Characterization of intact neo-glycoproteins by hydrophilic interaction liquid chromatography.

In this study, an HPLC HILIC-UV method was developed for the analysis of intact neo-glycoproteins. During method development the experimental conditions evaluated involved different HILIC columns (TSKgel Amide-80 and ZIC-pHILIC), and water-acetonitrile mixtures containing various types of acids and salts. The final selected method was based on a TSKgel Amide-80 column and a mobile phase composed of acetonitrile and water both containing 10 mM HClO4. The influence of temperature and sample preparation on the chromatographic performances of the HILIC method was also investigated. The method was applied to the separation of neo-glycoproteins prepared starting from the model protein RNase A by chemical conjugation of different glycans. Using the method here reported it was possible to monitor by UV detection the glycosylation reaction and assess the distribution of neo-glycoprotein isoforms without laborious sample workup prior to analysis.

Studies on the enantiomers of RC-33 as neuroprotective agents: isolation, configurational assignment, and preliminary biological profile.

In this study we addressed the role of chirality in the biological activity of RC-33, recently studied by us in its racemic form. An asymmetric synthesis procedure was the first experiment, leading to the desired enantioenriched RC-33 but with an enantiomeric excess (ee) not good enough for supporting the in vitro investigation. An enantioselective high-performance liquid chromatography (HPLC) procedure was then successfully carried out, yielding both RC-33 enantiomers in amounts and optical purity suitable for the pharmacological study. The absolute configuration of pure enantiomers was easily assigned exploiting the asymmetric synthesis previously devised. As emerged in the preliminary in vitro biological investigation, (S)- and (R)-RC-33 possess a comparable affinity towards the σ1 receptor and a very a similar behavior in the calcium influx assay, resulting in an equally effective σ1 receptor agonist. Overall, the results obtained so far suggest that the interaction with the biological target is nonstereoselective and leads us to hypothesize that there is a lack of stereoselectivity in the biological activity of RC-33.

Column comparison and method development for the analysis of short-chain carboxylic acids by zwitterionic hydrophilic interaction liquid chromatography with UV detection.

Short-chain carboxylic acids are relevant in pharmaceutical, food quality control, and biomedical analysis. In this study, 11 acids commonly found in drugs and in food products were selected. Wine was chosen as matrix for testing the method. The test compounds were used for comparing the selectivity of four 150 × 2.1 mm zwitterionic hydrophilic interaction LC (HILIC) columns (ZIC-HILIC 5 µm, 200 Å, and 3.5 µm, 100 Å, ZIC-pHILIC 5 µm, ZIC-cHILIC 3 µm, 100 Å) while varying the conditions to optimize for low UV wavelength detection and achieve high sensitivity. Retention using potassium phosphate and ammonium carbonate as mobile-phase components at pH 6.0, 7.5, and 8.5-8.9 was studied considering recent hypotheses on HILIC mechanism-related with the Hofmeister series effect and ion hydration. An isocratic method with UV detection at 200 nm and mobile phase consisting of 75% acetonitrile and 10 mM potassium phosphate at pH 6.0 applied to a ZIC-cHILIC column was found provisionally optimal and partially validated for the 11 analytes. Satisfactory results (R(2) from 0.9940 to >0.9999), and recoveries from 93-106% for all analytes evidenced the method as suitable for wine analysis. To the best of our knowledge, no previous study has reported on the direct ZIC-HILIC separation and UV detection of the acids considered here in wine.

Identification of a potent and selective σ1 receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells.

Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (σ) ligands based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was studied. Novel arylalkenylamines generally possess high σ(1) receptor affinity (K(i) values <25 nM) and good σ(1)/σ(2) selectivity (K(i)σ(2) >100). Particularly, the piperidine derivative (E)-17 and its arylalkylamine analog (R,S)-33 were observed to be excellent σ(1) receptor ligands (K(i)=0.70 and 0.86 nM, respectively) and to display significantly high selectivity over σ(2), µ-, and κ-opioid receptors and phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptors. Moreover in PC12 cells (R,S)-33 promoted the nerve growth factor (NGF)-induced neurite outgrowth and elongation. Co-administration of the selective σ(1) receptor antagonist BD-1063 totally counteracted this effect, confirming that σ(1) receptors are involved in the (R,S)-33 modulation of the NGF effect in PC12 cells and suggesting a σ(1) agonist profile. As a part of our work, a threedimensional σ(1) pharmacophore model was also developed employing GALAHAD methodology. Only active compounds were used for deriving this model. The model included two hydrophobes and a positive nitrogen as relevant features and it was able to discriminate between molecules with and without affinity toward σ(1) receptor subtype.

Design, synthesis and SAR analysis of novel selective sigma1 ligands (Part 2).

In order to investigate the molecular features involved in sigma receptors (sigma-Rs) binding, new compounds based on arylalkylaminoalcoholic, arylalkenyl- and arylalkylaminic scaffolds were synthesized and their affinity towards sigma(1)- and sigma(2)-Rs subtypes was evaluated. The most promising compounds were also screened for their affinity at micro-opioid, delta-opioid and kappa-opioid receptors. Biological results are herein presented and discussed.

Determination of N-acetylglucosamine in cosmetic formulations and skin test samples by hydrophilic interaction liquid chromatography and UV detection.

N-Acetylglucosamine is an ingredient in pharmaceuticals, nutritional supplements and in cosmetics. N-Acetylglucosamine in cosmetics is expected to improve skin hydration, reparation, and to contribute as anti-wrinkle agent. This study reports on the validation and application of an HPLC method based on HILIC and UV detection for determining N-acetylglucosamine in cosmetics and in samples obtained after testing the skin exposed to cosmetics formulations. The chromatographic column used is a ZIC(®)-pHILIC (150 mm × 4.6 mm, 5 µm particle size) on which a mobile phase containing acetonitrile-aqueous KH2PO4 (70:30, v/v) 15 mM was applied in isocratic elution mode injecting 20 µl of sample at 0.5 ml/min constant flow-rate and 10±1°C column temperature. Under these conditions the total run time was 10 min and N-acetylglucosamine eluted baseline separated from all other compounds in the samples. Calibration in the range from 40 to 80 µg/ml allowed to assess the method linearity (R(2)>0.999) in a concentration range corresponding to about 50% to 120% of the expected levels of N-acetylglucosamine in the formulations. Precision expressed by RSD% was always better than 2% in intra-day and inter-day assays of authentic samples. Accuracy was in all cases within 95-105% of the expected concentration value in formulations containing N-acetylglucosamine. The sensitivity of the method was at the level of 10 µg/ml as limit of detection, and at 40 µg/ml as limit of quantitation. The application of the method to formulations containing solid lipid nanoparticles documents its usefulness in cosmetic quality control. The results witness that the method is also suitable for the determination of N-acetylglucosamine in samples obtained from skin test strips.

Chemical, pharmacological, and in vitro metabolic stability studies on enantiomerically pure RC-33 compounds: promising neuroprotective agents acting as σ1 receptor agonists.

Our recent research efforts identified racemic RC-33 as a potent and metabolically stable σ1 receptor agonist. Herein we describe the isolation of pure RC-33 enantiomers by chiral chromatography, assignment of their absolute configuration, and in vitro biological studies in order to address the role of chirality in the biological activity of these compounds and their metabolic processing. The binding of enantiopure RC-33 to the σ1 receptor was also investigated in silico by molecular dynamics simulations. Both RC-33 enantiomers showed similar affinities for the σ1 receptor and appeared to be almost equally effective as σ1 receptor agonists. However, the R-configured enantiomer showed higher in vitro hepatic metabolic stability in the presence of NADPH than the S enantiomer. Overall, the results presented herein led us to select (R)-RC-33 as the optimal candidate for further in vivo studies in an animal model of amyotrophic lateral sclerosis.