Plasma Proprotein Convertase Subtilisin/kexin Type 9 Research Articles

How to verify the analytical and clinical performance of ELISA immunoanalysis in the real laboratory practice. PCSK9 as an example

BackgroundManufacturers and diagnostic companies often recommend on-site verification of analytical performance in the clinical laboratory. The validation process used by manufacturers is rarely described in detail, and certain information on analytical performance is missing from the product sheet, especially for immunoanalytical methods. We describe an approach to the detailed validation of an ELISA method for the measurement of proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma concentrations. We compared manufacturers' claims of analytical performance with data obtained in the field laboratory using several approaches. MethodsWe used the Human Proprotein Convertase 9/PCSK9 Quantikine ELISA diagnostic kit (R&D systems, Bio-Techne Ltd., Abingdon Science Park, Abingdon, UK) and three levels of quality control solution Quantikine Immunoassay Control Group 235 (R&D systems, Bio-Techne Ltd., Abingdon Science Park, Abingdon, UK) to verify precision. We measured the concentration of PCSK9 using the DS2 ELISA Reader (Dynex Technologies GmbH, Denkendorf, Germany). We used analysis of variance (ANOVA) and the R statistical package (R core team, version 1.4.5). Statistical analysis and terminology were performed according to protocol CLSI EP15-A3, and the reference interval was checked according to CLSI/IFCC C28-A3c. ResultsWe found a significant difference between the manufacturer's claims of analytical performance and real data measured in the routine clinical laboratory. The calculated CV (%) for repeatability (calculated by simple estimation of the mean and SD, as used by the manufacturer) was between 5.5% and 7.4%, but the manufacturer's claim was between 4.1% and 6.5%. Using ANOVA, the true repeatability was between 5.0% and 6.9%. Similarly, ANOVA revealed values of CV (%) for within-laboratory imprecision between 6.5% and 9.1%, while the manufacturer's claims were between 4.1% and 5.9%. The recovery ranged from 105.5% to 121.8%. The manufacturer's recommended reference interval was checked and we didn't find any significant difference between men and women. ConclusionsWe describe a comprehensive approach to verify the analytical performance of an ELISA method using the measurement of PCSK9 plasma concentration as an example. We found differences between the results of this approach based on the CLSI EP15-A3 protocol and data provided by the manufacturer. We recommend the verification of analytical performance by more complex statistical tools in laboratory practice.

Prognostic Value of PCSK9 Levels in Premenopausal Women at Risk of Breast Cancer-Evidence from a 17-Year Follow-Up Study.

The involvement of cholesterol in cancer development remains a topic of debate, and its association with breast cancer has yet to be consistently demonstrated. Considering that circulating cholesterol levels depend on several concomitant processes, we tested the liability of plasma levels of proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the key regulators of cholesterol levels, as a prognostic biomarker in the context of breast neoplastic events. Within a prospective randomized breast cancer prevention trial we measured baseline plasma levels of PCSK9. A total of 235 at-risk premenopausal women were randomized and followed up for 17 years. Participants enrolled in this placebo-controlled, phase II, double-blind trial were randomly assigned to receive either tamoxifen 5 mg/d or fenretinide 200 mg/d, both agents, or placebo for 2 years. The associations with breast cancer events were evaluated through competing risk and Cox regression survival models, adjusted for randomization strata (5-year Gail risk ≥ 1.3% vs. intraepithelial neoplasia or small invasive breast cancer of favorable prognosis), age, and treatment allocation. PCSK9 associations with biomarkers linked to breast cancer risk were assessed on blood samples collected at baseline. The plasmatic PCSK9 median and interquartile range were 207 ng/mL and 170-252 ng/mL, respectively. Over a median follow-up period of 17 years and 89 breast neoplastic events, disease-free survival curves showed a hazard ratio of 1.002 (95% CI: 0.999-1.005, p = 0.22) for women with PCSK9 plasma levels ≥ 207 ng/mL compared to women with levels below 207 ng/mL. No differences between randomization strata were observed. We found a negative correlation between PCSK9 and estradiol (r = -0.305), maintained even after partial adjustment for BMI and age (r = -0.287). Cholesterol (r = 0.266), LDL-C (r = 0.207), non-HDL-C (r = 0.246), remnant cholesterol (r = 0.233), and triglycerides (r = 0.233) also correlated with PCSK9. In premenopausal women at risk of early-stage breast cancer, PCSK9 did not appear to have a role as a prognostic biomarker of breast neoplastic events. Larger studies are warranted investigating patients in different settings.

