Synthesis and pharmacology of 3-hydroxy-Δ-isoxazoline-cyclopentane analogues of glutamic acid

The synthesis and pharmacology of two potential glutamic acid receptor ligands are described. Preparation of the bicyclic 3-hydroxy-Δ²-isoxazoline-cyclopentane derivatives (±)-7 and (±)-8 was accomplished via 1,3-dipolar cycloaddition of bromonitrile oxide to suitably protected 1-amino-cyclopent-3-enecarboxylic acids. Their structure was established using a combination of ¹H NMR spectroscopy and molecular mechanics calculations carried out on the intermediate cycloadducts (±)-11 and (±)-12. Amino acid derivatives (±)-7 and (±)-8 were assayed at ionotropic and metabotropic glutamic acid receptor subtypes and their activity compared with that of trans-ACPD and cis-ACPD. The results show that the replacement of the ω-carboxylic group of the model compounds with the 3-hydroxy-Δ²-isoxazoline moiety abolishes or reduces drastically the activity at the metabotropic glutamate receptors. Conversely, on passing from cis-ACPD to derivative (±)-8, the agonist activity at NMDA receptors is almost unaffected.     

Improvement of water solubility and dissolution rate of ursodeoxycholic acid and chenodeoxycholic acid by complexation with natural and modified β-cyclodextrins

The inclusion complexes of ursodeoxycholic and chenodeoxycholic acid with β-cyclodextrin, heptakis-(2,6-di-O-methyl)-β-cyclodextrin and soluble polymerized β-cyclodextrin were investigated in solution (¹H-NMR spectrometry) and solid state (FT-IR spectroscopy and differential scanning calorimetry). Stability constants were determined at pH 7.4 and different temperatures and consequently thermodynamic parameters were obtained. All cyclodextrins are able to increase water solubility of the bile acids, particularly polymerized β-cyclodextrin. All complexes show high dissolution rate at 37°C and pH 1.1 and in particular freeze-dried complexes.     

Preparation and characterization of β-cyclodextrin and poly(acrylic acid) microspheres

Abstract

A method for the preparation of poly(acrylic acid) (PAA) microspheres cross-linked with beta-cyclodextrin (β-CD) is described. The method is based on a water-in-oil (w/o) emulsion solvent evaporation technique which facilitates a condensation reaction between PAA and β-CD. Aqueous solutions of PAA and β-CD were used as the dispersed phase and food grade olive oil was used as the continuous phase. The effect of homogenization speed (used in the preparation of the emulsion), phase volume ratio and cyclodextrin-polymer load on the particle size of the microspheres produced was investigated in a replicated factorial design. Microspheres were sized by light microscopy. The particle size of the microspheres was influenced by all three variables with two significant first order interactions between the variables being observed (homogenization speed with phase volume ratio and homogenization speed with load). A second order interaction between the three principal factors was also observed. Particle size ranged from 16 to 150m, depending on the production variables employed. The yield for the technique was 69.5 × 9.5%. Using selected conditions, microspheres of 15–25 pm size were prepared from a range of PAA with different weight average molecular weights (w). These particles were then characterized for β-CD, free carboxylic acid group content and residual oleic acid.     

Preparation and characterization of solidified oleanolic acid–phospholipid complex aiming to improve the dissolution of oleanolic acid

The purpose of this study was to prepare the oleanolic acid–phospholipid complex (OA-PC) and then solidify it employing fumed silica by simple solvent evaporation technique to improve dissolution rate of oleanolic acid and oleanolic acid–phospholipid complex. The process of OA-PC was optimized and the type and proportion of fumed silica were studied by dissolution text. The structures of the phospholipid complex and solidified powder were also characterized by differential scanning calorimetry, X-ray diffraction, and scanning electron microscope. In the dissolution tests, OA from solidified powder was further released compared with that from pure OA and OA-PC in different kinds of dissolution media. These results suggest that the method of preparing solidified powder of oleanolic acid–phospholipid complex is suitable for enhancing the dissolution rate of OA and OA-PC.     

