Pierre Van Antwerpen

Research in Drug Development (RD3)

Pharmacognosy Bioanalysis and Drug Discovery

Research activities

His first research interests focus on the validation of myeloperoxidase as a therapeutical target. In this context, part of his research is also dedicated to the inhibition of myeloperoxidase by chemical screening and rational drug design approach. Since 2008, his main task has been the development of the analytical platform of the Faculty of Pharmacy (ULB) and especially the mass spectrometry. Beside the myeloperoxidase research topic, method validations suitable for the pharmaceutical and biomedical researches are the main part of his actual activities: oxidized peptide quantifications, glycobiology, proteomics, metabolomics, lipidomics, small molecules analysis...

Top ten articles

Articles dans des revues avec comité de lecture

A paraître

Targeted metabolomics and transcript profiling of methyltransferases in three coffee species

Xanthines are the most known alkaloids present in coffee plants. The studies about their metabolism and their pharmacological activities have started several years ago to provide more information about the chemical properties of the most popular beverages in the world, such as coffee or tea. This work consisted in performing metabolomic analysis based on LC-(HR)MS data to investigate the metabolism of xanthines in the leaves and in the fruits of Coffea anthonyi, Coffea arabica and Coffea canephora. While C. arabica and C. canephora have been widely studied, only few information is available for C. anthonyi. However, due to the wide climate changes, this species growing at low altitudes could be a promising species to make coffee beverages. The expression of genes for proteins involved in xanthines biosynthesis has also been investigated by performing RT-qPCR. An overview of the histolocalisation of xanthines in the different organs of Coffea arabica flowers was also provided.

2024

Development and validation of online SPE purification coupled to HILIC-fluorescence-MS analysis for the characterization of N-glycans

N-glycans of therapeutic glycoproteins is a critical quality attribute to be addressed. We developed a sensitive method for N-glycan characterization using procainamide (ProcA) labelling and online solid phase extraction (online SPE). N-glycans were enzymatically released, then labeled with ProcA and cleaned up via the online SPE using HILIC chemistry (online HILIC SPE). Two preparation protocols were optimized: a short one (1 h 30) and a long one (18 h). Furthermore, the developed approach was compared to RapiFluor-MS (RFMS) kit (from Waters) and to InstantPC kit (from Agilent) which both include a classical HILIC μElution plate SPE purification. Samples were analyzed using HILIC separation coupled to fluorescence and MS detection (HILIC-FLD-MS) with or without the online HILIC SPE. During the validation, repeatability, intermediate precision, stability, response function and injection volume were tested. Human IgG mix (Multigam®) and NIST mAb standard were used as references as their glycoprofiles are well described. A comparison of three batches of a rituximab biosimilar (Truxima®) and one batch of its originator (MabThera®) was also performed. Online HILIC SPE sample cleanup shows a higher sensitivity and repeatability compared to the classical HILIC μElution SPE. Our online HILIC SPE approach also offers the highest MS signal compared to both commercial kits. However, InstantPC shows the highest FLD signal. The analyses of rituximab samples were in line with the literature showing the efficiency of the method for N-glycan monitoring of biotherapeutics. In conclusion, the results demonstrated the usefulness and ease of application of the developed protocol with the online HILIC SPE purification.

No reliable clinical results without a proper description of medicinal herbs

2023

Untargeted Metabolomics Approach Correlated Enniatin B Mycotoxin Presence in Cereals with Kashin-Beck Disease Endemic Regions of China.

Kashin-Beck disease (KBD) is a multifactorial endemic disease that only occurs in specific Asian areas. Mycotoxin contamination, especially from the Fusarium spp., has been considered as one of the environmental risk factors that could provoke chondrocyte and cartilage damage. This study aimed to investigate whether new mycotoxins could be identified in KBD-endemic regions as a potential KBD risk factor. This was investigated on 292 barley samples collected in Tibet during 2009-2016 and 19 wheat samples collected in Inner Mongolia in 2006, as control, from KBD-endemic and non-endemic areas. The LC-HRMS(/MS) data, obtained by a general mycotoxin extraction technic, were interpreted by both untargeted metabolomics and molecular networks, allowing us to identify a discriminating compound, enniatin B, a mycotoxin produced by some Fusarium spp. The presence of Fusarium spp. DNA was detected in KBD-endemic area barley samples. Further studies are required to investigate the role of this mycotoxin in KBD development in vivo.

Hyperbaric oxygenation improve red blood cell deformability in patients with acute or chronic inflammation

Introduction: Red blood cells (RBC) are one of the key elements of the microcirculation. Their ability to pass through capillaries and to deliver oxygen to cells is due to their large degree of deformability linked to the characteristics of the RBC membrane. Alterations in RBC deformability as a result of membrane damage, linked in part to increased synthesis of reactive oxygen species (ROS), can be observed in several diseases, such as sepsis, and may contribute to the altered microcirculation observed in these pathologies. Hyperbaric oxygen therapy (HBOT), with inhalation of 100 % oxygen, has been proposed in several acute or chronic pathologies, including carbon monoxide poisoning. Objective: We investigated the effects of HBOT on oxidative stress from ROS produced by myeloperoxidase (MPO) and on RBC deformability in patients with acute or chronic inflammation (n = 10), in patients with acute carbon monoxide poisoning (n = 10), and in healthy volunteers (n = 10). Methods: RBC deformability was evaluated before and after HBOT in the various populations using the ektacytometry technique (Laser-assisted Optical Rotational Red Cell Analyzer - LORRCA). Deformability was determined by the elongation index (EI) in relation to the shear stress (SS) over a range of 0.3 to 50 Pa. Oxidative stress was estimated through changes in proteins (chlorotyrosine and homocitrulline) induced by MPO activity measured by liquid chromatography-tandem mass spectrometry analysis. Results: Before HBOT, EI was significantly lower in patients with acute or chronic inflammation than in healthy volunteers and patients with acute carbon monoxide poisoning for the majority of SS values studied. After one session of HBOT, the EI was significantly higher than before HBOT for SS values of 1.93 Pa or higher in patients with acute or chronic inflammation. This effect remains constant after 10 sessions. There were no differences before and after HBOT in protein or amino acid oxidation due to ROS generation mediated by MPO in the three populations. Conclusions: Our results confirm altered RBC deformability in patients with acute and chronic conditions associated with an underlying inflammatory process. HBOT improves deformability only after one session and therefore may improve microcirculation in this population. According to our results, this improvement does not seem mediated by the ROS pathway via MPO. These results need to be confirmed in a larger population.

Role of Myeloperoxidase in ROS Generation and Inflammation Response on Prostate Epithelial Cells

Myeloperoxidase (MPO) has been reported in prostate tissue, and considering its pro-oxidant properties, this location might be linked to prostate pathology. The possibility that the glandular prostatic tissue might be the source of MPO and its potential inflammatory effects must be tested. Human prostate material was obtained from prostate biopsies and radical prostatectomies. Immunohistochemistry was performed using MPO-specific human antibody. In situ hybridization using MPO-specific probes and laser-assisted microdissection for quantitative real-time RT-PCR were performed to observe whether MPO is being produced in prostate tissue. Mass spectrometry on prostate biopsies was used to detect products of MPO activity in nucleic acids (DNA/RNA). MPO contribution to intracellular accumulation of ROS and interleukin-8 in prostatic epithelial cells was monitored in vitro. Immunohistochemistry confirmed cellular localization of MPO in epithelial cells of the prostate. The staining varied from light to high intensity. In situ hybridization did not address the presence of mRNA coding for MPO. No MPO-specific modifications on nucleic acids were detected. Mox-LDL was a major factor inducing ROS and cytokines production in prostatic epithelial cells. We did not demonstrate that MPO was synthetized by prostatic epithelial cells. However, in vitro experiments showed the ability of MPO to potentiate the ROS production and inflammation on prostate epithelial cells. Results do not allow us to demonstrate a role of MPO in prostate to date but further studies are mandatory to focus on the potential impact of MPO in the development of prostatic diseases.

Two new siderophores produced by Pseudomonas sp. NCIMB 10586: The anti-oomycete non-ribosomal peptide synthetase-dependent mupirochelin and the NRPS-independent triabactin

Introduction Globisporangium ultimum is an oomycetal pathogen causing damping-off on over 300 different plant hosts. Currently, as for many phytopathogens, its control relies in the use of chemicals with negative impact on health and ecosystems. Therefore, many biocontrol strategies are under investigation to reduce the use of fungicides. Results In this study, the soil bacterium Pseudomonas sp. NCIMB 10586 demonstrates a strong iron-repressed in vitro antagonism against G. ultimum MUCL 38045. This antagonism does not depend on the secretion of the broad-range antibiotic mupirocin or of the siderophore pyoverdine by the bacterial strain. The inhibitor molecule was identified as a novel non-ribosomal peptide synthetase (NRPS) siderophore named mupirochelin. Its putative structure bears similarities to other siderophores and bioactive compounds. The transcription of its gene cluster is affected by the biosynthesis of pyoverdine, the major known siderophore of the strain. Besides mupirochelin, we observed the production of a third and novel NRPS-independent siderophore (NIS), here termed triabactin. The iron-responsive transcriptional repression of the two newly identified siderophore gene clusters corroborates their role as iron scavengers. However, their respective contributions to the strain fitness are dissimilar. Bacterial growth in iron-deprived conditions is greatly supported by pyoverdine production and, to a lesser extent, by triabactin. On the contrary, mupirochelin does not contribute to the strain fitness under the studied conditions. Conclusion Altogether, we have demonstrated here that besides pyoverdine, Pseudomonas sp. NCIMB 10586 produces two newly identified siderophores, namely mupirochelin, a weak siderophore with strong antagonism activity against G. ultimum, and the potent siderophore triabactin.

2022

A lipid transfer protein ensures nematode cuticular impermeability

The cuticle of C. elegans is impermeable to chemicals, toxins, and pathogens. However, increased permeability is a desirable phenotype because it facilitates chemical uptake. Surface lipids contribute to the permeability barrier. Here, we identify the lipid transfer protein GMAP-1 as a critical element setting the permeability of the C. elegans cuticle. A gmap-1 deletion mutant increases cuticular permeability to sodium azide, levamisole, Hoechst, and DiI. Expressing GMAP-1 in the hypodermis or transiently in the adults is sufficient to rescue this gmap-1 permeability phenotype. GMAP-1 protein is secreted from the hypodermis to the aqueous fluid filling the space between collagen fibers of the cuticle. In vitro, GMAP-1 protein binds phosphatidylserine and phosphatidylcholine while in vivo, GMAP-1 sets the surface lipid composition and organization. Altogether, our results suggest GMAP-1 secreted by hypodermis shuttles lipids to the surface to form the permeability barrier of C. elegans.

Delayed increase of plasma selenoproteins and absence of side effect induced by infusion of pharmacological dose of sodium selenite in septic shock: Secondary analysis of a multicenter, randomized controlled trial

Background: In sepsis, neutrophil respiratory bursts participate in endothelium damage, the first step to multiple organ failure. In plasma two antioxidant selenoenzymes, which protect the endothelium, decrease: selenoprotein-P, and to a lesser extent glutathione peroxidase (GPX3). Sodium selenite (Na2SeO3) is a Se donor, but also an oxidant chemotherapy drug depending on its concentration. In a previous published study, Na2SeO3 continuous infusion in septic shock patients at a pharmacological dose of 4 mg1 Se/day on day-1, followed by a high nutritional dose of 1 mg Se/day during 9 days, showed no beneficial effect on weaning of catecholamine nor on survival. In this ancillary study, we report clinical and biological effects of such continuous infusion of Na2SeO3. Methods: This was a multicenter, placebo-controlled, double-blind study on 60 patients. Na2SeO3 or placebo in continuous infusion as described above. Evolution with time of plasma Se, selenoprotein-P, GPX3, Organ dysfunction (sequential organ failure assessment SOFA scores, including PaO2/FiO2, for respiratory failure, and plasma lactate) and quality of life at 6 months (by SF36 scores) were analyzed using two-way (time, treatment) non-parametric repeated-measures analysis of variance (Friedman test). Main Results: At baseline, plasma Se was about a quarter of reference values. From baseline to day-4 plasma Se, selenoprotein-P and GPX3 significantly increased by 3.9, 2.7 and 1.8 respectively in the Na2SeO3 group as compared with placebo and remained elevated by 2.3, 2.7 and 2.1 at day-14 respectively (p < 0.001). Na2SeO3 did not affect global and organ by organ SOFA Scores and plasma lactate concentration at day-1 and later up to day-14. The evolution of PaO2/FiO2 until day-14 was similar in the two groups. Quality of life in the surviving patients at 6 months was similar between the two groups. Conclusion: Continuous infusion of Na2SeO3 at 4 mg Se at day-1 seems to have neither beneficial nor toxic effect at day-1 or later and induces a late increase of selenoprotein-P at day-4. Preclinical studies are required to confirm the use of Na2SeO3 as a cytotoxic drug against neutrophils and protection of the endothelium by selenoprotein-P.

Native glycosylation and binding of the antidepressant paroxetine in a low-resolution crystal structure of human myeloperoxidase.

Human myeloperoxidase (MPO) utilizes hydrogen peroxide to oxidize organic compounds and as such plays an essential role in cell-component synthesis, in metabolic and elimination pathways, and in the front-line defence against pathogens. Moreover, MPO is increasingly being reported to play a role in inflammation. The enzymatic activity of MPO has also been shown to depend on its glycosylation. Mammalian MPO crystal structures deposited in the Protein Data Bank (PDB) present only a partial identification of their glycosylation. Here, a newly obtained crystal structure of MPO containing four disulfide-linked dimers and showing an elaborate collection of glycans is reported. These are compared with the glycans identified in proteomics studies and from 18 human MPO structures available in the PDB. The crystal structure also contains bound paroxetine, a blocker of serotonin reuptake that has previously been identified as an irreversible inhibitor of MPO, in the presence of thiocyanate, a physiological substrate of MPO.

Targeted and Untargeted Mass Spectrometry-Based Metabolomics for Chemical Profiling of Three Coffee Species

While coffee beans have been studied for many years, researchers are showing a growing interest in coffee leaves and by-products, but little information is currently available on coffee species other than Coffea arabica and Coffea canephora. The aim of this work was to perform a targeted and untargeted metabolomics study on Coffea arabica, Coffea canephora and Coffea anthonyi. The application of the recent high-resolution mass spectrometry-based metabolomics tools allowed us to gain a clear overview of the main differences among the coffee species. The results showed that the leaves and fruits of Coffea anthonyi had a different metabolite profile when compared to the two other species. In Coffea anthonyi, caffeine levels were found in lower concentrations while caffeoylquinic acid and mangiferin-related compounds were found in higher concentrations. A large number of specialized metabolites can be found in Coffea anthonyi tissues, making this species a valid candidate for innovative healthcare products made with coffee extracts.

Unexpected Role of MPO-Oxidized LDLs in Atherosclerosis: In between Inflammation and Its Resolution

Inflammation and its resolution are the result of the balance between pro-inflammatory and pro-resolving factors, such as specialized pro-resolving mediators (SPMs). This balance is crucial for plaque evolution in atherosclerosis, a chronic inflammatory disease. Myeloperoxidase (MPO) has been related to oxidative stress and atherosclerosis, and MPO-oxidized low-density lipoproteins (Mox-LDLs) have specific characteristics and effects. They participate in foam cell formation and cause specific reactions when interacting with macrophages and endothelial cells. They also increase the production of intracellular reactive oxygen species (ROS) in macrophages and the resulting antioxidant response. Mox-LDLs also drive macrophage polarization. Mox-LDLs are known to be pro-inflammatory particles. However, in the presence of Mox-LDLs, endothelial cells produce resolvin D1 (RvD1), a SPM. SPMs are involved in the resolution of inflammation by stimulating efferocytosis and by reducing the adhesion and recruitment of neutrophils and monocytes. RvD1 also induces the synthesis of other SPMs. In vitro, Mox-LDLs have a dual effect by promoting RvD1 release and inducing a more anti-inflammatory phenotype macrophage, thereby having a mixed effect on inflammation. In this review, we discuss the interrelationship between MPO, Mox-LDLs, and resolvins, highlighting a new perception of the role of Mox-LDLs in atherosclerosis.

