References of "Servais, Anne-Catherine"
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See detailStudy of insulin aggregation by SEC and CGE
Demelenne, Alice ULiege; Napp, Aurore ULiege; Servais, Anne-Catherine ULiege et al

Conference (2016, October)

Introduction: Insulin is a widely used antidiabetic drug, which regulates carbohydrate and fat metabolism of human body. This hormone is mostly formulated in hexamer by addition of zinc as an excipient ... [more ▼]

Introduction: Insulin is a widely used antidiabetic drug, which regulates carbohydrate and fat metabolism of human body. This hormone is mostly formulated in hexamer by addition of zinc as an excipient but only the monomeric form is active once dissociated in the bloodstream. Insulin is prone to unfold when submitted to denaturating factors as temperature, ionic strength, agitation and pH. An accumulation of unfolded proteins results in a high tendency to aggregate and form amyloid fibrils. A deposit of those fibrils in the subcutaneous tissue leads to a complication called “insulin-derived amyloidosis”. Moreover, during its production, insulin is often subjected to extreme conditions making lack of aggregates an important parameter to be controlled during its quality control. United States and European Pharmacopoeias use both size exclusion chromatography (SEC) to assess the level of covalent high molecular weight species. This technique is reproducible, and easy to use but shows many drawbacks including possible changes in the aggregates composition by dilution into the HPLC system or adsorption of sample onto the stationary phase. For those reasons other techniques have been considered in the literature for studying aggregation of insulin. Optical microscopy, electron microscopy, dynamic light scattering, turbidimetry, Fourier Transform infrared spectroscopy, Raman spectroscopy, thioflavin T fluorescence and circular dichroism spectroscopy are some of them. In any cases, the use of orthogonal techniques is essential to assess the relevance of the results. Results: In this study, insulin aggregates were generated after optimization of incubation conditions (pH, temperature, agitation…). Those aggregates were then analyzed by SEC and capillary electrophoresis (CE). CE shows many advantages in terms of sample and solvent consumption and enables analysis of samples under their native form. We showed that capillary gel electrophoresis (CGE) is a promising technique to analyze covalent aggregates of insulin due to the fact that it separates the aggregates according to their size and not to their size/charge ratio. The use of a laser-induced fluorescence detector was also found attractive to enhance the sensitivity of the method. [less ▲]

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See detailCapillary electrophoresis method to determine siRNA complexation with cationic liposomes
Furst, Tania ULiege; Bettonville, Virginie ULiege; Farcas, Elena ULiege et al

in Electrophoresis (2016)

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally ... [more ▼]

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation are gel electrophoresis and RiboGreen® fluorescence-based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this work is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this research, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short-end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared to the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated to CE approach since no carcinogenic product is used. Finally, this methodology can also be extended to the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles. [less ▲]

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See detailApproches intégrées en Sciences Pharmaceutiques
Servais, Anne-Catherine ULiege; Ziemons, Eric ULiege

Conference (2016, May 13)

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See detailVolumetric Absorptive Microsampling for Hepcidin Peptide Extraction from Whole Blood
Houbart, Virginie ULiege; COBRAIVILLE, Gaël ULiege; Nys, Gwenaël ULiege et al

in LCGC North America (2016), 34(5),

Whole blood analysis is an emerging trend in the field of bioanalysis. We developed a fast and simple protocol to extract and analyze a peptide, hepcidin, from whole blood. Sampling and extraction were ... [more ▼]

Whole blood analysis is an emerging trend in the field of bioanalysis. We developed a fast and simple protocol to extract and analyze a peptide, hepcidin, from whole blood. Sampling and extraction were carried out using volumetric absorptive microsampling (VAMS), a novel blood collection method that allows the sampling of a known blood volume independently from hematocrit. The composition of the extraction medium was optimized using an experimental design to get the most intense signal of hepcidin, considering different organic solvents and acidic additives. [less ▲]

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See detailFully automated electrophoretically mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate approach
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Lamalle, Caroline ULiege et al

in Electrophoresis (2016), 37(2), 248-255

In this study, a fully automatized in-capillary system was developed to monitor the activity of CYP1A1 in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an in-line bioreaction ... [more ▼]

In this study, a fully automatized in-capillary system was developed to monitor the activity of CYP1A1 in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an in-line bioreaction in the presence of CYP1A1 supersomes and NADPH as co-factor, giving rise to hydroxycoumarin, the product that was assayed. The optimization of the experimental conditions was supported by the application of a design of experiment, providing a better understanding of electrophoretic mixing parameters that influence the metabolic reactions. The results obtained in optimal conditions were compared not only to those achieved after off-line metabolization but also with liver microsomes. Finally, inhibition studies were conducted showing an important decrease of hydroxycoumarin formation using apigenin as CYP1A1 potent inhibitor. This study demonstrates the usefulness of our in-line system for the fully automated in vitro metabolism studies and the screening of new CYP1A1 inhibitors. [less ▲]

