Background and Aims : A diet rich in eicosapentaenoic acid (EPA, C20:5 n-3) is cardioprotective. We studied in vitro the impact of EPA membrane incorporation on the anti-atherogenic ABCA1 efflux pathway in the absence or presence of LPS to create the inflammatory microenvironment observed within atherosclerotic plaques.

Porphyrins are a class of photosensitizers used in photodynamic therapy (PDT). Understanding the interaction of porphyrins with membrane cells components is important in order to improve this therapy. Many analytical methods can be used for this aim. High performance liquid chromatography (HPLC) was used for the separation of porphyrins on RP and HILIC stationary phases as well as on a biomimetic membrane IAM phase. Twenty-six tetraphenyl porphyrins (TPP) were successfully separated on an IAM column, a C18 Gravity RP column, a C8 Gravity RP column, a PolarTec RP column and a HILIC column. Stationary phases were chosen as the most appropriate to cover the study of different types of interactions. Elution was performed with a 45 min linear gradient. Obtained gradient retention times were converted to gradient chromatography hydrophobicity index (CHI) and to an apparent retention factor (kapp). The partition coefficients (logP) of the 26 compounds were measured in a 2-octanol/PBS system and estimated in silico. Correlation between kapp values was studied. Moreover, a multivariate analysis was performed to explain columns relationships. Obtained results show that porphyrins are separated mainly according to hydrophobic interactions that are relative to their structure (sugar number and the disposition around the porphyrin macrocycle).

This study focuses on the mechanism of sitamaquine-resistance in Leishmania donovani. Sitamaquine accumulated 10 and 1.4 fold more in cytosol than in membranes of wild-type (WT) and of sitamaquine-resistant (Sita-R160) L. donovani promastigotes, respectively. The sitamaquine accumulation was a concentration-dependent process in WT whereas a saturation occurred in Sita-R160 suggesting a reduced uptake or an increase of the sitamaquine efflux. Membrane negative phospholipids being the main target for sitamaquine uptake, a lipidomic analysis showed that sitamaquine-resistance did not rely on a decrease of membrane negative phospholipid rate in Sita-R160, discarding the hypothesis of reduced uptake. However, sterol and phospholipid metabolisms were strongly affected in Sita-R160 suggesting that sitamaquine-resistance could be related to an alteration of phosphatidylethanolamine-N-methyl-transferase and choline kinase activities and to a decrease in cholesterol uptake and of ergosterol biosynthesis. Preliminary data of proteomics analysis exhibited different protein profiles between WT and Sita-160R remaining to be characterized.

Visceral leishmaniasis is a life-threatening disease that affects nearly a million people every year. The emergence of Leishmania strains resistant to existing drugs complicates its treatment. The purpose of this study was to develop a new lipid formulation based on nanocochleates combining two active drugs: Amphotericin B (AmB) and Miltefosine (HePC). Nanocochleates composed of dioleoylphosphatidylserine (DOPS) and Cholesterol (Cho) and Ca2+, in which HePC and AmB were incorporated, were prepared. Properties such as particle size, zeta potential, drug payload, in-vitro drug release and storage stability were investigated. Moreover, in-vitro stability in gastrointestinal fluid was performed in view of an oral administration. AmB-HePC-loaded nanocochleates with a mean particle size of 250 +/- 2 nm were obtained. The particles displayed a narrow size distribution and a drug payload of 29.9 +/- 0.5 mg/g for AmB, and 14.0 +/- 0.9 mg/g for HePC. Drug release occurred preferentially in intestinal medium containing bile salts. Therefore, AmB-HePC-loaded nanocochleates could be a promising oral delivery system for the treatment of visceral leishmaniasis. (C) 2014 Elsevier B.V. and Societe francaise de biochimie et biologie Moleculaire (SFBBM). All rights reserved.