Synthesis and antitumor evaluation of α-methylene-γ-butyrolactone-linked to 5-Substituted uracil nucleic acid bases

Six, heretofore undescribed, 5′-Methyl-5′-(5-Substituted uracil-1-ylmethyl)-2′-oxo-3′-methylenetetrahydrofurans (F, Cl, Br, I, CH₃, H) (6a-f) were synthesized and evaluated against three cell lines (FM-3A, P-388 and U-937). For the preparation of α-methylene-γ-butyrolactone bearing 5-substituted uracils (6a-f), the efficient Reformatsky type reaction was employed which involves the treatment of ethyl α-(bromomethyl) acrylate and zinc with the respective 5-substituted uracil-1-ylacetones (5a-f). The acetone derivatives (5a-f) were directly obtained by the respective alkylation reaction of 5-substituted uracils with chloroacetone in the presence of K₂CO₃ (or NaH). These lactone compounds6a-f exhibited moderate to significant activity in all of the three cell lines, and6b, 6c and6e showed significant antitumor activities (inhibitory concentrations (IC₅₀) ranged from 1.3–3.8 μg/ml).     

Docking and scoring for nucleic acid–ligand interactions: Principles and current status

Nucleic acid (NA)–ligand interactions have crucial roles in many cellular processes and, thus, are increasingly attracting therapeutic interest in drug discovery. Molecular docking is a valuable tool for studying molecular interactions. However, because NAs differ significantly from proteins in both their physical and chemical properties, traditional docking algorithms and scoring functions for protein–ligand interactions might not be applicable to NA–ligand docking. Therefore, various sampling strategies and scoring functions for NA–ligand interactions have been developed. Here, we review the basic principles and current status of docking algorithms and scoring functions for DNA/RNA–ligand interactions. We also discuss challenges and limitations of current docking and scoring approaches.     

Chromatographic determination of thiodiglycolic acid — a metabolite of vinyl chloride

The determination of thiodiglycolic acid (TDGA) in urine by gas chromatography (GC) is described. The analytical procedure includes addition of internal standard (o-phthalic acid), ethyl acetate extraction, evaporation of the solvent and silylation of TDGA with N-trimethylsilyldiethylamine in pyridine (1 1).The results are calculated from the ratio of TDGA and internal standard peak heights. The detection limit of the method is 10 mg/l and the coefficient of variation is ±5%.     

Structure–activity relationship of GPR15L peptide analogues and investigation of their interaction with the GPR15 receptor

The 57-mer full-length GPR15L(25-81) peptide has been identified as the principal endogenous agonist of the G protein-coupled receptor GPR15. Its main activity resides in the C-terminal 11-mer GPR15L(71-81), which has full efficacy but ~40-fold lower potency than the full-length peptide. Here, we systematically investigated the structure–activity relationship of GPR15L(71-81) by truncations/extensions, alanine-scanning, and N- and C-terminal capping. The synthesized peptide analogues were tested at GPR15 stably expressed in HEK293A cells using a homogenous time-resolved Förster resonance energy transfer-based G_i cAMP functional assay. We show that the C-terminal α carboxyl group and the residues Leu⁷⁸, Pro⁷⁵, Val⁷⁴, and Trp⁷² are critical for receptor interaction and contribute significantly to the peptide potency. Furthermore, we tested the ability of GPR15L(71-81), C-terminally amidated GPR15L(71-81), and GPR15L(25-81) to activate the three GPR15 receptor mutants in a bioluminescence resonance energy transfer-based G protein activation assay. The results demonstrate that the Lys192 and Glu272 residues in GPR15 are important for the potency of the GPR15L peptide. Overall, our study identifies critical residues in the peptide and receptor sequences for future drug design.     

