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PubMed2004

Development of a liquid chromatography-electrospray mass spectrometric method for the simultaneous analysis of benzoxazolinones and their degradation products.

A new method for the simultaneous analysis of some benzoxazolinones, aminophenoxazinones and malonamic acids was developed based on liquid chromatography (LC) coupled to mass spectrometry (MS), using electrospray ionization (ESI) and operating in positive mode. Different ESI-MS parameters, such as fragmentor voltage, capillary voltage, drying gas flow, nebulizer gas pressure and drying gas temperature, were optimized in order to obtain structural information and to achieve maximum sensitivity. Chromatographic separation was performed by a reversed-phase LC column using a linear gradient of water and methanol. Quality assurance of the developed method was assessed by measuring parameters as linearity, sensitivity, repeatability and reproducibility. Quantification method based on the use of internal standard was developed, selecting synthetic 2-methoxy-2H-1,4-benzoxazin-3(4H)-one as internal standard. Good correlations were obtained for all analytes relative to this compound in the range of 0.05-1.5 ng/microL. Instrumental detection limits were between 0.02 and 0.2 ng/microL. Repeatability and reproducibility studies showed acceptable coefficient of variation values.

Benzoxazoles↗
PubMed2004

6"-Azidohex-2"-yne-cannabidiol: a potential neutral, competitive cannabinoid CB1 receptor antagonist.

Previous experiments with the mouse vas deferens have shown that cannabidiol produces surmountable antagonism of cannabinoid CB(1) receptor agonists at concentrations well below those at which it binds to cannabinoid CB(1) receptors and antagonizes alpha(1)-adrenoceptor agonists insurmountably. It also enhances electrically evoked contractions of this tissue. We have now found that subtle changes in the structure of cannabidiol markedly influence its ability to produce each of these effects, suggesting the presence of specific pharmacological targets for this non-psychoactive cannabinoid. Our experiments were performed with cannabidiol, 6"-azidohex-2"-yne-cannabidiol, abnormal-cannabidiol and 2'-monomethoxy- and 2',6'-dimethoxy-cannabidiol. Of these, 6"-azidohex-2"-yne-cannabidiol was as potent as cannabidiol in producing surmountable antagonism of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (R-(+)-WIN55212) in vasa deferentia. However, it produced this antagonism with a potency that matched its cannabinoid CB(1) receptor affinity, suggesting that, unlike cannabidiol, it is a competitive cannabinoid CB(1) receptor antagonist. Moreover, since it did not enhance the amplitude of electrically evoked contractions, it may be a neutral cannabinoid CB(1) receptor antagonist.

Adenosine Triphosphate↗
PubMed2004

Amphetamine effects on dopamine levels and behavior following cannabinoid exposure during adolescence.

The cannabis gateway hypothesis purports that early exposure to cannabis is a risk factor for subsequent use of other addictive drugs, e.g., psychostimulants. Neurobiological sensitization, consistent with a gateway hypothesis, was currently studied in regard to amphetamine response. Rats were exposed to the cannabinoid receptor agonist WIN 55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone] 1.25 mg/kg, intraperitoneally; i.p. for 5 days during early adolescence. Amphetamine (0.5 mg/kg, i.p.) or WIN 55,212-2 (1.25 mg/kg, i.p.) was administered in late adolescence and in vivo dopamine levels were simultaneously measured in the nucleus accumbens. Locomotor and stereotyped behaviors were also monitored in rats pretreated with WIN 55,212-2 (0.625, 1.25 or 2.5 mg/kg) or Delta-9-tetrahydrocannabinol (0.75, 1.5 or 3.0 mg/kg, i.p.) for 5 days during early adolescence and challenged with amphetamine (0.5 or 2.0 mg/kg) in late adolescence or as adults. Pretreatment with WIN 55,212-2 or Delta-9-tetrahydrocannabinol during early adolescence did not alter the dopaminergic or behavioral responses to amphetamine in adolescence or adulthood. In conclusion, these findings do not support the cannabis gateway hypothesis in regard to subsequent amphetamine exposure.

Age Factors↗
PubMed2005

Blockade of 5-HT(3) receptor with MDL 72222 and Y 25130 reduces beta-amyloid protein (25--35)-induced neurotoxicity in cultured rat cortical neurons.

