Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors: A Brief Overview

Proprotein convertase subtilisin/kexin type 9 inhibitors serve as a valuable addition to the armamentarium of lipid-lowering agents and have promising potential. By inhibiting the proprotein convertase subtilisin/kexin type 9 enzyme, this novel molecule leads to increased low-density lipoprotein receptor density and decreased circulation of low-density lipoprotein. The fact the agent is a monoclonal antibody has led to limited drug interactions and minimized adverse drug events. It is critical for all providers to have a basic understanding of these novel therapies with their introduction and use for treatment.     

Serum Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is Independently Associated with Insulin Resistance,Triglycerides, Lipoprotein(a) Levels but not Low-Density Lipoprotein Cholesterol Levels in a General Population

Aim: Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been identified as an important regulator of low-density lipoprotein (LDL) receptor processing. Evolocumab and alirocumab are PCSK9 inhibitors; however, little is known about the association between PCSK9 levels and lipid profiles in a general population. Because PCSK9 inhibitors have LDL-C lowering effects, we investigated whether there is a positive correlation between serum PCSK9 levels and LDL-C or lipoprotein(a) [Lp(a)].Methods: In Uku town, 674 residents (mean age; 69.2 ± 8.3 years) received health check-ups. The participants underwent a physical examination and blood tests, including PCSK9 and Lp(a). Serum PCSK9 and Lp(a) were measured by ELISA and Latex methods, respectively. HOMA-IR was calculated by fasting plasma glucose × insulin levels/405.Results: The mean (range) of PCSK9 and Lp(a) were 211.2 (49–601) ng/mL and 60 (1–107) mg/dL, respectively. Because of a skewed distribution, the log-transformed values were used. With univariate linear regression analysis, PCSK9 levels were associated with Lp(a) (p = 0.028), triglycerides (p < 0.001), and HOMA-IR (p < 0.001), but not with LDL-C (p = 0.138) levels. Multiple stepwise regression analysis revealed that serum PCSK9 levels were independently associated with triglycerides (p < 0.001), Lp(a) (p = 0.033) and HOMA-IR (p = 0.041).Conclusions: PCSK-9 is independently associated with triglycerides, Lp(a) levels, and HOMA-IR, but not LDL-C, in a relatively large general population sample.     

Association between causative mutations and response to PCSK9 inhibitor therapy in subjects with familial hypercholesterolemia: A single center real-world study

Background and aimsFamilial hypercholesterolemia (FH) is an autosomal dominant disease that leads to cardiovascular (CV) disease. Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9-I) demonstrated efficacy in low-density lipoprotein cholesterol (LDL-C) reduction and in prevention of CV events. The aim of our study is to evaluate the relationship between LDL receptor (LDLR) mutations and response to PCSK9-I therapy.Methods and resultsWe evaluated total cholesterol (TC), LDL-C, high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) in consecutive patients with FH before PCSK9-I treatment and after 12 (T12w) and 36 (T36w) weeks of treatment. We evaluated LDL-C target achievement according to different mutations in LDLR. Eighty FH subjects (mean age:54 ± 13.3 years), 39 heterozygous (He) with defective LDLR gene mutations, 30 He with null mutations and 11 compound-He or homozygous (Ho) were recruited. At baseline, 69 subjects were under maximal lipid lowering therapy (MLLT) and 11 subjects had statin-intolerance. From baseline to T36w we observed an overall 51% reduction in LDL-C. We found no difference in LDL-C changes between subjects with He-defective mutation and He-null mutations both at T12w (p = 1.00) and T36w (p = 0.538). At T36w, LDL-C target was achieved in 59% of He-defective mutations subjects and in 36% of He-null mutations subgroup (p = 0.069), whereas none of compound-He/Ho-FH achieved LDL-C target.ConclusionsAfter 36 weeks there were no differences in response to PCSK9-I therapy between different groups of He-FH subjects. Response to PCSK9-I was significantly lower in carriers of compound-He/Ho mutations.Registration number for clinical trials: NCT04313270 extension.     

