36 was -51.6% (95% CI, -64.9% to -38.2%) for the 10-mg Q12W dose, -89.7% (95% CI, -103.0% to -76.4%) for the 75-mg Q12W dose, -92.3% (95% CI, -105.6% to -78.9%) for the 225-mg Q12W dose, and -93.7% (95% CI, -107.1% to -80.3%) for the Q24W dose (P < .001 for all). These effects were maintained to week 48 (-50.8%, -100.2%, -104.7%, and -85.8%, respectively; P < .001 for all). There was a strong correlation between percentage reduction in Lp(a) and OxPL-apoB for patients treated with olpasiran (r = 0.79; P < .001). Olpasiran did not significantly impact hs-CRP or hs-IL-6 compared with placebo to weeks 36 or 48 (P > .05)., Conclusion and Relevance: In the OCEAN(a)-DOSE multicenter randomized clinical trial, olpasiran led to a significant and sustained reduction in OxPL-apoB but no significant effects on hs-CRP or hs-IL-6.

Background: Olpasiran, a small interfering RNA (siRNA), blocks lipoprotein(a) (Lp(a)) production by preventing translation of apolipoprotein(a) mRNA. In phase 2, higher doses of olpasiran every 12 weeks (Q12W) reduced circulating Lp(a) by >95%., Objectives: This study sought to assess the timing of return of Lp(a) to baseline after discontinuation of olpasiran, as well as longer-term safety., Methods: OCEAN(a)-DOSE (Olpasiran Trials of Cardiovascular Events And LipoproteiN[a] Reduction-DOSE Finding Study) was a phase 2, dose-finding trial that enrolled 281 participants with atherosclerotic cardiovascular disease and Lp(a) >150 nmol/L to 1 of 4 active doses of olpasiran vs placebo (10 mg, 75 mg, 225 mg Q12W, or an exploratory dose of 225 mg Q24W given subcutaneously). The last dose of olpasiran was administered at week 36; after week 48, there was an extended off-treatment follow-up period for a minimum of 24 weeks., Results: A total of 276 (98.2%) participants entered the off-treatment follow-up period. The median study exposure (treatment combined with off-treatment phases) was 86 weeks (Q1-Q3: 79-99 weeks). For the 75 mg Q12W dose, the off-treatment placebo-adjusted mean percent change from baseline in Lp(a) was -76.2%, -53.0%, -44.0%, and -27.9% at 60, 72, 84, and 96 weeks, respectively (all P < 0.001). The respective off-treatment changes in Lp(a) for the 225 mg Q12W dose were -84.4%, -61.6%, -52.2%, and -36.4% (all P < 0.001). During the extension follow-up phase, no new safety concerns were identified., Conclusions: Olpasiran is a potent siRNA with prolonged effects on Lp(a) lowering. Participants receiving doses ≥75 mg Q12W sustained a ∼40% to 50% reduction in Lp(a) levels close to 1 year after the last dose. (Olpasiran Trials of Cardiovascular Events And LipoproteiN[a] Reduction-DOSE Finding Study [OCEAN(a)-DOSE]; NCT04270760)., Competing Interests: Funding Support and Author Disclosures The OCEAN(a)-DOSE study was funded by Amgen. Dr O’Donoghue has received grant funding through Brigham and Women’s Hospital from Novartis, Amgen, AstraZeneca, and Janssen; and has received honoraria from Novartis, Amgen, AstraZeneca, and Janssen. Dr Rosenson has received institutional grant funding from Amgen, Arrowhead, Lilly, Novartis, and Regeneron; has received consulting fees from Amgen, Arrowhead, CRISPER Therapeutics, Lilly, Novartis, Precision Biosciences, Regeneron, and Ultragenyx; has received nonpromotional speaker fees from Amgen and Kowa; and has held stock in MediMergent. Dr López is an employee of Amgen; and/or has owned Amgen stock or stock options. Dr Lepor has received grant support from Amgen, Novartis, Regeneron, New Amsterdam, Merck, Arrowhead, and AstraZeneca; and has received speaker and/or consulting fees from Amgen, Novartis, Regeneron, Merck, AstraZeneca, and Boehringer Ingelheim. Dr Baum has received consulting fees from Altimmune, Amgen, Axcella, Boehringer Ingelheim, Lilly, Esperion, Ionis, Madrigal, Merck, Novartis, and Regeneron; and has received speaker fees from Amgen, Lilly, and Regeneron. Dr Gaudet has received grants and personal fees from Amgen during the conduct of the study; has received grants from Acasti, Kowa, Uniqure, Sanofi, Aegerion, Applied Therapeutics, AstraZeneca, Boehringer Ingelheim, Ceapro, Dalcor, Esperion, Arrowhead, Regeneron, Akcea, Allergan, Amryt, Ionis, Novartis, Novo Nordisk, Pfizer, Lilly, and The Medicine Company, outside the submitted work; and has received personal fees from Arrowhead, Regeneron, Akcea, Allergan, Amryt, Ionis, Novartis, Novo Nordisk, Pfizer, Lilly, CRISPR Therapeutics, Saliogen, and Ceapro, outside the submitted work. Dr Knusel is an employee of Amgen; and/or has owned Amgen stock or stock options. Ms Kuder has served as a member of the TIMI Study Group, which has received institutional research grant support through Brigham and Women’s Hospital from Abbott, Amgen, Anthos Therapeutics, AstraZeneca, Bayer HealthCare Pharmaceuticals, Daiichi-Sankyo, Eisai, Intarcia, MedImmune, Merck, Novartis, Pfizer, Quark Pharmaceuticals, Regeneron, Roche, Siemens Healthcare Diagnostics, The Medicines Company, and Zora Biosciences. Ms Murphy has served as a member of the TIMI Study Group, which has received institutional research grant support through Brigham and Women’s Hospital from Abbott, Amgen, Anthos Therapeutics, AstraZeneca, Bayer HealthCare Pharmaceuticals, Daiichi-Sankyo, Eisai, Intarcia, MedImmune, Merck, Novartis, Pfizer, Quark Pharmaceuticals, Regeneron, Roche, Siemens Healthcare Diagnostics, The Medicines Company, and Zora Biosciences. Dr H. Wang is an employee of Amgen; and/or has owned Amgen stock or stock options. Dr Wu is an employee of Amgen; and/or has owned Amgen stock or stock options. Dr Shah is an employee of Amgen; and has owned Amgen stock. Dr J. Wang is an employee of Amgen; and has owned Amgen stock. Dr Wilmanski is an employee of Amgen; and has owned Amgen stock. Dr Sohn is an employee of Amgen; and/or has owned Amgen stock or stock options; and has existing or pending patents related to olpasiran. Dr Kassahun is an employee of Amgen; and/or has owned Amgen stock or stock options; and has existing or pending patents related to olpasiran. Dr Sabatine has received research grant support through Brigham and Women’s Hospital from Abbott, Amgen, Anthos Therapeutics, Inc, AstraZeneca, Boehringer Ingelheim, Daiichi-Sankyo, Ionis, Merck, Novartis, Pfizer, Saghmos Therapeutics, and Verve Therapeutics; has served as a consultant for Amgen, AMPEL BioSolutions, Anthos Therapeutics, Inc, AstraZeneca, Beren Therapeutics, Boehringer Ingelheim, Dr Reddy’s Laboratories, Fibrogen, Merck, Moderna, Novo Nordisk, Precision BioSciences, and Silence Therapeutics; and has served as a member of the TIMI Study Group, which has also received institutional research grant support through Brigham and Women’s Hospital from ARCA Biopharma, Janssen Research and Development, Siemens Healthcare Diagnostics, Softcell Medical Limited, Regeneron, Roche, and Zora Biosciences. Dr Stout has reported that he has no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Lipoprotein(a) is a presumed risk factor for atherosclerotic cardiovascular disease. Olpasiran is a small interfering RNA that reduces lipoprotein(a) synthesis in the liver., Methods: We conducted a randomized, double-blind, placebo-controlled, dose-finding trial involving patients with established atherosclerotic cardiovascular disease and a lipoprotein(a) concentration of more than 150 nmol per liter. Patients were randomly assigned to receive one of four doses of olpasiran (10 mg every 12 weeks, 75 mg every 12 weeks, 225 mg every 12 weeks, or 225 mg every 24 weeks) or matching placebo, administered subcutaneously. The primary end point was the percent change in the lipoprotein(a) concentration from baseline to week 36 (reported as the placebo-adjusted mean percent change). Safety was also assessed., Results: Among the 281 enrolled patients, the median concentration of lipoprotein(a) at baseline was 260.3 nmol per liter, and the median concentration of low-density lipoprotein cholesterol was 67.5 mg per deciliter. At baseline, 88% of the patients were taking statin therapy, 52% were taking ezetimibe, and 23% were taking a proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitor. At 36 weeks, the lipoprotein(a) concentration had increased by a mean of 3.6% in the placebo group, whereas olpasiran therapy had significantly and substantially reduced the lipoprotein(a) concentration in a dose-dependent manner, resulting in placebo-adjusted mean percent changes of -70.5% with the 10-mg dose, -97.4% with the 75-mg dose, -101.1% with the 225-mg dose administered every 12 weeks, and -100.5% with the 225-mg dose administered every 24 weeks (P&lt;0.001 for all comparisons with baseline). The overall incidence of adverse events was similar across the trial groups. The most common olpasiran-related adverse events were injection-site reactions, primarily pain., Conclusions: Olpasiran therapy significantly reduced lipoprotein(a) concentrations in patients with established atherosclerotic cardiovascular disease. Longer and larger trials will be necessary to determine the effect of olpasiran therapy on cardiovascular disease. (Funded by Amgen; OCEAN[a]-DOSE ClinicalTrials.gov number, NCT04270760.)., (Copyright © 2022 Massachusetts Medical Society.)