The present study was performed to examine neuroprotective effects of 5-hydroxytryptamine (5-HT)(3) receptor antagonists against beta-amyloid protein (25--35)-, a synthetic 25--35 amyloid peptide, induced neurotoxicity using cultured rat cortical neurons. beta-Amyloid protein (25--35) produced a concentration-dependent reduction of cell viability, which was significantly reduced by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801), an N-methyl-d-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca(2+) channel blocker, and N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. The 5-HT(3) receptor antagonists, tropanyl-3,5-dichlorobenzoate (MDL-72222, 0.1--10 microM) and N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y 25130, 0.05--5 microM), decreased the beta-amyloid protein (25--35) (10 microM)-induced neuronal cell death as assessed by a colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. MDL 72222 and Y 25130 inhibited the beta-amyloid protein (25--35) (10 microM)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) and glutamate release, generation of reactive oxygen species, and caspase-3 activity. These neuroprotective effects of MDL 72222 (10 microM) and Y 25130 (5 microM) were completely blocked by the simultaneous treatment with 100 microM 1-phenylbiguanide, a 5-HT(3) receptor agonist, indicating that the protective effects of these compounds were due to 5-HT(3) receptor blockade. These results suggest that the activation of the 5-HT(3) receptor may be partially involved in beta-amyloid protein-induced neurotoxicity, by membrane depolarization for Ca(2+) influx. Therefore, the blockade of 5-HT(3) receptor with MDL 72222 and Y 25130, may ameliorate the beta-amyloid protein-induced neurotoxicity by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of reactive oxygen species and caspase-3 activity.

Amyloid beta-Peptides↗
PubMed2005

On the structural basis of the catalytic mechanism and the regulation of the alpha subunit of tryptophan synthase from Salmonella typhimurium and BX1 from maize, two evolutionarily related enzymes.

Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the alpha-subunit (TSA) of the bifunctional tryptophan synthase (TRPS) catalyzes the cleavage of indole 3-glycerol phosphate (IGP) to indole and d-glyceraldehyde 3-phosphate (G3P). Subsequently, indole diffuses through the connecting tunnel to the beta-active site where it is condensed with serine to form tryptophan and water. The maize enzyme, BX1, a homolog of TSA, also cleaves IGP to G3P and indole, and the indole is further converted to 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, a secondary plant metabolite. BX1 cleaves IGP significantly faster to G3P and indole than does TSA. In line with their different biological functions, these two evolutionary related enzymes differ significantly in their regulatory aspects while catalyzing the same chemistry. Here, the mechanism of IGP cleavage by TSA was analyzed using a novel transition state analogue generated in situ by reaction of 2-aminophenol and G3P. The crystal structure of the complex shows an sp3-hybridized atom corresponding to the C3 position of IGP. The catalytic alphaGlu49 rotates to interact with the sp3-hybridized atom and the 3' hydroxyl group suggesting that it serves both as proton donor and acceptor in the alpha-reaction. The second catalytic residue, alphaAsp60 interacts with the atom corresponding to the indolyl nitrogen, and the catalytically important loop alphaL6 is in the closed, high activity conformation. Comparison of the TSA and TSA-transition state analogue structures with the crystal structure of BX1 suggests that the faster catalytic rate of BX1 may be due to a stabilization of the active conformation: loop alphaL6 is closed and the catalytic glutamate is in the active conformation. The latter is caused by a substitution of the residues that stabilize the inactive conformation in TRPS.

Bacterial Proteins↗
PubMed2007

Isolation and characterization of some potential impurities in ropinirole hydrochloride.

Three impurities in ropinirole hydrochloride drug substance at levels approximately 0.06-0.15% were detected by reverse-phase high performance liquid chromatography (HPLC). These impurities were isolated from the drug substance. These impurities were analyzed using reverse-phase HPLC. Based on the spectral data (IR, NMR and MS), structures of these impurities were characterized as 4-[2-(propylamino) ethyl]-1,3-dihydro-2H-indol-2-one hydrochloride (impurity-A), 5-[2-(diropylamino) ethyl]-1,4-dihydro-3H-benzoxazin-3-one hydrochloride (impurity-B) and 4-[2-(diropylamino) ethyl]-1H-indol-2,3-dione hydrochloride (impurity-C). Synthesis of these impurities is discussed.

Antiparkinson Agents↗
PubMed2005

Blockade of 5-HT(3) receptor with MDL7222 and Y25130 reduces hydrogen peroxide-induced neurotoxicity in cultured rat cortical cells.