Article In Vitro Antioxidant and Anticancer Properties of Various E. senegalensis Extracts

Although Erythrina senegalensis is a plant widely used in traditional medicine in sub-Saharan Africa, its biological properties have been poorly investigated to date. We first characterized by conventional reactions the composition of several stem bark extracts and evaluated in acellular and cellular assays their pro-or antioxidant properties supported by their high phenolic and flavonoid content, particularly with the methanolic extract. The pro-or antioxidant effects observed did not correlate with their IC50 concentrations against five cancer cell lines determined by MTT assay. Indeed, the CH2 Cl2 extract and its ethyl acetate (EtOAc) subfraction appeared more potent although they harbored lower pro-or antioxidant effects. Nevertheless, at equipotent concentration, both extracts induced ER-and mitochondria-derived vacuoles observed by fluorescent microscopy that further led to non-apoptotic cell death. LC coupled to high resolution MS investigations have been performed to identify chemical compounds of the extracts. These investigations highlighted the presence of compounds formerly isolated from E. senegalensis including senegalensein that could be retrieved only in the EtOAc subfraction but also thirteen other compounds, such as 16:3-Glc-stigmasterol and hexadecanoic acid, whose anticancer properties have been previously reported. Nineteen other compounds remain to be identified. In conclusion, E. senegalensis appeared rich in compounds with antioxidant and anticancer properties, supporting its use in traditional practice and its status as a species of interest for further investigations in anticancer drug research.

Use and misuse of prescription stimulants by university students: a cross-sectional survey in the french-speaking community of Belgium, 2018.

Misuse of prescription stimulants (PS) has been reported among students to enhance academic performance in Flanders (Belgium). However, PS misuse among students in the French-speaking community is unknown. The main purpose of the study was to estimate the prevalence of medical use and misuse of PS by university students in the French-speaking community (Belgium), and to investigate the reasons and sources associated with PS misuse.

Effects of the flow diversion technique on nucleotide levels in intra-cranial aneurysms: A feasibility study providing new research perspectives.

The flow diverter stent (FDS) has become a first-line treatment for numerous intra-cranial aneurysms (IAs) by promoting aneurysm thrombosis. However, the biological phenomena underlying its efficacy remain unknown. We proposed a method to collect in situ blood samples to explore the flow diversion effect within the aneurysm sac. In this feasibility study, we assessed the plasma levels of nucleotides within the aneurysm sac before and after flow diversion treatment.

2021

Coffee Leaves: An Upcoming Novel Food?

Unlike those of coffee beans, the healthy properties of coffee leaves have been overlooked for a long time, even if they are consumed as a beverage by local communities of several African countries. Due to the presence of xanthines, diterpenes, xanthones, and several other polyphenol derivatives as main secondary metabolites, coffee leaves might be useful to prevent many daily disorders. At the same time, as for all bioactive molecules, careless use of coffee leaf infusions may be unsafe due to their adverse effects, such as the excessive stimulant effects on the central nervous system or their interactions with other concomitantly administered drugs. Moreover, the presence of some toxic diterpene derivatives requires careful analytical controls on manufactured products made with coffee leaves. Accordingly, knowledge about the properties of coffee leaves needs to be increased to know if they might be considered a good source for producing new supplements. The purpose of the present review is to highlight the biosynthesis, metabolism, and distribution of the 4 main classes of secondary metabolites present in coffee leaves, their main pharmacological and toxicological aspects, and their main roles in planta. Differences in coffee leaf chemical composition depending on the coffee species will also be carefully considered.

Anti-inflammatory, antioxidant effects, and bioaccessibility of Tigzirt propolis

This work aims to assess the anti-inflammatory effects of Tigzirt propolis native to Algeria. We divided 48 male Wistar rats into 8 groups. We orally administered ethyl acetate extract of propolis (EAP), pure polyphenols compounds, or diclofenac 5 days before induction of inflammation by of carrageenan (100 μg/ml, i.p.). We determined the development of paw edema, biological parameters, myeloperoxidase activity, TNF-α, and prostaglandin E2 and measured the oxidative status parameters, as well. Finally, we analyzed the absorption and bioaccessibility of propolis in rats' plasma using GC-MS after orally dosing rats (250 mg/kg). The pretreatment by 200 and 250 mg/kg of propolis significantly reduced the edema rates after the third hour. Propolis can restore the disruption of homeostasis as well as markers of inflammation induced by carrageenan in Wistar rats, and an increase of the enzymatic activities. Furthermore, the inflammation was better resolved in rats that received propolis than in those treated with pure polyphenols. Practical applications: Propolis is a natural mixture that bees produce by mixing gathered resin and gums to bee saliva and wax. Our research investigated the effect of Tigzirt propolis on the inhibition of biomarkers of inflammation and the development of paw edema. Propolis extract helped to reduce PGE2, TNF-α, myeloperoxidase, and malondialdehyde levels and increase the total antioxidant levels in plasma. Our findings emphasized the use of phenolic extract of propolis in industries such as nutraceuticals for the prevention of inflammatory diseases. It can also protect the body against damage under oxidative stress.

Untargeted metabolomics approach to discriminate mistletoe commercial products

Acute effects of hypouricemia on endothelium, oxidative stress, and arterial stiffness: A randomized, double‐blind, crossover study

We hypothesized acute moderate and drastic reductions in uric acid concentration exert different effects on arterial function in healthy normotensive and hypertensive adults. Thirty-six adults (aged 58 [55;63] years) with or without primary hypertension participated in a three-way, randomized, double-blind, crossover study in which [placebo] and [febuxostat] and [febuxostat and rasburicase] were administered. Febuxostat and rasburicase reduce the uric acid concentration by xanthine oxidoreductase inhibition and uric acid degradation into allantoin, respectively. Endothelial function was assessed in response to acetylcholine, sodium nitroprusside, heating (with and without nitric oxide synthase inhibition) using a laser Doppler imager. Arterial stiffness was determined by applanation tonometry, together with blood pressure, renin-angiotensin system activity, oxidative stress, and inflammation. Uric acid concentration was 5.1 [4.1;5.9], 1.9 [1.2;2.2] and 0.2 [0.2;0.3] mg/dL with [placebo], [febuxostat] and [febuxostat-rasburicase] treatments, respectively (p < 0.0001). Febuxostat improved endothelial response to heat particularly when nitric oxide synthase was inhibited (p < 0.05) and reduced diastolic and mean arterial pressure (p = 0.008 and 0.02, respectively). The augmentation index decreased with febuxostat (ANOVA p < 0.04). Myeloperoxidase activity profoundly decreased with febuxostat combined with rasburicase (p < 0.0001). When uric acid dropped, plasmatic antioxidant capacity markedly decreased, while superoxide dismutase activity increased (p < 0.0001). Other inflammatory and oxidant markers did not differ. Acute moderate hypouricemia encompasses minor improvements in endothelial function, blood pressure, and arterial stiffness. Clinical Trial Registration: NCT03395977, https://clinicaltrials.gov/ct2/show/NCT03395977.

Determination of the quality of metronidazole formulations by near-infrared spectrophotometric analysis

In the quality control of medicines and the fight against the phenomenon of poor quality medicines, there is an urgent need for rapid and broad spectrum methods for screening these types of medicines. In the present work, we have used near infrared spectroscopy combined with multivariate data analysis to develop chemometric models for the classification and quantification of metronidazole in Burkina Faso pharmaceutical formulations. For this purpose, drug samples were collected in drugstores located in different Burkina Faso border zones. Four product classes were defined based on the national nomenclature: 3 classes for the generic drugs (C1, C3, and C4) and one class for the reference (C2) drugs. The exploratory analysis using PCA identified two clusters of drugs within class C1. Discrimination was confirmed by the developed and optimised DD-SIMCA model, with only one target class. The quality control of the samples from product class C1 was proven to be very satisfactory with specificities and sensitivities of 100%. The quantification models developed with the PLS-R method were successfully applied for the determination of the active ingredient content in the samples, with acceptable relative bias between 0.15 and 12.7 % with respect to the dose determined by the HPLC method. The RMSEC was estimated at 13.57 (R2, 0.9937), the RMSECV at 18.07 (R2, 0.9888) and the RMSEP at 13.69 (R2, 0.9941).The models developed and the results obtained are promising for routine quality control of similar formulations of metronidazole.

Mass Spectrometry for the Monitoring of Lipoprotein Oxidations by Myeloperoxidase in Cardiovascular Diseases.

Oxidative modifications of HDLs and LDLs by myeloperoxidase (MPO) are regularly mentioned in the context of atherosclerosis. The enzyme adsorbs on protein moieties and locally produces oxidizing agents to modify specific residues on apolipoproteins A-1 and B-100. Oxidation of lipoproteins by MPO (Mox) leads to dysfunctional Mox-HDLs associated with cholesterol-efflux deficiency, and Mox-LDLs that are no more recognized by the LDL receptor and become proinflammatory. Several modification sites on apoA-1 and B-100 that are specific to MPO activity are described in the literature, which seem relevant in patients with cardiovascular risk. The most appropriate analytical method to assess these modifications is based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). It enables the oxidized forms of apoA-1and apoB-100 to be quantified in serum, in parallel to a quantification of these apolipoproteins. Current standard methods to quantify apolipoproteins are based on immunoassays that are well standardized with good analytical performances despite the cost and the heterogeneity of the commercialized kits. Mass spectrometry can provide simultaneous measurements of quantity and quality of apolipoproteins, while being antibody-independent and directly detecting peptides carrying modifications for Mox-HDLs and Mox-LDLs. Therefore, mass spectrometry is a potential and reliable alternative for apolipoprotein quantitation.

Selenocompounds and Sepsis: Redox Bypass Hypothesis for Early Diagnosis and Treatment: Part A - Early Acute Phase of Sepsis: An Extraordinary Redox Situation (Leukocyte/Endothelium Interaction Leading to Endothelial Damage)

Significance: Sepsis is a health disaster. In sepsis, an initial, beneficial local immune response against infection evolves rapidly into a generalized, dysregulated response or a state of chaos, leading to multiple organ failure. Use of life-sustaining supportive therapies creates an unnatural condition, enabling the complex cascades of the sepsis response to develop in patients who would otherwise die. Multiple attempts to control sepsis at an early stage have been unsuccessful. Recent Advances: Major events in early sepsis include activation and binding of leukocytes and endothelial cells in the microcirculation, damage of the endothelial surface layer (ESL), and a decrease in the plasma concentration of the antioxidant enzyme, selenoprotein-P. These events induce an increase in intracellular redox potential and lymphocyte apoptosis, whereas apoptosis is delayed in monocytes and neutrophils. They also induce endothelial mitochondrial and cell damage. Critical Issues: Neutrophil production increases dramatically, and aggressive immature forms are released. Leukocyte cross talk with other leukocytes and with damaged endothelial cells amplifies the inflammatory response. The release of large quantities of reactive oxygen, halogen, and nitrogen species as a result of the leukocyte respiratory burst, endothelial mitochondrial damage, and ischemia/reperfusion processes, along with the marked decrease in selenoprotein-P concentrations, leads to peroxynitrite damage of the ESL, reducing flow and damaging the endothelial barrier. Future Directions: Endothelial barrier damage by activated leukocytes is a time-sensitive event in sepsis, occurring within hours and representing the first step toward organ failure and death. Reducing or stopping this event is necessary before irreversible damage occurs.

Fc Glycosylation Characterization of Human Immunoglobulins G Using Immunocapture and LC-MS.

Immunoglobulins G (IgG) are proteins produced by the immune system of higher life forms that play a central role in the defense against microbial pathogens. IgG bind pathogens with the hypervariable Fab component and mediate a diversity of effector functions by binding to immune effector cells via their crystallizable (Fc) component. All IgG Fc carry a polymorphic N-glycan that regulates its binding properties and thereby its effector functions. The glycosylation profile of IgG Fc is modulated by physiological and pathological conditions, including infectious diseases and inflammatory disorders. Characterization of IgG Fc glycosylation profiles is a promising approach to understand the pathogenesis of diseases involving the immune system and to develop novel biomarkers of disease activity. Measuring the proportion of the different IgG Fc glycoforms remains an analytical challenge, that requires a sensitive and reproducible analytical approach.This chapter describes an optimized approach for the preparation and the analysis of Fc N-glycans from total serum or plasma IgG using magnetic beads, RapiFluor MS label©, and LC-MS.

Effects of hyperoxia and cardiovascular risk factors on myocardial ischaemia-reperfusion injury : a randomized, sham-controlled parallel study

Does the Phytochemical Diversity of Wild Plants Like the Erythrophleum genus Correlate with Geographical Origin?

Secondary metabolites are essential for plant survival and reproduction. Wild undomesticated and tropical plants are expected to harbor highly diverse metabolomes. We investigated the metabolomic diversity of two morphologically similar trees of tropical Africa, Erythrophleum suaveolens and E. ivorense, known for particular secondary metabolites named the cassaine-type diterpenoids. To assess how the metabolome varies between and within species, we sampled leaves from individuals of different geographic origins but grown from seeds in a common garden in Cameroon. Metabolites were analyzed using reversed phase LC-HRMS(/MS). Data were interpreted by untargeted metabolomics and molecular networks based on MS/MS data. Multivariate analyses enabled us to cluster samples based on species but also on geographic origins. We identified the structures of 28 cassaine-type diterpenoids among which 19 were new, 10 were largely specific to E. ivorense and five to E. suaveolens. Our results showed that the metabolome allows an unequivocal distinction of morphologically-close species, suggesting the potential of metabolite fingerprinting for these species. Plant geographic origin had a significant influence on relative concentrations of metabolites with variations up to eight (suaveolens) and 30 times (ivorense) between origins of the same species. This shows that the metabolome is strongly influenced by the geographical origin of plants (i.e., genetic factors).

2020

A patent review of myeloperoxidase inhibitors for treating chronic inflammatory syndromes (focus on cardiovascular diseases, 2013-2019)

Introduction: Myeloperoxidase (MPO) is an immune enzyme found in neutrophils and macrophages. It produces the highly oxidative compound HOCl from H2O2 and Cl− ions inside the phagosome of the neutrophil. Leakage of the enzyme outside the cell causes oxidative damages for the biomolecules promoting many inflammatory diseases such as atherosclerosis. Thus, there is a real interest to develop potent inhibitors of MPO as non-steroidal anti-inflammatory agents. This review highlights the several published MPO inhibitors, their activity, and the challenges met during the development of these compounds. Areas covered: This article covers the patent literature published on MPO inhibitors from 2013 to 2019, as well as the potential use of these compounds as therapeutics for inflammatory syndromes, especially that plays an important role in the initiation and progression of atherosclerosis. Expert opinion: To date, many MPO inhibitors with different structures have been studied, many of which have prominent inhibitory activities. Furthermore, the specificity of these drugs offers hope for the targeted therapy of inflammatory syndromes. Although many data have proved that MPO can contribute to several chronic inflammatory syndromes, the usefulness of MPO inhibitors in preventing and treating inflammatory disorders is still under investigation.

A new potential anti‐cancer beta‐carboline derivative decreases the expression levels of key proteins involved in glioma aggressiveness: A proteomic investigation

Gliomas remain highly fatal due to their high resistance to current therapies. Deregulation of protein synthesis contributes to cancer onset and progression and is a source of rising interest for new drugs. CM16, a harmine derivative with predicted high blood-brain barrier penetration, exerts antiproliferative effects partly through translation inhibition. We evaluated herein how CM16 alters the proteome of glioma cells. The analysis of the gel-free LC/MS and auto-MS/MS data showed that CM16 induces time- and concentration-dependent significant changes in the total ion current chromatograms. In addition, we observed spontaneous clustering of the samples according to their treatment condition and their proper classification by unsupervised and supervised analyses, respectively. A two-dimensional gel-based approach analysis allowed us to identify that treatment with CM16 may downregulate four key proteins involved in glioma aggressiveness and associated with poor patient survival (HspB1, BTF3, PGAM1, and cofilin), while it may upregulate galectin-1 and Ebp1. Consistently with the protein synthesis inhibition properties of CM16, HspB1, Ebp1, and BTF3 exert known roles in protein synthesis. In conclusion, the downregulation of HspB1, BTF3, PGAM1 and cofilin bring new insights in CM16 antiproliferative effects, further supporting CM16 as an interesting protein synthesis inhibitor to combat glioma.

2019

Severe Hypouricemia Impairs Endothelium-Dependent Vasodilatation and Reduces Blood Pressure in Healthy Young Men: A Randomized, Placebo-Controlled, and Crossover Study.