Maternal Obesity Modulates Cord Blood Concentrations of Proprotein Convertase Subtilisin/Kexin-type 9 Levels.

In utero exposure to maternal obesity or diabetes is considered a pro-inflammatory state. To evaluate whether cord blood proprotein convertase subtilisin/kexin-type 9 (PCSK9), which is regulated by inflammation and metabolic derangements, is elevated in neonates born to overweight, obese, or diabetic mothers. A retrospective study in full-term neonates born between 2010 and 2023, at Brigham and Women's Hospital. There were 116 neonates included in our study, of which 74 (64%) were born to overweight/obese mothers and 42 (36%) were born to nonoverweight/nonobese mothers. Neonates born to overweight/obese mothers had significantly higher cord blood concentrations of PCSK9 compared with neonates born to nonoverweight/nonobese group (323 [253-442] ng/mL compared with 270 [244-382] ng/mL, P = .041). We found no significant difference in cord blood concentrations of PCSK9 between neonates of diabetic mothers compared with neonates of nondiabetic mothers. In multivariate linear regression analysis, higher cord plasma PCSK9 concentration was significantly associated with maternal overweight/obesity status (b = 50.12; 95% CI, 4.02-96.22; P = .033), after adjusting for gestational age, birth weight, male sex, and intrauterine growth restriction. Neonates born to mothers with overweight/obesity have higher cord blood PCSK9 concentrations compared with the nonoverweight/nonobese group, and higher cord blood PCSK9 concentrations were significantly associated with maternal overweight/obesity status, after adjusting for perinatal factors. Larger longitudinal studies are needed to examine the role of PCSK9 in the development of metabolic syndrome in high-risk neonates born to overweight, obese, or diabetic mothers.

Geranylgeranyl isoprenoids and hepatic Rap1a regulate basal and statin-induced expression of PCSK9

LDL-C lowering is the main goal of atherosclerotic cardiovascular disease prevention, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is now a validated therapeutic strategy that lowers serum LDL-C and reduces coronary events. Ironically, the most widely used medicine to lower cholesterol, statins, has been shown to increase circulating PCSK9 levels, which limits their efficacy. Here, we show that geranylgeranyl isoprenoids and hepatic Rap1a regulate both basal and statin-induced expression of PCSK9 and contribute to LDL-C homeostasis. Rap1a prenylation and activity is inhibited upon statin treatment, and statin-mediated PCSK9 induction is dependent on geranylgeranyl synthesis and hepatic Rap1a. Accordingly, treatment of mice with a small-molecule activator of Rap1a lowered PCSK9 protein and plasma cholesterol and inhibited statin-mediated PCSK9 induction in hepatocytes. The mechanism involves inhibition of the downstream RhoA-ROCK pathway and regulation of PCSK9 at the post-transcriptional level. These data further identify Rap1a as a novel regulator of PCSK9 protein and show that blocking Rap1a prenylation through lowering geranylgeranyl levels contributes to statin-mediated induction of PCSK9.

Association of PCSK-9 with the Biomarkers of Type-2 Diabetes and its Complications in the Indian Population: a Pilot Study.