The present study was performed to examine the neuroprotective effects of 5-hydroxytryptamine (5-HT)(3) receptor antagonists against hydrogen peroxide (H(2)O(2))-induced neurotoxicity using cultured rat cortical neurons. Pretreatment of 5-HT(3) receptor antagonists, tropanyl-3,5-dichlorobenzoate (MDL72222, 0.1 and 1 microM) and N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130, 0.5 and 5 microM), significantly inhibited the H(2)O(2) (100 microM)-induced neuronal cell death as assessed by a MTT assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. The protective effects of MDL72222 (1 microM) and Y25130 (5 microM) were completely blocked by the simultaneous treatment with 100 microM 1-phenylbiguanide, a 5-HT(3) receptor agonist, indicating that the protective effects of these compounds were due to 5-HT(3) receptor blockade. In addition, MDL72222 (1 microM) and Y25130 (5 microM) inhibited the H(2)O(2) (100 microM)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) and glutamate release, generation of reactive oxygen species (ROS), and caspase-3 activity. These results suggest that the activation of the 5-HT(3) receptor may be partially involved in H(2)O(2)-induced neurotoxicity, by membrane depolarization for Ca(2+) influx. Therefore, the blockade of 5-HT(3) receptor with MDL72222 and Y25130 may ameliorate the H(2)O(2)-induced neurotoxicity by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of ROS and caspase-3 activity.

Animals↗
PubMed2003

Benzoxazinoids and iridoid glucosides from four Lamium species.

A new class of compounds for the plant family Lamiaceae, benzoxazinoids, was found in Lamium galeobdolon. From the aerial parts of the species were isolated the new 2-O-beta-D-glucopyranosyl-6-hydroxy-2H-1,4-benzoxazin-3(4H)-one (6-hydroxy blepharin) together with four known benzoxazinoids, DHBOA-Glc, blepharin, DIBOA, DIBOA-Glc, as well as harpagide, 8-O-acetyl-harpagide and salidroside. Eight known iridoid glucosides, 24-epi-pterosterone and verbascoside were isolated from Lamium amplexicaule, L. purpureum and L. garganicum. The iridoids, 5-deoxylamiol and sesamoside, as well as the phytoecdysone, 24-epi-pterosterone, were found for the first time for the genus Lamium. The phytochemical data are discussed from a systematic and evolutionary point of view.

Ecdysone↗
PubMed2000

Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) for the CB(1) cannabinoid receptor.

Two subtypes of cannabinoid receptors are currently recognized, CB(1), found in brain and neuronal cells, and CB(2), found in spleen and immune cells. We have characterized 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) as a novel aryl pyrazole antagonist for the CB(1) receptor. CP-272871 competed for binding of the cannabinoid agonist (3)H-labeled (-)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol ([(3)H]CP-55940) at the CB(1) receptor in rat brain membranes with a K(d) value 20-fold greater than that of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A). CP-272871 also competed for binding with the aminoalkylindole agonist (3)H-labeled (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1, 4-benzoxazin-6-yl](1-naphthyl)methanone ([(3)H]WIN-55212-2), as well as the aryl pyrazole antagonist [(3)H]SR141716A. Inverse agonist as well as antagonist properties were observed for both SR141716A and CP-272871 in signal transduction assays in biological preparations in which the CB(1) receptor is endogenously expressed. SR141716A augmented secretin-stimulated cyclic AMP (cAMP) accumulation in intact N18TG2 neuroblastoma cells, and this response was reversed by the agonist desacetyllevonantradol. CP-272871 antagonized desacetyllevonantradol-mediated inhibition of adenylyl cyclase in N18TG2 membranes, and increased adenylyl cyclase activity in the absence of agonist. SR141716A and CP-272871 antagonized desacetyllevonantradol-stimulated (35)S-labeled guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding to brain membrane G-proteins, and decreased basal [(35)S]GTPgammaS binding to G-proteins. K(+) enhanced CP-272871 and SR141716A inverse agonist activity compared with Na(+) or NMDG(+) in the assay. These results demonstrated that the aryl pyrazoles SR141716A and CP-272871 behave as antagonists and as inverse agonists in G-protein-mediated signal transduction in preparations of endogenously expressed CB(1) receptors.

Animals↗
PubMed2001

Cannabinoids inhibit GABAergic synaptic transmission in mice nucleus accumbens.

