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The inferior colliculus (IC) receives ascending and descending information from several convergent neural sources. As such, exploring the neural pathways that converge in the IC is crucial to uncovering their multi-varied roles in the integration of auditory and other sensory information. Among these convergent pathways, the IC commissural connections represent an important route for the integration of bilateral information in the auditory system. Here, we describe the preparation and validation of a novel in vitro slice preparation for examining the functional topography and synaptic properties of the commissural and intrinsic projections in the IC of the mouse. This preparation, in combination with modern genetic approaches in the mouse, enables the specific examination of these pathways, which potentially can reveal cell-type specific processing channels in the auditory midbrain., (Copyright © 2015 Elsevier B.V. All rights reserved.)

As a new type of antiparasitic drugs, ivermectin (IVM) has been widely applied in agriculture, stock raising and aquaculture in China because of its broad spectrum and high efficiency. In order to evaluate the IVM' s reproductive toxicity to male Crucian carp (Carassius auratus), IVM was orally given to the experimental fish with different dosages and the gonadosomatic index (GSI), sexual hormone contents (including testosterone and estradiol) in serum and testis, γ-aminobutyric acid content in serum and brain tissues, ultra-structure of spermatozoa and gonadal tissue in fish were determined in this study. The experimental fish were classified into A, B, C and D groups corresponding to the different dosages of IVM (0, 0.3, 0.9 and 1.5 mg . kg-1, respectively once a day for 3 days continuously). Several indices in fish were detected after 8 days self-purification. The results indicated that GSI gradually decreased with the increase of drug dosage, and GSI in groups C and D was significantly lower than that in group A. The contents of testosterone, estradiol and y-aminobutyric acid exhibited a trend of first increasing and then decreasing and reached the peak at group B. Sperm longevity gradually decreased and the motion time also decreased in II, III and IV level sperms with the increasing dosage of IVM, which appeared to be especially obvious in group C and D. No obvious differences were found in the ultra-structure of spermatozoa and gonadal tissues. In conclusion, this study suggested that IVM had no obvious reproductive toxicity to male Crucian carp at the normal therapeutic dosage but could cause serious potential reproductive toxicity to fish at a high concentration.

Objective: Melanocortin-1 (MC1) receptor is an attractive melanoma-specific target which has been used for melanoma imaging and therapy. In this work, a new lactam bridge α-MSH analog was labeled with (99m)Tc via HYNIC and EDDA/tricine as coligands including gamma aminobutyric acid (GABA) as a three carbon chain spacer between HYNIC and the N-terminus of the cyclic peptide. Also, stability in human serum, receptor bound internalization, in vivo tumor uptake, and tissue biodistribution were thoroughly investigated., Methods: HYNIC-GABA-Nle-CycMSHhept was synthesized using a standard Fmoc strategy. Labeling was performed at 95 °C and analysis involved instant thin layer chromatography and high performance liquid chromatography methods. The receptor bound internalization rate was studied in MC1 receptor expressing B16/F10 cells. Biodistribution of radiopeptide was studied in nude mice bearing B16/F10 tumor., Results: Labeling yield of >98 % (n = 3) was obtained corresponding to a specific activity of 81 MBq/nmol. Peptide conjugate showed efficient stability in the presence of human serum. The radioligand showed specific internalization into B16/F10 cells (12.45 ± 1.1 % at 4 h). In biodistribution studies, a receptor-specific uptake was observed in MC1 receptor-positive organs so that after 2 h the uptake in mouse tumor was 5.10 ± 0.08 % ID/g, while low accumulation in the kidney uptake was observed (4.58 ± 0.68 % ID/g at 2 h after injection)., Conclusions: The obtained results show that the presented new designed labeled peptide conjugate may be a suitable candidate for diagnosis of malignant tumors.

Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [(3)H]GABA uptake assay identified isatin derivatives as a new class of hGAT3 inhibitors. A subsequent structure-activity relationship (SAR) study led to the identification of hGAT3-selective inhibitors (i.e., compounds 20 and 34) that were superior to the reference hGAT3 inhibitor, (S)-SNAP-5114, in terms of potency (low micromolar IC50 values) and selectivity (>30-fold selective for hGAT3 over hGAT1/hGAT2/hBGT1). Further pharmacological characterization of compound 20 (5-(thiophen-2-yl)indoline-2,3-dione) revealed a noncompetitive mode of inhibition at hGAT3. This suggests that this compound class, which has no structural resemblance to GABA, has a binding site different from the substrate, GABA. This was supported by a molecular modeling study that suggested a unique binding site that matched the observed selectivity, inhibition kinetics, and SAR of the compound series. These compounds are the most potent GAT3 inhibitors reported to date that provide selectivity for GAT3 over other GABA transporter subtypes.

A one-pot synthesis of various GABA amides has been demostrated, employing the nucleophilic addition of primary and secondary amines across the double bond of cyclopropene-3-carboxamides, followed by ring-opening of the resulting donor-acceptor cyclopropanes and subsequent in situ reduction of enamine (imine) intermediates.