Background: Data support lipoprotein(a) (Lp[Lp(a)]) being a risk factor for atherosclerotic cardiovascular disease (ASCVD). Olpasiran is a small interfering RNA molecule that markedly reduces Lp(a) production in hepatocytes., Study Design: The Olpasiran trials of Cardiovascular Events And lipoproteiN(a) reduction-DOSE finding study is a multicenter, randomized, double-blind, placebo-controlled dose-finding study in 281 subjects with established ASCVD and Lp(a) > 150 nmol/L. Patients were randomly allocated to one of 4 active subcutaneous doses of olpasiran (10 mg q12 weeks, 75 mg q12 weeks, 225 mg q 12 weeks, or 225 mg q24 weeks) or matched placebo. The primary objective is to evaluate the effects of olpasiran dosed every 12 weeks compared with placebo on the percent change in Lp(a) from baseline at 36 weeks. Enrollment is now complete and follow-up is ongoing., Conclusions: OCEAN(a)-DOSE trial is assessing the Lp(a)-lowering efficacy and safety of olpasiran. These data will be used to determine optimal dosing and design for a cardiovascular outcomes trial., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Some trials have reported diminished efficacy for statins in the elderly, and in women compared with men. We examined the efficacy and safety of evolocumab by patient age and sex in the FOURIER trial, the first major cardiovascular outcome trial of a PCSK9 inhibitor., Methods and Results: FOURIER was a randomised, double blind trial, comparing evolocumab with placebo in 27,564 patients with atherosclerotic cardiovascular disease receiving statin therapy (median follow-up 2.2 years). The primary endpoint was cardiovascular death, myocardial infarction, stroke, hospitalisation for unstable angina or coronary revascularisation. Cox proportional hazards models were used to assess the efficacy of evolocumab versus placebo stratified by quartiles of patient age and by sex. There were small variations in the cardiovascular event rate across the age range (for the primary endpoint, Kaplan-Meier at 3 years 15.6%, >69 years, vs. 15.1%, ≤56 years, P = 0.45); however, the relative efficacy of evolocumab was consistent regardless of patient age (for the primary endpoint (Q1 hazard ratio, 95% confidence interval) 0.83, 0.72-0.96, Q2 0.88, 0.76-1.01, Q3 0.82, 0.71-0.95, Q4 0.86, 0.74-1.00; Pinteraction = 0.91), and the key secondary endpoint (cardiovascular death, myocardial infarction, stroke) (Q1 0.74 (0.61-0.89), Q2 0.83 (0.69-1.00), Q3 0.78 (0.65-0.94), Q4 0.82 (0.69-0.98)); Pinteraction = 0.81). Women had a lower primary endpoint rate than men (Kaplan-Meier at 3 years 12.5 vs. 15.3%, respectively, P < 0.001). Relative risk reductions in the primary endpoint and key secondary endpoint were similar in women (0.81 (0.69-0.95) and 0.74 (0.61-0.90), respectively) compared with men (0.86 (0.80-0.94) and 0.81 (0.73-0.90), respectively), Pinteraction = 0.48 and 0.44, respectively. Adverse events were more common in women and with increasing age but, with the exception of injection site reactions, there were no important significant differences reported by those assigned evolocumab versus placebo., Conclusions: The efficacy and safety of evolocumab are similar throughout a broad range of ages and in both men and women., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Background: Lipoprotein apheresis (LA) can effectively lower lipoproteins but is an invasive procedure., Objective: The objective of this study was to evaluate whether evolocumab can reduce LA requirement in patients undergoing chronic LA., Methods: Patients on regular weekly or every-2-week LA and moderate- to high-intensity statin (if tolerated) with pre-LA low-density lipoprotein cholesterol (LDL-C) levels ≥2.6 mmol/L (100 mg/dL) to ≤4.9 mmol/L (190 mg/dL) were randomized to continue the same LA frequency, or discontinue LA and receive evolocumab 140 mg every-2-weeks subcutaneously for 6 weeks. At week 6, all patients received only open-label evolocumab for 18 weeks. The primary endpoint was LA avoidance at the end of 6 weeks based on achieving pre-LA LDL-C <2.6 mmol/L at week 4., Results: Thirty-nine patients (mean [SD] age 62 [10] years, 59% male, 82% with familial hypercholesterolemia) were randomized (evolocumab, n = 19; LA, n = 20). At the end of 6 weeks, more patients receiving evolocumab avoided LA than those receiving LA (84% vs 10%; treatment difference, 74% [95% CI: 45, 87]; P < .0001). Thirty patients (77%) did not require LA at 24 weeks. Evolocumab reduced pre-LA LDL-C by 50% from the baseline to week 4 compared with a 3% increase in the LA arm. Pre-LA LDL-C <1.8 mmol/L (70 mg/dL) was achieved by 10 patients (53%) receiving evolocumab and none receiving LA (week 4). Safety was comparable between arms., Conclusion: Evolocumab treatment significantly reduced LA requirement in patients undergoing chronic LA. In addition, >50% of patients achieved LDL-C <1.8 mmol/L on evolocumab alone, demonstrating that in patients with pre-LA LDL-C ≤4.9 mmol/L, evolocumab may replace LA., (Copyright © 2019 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Background: Omecamtiv mecarbil (OM) is a selective cardiac myosin activator that increases myocardial function in healthy volunteers and in patients with chronic heart failure., Objectives: This study evaluated the pharmacokinetics, pharmacodynamics, tolerability, safety, and efficacy of OM in patients with acute heart failure (AHF)., Methods: Patients admitted for AHF with left ventricular ejection fraction ≤40%, dyspnea, and elevated plasma concentrations of natriuretic peptides were randomized to receive a double-blind, 48-h intravenous infusion of placebo or OM in 3 sequential, escalating-dose cohorts., Results: In 606 patients, OM did not improve the primary endpoint of dyspnea relief (3 OM dose groups and pooled placebo: placebo, 41%; OM cohort 1, 42%; cohort 2, 47%; cohort 3, 51%; p = 0.33) or any of the secondary outcomes studied. In supplemental, pre-specified analyses, OM resulted in greater dyspnea relief at 48 h (placebo, 37% vs. OM, 51%; p = 0.034) and through 5 days (p = 0.038) in the high-dose cohort. OM exerted plasma concentration-related increases in left ventricular systolic ejection time (p < 0.0001) and decreases in end-systolic dimension (p < 0.05). The adverse event profile and tolerability of OM were similar to those of placebo, without increases in ventricular or supraventricular tachyarrhythmias. Plasma troponin concentrations were higher in OM-treated patients compared with placebo (median difference at 48 h, 0.004 ng/ml), but with no obvious relationship with OM concentration (p = 0.95)., Conclusions: In patients with AHF, intravenous OM did not meet the primary endpoint of dyspnea improvement, but it was generally well tolerated, it increased systolic ejection time, and it may have improved dyspnea in the high-dose group. (Acute Treatment with Omecamtiv Mecarbil to Increase Contractility in Acute Heart Failure [ATOMIC-AHF]; NCT01300013)., (Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: This study sought to evaluate the efficacy and safety of subcutaneous evolocumab compared with oral ezetimibe in hypercholesterolemic patients who are unable to tolerate effective statin doses., Background: Statin intolerance, which is predominantly due to muscle-related side effects, is reported in up to 10% to 20% of patients. Evolocumab, a fully human monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), demonstrated marked reductions in plasma low-density lipoprotein cholesterol (LDL-C) in a phase 2 study in statin-intolerant patients., Methods: The GAUSS-2 (Goal Achievement after Utilizing an Anti-PCSK9 Antibody in Statin Intolerant Subjects) trial was a 12-week, double-blind study of randomized patients (2:2:1:1) to evolocumab 140 mg every two weeks (Q2W) or evolocumab 420 mg once monthly (QM) both with daily oral placebo or subcutaneous placebo Q2W or QM both with daily oral ezetimibe 10 mg. Co-primary endpoints were percent change from baseline in LDL-C at the mean of weeks 10 and 12, and at week 12., Results: Three hundred seven patients (age 62 ± 10 years; LDL-C 193 ± 59 mg/dl) were randomized. Evolocumab reduced LDL-C from baseline by 53% to 56%, corresponding to treatment differences versus ezetimibe of 37% to 39% (p <0.001). Muscle adverse events occurred in 12% of evolocumab-treated patients and 23% of ezetimibe-treated patients. Treatment-emergent adverse events and laboratory abnormalities were comparable across treatment groups., Conclusions: Robust efficacy combined with favorable tolerability makes evolocumab a promising therapy for addressing the largely unmet clinical need in high-risk patients with elevated cholesterol who are statin intolerant. (Goal Achievement After Utilizing an Anti-PCSK9 Antibody in Statin Intolerant Subjects-2; NCT01763905)., (Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases serum LDL-cholesterol (LDL-C) concentrations. We assessed the effects of AMG 145, a human monoclonal antibody against PCSK9, in patients with hypercholesterolaemia in the absence of concurrent lipid-lowering treatment., Methods: In a phase 2 trial done at 52 centres in Europe, the USA, Canada, and Australia, patients (aged 18-75 years) with serum LDL-C concentrations of 2·6 mmol/L or greater but less than 4·9 mmol/L were randomly assigned equally through an interactive voice response system to subcutaneous injections of AMG 145 70 mg, 105 mg, or 140 mg, or placebo every 2 weeks; subcutaneous AMG 145 280 mg, 350 mg, or 420 mg or placebo every 4 weeks; or oral ezetimibe 10 mg/day. The primary endpoint was percentage change from baseline in LDL-C concentration at week 12. Analysis was by modified intention to treat. Study personnel and patients were masked to treatment assignment of AMG 145 or placebo. Ezetimibe assignment was open label. This trial is registered with ClinicalTrials.gov, number NCT01375777., Findings: 406 patients were assigned to AMG 145 70 mg (n=45), 105 mg (n=46), or 140 mg (n=45) every 2 weeks; AMG 145 280 mg (n=45), 350 mg (n=45), or 420 mg (n=45) every 4 weeks; placebo every 2 weeks (n=45) or every 4 weeks (n=45); or ezetimibe (n=45). AMG 145 significantly reduced LDL-C concentrations in all dose groups (mean baseline LDL-C concentration 3·7 mmol/L [SD 0·6]; changes from baseline with every 2 weeks AMG 145 70 mg -41·0% [95% CI -46·2 to -35·8]; 105 mg -43·9% [-49·0 to -38·7]; 140 mg -50·9% [-56·2 to -45·7]; every 4 weeks AMG 145 280 mg -39·0% [-44·1 to -34·0]; 350 mg -43·2% [-48·3 to -38·1]; 420 mg -48·0% [-53·1 to -42·9]; placebo every 2 weeks -3·7% [-9·0 to 1·6]; placebo every 4 weeks 4·5% [-0·7 to 9·8]; and ezetimibe -14·7% [-18·6 to -10·8]; p<0·0001 for all doses vs placebo or ezetimibe). Treatment-emergent adverse events occurred in 136 (50%) of 271 patients in the AMG 145 groups, 41 (46%) of 90 patients in the placebo groups, and 26 (58%) of 45 patients in the ezetimibe group; no deaths or serious treatment-related adverse events were reported., Interpretation: The results of our study support the further assessment of AMG 145 in long-term studies with larger and more diverse populations including patients with documented statin intolerance., Funding: Amgen., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lowering low-density lipoprotein cholesterol (LDL-C) is a cornerstone for the prevention of atherosclerotic heart disease, improving clinical outcomes and reducing vascular mortality in patients with hypercholesterolemia. The clinical benefits of LDL-C reduction appear to extend even to patients starting with LDL-C as low as 60-80 mg/dL prior to initiating therapy. Statins are the first-line agents for treating hypercholesterolemia and are effective in reducing LDL-C, but many patients are unable to achieve their optimal lipid targets despite intensive statin therapy. Therefore, there has been a strong impetus for the development of novel pharmacologic agents designed to lower LDL-C further in patients already on statin therapy. Genetic mutations resulting in altered cholesterol homeostasis provide valuable information regarding novel approaches for treating hypercholesterolemia. To that end, mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) were linked to altered levels of LDL-C, illustrating this protein's role in lipid metabolism. PCSK9 promotes degradation of the LDL receptor, preventing its transport back to the cell surface and thereby increasing circulating LDL-C. Conversely, inhibition of PCSK9 can profoundly decrease circulating LDL-C, and thus is an attractive new target for LDL-C-lowering therapy. AMG 145 is a fully human monoclonal immunoglobulin G2 antibody that binds specifically to human PCSK9 and inhibits its interaction with the low-density lipoprotein receptor. In this manuscript, we describe the rationale and design of LDL-C Assessment with PCSK9 Monoclonal Antibody Inhibition Combined With Statin Therapy-Thrombolysis In Myocardial Infarction 57 (LAPLACE-TIMI 57; NCT01380730), a 12-week, randomized, double-blind, dose-ranging, placebo-controlled study designed to assess the safety and efficacy of AMG 145 when added to statin therapy in patients with hypercholesterolemia., (© 2012 Wiley Periodicals, Inc.)

