Detection of anti-Pneumocystis jirovecii antibodies in human serum using a recombinant synthetic multi-epitope kexin-based antigen.

Interest in the detection of specific anti-Pneumocystis jirovecii antibodies has emerged as less-invasive alternative diagnostic approaches. Here is presented the performance of an ELISA based on a recombinant synthetic multi-epitope kexin 1 (Kex1) antigen of P. jirovecii, previously developed. Results showed that IgM anti-Kex1 levels were found significantly increased in patients with Pneumocystis pneumonia (PcP) compared with non-PcP cases (p < 0.001), allowing a diagnostic performance of PcP with a 70.8% sensitivity and a 75.0% specificity. These results suggest that this Kex1-based ELISA is a promising tool toward the serodiagnosis of PcP when the standard methods are difficult to perform.

Human spermatozoa anti-proprotein convertase subtilisin/kexin type 4 synthesis using New Zealand rabbit for novel immunocontraception in males.

Purpose: Proprotein convertase subtilisin/kexin type 4 (PCSK4, a 54-kDa protease) is expressed in the plasma membrane of the acrosome in human spermatozoa. It plays a critical role in penetrating the zona pellucida. Synthesis of human anti-PCSK4 might be important for novel male immunocontraception. Materials and Methods: We used semen from adult males as the source of antigen (acrosomal PCSK4). Isolation and antigen characterization were done by immunohistochemistry followed by electrophoresis. Purification of PCSK4 was done by the electroelution method, followed by immunization with an animal model (Oryctolagus cuniculus). Antibody was collected, purified, and tested by using Western blot and dot blot. Antibody in vitro potential testing was performed on human spermatozoa by laser scanning microscopy with rhodamine stain under a light microscope and on rat spermatozoa. Results: Human anti-PCSK4 bound with PCSK4 on the head of human spermatozoa and could interfere with rat spermatozoa activity to penetrate the oocyte. Conclusions: The result of this study can be used as a basis to develop new immunocontraceptives targeted towards males. This study shows that antibodies from induction of PCSK4 from spermatozoa may hinder fertilization.

[Heterozygous familial hypercholesterolemia: the first challenge for anti-PCSK9 monoclonal antibodies]. / L'ipercolesterolemia familiare eterozigote: la prima sfida per gli anticorpi monoclonali anti-PCSK9.

Heterozygous familial hypercholesterolemia (HeFH) is characterized by a prevalence of 1/200 (higher than 1/500 as previously estimated): based on this updated prevalence, in Italy there are about 250-300 000 subjects with HeFH. Patients with HeFH are significantly underdiagnosed (in Italy only 4-5% of total estimated HeFH are properly diagnosed), undertreated (only 1 in 5 to 10 HeFH at target for LDL-cholesterol), and characterized by a high or very high cardiovascular risk. There are simple criteria for the diagnosis of familial hypercholesterolemia such as those issued by the Dutch Lipid Clinic Network (DLCN), easy to implement both in general practice as well as by the specialists. Genetic diagnosis is strongly suggested to support the diagnosis of familial hypercholesterolemia. Lipid-lowering therapy with high dose of highly effective statins, often associated with ezetimibe, should be initiated immediately at diagnosis in adults and at age 8-10 years in childhood. Evolocumab and alirocumab are monoclonal antibodies against PCSK9. They are a highly innovative lipid-lowering approach, characterized by a good safety profile and a remarkable LDL-cholesterol lowering effect when associated with the maximally tolerated dose of statins plus ezetimibe. Studies with alirocumab and evolocumab in HeFH patients show a further LDL-cholesterol decrease by 50-60% vs intensive lipid-lowering therapy with statins ± ezetimibe, with 70-80% of HeFH patients achieving their LDL-cholesterol targets.

[Management of hypercholesterolemia in patients with acute coronary syndrome: current mechanisms and future perspectives]. / La gestione del colesterolo nel paziente con sindrome coronarica acuta: dinamiche attuali e prospettive future.