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See detailEnantioselective capillary electrophoresis-mass spectrometry of amino acids in cerebrospinal fluid using a chiral derivatizing agent and volatile surfactant.
Prior, A.; Moldovan, Radu-Cristian ULiege; Crommen, Jacques ULiege et al

in Analytica Chimica Acta (2016), 940

The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new ... [more ▼]

The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new method is presented in which the use of a chiral selector is circumvented by employing (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral AA derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers. Efficient AA derivatization with FLEC was completed within 10 min. Infusion experiments showed that the APFO concentration hardly affects the MS response of FLEC-AAs and presents significantly less ion suppression than equal concentrations of ammonium acetate. The effect of the pH and APFO concentration of the BGE and the capillary temperature were studied in order to achieve optimized enantioseparation. Optimization of CE-MS parameters, such as sheath-liquid composition and flow rate, ESI and MS settings was performed in order to prevent analyte fragmentation and achieve sensitive detection. Selective detection and quantification of 14 chiral proteinogenic AAs was achieved with chiral resolution between 1.2 and 8.6, and limits of detection ranging from 130 to 630 nM injected concentration. Aspartic acid and glutamic acid were detected, but not enantioseparated. The optimized method was applied to the analysis of chiral AAs in cerebrospinal fluid (CSF). Good linearity (R(2) > 0.99) and acceptable peak area and electrophoretic mobility repeatability (RSDs below 21% and 2.4%, respectively) were achieved for the chiral proteinogenic AAs, with sensitivity and chiral resolution mostly similar to obtained for standard solutions. Next to l-AAs, endogenous levels of d-serine and d-glutamine could be measured in CSF revealing enantiomeric ratios of 4.8%-8.0% and 0.34%-0.74%, respectively, and indicating the method's potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs. [less ▲]

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See detailAnalysis of protamine peptides in insulin pharmaceutical formulations by capillary electrophoresis.
Lamalle, Caroline; Servais, Anne-Catherine ULiege; Demelenne, Alice ULiege et al

in Journal of Separation Science (2016), 39(6), 1189-94

Protamines are a group of highly basic peptides that are sometimes added to insulin formulations to prolong the pharmacological action. In this study, different methods were investigated to identify ... [more ▼]

Protamines are a group of highly basic peptides that are sometimes added to insulin formulations to prolong the pharmacological action. In this study, different methods were investigated to identify protamine in insulin formulations. Capillary electrophoresis in aqueous and non-aqueous media was tested to separate these peptides with very close amino acid sequences. Different buffers (phosphate or formate, both acidified) and various additives (principally negatively charged and neutral surfactants) were investigated to optimize peptide separation. Finally, a micellar electrokinetic capillary chromatography method using a capillary of 120 cm effective length and an aqueous background electrolyte made up of 100 mM phosphate buffer (pH 2) and 50 mM Thesit(R) gave the best results, providing the separation of the four major protamine peptides within 25 min. [less ▲]

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See detailLiquid chromatography separation of the chiral prodrug eslicarbazepine acetate and its main metabolites in polar organic mode. Application to their analysis after in vitro metabolism.
Servais, Anne-Catherine ULiege; Janicot, Bertrand; Takam, Arnold et al

in Journal of Chromatography. A (2016), 1467

A LC method using a chiral stationary phase (CSP) with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector in polar organic mode (POM) was developed for the separation of the ... [more ▼]

A LC method using a chiral stationary phase (CSP) with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector in polar organic mode (POM) was developed for the separation of the biopharmaceutic classification system (BCS) class II chiral prodrug eslicarbazepine acetate (ESL) and its main metabolites, namely eslicarbazepine, its optical antipode, (R)-licarbazepine, and the achiral oxcarbazepine (OXC). The percentage of methanol (MeOH) in the mobile phase containing acetonitrile (ACN) as the main solvent was found to significantly influence analyte retention and resolution. A reversal of elution order of OXC and (R)-licarbazepine was observed, depending on the MeOH percentage in the mobile phase. The optimized mobile phase consisted of ACN/MeOH/acetic acid/diethylamine (95/5/0.2/0.07; v/v/v/v). The potential of this chemo- and enantioselective LC method combined with solid-phase extraction (SPE) was then evaluated for in vitro metabolism studies using ESL as a model case. Only eslicarbazepine could be detected after incubation of ESL in human liver microsome systems. [less ▲]

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See detailStudy of intact virus-like particles of human papillomavirus by capillary electrophoresis
Bettonville, Virginie ULiege; Nicol, Jérôme ULiege; Thelen, Nicolas ULiege et al

in Electrophoresis (2016), 37

Virus-like particles of human papillomavirus (HPV-VLP), resulting from the self-assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native ... [more ▼]