Perfluorooctane sulfonate (PFOS) is a fluorinated organic compound. This chemical is neurotoxic and can alter the pituitary secretion. This is an initial study aimed at knowing the toxic effects of high doses of PFOS on prolactin secretion and the possible mechanisms involved in these alterations. For that, adult male rats were orally treated with 3.0 and 6.0 mg of PFOS/kg body weight (b.w.)/day for 28 days. At the end of the treatment, the serum levels of prolactin and estradiol as well as the concentration of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and gamma-aminobutyric acid (GABA) were quantified in the anterior and in the mediobasal hypothalamus. PFOS, at the administered doses, reduced prolactin and estradiol secretion, increased the concentration of dopamine and GABA in the anterior hypothalamus, and decreased the ratios DOPAC/dopamine and HVA/dopamine in this same hypothalamic area. The outcomes reported in this study suggest that (1) high doses of PFOS inhibit prolactin secretion in adult male rats; (2) only the periventricular-hypophysial dopaminergic (PHDA) neurons seem to be involved in this inhibitory effect but not the tuberoinfundibular dopaminergic (TIDA) and the tuberohypophysial dopaminergic (THDA) systems; (3) GABAergic cells from the paraventricular and supraoptic nuclei could be partially responsible for the PFOS action on prolactin secretion; and finally (4) estradiol might take part in the inhibition exerted by elevated concentration of PFOS on prolactin release., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Neuropathic pain is a manifestation of chronic pain that arises with damage to the somatosensory system. Pharmacologic treatment recommendations for alleviation of neuropathic pain are often multimodal, and the few reports communicating treatment of suspected neuropathic pain in avian patients describe the use of gabapentin as part of the therapeutic regimen. To determine the pharmacokinetics of gabapentin in Hispaniolan Amazon parrots ( Amazona ventralis ), compounded gabapentin suspensions were administered at 30 mg/kg IV to 2 birds, 10 mg/kg PO to 3 birds, and 30 mg/kg PO to 3 birds. Blood samples were collected immediately before and at 9 different time points after drug administration. Plasma samples were analyzed for gabapentin concentration, and pharmacokinetic parameters were calculated with both a nonlinear mixed-effect approach and a noncompartmental analysis. The best compartmental, oral model was used to simulate the concentration-time profiles resulting from different dosing scenarios. Mild sedation was observed in both study birds after intravenous injection. Computer simulation of different dosing scenarios with the mean parameter estimates showed that 15 mg/kg every 8 hours would be a starting point for oral dosing in Hispaniolan Amazon parrots based on effective plasma concentrations reported for human patients; however, additional studies need to be performed to establish a therapeutic dose.

Purpose: To provide clear information on the activation regions of itching, we investigated the functional activity of cerebral regions in the pruritus-induced rat model using manganese-enhanced magnetic resonance imaging (MEMRI)., Materials and Methods: Itching was induced via neonatal capsaicin treatment in Sprague-Dawley rats (itching rats), and scratching behavior of the control, itching, and gabapentin (GBP)-treated itching rats was compared. Then the activated or deactivated brain regions were investigated in the control, itching, and GBP-treated itching rats using a 4.7T MRI system., Results: While the itching rats engaged in vigorous scratching (121.2 ± 22.4 times), the scratching behavior was decreased in the GBP-treated itching rats (30.6 ± 8.8 times). GBP induced the attenuation of functional activity in two regions -7.10 mm from bregma, in one region -6.65 mm from bregma, and in one region -6.06 mm from bregma. The brain regions related to itching were as follows: parafascicular nucleus, thalamus, superior/inferior colliculus, periaqueductal gray, cingulate cortex, amygdala, midbrain regions, lateral habenula, and hypothalamic areas., Conclusion: Our MEMRI investigation indicates new functional activity of cerebral regions in rats due to the effect of itching or GBP. This information could be used to monitor the therapeutic effects of novel agents or for clinical strategies to treat pathological itch., (© 2014 Wiley Periodicals, Inc.)

This paper was designed to investigate anticonvulsant and sedative effects of eudesmin isolated from Acorus tatarinowii. The eudesmin (5, 10, and 20 mg/kg) was administered intraperitoneally (i.p.). The maximal electroshock test (MES) and pentylenetertrazole (PTZ)-induced seizures in male mice were used to evaluate anticonvulsant activities of eudesmin, and sedative effects of eudesmin were evaluated by pentobarbital sodium-induced sleeping time (PST) and locomotor activity in mice. Finally, the mechanisms of eudesmin were investigated by determining contents of glutamic acid (Glu) and gamma-aminobutyric acid (GABA) in epileptic mice, and expressions of glutamate decarboxylase 65 (GAD65), GABAA , Bcl-2, and caspase-3 in the brain of chronic epileptic rats. Results of MES and PTZ tests revealed that eudesmin possesses significant anticonvulsant effects, and the PST and locomotor activity tests demonstrated that eudesmin has significant sedative effects. Furthermore, our study revealed that after treatment with eudesmin, GABA contents increased, whereas Glu contents decreased, and ratio of Glu/GABA decreased. Our results also indicated that expressions of GAD65, GABAA, and Bcl-2 were up-regulated by treating with eudesmin, whereas the caspase-3 obviously was down-regulated. In conclusion, eudesmin has significant anticonvulsant and sedative effects, and the mechanism of eudesmin may be related to up-regulation of GABAA and GAD65 expressions, and anti-apoptosis of neuron the in brain., (Copyright © 2015 John Wiley & Sons, Ltd.)

A simple and reliable method based on capillary electrophoresis with laser-induced fluorescence detection was developed for the analysis of the antiepileptic drug Gabapentin in human plasma and urine. 4-Chloro-7-nitrobenzofurazan was used for precolumn derivatization of the drug. With an uncoated fused silica capillary (40.0 cm effective length, 50.2 cm total length and 75 μm internal diameter), optimal separation was achieved with 30 mM sodium dodecyl sulfate, 40 mM sodium borate (pH 10.25) and acetonitrile 10% (v/v) as running buffer. The applied voltage was 20 kV and the samples were injected by pressure (3.45 kPa × 3 s). The method was fully validated with regard to linear range, sensitivity, precision, limit of detection and limit of quantification in human plasma and urine samples. Linear ranges were 0.1-15 μg mL(-1) for plasma and urine. The intra- and interday precisions were ≤9.02 and 13.90%, respectively. The recoveries were 96.0-109.3% for plasma and 94.3-98.0% for urine. The method was successfully applied for the determination of Gabapentin in human plasma and urine., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Gamma-aminobutyric acid (GABA) has many biological functions, including the inhibition of blood pressure increases and acceleration of growth hormone secretion. In this study, we discovered the utility of measuring the concentration of carbon dioxide (CO2 ) dissolved in the reaction solution, for development of a real-time and convenient technique to estimate GABA production. In addition to mochi barley bran, we examined the polished grains of three species: mochi barley (a variant of hulless barley), barley, and Japanese millet, all soaked in l-glutamic acid (l-Glu) solution at pH 4.5. We found a positive correlation between GABA and CO2 concentrations, and the production of CO2 was suppressed in the absence of l-Glu at pH 4.5. These results suggest that GABA content can be easily predicted by measuring the aqueous CO2 content using a CO2 sensor, during the process of GABA production in polished mochi barley grains and bran., (© 2015 Institute of Food Technologists®)