Proprotein convertase subtilisin/kexin type-9 (PCSK-9) is a serine protease with profound effects on plasma LDL-C, the major risk factor for cardiovascular diseases (CVDs). However, plasma PCSK-9 level and its association with the biomarkers of CVDs, diabetes, and associated complications have not yet been reported in the northeastern population of India. Of the total cohort (n = 233), we analyzed healthy controls (HC; n = 50), freshly diagnosed type-2-diabetes mellitus (T2DM-FD; n = 46), T2DM treated (T2DM-T; n = 49), diabetic nephropathy (T2DM-N; n = 43), and diabetic dyslipidemia (T2DM-DL; n = 45) subjects. Plasma PCSK-9 and other biological determinants associated with T2DM, CVD, and nephrotic dysfunction were assessed. The level of plasma PCSK-9 in HC, T2DM-FD, T2DM-T, T2DM-N, and T2DM-DL groups was found to be 184.1 ± 13.83, 183.1 ± 24.4.3, 241.8 ± 75.42, 403.7 ± 85.94, and 641.3 ± 135.5 ng/mL, respectively, indicating its role in the severity of the here-mentioned complications. Moreover, plasma PCSK-9 levels further showed a significant correlation with the biomarkers of hyperglycemia, particularly HbA1c, as well as LDL-C in T2DM-FD, T2DM-N, and T2DM-DL subjects of the Indian population, while moderate association in T2DM-T subjects. Our first-of-its-kind clinical study aiming to quantify the circulatory PCSK-9 level in the Indian population concluded that elevated PCSK-9 was significantly associated with the severity of diabetes and associated complications. Moreover, such elevation in PCSK-9 might be attributed to the lipid- and glucoselowering medication-induced SREBP-2-dependent mechanisms. Since our conclusion is based on a pilot study, further cohort studies in larger populations of India are required to get a generalization regarding the role of PCSK-9 in DM and associated complications.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) as a marker of coronary lesion severity in stable coronary artery disease (CAD) patients.

Coronary artery disease (CAD) remains a significant global health concern with considerable high morbidity and mortality and its development is influenced by various genetic and environmental factors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a vital regulator of low-density lipoprotein receptor (LDLR) metabolism, directly impacting serum cholesterol levels. However, its role in development of CAD is not fully understood. The aim of this study was to assess the association between the level of PCSK9 and coronary lesion severity in patients with CAD. A case-control study using consecutive sampling was conducted among CAD patients at H. Adam Malik General Hospital and Murni Teguh Memorial Hospital, Medan, Indonesia. A total of 200 CAD patients were divided into two groups based on the SYNTAX score: control (score ≤22, n=100) and case (score >22, n=100). Plasma PCSK9 levels were measured from venous blood using quantitative sandwich enzyme immunoassay. The Chi-squared test was used to analyze the data. Our data suggested that PCSK9 level was associated with coronary lesion severity (p<0.001) of which high PCSK9 level was associated with severe coronary lesion. We also found that hypertension (p<0.001), smoking (p=0.072), diabetes (p<0.001), dyslipidemia (p<0.001), obesity (p=0.023), and family history (p=0.001) were associated with lesion severity. Using the receiver operating characteristic (ROC) curve analysis, the cut-off 70.35 ng/mL of PCSK9 had sensitivity 75% and specificity 78% to predict severe coronary lesion. This study highlights that PCSK9 level has moderate sensitivity and specificity to predict the coronary lesion severity among CAD patients.

The effect of proprotein convertase subtilisin/kexin type 9 inhibition on the plasma proteome: a SPIRE sub-study

Abstract Background The plasma proteome is an intermediate phenotype for disease and is a tool to inform on pharmacological mechanisms of effect. For selective low-density lipoprotein cholesterol (LDL-C) lowering by proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition, multiple studies have demonstrated that its potent LDL-C lowering effect coincides with marked reductions in both cellular and arterial plaque inflammation, despite unaltered high-sensitivity C-reactive protein (hs-CRP) levels. Purpose In the present study, we set out to investigate potential anti-inflammatory effects of PCSK9 inhibition using plasma proteomics. Methods In 190 patients participating in the SPIRE-1/2 trials, 184 targeted proteins were measured at baseline and after 12 months of bococizumab treatment using the Cardiovascular II and III panels (Sweden). Of the included patients, 96 patients received placebo and 94 received 150mg of bococizumab, once every two weeks. Considering the development of auto-antibodies in some patients, patients with LDL-C reduction below 15% were excluded. The primary outcome was the fold change in protein levels after 12 months compared to baseline. The Benjamini-Hochberg method was used to adjust for multiple comparisons with a false discovery rate (FDR) of 5%. The findings were compared to the LoDoCo2 sub-study which used the same proteomic panels. Results In the placebo group, there was no significant change in any of the proteins. In the bococizumab group (n=77, mean age 62.2±9.7 years and 70 [91%] male), LDL-C levels were reduced from 114±41 mg/dL to 47±34 mg/dL (mean reduction 66±29 mg/dL) on top of statin therapy while there was a neutral effect on hs-CRP (median change 0.0 mg/L IQR [-0.8,1.1]). PCSK9 was the only protein with change upon treatment with bococizumab (24.4±11.0 vs. 15.0±10.7; PFDR=0.033; Figure 1A). In comparison, treatment of colchicine in the LoDoCo2 sub-study was associated with a significant change in 60 proteins (Figure 1B). Conclusion Here, we show that PCSK9 inhibition with bococizumab resulted in a highly selective reduction of PCSK9 plasma levels without robustly affecting other circulating proteins in the measured panels, implying PCSK9 inhibition lowers ASCVD risk via pure lipid lowering pathways. Our study reinforces the neutral systemic inflammatory effect of PCSK9 inhibition on top of statin therapy. The local effects on cellular and vascular inflammation by PCSK9 inhibition observed in earlier studies most likely reflect a direct consequence of a reduced cholesterol content in inflammatory cells and plaques, subsequently leading to a local anti-inflammatory effect. These findings support the combination of targeted anti-inflammatory therapies on top of potent LDL-C lowering in order to further reduce the residual inflammatory risk.