Background Uric acid (UA) is a plasmatic antioxidant that has possible effects on blood pressure. The effects of UA on endothelial function are unclear. We hypothesize that endothelial function is not impaired unless significant UA depletion is achieved through selective xanthine oxidase inhibition with febuxostat and recombinant uricase (rasburicase). Methods and Results Microvascular hyperemia, induced by iontophoresis of acetylcholine and sodium nitroprusside, and heating-induced local hyperemia after iontophoresis of saline and a specific nitric oxide synthase inhibitor were assessed by laser Doppler imaging. Blood pressure and renin-angiotensin system markers were measured, and arterial stiffness was assessed. CRP (C-reactive protein), allantoin, chlorotyrosine/tyrosine ratio, homocitrulline/lysine ratio, myeloperoxidase activity, malondialdehyde, and interleukin-8 were used to characterize inflammation and oxidative stress. Seventeen young healthy men were enrolled in a randomized, double-blind, placebo-controlled, 3-way crossover study. The 3 compared conditions were placebo, febuxostat alone, and febuxostat together with rasburicase. The allantoin (μmol/L)/UA (μmol/L) ratio differed between sessions (P<0.0001). During the febuxostat-rasburicase session, heating-induced hyperemia became altered in the presence of nitric oxide synthase inhibition; and systolic blood pressure, angiotensin II, and myeloperoxidase activity decreased (P≤0.03 versus febuxostat). The aldosterone concentration decreased in the febuxostat-rasburicase group (P=0.01). Malondialdehyde increased when UA concentration decreased (both P<0.01 for febuxostat and febuxostat-rasburicase versus placebo). Other parameters remained unchanged. Conclusions A large and short-term decrease in UA in humans alters heat-induced endothelium-dependent microvascular vasodilation, slightly reduces systolic blood pressure through renin-angiotensin system activity reduction, and markedly reduces myeloperoxidase activity when compared with moderate UA reduction. A moderate or severe hypouricemia leads to an increase in lipid peroxidation through loss of antioxidant capacity of plasma. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT03395977.

Dysregulation of Macropinocytosis Processes in Glioblastomas May Be Exploited to Increase Intracellular Anti-Cancer Drug Levels: The Example of Temozolomide

Chemical composition of propolis extract and its effects on epirubicin-induced hepatotoxicity in rats

Propolis is a natural substance, produced by honeybees from the resin of various plants. The purpose of this study was to determine the chemical composition and evaluate the hepatoprotective effect of ethyl acetate extract of propolis from Tigzirt, against the toxicity induced by epirubicin which is a anticancer agent, and belongs to the family of antracyclines. The study included thirty male Wistar albino rats divided into five groups. The rats received the extraction of propolis or the quercetin orally for 15 days. The hepatotoxicity was promoted by injection epirubicin (i.v.) with a cumulative dose of 9 mg/kg. Several biological parameters were measured. Oxidative status was also assessed by evaluating antioxidant enzyme and histological study of some organs. Epirubicin caused oxidative stress by a significant decrease in hepatic antioxidant enzymes (gluthation peroxidase, catalase, superoxide dismutase), increased malondialdehyde and liver parameters (ASAT, ALAT, γGT, ALP) compared to the control. The histological study revealed major damage to the liver. Perturbations in this liver function, antioxidant status and damage to the liver by epirubicin have been repaired by the administration of propolis. Furthermore, epirubicin showed inflammatory effects induced by an increase in TNF-α and PGE2. Pretreatment with propolis to rats restored these inflammatory parameters. The chemical identification of extract of propolis by HPLC/UV shows the presence of polyphenolic compounds and many flavonoids. The propolis extract showed a significant reduction in oxidative damage from oxidative stress and a very important protective effect against epirubicin-induced hepatotoxicity.

The soluble curcumin derivative NDS27 inhibits superoxide anion production by neutrophils and acts as substrate and reversible inhibitor of myeloperoxidase.

A water-soluble curcumin lysinate incorporated into hydroxypropyl-β-cyclodextrin (NDS27) has been developed and shown anti-inflammatory properties but no comparative study has been made in parallel with its parent molecule, curcumin on polymorphonuclear neutrophils (PMNs) and myeloperoxidase (MPO) involved in inflammation. The effect of NDS27, its excipients (hydroxypropyl-β-cyclodextrin and lysine), curcumin lysinate and curcumin were compared on the release of superoxide anion by PMNs using a chemiluminescence assay and on the enzymatic activity of MPO. It was shown that curcumin and NDS27 exhibit similar inhibition activities on superoxide anion release by stimulated PMNs but also on MPO peroxidase and halogenation activities. The action mechanism of curcumin and NDS27 on the MPO activity was refined by stopped-flow and docking analyses. We demonstrate that both curcumin and NDS27 are reversible inhibitors of MPO by acting as excellent electron donors for redox intermediate Compound I (∼107 M-1 s-1) but not for Compound II (∼103 M-1 s-1) in the peroxidase cycle of the enzyme, thereby trapping the enzyme in the Compound II state. Docking calculations show that curcumin is able to enter the enzymatic pocket of MPO and bind to the heme cavity by π-stacking and formation of hydrogen bonds involving substituents from both aromatic rings. Hydroxypropyl-β-cyclodextrin is too bulky to enter MPO channel leading to the binding site suggesting a full release of curcumin from the cyclodextrin thereby allowing its full access to the active site of MPO. In conclusion, the hydroxypropyl-β-cyclodextrin of NDS27 enhances curcumin solubilization without affecting its antioxidant capacity and inhibitory activity on MPO.

Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices.

Nucleotides play a role in inflammation processes: cAMP and cGMP in the endothelial barrier function, ADP in platelet aggregation, ATP and UTP in vasodilatation and/or vasoconstriction of blood vessels, UDP in macrophages activation. The aim of this study is to develop and validate a LC/MS-MS method able to quantify simultaneously nine nucleotides (AMP, cAMP, ADP, ATP, GMP, cGMP, UMP, UDP and UTP) in biological matrixes (cells and plasma). The method we developed, has lower LOQ's than others and has the main advantage to quantify all nucleotides within one single injection in less than 10 min. The measured nucleotides concentrations obtained with this method are similar to those obtained with assay kits commercially available. Analysis of plasma and red blood cells from healthy donors permits to estimate the physiological concentration of those nucleotides in human plasma and red blood cells, such information being poorly available in the literature. Furthermore, the protocol presented in this paper allowed us to observe that AMP, ADP, ATP concentrations are modified in human red blood cells and plasma after a venous stasis of 4 min compared to physiological blood circulation. Therefore, this specific method enables future studies on nucleotides implications in chronic inflammatory diseases but also in other pathologies where nucleotides are implicated in.

2018

Apoliporotein L3 interferes with endothelial tube formation via regulation of ERK1/2, FAK and Akt signaling pathway.

Endothelial cells are main actors in vascular homeostasis as they regulate vascular pressure and permeability as well as hemostasis and inflammation. Disturbed stimuli delivered to and by endothelial cells correlate with the so-called endothelial dysfunction and disrupt this homeostasis. As constituents of the inner layer of blood vessels, endothelial cells are also involved in angiogenesis. Apolipoprotein Ls (APOL) comprise a family of newly discovered apolipoproteins with yet poorly understood function, and are suggested to be involved in inflammatory processes and cell death mechanisms. Here we investigate the role of APOLs in endothelial cells stimulated with factors known to be involved in atherogenesis and their possible contribution to endothelial dysfunction with an emphasis on inflammation driven-angiogenesis in vitro.

Myeloperoxidase promotes tube formation, triggers ERK1/2 and Akt pathways and is expressed endogenously in endothelial cells

Myeloperoxidase is a member of the mammalian peroxidase family, mainly expressed in the myeloblastic cell lineage. It is considered a major bactericidal agent as it is released in the phagosome where it catalyzes the formation of reactive oxygen species. It is also released in the extracellular spaces including blood where it is absorbed on (lipo)proteins and endothelial cell surface, interfering with endothelial function. We performed RNA sequencing on MPO-treated endothelial cells, analyzed their transcriptome and validated the profile of gene expression by individual qRT-PCR. Some of the induced genes could be grouped in several functional networks, including tubulogenesis, angiogenesis, and blood vessel morphogenesis and development as well as signal transduction pathways associated to these mechanisms. MPO treatment mimicked the effects of VEGF on several signal transduction pathways, such as Akt, ERK or FAK involved in angiogenesis. Accordingly MPO, independently of its enzymatic activity, stimulated tube formation by endothelial cells. RNA interference also pointed at a role of endogenous MPO in tubulogenesis and endothelium wound repair in vitro. These data suggest that MPO, whether from endogenous or exogenous sources, could play a role in angiogenesis and vascular repair in vivo.

Myeloperoxidase et Volume Prostatique: étude préliminaire

Objectives: Oxidative stress is associated with the development of BPH and might be modulated by several factors. Myeloperoxidase (MPO) has recently been observed in prostate tissue. Our goal was to investigate the correlation between MPO and the prostate volume. Material and methods: Hundred and twenty-one patients (48-70 years) with a filled IPSS were prospectively included. Blood sampling (PSA, testosterone, Angiotensin II (AngII), MPO, Mox-LDL) and transrectal ultrasound of the prostate were performed with total volume (TV) and transitional zone volume (TZ) measurements. For correlation, univariate analyses were depicted by Pearson's coefficient. Multilinear regression analysis used a stepwise backward selection of the explicative variables. Results: In multivariate analysis, the TV was positively correlated to the combination of age and Ang II but negatively to MPO specific activity (Std Coef = −0.272, P = 0.004). Significant correlations were confirmed between TZ, age and MPO specific activity but not with Ang II. A negative correlation between TZ and MPO specific activity was also observed (Std Coef = −0.21, P = 0.016). No correlation was found with Mox-LDL. Conclusions: Negative correlation between MPO and prostate volume was observed but careful interpretations may be endorsed and longitudinal study is necessary. It seems relevant to focus on the potential contribution of MPO in the development of prostatic diseases as this enzyme can also promote DNA oxidation. Level of evidence: 4.

Electrochemical studies of ethoxyquin and its determination in salmon samples by flow injection analysis with an amperometric dual detector

Ethoxyquin (EQ) is an antioxidant widely used in the food industry. Some concerns for human health have been reported since its utilization in animal feed may lead to residues in human food such as salmon samples. This work aimed to investigate the electrochemical behavior of EQ and its major oxidation products namely a dimer of EQ (EQDM) and ethoxyquin quinone imine (EQI) by cyclic voltammetry (CV) at a carbon screen printed electrode (cSPE). The stability of these products and their reactivity against glutathione (GSH) were also studied. Oxidized products and thiol adducts were identified by a microelectrolysis of an EQ solution onto a cSPE in the absence and in the presence of GSH. The products were analyzed off-line, by liquid chromatography coupled to a mass spectrometer (LC-MS). One of the generated product (EQI) was shown to be highly reactive towards GSH. Based on the redox pattern of EQ, a flow injection analysis with a dual cSPE was developed in order to detect in a rapid manner EQ in salmon samples. Since matrix effects were observed, matrix-matched calibration curves with spiked samples were built. A linear response was obtained between 20-100 μM and a limit of detection (LOD) of 7.5 μM (8.2 mg/kg of salmon). Trueness was assessed with recovery assays at three levels of concentration. The recovery was close to 100 %. Precision was determined as RSD (%) with values lower than 6 % in all cases.

The other myeloperoxidase: Emerging functions

Myeloperoxidase (MPO) is a member of the mammalian peroxidase family. It is mainly expressed in neutrophils, monocytes and macrophages. As a catalyzer of reactive oxidative species and radical species formation, it contributes to neutrophil bactericidal activity. Nevertheless MPO invalidation does not seem to have major health consequences in affected individuals. This suggests that MPO might have alternative functions supporting its conservation during evolution. We will review the available data supporting these non-canonical functions in terms of tissue specific expression, function and enzymatic activity. Thus, we discuss its cell type specific expression. We review in between others its roles in angiogenesis, endothelial (dys-) function, immune reaction, and inflammation. We summarize its pathological actions in clinical conditions such as cardiovascular disease and cancer.

Human peroxidasin 1 promotes angiogenesis through ERK1/2, Akt and FAK pathways.

The term angiogenesis refers to sprouting of new blood vessels from pre-existing ones. The angiogenic process involves cell migration and tubulogenesis requiring interaction between endothelial cells and the extracellular matrix. Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase found embedded in the basement membranes. As it promotes the stabilization of extracellular matrix, we investigated its possible role in angiogenesis both in vitro and in vivo.

Data on myeloperoxidase-oxidized low-density lipoproteins stimulation of cells to induce release of resolvin-D1

This article present data related to the publication entitled “Native and myeloperoxidase-oxidized low-density lipoproteins act in synergy to induce release of resolvin-D1 from endothelial cells” (Dufour et al., 2018). The supporting materials include results obtained by Mox-LDLs stimulated macrophages and investigation performed on scavenger receptors. Linear regressions (RvD1 vs age of mice and RvD1 vs CL-Tyr/Tyr) and Data related to validation were also presented. The interpretation of these data and further extensive insights can be found in Dufour et al. (2018) [1].

Native and myeloperoxidase-oxidized low-density lipoproteins act in synergy to induce release of resolvin-D1 from endothelial cells.

Oxidation of native low-density lipoproteins (LDLs-nat) plays an important role in the development of atherosclerosis. A major player in LDL-nat oxidation is myeloperoxidase (MPO), a heme enzyme present in azurophil granules of neutrophils and monocytes. MPO produces oxidized LDLs called Mox-LDLs, which cause a pro-inflammatory response in human microvascular endothelial cells (HMEC), monocyte/macrophage activation and formation of foam cells. Resolvin D1 (RvD1) is a compound derived from the metabolism of the polyunsaturated fatty acid DHA, which promotes resolution of inflammation at the ng/ml level.

Metabolomics fingerprint of coffee species determined by untargeted-profiling study using LC-HRMS

Coffee bean extracts are consumed all over the world as beverage and there is a growing interest in coffee leaf extracts as food supplements. The wild diversity in Coffea (Rubiaceae) genus is large and could offer new opportunities and challenges. In the present work, a metabolomics approach was implemented to examine leaf chemical composition of 9 Coffea species grown in the same environmental conditions. Leaves were analyzed by LC-HRMS and a comprehensive statistical workflow was designed. It served for univariate hypothesis testing and multivariate modeling by PCA and partial PLS-DA on the Workflow4Metabolomics infrastructure. The first two axes of PCA and PLS-DA describes more than 40% of variances with good values of explained variances. This strategy permitted to investigate the metabolomics data and their relation with botanic and genetic informations. Finally, the identification of several key metabolites for the discrimination between species was further characterized.

New Folate-Grafted Chitosan Derivative To Improve Delivery of Paclitaxel-Loaded Solid Lipid Nanoparticles for Lung Tumor Therapy by Inhalation.

Inhaled chemotherapy for the treatment of lung tumors requires that drug delivery systems improve selectivity for cancer cells and tumor penetration and allow sufficient lung residence. To this end, we developed solid lipid nanoparticles (SLN) with modified surface properties. We successfully synthesized a new folate-grafted copolymer of polyethylene glycol (PEG) and chitosan, F-PEG-HTCC, with a PEG-graft ratio of 7% and a molecular weight range of 211-250 kDa. F-PEG-HTCC-coated, paclitaxel-loaded SLN were prepared with an encapsulation efficiency, mean diameter, and zeta potential of about 100%, 250 nm, and +32 mV, respectively. The coated SLN entered folate receptor (FR)-expressing HeLa and M109-HiFR cells in vitro and M109 tumors in vivo after pulmonary delivery. The coated SLN significantly decreased the in vitro half-maximum inhibitory concentrations of paclitaxel in M109-HiFR cells (60 vs 340 nM, respectively). We demonstrated that FR was involved in these improvements, especially in M109-HiFR cells. After pulmonary delivery in vivo, the coated SLN had a favorable pharmacokinetic profile, with pulmonary exposure to paclitaxel prolonged to up to 6 h and limited systemic distribution. Our preclinical findings therefore demonstrated the positive impact of the coated SLN on the delivery of paclitaxel by inhalation.

2017

Relationship between oxidative stress and erectile function.

The aim of this study was to investigate markers of inflammation and oxidative stress in the corpus cavernosum (CC) and to compare levels of inflammatory markers recorded in CC to venous blood from the arm to examine the potential impact of inflammatory parameters on erectile function and endothelial dysfunction in vitro. Ninety-seven patients with no complaint of erectile dysfunction (ED) at inclusion were prospectively included and completed the Erectile Function domain of the IIEF questionnaire. Several parameters, including lipids, MPO-dependent oxidised LDL (Mox-LDL), IL-8, IL-18, were measured. After RNA extraction, the expression of eNOS was analysed. A paired t-test was used for comparisons between arm and CC blood results. A two-way ANOVA was used to estimate the effects of IL-18 and IL-8 on the IIEF score. Mean patient age was 59 ± 14.5 years. IL-18, Mox-LDL, and Mox-LDL/ApoB levels were significantly increased in CC compared to arm blood. The IIEF score was correlated with IL-18 levels in the venous blood (R = -0.31, p = .003) and in the CC (R = -0.37, p = .004) and with IL-8 (R = -0.31, p = .009 and R = -0.28, respectively, p = .02). There was a significant effect with the IL-18 on IIEF potentiated by high serum IL-8 concentrations. IL-18 and Mox-LDL significantly decreased eNOS mRNA expression in human aortic endothelial cell line (HAEC). These preliminary results address the importance of inflammation in the CC and highlight a difference in marker concentrations between venous and CC blood. However, they do not show any difference in terms of clinical erectile score predictivity. Involvement of inflammatory cytokines isolated in CC in the genesis of ED requires further studies.