We describe an analytical methodology to obtain high sensitivity and better resolution through the study of fluorometric excitation (λex) and emission (λem) spectrum wavelengths of OPA-amino acids. The spectrum emission study revealed a maximum signal peak at 450 nm for aspartate and glutamine. For glycine, taurine, and GABA, the maximum signal peak was at 448 and for glutamate at 452 nm. The remaining amino acids analyzed showed a maximum emission around 450 nm. The best signal obtained within the spectrum excitation experiments was using 229- to 450-nm λex-λem. The drawbacks observed at these wavelengths were a baseline drift and negative peaks occurrence. Thus, the excitation wavelength of 240 nm was chosen (240- to 450-nm λex-λem) as a compromise between a very good signal response and a baseline stability to resolve the 18 amino acids studied. Furthermore, this protocol was properly validated. On the other hand, the elution gradient program used for neuroactive amino acids (aspartate, glutamate, glycine, taurine and GABA) showed separation to the baseline, in a 15-min run in all of them. Other amino acids, up to 18, also exhibited a very good separation in a 25-min run. In conclusion, we propose the use of 240- to 450-nm λex-λem wavelengths, in OPA-amino acids analysis, as the most suitable protocol to obtain the best signal response, maintaining an optimum chromatographic resolution.

Chemical manufacturing is conducted using either batch systems or continuous-flow systems. Flow systems have several advantages over batch systems, particularly in terms of productivity, heat and mixing efficiency, safety, and reproducibility. However, for over half a century, pharmaceutical manufacturing has used batch systems because the synthesis of complex molecules such as drugs has been difficult to achieve with continuous-flow systems. Here we describe the continuous-flow synthesis of drugs using only columns packed with heterogeneous catalysts. Commercially available starting materials were successively passed through four columns containing achiral and chiral heterogeneous catalysts to produce (R)-rolipram, an anti-inflammatory drug and one of the family of γ-aminobutyric acid (GABA) derivatives. In addition, simply by replacing a column packed with a chiral heterogeneous catalyst with another column packed with the opposing enantiomer, we obtained antipole (S)-rolipram. Similarly, we also synthesized (R)-phenibut, another drug belonging to the GABA family. These flow systems are simple and stable with no leaching of metal catalysts. Our results demonstrate that multistep (eight steps in this case) chemical transformations for drug synthesis can proceed smoothly under flow conditions using only heterogeneous catalysts, without the isolation of any intermediates and without the separation of any catalysts, co-products, by-products, and excess reagents. We anticipate that such syntheses will be useful in pharmaceutical manufacturing.

Dominant strains of lactic acid bacteria (LAB) isolated from honey bees were evaluated for their γ-aminobutyric acid (GABA)-producing ability. Out of 24 strains, strain Taj-Apis362 showed the highest GABA-producing ability (1.76 mM) in MRS broth containing 50 mM initial glutamic acid cultured for 60 h. Effects of fermentation parameters, including initial glutamic acid level, culture temperature, initial pH and incubation time on GABA production were investigated via a single parameter optimization strategy. The optimal fermentation condition for GABA production was modeled using response surface methodology (RSM). The results showed that the culture temperature was the most significant factor for GABA production. The optimum conditions for maximum GABA production by Lactobacillus plantarum Taj-Apis362 were an initial glutamic acid concentration of 497.97 mM, culture temperature of 36 °C, initial pH of 5.31 and incubation time of 60 h, which produced 7.15 mM of GABA. The value is comparable with the predicted value of 7.21 mM.

Low levels of γ-aminobutyric acid (GABA), one of two major neurotransmitters that regulate brain neuronal activity, are associated with many neurological disorders, such as epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, and cocaine addiction. One of the main methods to raise the GABA level in human brain is to use small molecules that cross the blood-brain barrier and inhibit the activity of γ-aminobutyric acid aminotransferase (GABA-AT), the enzyme that degrades GABA. We have designed a series of conformationally restricted tetrahydrothiophene-based GABA analogues with a properly positioned leaving group that could facilitate a ring-opening mechanism, leading to inactivation of GABA-AT. One compound in the series is 8 times more efficient an inactivator of GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. Our mechanistic studies show that the compound inactivates GABA-AT by a new mechanism. The metabolite resulting from inactivation does not covalently bind to amino acid residues of GABA-AT but stays in the active site via H-bonding interactions with Arg-192, a π-π interaction with Phe-189, and a weak nonbonded S···O═C interaction with Glu-270, thereby inactivating the enzyme.

Nuclear magnetic resonance spectroscopy serves as an important tool for analyzing chemicals and biological metabolites. However, its performance is subject to the magnetic-field homogeneity. Under inhomogeneous fields, peaks are broadened to overlap each other, introducing difficulties for assignments. Here, we propose a method termed as line broadening interference (LBI) to provide high-resolution information under inhomogeneous magnetic fields by employing certain gradients in the indirect dimension to interfere the magnetic-field inhomogeneity. The conventional spectral-line broadening is thus interfered to be non-diagonal, avoiding the overlapping among adjacent resonances. Furthermore, an inhomogeneity correction algorithm is developed based on pattern recognition to recover the high-resolution information from LBI spectra. Theoretical deductions are performed to offer systematic and detailed analyses on the proposed method. Moreover, experiments are conducted to prove the feasibility of the proposed method for yielding high-resolution spectra in inhomogeneous magnetic fields.