Aims: To assess the safety and tolerability of darbepoetin alfa (DA) in the treatment of anaemia in heart failure (HF)., Methods and Results: In this pooled analysis of three randomized, double-blind, placebo-controlled studies of anaemic [haemoglobin (Hb) < or =12.0 g/dL or < or =12.5 g/dL] symptomatic HF subjects, DA was administered subcutaneously once every 2 weeks and titrated to achieve and maintain a target Hb of 14.0 +/- 1.0 g/dL. In total, 516 subjects were randomized; 231 (44.8%) to placebo, 285 (55.2%) to DA. Darbepoetin alfa was well tolerated, with an adverse event (AE) profile similar to placebo. Most subjects (placebo, 85%; DA, 87%) experienced at least one AE. There was a lower incidence of serious AEs in the DA group (placebo, 43%; DA, 37%) with the most frequent being worsening HF (placebo, 19%; DA, 11%). Treatment-related AEs were reported for 9% and 12% in placebo and DA subjects, respectively. Fewer deaths were reported in DA group (6%) vs. placebo (8%)., Conclusion: Darbepoetin alfa was well tolerated with an AE profile similar to placebo in HF subjects treated to a target Hb of 14.0 +/- 1.0 g/dL. Contrary to recent data in other patient populations, there was no evidence of increased risk of mortality or cardiovascular events.