Antioxidant effects and bioavailability evaluation of propolis extract and its content of pure polyphenols

Propolis is a resinous mixture which honey bees collect from buds and bark of some trees. It contains several antioxidant compounds such as polyphenols and flavonoids. This paper describes the antioxidant capacity of propolis extracts and pure polyphenols in vivo. Several biological parameters were measured after promoting oxidative stress. All the homeostasis changes and liver, kidney, and lung damage were restored by propolis extract. Propolis extracts directly modulated lipid peroxidation where LDL level decreased from 1.3 to 0.8 g/L after giving propolis, illustrating the antioxidant contribution of propolis. Finally, orally administered pure antioxidant compounds such as caffeic acid, quercetin, and kaempferol at the doses corresponding to in vitro antioxidant capacity of the propolis extract did not restore the physiological parameters. Bioavailability study demonstrated that the isolated polyphenols have lower absorption (plasma Cmax of polyphenols = 15-20 µg/mL) than the complex extract of propolis (plasma Cmax of polyphenols = 40-50 µg/mL). Practical applications: Extracts of propolis showed significant reduction of oxidative damages from oxidative stress. The complexity of propolis has an antioxidant capacity more than its isolated polyphenols. Monitoring the concentrations of the compounds in serum after orally administering of propolis and isolated polyphenols demonstrated that these antioxidant compounds have higher bioavailability when applied as crude extracts. These results make the propolis extracts a promising antioxidant which could be used as a food supplement.

Batch-to-batch N-glycosylation Study of Infliximab, Trastuzumab, and Bevacizumab, and Stability Study of Bevacizumab

Objectives Infliximab, trastuzumab and bevacizumab are among the most frequently prescribed therapeutic proteins, and like most other therapeutic proteins, are glycosylated. As differences in glycosylation may significantly change the safety and efficacy of therapeutic glycoproteins, it is extremely important to control N-glycosylation consistency. In the first part of this study, the batch-to-batch consistency of the N-glycosylation of infliximab, trastuzumab and bevacizumab was analysed. In the second part, the consistency of the N-glycosylation of bevacizumab stored in polycarbonate syringes (for off-label drug use) for 3 months was examined. Methods N-glycans were (i) enzymatically released using peptide-N-glycosidase F, (ii) reduced, and (iii) analysed using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry. Mass spectrometry data were interpreted using principal component analysis combined with two-way analysis of variance and Tukey post hoc tests. The biological activity of infliximab and trastuzumab was examined using enzyme-linked immunosorbent assays. Results The results of both studies make important contributions to the field of hospital pharmacy. All batches of the studied therapeutic glycoproteins (infliximab, trastuzumab and bevacizumab) varied considerably (especially in galactosylation), while the N-glycosylation of bevacizumab remained unchanged during 3-month storage. Conclusions Threshold values for batch-to-batch N-glycosylation variations should be established and batch-to-batch glycosylation consistency should be regularly tested. In our study, samples with significantly different N-glycosylation profiles showed no significant variations in biological activity, suggesting that the differences are probably not therapeutically significant.

Discovery of Novel Potent Reversible and Irreversible Myeloperoxidase Inhibitors Using Virtual Screening Procedure

The heme enzyme myeloperoxidase (MPO) participates in innate immune defense mechanism through formation of microbicidal reactive oxidants. However, evidence has emerged that MPO-derived oxidants contribute to propagation of inflammatory diseases. Because of the deleterious effects of circulating MPO, there is a great interest in the development of new efficient and specific inhibitors. Here, we have performed a novel virtual screening procedure, depending on ligand-based pharmacophore modeling followed by structure-based virtual screening. Starting from a set of 727842 compounds, 28 molecules were selected by this virtual method and tested on MPO in vitro. Twelve out of 28 compounds were found to have an IC50 less than 5 μM. The best inhibitors were 2-(7-methoxy-4-methylquinazolin-2-yl)guanidine (28) and (R)-2-(1-((2,3-dihydro-1H-imidazol-2-yl)methyl)pyrrolidin-3-yl)-5-fluoro-1H-benzo[d]imidazole (42) with IC50 values of 44 and 50 nM, respectively. Studies on the mechanism of inhibition suggest that 28 is the first potent mechanism-based inhibitor and inhibits irreversibly MPO at nanomolar concentration.

The waste of saffron crop, a cheap source of bioactive compounds

The present study aimed to estimate the biological valorization potential of food saffron by-products through biological activities of phenol content in alcoholic and ethyl acetate extracts of five by-products of Crocus sativus L.: leaves, tepals, spaths, corm, and tunics. The results evidence the presence of products with bioactive capacities in cheap by-products like spaths, leaves and corms. The LC-MS/MS analysis showed methanolic fraction of stigmas contained as major products the crocins while fractions of spaths and leaves contained predominantly flavonoids. We also showed the presence of flavonols and flavones which exhibit antioxidant capacities, activate proteasome in human dermal fibroblast cells from aged donors and inhibit the growth and proteasome activity of cancer cells. Altogether, our data indicate that compounds present in saffron's by-products, such as the leaves or spaths, might be valuable for food valorization and should be studied as a useful biological product for its anti-aging and anti-cancer abilities.

Tannins and Tannin-Related Derivatives Enhance the (Pseudo-)Halogenating Activity of Lactoperoxidase

Several hydrolyzable tannins, proanthocyanidins, tannin derivatives, and a tannin-rich plant extract of tormentil rhizome were tested for their potential to regenerate the (pseudo-)halogenating activity, i.e., the oxidation of SCN- to hypothiocyanite -OSCN, of lactoperoxidase (LPO) after hydrogen peroxide-mediated enzyme inactivation. Measurements were performed using 5-thio-2-nitrobenzoic acid in the presence of tannins and related substances in order to determine kinetic parameters and to trace the LPO-mediated -OSCN formation. The results were combined with docking studies and molecular orbital analysis. The -OSCN-regenerating effect of tannin derivatives relates well with their binding properties toward LPO as well as their occupied molecular orbitals. Especially simple compounds like ellagic acid or methyl gallate and the complex plant extract were found as potent enzyme-regenerating compounds. As the (pseudo-)halogenating activity of LPO contributes to the maintenance of oral bacterial homeostasis, the results provide new insights into the antibacterial mode of action of tannins and related compounds. Furthermore, chemical properties of the tested compounds that are important for efficient enzyme-substrate interaction and regeneration of the -OSCN formation by LPO were identified.

A physical description of the adhesion and aggregation of platelets

The early stages of clot formation in blood vessels involve platelet adhesion-aggregation. Although these mechanisms have been extensively studied, gaps in their understanding still persist. We have performed detailed in vitro experiments, using the well-known Impact-R device, and developed a numerical model to better describe and understand this phenomenon. Unlike previous studies, we took into account the differential role of pre-activated and non-activated platelets, as well as the three-dimensional nature of the aggregation process. Our investigation reveals that blood albumin is a major parameter limiting platelet aggregate formation in our experiment. Simulations are in very good agreement with observations and provide quantitative estimates of the adhesion and aggregation rates that are hard to measure experimentally. They also provide a value of the effective diffusion of platelets in blood subject to the shear rate produced by the Impact-R.

From Dynamic Combinatorial Chemistry to in Vivo Evaluation of Reversible and Irreversible Myeloperoxidase Inhibitors.

The implementation of dynamic combinatorial libraries allowed the determination of highly active reversible and irreversible inhibitors of myeloperoxidase (MPO) at the nanomolar level. Docking experiments highlighted the interaction between the most active ligands and MPO, and further kinetic studies defined the mode of inhibition of these compounds. Finally, in vivo evaluation showed that one dose of irreversible inhibitors is able to suppress the activity of MPO after inducing inflammation.

LC-MS analysis combined with principal component analysis and soft independent modelling by class analogy for a better detection of changes in N-glycosylation profiles of therapeutic glycoproteins.

Therapeutic proteins are among the top selling drugs in the pharmaceutical industry. More than 60 % of the approved therapeutic proteins are glycosylated. Nowadays, it is well accepted that changes in glycosylation may affect the safety and the efficacy of the therapeutic proteins. For this reason, it is important to characterize both the protein and the glycan structures. In this study, analytical and data processing methods were developed ensuring an easier characterization of glycoprofiles. N-glycans were (i) enzymatically released using peptide-N-glycosidase F (PNGase F), (ii) reduced, and (iii) analyzed by hydrophilic interaction liquid chromatography coupled to a high-resolution mass spectrometer (HILIC-HRMS). Glycosylation changes were analyzed in human plasma immunoglobulin G samples which had previously been artificially modified by adding other glycoproteins (such as ribonuclease B and fetuin) or by digesting with enzyme (neuraminidase). Principal component analysis (PCA) and classification through soft independent modelling by class analogy (SIMCA) were used to detect minor glycosylation changes. Using HILIC-MS-PCA/SIMCA approach, it was possible to detect small changes in N-glycosylation, which had not been detected directly from the extracted-ion chromatograms, which is current technique to detect N-glycosylation changes in batch-to-batch analysis. The HILIC-MS-PCA/SIMCA approach is highly sensitive approach due to the sensitivity of MS and appropriate data processing approaches. It could help in assessing the changes in glycosylation, controlling batch-to-batch consistency, and establishing acceptance limits according to the glycosylation changes, ensuring safety and efficacy. Graphical abstract N-glycosylation characterization using LC-MS-PCA approach.

The presence of modified nucleosides in extracellular fluids leads to the specific incorporation of 5-chlorocytidine into RNA and modulates the transcription and translation

Myeloperoxidase (MPO) is able to promote several kinds of damage and is involved in mechanisms leading to various diseases such as atherosclerosis or cancers. An example of these damages is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity. Since 5-chlorocytidine has been recently shown in healthy donor plasmas, this study aimed at discovering if these circulating modified nucleosides could be incorporated into RNA and DNA and if their presence impacts the ability of enzymes involved in the incorporation, transcription, and translation processes. Experimentations, which were carried out in vitro with endothelial and prostatic cells, showed a large penetration of all chloronucleosides but an exclusive incorporation of 5-chlorocytidine into RNA. However, no incorporation into DNA was observed. This specific incorporation is accompanied by an important reduction of translation yield. Although, in vitro, DNA polymerase processed in the presence of chloronucleosides but more slowly than in control conditions, ribonucleotide reductase could not reduce chloronucleotides prior to the replication. This reduction seems to be a limiting step, protecting DNA from chloronucleoside incorporation. This study shows the capacity of transcription enzyme to specifically incorporate 5-chlorocytidine into RNA and the loss of capacity—complete or partial—of different enzymes, involved in replication, transcription or translation, in the presence of chloronucleosides. Questions remain about the long-term impact of such specific incorporation in the RNA and such decrease of protein production on the cell viability and function.

2016

Antimicrobial effects of six Algerian propolis extracts

This study aimed to study the antimicrobial effect of propolis and determine the essential compounds which give it the antimicrobial activity. The antimicrobial effect was investigated for six ethyl acetate extracts of propolis (EAPs) collected from different regions of Algeria on four pathogenic strains (Shigella dysenteriae, Shigella sonnei, Staphylococcus aureus and Escherichia coli) and two benefit strains (Bifidobacterium animalis and Lactobacillus rhamnosus). The results obtained in this study indicated that the different EAPs showed potential inhibitory effects on tested pathogenic strains with variable minimum inhibitory concentration (MIC) values of 0.3 and 9 mg ml−1, while the beneficial bacteria showed resistance against EAPs of propolis. The results demonstrated also that the flavonoid compounds have very low MIC values, while phenolic acid compounds have variables MIC values of 1-10 mg ml−1. These results indicated that propolis contains proven substances with antimicrobial activity and are a prelude to the investigations aimed at empowerment of the bee substance as a potential source of antimicrobial agents with multiple outlets.

D3. Early prediction of preeclampsia risk assessment: analytical determination for marinobufagenin in pregnant women.

Marinobufagenin (MBG), an endogenous bufadienolide cardiac inotrope, is responsible for vasoconstriction and natriuresis [1,2]. Growing interest by its implication in volume expansion mediated hypertensive states such as essential hypertension, congestive heart failure, chronic renal failure, primary aldosterism and preeclampsia (PE). Elevated endogenous MBG levels have been described early in preeclamptic patients [2-5].

Characterization of chemical features of potent myeloperoxidase inhibitors.

Despite its important role in the immune system, myeloperoxidase (MPO) is implicated in a wide range of inflammatory syndromes due to its oxidative product HOCl. The oxidative damages caused by MPO make it a new target for developing promising anti-inflammatory agents. In this paper, we tried to understand the mechanism of MPO inhibition in order to facilitate the drug design, to develop more accurate virtual tests and to understand the structure-activity relationship.

Liquid chromatography-quadrupole time of flight tandem mass spectrometry-based targeted metabolomic study for varietal discrimination of grapes according to plant sterols content

Grapevine and derived products are rich in a wide range of compounds and its quality mainly depends on its metabolites, as a result of viticulture practices. Plant sterols, also called phytosterols (PS), are secondary metabolites regarded as bioactive substance present in grape berries and other plant-based food. The present study deals with a metabolomic approach focusing on phytosterols family in six varieties of Rioja grapes (Cabernet Sauvignon, Tempranillo, Graciano, Garnacha, White Garnacha and Viura), in order to find significant differences among them. Liquid chromatography- mass spectrometry with a quadrupole-time of flight mass analyzer (LC-QTOF) was used to find as many metabolites as possible in the different grape berry fractions, and using statistics to help finding significant clustering of the metabolic profile of pulp, peel and seeds in relation to the variety. The best chromatographic and detection conditions were achieved by gas phase ionization via atmospheric pressure chemical ionization (APCI) in positive mode. Furthermore, analysis with electrospray (ESI) is also needed for phytosterol derivatives confirmation. Putative compounds of interest in the analyzed samples were found by an automated compound extraction algorithm (Molecular Feature Extraction, MFE) and an initial differential expression from the data was created with the aid of commercial software. Once the data were collected, the results were filtered, aligned and normalized, and evaluating applying one-way analysis of variance (ANOVA) with a 95% significance level. For sample class prediction, partial least square-discriminant analysis (PLS-DA) is used as a supervised pattern recognition method and excellent separation among the grape varieties is shown. An overall accuracy of 93.3% (pulp samples), 100.0% (peel) or 96.7% (seeds) in discriminating between grape varieties was achieved when comparing the different fractions. In general, 7 PS derivatives were identified with ID scores higher than 84%.

Validation of a sensitive LC/MSMS method for chloronucleoside analysis in biological matrixes and its applications

Myeloperoxidase promotes several kinds of damage and is involved in the development of various diseases (as atherosclerosis and cancers). An example of these damage is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity on those acids. This study aimed to develop and validate a method to analyze oxidized and MPO-specific chlorinated nucleosides in biological matrixes (cells, tissues and plasma). Although a lot of methods to quantify oxidized or chlorinated nucleosides have already been established, none of them took into account all these derivatives together. The new method used a Triple Quadrupole mass spectrometer fitted with a Jet Stream electrospray ionization source. This approach has two advantages compared with existing LC/MSMS analyses: it includes MPO-induced modifications in a unique analysis and obtains a better sensitivity. Our optimized method reached LOQs of 1.50 pg and 1.42 pg respectively for oxoG and oxo(d)G, being 4 times more sensitive than previous methods, and LOQs of 1.39 pg, 1.30 pg and 63.4 fg respectively for 5-chlorocytidine, 5-chloro-2′-deoxycytidine and 8-chloroguanosine. Developed method is also 25 times more sensitive for chloroguanosine than the best existing method. Nevertheless, this method is not specific enough for 8-chloro-(2′-deoxy)adenosine analysis. Examples of applications demonstrate the interest of this validated method. Indeed analysis of plasma from healthy donors highlighted exclusively the presence of 5-chlorocytidine (1.0±0.2 nM) whereas analysis of treated endothelial cells by HOCl showed chlorination of guanosine and cytidine in cytoplasmic pools and chlorination of (deoxy)cytidine in DNA and RNA. In conclusion, this study shows that 5-chloro-2′-deoxycytidine, 5-chlorocytidine and 8-chloroguanosine are good markers allowing us to detect the MPO activity in biological fluids. The robust, specific and sensitive developed method enables future studies on MPO implications in human diseases.