Virus-like particles of human papillomavirus (HPV-VLP), resulting from the self-assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil® and Cervarix®, are based on HPV-VLP L1. Analytical techniques currently used to characterize HPV-VLP, such as SDS-PAGE, Western blot, ELISA, are time-consuming and semi-quantitative. In this study, capillary electrophoresis (CE) was evaluated for the analysis of intact HPV16-VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH and detergent additives were investigated. Reproducible HPV-VLP analysis in CE was achieved using poly(ethylene oxide) coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV-VLP detection in less than 10 min (migration times RSD : 1.6 %). The identity of HPV-VLP peak was confirmed by comparison with a sample obtained from a wild-type baculovirus and with VLP-based vaccine, Gardasil®, after adjuvant dissolution. Finally, we applied the developed methodology to VLP-based vaccines, demonstrating that CE could be successfully used for vaccine quality control. [less ▲]

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See detailFULLY AUTOMATED ELECTROPHORETICALLY MEDIATED MICROANALYSIS SYSTEM FOR CYP1A1 ACTIVITY MONITORING
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Lamalle, Caroline ULiege et al

Poster (2015, June 23)

Introduction Since the efficacy and toxicity of drugs are closely related to their pharmacokinetics, a good understanding of metabolic pathways is important at an early stage of development. The ... [more ▼]

Introduction Since the efficacy and toxicity of drugs are closely related to their pharmacokinetics, a good understanding of metabolic pathways is important at an early stage of development. The identification of the enzymes involved in drug metabolism is thus of critical importance for the design of further clinical studies. The availability of specifically expressed human CYPs, namely supersomes, allows the investigation of the contribution of a single metabolic enzyme to the biotransformation pathway of the compound under investigation. CYP1A1, a member of the cytochrome P450 superfamily, was studied in this project. Interestingly, it has been described to be over expressed in various types of cancer. Consequently, CYP1A1 has emerged as a particularly interesting target for cancer therapy. Methods All the experiments were carried out on a HP3DCE system equipped with an on-column DAD. The EMMA procedure was performed by injecting a plug containing CYP1A1 supersomes, followed by a plug that contained the co-factor and the substrate, then another plug of CYP1A1 supersomes. The reaction was triggered by the application of a voltage switch. The voltage was then turned off to allow the metabolic reaction to occur. The separation of the components was then performed at -25 kV. Results The present study describes the development of a fully automatized in-capillary method to follow metabolization of 7-hydroxycoumarin and screen CYP1A1 inhibitors. After preliminary studies, satisfying results were obtained using CYP1A1 at a concentration of 200 pmol/mL, while the incubation time was settled to 15 min. Equal reactant plugs were injected at -50 mbar for 6 sec. The short-end injection performed gave rise to a baseline separation of the molecules (substrate, product, CYP1A1 and NADPH) in less than 2 minutes. Adequate plugs overlap was obtained using electrophoretic mixing. The DoE performed highlighted that the voltage switch has a great impact on the metabolite formation. The amount of product obtained in the optimal conditions was found to be comparable to the one detected after conventional off-line metabolization. Besides the interest of developing an automatized CE approach for metabolisation studies, we also wanted to investigate the potentiality of this approach to screen CYP1A1 inhibitors. The ability of our system to monitor CYP1A1 inhibition was undertaken with apigenin, a well-known inhibitor. It is noteworthy that the compatibility of our system with MEKC ensures its applicability to a large variety of molecules. Novel aspect Monitoring CYP1A1 activity using a rapid and fully automated EMMA method that could be used for new anticancer agents screening. [less ▲]

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See detailFully automated electrophoretically mediated microanalysis system for CYP1A1 activity monitoring
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Lamalle, Caroline ULiege et al

Conference (2015, May 28)

In order to evaluate the potentiality of capillary electrophoresis for CYP1A1 activity monitoring, an in-line method was developed with the well-known 7-ethoxycoumarin substrate. The electrophoretically ... [more ▼]

In order to evaluate the potentiality of capillary electrophoresis for CYP1A1 activity monitoring, an in-line method was developed with the well-known 7-ethoxycoumarin substrate. The electrophoretically mediated microanalysis approach was used with CYP1A1 supersomes to provide a rapid and fully automated method. The in-line homogenous enzyme assay was performed in physiological conditions (pH 7.4), whereas a MEKC buffer was used as background electrolyte. In order to reduce the analysis time, the short end injection was performed. Firstly a plug containing CYP1A1 supersomes was hydrodynamically injected into a fused silica capillary, followed by a plug of co-factor (NADPH) and substrate (7-ethoxycoumarin) and finally another plug of CYP1A1 (sandwich mode). The experimental conditions were finely investigated and tuned by design of experiment methodology. The metabolization rate measured in the optimized conditions was comparable with the one obtained after off-line metabolization. Finally, inhibition studies were conducted and a significant decrease of 7-hydroxycoumarin formation was observed using apigenin as CYP1A1 potent inhibitor. [less ▲]

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