Background Due to repetitive and tedious handling tasks, production of anticancer drugs for infusion is associated with a high risk of non-conformity. Thus, on-line quality control is necessary to improve the quality of preparation. Since the quantities produced are ever growing, very fast analytical methods of control are needed to minimise the delay before release. Purpose A high-performance liquid chromatography method has been developed for quality control of vinca alkaloid infusion bags (vindesine, vincristine, vinorelbine and vinblastine). Materials and Methods The separation was optimised by a Doehlert experimental design using a mixture of those 4 alkaloids. Chromatography was performed using Prostar Varian chromatographic equipment with a Photodiode array Detector. A short Polaris C18 column (3 µm, 50 mm × 4.6 mm) was used for all separations. The optimization varied 3 parameters: pH of the phosphate buffer 25 mM (7.0–7.6), flow rate of the mobile phase (0.7–1.3 mL.min−1) and proportion of acetonitrile (47–53%). 36 trials were necessary. The target response was the shortest run time giving a minimal resolution score of 1.5 for the most critical pair of peaks. Results For vinorelbine, pH had a major effect on resolution. Optimal resolutions were obtained with a pH of 7.25. Then, the flow rate was set at 1.6 mL.min−1 with a mobile phase consisting of water-acetonitrile (47–53 v/v). Under these conditions, resolution was at least 1.6 with an analysis time less than 2.0 min. Retention times were 1.03, 1.27, 1.39 and 1.68 minutes for vindesine, vincristine, vinblastine and vinorelbine respectively. Methods were validated according to ICH criteria and are now routinely used without troubleshooting. Conclusions This method allows in-line quality control of 4 vinca alkaloids in a very short time (less than 2 minutes) and constitutes a suitable and safe tool for chemotherapy preparation. No conflict of interest.

Leishmaniasis is a widespread parasitic disease principally treated by intravenous drugs. Hexadecylphosphocholine (miltefosine) has recently proved its efficacy by oral route. Although its mechanism of action has been investigated, and principally relies on perturbations of the metabolism of lipids and especially phospholipids, further studies need to be conducted to detect precisely which metabolic pathways are impacted. For this purpose, the present work proposes a complete lipidomic study focused on membrane phospholipids of clones of Leishmania donovani non-treated (NT), treated (T) and resistant (R) to miltefosine. Firstly, a separation of phospholipids in normal phase high-performance liquid chromatography (NP-HPLC) was coupled to a mass spectrometer (MS) equipped with an electrospray (ESI) ion source, and response was compared to evaporative light scattering detection (ELSD). Secondly, a quantification of phospholipid classes was performed using NP-HPLC/ESI/MS on NT, T and R clones of L. donovani . Thirdly, full-scan acquisitions of analyzed samples were compared using orthogonal signal correction-partial least square-discriminant analysis (OSC-PLS-DA) to highlight phospholipid molecular species of interest between the three types of clones. Structural determination of the most relevant species has finally been performed using tandem mass spectrometry. A first hypothesis on the effect of miltefosine on lipid metabolic pathways is then proposed.

Beads made of alpha-cyclodextrin and soybean oil are an efficient system for the encapsulation of lipophilic drugs. In this work we investigated the impact of vegetable oil composition and physicochemical properties on bead feasibility and properties. Vegetable oils used in the formulation of dermatological medicines were selected. Beads were successfully formed using wheat germ and sweet almond oils. The oils which could be formulated as beads had low acid value and contained at least 99 % of triglycerides. Bead yield, crystalline organization and rheological behavior of these formulations were similar. Beads formed from soybean oil were larger than those from wheat germ and sweet almond oils due to some traces of triglyceride degradation products. Borage oil led only to a fluid mixture which did not evolve into beads. The acid value of this oil was high and total triglyceride content was 68 % demonstrating the presence of a significant amount of triglyceride degradation products. The rheological behavior and X-ray diffraction pattern of the borage oil-based formulation were different from the other oils. Beads can be successfully prepared with various vegetable oils without modifying the conditions optimized with soybean oil. However, triglyceride degradation products can either increase bead size or prevent their formation.