Gamma-aminobutyric acid (GABA) may be synthesized in plant tissues when the organism is under stressful conditions. Rice bran byproduct obtained from the milling of brown rice was treated under anaerobic storage with nitrogen at different temperatures (20-60 °C) and moisture contents (10-50%) up to 12h. For the GABA synthesis, the storage at 30% moisture content and 40 °C appeared optimal. Utilisation of an electrolyzed oxidizing water (EOW, pH 3.3) for moisture adjustment and addition of glutamic acid increased the GABA content in rice bran. The maximum GABA content in rice bran (523 mg/100g) could be achieved by the anaerobic storage at 30% EOW for 5h at 40 °C after an addition of glutamic acid (5mM). This amount was approximately 17 times higher than that in the control (30 mg/100g). The use of EOW also prevented bacterial growth by decreasing the colony counts almost by half., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A RP-LC-FL detection method has been developed to identify and quantitate four amino acid neurotransmitters including glutamic acid, glycine, taurine and γ-aminobutyric acid in rat and mouse spinal cord tissue. 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) was employed for the derivatization of these neurotransmitters prior to RP-LC-FL analysis. Different parameters which influenced separation and derivatization were optimized. Under optimum conditions, linearity was achieved within the concentration ranges of 0.50-50.00 μM for all analytes with correlation coefficients from 0.9912 to 0.9997. The LODs ranged from 0.03 μM to 0.06 μM. The proposed method has been successfully applied to the determination of amino acid neurotransmitters in biological samples such as rat and mouse spinal cord with satisfactory recoveries., (Copyright © 2014 Elsevier B.V. All rights reserved.)

PdCl2(CH3CN)2-catalyzed arylation of unactivated C(sp(3))-H bonds using (diacetoxyiodo)arenes as arylation reagents is reported. The reactivity of (diacetoxyiodo)arenes as arylation reagents is enabled in the presence of Cs2CO3 under the reaction conditions. This arylation method is highly efficient and occurs without the use of silver salt. The reaction tolerates a broad substrate scope that was not demonstrated by other silver salt-free C(sp(3))-H bond arylation conditions. The synthetic utility of the method is further illustrated in the synthesis of the psychotropic drug phenibut. A detailed mechanism study has been conducted to understand the reaction pathway.

The accumulation of proline (Pro) in plants exposed to biotic/abiotic stress is a well-documented and conserved response in most vegetal species. Stress conditions induce the overproduction of reactive oxygen species which can lead to cellular damage. In vitro assays have shown that enzyme inactivation by hydroxyl radicals (·OH) can be avoided in presence of Pro, suggesting that this amino acid could act as an ·OH scavenger. We applied Density Functional Theory coupled with a polarizable continuum model to elucidate how Pro reacts with ·OH. In this work we suggest that Pro reacts favourably with ·OH by H-abstraction on the amine group. This reaction produces the spontaneous decarboxylation of Pro leading to the formation of pyrrolidin-1-yl. In turn, pyrrolidin-1-yl can easily be converted to Δ1-pyrroline, the substrate of the enzyme Δ1-pyrroline dehydrogenase, which produces γ-aminobutyric acid (GABA). GABA and Pro are frequently accumulated in stressed plants and several protective roles have been assigned to these molecules. Thereby we present an alternative non-enzymatic way to synthetize GABA under oxidative stress. Finally this work sheds light on a new beneficial role of Pro accumulation in the maintenance of photosynthetic activity.

We report isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs from the bioluminescent earthworm Fridericia heliota. They were found to be highly unusual modified peptides, comprising either of the two tyrosine-derived chromophores, CompX or CompY and a set of amino acids, including threonine, gamma-aminobutyric acid, homoarginine, and unsymmetrical N,N-dimethylarginine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and rise novel questions concerning their biosynthetic origin., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Purpose: The important pathological consequences of ischaemic heart disease arise from the detrimental effects of the accumulation of long-chain acylcarnitines in the case of acute ischaemia-reperfusion. The aim of this study is to test whether decreasing the L-carnitine content represents an effective strategy to decrease accumulation of long-chain acylcarnitines and to reduce fatty acid oxidation in order to protect the heart against acute ischaemia-reperfusion injury., Key Results: In this study, we used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (Methyl-GBB), which inhibits γ-butyrobetaine dioxygenase (IC₅₀ 3 μM) and organic cation transporter 2 (OCTN2, IC₅₀ 3 μM), and, in turn, decreases levels of L-carnitine and acylcarnitines in heart tissue. Methyl-GBB reduced both mitochondrial and peroxisomal palmitate oxidation rates by 44 and 53% respectively. In isolated hearts treated with Methyl-GBB, uptake and oxidation rates of labelled palmitate were decreased by 40%, while glucose oxidation was increased twofold. Methyl-GBB (5 or 20 mg·kg(-1)) decreased the infarct size by 45-48%. In vivo pretreatment with Methyl-GBB (20 mg·kg(-1)) attenuated the infarct size by 45% and improved 24 h survival of rats by 20-30%., Conclusions and Implications: Reduction of L-carnitine and long-chain acylcarnitine content by the inhibition of OCTN2 represents an effective strategy to protect the heart against ischaemia-reperfusion-induced damage. Methyl-GBB treatment exerted cardioprotective effects and increased survival by limiting long-chain fatty acid oxidation and facilitating glucose metabolism., (© 2014 The British Pharmacological Society.)