Excess neuronal activity upregulates the expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in adult hippocampus. Nerve growth factor has been shown to contribute the induction of aberrant hippocampal mossy fiber sprouting in the inner molecular layer of the dentate gyrus, however the role of prolonged brain-derived neurotrophic factor exposure is uncertain. We examined the distribution and plasticity of mossy fibers in transgenic mice with developmental overexpression of brain-derived neurotrophic factor. Despite 2--3-fold elevated BDNF levels in the hippocampus sufficient to increase the intensity of neuropeptide Y immunoreactivity in interneurons, no visible changes in mossy fiber Timm staining patterns were observed in the inner molecular layer of adult mutant hippocampus compared to wild-type mice. In addition, no changes of the mRNA expression of two growth-associated proteins, GAP-43 and SCG-10 were found. These data suggest that early and persistent elevations of brain-derived neurotrophic factor in granule cells are not sufficient to elicit this pattern of axonal plasticity in the hippocampus., (Copyright 2001 Wiley-Liss, Inc.)

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning.

In the spontaneous ataxic mutant mouse stargazer, there is a selective reduction of brain-derived neurotrophic factor (BDNF) mRNA expression in the cerebellum. BDNF protein levels in the cerebellum are reduced by 70%. Despite normal levels of full-length and truncated TrkB receptor, constitutive and neurotrophin-4/5-induced tyrosine phosphorylation was significantly reduced in several signal transduction molecules, including phospholipase-Cgamma1, erk1, and erk2. Morphological examination revealed an increased number of external granule cells at postnatal day 15 and the presence of abnormal neurons resembling immature granule cells in the adult. These abnormalities are associated with a severe impairment in the acquisition of classical eyeblink conditioning, indicating cerebellar malfunction. Our data suggest that normal BDNF expression and TrkB signal transduction in the cerebellum are necessary for learning and plasticity in this model.

Purpose: Recent studies show that exogenous brain-derived neurotrophic factor (BDNF) can promote retinal ganglion cell survival in vivo and in vitro. BDNF is expressed by a subpopulation of cells in the ganglion cell layer (GCL). To investigate whether endogenous BDNF may play a role in neuronal protection after ganglion cell trauma, BDNF expression in the retina was examined after optic nerve (ON) injury., Methods: The optic nerve in Sprague-Dawley rats was crushed intraorbitally posterior to the optic disc. For controls, the optic nerve on the opposite side in each animal was similarly exposed but was not crushed. After intervals of 6 hours to 6 weeks, eye tissues were processed for in situ hybridization, Northern blot, and RNase protection assay using radiolabeled rat riboprobes., Results: After ON injury, BDNF expression was significantly elevated in cells restricted to the GCL, and more cells demonstrated expression of BDNF than were observed in the controls. Elevated BDNF expression was first observed at 24 hours, peaked at 48 hours, and declined to the basal level 2 weeks after ON injury. Quantitative analysis showed a fivefold to sixfold increase in the number of BDNF-positive cells and a 54% increase in BDNF signal intensity in individual cells in the GCL 48 hours after ON injury. In control retinas without ON injury, BDNF expression was localized to some cells in the GCL, as was observed in normal eyes without surgery. Northern blot and RNase protection assay demonstrated a 38% elevation in BDNF expression above control levels 48 hours after ON injury., Conclusions: These results indicate that cells in the GCL can upregulate gene expression of BDNF in response to ganglion cell axonal injury and suggest that endogenous BDNF may contribute to a natural neuroprotective process after ON injury.