The stratum corneum owns a barrier function, which can be altered by a modification of its lipid composition. In this work, a new simple method was developed in normal phase liquid chromatography to separate the three major lipid classes of the stratum Corneum, fatty acids, ceramides, and cholesterol. A quantitative investigation was carried out, comparing an internal normalisation and an external standardisation with two universal detectors: an evaporating light scattering detector and a charged aerosol detector, the Corona. The study points out the fact that the external standardisation is suitable for a quantitative study of the different lipid classes, using a representative standard for each class. Moreover, the use of the Corona is more adapted for further investigation, due to a better repeatability, accuracy, and precision. A test made on a lipid extract from the forearm of volunteers shows that the present method is appropriate for biological extract analysis.

OBJECTIVES This study focuses on the mechanism of interaction of sitamaquine with Leishmania donovani membranes, and its accumulation within the parasites. METHODS A biomimetic model of the outer layer of a Leishmania plasma membrane was used to examine the interactions of sitamaquine with lipids. The plasma membranes of L. donovani promastigotes were depleted of sterol using cholesterol oxidase, in order to assess the importance of sterols in drug-membrane interactions. Sterols were quantified and sitamaquine susceptibility was assessed using the MTT test. Kinetics of sitamaquine accumulation and efflux were measured under different conditions. RESULTS Sitamaquine interacts first with phospholipid anionic polar head groups and then with phospholipid acyl chains to insert within biological membranes and accumulates rapidly in the Leishmania cytosol according to a sterol-independent process. The rapid sitamaquine efflux observed was related to an energy-dependent mechanism since the intracellular amount of sitamaquine was enhanced three times in the absence of glucose and the efflux was inhibited in energy-depleted conditions. (1)H NMR analysis of motile lipid showed that sitamaquine did not affect lipid trafficking in Leishmania. CONCLUSIONS We propose that sitamaquine rapidly accumulates in Leishmania by diffusion along an electrical gradient and is concentrated in the cytosol by an energy- and sterol-independent process. The affinity of sitamaquine for membranes was transitory and an energy-dependent efflux was demonstrated, suggesting the presence of an as yet uncharacterized transporter.

Les cyclodextrines naturelles, qui sont des oligosaccharides cycliques, peuvent être modifiées pour obtenir des dérivés plus solubles ou insolubles dans l’eau. Leur intérêt majeur est l’encapsulation moléculaire de composés hydrophobes au sein de leur cavité pour former un complexe d’inclusion. Cette propriété a été mise à profit en pharmacie galénique, pour administrer des actifs difficiles à formuler et/ou fragiles. Une application plus récente et plus originale des cyclodextrines consiste à les utiliser pour formuler des systèmes dispersés comme les micro- et nanoparticules ou bien encore les liposomes. L’apport des cyclodextrines est alors multiple : amélioration de la stabilité (molécules fragiles) et du taux d’encapsulation, modification des caractéristiques des particules… De façon plus originale, les cyclodextrines peuvent aussi être employées comme excipient constitutif du système dispersé (émulsionnant, matériau). Par exemple, les interactions entre les cyclodextrines et les triglycérides, qui sont les composés majoritaires des huiles végétales, permettent à la fois de stabiliser des émulsions simples ou multiples mais aussi de former des particules appelées « billes ». Très riches en huile, le potentiel de ces particules pour la formulation de molécules lipophiles est intéressant aussi bien pour leur administration par voie orale que cutanée. Ces deux applications sont plus particulièrement développées dans cet article.

This study was initiated by the laboratories and control department of the French Health Products Safety Agency (AFSSAPS) as part of the fight against the public health problem of rising counterfeit and imitation medicines. To test the discriminating ability of Near InfraRed Spectroscopy (NIRS), worse cases scenarios were first considered for the discrimination of various pharmaceutical final products containing the same Active Pharmaceutical Ingredient (API) with different excipients, such as generics of proprietary medicinal products (PMP). Two generic databases were explored: low active strength hard capsules of Fluoxetine and high strength tablets of Ciprofloxacin. Then 4 other cases involving suspicious samples, counterfeits and imitations products were treated. In all these cases, spectral differences between samples were studied, giving access to API or excipient contents information, and eventually allowing manufacturing site identification. A chemometric background is developed to explain the optimisation methodology, consisting in the choices of appropriate pretreatments, algorithms for data exploratory analyses (unsupervised Principal Component Analysis), and data classification (supervised cluster analysis, and Soft Independent Modelling of Class Analogy). Results demonstrate the high performance of NIRS, highlighting slight differences in formulations, such as 2.5% (w/w) in API strength, 1.0% (w/w) in excipient and even coating variations (<1%, w/w) with identical contents, approaching the theoretical limits of NIRS sensitivity. All the different generic formulations were correctly discriminated and foreign PMP, constituted of formulations slightly different from the calibration ones, were also all discriminated. This publication addresses the ability of NIRS to detect counterfeits and imitations and presents the NIRS as an ideal tool to master the global threat of counterfeit drugs.

