Proprotein Convertase Subtilisin/kexin 9 Inhibition Research Articles

Causal relationship between PCSK9 inhibitor and primary glomerular disease: a drug target Mendelian randomization study.

Successive observational studies have highlighted low-density lipoprotein cholesterol (LDL-C) as a standalone risk factor for the progression of chronic kidney disease (CKD) to end-stage renal disease. Lowering LDL-C levels significantly reduces the incidence of atherosclerotic events in patients with progressive CKD. Recent research indicates that proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors not only effectively lower LDL-C levels in CKD patients but also exhibit therapeutic potential for autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and ulcerative colitis. However, the role of PCSK9 inhibitors (PCSK9i) in treating CKD beyond lowering LDL-C levels remains uncertain. Therefore, this study employs drug-targeted Mendelian randomization (MR) to investigate the causal impact of PCSK9i on primary glomerular diseases such as IgA nephropathy (IgAN), membranous nephropathy (MN), and nephrotic syndrome (NS). Single-nucleotide polymorphisms (SNPs) linked to LDL-C were sourced from the Global Lipids Genetics Consortium genome-wide association study (GWAS). Genes situated in proximity to 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), and PCSK9 served as proxies for therapeutic inhibition of these targets. The causal link between PCSK9i and the risk of primary glomerular disorders was discovered using drug-target MR studies. The HMGCR inhibitor, a drug target of statins, was utilized for comparative analysis with PCSK9i. Primary outcomes included the risk assessment for IgAN, MN, and NS, using the risk of coronary heart disease as a positive control. The inhibition of PCSK9, as proxied genetically, was found to significantly reduce the risk of IgAN [odds ratio, OR (95% confidence interval, CI) = 0.05 (-1.82 to 1.93), p = 2.10 × 10-3]. Conversely, this inhibition was associated with an increased risk of NS [OR (95% CI) = 1.78 (1.34-2.22), p = 0.01]. Similarly, HMGCR inhibitors (HMGCRi) demonstrated a potential reduction in the risk of IgAN [OR (95%CI) = 0.0032 (-3.58 to 3.59), p = 1.60 × 10-3). PCSK9i markedly decreased the risk of IgAN, suggesting a potential mechanism beyond their primary effect on LDL-C. However, these inhibitors were also associated with an increased risk of NS. On the other hand, HMGCRi appears to serve as a protective factor against IgAN. Conversely, PCSK9i may pose a risk factor for NS, suggesting the necessity for cautious application and further research into their impacts on various glomerular diseases.

PCSK9 activation promotes early atherosclerosis in a vascular microphysiological system.

Atherosclerosis is a primary precursor of cardiovascular disease (CVD), the leading cause of death worldwide. While proprotein convertase subtilisin/kexin 9 (PCSK9) contributes to CVD by degrading low-density lipoprotein receptors (LDLR) and altering lipid metabolism, PCSK9 also influences vascular inflammation, further promoting atherosclerosis. Here, we utilized a vascular microphysiological system to test the effect of PCSK9 activation or repression on the initiation of atherosclerosis and to screen the efficacy of a small molecule PCSK9 inhibitor. We have generated PCSK9 over-expressed (P+) or repressed (P-) human induced pluripotent stem cells (iPSCs) and further differentiated them to smooth muscle cells (viSMCs) or endothelial cells (viECs). Tissue-engineered blood vessels (TEBVs) made from P+ viSMCs and viECs resulted in increased monocyte adhesion compared to the wild type (WT) or P- equivalents when treated with enzyme-modified LDL (eLDL) and TNF-α. We also found significant viEC dysfunction, such as increased secretion of VCAM-1, TNF-α, and IL-6, in P+ viECs treated with eLDL and TNF-α. A small molecule compound, NYX-1492, that was originally designed to block PCSK9 binding with the LDLR was tested in TEBVs to determine its effect on lowering PCSK9-induced inflammation. The compound reduced monocyte adhesion in P+ TEBVs with evidence of lowering secretion of VCAM-1 and TNF-α. These results suggest that PCSK9 inhibition may decrease vascular inflammation in addition to lowering plasma LDL levels, enhancing its anti-atherosclerotic effects, particularly in patients with elevated chronic inflammation.

Identification and validation of PCSK9 as a prognostic and immune-related influencing factor in tumorigenesis: a pan-cancer analysis.