Exploring the Link between Plasma Levels of PCSK9, Immune Dysregulation and Atherosclerosis in Patients with Primary Sjögren's Syndrome.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates lipid metabolism contributing to cardiovascular (CV) risk in the general population. The relationship between PCSK9 and CV risk in systemic autoimmune diseases has been poorly explored. We investigated the association between plasma PCSK9, measures of immune-inflammatory status and markers of atherosclerosis in 52 consecutive patients with primary Sjögren's syndrome (pSS) in comparison to healthy controls (HCs). Median plasma PCSK9 levels were significantly higher in pSS patients versus HCs (162 (79-255) vs. 53 (39-99) ng/mL). Significantly higher prevalence of subclinical atherosclerosis and lower of dyslipidaemia (61% vs. 85%, p = 0.042) characterized pSS patients versus HCs. In pSS, no significant correlation emerged between PCSK9 and disease activity, atherosclerosis and lipid levels. In HCs, PCSK9 significantly correlated with lipid levels and atherosclerosis. Interestingly, significantly higher PCSK9 levels were found in HCs with high-to-very-high as compared to low-to-moderate CV risk (p = 0.018) while a non-significant trend towards higher PCSK9 levels was detected in pSS patients with low-to-moderate as compared to high-to-very-high CV risk (p = 0.060). This is the first demonstration that pSS patients, despite lower prevalence of dyslipidaemia and higher CV risk profile, are characterized by a 3-fold increase in PCSK9 levels in comparison to HCs. As PCSK9 does not correlate with measures of CV risk, its role in CV morbidity in pSS needs further investigation.

PCSK9 in extrahepatic tissues: What can we expect from its inhibition?

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that belongs to the serine protease family and plays a key role in regulating low-density lipoprotein cholesterol (LDL-C) levels in the blood. PCSK9 binds to the LDL receptor (LDLR), targeting it for degradation, resulting in an increase in circulating LDL-C levels. Loss-of-function mutations in the PCSK9 gene are associated with lower LDL-C levels and lower cardiovascular risk; in contrast, gain-of-function mutations are a cause of familial hypercholesterolaemia. The identification of PCSK9 as a pharmacological target led to the development of inhibitors for the treatment of hypercholesterolaemia. To date, the monoclonal antibodies evolocumab and alirocumab (which target plasma PCSK9) and the small-interfering RNA inclisiran (which targets hepatic PCSK9 mRNA) have been approved for the treatment of hypercholesterolaemia. Although hepatic PCSK9 plays a central role in regulating plasma LDL-C levels, this protein is also expressed in other tissues, including the brain, pancreas, heart, kidney, intestine and adipose tissue. In extrahepatic tissues, the functions of PCSK9 are both dependent and independent of LDLR and not necessarily harmful. For this reason, it is essential to uncover any potentially harmful effects of therapies that inhibit PCSK9, beyond their known LDL-C-lowering and CV risk-reducing effects.