The objective of the present study was to transfer the concept of library screening by MS binding assays, so far applied to pseudostatic hydrazine libraries, to static oxime libraries to screen for new potent inhibitors of mGAT1, the most abundant GABA transporter in the central nervous system that represents a validated drug target for the treatment of epilepsy. Library generation was performed by reaction of guvacine derivatives possessing a hydroxylamine functionality with various sets of four aldehydes. After dilution, the libraries were screened by competitive MS binding assays. Deconvolution experiments allowed hits in the most active libraries to be identified, and they were resynthesized for biological evaluation. That way a series of compounds was identified that displayed binding affinities ≥8.00 (pKi ) at mGAT1, one of which was found to be the most potent mGAT1 inhibitor known to date in a functional GABA uptake assay with a pIC50 value of 8.27 ± 0.03., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new radical-based coupling method has been developed for the single-step generation of various γ-amino acids and α,β-diamino acids from α-aminoacyl tellurides. Upon activation by Et3 B and O2 at ambient temperature, α-aminoacyl tellurides were readily converted into α-amino carbon radicals through facile decarbonylation, which then reacted intermolecularly with acrylates or glyoxylic oxime ethers. This mild and powerful method was effectively incorporated into expeditious synthetic routes to the pharmaceutical agent gabapentin and the natural product (-)-manzacidin A., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Crystal structure of a lectin purified from Butea monosperma seeds was determined by Molecular Replacement method. Its primary structure was determined by Tandem Mass Spectroscopy and electron density maps from X-ray diffraction data. Its quaternary structure was tetrameric, formed of two monomers, α and β, β appearing as truncated α. The occurrence of two tetramers in the asymmetric unit of the crystal might be a consequence of asymmetric contacts due to difference in glycosylation and variable loops structures, to form an 'octamer-structure'. The crystal structure showed binding pockets for γAbu, having a proposed role in plant defense, at the interface of canonical dimer-partners. Hemagglutination studies, enzyme kinetics, isothermal titration calorimetry and molecular dynamics showed that the lectin is specific to N-acetyl d-galactosamine, galactose and lactose in decreasing order, and α-amylase inhibitor.

We report here a novel method for the synthesis of a pH-responsive composite using visible light. Formation of the pH-responsive layer is based on poly(methacrylic acid-g-ethylene glycol) as the macromer, eosin Y as the photoinitiator and triethanolamine as the co-initiator. The hydrogel was functionalized with hydrophobic domains through incorporation of crosslinked styrene-butadiene-styrene (SBS) copolymer into the pH-responsive prepolymer. Swelling ratios were decreased with the addition of SBS, and resulted in high hydrogel crosslink density. The composite allowed for controlled release of an anticonvulsant model drug, pregabalin, under neutral pH condition and the release was analyzed to describe the mode of transport through the network. In vitro human fibroblast survival assay and in vivo rabbit implantation experiments demonstrated that this hybrid network is not toxic and has desirable biocompatibility properties. This is the first report about the synthesis of a pH-responsive network incorporating crosslinked SBS synthesized under visible light. The approach for multifunctional membranes could allow the incorporation of molecules with specific functionalities so that sequential molecule delivery in response to specific stimuli could be achieved., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Amidated kyotorphin (L-Tyr-L-Arg-NH2; KTP-NH2) causes analgesia when systemically administered. The lipophilic ibuprofen-conjugated derivative of KTP-NH2 has improved analgesic efficacy. However, fast degradation by peptidases impacts negatively in the pharmacodynamics of these drugs. In this work, selected derivatives of KTP and KTP-NH2 were synthesized to combine lipophilicity and resistance to enzymatic degradation. Eight novel structural modifications were tested for the potential to transverse lipid membranes and to evaluate their efficacy in vivo. The rationale behind the design of the pool of the eight selected molecules consisted in the addition of individual group at the N-terminus, namely the tert-butyloxycarbonyl (Boc), γ-aminobutyric acid (GABA), acetyl, butanoyl, and propanoyl or in the substitution of the tyrosine residue by an indole moiety and in the replacement of the peptidic bond by a urea-like bond in some cases. All the drugs used in the study are intrinsically fluorescent, which enables the use of spectrofluorimetry to sample the drugs in the permeation assays. The results show that the BOC and indolyl derivatives of KTP-NH2 have maximal ability to permeate membranes with concomitant maximal analgesic power. Overall, the results demonstrate that membrane permeation is correlated with analgesic efficacy. However, this is not the only factor accounting for analgesia. KTP-NH2 for instance has low passive permeation but is known to have central action. In this case, hypothetical transcytosis over the blood-brain barrier seems to depend on dipeptide transporters., (© 2014 Wiley Periodicals, Inc.)

Context: Gabapentin was selected to formulate oral controlled release dry suspension because of short biological half life of 5-7 h and low bioavailability (60%). Gabapentin is a bitter drug so an attempt was made to mask its taste., Objective: To formulate and evaluate controlled release dry suspension for reconstitution to increase the bioavailability and to control bitter taste of drug., Materials and Methods: Cyclodextrin based nanosponges were synthesized by previously reported melt method. The nanosponge-drug complexes were characterized by FTIR, DSC and PXRD as well as evaluated for taste and saturation solubility. The complexes were coated on Espheres by a suspension layering technique followed by coating with ethyl cellulose and Eudragit RS-100. A dry powder suspension for reconstitution of the microspheres was formulated and evaluated for taste, redispersibility, in vitro dissolution, sedimentation volume, leaching and pharmacokinetics., Results and Discussion: The complexes showed partial entrapment of drug nanocavities. Significant decrease in solubility (25%) was observed in the complexes than pure drug in different media. The microspheres of nanosponge complexes showed desired controlled release profile for 12 h. Insignificant drug leaching was observed in reconstituted suspension during storage for 7 days at 45 °C/75% RH. Nanosponges effectively masked the taste of Gabapentin and the coating polymers provided controlled release of the drug and enhanced taste masking. The results of in vivo studies showed increase in bioavailability of controlled release suspension by 24.09% as compared to pure drug., Conclusion: The dry powder suspension loaded with microspheres of nanosponges complexes can be proposed as a suitable controlled release drug delivery for Gabapentin.

γ-Aminobutyric acid type A and glycine receptors (GABA(A)Rs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABA(A)Rs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin-GABA(A)R α3 complex, revealing that the N-terminal region of the α3 peptide occupies the same binding site as the GlyR β subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABA(A)R α subunits, engages in unique interactions. Thermodynamic dissections of the gephyrin-receptor interactions identify two residues as primary determinants for gephyrin's subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABA(A)Rs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin-GABA(A)R interaction.