Proprotein convertase subtilisin/kexin-9 (PCSK9) has been primarily studied in the cardiovascular field however, its role in cancer pathophysiology remains incompletely defined. Recently, a pivotal role for PCSK9 in cancer immunotherapy was proposed based on the finding that PCSK9 inhibition was associated with enhancing the antigen presentation efficacy of target programmed cell death-1 (PD-1). Herein, we provide results of a comprehensive pan-cancer analysis of PCSK9 that assessed its prognostic and immunological functions in cancer. Using a variety of available online cancer-related databases including TIMER, cBioPortal, and GEPIA, we identified the abnormal expression of PCSK9 and its potential clinical associations in diverse cancer types including liver, brain and lung. We also validated its role in progression-free survival (PFS) and immune infiltration in neuroblastoma. Overall, the pan-cancer survival analysis revealed an association between dysregulated PCSK9 and poor clinical outcomes in various cancer types. Specifically, PCSK9 was extensively genetically altered across most cancer types and was consistently found in different tumor types and substages when compared with adjacent normal tissues. Thus, aberrant DNA methylation may be responsible for PCSK9 expression in many cancer types. Focusing on liver hepatocellular carcinoma (LIHC), we found that PCSK9 expression correlated with clinicopathological characteristics following stratified prognostic analyses. PCSK9 expression was significantly associated with immune infiltrate since specific markers of CD8+ T cells, macrophage polarization, and exhausted T cells exhibited different PCSK9-related immune infiltration patterns in LIHC and lung squamous cell carcinoma. In addition, PCSK9 was connected with resistance of drugs such as erlotinib and docetaxel. Finally, we validated PCSK9 expression in clinical neuroblastoma samples and concluded that PCSK9 appeared to correlate with a poor PFS and natural killer cell infiltration in neuroblastoma patients. PCSK9 could serve as a robust prognostic pan-cancer biomarker given its correlation with immune infiltrates in different cancer types, thus potentially highlighting a new direction for targeted clinical therapy of cancers.

Assessing the Impact of PCSK9 and HMGCR Inhibition on Liver Function: Drug-Target Mendelian Randomization Analyses in Four Ancestries

Observational studies have linked lipid-lowering drug targets pro-protein convertase subtilisin/kexin 9 (PCSK9) and HMG-CoA reductase (HMGCR) with adverse liver outcomes; however, liver disease incidence varies across diverse populations, and the long-term hepatic impact of these lipid-lowering drugs among non-white Europeans remains largely unknown. We use single nucleotide polymorphisms (SNPs) in PCSK9 and HMGCR loci from genome-wide association study data of low-density lipoprotein cholesterol in 4 populations (East Asian [EAS], South Asian [SAS], African [AFR], and European [EUR]) to perform drug-target Mendelian randomization investigating relationships between PCSK9 and HMGCR inhibition and alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), and bilirubin. Analyses of PCSK9 instruments, including functional variants R46L and E670G, failed to find evidence for relationships of low-density lipoprotein cholesterol lowering via PCSK9 variants and adverse effects on ALT, AST, GGT, or ALP among the cohorts. PCSK9 inhibition was associated with increased direct bilirubin levels in EUR (β= 0.089; P value= 5.69× 10-6) and, nominally, in AFR (β= 0.181; P value= .044). HMGCR inhibition was associated with reduced AST in SAS (β= -0.705; P value= .005) and, nominally, reduced AST in EAS (β= -0.096; P value= .03), reduced ALP in EUR (β= -2.078; P value= .014), and increased direct bilirubin in EUR (β= 0.071; P value= .032). Sensitivity analyses using genetic instruments derived from circulating PCSK9 protein levels, tissue-specific PCSK9 expression, and HMGCR expression were in alignment, strengthening causal inference. We did not find ALT, AST, GGT, or ALP associated with genetically proxied PCSK9 and HMGCR inhibition across ancestries. We identified possible relationships in several ancestries between PCSK9 and increased direct and total bilirubin and between HMGCR and reduced AST. These findings support long-term safety profiles and low hepatotoxic risk of PCSK9 and HMGCR inhibition in diverse populations.

Computational Design, Synthesis, and Biological Evaluation of Diimidazole Analogues Endowed with Dual PCSK9/HMG-CoAR-Inhibiting Activity.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is responsible for the degradation of the hepatic low-density lipoprotein receptor (LDLR), which regulates circulating cholesterol levels. Consequently, the PCSK9 inhibition is a valuable therapeutic approach for the treatment of hypercholesterolemia and cardiovascular diseases. In our studies, we discovered Rim13, a polyimidazole derivative reducing the protein-protein interaction between PCSK9 and LDLR with an IC50 of 1.6 μM. The computational design led to the optimization of the shape of the PCSK9/ligand complementarity, enabling the discovery of potent diimidazole derivatives. In fact, carrying out biological assays to fully characterize the cholesterol-lowering activity of the new analogues and using both biochemical and cellular techniques, compound Dim16 displayed improved PCSK9 inhibitory activity (IC50 0.9 nM). Interestingly, similar to other lupin-derived peptides and their synthetic analogues, some compounds in this series showed dual hypocholesterolemic activity since some of them complementarily inhibited the 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Abstract 9624: Major Adverse Cardiac Events of PCSK9 Modulation by Monoclonal Antibodies vs Small Interfering Ribonucleic Acid Inhibitors. A Meta-Analysis of 92,775 Patients