Novel bis-arylalkylamines as myeloperoxidase inhibitors: Design, synthesis, and structure-activity relationship study.

Human myeloperoxidase (MPO) plays an important role in innate immunity but also aggravates tissue damage by oxidation of biomolecules at sites of inflammation. As a result from a recent high-throughput virtual screening approach for MPO inhibitors, bis-2,2'-[(dihydro-1,3(2H,4H) pyrimidinediyl)bis(methylene)]phenol was detected as a promising lead compound for inhibition of the MPO-typical two-electron oxidation of chloride to hypochlorous acid (IC50 = 0.5 μM). In the present pharmacomodulation study, 37 derivatives of this lead compound were designed and synthesized driven by comprehensive docking studies and the impact on the chlorination activity of MPO. We describe the structural requirements for optimum (i) binding to the heme periphery and (ii) inhibition capacity. Finally, the best three inhibitors (bis-arylalkylamine derivatives) were probed for interaction with the MPO redox intermediates Compound I and Compound II. Determined apparent bimolecular rate constants together with determination of reduction potential and nucleophilicity of the selected compounds allowed us to propose a mechanism of inhibition. The best inhibitor was found to promote the accumulation of inactive form of MPO-Compound II and has IC50 = 54 nM, demonstrating the successful approach of the drug design. Due to the similarity of ligand interactions between MPO and serotonine transporter, the selectivity of this inhibitor was also tested on the serotonin transporter providing a selectivity index of 14 (KiSERT/IC50MPO).

Flavonoids as promoters of the (pseudo-)halogenating activity of lactoperoxidase and myeloperoxidase.

In this study several flavonoids were tested for their potential to regenerate the (pseudo-)halogenating activity (hypothiocyanite formation) of the heme peroxidases lactoperoxidase (LPO) and myeloperoxidase (MPO) after hydrogen peroxide-mediated enzyme inactivation. Several flavonoid subclasses with varying hydroxylation patterns (especially of the flavonoid B-ring) were examined in order to identify structural properties of efficient enzyme regenerators. Kinetic parameters and second-order rate constants were determined. A 3',4'-dihydroxylated B-ring together with C-ring saturation and hydroxylation were found to be important structural elements, which strongly influence the flavonoid binding and oxidizability by the LPO/MPO redox intermediates Compounds I and II. In combination with docking studies these results allow an understanding of the differences between flavonoids that promote the hypothiocyanite production by LPO and MPO and those that inhibit this enzymatic reaction.

Myeloperoxidase as a Target for Treatment of Inflammatory Syndromes: Mechanisms and Structure Activity Relationships of Inhibitors.

Inflammation is the initial response of the body to the harmful stimuli and it is achieved by the increased movement of leukocytes (especially granulocytes) from blood into injured tissues. It is required for healing wounds and infections. Despite their indispensable role in microbial killing, the inflammation reactions may also cause diseases to a host such as hay fever, atherosclerosis, and rheumatoid arthritis. The enzymes and oxidizing species released during the inflammatory process can cause damages to the host tissues which lead to inflammatory syndromes. The role of myeloperoxidase (MPO) in the inflammatory reactions is well documented. It contributes in killing the pathogens but it is also implicated in several inflammatory syndromes such as Parkinson's disease, Alzheimer's disease and atherosclerosis. Thus, this enzyme attracted more and more attention of the scientists and it became a target for drug designing. In the last decade, several reversible and irreversible MPO inhibitors were identified as very high potent inhibitors such as fluoroalkylindole, aromatic hydroxamic acid, thioxanthine and benzoic acid hydrazide derivatives. In this review, we tried to illustrate the MPO inhibitors and highlight their structure activity relationship (SAR). In this paper we also discussed the mechanism of the inhibitory effect of the most potent compounds.

Glycan characterization of biopharmaceuticals: Updates and perspectives.

Therapeutic proteins are rapidly becoming the most promising class of pharmaceuticals on the market due to their successful treatment of a vast array of serious diseases, such as cancers and immune disorders. Therapeutic proteins are produced using recombinant DNA technology. More than 60% of therapeutic proteins are posttranslationally modified following biosynthesis by the addition of N- or O-linked glycans. Glycosylation is the most common posttranslational modifications of proteins. However, it is also the most demanding and complex posttranslational modification from the analytical point of view. Moreover, research has shown that glycosylation significantly impacts stability, half-life, mechanism of action and safety of a therapeutic protein. Considering the exponential growth of biotherapeutics, this present review of the literature (2009-2015) focuses on the characterization of protein glycosylation, which has witnessed an improvement in methodology. Furthermore, it discusses current issues in the fields of production and characterization of therapeutic proteins. This review also highlights the problem of non-standard requirements for the approval of biosimilars with regard to their glycosylation and discusses recent developments and perspectives for improved glycan characterization.

Identification of compounds with anti-proliferative activity from the wood of Ficus elastica Roxb. ex Hornem. aerial roots.

The natural products fromplants still remain an important source of pharmaceuticals. In the current study, methanolicwood extracts of Ficus elastica Roxb. ex Hornem. (Moraceae) aerial roots were screened for anti-cancer activity. Using bioassay-guided fractionation, three new compounds, elasticamide (1), elastiquinone (2) and ficusoside B (3), together with four known compounds, were isolated. The structures of the new compounds were established by means of extensive spectroscopic analyses (1D and 2D NMR, HR-ESI-MS, as well as IR and UV) and by comparison of their spectroscopic data with those reported for structurally related molecules. The isolated compounds were afterwards evaluated for their anti-proliferative effect on six human cancer cell lines (U373n and Hs683 glioblastoma, MCF7 and A549 NSCLC carcinoma, and B16F10 and SK-MEL-28 melanoma) using colorimetric MTT assay. Most notably, elastiquinone (2) showed cytotoxic activity with IC50 = 14 μM against B16F10 melanoma cells, whereas the peracetylated form of ficusoside B (3a) displayed the lowest IC50 value (11 μM) against U373n gliomacell lines. Elasticamide (1) and ficusoside B (3) exhibited aweak cytotoxicity with IC50 values ≥88 μM. The results of this investigation demonstrate that the wood of F. elastica aerial roots might be a potential source for identifying new compounds with potent anti-proliferative activity.

2015

Type 2 17-β hydroxysteroid dehydrogenase as a novel target for the treatment of osteoporosis

Low estradiol level in postmenopausal women is implicated in osteoporosis, which occurs because of the high bone resorption rate. Estrogen formation is controlled by 17-β hydroxysteroid dehydrogenase 17-β HSD enzymes, where 17-β HSD type 1 contributes in the formation of estradiol, while type 2 catalyzes its catabolism. Inhibiting 17-β HSD2 can help in increasing estradiol concentration. Several promising 17-β HSD2 inhibitors that can act at low nanomolar range have been identified. However, there are some specific challenges associated with the application of these compounds. Our review provides an up-to-date summary of the current status and recent progress in the production of 17-β HSD2 inhibitors as well as the future challenges in their clinical application.

New dry powders for inhalation containing temozolomide-based nanomicelles for improved lung cancer therapy.

Besides the numerous advantages of a chemotherapy administered by the inhalation route for lung cancer therapy, dry powder for inhalation (DPI) offers many advantages compared to other techniques and seems to be a technique that is well-adapted to an anticancer treatment. DPI formulations were developed using the cytotoxic drug temozolomide and a new folate-grafted self-assembling copolymer, a conjugate of three components, folate-polyethylene glycol-hydrophobically-modified dextran (F-PEG-HMD). F-PEG-HMD was synthesized using carbodiimide-mediated coupling chemistry in three main steps. F-PEG-HMD was characterized by 1H-NMR, mass spectrometry and thermal analysis. F-PEG-HMD presented a critical micellar concentration in water of 4x10-7 M. F-PEG-HMD nanomicelles were characterized by a trimodal particle size distribution with Z-average diameter of 83±1 nm in water. Temozolomide-loaded nanomicelles were prepared by solubilization of F-PEG-HMD in the presence of temozolomide. Temozolomide solubility in water was increased in the presence of F-PEG-HMD (2-fold increase in molar solubility) which could potentially lead to increased local concentrations in the tumor site. The temozolomide-loaded F-PEG-HMD nanomicelles were characterized by a Z-average diameter of ~50 to ~60 nm, depending on the F-PEG-HMD concentration used. The nanomicelles were then spray-dried to produce dry powders. Temozolomide remained stable during all the formulation steps, confirmed by similar in vitro anticancer properties for the DPI formulations and a raw temozolomide solution. Two of the developed DPI formulations were characterized by good aerodynamic properties (with a fine particle fraction of up to 50%) and were able to release the F-PEG-HMD nanomicelles quickly in aqueous media. Moreover, in vitro, the two DPI formulations showed wide pulmonary deposition in the lower respiratory tract where adenocarcinomas are more often found. The present study, therefore, shows that F-PEG-HMD-based dry powders for inhalation could constitute an interesting drug delivery system able to release nanomicelles that are useful in adenocarcinomas that overexpress folate receptors.

Special issue on "Peroxidase"

An immunological method to combine the measurement of active and total myeloperoxidase on the same biological fluid, and its application in finding inhibitors which interact directly with the enzyme.

Myeloperoxidase (MPO) is a pro-oxidant enzyme involved in inflammation, and the measurement of its activity in biological samples has emerged essential for laboratory and clinical investigations. We will describe a new method which combines the SIEFED (specific immunological extraction followed by enzymatic detection) and ELISA (ELISAcb) techniques to measure the active and total amounts of MPO on the same human sample and with the same calibration curve, as well as to define an accurate ratio between both the active and total forms of the enzyme. The SIEFED/ELISAcb method consists of the MPO extraction from aqueous or biological samples by immobilized anti-MPO antibodies coated onto microplate wells. After a washing step to eliminate unbound material, the activity of MPO is measured in situ by adding a reaction solution (SIEFED). Following aspiration of the reaction solution, a secondary anti-MPO antibody is added into the wells and the ELISAcb test is carried out in order to measure the total MPO content. To validate the combined method, a comparison was made with SIEFED and ELISA experiments performed separately on plasma samples isolated from human whole blood, after a neutrophil stimulation. The SIEFED/ELISAcb provides a suitable tool for the measurement of specific MPO activity in biological fluids and for the estimation of the inhibitory potential of a fluid. The method can also be used as a pharmacological tool to make the distinction between a catalytic inhibitor, which binds to MPO and inhibits its activity, and a steric inhibitor, which hinders the enzyme and prevents its immunodetection.

Rational drug design applied to myeloperoxidase inhibition

Rational drug design is a general approach using protein-structure technique in which the discovery of a ligand can be driven either by chance, screening, or rational theory. Myeloperoxidase (MPO) was rapidly identified as a therapeutical target because of its involvement in chronic inflammatory syndromes. In this context, the research of MPO inhibitors was intensified and development of new chemical entities was rationally driven by the research of ligands that enter into the MPO catalytic pocket. Actually, as soon as crystallography data of MPO have become available and its structure was virtually designed, the rational drug design has been applied to this peroxidase. Pharmaceutical industries and academic laboratories apply rational drug design on MPO by either optimizing known inhibitors or searching new molecules by high-throughput virtual screening. By these ways, they were able to find efficient MPO inhibitors and understand their interactions with the enzyme. During this quest of MPO inhibition, it appears that Glu268 is a crucial residue in order to optimize ligandtarget interaction. This amino acid should be carefully considered by medicinal chemist when they design inhibitors interfering with MPO activity.

Advancement in stationary phase for peptide separation helps in protein identification

In the last decades, proteomics has largely progressed. Mass spectrometry and liquid chromatography (LC) are generally used in proteomics. These techniques enable proper separation of peptides and good identification and/or quantification of them. Later, nano-scaled liquid chromatography, improvements of mass spectrometry resolution and sensitivity brought huge advancements. Enhancements in chemistry of chromatographic columns also brought interesting results. In the present work, the potency of identification of proteins by different nano-chip columns was studied and compared with classical LC column. The present study was applied to cardiovascular field where proteomics has shown to be highly helpful in research of new biomarkers. Protein extracts from atheroma plaques were used and proteomics data were compared. Results show that fewer spectra were acquired by the mass spectrometer when nano-chip columns were used instead of the classical ones. However, approximately 40% more unique peptides were identified by the recently optimized chip named Polaris-HR-chip-3C18 column, and 20% more proteins were identified. This fact leads to the identification of more low-abundance proteins. Many of them are involved in atheroma plaque development such as apolipoproteins, ceruloplasmin, etc. In conclusion, present data shows that recent developments of nanoLC column chemistry and dimensions enabled the improved detection and identification of low-abundance proteins in atheroma plaques. Several of them are of major interest in the field of cardiovascular disease.

Multidomain Human Peroxidasin 1 is a Highly Glycosylated and Stable Homotrimeric High-Spin Ferric Peroxidase.

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine crosslinks. The latter confer critical structural reinforcement to collagen IV scaffolds. Here hsPxd01 and various truncated variants lacking non-enzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure and unfolding pathway are reported. The homotrimeric ironprotein contains a covalently-bound ferric high-spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of -233 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (kcat/KM) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV crosslinking.

2014

Nanoimmunoassay onto a screen printed electrode for HER2 breast cancer biomarker determination

A chip format sandwich-type immunoassay based on Nanobodies® (Nbs) with the Human Epidermal Growth Factor Receptor (HER2) extracellular domain as antigen model has been developed. The HER2 is considered as an important biomarker because its overexpression causes an aggressive type of breast cancer. Nbs are single domain antigen-binding fragments derived from camelid heavy-chain antibodies. The strategy of the presently developed sandwich immunoassay takes advantage of the small size of Nbs for the detection of the electroactive redox tracer onto the screen printed electrode (SPE). A capture anti HER2 Nb was covalently immobilized onto the SPE, and the detection Nb, raised against another epitope of HER2, was labeled with horseradish peroxidase (HRP). The biosensor signal corresponded to the electroreduction of para-quinone generated at the SPE by the HRP in the presence of hydroquinone and hydrogen peroxide. The best performing and optimized immunoassay conditions consisted of 2 and 20 min for the first and the second incubation times, respectively. The amperometric signal obtained was proportional to the logarithm of HER2 concentration between 1 and 200 μg/mL and the modified SPE storage stability lasted for at least three weeks. Determination of HER2 in human cells has been realized. © 2014 Elsevier B.V.

Hybrid molecules inhibiting myeloperoxidase activity and serotonin reuptake: a possible new approach of major depressive disorders with inflammatory syndrome.

Major depressive disorder (MDD) is accompanied with an imbalance in the immune system and cardiovascular impairments, such as atherosclerosis. Several mechanisms have been pointed out to underlie this rather unexpected association, and among them the activity of myeloperoxidase (MPO). The aim of our study was to find compounds that inhibit both MPO and serotonin transporter (SERT) for treating MDD associated with cardiovascular diseases.

Impact of interaction between myeloperoxidase and low-density lipoprotein on the specific activity of the enzyme and subsequent post-translational oxidative modifications of apolipoprotein B-100.

Oxidation of low-density lipoprotein (LDL) by myeloperoxidase (MPO)-H2O2-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing post-translational modifications on apolipoprotein-B-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using mass spectrometry we further analyzed in vitro modifications of apolipoprotein-B-100 by HOCl generated by the MPO-H2O2-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H2O2-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several of HOCl-mediated modifications of apolipoprotein-B-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.

MYELOPEROXIDASE AND ITS PRODUCTS IN SYNOVIAL FLUID OF PATIENTS WITH TREATED OR UNTREATED RHEUMATOID ARTHRITIS.

ABSTRACT Objective Plasma and synovial myeloperoxidase (MPO) and its products were strongly associated with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, it is well known that there is a link between oxidative stress and cytokines. The present study aims at investigating the link between synovial MPO (and its products), IL-18, which is involved in the degradation of articular cartilage in RA, and IL-8, which is involved in recruitment and activation of neutrophils during inflammation. Effects of the treatment of RA on the biological parameters were also investigated. Methods Patients (n=105) were studied including 39 patients with OA, 33 with RA and 33 with RA receiving a specific treatment. DAS-28 was calculated whereas MPO antigen/activity, neutrophils, chloro-tyrosine, homocitrulline, IL-8 and IL-18 were measured in synovial fluid (SF) and CRP was measured in serum. Results DAS-28 and CRP level were not significantly different between groups. MPO activity, and MPO, chloro-tyrosine and homocitrulline levels were significantly higher in SF of RA patients than OA patients. MPO specific activity (MPO activity/antigen ratio) was significantly lower in treated than in untreated RA patients as was IL-8. MPO activity and concentration were correlated with IL-8 and IL-18 in untreated but not in treated RA patients. Conclusions MPO level is related to IL-8 and IL-18 levels in untreated RA patients. A link has been shown between treatment and decrease of IL-8, MPO specific activity and homocitrulline in SF. The causal role of MPO in SF inflammation and how treatment can affect MPO specific activity need further investigations.

