Proprotein Convertase Subtilisin/kexin type 9 Inhibition in Cardiovascular Prevention.

Elevated levels of Low Density Lipoprotein cholesterol (LDL-C) are directly associated with increased risk for atherosclerotic cardiovascular and cerebrovascular events. Statins have been used to control serum LDLC and this has translated into reduction in cardiovascular and cerebrovascular events. However, despite high dose statin therapy, LDL-C control may remain inadequate in some patients, particularly those with familial hypercholesterolemia. A new therapeutic approach has emerged in recent years with proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In this review, we describe the development and the use of this new class of drugs.

Role of lipoproteins and proprotein convertase subtilisin/kexin type 9 in endotoxin clearance in sepsis.

PURPOSE OF REVIEW: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is a recent high-impact cardiovascular intervention aimed at reducing low-density lipoprotein (LDL) cholesterol levels. Notably, pathogen lipids are also carried in lipoprotein particles and are cleared by hepatocyte LDL receptors. Therefore, the role of PCSK9 in sepsis is reviewed. RECENT FINDINGS: Endogenous PCSK9 decreases clearance of LDL cholesterol by decreasing the number of LDL receptors on hepatocytes. Similarly, PCSK9 decreases clearance of pathogen lipids, such as endotoxin, carried in LDL. Pathogen lipids, such as lipopolysaccharide (LPS) from gram-negative organisms or lipoteichoic acid from gram-positive organisms, are carried in high-density lipoprotein, LDL, and very low-density lipoprotein particles. Transfer proteins that handle pathogen lipids (e.g., LPS binding protein) are homologous to transfer proteins that handle cholesterol (e.g., phospholipid transfer protein, cholesterol ester transfer protein). Reduction in PCSK9 function results in increased LPS clearance, a decreased inflammatory response, and improved clinical outcomes in mice. PCSK9 inhibition improves survival in septic mice. Similarly, humans who carry loss-of-function variants of the PCSK9 gene have increased survival in sepsis. SUMMARY: PCSK9 inhibition may be a useful strategy to increase pathogen lipid clearance in the treatment of patients with sepsis.

The Central Role of Proprotein Convertase Subtilisin/Kexin Type 9 in Septic Pathogen Lipid Transport and Clearance.

Microbial cell walls contain pathogenic lipids, including LPS in gram-negative bacteria, lipoteichoic acid in gram-positive bacteria, and phospholipomannan in fungi. These pathogen lipids are major ligands for innate immune receptors and figure prominently in triggering the septic inflammatory response. Alternatively, pathogen lipids can be cleared and inactivated, thus limiting the inflammatory response. Accordingly, biological mechanisms for sequestering and clearing pathogen lipids from the circulation have evolved. Pathogen lipids released into the circulation are initially bound by transfer proteins, notably LPS binding protein and phospholipid transfer protein, and incorporated into high-density lipoprotein particles. Next, LPS binding protein, phospholipid transfer protein, and other transfer proteins transfer these lipids to ApoB-containing lipoproteins, including low-density (LDL) and very-low-density lipoproteins and chylomicrons. Pathogen lipids within these lipoproteins and their remnants are then cleared from the circulation by the liver. Hepatic clearance involves the LDL receptor (LDLR) and possibly other receptors. Once absorbed by the liver, these lipids are then excreted in the bile. Recent evidence suggests pathogen lipid clearance can be modulated. Importantly, reduced proprotein convertase subtilisin/kexin type 9 activity increases recycling of the LDLR and thereby increases LDLR on the surface of hepatocytes, which increases clearance by the liver of pathogen lipids transported in LDL. Increased pathogen lipid clearance, which can be achieved by inhibiting proprotein convertase subtilisin/kexin type 9, may decrease the systemic inflammatory response to sepsis and improve clinical outcomes.

A pilot study on the serum pharmacokinetics of nattokinase in humans following a single, oral, daily dose.