Acute coronary syndromes (ACS) are a major health problem both in industrialized countries and in developing ones, and a leading cause of death and disability. The pathogenesis of ACS is multifactorial and complex, with approximately 65-70% of cases caused by the abrupt occlusion of a coronary vessel. This usually occurs as a result of thrombus formation over a vulnerable, lipid-rich atherosclerotic plaque, which undergoes rupture or erosion. High levels of LDL-cholesterol (LDL-C) are a well known risk factor for the development of atherosclerosis, and the reduction in plasma LDL-C is a fundamental treatment both in primary and secondary prevention. Statins are the most extensively used lipid-lowering drugs. They have been associated with reduced progression of coronary atherosclerosis and a decreased incidence of new ACS episodes or post-ACS major cardiovascular events. For ACS patients, the European Society of Cardiology (ESC) has suggested an early treatment - starting with the acute phase - together with a well defined target value of LDL-C level, which should be achieved during the follow-up. While statin therapy has significantly lowered cardiovascular risk, several cardiovascular events are still not prevented and a residual risk remains also after intensive therapy. In addition, a significant proportion of high-risk patients do not achieve the LDL-C target recommended by the ESC guidelines or present with statin intolerance, which does not allow a continuative and effective treatment. This is the main reason why innovative lipid-lowering therapies might become a new opportunity in ACS patients. The recently published results of the IMPROVE-IT trial have shown that the association statin + ezetimibe is superior to statin alone in preventing cardiovascular death, non-fatal myocardial infarction, rehospitalization for unstable angina, coronary revascularization and stroke in a population of medium to high-risk patients stabilized after ACS. Monoclonal antibodies targeting human PCSK9 are currently being tested on top of statins in patients with ACS, to investigate their superiority in reducing major cardiovascular adverse events as compared with statins alone at the maximally tolerated dose. The results, expected for 2017, will hopefully benefit the treatment of patients in secondary prevention and contribute to a better outcome of ACS patients worldwide.

Evolocumab: A Review in Hyperlipidemia.

Evolocumab (Repatha(®)) is a monoclonal antibody targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) that is administered subcutaneously at a dosage of 140 mg every 2 weeks or 420 mg once monthly. Across 12-week phase III trials in patients with primary hypercholesterolemia or mixed dyslipidemia, evolocumab was more effective than placebo (treatment difference -54.8 to -76.3%) and/or ezetimibe (treatment difference -36.9 to -47.2%) at reducing low-density lipoprotein cholesterol (LDL-C) levels, including when added to statin therapy, when administered to statin-intolerant patients, when administered as monotherapy, and in patients with heterozygous familial hypercholesterolemia who were receiving statins with or without other lipid-lowering drugs. Evolocumab also significantly lowered LDL-C levels (treatment difference of ≈30% vs. placebo) in patients with homozygous familial hypercholesterolemia when added to statins with or without ezetimibe in a 12-week phase III trial. The efficacy of evolocumab was maintained in the longer term, and it was well tolerated. In conclusion, subcutaneous evolocumab is a valuable new treatment for use in primary hypercholesterolemia or mixed dyslipidemia and homozygous familial hypercholesterolemia, particularly in patients unable to reach LDL-C goals despite treatment with statins with or without other lipid-lowering therapies and in patients who do not tolerate or are not able to receive statins.

Atherosclerosis stabilization with PCSK-9 inhibition: An evolving concept for cardiovascular prevention.

Monoclonal antibodies (mAbs) to proprotein convertase subtilisin/kexin type 9 (PCSK-9) can further lower LDL-C by ≥60% in statin-treated patients. Preliminary data suggest they may reduce cardiovascular (CVD) events. Ongoing PCSK-9 mAb cardiovascular outcomes trials could provide the opportunity to determine whether a "legacy effect" similar to that observed for statins will occur over the post-trial observation period. We hypothesize these trials could demonstrate that (1) very aggressive LDL-C lowering with PCSK-9 mAbs added to background statin therapy will induce extensive atherosclerosis stabilization and regression in the large majority of treated patients, and (2) continued maintenance therapy with high intensity statin therapy (with or without ezetimibe) should then inhibit new plaque formation, with a long-term prevention of CVD events. The necessity of expensive lifetime treatment with PCSK-9 inhibitors could then be avoided in all but a small subset of patients who could benefit from longer treatment.