Introduction: Incliciran, a small interfering ribonucleic acid (siRNA), is the new modulator of the proprotein convertase subtilisin kexin 9 (PCSK9) that has proven safe and effective in reducing LDL-C and ASCVD. However, it is unclear if incliciran is superior to the already established PCSK9 monoclonal antibodies (mAb) in reducing major adverse cardiac events (MACE). Methods: We performed a meta-analysis of randomized controlled trials of patients with high cardiovascular risk who underwent PCSK9 inhibition by either mAb or siRNA. The primary outcome was the reduction of MACE. Results: Thirty RCTs with a total of 92,775 patients were included. For patients with a high cardiovascular risk profile, the modulation of PCSK9 by mAb decreases the risk of MACE (HR 0.84, 95% CI 0.8 - 0.89, p<0.0001) compared with statins and/or ezetimibe. Similar effects occurred when siRNA (HR 0.78, 95% CI 0.63 - 0.97, p=0.024) was compared to statins alone. The impact on MACE was no different between mAb and siRNA modulation of PCSK9 (p=0.49) (figure 1). When PCSK9 inhibitors were compared to statins monotherapy, only evolocumab and incliciran were statistically significant in reducing MACE (p<0.0001 and p=0.024, respectively). An additional analysis of all studies, including those with statins plus ezetimibe, showed that all therapies except bococicimab (p=0.309) reduced MACE (figure 2). Conclusions: This study suggests that PCSK9 inhibition by siRNA is as effective as mAb PCSK9 for reducing MACE in patients with a high cardiovascular risk profile, with the convenience of being administered twice a year.

Evaluation of the effects of immunization against PCSK9 on selected microRNAs involved in cholesterol homeostasis

Abstract Background MicroRNAs (miRNAs) have been discovered as novel modulators of gene expression. These molecules are involved in regulating lipid metabolism through a complicated interactive mechanism involving gene regulatory networks and can play a role in metabolic diseases. PCSK9 (proprotein convertase subtilisin/Kexin 9) is a critical regulator in cholesterol homeostasis. PCSK9 inhibition is an effective therapeutic strategy for the treatment of dyslipidemia. Accordingly, it is essential to identify the controlling mechanisms of the PCSK9 and the effects of its inhibitors on microRNAs involved in cholesterol homeostasis. Purpose Considering the role that miR-27a, miR-30c, and miR-191 play in regulating genes involved in cholesterol homeostasis, we investigated the effect of immunization against PCSK9 on the mentioned miRNAs. Methods A PCSK9 immunogenic peptide constructed by linking a short PCSK9 mimotope peptide to a tetanus peptide was used for immunization. The PCSK9 immunogenic peptide construct was mixed homogeneously with 0.4% alum adjuvant at a ratio of 1:1 for use as vaccine formulation. Mice in the vaccinated group were inoculated subcutaneously in four terms with bi-weekly intervals. In order to assess the expression of miRNAs in vaccinated mice' livers, sampling was carried out two weeks after the last injection. The levels of miR-27a, miR-30c, and miR-191 expression in the liver of vaccinated mice were evaluated using real-time quantitative PCR, and relative expression levels were presented as fold change normalized to U6 as the internal control. Results Hepatic miR-27a expression was significantly decreased in vaccinated mice compared with the control mice (fold change: 0.731±0.1, p=0.027). There was a borderline nonsignificant reduction in the hepatic expression of miR-30c in the vaccine group compared to the control group (fold change: 0.569±0.1, p=0.078). No significant difference was detected in the hepatic expression of miR-191 between the vaccine and control groups (fold change: 0.852±0.1, p=0.343). Conclusions Immunization against PCSK9 leads to decreased hepatic expression of miR-27a. Since miR-27a is involved in the regulation of targets like angiopoietin-like 3 and glycerol-3-phosphate acyltransferase 1, its reduction may explain part of the lipid-lowering activity of PCSK9 vaccine. Funding Acknowledgement Type of funding sources: None.

Sex differences in efficacy and side effects of proprotein convertase subtilisin / kexin 9 (PCSK9) inhibitors in real world data

Background and Aims : Proprotein convertase subtilisin/Kexin 9 (PCSK9) effectively reduce low-density lipoprotein (LDLC) and have a favorable safety profile. Previously FOURIER data showed that LDLC reduction was lower in women compared to men. The aim of this study was to assess efficacy and safety in women and men who use PCSK9 inhibitors in real-world setting.Methods: In this prospective registry of all consecutive patients started with a PCSK9 inhibitor at a lipid clinic of a university hospital. We collected clinical information, including baseline and follow-up mean LDLC levels at 6 months after initiation of PCSK9 inhibitor treatment. Side effects, injection site reactions and drug discontinuation were recorded.Results: We analyzed 335 patients (154 women), mean age of 58±11 years. Women were older (60 vs 57 yrs P=0.03) and had less often cardiovascular events (58% vs 71% P<0.01). Women had higher baseline LDLC levels compared to men (4.8 vs 4.1 mmol/L, P<0.001) and higher on-treatment LDLC levels after 6 months (2.2 vs 1.6 mmol/L, P<0.001). LDLC reduction was 58%±19%. Women had lower LDLC reduction compared to men (53% vs 61% P<0.001). Women more often reported side effects than men (55% vs 45% P=0.03, while injection site reactions ( 10% vs 6%, P=NS) and drug discontinuation (6%vs 8%, P=NS) were similar.Conclusions: In line with the FOURIER study, we found that women had lower LDL-C reduction compared to men after initiation of PCSK9 inhibition. Moreover women reported more side effects. Future research is needed to assess reasons for these sex differences. Background and Aims : Proprotein convertase subtilisin/Kexin 9 (PCSK9) effectively reduce low-density lipoprotein (LDLC) and have a favorable safety profile. Previously FOURIER data showed that LDLC reduction was lower in women compared to men. The aim of this study was to assess efficacy and safety in women and men who use PCSK9 inhibitors in real-world setting. Methods: In this prospective registry of all consecutive patients started with a PCSK9 inhibitor at a lipid clinic of a university hospital. We collected clinical information, including baseline and follow-up mean LDLC levels at 6 months after initiation of PCSK9 inhibitor treatment. Side effects, injection site reactions and drug discontinuation were recorded. Results: We analyzed 335 patients (154 women), mean age of 58±11 years. Women were older (60 vs 57 yrs P=0.03) and had less often cardiovascular events (58% vs 71% P<0.01). Women had higher baseline LDLC levels compared to men (4.8 vs 4.1 mmol/L, P<0.001) and higher on-treatment LDLC levels after 6 months (2.2 vs 1.6 mmol/L, P<0.001). LDLC reduction was 58%±19%. Women had lower LDLC reduction compared to men (53% vs 61% P<0.001). Women more often reported side effects than men (55% vs 45% P=0.03, while injection site reactions ( 10% vs 6%, P=NS) and drug discontinuation (6%vs 8%, P=NS) were similar. Conclusions: In line with the FOURIER study, we found that women had lower LDL-C reduction compared to men after initiation of PCSK9 inhibition. Moreover women reported more side effects. Future research is needed to assess reasons for these sex differences.