Preparation and characterization of adefovir dipivoxil–stearic acid cocrystal with enhanced physicochemical properties

Abstract

The objectives of this study were to prepare cocrystal composed of adefovir dipivoxil (AD) and stearic acid (SA) and to investigate the enhanced properties of the cocrystal. The cocrystal was prepared by antisolvent precipitation and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRPD), and differential scanning calorimetry (DSC). The enhanced properties were evaluated by dissolution testing, permeability studies, and powder rheology analysis. The AD raw material has a cuboid-like crystal and the cocrystal has a needle shape. In the FT-IR study, there were bathochromic shifts caused by the hydrogen bonding. The melting point of the cocrystal was 52.9?°C, which was lower than that of AD. The XRPD pattern also had distinct differences, supporting the formation of a new crystalline form. The cocrystal showed changes in the lattice energy and the solvation strength, which caused an enhanced dissolution. The permeability was increased due to the SA, which acts as a P-gp inhibitor. The tabletability was enhanced due to the altered crystal habit. In conclusion, cocrystal containing AD and SA was successfully prepared, presenting advantages such as enhanced solubility, tabletability, and permeability. The use of the cocrystal is a desirable approach for the improved physicochemical properties.     

Genetically modified microorganism Spingomonas paucimobilis UT26 for simultaneously degradation of methyl-parathion and γ-hexachlorocyclohexane

Bioremediation of pesticide residues by bacteria is an efficient and environmentally friendly method to deal with environmental pollution. In this study, a genetically modified microorganism (GMM) named UT26XEGM was constructed by introducing a parathion hydrolase gene into an initially γ-hexachlorocyclohexane (γ-HCH) degrading bacterium Spingomonas paucimobilis UT26. In order to reduce its potential risk of gene escaping into the environment for the public concern on biosafety, a suicide system was also designed that did not interfere with the performance of the GMM until its physiological function was activated by specific signal. The system was designed with circuiting suicide cassettes consisting of killing genes gef and ecoRIR from Escherichia coli controlled by Pm promoter and the xylS gene. The cell viability and original degradation characteristics were not affected by the insertion of exogenous genes. The novel GMM was capable of degrading methyl-parathion and γ-HCH simultaneously. In laboratory scale testing, the recombinant bacteria were successfully applied to the bioremediation of mixed pesticide residues with the activity of self-destruction after 3-methylbenzoate induction.     

Vertebrate cyclodiene insecticide resistance: Role of γ-aminobutyric acid and diazepam binding sites

Certain populations of the mosquitofish (Gambusia affinis) are highly resistant to cyclodiene and cyclodiene-type insecticides that competitively interact with the picrotoxinin binding site of the -aminobutyric acid (GABA) receptor-ionophore complex in the central nervous system. Resistance involves a reduction in affinity of the picrotoxinin binding site. The present study reports that GABA receptor binding is increased in resistant brain membranes compared to membranes from susceptible fish at concentrations of free radioligand above 0.2 M. The increase appears to be due to a greater number of binding sites (B_max) in the resistant population. Diazepam binding affinity (K_d) and B_max were not different in membranes from resistant fish compared to those from susceptible fish. Up-regulation of GABA binding sites in the resistant fish population may compensate for a possible reduction of GABAergic transmission caused by chronic environmental exposure to cyclodiene insecticides. However, a lack of cross-resistance to bicuculline (a competitive GABA antagonist) indicates that an increase in GABA sites is not a mechanism of cyclodiene resistance.This work was supported by National Institute of Environmental Health Sciences (NIEHS) Grant ES03069     

Preparation,release and physicochemical characterisation of ethyl butyrate and hexanal inclusion complexes with β- and γ-cyclodextrin

Abstract

Complexes of ethyl butyrate and hexanal encapsulated by β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) were prepared by coprecipitation, and gas chromatography was used to quantity the flavour compounds in the complexes. The ethyl butyrate–γ-CD complex had the highest inclusion ratio (12.20%) followed by the ethyl butyrate-β-CD, hexanal-β-CD and hexanal-γ-CD complexes (11.29, 4.41 and 3.33%, respectively). Release experiments were performed under different relative humidities (RH 93, 75 and 52%) and temperatures (4 and 25?°C). The flavour release behaviours of the complexes were described by the Avrami equation. The rate of flavour release was enhanced with both increasing temperature and RH, although the effect of RH was stronger. Physicochemical characterisation using FT-IR, XRD, DSC and SEM analyses demonstrated that crystalline complexes were formed. Both β-CD and γ-CD were able to encapsulate ethyl butyrate and hexanal, and lower RH and temperature were more suitable for the storage of these complexes.     