Therapeutic Potential and Critical Analysis of the PCSK9 Monoclonal Antibodies Evolocumab and Alirocumab.

OBJECTIVE: To review the mechanism of action for PCSK9 monoclonal antibodies and critically evaluate the therapeutic potential of evolocumab and alirocumab in the treatment of hypercholesterolemia. DATA SOURCES: Ovid MEDLINE search from 1980 to August 2015 using the terms PCSK9, evolocumab, and alirocumab with forward and backward citation tracking. STUDY SELECTION AND DATA EXTRACTION: English-language trials and studies assessing the mechanism, efficacy, or safety of PCSK9 monoclonal antibodies were included. DATA SYNTHESIS: PCSK9 monoclonal antibodies have a potent ability to reduce low-density lipoprotein (LDL) by almost 50% in controlled trials: -47.49% (95% CI = -69.6% to -25.4%). They have an acceptable safety profile with no significant elevations in Creatine Kinase (CK) (odds ratio [OR] = 0.72; 95% CI = 0.54 to 0.96) or serious adverse events (OR = 1.01; 95% CI = 0.87 to 1.18), and preliminary evidence suggests reductions in myocardial infarction (OR = 0.49; 95% CI = 0.26 to 0.93). Although it is effective in several familial hypercholesterolemia (FH) patient types, it does not work in homozygous patients with dual allele LDL receptor negative polymorphisms or those who are homozygous for autosomal recessive hypercholesterolemia. CONCLUSIONS: Although not preferred over statins because of limited clinical trial evidence of cardiovascular event reductions, dosing convenience, and expense, PCSK9 monoclonal antibodies will have a prominent role to play in the treatment of hypercholesterolemia, especially in patients needing large LDL reductions, including patients with many types of FH.

A cholesterol-lowering VLP vaccine that targets PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that controls cholesterol homeostasis by enhancing endosomal and lysosomal degradation of the low-density lipoprotein receptor (LDL-R). Mutations that cause increased activity of PCSK9 are associated with hypercholesterolemia, atherosclerosis and early cardiovascular disease (CVD), whereas individuals with loss-of-function mutations in PCSK9 are apparently healthy but are hypocholesterolemic and have a dramatically decreased risk of CVD. In this study, we generated virus-like particle (VLP)-based vaccines targeting PCSK9. Mice and macaques vaccinated with bacteriophage VLPs displaying PCSK9-derived peptides developed high titer IgG antibodies that bound to circulating PCSK9. Vaccination was associated with significant reductions in total cholesterol, free cholesterol, phospholipids, and triglycerides. A vaccine targeting PCSK9 may, therefore, be an attractive alternative to monoclonal antibody-based therapies.

Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody.

Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site. In vitro exposure of PCSK9 to furin resulted in degradation of PCSK9 bound to LY, whereas cleavage was blocked by other mAbs. These other mAbs caused a significant accumulation of serum PCSK9 and displayed a shorter duration of LDL-cholesterol lowering than LY when administered to mice expressing the WT human PCSK9. In mice expressing a noncleavable variant of human PCSK9, LY behaved like a cleavage-blocking mAb, in that it caused significant PCSK9 accumulation, its duration of LDL lowering was reduced, and its clearance (CL) from serum was accelerated. Thus, LY neutralizes PCSK9 and allows its proteolytic degradation to proceed, which limits PCSK9 accumulation, reduces the CL rate of LY, and extends its duration of action. PCSK9 mAbs with this property are likely to achieve longer durability and require lower doses than mAbs that cause antigen to accumulate.

Efficiency and safety of proprotein convertase subtilisin/kexin 9 monoclonal antibody on hypercholesterolemia: a meta-analysis of 20 randomized controlled trials.