Effects of PCSK-9 Inhibition by Alirocumab Treatments on Biliary Cirrhotic Rats.

Hyperlipidemia and oxidative stress with elevated oxidized low-density lipoprotein (ox-LDL) exacerbate hepatic inflammation and fibrosis. The plasma level of low-density lipoprotein (LDL) is controlled by proprotein convertase subtilisin/kexin 9 (PCSK9). Alirocumab is a monoclonal antibody that decreases LDL via inhibiting PCSK9 function. Apart from lipid-lowering effects, alirocumab exerts anti-inflammation, anti-angiogenesis and anti-oxidant effects. This study aims to investigate the impact of alirocumab treatment on common bile duct ligation (BDL)-induced biliary cirrhotic rats. After a 4-week treatment of alirocumab, the hemodynamic data, blood biochemistry, ox-LDL level, oxidative stress markers, severity of hepatic encephalopathy and abnormal angiogenesis of BDL rats were measured and compared to the control group. BDL rats presented cirrhotic pictures and elevated ammonia, total cholesterol, LDL and ox-LDL levels compared to the control group. Alirocumab decreased plasma levels of total cholesterol, LDL, and oxidative stress markers; however, it did not affect the hemodynamics, liver and renal biochemistry, and the plasma levels of ammonia and ox-LDL. The motor activities, portal-systemic collaterals and mesenteric vascular density were not significantly different between alirocumab-treated and control groups. In addition, it did not affect hepatic inflammation, intrahepatic angiogenesis, liver fibrosis and free cholesterol accumulation in the liver of BDL rats. In conclusion, PCSK9 inhibition by alirocumab treatment ameliorates hyperlipidemia and systemic oxidative stress in biliary cirrhotic rats. However, it does not affect the plasma level of ox-LDL, intrahepatic inflammation and fibrosis. In addition, PCSK9 inhibition has a neutral effect on abnormal angiogenesis and hepatic encephalopathy in biliary cirrhotic rats.

Abstract 350: Disruption Of Proprotein Convertase Subtilisin/Kexin 9 Suppresses Inflammation And Attenuates Abdominal Aortic Aneurysm Formation

Objective: Abdominal aortic aneurysm (AAA) is a degenerative inflammatory disease, with its rupture highly lethal. There have been no effective pharmacological medications. Proprotein convertase subtilisin/kexin 9 (PCSK9), which is known to modulate cholesterol metabolism, has recently been associated with AAA in human genome-wide association studies. However, its role in AAA pathogenesis is unknown. Approach and Results: Histology staining of mouse aortic aneurysmal tissue showed PCSK9 expression and its colocalization with macrophages. To investigate the role of PCSK9 in AAA formation, mice deficient in PCSK9 were crossed with ApoE SA/SA mice, which are hypercholesterolemic and inducible to develop angiotensin II-mediated hypertension upon doxycycline (Dox) administration. Deletion of PCSK9 reduced both incidence and severity of AAA and increased survival rate, without changing plasma cholesterol levels. This was associated with decreased aortic expression and activity of matrix metalloproteinase 9 (MMP9), less elastin breakdown, and reduced aortic expansion and aneurysm rupture. Additionally, PCSK9 deficiency suppressed systemic inflammatory response and macrophage infiltration into AAA lesions. In isolated macrophages stimulated with LPS, the upregulated expression of pro-inflammatory cytokines ( Tnfa, Il1b, Il6 ) was dramatically diminished in PCSK9-deficient macrophages. Furthermore, administration of PCSK9-specific siRNA in mice resulted in less macrophage accumulation in the aneurysmal aortic wall, decreased MMP9-mediated elastin degradation, and substantially constrained AAA formation. Conclusions: Endogenous PCSK9 promotes AAA formation, consistent with its novel action in mediating systemic and local inflammation, particularly, the macrophage-dependent MMP9 activity. Pharmacological inhibition of PCSK9 might be a promising strategy to curtail AAA. Key Words: aneurysm, PCSK9, elastin, inflammation, macrophages

Role of PCSK9 in Homocysteine-Accelerated Lipid Accumulation in Macrophages and Atherosclerosis in ApoE-/- Mice.

