Emmanuelle Lipka

Preparative supercritical fluid chromatography: A powerful tool for chiral separations.

In 2012, the 4 biggest pharmaceutical blockbusters were pure enantiomers and separating racemic mixtures is now frequently a key step in the development of a new drug. For a long time, preparative liquid chromatography was the technique of choice for the separation of chiral compounds either during the drug discovery process to get up to a hundred grams of a pure enantiomer or during the clinical trial phases needing kilograms of material. However the advent of supercritical Fluid Chromatography (SFC) in the 1990s has changed things. Indeed, the use of carbon dioxide as the mobile phase in SFC offers many advantages including high flow rate, short equilibration time as well as low solvent consumption. Despite some initial teething troubles, SFC is becoming the primary method for preparative chiral chromatography. This article will cover recent developments in preparative SFC for the separation of enantiomers, reviewing several aspects such as instrumentation, chiral stationary phases, mobile phases or purely preparative considerations including overloading, productivity or large scale chromatography.

Involvement of the P2X7 Purinergic Receptor in Inflammation: An Update of Antagonists Series Since 2009 and their Promising Therapeutic Potential

The purinergic receptor P2X7 is highly expressed in immune peripheral and central cells suggesting its important role in numerous diseases characterized by inflammatory processes like cancer, or neurodegenerative pathologies in relation with modulation of the immune system. Thereby, antagonization of this receptor may be a hopeful therapeutic strategy to treat a large range of diseases. Indeed, selective P2X7 antagonists display beneficial anti-inflammatory, analgesic, and in some cases, anticancer properties. This article will review the involvement of P2X7 in the immune system, the update of P2X7 antagonists series since 2009 and their promising therapeutic potential for the treatment of several immune- related diseases.

Dual CD system in capillary electrophoresis for direct separation of the four stereoisomers of agonist and antagonist melatoninergic ligands

In this study, baseline separation of the stereoisomers of six tetrahydronaphthalenic derivatives (agonists and antagonists for the melatonin (N‐acetyl‐5‐methoxytryptamin) binding sites) was successfully achieved using CE and CDs as chiral selectors. The method for the simultaneous chiral separation of the four stereoisomers uses a capillary dynamically coated with polyethylene oxide and a dual CD system. Optimisation was performed first upon the constituents of the CD system, by varying neutral and anionic CD type, size and concentration, at first in mono‐CD systems and subsequently in dual neutral/anionic CD systems. Once these characteristics of the dual CD system were established, operational parameters such as voltage and temperature were then optimised. Under the optimal conditions (i.e. 1.5% w/v of highly S‐β‐CD and 10 mM of γ‐CD in 25 mM phosphate buffer (pH 2.5) as the BGE, separation voltage 20 kV and a temperature of 25°C), complete resolution of the six molecules was accomplished. Preliminary results for repeatability and the migration order of the optimised method are described.

4-Oxo-1,4-dihydropyridines as selective CB₂ cannabinoid receptor ligands. Part 2: discovery of new agonists endowed with protective effect against experimental colitis

Further on to our earlier work on the 4-oxo-1,4-dihydropyridine, we describe herein our strategy to get access to potent selective CB₂ receptor agonists. Thus, we designed and synthesized 29 compounds, evaluated on both hCB₁ and hCB₂ cannabinoid receptors, and assessed 11 of them in the TNBS-induced colitis model in mice. Compound 48 was found to be the most efficient of our series, exhibiting an exquisite protection against experimental colitis, superior to the one observed after treatment with Pentasa.

Enantioseparation of pyroglutamide derivatives on polysaccharide based chiral stationary phases by high-performance liquid chromatography and supercritical fluid chromatography: a comparative study.

Analytical enantioseparation of three pyroglutamide derivatives with pharmacological activity against the purinergic receptor P2X7, was run in both high-performance liquid chromatography and supercritical fluid chromatography. Four polysaccharide based chiral stationary phases, namely amylose and cellulose tris (3,5-dimethylphenylcarbamate), amylose tris ((S)-α-methylbenzylcarbamate) and cellulose tris (4-methylbenzoate) with various mobile phases consisted of either heptane/alcohol (ethanol and 2-propanol) or carbon dioxide/alcohol (methanol or ethanol) mixtures, were investigated. After analytical screenings, the best conditions were transposed, for compound 1, to semi-preparative scale. Each approach was fully validated to meet the International Conference on Harmonisation requirements and compared. Whereas the limits of detection and quantification were near six-fold better in HPLC than in SFC (respectively 0.20 and 0.66 μM versus 1.11 and 3.53 μM for one of the enantiomers), in terms of low solvent consumption (7.2 mL of EtOH versus 3.2 mL of EtOH plus 28.8 mL of toxic and inflammable heptane per injection in SFC and HPLC, respectively), time effective cost (9 min versus 40 min per injection in SFC and HPLC, respectively) and yields (98% versus 71% in SFC and HPLC, respectively), the latter method proved its ecological superiority.

Enantioseparation of chiral benzimidazole derivatives by electrokinetic chromatography using sulfated cyclodextrins.