Effect of baclofen and haloperidol on γ-aminobutyric acid and dopamine systems in an animal model of tardive dyskinesia

1. The effects of 5 days treatment with baclofen (20 mg/kg, i.p.) or high doses of haloperidol (10 mg/kg, i.p.) were studied on dopamine and γ-aminobutyric acid levels of corpus striatum, frontal cortex and mid-brain of normal rats as well as those withdrawn for 4 days after 30 days of daily treatment with haloperidol (1 mg/kg).2. Baclofen treatment slightly but significantly increased (by 13%) GABA levels in the corpus striatum of normal rats. This, by inhibitory effects, probably depleted dopamine level in corpus striatum.3. In the mid-brain, no change in GABA level was observed after baclofen treatment. The frontal cortex, which receives dopaminergic nerve endings from cell bodies lying in the ventral tegmental area, showed high levels of dopamine.4. Administration of baclofen to haloperidol-withdrawn rats tended to further increase GABA levels in striatum; however, the change was not statistically significant. In contrast, mid-brain GABA levels were decreased by 21% when compared with haloperidol-withdrawn values taken as 100%.5. Baclofen treatment increased DA level by 66% in the frontal cortex and decreased it by 35% in striatum of rats.6. In normal rats, administration of high doses of haloperidol (10 mg/kg) increased GABA levels of the corpus striatum and the frontal cortex by 48 and 16%, respectively.7. In mid-brain, a slight increase (11%) was reported in GABA level.8. Haloperidol treatment decreased DA in striatum, but increased it in the mid-brain and the frontal cortex. High doses of haloperidol for 5 days to rats previously treated with haloperidol (1 mg/kg) daily for 30 days and subsequently withdrawn for 4 days produced no further change in GABA levels of various brain areas examined. In mid-brain, a slight decrease was seen in GABA level.9. Haloperidol treatment in high doses of haloperidol-withdrawn rats decreased DA level in corpus striatum by 43%, but increased it in the frontal cortex and mid-brain as was seen in normal animals.10. Our data demonstrated that high doses of haloperidol, like baclofen, enhanced the functioning of GABAergic neurons in striatum, which in turn, influenced the DAergic system in this brain area.     

Fatty acid esters as novel metabolites of γ-hydroxybutyric acid: A preliminary investigation

γ-Hydroxybutyric acid (GHB) is a substance frequently abused as a knockout agent. Because of possible amnesia experienced by victims of GHB exposure and the short detection time of GHB in biological samples, the proof of GHB uptake is often challenging for forensic toxicologists. For this reason, various approaches have been evaluated to prolong the detection of GHB intake. In the present study, a fatty acid ester of GHB (4-palmitoyloxy butyrate [GHB-Pal; 3-carboxypropyl hexadecanoate]) was synthesized with the intent of examining whether such esters could be detected as metabolites of GHB in blood samples. Using the structurally elucidated synthesis product (structural elucidation by means of high performance liquid chromatography quadrupole time of flight mass spectrometry [LC-QToF-MS]), an LC triple quadrupole mass spectrometric (LC–MS/MS) method was established for the detection of GHB-Pal. Blood (plasma) samples from four cases in which GHB was previously detected at relevant concentrations (56.1–96.5 μg/ml) were analyzed with respect to GHB-Pal. Signals for GHB-Pal, as well as possible signals for other fatty acid esters of GHB, were detectable in these specimens. (Negative) control samples (20 plasma samples and 20 red blood cell/blood clot samples; from cases in which an intake of GHB or its precursors was not assumed) were all negative for GHB-Pal. To evaluate a possible forensic benefit of GHB fatty acid esters (prolongation of the detection window of a GHB uptake), the analysis of additional plasma samples collected after GHB uptake (or controlled GHB administration) and quantification of GHB fatty acid esters are needed.