Background: Homocysteine (Hcy) has been established as an independent risk factor for atherosclerosis, and the involvement of hyperhomocysteinemia (HHcy) in atherosclerotic lesions is complex. Proprotein convertase subtilisin kexin 9 (PCSK9) has vital importance in lipid metabolism, and its inhibitors have intense lipid-lowering and anti-atherosclerotic effects. However, the underlying effect of PCSK9 on HHcy-accelerated dyslipidemia of macrophages is still uncertain. The purpose of this study was to investigate the potential role of PCSK9 in Hcy-induced lipid accumulation and atherosclerotic lesions.Methods: In vitro, gene and protein expressions were assessed by real-time quantitative PCR and western blot in THP-1 macrophages with Hcy incubation. Lipid accumulation and cholesterol efflux were evaluated with Hcy treatment. SBC-115076 was used to examine the role of PCSK9 in ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1)-dependent cholesterol efflux. In vivo, lesion area, lipid deposition and collagen contents were determined in aortas of ApoE−/− mice under a methionine diet. SBC-115076 was subcutaneously injected to explore the potential effects of PCSK9 inhibition on alleviating the severity of HHcy-related atherosclerotic lesions.Results: In THP-1 macrophages, Hcy dose- and time-dependently promoted PCSK9 gene and protein levels without regulating the translation of Low-density lipoprotein receptor (LDLR). SBC-115076 used to inhibit PCSK9 largely alleviated lipid accumulation and reversed the cholesterol efflux to apolipoprotein-I(apoA-I) and high-density lipoprotein (HDL) mediated by ABCA1 and ABCG1. In ApoE−/− mice, methionine diet induced HHcy caused larger lesion area and more lipid accumulation in aortic roots. SBC-115076 reduced atherosclerotic severity by reducing the lesion area and lipid accumulation and increasing expressions of ABCA1 and ABCG1 in macrophages from atherosclerotic plaque. In addition, SBC-115076 decreased plasma Hcy level and lipid profiles significantly.Conclusion: PCSK9 promoted lipid accumulation via inhibiting cholesterol efflux mediated by ABCA1 and ABCG1 from macrophages and accelerated atherosclerotic lesions under HHcy treatment. Inhibiting PCSK9 may have anti-atherogenic properties in HHcy-accelerated atherosclerosis.

Impact of PCSK9 on CTRP9-Induced Metabolic Effects in Adult Rat Cardiomyocytes.

The adipocytokine adiponectin and its structural homologs, the C1q/TNF-related proteins (CTRPs), increase insulin sensitivity, fatty acid oxidation and mitochondrial biogenesis. Adiponectin- and CTRP-induced signal transduction has been described to involve the adiponectin receptors and a number of co-receptors including the Low density lipoprotein receptor-related protein 1 (LRP1). LRP1 is another target of the proprotein convertase subtilisin/kexin-9 (PCSK9) in addition to the LDL-receptor (LDL-R). Here, we investigated the influence of PCSK9 on the metabolic effects of CTRP9, the CTRP with the highest homology to adiponectin. Knockdown of LRP1 in H9C2 cardiomyoblasts blunts the effects of CTRP9 on signal transduction and mitochondrial biogenesis, suggesting its involvement in CTRP9-induced cellular effects. Treatment of adult rat cardiomyocytes with recombinant PCSK9 but not knockdown of endogenous PCSK9 by siRNA results in a strong reduction in LRP1 protein expression and subsequently reduces the mitochondrial biogenic effect of CTRP9. PCSK9 treatment (24 h) blunts the effects of CTRP9-induced signaling cascade activation (AMP-dependent protein kinase, protein kinase B). In addition, the stimulating effects of CTRP9 on cardiomyocyte mitochondrial biogenesis and glucose metabolism (GLUT-4 translocation, glucose uptake) are largely blunted. Basal fatty acid (FA) uptake is strongly reduced by exogenous PCSK9, although protein expression of the PCSK9 target CD36, the key regulator of FA transport in cardiomyocytes, is not altered. In addition, only minor effects of PCSK9 were observed on CTRP9-induced FA uptake or the expression of genes involved in FA metabolism or uptake. Finally, this CTRP9-induced increase in CD36 expression occurs independent from LRP1 and LDL-R. In conclusion, PCSK9 treatment influences LRP1-mediated signaling pathways in cardiomyocytes. Thus, therapeutic PCSK9 inhibition may provide an additional benefit through stimulation of glucose metabolism and mitochondrial biogenesis in addition to the known lipid-lowering effects. This could be an important beneficial side effect in situations with impaired mitochondrial function and reduced metabolic flexibility thereby influencing cardiac function.

New Pharmacological Approaches to Target PCSK9.