Baseline separation of 18 new substituted benzimidazole derivatives, potent AMP-activated protein kinase (AMPK) activators, with one chiral center, was achieved by CD-EKC using sulfated and highly sulfated CDs (SCDs and HS-CDs) as chiral selectors. The influence of the type and concentration of the chiral selectors on the enantioseparations was investigated. The SCDs exhibit a very high enantioselectivity power since they allow excellent enantiomeric resolutions compared to those obtained with the neutral CDs. The enantiomers were resolved with analysis times around 6 min using 25 mM phosphate buffer at pH 2.5 containing either beta-S-CD, HS-beta-CD, HS-gamma-CD (3 or 4% w/v) at 25 degrees C, with a voltage of 20 kV. The apparent association constants of the inclusion complexes were calculated. The study of the solute structure-enantioseparation relationships seems to show the high contribution of the interactions between the solutes phenyl ring and the CDs to the enantiorecognition process. The optimized method was briefly validated (LOD less than 1%) and the purity of enantiomers of compound 3 was determined. The enantiomer migration shows reversal order depending on the kind of CD.

Enhanced detection for determination of enantiomeric purity of novel agomelatine analogs by EKC using single and dual cyclodextrin systems

Performing CD‐EKC, baseline separation of five agomelatine analogs, potential antidepressant compounds, was achieved. A method for the enantioresolution and determination of enantiomeric purity of these naphthalene derivatives was developed using capillaries dynamically coated with polyethylene oxide and anionic cyclodextrins (highly sulfated CD) as chiral selectors. Operational parameters such as the nature and concentration of the cyclodextrins were investigated. In a second step the implementation of a dual cyclodextrin system was found to strongly enhance the LOD of the analytes. After optimization, best conditions were a 25 mM phosphate buffer at pH 2.5 containing 5% w/v (i.e. 19.7 mM) of highly sulfated‐γ‐CD and 10 mM of 6‐monodeoxy‐6‐monoamino‐β‐CD dual system, leading to resolution of, at least, 3.6 in 35 min. A preliminary validation of the developed method was undertaken: linearity, precision, and LOD and LOQ were evaluated. The latest ones were found equal to 0.25 and 0.82 μM and to 0.31 and 0.96 μM respectively for the first and the second enantiomer of compound 1.

Determination of pKa values of benzimidazole derivatives from mobility obtained by capillary electrophoresis.

Dissociation constants of benzimidazole derivatives have been determined using capillary zone electrophoresis (CZE). Since CZE is a separation method, high purity and known concentration for the samples is not necessary because only mobilities are measured. The precision of pK(a) measurements of seven compounds is useful to observe pK(a) shifts induced by chemical variations. Some of them were compared to potentiometry and spectroscopy experiments. Good correlated pK(a) values are observed between the three analytical techniques.

Enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors, potential adrenergic ligands, by analytical and preparative liquid chromatography on amylose chiral stationary phases and characterization of the enantiomers

To obtain milligram amounts of the enantiomers of benzoxazolinone derivatives to be tested for binding to adrenergic sites, analytical HPLC methods using derivatized amylose chiral stationary phases were developed for the direct enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors, derivatives with one or two chirals centers. The separations were made using normal phase methodology with a mobile phase of n-hexane-alcohol (ethanol, 1-propanol, or 2-propanol) in various proportions, and silica-based amylose (tris-3, 5-dimethylphenylcarbamate) Chiralpak AD and (tris-(S)-1-phenylethylcarbamate) Chiralpak AS columns. The effects of concentration of various aliphatic alcohols in the mobile phase were studied. The best separation was achieved on Chiralpak AS, so preparative HPLC was set up with this chiral stationary phase using a mobile phase consisting of n-hexane-alcohol using isocratic conditions and multiple repetitive injections. Physicochemicals properties of enantiomers were reported The effect of structural features of the solutes on discrimination between the enantiomers was examined. Limit of detection (LD) and limit of quantification (LQ) were determined using both ultra-violet (UV) and evaporative light-scattering detection (ELSD).

Evolution of availability of curcumin inside poly-lactic-co-glycolic acid nanoparticles: impact on antioxidant and antinitrosant properties

Purpose Curcumin exhibits antioxidant properties potentially beneficial for human health; however, its use in clinical applications is limited by its poor solubility and relative instability. Nanoparticles exhibit interesting features for the efficient distribution and delivery of curcumin into cells, and could also increase curcumin stability in biological systems. There is a paucity of information regarding the evolution of the antioxidant properties of nanoparticle-encapsulated curcumin. Method We described a simple method of curcumin encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles without the use of detergent. We assessed, in epithelial cells and in an acellular model, the evolution of direct antioxidant and antinitrosant properties of free versus PLGA-encapsulated curcumin after storage under different conditions (light vs darkness, 4°C vs 25°C vs 37°C). Results In epithelial cells, endocytosis and efflux pump inhibitors showed that the increased antioxidant activity of PLGA-encapsulated curcumin relied on bypassing the efflux pump system. Acellular assays showed that the antioxidant effect of curcumin was greater when loaded in PLGA nanoparticles. Furthermore, we observed that light decreased, though heat restored, antioxidant activity of PLGA-encapsulated curcumin, probably by modulating the accessibility of curcumin to reactive oxygen species, an observation supported by results from quenching experiments. Moreover, we demonstrated a direct antinitrosant activity of curcumin, enhanced by PLGA encapsulation, which was increased by light exposure. Conclusion These results suggest that the antioxidant and antinitrosant activities of encapsulated curcumin are light sensitive and that nanoparticle modifications over time and with temperature may facilitate curcumin contact with reactive oxygen species. These results highlight the importance of understanding effects of nanoparticle maturation on an encapsulated drug’s activity.