Plasma Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) as a Possible Biomarker for Severe COVID-19.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) reduces low density lipoprotein (LDL) uptake, leading to increased plasma levels of LDL. In addition, PCSK9 has been implicated in inflammation independently of the effects on cholesterol metabolism. The current analysis showed that our 156 patients with systemic inflammatory response syndrome (SIRS) or sepsis had higher plasma PCSK9 levels in contrast with the 68 healthy controls. COVID-19 sepsis patients had increased plasma PCSK9 levels in comparison to sepsis patients not infected by SARS-CoV-2. For further analysis, patients were divided in two groups based on COVID-19. In both sub-cohorts, plasma PCSK9 levels did not correlate with C-reactive protein, leukocyte count, and procalcitonin. Plasma PCSK9 levels of both patient groups did not significantly differ among SIRS/sepsis patients with and without dialysis and patients with and without ventilation. Furthermore, vasopressor therapy was not significantly associated with altered plasma PCSK9 levels. In the non-COVID-19 SIRS/sepsis group, patients with Gram-negative and Gram-positive infections had similar plasma PCSK9 levels as patients without a detectable pathogen in their blood. In conclusion, the current study suggests PCSK9 as a possible biomarker for COVID-19, but this needs to be validated in larger cohorts.

Association of circulating proprotein convertase subtilisin/kexin type 9 concentration with coagulation abnormalities in patients with primary membranous nephropathy

Objectives The aims of the study were to explore the potential associations between plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) and coagulation indexes in patients with primary membranous nephropathy (PMN). Methods A total of 87 patients diagnosed with PMN were enrolled in our study. 30 healthy participants were recruited to match PMN participants. Plasma PCSK9 concentrations were tested by enzyme-linked immunosorbent assay (ELISA). Correlations between PCSK9 and coagulation abnormalities in patients with PMN were analyzed using univariate and multiple linear regression analysis. Results Plasma PCSK9 levels in patients with PMN were significantly higher than that in healthy controls [232.0 (143.5, 359.5) ng/mL vs. 166.8 (129.7, 199.7) ng/mL; p = 0.001]. Plasma levels of PCSK9 were positively correlated with factor VIII, factor IX, factor XI, log-transformed tissue factor, protein C and protein S (r = 0.267, p = 0.013; r = 0.496, p < 0.001; r = 0.217, p = 0.045; r = 0.584, p < 0.001; r = 0.372, p = 0.001; r = 0.282, p = 0.011). In multiple linear regression analysis, PCSK9 concentration was independently and positively correlated with factor VIII, factor IX, and tissue factor (β = 0.186, p = 0.047; β = 0.325, p = 0.001; β = 0.531, p < 0.001; respectively). PCSK9 concentration was independently and negatively correlated with PT (β= −0.343, p = 0.011). Conclusion Plasma PCSK9 levels had good positive correlations with procoagulant clotting factors and negative correlations with PT in PMN, which might provide novel information with regard to PCSK9 and hypercoagulability in PMN.

Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

The clinical effects of inclisiran, a first-in-class LDL-C lowering siRNA therapy, on the LDL-C levels in Chinese patients with hypercholesterolemia

Inclisiran is a novel siRNA therapy that inhibits the synthesis of proprotein convertase subtilisin-kexin type 9 (PCSK9) by targeting the PCSK9 mRNA, consequently, decreases low-density lipoprotein cholesterol (LDL-C). To assess the safety, PK and LDL-C lowering effects of inclisiran in the Chinese patients with elevated LDL-C despite treatment with maximally tolerated LDL-C lowering therapies. Forty Chinese patients with hypercholesterolemia (LDL-C ≥100 mg/dL) who were on maximally tolerated statin were randomized to receive a single dose of either inclisiran sodium 100 or 300mg s.c. injection (each for 15 patients) or placebo (10 patients). Safety, pharmacokinetics and pharmacodynamics (i.e., PCSK9 and LDL-C levels) were evaluated for up to 90 days after the s.c. injection of study drug. Following single subcutaneous injections inclisiran sodium at 100 mg or 300 mg, inclisiran has a relative short elimination half-life (T1/2, 6.5 hours). Both plasma PCSK9 and serum LDL-C decreased rapidly and consistently, with the maximal reduction between Day 30 and Day 60; then the decreases of PCSK9 and LDL-C were generally maintained up to 56.4% and 49.6% of 100 mg, 74.9% and 58.3% of 300 mg, respectively, at day 90. All adverse events were mild or moderate in severity, and no discontinuations due to adverse events. There were no serious adverse events being reported. Inclisiran was generally safe and well tolerated. Single dose of both Inclisiran 100 and 300 mg significantly reduced PCSK9 and LDL-C levels in Chinese patients up to Day 90. The greatest reductions were observed with the 300 mg regimen of Inclisiran. ClinicalTrials.gov: NCT04774003.