Proprotein convertase subtilisin kexin 9 (PCSK9) plays a crucial role in regulating circulating levels of LDL-C as a consequence of its ability to inhibit LDL receptor recycling in the liver. Loss of function variants in the PCSK9 gene result in low LDL-C levels and associate with reduced cardiovascular risk, whereas gain of-function variants associate with hypercholesterolemia and increased risk of early cardiovascular events. Thus, PCSK9 inhibition has been established as an additional approach for the treatment of hypercholesterolemia. The aim of this review is to provide a brief overview of current strategies targeting PCSK9 and discuss clinical results of the emerging approaches. Two monoclonal antibodies targeting circulating PCSK9 (evolocumab and alirocumab) have been approved for the treatment of hypercholesterolemia and cardiovascular disease. Later, a gene silencing approach (inclisiran), which inhibits hepatic PCSK9 synthesis, was shown to be as effective as monoclonal antibodies but with a twice a year injection and is currently under evaluation for approval. Due to the elevated costs of such therapies, several other approaches have been explored, including peptide-based anti PCSK9 vaccination, and small oral PCSK9 inhibitors, which are still in preclinical phase. In the coming years, we will assist to a progressive introduction of novel anti-PCSK9 approaches in the clinical practice for the treatment of patients with hypercholesterolemia as well as patients at high cardiovascular risk.

Lipid Lowering Treatment and Eligibility for PCSK9 Inhibition in Post-Myocardial Infarction Patients in Italy: Insights from Two Contemporary Nationwide Registries.

Introduction The current use of lipid lowering therapies and the eligibility for proprotein convertase subtilisin/kexin-9 (PCSK9) inhibitors of patients surviving a myocardial infarction (MI) is poorly known. Methods Using the data from two contemporary, nationwide, prospective, real-world registries of patients with stable coronary artery disease, we sought to describe the lipid lowering therapies prescribed by cardiologists in patients with a prior MI and the resulting eligibility for PCSK9 inhibitors according to the European Society of Cardiology (ESC)/European Atherosclerosis Society (EAS) and the Italian regulatory agency (Agenzia Italiana del Farmaco; AIFA) criteria. The study cohort was stratified according to the following low-density lipoprotein cholesterol (LDL-C) levels at the time of enrolment: <70 mg/dl; 70–99 mg/dl and ≥100 mg/dl. Results Among the 3074 post-MI patients with LDL-C levels available, a target level of LDL-C < 70 mg/dl was present in 1186 (38.6%), while 1150 (37.4%) had LDL-C levels ranging from 70 to 99 mg/dl and the remaining 738 (24.0%) an LDL-C ≥ 100 mg/dl. A statin was prescribed more frequently in post-MI patients with LDL-C levels <70 mg/dl (97.1%) compared to the other LDL-C groups (p < 0.0001). A low dose of statin was prescribed in 9.3%, while a high dose in 61.4% of patients. Statin plus ezetimibe association therapy was used in less than 18% of cases. In the overall cohort, 293 (9.8%) and 450 (22.2%) resulted eligible for PCSK9 inhibitors, according to ESC/EAS and AIFA criteria, respectively. Conclusions Post-MI patients are undertreated with conventional lipid lowering therapies. A minority of post-MI patients would be eligible to PCSK9 inhibitors according to ESC/EAS guidelines and Italian regulatory agency criteria.

Effects of immunisation against PCSK9 in mice bearing melanoma

IntroductionInhibition of proprotein convertase subtilisin/kexin 9 (PCSK9) is an established modality for the treatment of hypercholesterolaemia. However, the impact of PCSK9 inhibition in other situations such as cancer remains largely unknown. The current study was conducted to study the effects of PCSK9 inhibition on cancer endpoints in mice bearing melanoma.Material and methodsTo generate antiPCSK9 antibody in vivo, a nanoliposomal antiPCSK9 vaccine adsorbed to 0.4% Alum adjuvant was subcutaneously injected in C57BL/6 mice four times with bi-weekly intervals. Two weeks after the last immunisation, mice were subcutaneously inoculated with B16F0 melanoma cells. After a tumour mass was palpable (approximately 10 mm3), the mice were randomly divided into four groups and subjected to different treatment protocols: (1) PBS (untreated control), (2) vaccine group, (3) the combination of vaccine and a single dose of liposomal doxorubicin (Doxil®), and (4) liposomal doxorubicin (positive control) group. To determine therapeutic efficacy, mouse body weight, tumour size, and survival were monitored every three days for 36 days.ResultsThe nanoliposomal antiPCSK9 vaccine was found to efficiently induce specific antibodies against PCSK9 in C57BL/6 mice, thereby reducing plasma levels and function of PCSK9. Tumour volumes in the vaccinated group were not significantly different from those in the liposomal doxorubicin, combination, and control groups. The time to reach endpoint (TTE) values of the vaccine (28 ±5 days), combination (30 ±6 days), liposomal doxorubicin (34 ±2 days), and control (31 ±2 days) groups were not significantly different, either. Furthermore, the tumour growth delay (TGD) values of the vaccine (–11.5 ±15.4%), liposomal doxorubicin (7.75 ±6.5%), combination (–6 ±20.77%), and control (0 ±7.5) groups were not significantly different. Finally, there was no significant difference between the median survival time and lifespan of the vaccinated versus other tested groups.ConclusionsThe nanoliposomal PCSK9 vaccine did not adversely affect the growth of melanoma tumour nor the survival of tumour-bearing mice.