The laminin-alpha2 chain is a component of brain capillary basement membranes and appears also to be present in neurons of rat, rabbit, pig and non-human primate brain as evidenced by immunohistochemistry. In the present study, we have further characterized this very distinct neuronal laminin-alpha2 chain-like immunoreactivity in the hippocampus of various species. Immunoelectron microscopy with poly- and monoclonal antibodies to the laminin-alpha2 chain G-domain localized laminin-alpha2 chain immunoreactivity in adult rat and rabbit hippocampus to dendritic processes, primarily to dendritic spines. In the developing rat hippocampus, spine-associated laminin-alpha2 chain-like immunoreactivity first appeared at a time corresponding to that of active synaptogenesis. After an entorhinal cortex lesion in adult rats, the time course of denervation-induced loss and reactive reappearance of spines in the molecular layer of the dentate gyrus was correlated closely to the loss and reappearance of laminin-alpha2 chain immunoreactivity. Immunoblot analysis of normal adult rat, rabbit and pig brain revealed a protein similar in size to the reported 80-kDa laminin-alpha2 chain fragment of human placenta as well as 140/160-kDa proteins. These results suggest the presence of proteins with antigenic homology to the laminin-alpha2 chain and/or laminin-alpha2 isoforms in dendrites and dendritic spines in selected areas of the brain, predominately in the hippocampus and other limbic structures. Given the adhesion and neurite promoting functions of laminins, it is possible that neuronal laminin-alpha2 chain-like proteins play a role in synaptic function and plasticity in the CNS.

Glutathione (GSH) is considered the primary molecule responsible for peroxide removal from the brain. Inhibition of its rate-limiting synthetic enzyme, glutamylcysteine synthetase (GCS), results in morphological damage to both cortical and nigral neurons in rodents. Here, we report cloning of the catalytic heavy chain GCS mRNA from mouse and its localization in the murine brain. Heavy chain GCS appears to be localized in glial populations in the hippocampus, cerebellum and olfactory bulb, with lower levels of expression in the cortex and substantia nigra. Variations in GCS levels and subsequent GSH synthesis may explain differences in susceptibility to neuropathology associated with oxidative stress noted in these various brain regions.

Neurotrophic factors, particularly the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and related molecules are proposed for the experimental treatment of neurode-generative disease. Earlier observations had suggested down-regulation of the neurotrophin receptor response with chronic stimulation. We therefore tested for effects of acute and chronic NGF treatment in vivo on the tyrosine phosphorylation response of Trk-type neurotrophin receptors in adult and aged rats. Rats were treated for 1 week with daily injections of NGF directly into the striatum. Surprisingly, this chronic neurotrophin treatment induced long-lasting tyrosine phosphorylation of Trk type receptors beyond the last injection. A similar result was obtained with 1 week of daily injections of BDNF into the hippocampus. Persistent TRK tyrosine phosphorylation was also observed after single neurotrophin injections. With 1 microgram of NGF injected, Trk-type receptors were maximally stimulated from immediately after the injection until 3 days after the treatment. Maintaining Trk tyrosine phosphorylation required maintained energy levels in the tissue. Incubation of microslices of brain tissue from NGF-injected animals in glucose-free buffer completely abolished all Trk tyrosine phosphorylation signals. Recovery of tissue in presence of glucose restored the signals in microslices derived from NGF-injected animals, in absence of acute NGF treatment. This result, together with dose-response comparisons after 2-h and 2-day survival times suggest that Trk protein remains tyrosine phosphorylated due to trophic protein which is only slowly being cleared out of the tissue during several days after the injection. Experiments with aged rats indicated similar extent and duration of Trk receptor activation after NGF administration in young adult and in aged brain.

Penetration into the brain is an important consideration in the pharmacological use of neurotrophic factors for the treatment of brain neurodegeneration, e.g., in Alzheimer's disease. Furthermore, intracerebroventricular treatment with nerve growth factor (NGF) has been found to induce side effects, including aberrant sympathetic sprouting and weight loss. Such findings suggest that direct intraparenchymal application of minimal amounts of trophic factors might be therapeutically desirable. We compared the effectiveness of intrastriatal and intracerebroventricular administrations of NGF on striatal cholinergic neurons in adult rats. Daily intrastriatal administration for 1 week of > or = 50 ng of NGF resulted in an increase in mRNA levels for choline acetyltransferase (ChAT) in striatal cholinergic cells to approximately 2-fold over control. A daily intraventricular dose of 4.5 micrograms of NGF was required for a similar response. Both 5 and 50 ng of NGF/day failed to induce an effect on transmembrane protein tyrosine kinase trkA mRNA levels, but injections of 750 or 1500 ng/day of NGF up-regulated trkA mRNA expression to approximately 2-fold of control. NGF delivered intracerebroventricularly failed to induce an observable change in striatal trkA mRNA, even at a dosage of 4.5 micrograms of NGF/day. These quantitative differences in NGF actions were reflected at the level of NGF receptors. Using Western blotting procedures, we found pronounced tyrosine phosphorylation of Trk-type proteins 2 hr after intrastriatal injection of 50 ng of NGF. Maximal responses were seen with either 150 or 750 ng of NGF. For maximal activation of Trks by intraventricular NGF injection, 4.5 micrograms of NGF was required. Taken together, our results strongly favor intraparenchymal injections or infusions of NGF, and possibly other trophic factors, for therapeutical applications to maximize the effects on the targeted neuronal populations and to minimize undesirable side effects.