The N-Acetylgalactosamine-conjugated small interfering RNA inclisiran can be coadministered safely with atorvastatin in cynomolgus monkeys resulting in additive low-density lipoprotein cholesterol reductions.

Inclisiran, a small interfering RNA, selectively inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9) synthesis in the liver and has been shown to reduce low-density lipoprotein cholesterol (LDL-C) by ≥50% in patients with hypercholesterolemia receiving maximally tolerated statins. The toxicokinetic, pharmacodynamic, and safety profiles of inclisiran when coadministered with a statin were characterized in cynomolgus monkeys. Six cohorts of monkeys were administered either atorvastatin (40 mg/kg, reduced to 25 mg/kg during the study, daily, oral gavage), inclisiran (300 mg/kg every 28 days, subcutaneous administration), atorvastatin (40/25 mg/kg) and inclisiran combinations (30, 100, or 300 mg/kg), or control vehicles over 85 days followed by 90 days' recovery. Inclisiran and atorvastatin toxicokinetic parameters were similar in cohorts administered either agent alone or in combination. Inclisiran exposure increased in a dose-proportional manner. At Day 86, atorvastatin increased plasma PCSK9 levels four-fold from pretreatment levels but did not significantly lower serum LDL-C levels. Inclisiran (alone or in combination) reduced PCSK9 (mean decrease 66-85%) and LDL-C (mean decrease 65-92%) from pretreatment levels at Day 86; levels were significantly lower than the control group (p ≤ .05) and remained decreased during the 90-day recovery. Coadministration of inclisiran with atorvastatin resulted in greater reductions in LDL-C and total cholesterol compared with either drug alone. No toxicities or adverse effects were observed in any cohort receiving inclisiran, either alone or in combination. In summary, inclisiran significantly inhibited PCSK9 synthesis and decreased LDL-C in cynomolgus monkeys without increasing the risk of adverse effects when coadministered with atorvastatin.

PCSK9 Levels Are Increased in Type 2 Diabetes Mellitus with, and without Statin Therapy in Thai Subjects

Background: Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) levels were found associated with not only lipid profiles, but also glucose homeostasis. Nevertheless, the relationship between PCSK9 levels, and diabetes was inconsistent in various studies. Objective: To investigate the plasma PCSK9 levels among normoglycemia, pre-diabetes, and T2DM patients with, and without statin therapy in Thais. Materials and Methods: Five hundred fifty-three subjects including 213 normoglycemia, 176 pre-diabetes, 46 T2DM without statin therapy, 40 T2DM with simvastatin therapy at 20 mg/day, and 78 T2DM with simvastatin therapy at 40 mg/day were recruited. Anthropometric data, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting blood sugar (FBS), and PCSK9 levels were measured. Results: PCSK9 levels were significantly higher in T2DM without statin therapy compared with normoglycemic subjects. In addition, T2DM with simvastatin therapy (40 mg/day) had significantly higher PCSK9 levels than T2DM without statin therapy, and T2DM with simvastatin therapy (20 mg/day). Serum PCSK9 levels were positively correlated with several metabolic parameters including age, body mass index (BMI), systolic blood pressure (SBP), TC, TG, and FBS (p&lt;0.05) in the present study subjects. Conclusion: PCSK9 levels were modulated by glycemic status, and statin therapy in Thai subjects. Elevation of plasma PCSK9 levels in T2DM with, and without statin therapy may increase the risk for dyslipidemia, and cardiovascular disease among T2DM patients. Keywords: PCSK9, Pre-diabetes, T2DM, FBS, Statin

Effect of PCSK9 on atherosclerotic cardiovascular diseases and its mechanisms: Focus on immune regulation.