CONTEXT: Nattokinase is a serine protease and is derived from natto, a traditional Japanese, fermented, soybean food meal. Multiple authors have described the significant fibrinolytic, antithrombotic, and antihypertensive effects of natto. Nattokinase has been growing in popularity for use as a dietary supplement for the benefit of cardiovascular health. Little is known regarding the pharmacokinetic and pharmacodynamic properties of this enzyme, and the bioavailability of nattokinase is currently unknown. OBJECTIVE: This study intended to (1) detect nattokinase directly and immunologically, (2) show that nattokinase and/or its metabolites were detectable in human blood following ingestion of a commercial preparation, and (3) chart a pharmacokinetic dosing effect for nattokinase. DESIGN: The research team designed the pilot study as an in vivo, human clinical trial. Healthy human subjects responded to an advertisement and were screened. Subjects who satisfied both inclusion and exclusion criteria were enrolled into the study. Subjects were then instructed to orally ingest a single capsule containing a known concentration of nattokinase immediately following a baseline blood draw. Subsequent blood draws occurred over a 24-h period. SETTING: This study was conducted in Oakland, California, at a clinical reference laboratory and was performed with the approval of an institutional review board (IRB) to ensure that appropriate ethical standards were met. PARTICIPANTS: Eleven healthy participants (five male, six female, ages 21-65), who met eligibility criteria, were enrolled. INTERVENTION(S): Administration of nattokinase occurred orally with the ingestion of a single daily dose (2000 FU) of nattokinase. Capsules, each containing approximately 100 mg of nattokinase, in softgel form (NSK-SD, Japan Bio Science Laboratory, Osaka, Japan), were used in the study. OUTCOME MEASURE(S): Baseline blood samples were collected, and participants were observed swallowing a single capsule of the nattokinase supplement before returning at 2, 4, 8, 12, 24, and 48 h post ingestion for subsequent blood draws. The presence of nattokinase in serum was measured by an enzyme-linked immunosorbent assay (ELISA), using a rabbit, polyclonal, antinattokinase-capture antibody. A pharmacokinetic pattern was observed for nattokinase between baseline and 48 h postdose. RESULTS: Peak serum levels of nattokinase were observed at approximately 13.3 h ± 2.5 h (mean ± standard error) postdose. Statistically significant increases in binding were detectable from baseline when comparing averaged data at time points t = 2 h-t = 24 h. CONCLUSIONS: These results provided the first evidence that nattokinase can be measured directly in the blood of humans. The results further suggest that a larger, more extensive, pharmacokinetic study of nattokinase is warranted. Additionally, looking for intact enzyme and bioactive nattokinase peptides should be a consideration for future studies investigating the pharmacokinetic profile of nattokinase.

Automated determination of drugs in blood samples after enzymatic hydrolysis using precolumn switching and post-column reaction detection.

Enzymatic hydrolysis of blood samples with subtilisin-A releases protein-bound drugs and permits the repeated (10-50 times) injection of up to 1-ml volumes on short (2-30 mm) precolumns without appreciable build-up of pressure or loss of performance of the precolumn. The principle of fully automated serum and plasma analysis is demonstrated with the drug secoverine as a model compound. After enzymatic hydrolysis of the sample with an equal volume of a 1 mg/ml solution of subtilisin-A for 15 min at 55 degrees C, the model compound is preconcentrated using a microprocessor-controlled column switching unit. Separation occurs in a reversed-phase liquid chromatographic system using a CN-type stationary phase and a buffered aqueous dioxane solution as mobile phase. Detection is done by UV spectrophotometry or fluorometrically after post-column ion-pairing reaction with dimethoxyanthracenesulphonate. The relative standard deviation of the procedure is less than +/- 6% (n = 10).

Elevated titers of serum agglutinators. A serologic indicator of infection.

Elevated titers of serum agglutinators (anti-FabIgG) are associated with significant and severe suppurative infection. Immunosuppression and long-term antibiotic therapy, however, may lead to negative tests.