Adrenalectomy (ADX) in rats has been shown to induce apoptosis of hippocampal granule cells. We tested whether neurotrophins are able to protect hippocampal neurons in this neurodegeneration model. Acid fucshsin stain was used to identify pyknotic cells in ADX rats treated for 4 days with NT-3, NT-4-5 or cytochrome-C, as a control protein. Cytochrome-C injections slightly decreased cell death on the ipsilateral side. NT-3 did not further promote this effect. Significantly less cell death was observed bilaterally in hippocampus treated with NT-4/5. TUNEL end labeling also confirmed the results. Our results demonstrated that NT-4/5, but not NT-3, promotes hippocampal neuron survival in adrenalectomized rats. They further show that injections of a control solution can induce a local protective effect.

In search of the possible involvement of neurotrophic factors in inherited neurological disease, we examined brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) mRNA expression patterns in the ataxic mutant mouse stargazer (stg). Using in situ hybridization, we found a selective and near total reduction in BDNF mRNA in the cerebellar granule cell layer. NT-3 or NGF mRNA expression in the cerebellum was normal. Northern blot analysis demonstrated a 70% reduction in BDNF mRNA in the whole cerebellum. BDNF mRNA levels in other mutant brain regions were unchanged. Absence of BDNF mRNA in granule cells was observed at postnatal age (P15), coincident with the onset of ataxia, and expression levels failed to follow the developmental increase found in the wild type at later ages (P20 and P30). Despite the severe BDNF reduction, in situ hybridization patterns for both the full-length and the truncated BDNF TrkB receptor mRNA were unaltered. No major cytoarchitectural abnormalities were apparent in the stg/stg cerebellum. BDNF expression in a related ataxic mutant, tottering, was unaltered. These data show that BDNF can be regulated selectively in distinct brain regions, possibly by differential activation of its multiple promoters. Absence of cerebellar granule cell BDNF mRNA in stg/stg mice demonstrates that sustained expression of this neurotrophin is not required for cell survival in the developing cerebellar cortex. Our data, in contrast, suggest a role of BDNF in maturation of specific cerebellar neurons and pathways. Early failure of cerebellar BDNF expression may be related to the ataxic phenotype in stg mice.

Improvement of the cholinergic deficit in Alzheimer's disease (AD) by intracerebral application of nerve growth factor (NGF) serves as a paradigmatic example for a novel approach to the treatment of neurodegeneration. The first part of this paper presents and discusses experiments which were performed in our laboratory to study the NGF receptor response after intracerebral NGF treatment in vivo. We found that intraparenchymal NGF elicits prolonged tyrosine phosphorylation of Trk type NGF receptors. Our results indicate that intraparenchymal injections are preferable to intraventricular application for targeting specific neuronal populations with minimal side effects. Besides the cholinergic deficit, severely disturbed brain energy metabolism, particularly in cortical association areas, is another consistent feature of AD. Metabolic hypofunction is observed early in the disease progression and correlates with the cognitive impairment. Cell culture findings are presented which indicate that brain-derived neurotrophic factor (BDNF), and other neurotrophins with activity on the TrkB tyrosine kinase receptor, increase mRNA levels and biochemical activity of enzymes of the glycolytic pathway in brain cells. Treatment with these factors was also found to stimulate glucose utilization in rat embryonic cortex cells in primary cultures. Our observations suggest that selected neurotrophins should become useful not only for the treatment of the cholinergic deficit in AD, but also of the cortical metabolic hypofunction associated with this disease.

Neurotrophins are a family of highly conserved proteins that affect the development and maintenance of distinct neuronal populations. Neurotrophins exist in vivo as homodimers, but we show that neurotrophins can exist as heterodimers in vitro and are pluripotent, being able to bind and to activate different Trk tyrosine kinase receptors as well as promote neuronal differentiation in PC12 cells as effectively as wild type homodimers. These asymmetric neurotrophin dimers allow unique characterization of neurotrophin structure-function relationships with Trk receptors. The chimeric Trk activities of these heterodimers suggest an alternative model of neurotrophin-Trk receptor activation in which the critical Trk-interacting elements may be attributed to a single protomer.

This study examined how possible nerve growth factor (NGF)-induced behaviour changes after septal damage might be modulated by the lesion extent, the dose of NGF administered and the delay between surgery and the onset of testing. In a first experiment, young rats which received electrolytic septal lesions of high or low intensity (inducing respectively large and mild lesions) were treated with 10 or 30 micrograms NGF administered intrahippocampally in a single injection. They were tested 4 months postoperatively for open field ambulation, spontaneous alternation and radial maze performance. It was observed that irrespective of the severity of the lesions rats were impaired in the spontaneous alternation and radial maze tests; however, no obvious changes appeared in the open field test. While an NGF injection did not affect behavioural performances in rats with large lesions, it was capable of ameliorating behavioural deficits in the spontaneous alternation and radial maze tests of rats with mild lesions in both NGF dosage groups. It was also seen that lesions produced a general decrease in hippocampal choline acetyltransferase (ChAT) activity, which was not significantly affected by an NGF administration. There was no significant correlation between ChAT activity and behavioural performance of NGF-treated rats. In a second experiment, young rats received mild septal lesions and were treated with 10 micrograms NGF. These rats were tested 2 weeks postoperatively for radial maze performance. NGF rats exhibited similar behaviour to controls with regard to all of the variables measured. The present results suggest that a single NGF administration spares some abilities to use spatial information efficiently providing lesions are partial.