Natural cyclodextrins are cyclic oligosaccharides which can be modified to obtain more water soluble or insoluble derivatives. The main interest of cyclodextrins results from their ability to form an inclusion complex with hydrophobic molecules. Inclusion constitutes a true molecular encapsulation. This property is employed in pharmaceutical industry to facilitate the formulation of poorly water soluble and/or fragile drugs. A more recent application of cyclodextrins consists in their use in the preparation of dispersed systems such as micro- and nanoparticles or even liposomes. When incorporated in dispersed systems, cyclodextrin can enhance drug solubility, drug stability and drug loading. Interestingly, cyclodextrins themselves can also be employed to form or stabilise dispersed systems (material or emulsifying agent). For example, the interactions between cyclodextrins with components of the vegetable oils (more especially with triglycerides) allow to stabilise simple or multiple emulsions but also to form particles called "beads". Very rich in oil, this novel lipid carrier presents an important potential for the encapsulation of highly lipophilic compounds and their delivery by topical and oral routes. These two applications are more particularly developed in the present paper.

A rapid and simple method for the simultaneous determination of tobramycin and colistin sulphate in a pharmaceutical formulation by reversed phase HPLC and evaporative light scattering detection is described. Chromatographic separation was carried out in gradient mode using a Zorbax SB C18 column (150mm×4mm, 3.5μm) with mobile phases of acetonitrile and water containing trifluoroacetic at 1ml/min. The method was validated using methodology described by the International Conference of Harmonization. The method was shown to be specific, precise, accurate and linear. Real samples were analyzed to demonstrate the applicability of the chromatographic method in a routine use.

The performance of charged aerosol detection (CAD) was compared to evaporative light scattering detection (ELSD) for the analysis of Leishmania membrane phospholipid (PL) classes by NP-HPLC. In both methods, a PVA-Sil column was used for the determination of the major Leishmania membrane PLs, phosphatidic acid, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylethathanolamine, phosphatidylserine, lysophosphatidylethathanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine in the same analysis. Although the response of both detection methods can be fitted to a power function, CAD response can also be described by a linear model with determination coefficients (R(2)) ranging from 0.993 to 0.998 for an injected mass of 30 ng to 20.00 microg. CAD appeared to be directly proportional when a restricted range was used and it was found to be more sensitive at lowest mass range than ELSD. With HPLC-ELSD the limits of detection (LODs) were between 71 and 1195 ng and the limits of quantification (LOQs) were between 215 and 3622 ng. With HPLC-CAD, the LODs were between 15 and 249 ng whereas the limits of quantification (LOQs) were between 45 and 707 ng. The accuracy of the methods ranged from 62.8 to 115.8% and from 58.4 to 110.5% for ELSD and CAD, respectively. The HPLC-CAD method is suitable to assess the influence of miltefosine on the composition of Leishmania membrane phospholipids.