Influence of Single Low‐Density Lipoprotein Apheresis on the Adhesion Molecules Soluble Vascular Cellular Adhesion Molecule‐1, Soluble Intercellular Adhesion Molecule‐1, and P‐Selectin

Abstract: The aim of our study was to investigate the influence of single low‐density lipoprotein apheresis (heparin extracorporeal low‐density lipoprotein precipitation [HELP]procedure) on plasma concentrations of soluble adhesion molecules (sAMs) such as soluble vascular cellular adhesion molecule‐1 (sVCAM‐1), soluble intercellular adhesion molecule‐1 (sICAM‐1), and P‐selectin in patients with familial heterozygous hypercholesterolemia and documented coronary artery disease enrolled in a chronic weekly HELP apheresis. Before HELP apheresis, the mean plasma concentration of sVCAM‐1 was 515 ± 119 ng/ml, 204 ± 58 ng/ml for sICAM‐1, and 112 ± 45 ng/ml for P‐selectin. After single HELP apheresis, plasma concentrations of sAM declined significantly by 32 ± 7%, 18 ± 15%, and 33 ± 25% for sVCAM‐ 1,sICAM‐1 and P‐selectin, respectively. After a 1 week interval, sAM concentrations rose to approximately the initial values. The concentrations of all sAMs studied were significantly lower in the plasma leaving than entering the filter. Due to filtration, the decline in plasma level of sVCAM‐1, sICAM‐1, and P‐selectin was 62 ± 19%, 51 ± 39%, and 67 ± 22%, respectively. In addition to lipid reduction, single HELP apheresis significantly lowers plasma concentrations of sVCAM‐1, sICAM‐1, and P‐selectin.     

冠心病患者血清前蛋白转化酶枯草溶菌素9与小而密低密度脂蛋白胆固醇的相关性研究

目的探讨冠心病患者血清前蛋白转化酶枯草溶菌素9(PCSK9)与小而密低密度脂蛋白胆固醇(sd LDLC)的关系。方法收集2014年3月至2014年12月在山西医科大学第一医院住院且行冠状动脉造影术确诊为冠心病的患者100例作为冠心病组,同期健康体检者67例作为对照组。Lipoprint脂蛋白分析仪检测LDLC颗粒大小、sd LDLC颗粒数及sd LDLC所占LDLC的百分比(简称sd LDLC百分比);酶联免疫吸附法测定血清PCSK9。结果冠心病组LDLC颗粒大小低于对照组(264.07±6.78比267.37±5.15,P0.01),sd LDLC颗粒数多于对照组(5.0±9.5比4.0±5.0,P0.05),sd LDLC百分比大于对照组(5.95%±10.50%比3.70%±5.85%,P0.01)。冠心病组血清PCSK9显著高于对照组(15.48μg/L比14.95μg/L,U=-2.74,P=0.006)。冠心病组血清PCSK9与sd LDLC百分比、LDLC呈正相关(r=0.212,P=0.034;r=0.202,P=0.032)。结论冠心病患者血清PCSK9与sd LDLC百分比呈正相关,抑制PCSK9可以预防冠心病。     

Heparin‐Induced Extracorporeal Low‐Density Lipoprotein Precipitation Futura,a New Modification of HELP Apheresis: Technique and First Clinical Results

Abstract: Heparin extracorporeal low‐density lipoprotein precipitation (HELP) Futura is a new development in the standard HELP Secura system that has been in clinical practice for more than 15 years. Based on this experience, a HELP Upgrading system was first evaluated in a clinical trial to confirm the same clinical results as in the standard system. The upgraded procedure worked with industrially prepared sterile dialysis solutions instead of the reverse osmosis device. The conclusion of the study was that the reductions of low‐density lipoprotein, lipoprotein (a), and fibrinogen and the correction of the acid‐base balance were comparable with the results of the standard system. As a second step, the HELP Futura system was evaluated in a clinical acceptance test and in a field test in the daily routine. After approximately 2,000 treatments on 35 patients, we can conclude that the HELP Futura procedure has reached a mature phase with a high degree of clinical safety and flexibility.     