BACKGROUND: Proprotein convertase subtilisin/kexin9 (PCSK9) monoclonal antibody significantly reduces low-density lipoprotein cholesterol level in patients with hypercholesterolemia. The goal of this study was to review recently reported randomized controlled trials to investigate the therapeutic effects and safety of PCSK9 inhibitors. METHODS AND RESULTS: The clinical randomized controlled trials published from inception to March 19, 2015 were identified from The Cochrane Library databases, PUBMED, and EBASE. Randomized controlled trials of at least 8 weeks duration using PCSK9 inhibitors in treating patients with hypercholesterolemia were included. Mean difference (MD) with a 95% CI was used to calculate the continuous data, the standardized mean difference with a 95% CI was used when the unit was not unified, and risk ratio with a 95% CI was used for dichotomous data. After screening, 20 trials fulfilled the inclusion criteria. PCSK9 inhibitors significantly decreased the levels of low-density lipoprotein cholesterol (MD=-65.29 mg/dL, 95% CI: -72.08 to -58.49), total cholesterol (MD=-60.04 mg/dL, 95% CI: -69.95 to -50.13), triglycerides (MD=-12.21 mg/dL, 95% CI: -16.21 to -8.22) and apolipoprotein-B (MD=-41.01 mg/dL, 95% CI: -46.07 to -35.94), lipoprotein(a) (standardized mean difference=-0.94, 95% CI: -1.12 to -0.77) and increased the levels of high-density lipoprotein cholesterol (MD=3.40 mg/dL, 95% CI: 3.12 to 3.68) and apolipoprotein-A1 (MD=6.75 mg/dL, 95% CI: 4.64 to 8.86). There was no significant difference in the incidence of treatment-emergent adverse events (risk ratio=1.01, 95% CI: 0.98 to 1.04), serious treatment-emergent adverse events (risk ratio=1.01, 95% CI: 0.88 to 1.17), and the discontinuation of treatment between the 2 groups (risk ratio=1.07, 95% CI: 0.86 to 1.34). CONCLUSIONS: The meta-analysis indicated that PCSK9 inhibitors had a strong effect in lowering low-density lipoprotein cholesterol and other lipid levels with satisfactory safety and tolerability in patients with hypercholesterolemia.

Selection and characterization of human PCSK9 antibody from phage displayed antibody library.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), which involves in low-density lipoprotein cholesterol (LDL-C) metabolism by interacting with the LDL receptor, is considered as a potent therapeutic target for treating hypercholesterolemia. Here, a fab antibody phage display library was constructed and employed for bio-panning against recombinant PCSK9. A Fab fragment (designated PA4) bound with high affinity to PCSK9 was isolated after four rounds of panning. The fully human antibody IgG1-PA4 bound specifically to PCSK9 with nanomolar affinity. In vitro, IgG1-PA4 inhibited PCSK9 binding to LDLR and attenuated PCSK9-mediated degradation of LDLR on the HepG2 cell surface. In C57BL/6 mice, administration of IgG1-PA4 at 30 mg/kg increased hepatic LDLR protein levels by as much as 3 fold when compared with control. Taken together, these results suggested that the IgG1-PA4 can be served as a potential candidate for the treatment of hypercholesterolemia by inhibiting PCSK9-mediated degradation of cell surface LDLRs.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibition and the Future of Lipid Lowering Therapy.

Low-density lipoprotein cholesterol (LDL-C) reduction with statins is the cornerstone of atherosclerotic cardiovascular disease (CVD) prevention. The LDL-C lowering non-statin therapy ezetimibe also modestly reduces CVD risk when added to statin therapy. There remains a clinical need for additional LDL-C lowering agents to reduce CVD risk in patients with genetic hypercholesterolemia, statin intolerance, or who are at high risk due to clinical CVD or diabetes. In clinical trials, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition using monoclonal antibodies has demonstrated robust LDL-C lowering efficacy of 50-65% and a favorable safety profile. These agents are a promising therapeutic strategy for addressing the unmet needs for additional CVD risk reduction. Regulatory approval for PCSK9 monoclonal antibodies may occur in the near future, and additional agents for PCSK9 inhibition are under development. This review focuses on the mechanism of LDL-C reduction using PCSK9 inhibition, as well as the phase I to III clinical trials of PCSK9 inhibitors. Results of the ongoing phase III CVD outcome trials are eagerly awaited.

