Development and optimization of an ultra-fast microextraction followed by HPLC-UV of tetracycline residues in milk products.

The present study developed a new ultra-fast microextraction (within 8 min) with acetonitrile followed by gradient reversed-phase HPLC-UV method to determinate six tetracyclines simultaneously in various milk products with HPLC analysis time of 9 min. Chromatographic separations were achieved with a phenomenex™ Synergi 4 µm Fusion-RP (150 × 4.60 mm) column at 220 nm, using acetonitrile/KH2PO4 buffer (4 mmol·L-1, pH 2.5) as mobile phase. The HPLC method showed excellent peak resolutions (3.3-8.8) and peak symmetry (0.99-1.11) inspite of short analysis time. The extraction rates from milk products showed consistently very good values over all tetracyclines (milk 80.58 ± 5.39 %, yoghurt 82.61 ± 5.63 %, cream cheese 80.13 ± 6.32 %, buttermilk 81.07 ± 6.49 %, kefir 79.69 ± 6.51 %, skyr 78.12 ± 5.22 %, quark 65.37 ± 4.72 %). The optimized method was found to be specific, reproducible, robust. This study combines for the first time a fast, cheap quantification of six tetracyclines via HPLC-UV with a reliable microextraction applicable to various milk products using only standard laboratory equipment.

Encapsulation of the Anti-inflammatory Dual FLAP/sEH Inhibitor Diflapolin Improves the Efficiency in Human Whole Blood.

Diflapolin is a dual FLAP/sEH inhibitor with potent anti-inflammatory efficiency in cellular assays and experimental in vivo studies. Despite these outstanding characteristics, its high lipophilicity and plasma protein binding hamper the bioactivity in blood. To overcome these limitations, diflapolin was encapsulated in poly(lactic-co-glycolic acid) nanoparticles to develop an efficient and biocompatible drug delivery system. Two different cosolvent approaches were tested showing the possibility to exchange dimethyl sulfoxide in the organic phase by the sustainable 400 g/mol poly(ethylene glycol). A particle size of 220 nm and the amorphous encapsulation of diflapolin in high amounts rendered the nanoparticles appropriate for the intended application. Excellent biocompatibility of the nanoparticles was demonstrated in an ex ovo hen's egg model. The potent suppression of FLAP-dependent 5-lipoxygenase product formation by the nanoparticles in human whole blood, superior to the free drug, makes them to a promising drug delivery system to improve the bioactivity of diflapolin.

Discovery of Novel 5-Lipoxygenase-Activating Protein (FLAP) Inhibitors by Exploiting a Multistep Virtual Screening Protocol.

Leukotrienes (LTs) are proinflammatory mediators derived from arachidonic acid (AA), which play significant roles in inflammatory diseases. The 5-lipoxygenase-activating protein (FLAP) is an integral membrane protein, which is essential for the initial step in LT biosynthesis. The aim of this study was to discover novel and chemically diverse FLAP inhibitors for treatment of inflammatory diseases requiring anti-LT therapy. Both ligand- and structure-based approaches were applied to explain the activities of known FLAP inhibitors in relation to their predicted binding modes. We gained valuable insights into the binding modes of the inhibitors by molecular modeling and generated a multistep virtual screening (VS) workflow in which 6.2 million compounds were virtually screened, and the molecular hypotheses were validated by testing VS-hit compounds biologically. The most potent hit compounds showed significant inhibition of FLAP-dependent cellular LT biosynthesis with IC50 values in the range from 0.13 to 0.87 µM. Collectively, this study provided novel bioactive chemotypes with potential for further development as effective anti-inflammatory drugs.

In Vitro Studies on Physiological and Chemical Stability of New LE404-Derivatives with Extended Half-Life.

Dibenzoazecines are a class of potential neuroleptics with high affinity to dopamine and serotonin receptors. The efficacy and high therapeutic range has already been demonstrated in vivo with the lead structure 7-methyl-5,6,7,8,9,14-hexahydrodibenzo[d,g]azecin-3-ol (LE404: ) and selected derivatives. There is a variety of new synthesized structurally different dibenzoazecine derivatives with the aim to improve pharmacokinetic parameters, all of which contain the lead structure LE404: . For a multitude of these substances is still a lack of information, inclusive of stability, physicochemical parameters, pharmacokinetics and metabolism. Therefore, the present study investigated the stability properties of 17 new azecine derivatives, including esterase cleavage, stability in simulated gastrointestinal fluid, stability at different pH-values and determination of octanol/water-partition coefficients. These findings, in correlation to the properties and efficacy of the already in vivo tested substances, will be useful for safety and efficacy in further in vivo tests.

Synthesis and Characterization of new Azecine-Derivatives as Potential Neuroleptics.

Dibenzo- and benzindolo-azecines represent a class of potential neuroleptics. To characterize the effectiveness at the dopamine and 5-HT2A-receptor representative structures were synthesized and tested by radio ligand binding studies, in vivo and in vitro studies.Neuroleptic potency and the risk of side effects of the prodrug 7-methyl-5,6,7,8,9,14-hexahydrodibenzo[d,g]azecin-3-yl isobutyrate, an ester derivative of the most promising azecine 7-methyl-5,6,7,8,9,14-hexahydrodibenzo[d,g]azecin-3-ol (LE404), was tested in vivo concerning conditioned avoidance response inhibition, locomotor activity and triggering of catalepsy vs. haloperidol as a reference. Also ester hydrolysis was examined using porcine liver esterase to thereby obtain an indication of the stability of the prodrug in vivo. An HPLC method was developed for purity control and determination of octanol/water-distribution coefficients.It has been shown that the tested substances in their efficacy are comparable to haloperidol and risperidone, but the therapeutic index in most cases is larger. Esterification as a prodrug principle leads to significantly prolonged effectiveness.