NMR‐based lipoprotein analysis for patients with severe hypercholesterolemia undergoing lipoprotein apheresis or PCSK9‐inhibitor therapy (NAPALI‐Study)

Taking into account the discordance between low‐density lipoprotein cholesterol (LDL‐C) and LDL particle (LDL‐P) number, cardiovascular risk more closely correlates with LDL‐P in patients. The aim of our study was to evaluate the number of lipid particles in patients with severe hypercholesterolemia treated with different lipid‐lowering regimens. Four groups of patients differing with respect to lipid‐lowering therapy were recruited from hypercholesterolemic outpatients and lipoprotein apheresis (LA) facilities, and were treated with statins alone (group A), with statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors (PCSK9i) (group B), with statins and LA (group C), or with statins, PCSK9i, and LA (group D). Cholesterol, triglycerides, LDL‐C, high‐density lipoprotein cholesterol (HDL‐C), LDL‐P number and size, HDL‐P number and size were determined using nuclear magnetic resonance spectroscopy. The lowest LDL‐P number was achieved at the end of LA sessions in combination with statins or in combination with statins and a monoclonal PCSK9i (median; 25th and 75th percentile) (group C: 244 nmoL/L: 237, 244, P < 0.05; group D: 244 nmoL/L: 99, 307, P < 0.05). Comparing LDL‐P number at the start of LA (group C: 978 nmoL/L: 728, 1404; group D: 954 nmoL/L: 677, 1521) to the other patient groups (groups A and B), the lowest LDL‐P number was measured in patients treated with PCSK9i and a statin (group B): LDL‐P (762 nmoL/L: 604, 1043, P < 0.05), large LDL‐P (472 nmoL/L: 296, 574, P < 0.05), and small LDL‐P (342 nmoL/L: 152, 494, P < 0.05). Very low‐density lipoprotein and HDL particle sizes remained approximately the same in all groups. LA in combination with statins or in combination with statins and PCSK9i most reduced LDL‐P numbers in hypercholesterolemic patients.     

Influence of Atorvastatin Versus Simvastatin on Fibrinogen and Other Hemorheological Parameters in Patients with Severe Hypercholesterolemia Treated with Regular Low‐Density Lipoprotein Immunoadsorption Apheresis

Abstract: Low‐density lipoprotein (LDL) apheresis is a treatment option in patients with coronary artery disease and elevated LDL cholesterol concentrations if maximal drug therapy fails to achieve adequate LDL cholesterol reduction. This therapy is more effective when combined with strong lipid‐lowering drugs, such as atorvastatin. However, conflicting data have been published concerning the effect of atorvastatin on fibrinogen concentration. Therefore, we investigated the effect of atorvastatin compared to simvastatin on fibrinogen concentration and other hemorheological parameters in patients treated by weekly LDL apheresis. Hemorheological parameters were, studied twice in 9 patients (4 female, 5 male, 54.0 ± 8.9 years) with coronary artery disease treated by weekly LDL immunoadsorption, once during concomitant simvastatin therapy (40 mg daily) and once during atorvastatin therapy (40 mg daily). Fibrinogen concentration, plasma and blood viscosity at different shear rates, parameters of red cell aggregation at stasis and shear rate 3/s, and erythrocyte filterability were determined 7 days after the last LDL apheresis after each drug had been given for a minimum for 8 weeks. Fibrinogen concentration did not show any statistically significant difference during therapy with atorvastatin (3.09 ± 0.36 g/L) compared to simvastatin (3.13 ± 0.77 g/L). Plasma and blood viscosity as well as erythrocyte filterability were also unchanged. The increase in red cell aggregation at stasis during atorvastatin treatment (5.82 ± 1.00 U versus 4.89±0.48 U during simvastatin; p < 0.05) was inversely correlated with a lower high‐density liprotein (HDL) cholesterol concentration (1.17 ± 0.21 mmol/L versus 1.31 ± 0.30 mmol/L during simvastatin; p < 0.05). LDL cholesterol showed a strong trend to lower concentrations during atorvastatin (4.14 ± 0.61 mmol/L versus 4.56 ± 0.66 mmol/L during simvastatin; p = 0.07), despite a reduced plasma volume treated (3,547 ± 1,239 ml during atorvastatin versus 3,888 ± 1,206 mL during simvastatin; p < 0.05). In conclusion, fibrinogen concentration and other hemorheological parameters were unchanged during atorvastatin compared to simvastatin therapy with the exception of a higher red cell aggregation at stasis. Therefore, with respect to hemorheology, we conclude that atorvastatin should not be withheld from hypercholesterolemic patients regularly treated with LDL immunoadsorption.     