Characterization and antioxidant properties of six algerian propolis extracts: ethyl acetate extracts inhibit myeloperoxidase activity.

Because propolis contains many types of antioxidant compounds such as polyphenols and flavonoids, it can be useful in preventing oxidative damages. Ethyl acetate extracts of propolis from several Algerian regions show high activity by scavenging free radicals, preventing lipid peroxidation and inhibiting myeloperoxidase (MPO). By fractioning and assaying ethyl acetate extracts, it was observed that both polyphenols and flavonoids contribute to these activities. A correlation was observed between the polyphenol content and the MPO inhibition. However, it seems that kaempferol, a flavonoid, contributes mainly to the MPO inhibition. This molecule is in a high amount in the ethyl acetate extract and demonstrates the best efficiency towards the enzyme with an inhibiting concentration at 50% of 4 ± 2 µM.

Myeloperoxidase oxidized LDL interferes with endothelial cell motility through miR-22 and heme oxygenase 1 induction: possible involvement in reendothelialization of vascular injuries.

Cardiovascular disease linked to atherosclerosis is the leading cause of death worldwide. Atherosclerosis is mainly linked to dysfunction in vascular endothelial cells and subendothelial accumulation of oxidized forms of LDL. In the present study, we investigated the role of myeloperoxidase oxidized LDL (Mox-LDL) in endothelial cell dysfunction. We studied the effect of proinflammatory Mox-LDL treatment on endothelial cell motility, a parameter essential for normal vascular processes such as angiogenesis and blood vessel repair. This is particularly important in the context of an atheroma plaque, where vascular wall integrity is affected and interference with its repair could contribute to progression of the disease. We investigated in vitro the effect of Mox-LDL on endothelial cells angiogenic properties and we also studied the signalling pathways that could be affected by analysing Mox-LDL effect on the expression of angiogenesis-related genes. We report that Mox-LDL inhibits endothelial cell motility and tubulogenesis through an increase in miR-22 and heme oxygenase 1 expression. Our in vitro data indicate that Mox-LDL interferes with parameters associated with angiogenesis. They suggest that high LDL levels in patients would impair their endothelial cell capacity to cope with a damaged endothelium contributing negatively to the progression of the atheroma plaque.

Inhibition of myeloperoxidase activity by the alkaloids of Peganum harmala L. (Zygophyllaceae).

Seeds and aerial parts of Peganum harmala L. (Peganum harmala) are widely used in Algeria as anti-inflammatory remedies. Evaluation of Peganum harmala total alkaloids extracts and pure β-carboline compounds as an anti-inflammatory treatment by the inhibition of an enzyme key of inflammatory, myeloperoxidase (MPO) and HPLC quantification of the alkaloids from the different parts of plant.

Ferulaldehyde and lupeol as direct and indirect antimicrobial compounds from Cordia gilletii (Boraginaceae) root barks

Cordia gilletii De Wild (Boraginaceae), a medicinal plant used against infectious diseases in the Democratic Republic of Congo, was investigated for direct and indirect antimicrobial properties. On one hand, the methanol extract is active against many pathogenic bacteria, including resistant strains. Its bio-guided fractionation led to the isolation of ferulaldehyde; this compound showed antimicrobial and antioxidant properties that may support the activity we observed for the methanol extract and some of the traditional uses of C. gilletii. On the other hand, the n-hexane extract of root barks possesses indirect antimicrobial properties, enhancing the activity of antibiotics against methicillin-resistant Staphylococcus aureus (MRSA). The fractionation of this extract led to the isolation of lupeol, which decreases the minimum inhibitory concentration of several antibiotics (4 to 8 fold) against MRSA and contributes to the effects observed for the raw n-hexane extract.

2013

Ophiobolin A, a sesterterpenoid fungal phytotoxin, displays higher in vitro growth-inhibitory effects in mammalian than in plant cells and displays in vivo antitumor activity.

Ophiobolin A, a sesterterpenoid produced by plant pathogenic fungi, was purified from the culture extract of Drechslera gigantea and tested for its growth-inhibitory activity in both plant and mammalian cells. Ophiobolin A induced cell death in Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells at concentrations ≥10 µM, with the TBY-2 cells showing typical features of apoptosis-like cell death. At a concentration of 5 µM, ophiobolin A did not affect plant cell viability but prevented cell proliferation. When tested on eight cancer cell lines, concentrations <1 µM of ophiobolin A inhibited growth by 50% after 3 days of culture irrespective of their multidrug resistance (MDR) phenotypes and their resistance levels to pro-apoptotic stimuli. It is, thus, unlikely that ophiobolin A exerts these in vitro growth-inhibitory effects in cancer cells by activating pro-apoptotic processes. Highly proliferative human keratinocytes appeared more sensitive to the growth-inhibitory effects of ophiobolin A than slowly proliferating ones. Ophiobolin A also displayed significant antitumor activity at the level of mouse survival when assayed at 10 mg/kg in the B16F10 mouse melanoma model with lung pseudometastases. Ophiobolin A could, thus, represent a novel scaffold to combat cancer types that display various levels of resistance to pro-apoptotic stimuli and/or various MDR phenotypes.

4-Bromo-2-(piperidin-1-yl)thiazol-5-yl-phenyl methanone (12b) inhibits Na(+)/K(+)-ATPase and Ras oncogene activity in cancer cells.

The in vitro growth inhibitory activity of 26 thiazoles (including 4-halogeno-2,5-disubtituted-1,3-thiazoles) and 5 thienothiazoles was assessed on a panel of 6 human cancer cell lines, including glioma cell lines. (4-Chloro-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12a) and (4-bromo-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12b) displayed ∼10 times greater in vitro growth inhibitory activity than perillyl alcohol (POH), which therapeutically benefits glioma patients through the inhibition of both alpha-1 Na(+)/K(+)-ATPase (NAK) and Ras oncogene activity. The in vitro cytostatic activities (as revealed by quantitative videomicroscopy) displayed by 12a and 12b were independent of the intrinsic resistance to pro-apoptotic stimuli associated with cancer cells. Compounds 12a and 12b displayed relatively similar inhibitory activities on purified guinea pig brain preparations that mainly express NAK alpha-2 and alpha-3 subunits, whereas only compound 12b was efficacious against purified guinea pig kidney preparations that mainly express the NAK alpha-1 subunit, which is also expressed in gliomas, melanomas and non-small-cell lung cancers NSCLCs.

Design, synthesis, and structure-activity relationship studies of novel 3-alkylindole derivatives as selective and highly potent myeloperoxidase inhibitors.

Due to its production of potent antimicrobial oxidants including hypochlorous acid, human myeloperoxidase (MPO) plays a critical role in innate immunity and inflammatory diseases. Thus MPO is an attractive target in drug design. (Aminoalkyl)fluoroindole derivatives were detected to be very potent MPO inhibitors; however, they also promote inhibition of the serotonin reuptake transporter (SERT) at the same concentration range. Via structure-based drug design, a new series of MPO inhibitors derived from 3-alkylindole were synthesized and their effects were assessed on MPO-mediated taurine chlorination and low-density lipoprotein oxidation as well as on inhibition of SERT. The fluoroindole compound with three carbons in the side chain and one amide group exhibited a selectivity index of 35 (Ki/IC50) with high inhibition of MPO activity (IC50 = 18 nM), whereas its effect on SERT was in the micromolar range. Structure-function relationships, mechanism of action, and safety of the molecule are discussed.

Variations in the chemical profile and biological activities of licorice (Glycyrrhiza glabra L.), as influenced by harvest times

This study investigates the variations in the chemical profile, free radical scavenging, antioxidant and gastroprotective activities of licorice extracts (LE) from plants harvested during the months of February to November. Correlations between biological properties and the chemical composition of LE were also investigated. The results showed that the total contents of phenols, flavonoids and tannins in LE varied at different harvest times. Liquiritin and glycyrrhizin, the major components of LE, varied in the range of 28.65-62.80 and 41.84-114.33 mg g−1, respectively. The relative proportion of glycyrrhizin derivative (3), glabridin (4), glabrene (5) and liquiritigenin derivative (6), varied in the range of 0.88-11.38 %, 1.86-10.03 %, 1.80-18.40 % and 5.53-16.31 %, respectively. These fluctuations correlated positively with changes in the antioxidant and free radical scavenging activities of licorice. In general, the samples from May and November showed the most favorable free radical scavenging and antioxidant effects, whereas the best gastroprotective effect was in May. Liquiritin and glycyrrhizin, the major constituents in the February and May LE, appeared to contribute to the superoxide radical scavenging and gastroprotective effects. Glabridin and glabrene, the compounds with the highest relative proportion in the November LE, accounted for the antioxidant and DPPH scavenging activities of licorice. It is concluded that the chemical profile of licorice quantitatively varied at different harvest times and these fluctuations determined changes in its bioactivities. These data could pave the way to optimize harvesting protocols for licorice in relation with its health-promoting properties.

Vasorelaxant and antihypertensive effects of methanolic extracts from Hymenocardia acida Tul.

In Congolese traditional medicine, decoctions of Hymenocardia acida root bark (HaRB) and trunk bark (HaTrB) are used for the treatment of conditions assumed to be hypertension. In this work, we propose to study the vasorelaxant effect of HaRB and HaTrB methanolic extracts on isolated rat thoracic aorta, to characterize the group of molecules responsible for the observed vasorelaxant activity, to evaluate the in vitro antioxidant activity of these extracts and to determine the antihypertensive activity of the HaRB extract on spontaneously hypertensive rats (SHR).

Assessment of oxidative stress in tumors and histologically normal mucosa from patients with head and neck squamous cell carcinoma: a preliminary study.

Over 90% of head and neck cancers are squamous cell carcinomas (HNSCC) and the overall 5-year survival rate is up to 50%. The redox status of these cancers is an important factor in carcinogenesis and plays a role in radioresistance and therefore locoregional recurrences. However, knowledge of the redox status is rather limited. Glutathione is the major reactive oxygen species scavenger in normal cells. We compared the levels of tissue redox potential in HNSCC tumor tissue and compared them with those of the adjacent, histologically cancer-free, mucosa. A total of 36 patients with HNSCC were included in the study. The redox status of tumor and normal adjacent tissue was measured by the oxidized/reduced glutathione (GSSG/GSH) ratio in capillary electrophoresis. The GSSG/GSH ratio in the tumor tissue was lower compared with adjacent normal tissue in 38% of the patients. Pretherapy HNSCC tumor tissue has variable GSH levels compared with adjacent cancer-free mucosa. This difference was not related to clinical and pathological parameters. Further studies are required to determine whether the GSSG/GSH ratio plays a role in carcinogenesis and could predict radioresistance.

Efficient 'one-pot' methodology for the synthesis of novel tetrahydro-β-carboline, tetrahydroisoquinoline and tetrahydrothienopyridine derivatives

A simple and efficient 'one-pot' methodology was developed to generate a new series of tetrahydro-β-carboline (THBC), tetrahydroisoquinoline (THIQ) and tetrahydrothienopyridine (THTP) derivatives. The key step of the methodology is based on a Pictet-Spengler type cyclization of a reactive N-carbamyliminium ion. This methodology was applied to the synthesis of a library of 32 compounds with potential anti-tumoral activity. © 2013 Elsevier Ltd. All rights reserved.

A new device to mimic intermittent hypoxia in mice.

Intermittent hypoxia (hypoxia-reoxygenation) is often associated with cardiovascular morbidity and mortality. We describe a new device which can be used to submit cohorts of mice to controlled and standardised hypoxia-normoxia cycles at an individual level. Mice were placed in individual compartments to which similar gas flow parameters were provided using an open loop strategy. Evaluations made using computational fluid dynamics were confirmed by studying changes in haemoglobin oxygen saturation in vivo. We also modified the parameters of the system and demonstrated its ability to generate different severities of cyclic hypoxemia very precisely, even with very high frequency cycles of hypoxia-reoxygenation. The importance of the parameters on reoxygenation was shown. This device will allow investigators to assess the effects of hypoxia-reoxygenation on different pathological conditions, such as obstructive sleep apnoea or chronic obstructive pulmonary disease.

Low-density lipoprotein modified by myeloperoxidase in inflammatory pathways and clinical studies.

Oxidation of low-density lipoprotein (LDL) has a key role in atherogenesis. Among the different models of oxidation that have been studied, the one using myeloperoxidase (MPO) is thought to be more physiopathologically relevant. Apolipoprotein B-100 is the unique protein of LDL and is the major target of MPO. Furthermore, MPO rapidly adsorbs at the surface of LDL, promoting oxidation of amino acid residues and formation of oxidized lipoproteins that are commonly named Mox-LDL. The latter is not recognized by the LDL receptor and is accumulated by macrophages. In the context of atherogenesis, Mox-LDL accumulates in macrophages leading to foam cell formation. Furthermore, Mox-LDL seems to have specific effects and triggers inflammation. Indeed, those oxidized lipoproteins activate endothelial cells and monocytes/macrophages and induce proinflammatory molecules such as TNF α and IL-8. Mox-LDL may also inhibit fibrinolysis mediated via endothelial cells and consecutively increase the risk of thrombus formation. Finally, Mox-LDL has been involved in the physiopathology of several diseases linked to atherosclerosis such as kidney failure and consequent hemodialysis therapy, erectile dysfunction, and sleep restriction. All these issues show that the investigations of MPO-dependent LDL oxidation are of importance to better understand the inflammatory context of atherosclerosis.

Myeloperoxidase-Dependent LDL Modifications in Bloodstream Are Mainly Predicted by Angiotensin II, Adiponectin, and Myeloperoxidase Activity: A Cross-Sectional Study in Men.

The present paradigm of atherogenesis proposes that low density lipoproteins (LDLs) are trapped in subendothelial space of the vascular wall where they are oxidized. Previously, we showed that oxidation is not restricted to the subendothelial location. Myeloperoxidase (MPO), an enzyme secreted by neutrophils and macrophages, can modify LDL (Mox-LDL) at the surface of endothelial cells. In addition we observed that the activation of the endothelial cells by angiotensin II amplifies this process. We suggested that induction of the NADPH oxidase complex was a major step in the oxidative process. Based on these data, we asked whether there was an independent association, in 121 patients, between NADPH oxidase modulators, such as angiotensin II, adiponectin, and levels of circulating Mox-LDL. Our observations suggest that the combination of blood angiotensin II, MPO activity, and adiponectin explains, at least partially, serum Mox-LDL levels.

2012

Ceramide, cerebroside and triterpenoid saponin from the bark of aerial roots of Ficus elastica (Moraceae).

Three compounds, ficusamide (1), ficusoside (2) and elasticoside (3), were isolated from the bark of aerial roots of Ficus elastica (Moraceae), together with nine known compounds, including four triterpenes, three steroids and two aliphatic linear alcohols. The chemical structures of the three compounds were established by extensive 1D and 2D NMR spectroscopy, mass spectrometry and by comparison with published data. The growth inhibitory effect of the crude extract and isolated compounds was evaluated against several microorganisms and fungi. The cytotoxicity against human cancer cell lines was also assessed. Ficusamide (1) displayed a moderate in vitro growth inhibitory activity against the human A549 lung cancer cell line and a strong activity against Staphylococcus saprophyticus, while elasticoside (3) showed a potent activity on Enterococcus faecalis.

Simultaneous measurement of protein-bound 3-chlorotyrosine and homocitrulline by LC-MS/MS after hydrolysis assisted by microwave: Application to the study of myeloperoxidase activity during hemodialysis.

A high degree of uremia is common in patients with end-stage renal disease and has been linked to the development of chronic inflammation and cardiovascular diseases. In conditions where transplantation is not possible, uremia can be reduced by hemodialysis although the repeated interventions have been implicated in loss of renal function, partially as a result of chronic inflammation and/or oxidative stress processes. In this context, it has been suggested that myeloperoxidase (MPO) can contribute to the oxidative stress during hemodialysis and to the cardiovascular risk. Protein damages due to MPO activity have never been assessed during hemodialysis although two of its reaction products, 3-chlorotyrosine and homocitrulline, are of interest. Indeed, the first one is a specific product of MPO activity and the formation of the second one could be catalyzed by MPO. In order to analyze these products in plasma proteins, a total hydrolysis method followed by liquid chromatography mass spectrometry analysis was developed. Different conditions of hydrolysis were tested and the optimized procedure was assessed for complete hydrolysis and artifactual chlorination. Finally, the method was used for analyzing 3-chlorotyrosine and homocitrulline in plasma proteins during a hemodialysis session in fifteen patients and data were related to measurements of MPO concentration and activity. Both increases in MPO activity and protein-bound 3-chlorotyrosine were observed, highlighting the involvement of MPO in oxidative stress during hemodialysis and further demonstrating the link between hemodialysis and cardiovascular diseases.