Nerve growth factor (NGF) increases the activity of choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine, in rat basal forebrain neurons both in vivo and in vitro. In poly(A)+ RNA isolated from cultures prepared from the embryonic (E15) rat basal forebrain, radiolabeled probes from the human ChAT gene detected a 3,700 nt and a less abundant 2,300 nt transcript. After growth in the presence of NGF, the abundance of both mRNAs was increased approximately twofold, paralleling the increase in ChAT enzyme activity. In vivo, the human ChAT probes detected a single 3,700 nt form of ChAT mRNA in both embryonic and adult rat basal forebrain. These results suggest that the NGF-mediated increase in ChAT activity in basal forebrain cultures is regulated at the transcriptional level.

To enable us to study expression of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] as a measure of dopaminergic neuron function in future experiments, methods were developed to quantify TH mRNA levels in cultures of dopaminergic mesencephalic cells. The model of selective dopaminergic toxicity of 1-methyl-4-phenylpyridinium (MPP+) was used to verify the specificity of our methods. Fetal (embryonic day 15) rat ventral mesencephalic cell cultures were treated with 15 microM MPP+ for 48 h, conditions previously shown to reduce the number of TH-immunoreactive neurons, TH activity, and dopamine uptake to 5-10% of control values. This treatment decreased the number of neurons labeled by TH in situ hybridization to 9% of untreated controls and caused a strong reduction of the abundance of TH mRNA in Northern blots. Our findings establish TH mRNA expression as a parameter for future studies of toxic and trophic effects on cultured dopaminergic neurons, and they support the view that MPP+ destroys dopaminergic neurons.

Severe disruption of certain cholinergic pathways is a characteristic feature of Alzheimer's disease. Attempts to establish animal models by interfering with cholinergic function have not been very successful. We now present data which show a substantial and progressive replacement of free and phospholipid-bound choline by the novel choline isostere N-amino-N,N-dimethylaminoethanol during its dietary administration in place of choline. Free choline in blood fell to approximately 20% of controls after 10 to 30 days on diet. Phospholipid-bound choline in plasma was reduced to less than 15%, and in erythrocytes to about 22%. After 120 days of diet free and bound choline were reduced in most tissues to approximately 30% of controls. Only liver retained more than 80% of free choline. Acetylcholine was decreased to 33 to 50% of control. Total true and false transmitter in experimental animals was in all tissues less that acetylcholine in controls, suggesting that muscarinic transmission would be impaired. Moderate reduction of choline acetyltransferase activity was seen in striatum and myenteric plexus, and of QNB-binding in hippocampus, striatum and myenteric plexus.

To study the selectivity of neurotrophic actions in the brain, we analyzed the actions of several known growth factors on septal cholinergic, pontine cholinergic, and mesencephalic dopaminergic neurons in culture. Similar to nerve growth factor (NGF), basic fibroblast growth factor (bFGF) stimulated choline acetyltransferase activity in septal cultures. In contrast to NGF, bFGF also enhanced dopamine uptake in mesencephalic cultures and stimulated cell proliferation in all 3 culture types. Insulin and the insulin-like growth factors I and II stimulated transmitter-specific differentiation and cell proliferation in all culture types. Epidermal growth factor (EGF) produced a small increase in dopamine uptake by mesencephalic cells and stimulated cell proliferation in all culture types. In septal cultures, bFGF was most effective when given at early culture times, NGF at later times. The stimulatory actions of bFGF and insulin did not require the presence of glial cells and were not mediated by NGF. In mesencephalic cultures, the stimulation of dopamine uptake by bFGF and EGF was dependent on glial proliferation. The results suggest different degrees of selectivity of the neurotrophic molecules. NGF and, very similarly, bFGF seem to influence septal cholinergic neurons directly and rather selectively, whereas the neurotrophic actions of insulin and the insulin-like growth factors appear to be more general.

Intracerebral chronic administration of nerve growth factor (NGF) is able to counteract age-related cholinergic dysfunction. This finding suggests that intracerebral administration of neurotrophic factors may become a generally valuable approach when attempting to ameliorate age-related neuronal deficits in experimental animals and humans. This commentary briefly discusses possible strategies for the development of such "neurotrophic factor treatments."

N-aminodeanol is an analog of choline that serves as a less effective substrate in all of its known enzymatic and transport mechanisms. It was utilized to test the hypothesis that the selective vulnerability of cholinergic neurones in Alzheimer's disease is due to competition for the available choline between pathways for acetylcholine and phospholipid synthesis. Rats placed on a choline free diet containing an equivalent amount of N-aminodeanol develop a model hypocholinergic state comprising hyperreactivity, hyperalgesia, aggressive behavior and a deficit in learning and memory. These effects are associated with a progressive replacement of free and lipid-bound choline and acetylcholine with N-aminodeanol and its corresponding esters. Choline acetyltransferase is reduced in some brain regions, suggesting a loss of cholinergic neurones. We propose that this represents a potentially useful animal model of Alzheimer's disease which deserves further investigation.