Beneficial Effect of Aggressive Low‐Density Lipoprotein Apheresis in a Familial Hypercholesterolemic Patient with Severe Diabetic Scleredema

Abstract: We present a 59‐year‐old woman with severe diabetic scleredema (DS) associated with heterozygous familial hypercholesterolemia (FH). She had been treated with drugs to lower blood glucose, with insulin for diabetes mellitus (DM), and with low‐density lipoprotein (LDL) apheresis therapy monthly or every 2 weeks in addition to drugs to lower serum lipids for FH. However, her scleredema had not improved. After we had tried weekly LDL apheresis therapy for a period of 3 years to treat her hyperlipidemia, the levels of her serum lipids were reduced to normal ranges, and scleredema in her nape improved. We also demonstrated the histopathological improvement in dermis of her cervical skin. We conclude that weekly LDL apheresis therapy is effective for diabetic scleredema that is resistant to conventional treatments.     

Effect of Heparin‐Induced Extracorporeal Low‐Density Lipoprotein Precipitation (HELP) Apheresis on Hepatitis C Plasma Virus Load

Abstract: Association of the hepatitis C virus (HCV) with apolipoprotein B containing lipoproteins has been suggested, and this led to the concept that the low‐density lipoprotein (LDL) receptor may also serve as a candidate receptor for HCV uptake into the liver. We have investigated whether heparin‐induced extracorporeal LDL precipitation (HELP) LDL apheresis treatment reduces HCV plasma load in 6 patients, all infected for more than 4 years with HCV and resistant against established anti‐HCV therapy. HELP apheresis treatment caused an HCV‐RNA decrease of 77.3% in mean. This decline was not correlated with LDL‐cholesterol reduction. HCV‐RNA was retained on the HELP filter as shown for 1 patient. The effect of RNA lowering was only transient due to the high turnover of HCV. However, HELP apheresis may open a window of opportunity for an immune‐modulating and antiviral therapy in the interval between two apheresis procedures in patients with high virus load.     

Effects of Direct Adsorption of Lipoproteins Apheresis on Lipoproteins,Low‐Density Lipoprotein Subtypes,and Hemorheology in Hypercholesterolemic Patients with Coronary Artery Disease

Abstract: Direct adsorption of lipoproteins (DALI) apheresis has been shown to reduce effectively low‐density lipoprotein (LDL) cholesterol and lipoprotein (a) concentrations. However, the effects on nontraditional risk indicators such as hemorheology and LDL subtypes have not been investigated so far. Five patients (2 women, 3 men, age 53 ± 8 years) with coronary artery disease and severe LDL hypercholesterolemia regularly treated with other LDL apheresis devices entered the study and were then treated with DALI for the first time. Hemorheological and lipoprotein parameters were measured before and immediately after the initial DALI apheresis as well as before the fourth DALI apheresis. Compared to baseline (before the first DALI apheresis), the following parameters were significantly improved (p < 0.05) after the first DALI apheresis: LDL cholesterol (69 ± 28 versus 208 ± 82 mg/dl) and cholesterol in each LDL subfraction as well as plasma viscosity (1.23 ± 0.04 versus 1.37 ± 0.06 mPa), C‐reactive protein, native blood viscosity, red cell aggregation, and red cell deformability. When parameters before the fourth DALI apheresis were compared to baseline, LDL cholesterol was still lower, and red cell deformability was still improved while cholesterol in each subfraction showed a statistical trend to lower concentrations (0.08 < p < 0.14). In conclusion, DALI apheresis not only reduces LDL cholesterol but also induced a significant reduction of cholesterol in all LDL subfractions and improved various hemorheological parameters.     

Chronic Alcohol Consumption Disrupted Cholesterol Homeostasis in Rats: Down‐Regulation of Low‐Density Lipoprotein Receptor and Enhancement of Cholesterol Biosynthesis Pathway in the Liver