PCSK9 antibodies: A new class of lipid-lowering drugs.

The serine protease proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low-density lipoprotein (LDL) receptor (LDLR) and directs it to the lysosome for degradation. This results in decreased numbers of LDLR available on the cell surface to bind LDL particles and remove them from the circulation and a subsequent increase in circulating LDL-cholesterol (LDL-C) concentrations. Since the role PCSK9 plays in LDL-C metabolism has been discovered in 2003, there have been major efforts in finding efficient and safe methods to inhibit it. Amongst those, the PCSK9 antibodies are the furthest in development, with multiple phase III and cardiovascular endpoint trials already underway. These fully human monoclonal antibodies have been extensively studied in a wide range of subjects such as in those with statin intolerance, as add-on to statin therapy, as monotherapy and in patients with familial hypercholesterolemia. PCSK9 antibodies have shown to be associated with a consistent robust additional decrease in LDL-C concentrations of around 50-70%. If the safety data from the ongoing phase III trials remain as reassuring as the data available till now, PCSK9 antibodies are going to offer a new, powerful therapeutic option to decrease LDL-C concentrations and hopefully cardiovascular risk.

PCSK9 inhibitors: Novel therapeutic agents for the treatment of hypercholesterolemia.

Reducing plasma levels of low-density lipoprotein cholesterol (LDL-c) remains the cornerstone in the primary and secondary prevention of cardiovascular disease. However, a substantial proportion of patients fail to achieve acceptable LDL-c levels with currently available lipid-lowering drugs. Over the last decade, inhibition of Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) has emerged as a promising target to reduce residual cardiovascular disease risk. Binding of PCSK9 to the LDL receptor targets the LDL receptor for lysosomal degradation. Inhibition of PCSK9 increases expression of the LDL receptor. This observation has led to the development of a number of approaches to directly target PCSK9. Three monoclonal antibodies against PCSK9 are currently being evaluated in phase 3 trials involving various patient categories on different background lipid lowering therapies. Current evidence shows reductions in LDL cholesterol levels of up to 70%, independent of background statin therapy. The results of phase 3 trials will demonstrate the long-term efficacy and safety of PCSK9 inhibition, and will indicate whether LDL-c lowering induced by this novel approach translates into beneficial effects on CVD outcome.

PCSK9 inhibitors.

The discovery of proprotein convertase subtilisin kexin 9 (PCSK9) has considerably changed the therapeutic options in the field of lipid management. PCSK9 reduces LDL receptor recycling, leading to a decrease of low-density lipoprotein cholesterol (LDL-C) receptors on the surface of hepatocytes and a subsequent increase of circulating LDL-C levels. In observational studies, the loss-of-function mutations of PCSK9 have been associated with a reduction of LDL-C levels and cardiovascular disease (CVD) events. In contrast, humans with high levels of PCSK9 have higher level of plasma LDL-C and significantly enhanced CVD risk during their lifetime, gain-of-function mutations on PCSK9 are, for instance, causatively associated with familial hypercholesterolaemia (FH). Inhibition of PCSK9 is therefore a promising therapeutic option for the lowering of LDL-C levels. The clinical development of human monoclonal antibodies against PCSK9 has progressed, with promising results reported from phase 2 clinical studies in patients with FH or intolerant to statin with LDL-C levels not on target levels. Phase I studies demonstrated safety and efficacy. In phase II, a 60%-70% reduction in LDL-C was observed, especially when subcutaneous injections were performed regularly every two weeks. No significant side effects were observed, with the exception of injection site reactions. Three large phase III programmes with the new anti PCSK9 antibodies are currently underway in patients with acute coronary syndrome (ACS) and LDL-C inadequately controlled by standard treatments. The main objective of these studies is to evaluate the effect of PCSK9 inhibition on the occurrence of CVD events in patients with ACS.