Atherosclerosis is a basic pathological characteristic of many cardiovascular diseases, and if not effectively treated, patients with such disease may progress to atherosclerotic cardiovascular diseases (ASCVDs) and even heart failure. The level of plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) is significantly higher in patients with ASCVDs than in the healthy population, suggesting that it may be a promising new target for the treatment of ASCVDs. PCSK9 produced by the liver and released into circulation inhibits the clearance of plasma low-density lipoprotein-cholesterol (LDL-C), mainly by downregulating the level of LDL-C receptor (LDLR) on the surface of hepatocytes, leading to upregulated LDL-C in plasma. Numerous studies have revealed that PCSK9 may cause poor prognosis of ASCVDs by activating the inflammatory response and promoting the process of thrombosis and cell death independent of its lipid-regulatory function, yet the underlying mechanisms still need to be further clarified. In patients with ASCVDs who are intolerant to statins or whose plasma LDL-C levels fail to descend to the target value after treatment with high-dose statins, PCSK9 inhibitors often improve their clinical outcomes. Here, we summarize the biological characteristics and functional mechanisms of PCSK9, highlighting its immunoregulatory function. We also discuss the effects of PCSK9 on common ASCVDs.

Association between plasma PCSK9 levels and lipid profile in patients with Parkinson's disease and comparison with healthy subjects.

Background: Up to know, limited and contradictory results have been published about the role of prognostic values of lipid profile and proprotein convertase subtilisin/kexin type 9 (PCSK9) in Parkinson's disease (PD). The aim of the present study is to investigate the role of lipid profile and PCSK9 in patients with PD and compare it with healthy individuals. Methods: In this case-control study, 31 individuals diagnosed with PD were compared with 31 healthy individuals. The lipid profile and PCSK9 of research participants were measured and the resulting data were analyzed using SPSS software. The P-values smaller than 0.05 were considered significant. Results: The mean age of participants in the PD and control group was 56.9 ± 8.8 and 53.7 ± 10.1 years, respectively (P > 0.050). 27 individuals (87.1%) in the PD group and 13 individuals (41.9%) in the control group were men. Low-density lipoprotein (LDL) level (84.2 ± 24.9 ml/dl vs. 105.5 ± 16.8, P < 0.001), high-density lipoprotein (HDL) level (45.5 ± 8.7 ml/dl vs. 51.1 ± 9.5 ml/dl, P < 0.001), and total cholesterol (155.3 ± 31.2 ml/dl vs. 192.8 ± 32.5 ml/dl, P < 0.001) were lower and triglyceride (TG) level was higher in the PD group (133.3 ± 79.3 ml/dl vs. 131.2 ± 58.6 ml/dl, P = 0.900) compared with the control group. PCSK9 level was higher in the PD group, but no significant difference was found (141.6 ± 70.0 vs. 129.7 ± 51.0 ng/ml, P = 0.500) compared to healthy subjects. Moreover, there was no relation between PCSK9 and severity of PD. Conclusion: Our findings showed that individuals with PD had lower levels of HDL, LDL, and total cholesterol compared with the control group. However, higher concentrations of PCSK9 were observed in patients with PD compared with healthy volunteers.

Clearance of plasma PCSK9 via the asialoglycoprotein receptor mediated by heterobifunctional ligands

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting the degradation of hepatic LDL receptors (LDLRs). Current therapeutic approaches use antibodies that disrupt PCSK9 binding to LDLR to reduce circulating LDL-C concentrations or siRNA that reduces PCSK9 synthesis and thereby levels in circulation. Recent reports describe small molecules that, like therapeutic antibodies, interfere with PCSK9 binding to LDLR. We report an alternative approach to decrease circulating PCSK9 levels by accelerating PCSK9 clearance and degradation using heterobifunctional molecules that simultaneously bind to PCSK9 and the asialoglycoprotein receptor (ASGPR). Various formats, including bispecific antibodies, antibody-small molecule conjugates, and heterobifunctional small molecules, demonstrate binding invitro and accelerated PCSK9 clearance invivo. These molecules showcase a new approach to PCSK9 inhibition, targeted plasma protein degradation (TPPD), and demonstrate the feasibility of heterobifunctional small molecule ligands to accelerate the clearance and degradation of pathogenic proteins in circulation.