Reaction of γ-Boc-GABA, prepared by protecting the γ-amino moiety of the amino butyric acid with the tert-butyloxycarbonyl (Boc) protecting group, with 4-methyl/ethyl benzenesulfonamide, followed by removal of the Boc protecting group in 3 M HCl afforded the corresponding hydrochlorides, which were further derivatized by reaction with a varying of aryl isocyanates to give a new classes of ureido substituted benzenesulfonamide containing a GABA moiety. Inhibition studies of the human carbonic anhydrase(CA, EC 4.2.1.1) isoforms, CA I–XIV with these new compounds revealed that they possess moderate-weak inhibition potency against hCA III, IV, VA, VI and XIII, rather efficient inhibitory power against hCA I, VI, and IX, and excellent inhibition of the physiologically relevant hCA II and VII, as well as of the two tumor-associated isoforms CA IX and XII. The inhibition profile of the new ureido-substituted benzenesulfonamides reported here is thus very different from the corresponding ureido-substituted analogs incorporating sulfanilamide, which were previously investigated as inhibitors of some of these enzymes.

Recurrent axon collaterals are a major means of communication between spiny projection neurons (SPNs) in the striatum and profoundly affect the function of the basal ganglia. However, little is known about the molecular and cellular mechanisms that underlie this communication. We show that intrastriatal nitric oxide (NO) signaling elevates the expression of the vesicular GABA transporter (VGAT) within recurrent collaterals of SPNs. Down-regulation of striatal NO signaling resulted in an attenuation of GABAergic signaling in SPN local collaterals, down-regulation of VGAT expression in local processes of SPNs, and impaired motor behavior. PKG1 and cAMP response element-binding protein are involved in the signal transduction that transcriptionally regulates VGAT by NO. These data suggest that transcriptional control of the vesicular GABA transporter by NO regulates GABA transmission and action selection.

A simple and sensitive gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) method using dried plasma spot testing cards was developed for determination of valproic acid and gabapentin concentrations in human plasma from patients receiving in-home medical care. We have proposed that a simple, easy and dry sampling method is suitable for in-home medical patients for therapeutic drug monitoring. Therefore, in the present study, we used recently developed commercially available easy handling cards: Whatman FTA DMPK-A and Bond Elut DMS. In-home medical care patients can collect plasma using these simple kits. The spots of plasma on the cards were extracted into methanol and then evaporated to dryness. The residues were trimethylsilylated using N-methyl-N-trimethylsilyltrifluoroacetamide. For GC-EI-MS analysis, the calibration curves on both cards were linear from 10 to 200 µg/mL for valproic acid, and from 0.5 to 10 µg/mL for gabapentin. Intra- and interday precisions in plasma were both ≤13.0% (coefficient of variation), and the accuracy was between 87.9 and 112% for both cards within the calibration curves. The limits of quantification were 10 µg/mL for valproic acid and 0.5 µg/mL for gabapentin on both cards. We believe that the present method will be useful for in-home medical care., (Copyright © 2014 John Wiley & Sons, Ltd.)

We have performed a series of molecular dynamics simulations to study the interactions between the neurotransmitters (NTs) γ-aminobutyrate (GABA), glycine (GLY), acetylcholine (ACH) and glutamate (GLU) as well as the amidated/acetylated γ-aminobutyrate (GABA(neu)) and the osmolyte molecule glycerol (GOL) with a dipalmitoylphosphatidylcholine (DPPC) bilayer. In agreement with previously published experimental data, we found the lowest membrane affinity for the charged molecules and a moderate affinity for zwitterionic and polar molecules. The affinity can be ranked as follows: ACH-GLU<

GAT-1 is a sodium- and chloride-coupled GABA transporter and a member of the neurotransmitter:sodium:symporters, which are crucial for synaptic transmission. The structure of bacterial homologue LeuT shows a thin extracellular gate consisting of a charge and an aromatic pair. Here we addressed the question of whether mutation of the aromatic and charge pair residues of GAT-1 has similar consequences. In contrast to charge pair mutants, significant radioactive GABA transport was retained by mutants of the aromatic pair residue Phe-294. Moreover, the magnitude of maximal transport currents induced by GABA by these mutants was comparable with those by wild type GAT-1. However, the apparent affinity of the nonconserved mutants for GABA was reduced up to 20-fold relative to wild type. The voltage dependence of the sodium-dependent transient currents of the Phe-294 mutants was similar to that of the wild type. On the other hand, the conserved charge pair mutant D451E exhibited a right-shifted voltage dependence, indicating an increased apparent affinity for sodium. In further contrast to D451E, whereas the extracellular aqueous accessibility of an endogenous cysteine residue to a membrane-impermeant sulfhydryl reagent was increased relative to wild type, this was not the case for the aromatic pair mutants. Our data indicate that, in contrast to the charge pair, the aromatic pair is not essential for gating. Instead they are compatible with the idea that they serve to diminish dissociation of the substrate from the binding pocket., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The study was aimed to prepare and evaluate gabapentin loaded albumin nanoparticles and to find out their effectiveness in treating epilepsy. Albumin nanoparticles of gabapentin were prepared by pH-coacervation method. The drug was administered into animals as free drug, gabapentin bound with nanoparticles, and gabapentin bound with nanoparticles coated with polysorbate 80. The polysorbate 80 coated nanoparticles increased the gabapentin concentration in the brain about 3 fold in comparison with the free drug. Moreover, the polysorbate 80 coated nanoparticles significantly reduced the duration of all phases of convulsion in both maximal electroshock induced and pentylenetetrazole induced convulsion models in comparison with free drug and drug bound with nanoparticle formulations, which indicates the ability of polysorbate 80 coated nanoparticles to enhance the gabapentin concentration in the brain., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-L-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC50 of 3.2 ± 0.3 μM and 59 ± 18 nM, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Parkinson's disease is a multifactorial neurodegenerative disorder, characterized by a reduction of dopamine (DA) levels. The molecular pathways involved in the pathogenesis of disease have not yet been fully disclosed. Therefore, developing new diagnostic methods and tools to evaluate the depletion of DA and of some of its metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine) is of outstanding importance for biochemical evaluations. Moreover, neurons responsible for DA release also produce the neurotransmitter gamma-aminobutyric acid (GABA), thus, quantitative measurements of GABA levels can have a relevant impact for a further understanding of the biochemical processes involved in the neurodegenerative event. In the present study, two HPLC methods based on the reversed-phase ion-pairing chromatography (RP-IPC) and the hydrophilic liquid interaction chromatography (HILIC) concepts were developed to allow the quantification of DA and its metabolites as well as GABA levels in mouse striatal and cortical tissue homogenates. The two fairly orthogonal HPLC methods were directly applied to the biological samples, without preliminary derivatization of the compounds of interest. A high level of selectivity was obtained for DA metabolites and GABA by running the gradient RP-IPC method with a volatile ion-pairing reagent, which makes it suitable for the quantitative assay of four out of five compounds. Matrix deriving interferences unabled the base-line separation of DA which was instead successfully achieved with the HILIC-based method. To avail of HPLC methods providing distinct selectivity profiles, makes possible the correct species quantification and allows to compensate the intrinsic limits characterizing all chromatographic methods., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Six series of 2-substituted 4-aminobutanamide derivatives were synthesized and evaluated for their ability to inhibit GABA transport proteins mGAT1-4 stably expressed in HEK-293 cell lines. The pIC50 values determined were in the range 4.23-5.23. Two compounds (15b and 15c) were selected for further in vitro studies. These compounds were also subjected to preliminary behavioral studies to evaluate their anticonvulsant, antidepressant-like, and antinociceptive activities in mice. Their influence on motor coordination was also assessed. We report that, among a spectrum of in vivo activities, both 15b and 15c displayed significant activity against pentylenetetrazole (PTZ)-induced seizures., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