Evaluation of new scaffolds of myeloperoxidase inhibitors by rational design combined with high-throughput virtual screening

Myeloperoxidase (MPO) is a major player of the innate immune defense system of human neutrophils and catalyzes the prodn. of strong oxidizing and halogenating antimicrobial products. Because of its role in pathogenesis of many (inflammatory) diseases, there is great interest in the development of efficient and specific inhibitors. Here, using the X-ray structure of MPO, high-throughput mol. docking of 1350000 compds. was performed. From this virtual screening process, 81 were tested for inhibition of the chlorination activity of MPO, finally ending up with eight inhibiting candidates of different chem. structures. These were tested for inhibiting MPO-mediated low-d. lipoprotein oxidn. and for interacting with the relevant redox intermediates of MPO. The best inhibitors were bis-2,2'-[(dihydro-1,3(2H,4H)-pyrimidinediyl)bis(methylene)]phenol and 8-[(2-aminoethyl)amino]-3,7-dihydro-3-methyl-7-(3-phenoxypropyl)-1H-purine-2,6-dione. Both did not irreversibly inactivate the enzyme but efficiently trapped it in its compd. II state. We discuss the mechanism of inactivation as well as pros and cons of the performed selection process.

Exposure of endothelial cells to physiological levels of myeloperoxidase-modified LDL delays pericellular fibrinolysis.

Blood fluidity is maintained by a delicate balance between coagulation and fibrinolysis. The endothelial cell surface is a key player in this equilibrium and cell surface disruptions can upset the balance. We investigated the role of pericellular myeloperoxidase oxidized LDLs (Mox-LDLs) in this balance.

Myeloperoxidase activity and its products during hemodialysis

Myeloperoxidase activity increases at the surface of LDL

2011

Utility of screen printed electrodes for in vitro metabolic stability assays: Application to acetaminophen and its thioconjugates

Intriguing location of myeloperoxidase in the prostate

Background: Myeloperoxidase (MPO) is a member of the peroxidase- cyclooxygenase superfamily, which is secreted from stimulated leucocytes at inflammatory sites. It is well known that MPO catalyses oxidation reactions via the release of reactive halogenating and nitrating species and thus induces tissue damage. Several studies have already implicated MPO in the development of neoplasia. Chronic or recurrent prostatic inflammation has long been recognized as having the potential to initiate and promote the development of prostate cancer. The objective was to investigate whether MPO is present in the prostate. METHODS Human prostate material was obtained from biopsies, transurethral resections of the prostate (TURP), prostatic adenomectomies, and retropubic radical prostatectomies. Twenty-nine slides of normal prostate tissue, benign prostatic hyperplasia (BPH), and prostate cancer were reviewed by a pathologist. Immunohistochemical analysis using MPO-specific human antibody was performed to detect MPO in the prostate tissue. Results: Immunocytohistochemistry showed cellular colocalization of MPO in the secretory epithelial cells of the prostate with staining varying from light to strong intensity. Staining in the glandular apical snouts was often reinforced although staining of basal as well as of luminal glandular cells was also present. Conclusions: We identified, for the first time, the presence of MPO at the surface of prostatic epithelial cells. In view of the pro-oxidant properties of this enzyme, further research is needed to define whether MPO contributes to the development of prostatic lesions. © 2011 Wiley Periodicals,Inc.

In vitro anticancer activity, toxicity and structure-activity relationships of phyllostictine A, a natural oxazatricycloalkenone produced by the fungus Phyllosticta cirsii.

The in vitro anticancer activity and toxicity of phyllostictine A, a novel oxazatricycloalkenone recently isolated from a plant-pathogenic fungus (Phyllosticta cirsii) was characterized in six normal and five cancer cell lines. Phyllostictine A displays in vitro growth-inhibitory activity both in normal and cancer cells without actual bioselectivity, while proliferating cells appear significantly more sensitive to phyllostictine A than non-proliferating ones. The main mechanism of action by which phyllostictine displays cytotoxic effects in cancer cells does not seem to relate to a direct activation of apoptosis. In the same manner, phyllostictine A seems not to bind or bond with DNA as part of its mechanism of action. In contrast, phyllostictine A strongly reacts with GSH, which is a bionucleophile. The experimental data from the present study are in favor of a bonding process between GSH and phyllostictine A to form a complex though Michael attack at C=C bond at the acrylamide-like system. Considering the data obtained, two new hemisynthesized phyllostictine A derivatives together with three other natural phyllostictines (B, C and D) were also tested in vitro in five cancer cell lines. Compared to phyllostictine A, the two derivatives displayed a higher, phyllostictines B and D a lower, and phyllostictine C an almost equal, growth-inhibitory activity, respectively. These results led us to propose preliminary conclusions in terms of the structure-activity relationship (SAR) analyses for the anticancer activity of phyllostictine A and its related compounds, at least in vitro.

Optimization of apolipoprotein-B-100 sequence coverage by liquid chromatography-tandem mass spectrometry for the future study of its posttranslational modifications.

Proteomic applications have been increasingly used to study posttranslational modifications of proteins (PTMs). For the purpose of identifying and localizing specific but unknown PTMs on huge proteins, improving their sequence coverage is fundamental. Using liquid chromatography coupled to mass spectrometry (LC-MS/MS), peptide mapping of the native apolipoprotein-B-100 was performed to further document the effects of oxidation. Apolipoprotein-B-100 is the main protein of low-density lipoprotein particles and its oxidation could play a role in atherogenesis. Because it is one of the largest human proteins, the sequence recovery rate of apolipoprotein-B-100 only reached 1% when conventional analysis parameters were used. The different steps of the peptide mapping process-from protein treatment to data analysis-were therefore reappraised and optimized. These optimizations allowed a protein sequence recovery rate of 79%, a rate which has never been achieved previously for such a large human protein. The key points for improving peptide mapping were optimization of the data analysis software; peptide separation by LC; sample preparation; and MS acquisition. The new protocol has allowed us to increase by a factor of 4 the detection of modified peptides in apolipoprotein-B-100. This approach could easily be transferred to any study of PTMs using LC-MS/MS.

Temporal dissociation between myeloperoxidase (MPO)-modified LDL and MPO elevations during chronic sleep restriction and recovery in healthy young men.

Many studies have evaluated the ways in which sleep disturbances may influence inflammation and the possible links of this effect to cardiovascular risk. Our objective was to investigate the effects of chronic sleep restriction and recovery on several blood cardiovascular biomarkers.

2010

Structure-based design, synthesis, and pharmacological evaluation of 3-(Aminoalkyl)-5-fluoroindoles as myeloperoxidase inhibitors.

Oxidized low-density lipoproteins (LDLs) accumulate in the vascular wall and promote local inflammation, which contributes to the progression of the atheromatous plaque. The key role of myeloperoxidase (MPO) in this process is related to its ability to modify APO B-100 in the intima and at the surface of endothelial cells. A series of 3-(aminoalkyl)-5-fluoroindole analogues was designed and synthesized by exploiting the structure-based docking of 5-fluorotryptamine, a known MPO inhibitor. In vitro assays were used to study the effects of these compounds on the inhibition of MPO-mediated taurine chlorination and oxidation of LDLs. The kinetics of the interaction between the MPO redox intermediates, Compounds I and II, and these inhibitors was also investigated. The most potent molecules possessed a 4- or 5-carbon aminoalkyl side chain and no substituent on the amino group. The mode of binding of these analogues and the mechanism of inhibition is discussed with respect to the structure of MPO and its halogenation and peroxidase cycles.

Targeting of eEF1A with Amaryllidaceae isocarbostyrils as a strategy to combat melanomas.

Melanomas display poor response rates to adjuvant therapies because of their intrinsic resistance to proapoptotic stimuli. This study indicates that such resistance can be overcome, at least partly, through the targeting of eEF1A elongation factor with narciclasine, an Amaryllidaceae isocarbostyril controlling plant growth. Narciclasine displays IC(50) growth inhibitory values between 30-100 nM in melanoma cell lines, irrespective of their levels of resistance to proapoptotic stimuli. Normal noncancerous cell lines are much less affected. At nontoxic doses, narciclasine also significantly improves (P=0.004) the survival of mice bearing metastatic apoptosis-resistant melanoma xenografts in their brain. The eEF1A targeting with narciclasine (50 nM) leads to 1) marked actin cytoskeleton disorganization, resulting in cytokinesis impairment, and 2) protein synthesis impairment (elongation and initiation steps), whereas apoptosis is induced at higher doses only (≥200 nM). In addition to molecular docking validation and identification of potential binding sites, we biochemically confirmed that narciclasine directly binds to human recombinant and yeast-purified eEF1A in a nanomolar range, but not to actin or elongation factor 2, and that 5 nM narciclasine is sufficient to impair eEF1A-related actin bundling activity. eEF1A is thus a potential target to combat melanomas regardless of their apoptosis-sensitivity, and this finding reconciles the pleiotropic cytostatic of narciclasine. -

Copper and myeloperoxidase-modified LDLs activate Nrf2 through different pathways of ROS production in macrophages.

Low-density lipoprotein (LDL) oxidation is a key step in atherogenesis, promoting the formation of lipid-laden macrophages. Here, we compared the effects of copper-oxidized LDLs (OxLDLs) and of the more physiologically relevant myeloperoxidase-oxidized LDLs (MoxLDLs) in murine RAW264.7 macrophages and in human peripheral blood monocyte-derived macrophages. Both oxidized LDLs, contrary to native LDLs, induced foam cell formation and an intracellular accumulation of reactive oxygen species (ROS). This oxidative stress was responsible for the activation of the NF-E2-related factor 2 (Nrf2) transcription factor, and the subsequent Nrf2-dependent overexpression of the antioxidant genes, Gclm and HO-1, as evidenced by the invalidation of Nrf2 by RNAi. MoxLDLs always induced a stronger response than OxLDLs. These differences could be partly explained by specific ROS-producing mechanisms differing between OxLDLs and MoxLDLs. Whereas both types of oxidized LDLs caused ROS production partly by NADPH oxidase, only MoxLDLs-induced ROS production was dependent on cytosolic PLA2. This study highlights that OxLDLs and MoxLDLs induce an oxidative stress, through distinct ROS-producing mechanisms, which is responsible for the differential activation of the Nrf2 pathway. These data clearly suggest that results obtained until now with copper oxidized-LDLs should be carefully reevaluated, taking into consideration physiologically more relevant oxidized LDLs.

Free radical-scavenging, antioxidant and immunostimulating effects of a licorice infusion (Glycyrrhiza glabra L.)

To contribute to the understanding of the mechanisms underlying the beneficial effects of licorice, the antioxidant, free radical-scavenging and immunostimulating effects of a licorice infusion (LI) were investigated, and its chemical profile was determined. Two major components of LI were identified as (1) liquiritin and (2) glycyrrhizin. LI weakly scavenged DPPH{radical dot}, and compounds 1 and 2 showed negligible effects. Both LI and 2 substantially scavenged superoxide radicals. The β-carotene bleaching was inhibited by LI, but compounds 1 and 2 showed no effect. The LI, 1, and 2 exhibited no meaningful activities against HOCl, and they showed pro-oxidant effects in the MPO-chlorinating system. Granulocytes and NK cells were markedly activated by LI, whereas 1 and 2 were inactive. The LI, 1, and 2 showed no effects on the lymphocyte cell cycle. These results support, in part, the traditional use of licorice to treat and prevent diseases in which oxidants or free radicals are implicated and suggest that LI could be used as a potential non-specific immune stimulator. © 2010 Elsevier Ltd. All rights reserved.

Benefits of napping and an extended duration of recovery sleep on alertness and immune cells after acute sleep restriction.

Understanding the interactions between sleep and the immune system may offer insight into why short sleep duration has been linked to negative health outcomes. We, therefore, investigated the effects of napping and extended recovery sleep after sleep restriction on the immune and inflammatory systems and sleepiness. After a baseline night, healthy young men slept for a 2-h night followed by either a standard 8-h recovery night (n=12), a 30-min nap (at 1p.m.) in addition to an 8-h recovery night (n=10), or a 10-h extended recovery night (n=9). A control group slept 3 consecutive 8-h nights (n=9). Subjects underwent continuous electroencephalogram polysomnography and blood was sampled every day at 7a.m. Leukocytes, inflammatory and atherogenesis biomarkers (high-sensitivity C-reactive protein, interleukin-8, myeloperoxidase, fibrinogen and apolipoproteins ApoB/ApoA), sleep patterns and sleepiness were investigated. All parameters remained unchanged in the control group. After sleep restriction, leukocyte and - among leukocyte subsets - neutrophil counts were increased, an effect that persisted after the 8-h recovery sleep, but, in subjects who had a nap or a 10-h recovery sleep, these values returned nearly to baseline. Inflammatory and atherogenesis biomarkers were unchanged except for higher myeloperoxidase levels after sleep restriction. The increased sleepiness after sleep restriction was reversed better in the nap and extended sleep recovery conditions. Saliva cortisol decreased immediately after the nap. Our results indicate that additional recovery sleep after sleep restriction provided by a midday nap prior to recovery sleep or a sleep extended night can improve alertness and return leukocyte counts to baseline values.

Enzyme immobilized magnetic nanoparticles for in-line capillary electrophoresis and drug biotransformation studies: application to paracetamol.

Enzyme Immobilized Magnetic Nanoparticles (EMNPs) were injected and magnetically retained, as a microreactor, in the capillary of a capillary electrophoresis (CE) setup with UV detection. The enzyme horseradish peroxidase (HRP) was chemically immobilized onto commercially available magnetic 300 nm diameter nanoparticles. Paracetamol (acetaminophen: N-acetyl-p-aminophenol), a common analgesic drug, was used as model drug compound. The enzymatic reaction was studied in-line by CE in 12.5 mM phosphate buffer pH 7.4 containing 20 mg/ml sulfated- beta -cyclodextrin and 0.1 mM hydrogen peroxide. By means of the developed setup, the apparent Michaelis Menten constant between HRP and acetaminophen (APAP) was determined as K(m)(app) = 53+/-5 microM. This approach was found to be of interest for enzyme kinetics studies with short time resolution condition. Based on our results and from literature data, it was possible to infer that the in-line generated product was an APAP dimer. Higher enzyme immobilized beads loading in the CE setup generated the APAP dimer with two additional minor peaks likely attributed to APAP trimer and tetramer. N-acetyl-p-benzoquinone imine (NAPQI) was not generated during APAP short time migration through the in-line microreactor.

Simple di- and trivanillates exhibit cytostatic properties toward cancer cells resistant to pro-apoptotic stimuli

Specific oxidation of apolipoprotein-B-100 by myeloperoxidase

Oxidation of cyanide to cyanate by myeloperoxidase: A new route for apolipoprotein carbamylation?

Glycosylation pattern of mature dimeric leukocyte and recombinant monomeric myeloperoxidase: glycosylation is required for optimal enzymatic activity.

The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro- and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the three-dimensional structure of MPO.

2009

A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts.

The SIEFED ("Specific Immunological Extraction Followed by Enzymatic Detection") method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by a washing (to eliminate the extraction medium and the biological fluid with their possible interfering molecules) and the measurement of the activity of MPO with a detection system containing a fluorogenic substrate, H(2)O(2) and nitrite ions as reaction enhancer. The SIEFED was applied to study active MPO in human biological fluids (plasma, bronchoalveolar lavage fluid and supernatant from carotids extracts). The SIEFED for human MPO has a sensitivity limit of 0.080 mU/mL and showed good precision with intra- and inter-assay coefficients of variation below 10 and 20% respectively within a broad range of MPO activities establish from 0.156 to 473 mU/mL. The SIEFED for human MPO will be useful for the specific detection of active MPO in complex fluids and can be complementary to an ELISA to determine an active/total MPO ratio in healthy volunteers and patients especially in case of chronic or acute inflammatory diseases.

Maladies cardiovasculaires, dysfonction érectile et myéloperoxydase

Coronary stenting is associated with an acute increase in plasma myeloperoxidase in stable angina patients but not in patients with acute myocardial infarction.