Background: Chronic alcohol consumption causes alcoholic liver disease, which is associated, or initiated, with dysregulated lipid metabolism. Very recent evidence suggested that dysregulated cholesterol metabolism plays an important role in the pathogenesis of alcoholic fatty liver diseases, however, the effects of chronic alcohol exposure on cholesterol homeostasis have not been well studied and underlying mechanisms behind are still elusive. Methods: Male Sprague–Dawley rats weighing 250 ± 5.5 g (mean ± SEM) divided into 2 groups (8 rats per group) and pair‐fed with liquid diets containing (in percent of energy intake) 18% protein, 35% fat, 12% carbohydrate, and 35% either ethanol (ethanol diet) or an isocaloric maltose‐dextrin mixture (control diet), according to Lieber and De Carli, for 4 weeks. Results: Long‐term excessive alcohol feeding to rats caused fatty liver and liver injury, which was associated with disrupted cholesterol homeostasis, characterized by increased hepatic cholesterol levels and hypercholesterolemia. Hepatic cholesterol increases were concomitant with constantly activated sterol regulatory element‐binding protein‐2 (SREBP‐2) in the liver and increased expression of 3‐hydroxy‐3‐methyl‐glutaryl‐CoA (HMG‐CoA) reductase, a rate‐limiting enzyme for cholesterol de novo synthesis, indicating enhanced cholesterol biosynthesis. Alcohol‐induced hypercholesterolemia was accompanied by decreased LDL receptor (LDLr) levels in the liver. Further investigations revealed that chronic alcohol exposure increased hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) contents to down‐regulate LDLr via a post‐translational mechanism. Moreover, alcohol feeding suppressed extracellular signal‐regulated kinase (ERK) activation in the liver. In vitro studies showed that inhibition of ERK activation was associated with decreased LDLr expression in HepG2 cells. Conclusions: Our study provides the first evidence that both increased PCSK9 expression and suppressed ERK activation in the liver contributes to alcohol‐induced hypercholesterolemia in rats.     

Development of Selective Low‐Density Lipoprotein (LDL) Apheresis System: Immobilized Polyanion as LDL‐Specific Adsorption for LDL Apheresis System

Abstract: Guest Editor's Introduction: The binding site of the human LDL receptor is rich in anionic amino acids, and interacts with apolipoprotein‐B by the electrostatic force. Therefore, anionic ligands could be used for the selective adsorption of LDL without HDL adsorption. Based upon this concept, Kaneka Co. developed LDL adsorbent. The adsorbent named Loposorber consists of microporous cellulose beads which immobilized dextrane sulfate with a molecular weight of several thousands. This paper describes the basic study for the selection of polyanions, in vitro adsorption characteristics, and the references of initial clinical applications. This paper was printed in Artificial Organs, vol. 20, page 922–929 (1996), and reprinted here with permission. Low‐density lipoprotein (LDL) is widely recognized as one of the major risk factors for developing coronary heart diseases. Despite intensive development of LDL‐lowering drugs, there still exist those patients with refractory hyperlipidemia whose plasma LDL levels are not sufficiently lowered by drugs. LDL apheresis, direct removal of plasma LDL from circulating blood, is thought to be the most promising treatment for such refractory patients. Various techniques, such as the use of an immunoadsorbent utilizing an anti‐LDL antibody, have been used in an attempt to achieve the selective removal of LDL. However, none were widely used because of complications, poor selectivity, and so forth. To establish a safe and effective LDL apheresis system, we chose a synthetic affinity adsorbent as the LDL‐removing device. Synthetic polyanion compounds were used as the affinity ligands for LDL adsorbent to simulate the anion‐rich sequence of LDL binding sites in the human LDL receptor. Among various polyanion compounds, those polyanions with sulfate or sulfonate groups and hydrophilic backbone were found to have a strong affinity for LDL. In contrast, polyanions with carboxyl groups showed poor affinity. Dextran sulfate (DS) was selected as the affinity ligand of LDL adsorbent for its high affinity and low toxicity. The influence of its charge density and molecular weight on its affinity for LDL was suitable. The affinity rapidly increased as the charge density increased, then, reached a constant value. Little affinity was found for either the DS monomer (glucose sulfate) or DS with a molecular weight higher than 10⁴ daltons whereas DS with molecular weights in the midrange showed strong affinity. DS with a midrange molecular weight was immobilized on cellulose hard gel to give LDL adsorbent clinical application. The adsorbent demonstrated an excellent selectivity for LDL and very low density lipoprotein (VLDL) in vitro. Adsorption of high‐density lipoprotein and major plasma proteins was almost negligible. Additional study of the LDL‐binding mechanism revealed that DS directly interacts with positively charged sites on LDL, which demonstrates that the nature of the interaction is the same as that of the LDL receptor. An LDL adsorption column (Liposorber) packed with an LDL adsorbent and polysulfone hollow‐fiber plasma separator (Sulflux) was developed as an efficient LDL apheresis system. Clinical investigation proved that this system is capable of intensively lowering the plasma LDL level without affecting major plasma components.