4120Anti-tumor activity of a nanoliposomal anti-PCSK9 vaccine in BALB/c mice bearing 4T1 breast cancer

Abstract Background Although suppression of proprotein convertase subtilisin/kexin 9 (PCSK9) has been known to be an effective lipid-lowering approach, the efficacy and safety of PCSK9 inhibitors in non-cardiovascular diseases, particularly cancer, are yet unknown. Purpose The present study aimed to evaluate effect of PCSK9 inhibition on cancer behavior and endpoints in mice model of breast tumor, using a nanoliposomal anti-PCSK9 vaccine. Methods The nanoliposomal anti-PCSK9 vaccine was formulated via conjugation of immunogenic PCSK9 peptide to the surface of nanoliposome particles by using DSPE-PEG-Maleimide lipid. The prepared vaccine was adsorbed to Alum adjuvant (L-IFPTA+) and immunized subcutaneously four times with a bi-weekly interval in BALB/c mice.Two weeks after the last immunization, the vaccinated and unvaccinated mice were subcutaneously inoculated with 4T1 breast carcinoma cells into the right flank. After tumor mass was palpable, the mice were randomly divided into four groups and subjected to different treatment protocols: (1) PBS (untreated control), (2) vaccine group, (3) the combination group which involved vaccinated tumor-bearing mice who received single tail vein injection of Doxil®, and (4) Doxil® (positive control) group which involved unvaccinated tumor-bearing mice who received Doxil®. To evaluate therapeutic efficacy, mouse body weight, tumor size, and survival were monitored every other day for 60 days. Results The vaccine was found to efficiently generate specific antibodies against PCSK9 in BALB/c mice, and thereby reduce concentration of plasma PCSK9 and impede its function. Evaluation of tumor size demonstrated that time to reach endpoint (TTE) of the vaccine, combination, Doxil, and control groups was 47±10, 57±4, 60±4, 39±9 days, respectively. Rate of tumor growth in vaccine, combination, and Doxil groups was decreased by 21, 48, 53%, respectively, when compared with the control group (Figure). In the vaccine group life span was increased by 4.2%, while the survival in the combination and Doxil group was significantly lower than the control group. Figure 1. Anti-PCSK9 vaccine efficacy. Conclusions Our results reveal that PCSK9 inhibition not only exerts no harmful effects, but also can moderately improve breast cancer behavior in animal model.

Computationally Driven Structure Optimization, Synthesis, and Biological Evaluation of Imidazole-Based Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Inhibitors.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is responsible for the degradation of the hepatic low-density lipoprotein receptor (LDLR), which in turn regulates the circulating low-density lipoprotein cholesterol (LDL-C) level. For this reason, the PCSK9 inhibition, by small molecules or peptides, is a validated therapeutic approach for fighting hypercholesterolemia and cardiovascular diseases. In this field, we have recently reported an imidazole-based peptidomimetic that has shown PCSK9 inhibitory activity in the micromolar range. Here, by applying advanced computational techniques, the binding mechanism of that imidazole peptidomimetic was predicted. Then, among a small set of poly-imidazole analogs, compounds showing the highest theoretical affinity were suitably synthesized, relying on a van Leusen reaction based multicomponent strategy. One compound (named RIm13) displayed a PCSK9 inhibitory activity 10-fold lower than the template compound, and, remarkably, at a concentration of 1 μM, it successfully prevented the LDLR degradation mediated by PCSK9 on HepG2 cells. As well as increasing the LDL uptake at the same concentration, RIm13 represents currently one of the most potent small molecules targeting the PCSK9/LDLR protein-protein interaction.

Efficacy and safety of proprotein convertase subtilisin/kexin 9 inhibitors in people with diabetes and dyslipidaemia.

Diabetic dyslipidaemia, characterized by quantitative, qualitative and kinetic changes in all major circulating lipids, contributes to the increased cardiovascular risk in patients with type 2 diabetes mellitus (T2DM). A promising therapeutic avenue is the inhibition of the proprotein convertase subtilisin kexin 9 (PCSK9) with human monoclonal antibodies (mAbs) that potently reduce plasma low-density lipoprotein cholesterol (LDL-C) levels on top of statin treatment. The aim of this review is to evaluate the efficacy of PCSK9 inhibitors to lower the residual cardiovascular risk of T2DM patients and to discuss the safety of PCSK9 inhibition in these patients. PCSK9 inhibitors potently lower plasma LDL-C levels in T2DM patients and reduce risk for the development of cardiovascular disease. Anti-PCSK9 mAbs are generally not more or less effective in T2DM patients compared to a general high-risk population. Nevertheless, due to their higher cardiovascular risk, the absolute risk reduction of major cardiovascular events is more significant in T2DM patients. This suggests that treatment of T2DM patients with anti-PCSK9 mAbs could be attractive from a cost-effectiveness perspective. Treatment with anti-PCSK9 mAbs did not result in significant treatment-emergent adverse effects. While genetic studies suggest a potential link between PCSK9 inhibition and glucose homeostasis, anti-PCSK9 mAbs did not worsen glycaemic control in T2DM patients, but their safety should be verified after a longer-term follow-up.