The direct application of carboxylic acids as a traceless activation group for radical Michael additions has been accomplished via visible light-mediated photoredox catalysis. Photon-induced oxidation of a broad series of carboxylic acids, including hydrocarbon-substituted, α-oxy, and α-amino acids, provides a versatile CO2-extrusion platform to generate Michael donors without the requirement for organometallic activation or propagation. A diverse array of Michael acceptors is amenable to this new conjugate addition strategy. An application of this technology to a three-step synthesis of the medicinal agent pregabalin (commercialized by Pfizer under the trade name Lyrica) is also presented.

The crystal structure of N-[(1-{2-oxo-2-[2-(pyrazin-2-ylcarbonyl)hydrazin-1-yl]ethyl}cyclohexyl)methyl]pyrazine-2-carboxamide monohydrate (Pyr-Gpn-NN-NH-Pyr·H2O), C19H23N7O3·H2O, reveals an unusual trans-gauche (tg(-)) conformation for the gabapentin (Gpn) residue around the C(γ)-C(β) (θ1) and C(β)-C(α) (θ2) bonds. The molecular conformation is stabilized by intramolecular N-H...N hydrogen bonds and weak C-H...O interactions. The packing of the molecules in the crystal lattice shows a network of strong N-H...O and O-H...O hydrogen bonds together with weak C-H...O and π-π inteactions.

A new and atom-economic palladium-catalyzed aminomethylamination of allenes with aminals by CN bond activation is described. This direct and operationally simple method provides a fundamentally novel approach for the synthesis of 1,3-diamines. Mechanistic studies suggest that a unique cationic π-allylpalladium complex containing an aminomethyl moiety is generated as a key intermediate through the carbopalladation of the allene with a cyclometalated palladium-alkyl species., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This paper investigated the influence of gamma-aminobutyric acid (GABA) on GABA metabolism and amino acid content under hypoxia stress by accurately controlling the level of dissolved oxygen in hydroponics, using the roots of melon 'Xiyu 1' seedlings as the test material. The results showed that compared with the control, the growth of roots was inhibited seriously under hypoxia stress. Meanwhile, the hypoxia-treated roots had significantly higher activities of glutamate decarboxylase (GAD), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), glutamine synthetase (GS), alanine aminotransferase (ALT), aspartate aminotransferase (AST) as well as the contents of GABA, pyruvic acid, alanine (Ala) and aspartic acid (Asp). But the contents of glutamic acid (Glu) and alpha-keto glutaric acid in roots under hypoxia stress was obviously lower than those of the control. Exogenous treatment with GABA alleviated the inhibition effect of hypoxia stress on root growth, which was accompanied by an increase in the contents of endogenous GABA, Glu, alpha-keto glutaric acid and Asp. Furthermore, under hypoxia stress, the activities of GAD, GDH, GOGAT, GS, ALT, AST as well as the contents of pyruvic acid and Ala significantly decreased in roots treated with GABA. However, adding GABA and viny-gamma-aminobutyric acid (VGB) reduced the alleviation effect of GABA on melon seedlings under hypoxia stress. The results suggested that absorption of GABA by roots could alleviate the injury of hypoxia stress to melon seedlings. This meant that GABA treatment allows the normal physiological metabolism under hypoxia by inhibiting the GAD activity through feedback and maintaining higher Glu content as well as the bal- ance of carbon and nitrogen.

The pharmacokinetics of 2 brands of pregabalin 300 mg capsules were compared in 23 healthy human volunteers after a single oral dose in a randomized cross-over study. The study protocol was prepared with relevance to the requirements set in the US FDA and the EMA guidances for conduction of bioequivalence studies. Reference (Lyrica(®), Pfizer, France) and test (Neurexal, Pharmaline, Lebanon) products were administered to fasted volunteers. Blood samples were collected up to 48 h and assayed for pregabalin using a validated LC-MS/MS method. The pharmacokinetic parameters AUC0-t, AUC0-∞, Cmax, Tmax, T1/2 and elimination rate constant were determined from plasma concentration-time profile by non-compartmental analysis method using WinNonlin V5.2. The analysis of variance did not show any significant difference between the 2 formulations and 90% confidence intervals fell within the acceptable range for bioequivalence: 80-125%. It was concluded that the 2 brands exhibited comparable pharmacokinetic profiles and that Pharmaline's Neurexal is bioequivalent to Lyrica(®) of Pfizer, France., (© Georg Thieme Verlag KG Stuttgart · New York.)