Heparin Does Not Regulate Circulating Human PCSK9 (Proprotein Convertase Subtilisin-Kexin Type 9) in a General Population-Brief Report.

PCSK9 (proprotein convertase subtilisin-kexin type 9) chaperones the hepatic LDLR (low-density lipoprotein receptor) for lysosomal degradation, elevating serum LDL (low-density lipoprotein) cholesterol and promoting atherosclerotic heart disease. Though the major effect on the hepatic LDLR comes from secreted PCSK9, the details of PCSK9 reuptake into the hepatocyte remain unclear. In both tissue culture and animal models, HSPGs (heparan sulfate proteoglycans) on hepatocytes act as co-receptors to promote PCSK9 reuptake. We hypothesized that if this PCSK9:HSPG interaction is important in humans, disrupting it with unfractionated heparin (UFH) would acutely displace PCSK9 from the liver and increase plasma PCSK9. We obtained remnant plasma samples from 160 subjects undergoing cardiac catheterization before and after administration of intravenous UFH. PCSK9 levels were determined using a commercial enzyme-linked immunosorbent assay. Median plasma PCSK9 was 113 ng/mL prior to UFH and 119 ng/mL afterward. This difference was not significant (P=0.83 [95% CI, -6.23 to 6.31 ng/mL]). Equivalence testing provided 95% confidence that UFH would not raise plasma PCSK9 by > 4.7%. Among all subgroups, only subjects with the lowest baseline PCSK9 concentrations exhibited a response to UFH (8.8% increase, adj. P=0.044). A modest correlation was observed between baseline plasma PCSK9 and the change in plasma PCSK9 due to UFH (RS=-0.3634; P<0.0001). Administration of UFH does not result in a clinically meaningful effect on circulating PCSK9 among an unselected population of humans. The results cast doubt on the clinical utility of disrupting the PCSK9:HSPG interaction as a general therapeutic strategy for PCSK9 inhibition. However, the observations suggest that in selected populations, disrupting the PCSK9:HSPG interaction could still affect PCSK9 reuptake and offer a therapeutic benefit.

A Novel, Orally Bioavailable, Small-Molecule Inhibitor of PCSK9 With Significant Cholesterol-Lowering Properties In Vivo

Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibits the clearance of low-density lipoprotein (LDL) cholesterol (LDL-C) from plasma by directly binding with the LDL receptor (LDLR) and sending the receptor for lysosomal degradation. As the interaction promotes elevated plasma LDL-C levels, and therefore a predisposition to cardiovascular disease, PCSK9 has attracted intense interest as a therapeutic target. Despite this interest, an orally bioavailable small-molecule inhibitor of PCSK9 with extensive lipid-lowering activity is yet to enter the clinic. We report herein the discovery of NYX-PCSK9i, an orally bioavailable small-molecule inhibitor of PCSK9 with significant cholesterol-lowering activity in hyperlipidemic APOE∗3-Leiden.CETP mice. NYX-PCSK9i emerged from a medicinal chemistry campaign demonstrating potent disruption of the PCSK9-LDLR interaction in vitro and functional protection of the LDLR of human lymphocytes from PCSK9-directed degradation ex vivo. APOE∗3-Leiden.CETP mice orally treated with NYX-PCSK9i demonstrated a dose-dependent decrease in plasma total cholesterol of up to 57%, while its combination with atorvastatin additively suppressed plasma total cholesterol levels. Importantly, the majority of cholesterol lowering by NYX-PCSK9i was in non-HDL fractions. A concomitant increase in total plasma PCSK9 levels and significant increase in hepatic LDLR protein expression strongly indicated on-target function by NYX-PCSK9i. Determinations of hepatic lipid and fecal cholesterol content demonstrated depletion of liver cholesteryl esters and promotion of fecal cholesterol elimination with NYX-PCSK9i treatment. All measured in vivo biomarkers of health indicate that NYX-PCSK9i has a good safety profile. NYX-PCSK9i is a potential new therapy for hypercholesterolemia with the capacity to further enhance the lipid-lowering activities of statins.