Lipoprotein(a), PCSK9 Inhibition, and Cardiovascular Risk.

Lipoprotein(a) [Lp(a)] may play a causal role in atherosclerosis. PCSK9 (proprotein convertase subtilisin/kexin 9) inhibitors have been shown to significantly reduce plasma Lp(a) concentration. However, the relationship between Lp(a) levels, PCSK9 inhibition, and cardiovascular risk reduction remains undefined. Lp(a) was measured in 25 096 patients in the FOURIER trial (Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk), a randomized trial of evolocumab versus placebo in patients with established atherosclerotic cardiovascular disease (median follow-up, 2.2 years). Cox models were used to assess the independent prognostic value of Lp(a) and the efficacy of evolocumab for coronary risk reduction by baseline Lp(a) concentration. The median (interquartile range) baseline Lp(a) concentration was 37 (13-165) nmol/L. In the placebo arm, patients with baseline Lp(a) in the highest quartile had a higher risk of coronary heart disease death, myocardial infarction, or urgent revascularization (adjusted hazard ratio quartile 4: quartile 1, 1.22; 95% CI, 1.01-1.48) independent of low-density lipoprotein cholesterol. At 48 weeks, evolocumab significantly reduced Lp(a) by a median (interquartile range) of 26.9% (6.2%-46.7%). The percent change in Lp(a) and low-density lipoprotein cholesterol at 48 weeks in patients taking evolocumab was moderately positively correlated ( r=0.37; 95% CI, 0.36-0.39; P<0.001). Evolocumab reduced the risk of coronary heart disease death, myocardial infarction, or urgent revascularization by 23% (hazard ratio, 0.77; 95% CI, 0.67-0.88) in patients with a baseline Lp(a) >median, and by 7% (hazard ratio, 0.93; 95% CI, 0.80-1.08; P interaction=0.07) in those ≤median. Coupled with the higher baseline risk, the absolute risk reductions, and number needed to treat over 3 years were 2.49% and 40 versus 0.95% and 105, respectively. Higher levels of Lp(a) are associated with an increased risk of cardiovascular events in patients with established cardiovascular disease irrespective of low-density lipoprotein cholesterol. Evolocumab significantly reduced Lp(a) levels, and patients with higher baseline Lp(a) levels experienced greater absolute reductions in Lp(a) and tended to derive greater coronary benefit from PCSK9 inhibition. URL: https://www.clinicaltrials.gov . Unique identifier: NCT01764633.

Non-antibody Approaches to Proprotein Convertase Subtilisin Kexin 9 Inhibition: siRNA, Antisense Oligonucleotides, Adnectins, Vaccination, and New Attempts at Small-Molecule Inhibitors Based on New Discoveries.

Low-density lipoprotein (LDL) is one of the principal risk factors for atherosclerosis. Circulating LDL particles can penetrate into the sub-endothelial space of arterial walls. These particles undergo oxidation and promote an inflammatory response, resulting in injury to the vascular endothelial wall. Persistent elevation of LDL-cholesterol (LDL-C) is linked to the progression of fatty streaks to lipid-rich plaque and thus atherosclerosis. LDL-C is a causal factor for atherosclerotic cardiovascular disease and lowering it is beneficial across a range of conditions associated with high risk of cardiovascular events. Therefore, all guidelines-recommended initiations of statin therapy for patients at high cardiovascular risk is irrespective of LDL-C. In addition, intensive LDL-C lowering therapy with statins has been demonstrated to result in a greater reduction of cardiovascular event risk in large clinical trials. However, many high-risk patients receiving statins fail to achieve the guideline-recommended reduction in LDL-C levels in routine clinical practice. Moreover, low levels of adherence and often high rates of discontinuation demand the need for further therapies. Ezetimibe has typically been used as a complement to statins when further LDL-C reduction is required. More recently, proprotein convertase subtilisin kexin 9 (PCSK9) has emerged as a novel therapeutic target for lowering LDL-C levels, with PCSK9 inhibitors offering greater reductions than feasible through the addition of ezetimibe. PCSK9 monoclonal antibodies have been shown to not only considerably lower LDL-C levels but also cardiovascular events. However, PCSK9 monoclonal antibodies require once- or twice-monthly subcutaneous injections. Further, their manufacturing process is expensive, increasing the cost of therapy. Therefore, several non-antibody treatments to inhibit PCSK9 function are being developed as alternative approaches to monoclonal antibodies. These include gene-silencing or editing technologies, such as antisense oligonucleotides, small interfering RNA, and the clustered regularly interspaced short palindromic repeats/Cas9 platform; small-molecule inhibitors; mimetic peptides; adnectins; and vaccination. In this review, we summarize the current knowledge base on the role of PCSK9 in lipid metabolism and an overview of non-antibody approaches for PCSK9 inhibition and their limitations. The subsequent development of alternative approaches to PCSK9 inhibition may give us more affordable and convenient therapeutic options for the management of high-risk patients.