Noninvasive methods for efficient drug delivery to the brain is an unmet need. Molecular access to the brain is regulated by the blood-brain barrier (BBB) established by the endothelial cells of brain vessels. Passive diffusion is one of the main mechanisms that organic compounds use to travel through these endothelial cells. This passage across the BBB is determined mainly by certain physicochemical properties of the molecule such as lipophilicity, size, and the presence of hydrogen bond donors and acceptors. One emerging strategy to facilitate the passage of organic compounds across the BBB is the use of peptide shuttles.1 In using this approach the permeability in front the BBB is, clearly, determined by the combined physicochemical properties of both the cargo and the shuttle. Herein we report the synthesis of a series of variations of one of the more efficient peptide shuttles, (N-MePhe)n . These include diverse structural features such as various backbone stereochemistries or the presence of non-natural amino acids, including halogenated residues. In several cases, we assessed the BBB permeability of both the shuttles alone and linked to a few cargos. Our results show how factors such as stereochemistry or halogen content influences the passage across the BBB and, more importantly, opens the way to a strategy of peptide shuttles 'à la carte', in which a particular fine-tuned shuttle is used for each specific cargo., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

CaV1 and CaV2 voltage-gated calcium channels are associated with β and α2δ accessory subunits. However, examination of cell surface-associated CaV2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged CaV2 channels. Here we report the development of fully functional CaV2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neuronal cell line and neurons. We first examined the effect of the auxiliary subunits. Although α2δ subunits copurify with CaV2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether α2δ subunits are associated with these channels at the cell surface. We found that, whereas α2δ-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with CaV2.2/β1b, despite our finding that α2δ-1 increases plasma-membrane CaV2.2 expression. However, this was due to occlusion of the antigenic epitope by association with CaV2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between α2δ-1 and the α1 subunit at the plasma membrane. We further show that, although CaV2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type α2δ-1 (but not a gabapentin-insensitive α2δ-1 mutant), the interaction between CaV2.2 and α2δ-1 is not disrupted by gabapentin. Altogether, these results demonstrate that CaV2.2 and α2δ-1 are intimately associated at the plasma membrane and allow us to infer a region of interaction.

We study the conformation and reorientation dynamics of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) under neutral and acidic conditions using a combination of broadband dielectric relaxation spectroscopy and polarization-resolved femtosecond mid-infrared pump-probe spectroscopy. We find that both zwitterionic and cationic GABA adopt nearly linear conformations in aqueous solution, meaning that the two charged functional groups of the GABA zwitterion are hydrated separately.

Context: Gabapentin follows saturation kinetics for absorption because of carrier-mediated transport and narrow absorption window in stomach. There is need to develop a gastroretentive formulation to maximize the absorption without crossing the saturation threshold for absorption., Objective: The aim was to develop a gastroretentive formulation of gabapentin to increase the fraction of drug absorbed in stomach., Materials and Methods: Sodium alginate and sodium carboxymethylcellulose were used to formulate the microsphere by ionotropic gelation with calcium chloride. The formulation was optimized using a three-factor, three-level Box-Behnken design., Results: The particle size varied from 559.50 to 801.10 μm, entrapment efficiency from 61.29 to 81.00% and in vitro release from 69.40 to 83.70%. The optimized formulation was found using point-prediction, and formulation OF-3 showed optimum results at 608.21 μm size, 79.65% entrapment efficiency and 82.72% drug release and 81% mucoadhesion up to 10 h. The drug release was controlled for more than 12 h., Discussion: The particle size was most influenced by sodium alginate while entrapment efficiency and drug release depended upon both polymers. The release followed Higuchi model., Conclusion: Gastroretentive formulation was successfully optimized by a three-factor, three-level Box-Behnken design and found to be useful.

The ionotropic GABAA receptors (GABAARs) are widely distributed in the central nervous system where they play essential roles in numerous physiological and pathological processes. A high degree of structural heterogeneity of the GABAAR has been revealed and extensive effort has been made to develop selective and potent GABAAR agonists. This review investigates the use of heterocyclic carboxylic acid bioisosteres within the GABAAR area. Several heterocycles including 3-hydroxyisoxazole, 3-hydroxyisoxazoline, 3-hydroxyisothiazole, and the 1- and 3-hydroxypyrazole rings have been employed in order to map the orthosteric binding site. The physicochemical properties of the heterocyclic moieties making them suitable for bioisosteric replacement of the carboxylic acid in the molecule of GABA are discussed. A variety of synthetic strategies for synthesis of the heterocyclic scaffolds are available. Likewise, methods for introduction of substituents into specific positions of the heterocyclic scaffolds facilitate the investigation of different regions in the orthosteric binding pocket in close vicinity of the core scaffolds of muscimol/GABA. The development of structural models, from pharmacophore models to receptor homology models, has provided more insight into the molecular basis for binding. Similar binding modes are proposed for the heterocyclic GABA analogues covered in this review by use of ligand-receptor docking into the receptor homology model and the presented structure-activity relationships. A network of interactions between the analogues and the binding pocket is leaving no room for substituents and underline the limited space in the GABAAR orthosteric binding site when in the agonist conformation.

An electrospray ionization-tandem mass spectrometry (ESI-tqMS) method was developed to identify the location of chlorine substitution during the chlorination of model organic compounds. The chlorine substitution in the aliphatic part and that in the benzene ring of an organic molecule can be differentiated by their corresponding ranges of optimum collision energies, 5-7 eV and over 15 eV, respectively, in the precursor ion scan of m/z 35. The method was applied to predict the structures of intermediates and reveal the transformation pathways during the chlorination of 4-amino-2-chlorobenzoic acid and phenylalanine as a function of reaction time and the chlorine-to-precursor ratio. In the case of phenylalanine, chlorine was found to replace one hydrogen atom attached to the aliphatic nitrogen; in the case of 4-amino-2-chlorobenzoic acid, chlorine was found to replace the hydrogen atoms attached to the aromatic rings.