Polyethylene glycol (PEG) chains covalently linked to phospholipids are often used in the preparation of lipid or even polymer colloidal particles to avoid recognition and clearance by the reticuloendothelial system and to increase their plasmatic half-life. To the best of our knowledge, no direct method allows yet to quantify these pegylated phospholipids. The aim of this work was to develop a method for the quantification of a typical pegylated phospholipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], DSPE-PEG2000, associated to polymeric microcapsules of perfluorooctyl bromide (PFOB). Reverse phase high-performance liquid chromatography (HPLC) was used, coupled with a corona charged aerosol detection (HPLC–CAD). Calibrations standards consisted of plain microcapsules and pegylated phospholipids (DSPE-PEG2000) in the concentration range of 2.23–21.36μg/mL (0.22–2.14μg injected). Calibration curve was evaluated with two different model, linear and power model. The power model describes experimental values better than the linear model, for pegylated phospholipids with the CAD detector. The correlation coefficient for the power model was 0.996, and limits of detection and quantification obtained were 33 and 100ng, respectively. This method proved to be selective and sensitive; the accuracy of the method ranged from 90 to 115% and the relative standard deviation was ≤5.3%. Pegylated phospholipids associated to microcapsules, as well as the phospholipids and total phospholipids in the suspensions were successfully quantified in three different preparations of microcapsules.

Near InfraRed Spectroscopy (NIRS) is a potentially powerful tool for assessing the homogeneity of industrial powder blends. In the particular context of hospital manufacturing, we considered the introduction of the technique at a small pharmaceutical process scale, with the objective of following blend homogeneity in mixtures of seven components. This article investigates the performance of various NIRS-based methodologies to assess powder blending. The formulation studied is prescribed in haematology unit, as part of the treatment for digestive decontamination in children receiving stem-cell transplantation. It is composed of the active pharmaceutical ingredients (APIs) colimycin and tobramycin and five excipients. We evaluated 39 different blends composing 14 different formulations, with uncorrelated proportions of constituents between these 14 formulations. The reference methods used to establish the NIRS models were gravimetry and a High Performance Liquid Chromatography method coupled to an Evaporative Light Scattering Detection. Unsupervised and supervised qualitative and quantitative chemometric methods were performed to assess powder blend homogeneity using a bench top instrument equipped with an optical fibre. For qualitative evaluations, unsupervised Moving Block Standard Deviation, autocorrelation functions and Partial Least Square Discriminant Analysis (PLS-DA) were used. For quantitative evaluations, Partial Least Square Cross-Validated models were chosen. Results are expressed as API, and major excipient percentages of theoretical values as a function of blending time. The 14 different formulations were only satisfactorily discriminated by supervised algorithms, such as an optimised PLS-DA model. The homogeneity state was demonstrated after 16 min of blending, quantifying three components with a precision between 1.2% and 1.4% w/w. This study demonstrates, for the first time, the effective implementation of NIRS for blend homogeneity evaluation, as early as the preformulation step in a small hospital manufacturing unit. It shows how NIRS involving sampling with an optic fibre can be useful to characterise, optimise and control a small-scale mixing processes on the basis of the distribution of APIs and excipients during blending.

This study compares, through micro high-temperature liquid chromatography (μHTLC), three commercial universal detectors that allow a direct detection of lipids. The detectors are: the charged aerosol detector (CAD), the evaporative light-scattering detector (ELSD) and the ion trap mass spectrometer with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) sources (APCI-MS and ESI-MS). This study shows the feasibility to use the high temperature with these detectors and hybrid behavior between concentration and mass flow rate detector in μHTLC. The detectors were compared in terms of response intensity, linearity and limit of detection for different high temperatures. The charged aerosol detector shows a linear response from 5 to 500 μg/mL and the correlation coefficients (r2) obtained for squalene, cholesterol and ceramide IIIB exceed 0.99.

The mechanism of action of antifungal canthin-6-one series was investigated in Saccharomyces cerevisiae. After a rapid uptake, a preferential accumulation of the drug within lipid droplets was observed. The antifungal action of canthin-6-one was found as reversible. Canthin-6-one did not exhibit affinity for sterols, and membrane ergosterol was not necessary for the antifungal activity since the MICs were similar on an ergosterol-deleted and the wild-type S. cerevisiae clones. Relative amount of unsaturated alkyl chain fatty acids was significantly enhanced suggesting a stimulation of desaturase enzyme systems. No synergistic effect was observed between canthin-6-one and amphotericin B, ketoconazole and caspofungine. Canthin-6-one should now be evaluated in vivo against fungal pathogens.