BACKGROUND: Myeloperoxidase (MPO) has emerged as a critical mediator in the physiopathology of atherosclerosis from plaque formation and growth until destabilization and rupture leading to acute coronary syndrome (ACS). Using coronary stenting as a model of plaque injury, we aimed to determine the evolution of systemic MPO levels following coronary stenting in stable angina patients and in patients with acute myocardial infarction (AMI). METHODS: Plasma levels of MPO, lactoferrin, interleukin (IL)-6, C-reactive protein and PMN counts were assessed in 13 patients with Non-ST-elevation myocardial infarction (NSTEMI) (Group A) and in 29 patients with stable angina pectoris (Group B), undergoing coronary stenting. Serial blood samples were taken before angioplasty (baseline) and at 1, 6 and 24 h following initial balloon inflation. RESULTS: Following angioplasty, the overall plasma MPO levels significantly increased at 1 h in group B (120.5+/-79.0 to 166+/-79.5, p=0.003) but not in group A (121+/-63.4 to 124.7+/-76.9, p=0.753). In Group B, the increase in MPO levels at 1 h were significantly higher in the presence of complex lesions compared to patients with simple lesions (p=0.023). Lactoferrin levels showed no change over time except for a significant decrease at 6 h in group B. CONCLUSIONS: In stable angina patients, coronary stenting is associated with an acute and transient increase in plasma MPO levels, but not in lactoferrin levels, with an enhanced response in the presence of complex lesions. In contrast, we observed no changes in plasma MPO and lactoferrin levels following stenting in patients with AMI. Given its pro-inflammatory properties, the potential implication of MPO release on clinical outcome in stable patients undergoing stenting needs further investigation.

A large-bolus injection, but not continuous infusion of sodium selenite improves outcome in peritonitis.

Administration of sodium selenite in septic shock has been associated with apparently conflicting results that may be related to different dosing schedules. Bolus administration, leading to a transient pro-oxidative effect, could limit the inflammatory reaction and improve outcomes. We studied 21 anesthetized, mechanically ventilated, invasively monitored, and fluid-resuscitated sheep. Nine hours after inducing peritonitis by injection of autologous feces, the animals were randomized into three groups: (i) bolus injection (2 mg selenium as selenite, followed by 0.06 microg . kg-1 . h-1, n = 7); (ii) continuous infusion (4 microg . kg-1 . h-1 selenium, n = 7), or (iii) control (n = 7). No vasopressors or antibiotics were administered. All animals were monitored until spontaneous death. Peak plasma selenium values reached 4 to 14 micromol . L-1. Compared with the other groups, sheep given a bolus of sodium selenite had delayed hypotension with better maintained cardiac index, delayed hyperlactatemia, fewer sepsis-induced microvascular alterations, and a prolonged survival time (21.9 [bolus group] vs. 18.4 [continuous group] and 18.3 h [control group], P < 0.05). Hence, in this model of septic shock, the administration of a large bolus of sodium selenite (rather than a continuous administration) resulted in beneficial effects, probably by a transient oxidative effect.

2008

Composés séléniés et selenium, quelle place en réanimation

L'atteinte de l'endothélium et le stress oxydant occupent une place majeure dans la dysfonction de la microcirculation précédant les défaillances multiviscérales du choc septique et des syndromes voisins. Le sélénium (Se) est un atome à la fois oxydant et antioxydant. En tant qu'élément trace essentiel, indispensable aux enzymes séléniés, il a un rôle antioxydant majeur chez l'homme. Par ailleurs, les composés non organiques séléniés, comme le sélénite de sodium, sont des oxydants à des degrés variables et sont dès lors des poisons dangereux. Chez le sujet sain, 55 % du Se plasmatique est incorporé dans la sélénoprotéine-P (Sel-P). La Sel-P se fixerait sur l'endothélium activé pour le protéger, d'où la diminution précoce de la sélénémie dans le choc septique. Il reste à prouver formellement que la Sel-P est, dans le sepsis, un marqueur précoce de l'atteinte de l'endothélium et des défaillances viscérales. En réanimation, lors d'un choc septique, l'apport précoce recommandé est de 50 à 200 μg/j de Se, en association avec d'autres micronutriments. Certains recommandent d'étendre, sans danger, la borne supérieure à 400 μg/j (tolerable upper intake level). L'intérêt d'un apport de 200 à 800 μg/j fait l'objet d'études. L'apport de doses supérieures à 800 μg/j (low adverse effect level) et particulièrement en bolus, ouvre une approche thérapeutique nouvelle du choc septique (hyperoxydation paradoxale initiale pour mieux contrôler l'inflammation). S'il existe des arguments cliniques et expérimentaux, en faveur de l'administration en bolus de doses médicamenteuses supérieures à 800 µg/j, des études de toxicité et d'efficacité préalables sont impératives.

Development and validation of a screening procedure for the assessment of inhibition using a recombinant enzyme.

Myeloperoxidase (MPO, E.C. 1.1.11.7) is a heme-containing enzyme that catalyses the synthesis of hypochlorous acid (HOCl) in the presence of hydrogen peroxide (H2O2) and chlorine anions. The production of HOCl is kept under strict control of neutrophils. However, in several pathological conditions, MPO is leaked in the extracellular fluid, which involves an over-production of reactive oxygen species like HOCl and promotes the damages caused by neutrophils. As a consequence, the inhibition of MPO by various agents has been investigated and a variety of molecules have been evaluated for this activity in different models. The present study aims to describe and validate a rapid screening method based on the taurine assay and using a recombinant MPO. After validation of the stock solutions used during the experiments, the amount of MPO for the completion of the reaction was measured and fixed to an optimal value. The inhibiting concentration at 50% of flufenamic acid (taken as a reference molecule) was then assessed in both a simple tube test and a microplate test and delivered similar results (1.3+/-0.2 microM vs 1.4+/-0.2 microM, P=0.2). Finally, different molecules able to inhibit MPO were evaluated in this rapid assay system providing results comparable to literature. The high throughput screening is undoubtedly a first line assessment method which affords the selection of inhibitors and permits to reduce the number of candidates for a further elucidation of the mechanism of MPO inhibition.

Conception of myeloperoxidase inhibitors derived from flufenamic acid by computational docking and structure modification

The development of myeloperoxidase (MPO) inhibitors has been conducted using flufenamic acid as a lead compound. Computational docking of the drug and its analogs in the MPO active site was first attempted. Several molecules were then synthesized and assessed using three procedures for the measurement of their inhibiting activity: (i) the taurine assay, (ii) the accumulation of compound II, and (iii) the LDL oxidation by ELISA. Most of the synthesized molecules had an activity in the same range as flufenamic acid but none of them were able to inhibit the MPO-dependent LDL oxidation. The experiments however gave some useful indications for a rational conception of MPO inhibitors. © 2007 Elsevier Ltd. All rights reserved.

Monocyte-platelet complexes on CD14/CD16 monocyte subsets: relationship with ApoA-I levels. A preliminary study.

The adhesion of the monocytes to the endothelium and their extravasation into the intima are key steps in atherogenesis. Studies showed the essential role of L-selectin (CD62-L), expressed by the monocytes, and the platelets by forming complexes with monocytes. The delipided apolipoprotein (Apo) A or high-density lipoprotein (HDL) has antiinflammatory effects on monocytes and can bind platelets (monocyte-platelet complexes [MPCs]). The aim of this study was to identify a possible relationship between the MPCs, the monocyte subset, and ApoA-I/HDL serum levels in vivo. Platelet-monocyte complexes were estimated by flow cytometry in 16 volunteers. Monocyte-platelet interaction was characterized by the percentage of monocytes coexpressing the constitutive platelet marker, glycocalicin gpIb-alpha (CD42b; CD42b+monocytes in %, MPC%). Monocytes were divided into four subsets based on lipopolysaccharide receptor (CD14) and FcgammaIII receptor (CD16) expression (CD14++/CD16-, G1; CD14++/CD16+, G2; CD14+/CD16-, G3; and CD14+/CD16+, G4). HDL and ApoA-I levels were measured by routine laboratory techniques. MPC% in the different subsets were G1=8.1+/-3.4%, G2=21.2+/-14%, G3=18+/-12.6%, and G4=22.3+/-14.3% (analysis of variance: P<.001). MPC% in the entire monocyte population was negatively correlated to ApoA-I (R=-0.71, P=.001). The relationship between ApoA-I and MPC% was found mainly in the subsets G1 (R=-0.67, P=.001) and G2 (R=-0.61, P=.01). MPC% was not correlated with any other lipids or lipoprotein or high-sensitivity C-reactive protein. When whole blood was incubated with HDL/ApoA-I, no modification of platelet CD42b fluorescence was observed, indicating that there is no direct interaction between the HDL/ApoA-I and the CD42b fluorescence. Among the monocytes, the G2 subset appeared to have the highest extravasation potential. Indeed, we previously showed that those cells overexpressed CD62-L, and we observed in this work that they were coated with platelets more than the G1 cells. The G2 subset could be more directly involved in the development of atherosclerotic lesions.

2007

Resveratrol inhibits the activity of equine neutrophil myeloperoxidase by a direct interaction with the enzyme

Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs: flufenamic acid and its 5-chloro-derivative directly interact with a recombinant human myeloperoxidase to inhibit the synthesis of hypochlorous acid.

The present in vitro study was designed to assess the inhibition of the myeloperoxidase (MPO)/H(2)O(2)/Cl(-) system by several non steroidal anti-inflammatory drugs (NSAIDs) of the oxicam family and of nimesulide and to compare their effect with flufenamic acid in order to investigate their influence on the chlorinating activity of MPO as a protective mechanism during chronic inflammatory syndromes. The inhibition of the system was assessed by measurement of the taurine chlorination while the accumulation of compound II was used to investigate the mechanism of inhibition. The oxidation products of NSAIDs by the MPO/H(2)O(2)/Cl(-) system were identified and flufenamic acid and derivatives were also assessed in the inhibition of LDL oxidation in two models. Flufenamic acid (IC(50) = 1.1+/-0.3 microM) is the most efficient inhibitor of the MPO/H(2)O(2)/Cl(-) system and nimesulide (IC(50) = 2.1+/-0.3 microM) is more active than the other NSAIDs of the oxicam family (IC(50) = 8-12 microM). The accumulation of compound II revealed that flufenamic acid acts as an electron donor while the other NSAIDs are antagonists of chloride anions. The identification of the oxidation products confirms that flufenamic behaves like an electron donor and is directly oxidized in the 5-hydroxy-derivative but gives also the 5-chloro-derivative which similarly inhibits the MPO/H(2)O(2)/Cl(-) system. Flufenamic acid has the best inhibiting activity towards the MPO/H(2)O(2)/Cl(-) system. However, in models that assess the LDL oxidation, flufenamic acid and its derivatives were unable to properly inhibit MPO activity as the enzyme is adsorbed on macrostructures such as LDL molecules.

Probucol does not inhibit myeloperoxidase-dependent low-density lipoprotein oxidation as a potent protective effect in atherosclerosis

2006

Triggering of inflammatory response by myeloperoxidase-oxidized LDL.

The oxidation theory proposes that LDL oxidation is an early event in atherosclerosis and that oxidized LDL contributes to atherogenesis in triggering inflammation. In contrast to the copper-modified LDL, there are few studies using myeloperoxidase-modified LDL (Mox-LDL) as an inflammation inducer. Our aim is to test whether Mox-LDL could constitute a specific inducer of the inflammatory response. Albumin, which is the most abundant protein in plasma and which is present to an identical concentration of LDL in the intima, was used for comparison. The secretion of IL-8 by endothelial cells (Ea.hy926) and TNF-alpha by monocytes (THP-1) was measured in the cell medium after exposure of these cells to native LDL, native albumin, Mox-LDL, or Mox-albumin. We observed that Mox-LDL induced a 1.5- and 2-fold increase (ANOVA; P < 0.001) in IL-8 production at 100 microg/mL and 200 microg/mL, respectively. The incubation of THP-1 cells with Mox-LDL (100 microg/mL) increased the production of TNF-alpha 2-fold over the control. Native LDL, albumin, and Mox-albumin showed no effect in either cellular types. The myeloperoxidase-modified LDL increase in cytokine release by endothelial and monocyte cells and by firing both local and systemic inflammation could induce atherogenesis and its development.

The pleiotropic effect of statins in haemodialysis patients is not the consequence of an inhibition of LDL oxidation by myeloperoxidase.

Captopril inhibits the oxidative modification of apolipoprotein B-100 caused by myeloperoxydase in a comparative in vitro assay of angiotensin converting enzyme inhibitors

The oxidative modification of low-density lipoproteins (LDL) is a key event in the formation of atheromatous lesions. Indeed, oxidized derivatives accumulate in the vascular wall and promote a local inflammatory process which triggers the progression of the atheromatous plaque. Myeloperoxidase (MPO) has been mentioned as a major contributor to this oxidative process. It takes part in the oxidation both of lipids by chlorination and peroxidation and of apolipoprotein B-100. Based on recent observations with several anti-inflammatory and thiol-containing drugs, the present study was designed to test the hypothesis that anti-hypertensive agents from the angiotensin converting enzyme (ACE) inhibitors group inhibit the oxidative modifications of Apo B-100 caused by MPO. Captopril, ramipril, enalapril, lisinopril and fosinopril were assessed by measuring: their inhibiting effect on the MPO / H2O2 / Cl- system, the accumulation of compound II, which reflects the inhibition of the synthesis of HOCl and the LDL oxidation by MPO in presence of several concentrations of ACE inhibitors. Only captopril, a thiol-containing ACE inhibitor, was able to significantly decrease the oxidative modification of LDL in a dose dependent manner and this by scavenging HOCl. This efficient anti-hypertensive drug therefore appears to also protect against the atherosclerotic process by this newly documented mechanism. © 2006 Elsevier B.V. All rights reserved.

2005

Thiol-containing molecules interact with the myeloperoxidase/H 2O2/chloride system to inhibit LDL oxidation

Oxidized low-density lipoproteins (LDL) accumulate in the vascular wall and promote a local inflammatory process contributing to the progression of atheromatous plaque. The key role of myeloperoxidase (MPO) in this process has been documented and the enzyme has been involved in the oxidative modification of apolipoprotein B-100 in the intima and at the surface of endothelial cells. As the inhibition of this last phenomenon could be of relevance in pharmacological interventions, thiol-containing molecules such as glutathione, captopril, and N-acetylcysteine (NAC) and its lysinate salt (NAL) were tested in this system and their properties were compared with those of flufenamic acid (control). This last compound already demonstrated an inhibition of the production of HOCl by MPO and a more intense inhibition of MPO activity than glutathione, NAC, NAL, and captopril. However, NAC and NAL inhibited the oxidative modification of LDL more intensively than captopril and glutathione whereas flufenamic acid had no comparable inhibiting effect. This could be related to the presence of LDL close to the catalytic site of the enzyme. NAC and NAL therefore appeared as the most efficient inhibitors probably as a consequence of their relatively small size. The relevance of such effects has to be documented by in vivo studies. © 2005 Elsevier Inc. All rights reserved.

2004

The reactions of oxicam and sulfoanilide non steroidal anti-inflammatory drugs with hypochlorous acid: Determination of the rate constants with an assay based on the competition with para-aminobenzoic acid chlorination and identification of some oxidation products

In vitro comparative assessment of the scavenging activity against three reactive oxygen species of non-steroidal anti-inflammatory drugs from the oxicam and sulfoanilide families

The study of the interaction of non-steroidal anti-inflammatory drugs (NSAIDs) with several reactive oxygen species is of great interest in inflammatory conditions where an uncontrolled release of these potentially damaging intermediates has been documented. This study focused on the scavenging of three species (hydroxyl radical, hydrogen peroxide and hypochlorous acid) with several members of the oxicam family and with the sulfoanilide nimesulide. Reaction with hydroxyl radical was assessed by the modified deoxyribose assay, and rate constants were calculated showing values between 0.8 and 1.1×1010 M-1 s-1 for oxicams and of about 0.9×1010 M-1 s-1 for nimesulide and ibuprofen. These were consistent with those of the literature but in the same range as those for other NSAIDs and for several thiol-containing molecules. The study of hydrogen peroxide scavenging by the horseradish peroxidase (HRP) assay lacked specificity but no interaction could be evidenced by the glutathione peroxidase assay. The scavenging of hypochlorous acid was finally investigated by the recently developed para-aminobenzoic acid assay which demonstrated better performances for meloxicam (1.7×104 M-1 s -1) as compared to the other oxicams (tenoxicam: 4.0×10 4 M-1 s-1, piroxicam: 3.6×104 M-1 s-1, lornoxicam: 4.3×104 M -1 s-1) and nimesulide (2.3×103 M -1 s-1). These rate constants were, however, lower than those for thiol-containing molecules and ascorbate. These results suggest that the antioxidant properties of NSAIDs could be influenced by a proper pharmacomodulation as far as the scavenging of hypochlorous acid is concerned while the interest is quite limited for the scavenging of hydroxyl radical. © 2004 Elsevier B.V. All rights reserved.