In mice nucleus accumbens slices, whole-cell patch clamp recording of medium-spiny neurons revealed that cannabimimetics ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone) (WIN-2) and ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) inhibit stimulus-evoked gamma-aminobutyric acid mediated inhibitory post-synaptic currents (IPSC). The actions of WIN-2 were reversed by the selective cannabinoid CB(1) receptor antagonist [N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride] (SR141716A). WIN-2 modified paired-pulse ratio of evoked IPSCs and decreased miniature IPSC frequency indicating a presynaptic localization of cannabinoid CB(1) receptors.

Analgesics↗
PubMed2002

Contractile response to a cannabimimetic eicosanoid, 2-arachidonoylglycerol, of longitudinal smooth muscle from the guinea-pig distal colon in vitro.

The effect of 2-arachidonoylglycerol, a cannabimimetic eicosanoid, was studied on mucosa-free longitudinal muscle strips isolated from the guinea-pig distal colon. In the presence of indomethacin (3 microM) and N(G)-nitro-L-arginine (100 microM), 2-arachidonoylglycerol (10 nM-10 microM) produced concentration-dependent and tetrodotoxin (1 microM)-sensitive contractions of the longitudinal muscle strips. The contractions were markedly attenuated in the presence of atropine (0.2 microM), and partially by hexamethonium (100 microM) pretreatment. The response to 2-arachidonoylglycerol was mimicked with N-arachidonoylethanolamine (anandamide, 0.1-30 microM), another cannabimimetic eicosanoid, but the cannabinoid CB(1)/CB(2) receptor agonist, R-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2) (0.1-10 microM), and the vanilloid receptor agonist, (all Z)-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM 404) (10-30 microM), were without effect. The cannabinoid CB(1) receptor antagonist, N-piperidino-5-(4-chlorophenyl)-l-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) (1 microM), the cannabinoid CB(2) receptor antagonist, [N-[1S]-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-l-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 microM), and the vanilloid receptor antagonist, capsazepine (10 microM), did not shift the concentration-response curve for 2-arachidonoylglycerol to the right. The contractile action of 2-arachidonoylglycerol was also partially attenuated in the presence of nordihydroguaiaretic acid (10 microM), a lipoxygenase inhibitor. These results indicate that 2-arachidonoylglycerol produces contraction of longitudinal muscle of the guinea-pig distal colon via mainly stimulation of myenteric cholinergic neurones, and that neither cannabinoid CB(1)/CB(2) receptors nor vanilloid receptors contributed to the response. The present results suggest the possibility that lipoxygenase metabolites may also contribute, at least in part, to the contractile action of 2-arachidonoylglycerol.

Animals↗
PubMed1998

Cannabinoids activate mesolimbic dopamine neurons by an action on cannabinoid CB1 receptors.

The present study was designed to determine if cannabinoids share with other drugs of abuse the ability to stimulate mesolimbic dopaminergic neurons and if this effect is mediated by cannabinoid receptors. To this end, the effects of the prototypical cannabinoid, delta9 tetrahydrocannabinol ¿(-)-trans-(6aR,10aR)-6a,7,8,10a-tetrahydro-6,6,9-trimethyl- 3-pentyl-6H-dibenzo[b,d]pyran-1-ol¿, and the two highly potent synthetic cannabinoids, ¿(R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)-methyl]pyrrolo[1,2,3-d e]-1,4-benzoxazin-6-yl, +(1-naphtalenyl)methanone¿ WIN 55,212-2 and ¿(-)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl )-cicloexan-1-ol¿ CP 55,940, on the spontaneous discharge rate of meso-accumbens dopamine (A10 dopamine) neurons were studied in rats. The intravenous administration of delta9-tetrahydrocannabinol, WIN 55,212-2 and CP 55,940 (0.0625-1.0 mg/kg) produced a dose-dependent increase in the spontaneous firing of A10 dopamine neurons both in non-anesthetized and anesthetized rats, with a maximal percent increase of 120, 187 and 155 in non-anesthetized and 33, 102 and 52, respectively, in anesthetized rats. The stimulant response to cannabinoids was suppressed by the specific cannabinoid receptor antagonist ¿N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide¿ SR 141716A, indicating a cannabinoid receptor-mediated effect. These findings support the contention that cannabinoids regulate mesolimbic dopamine transmission and may help to explain the addictive properties of marijuana.

Action Potentials↗
PubMed1999

Comparison of vasopressin binding sites in human uterine and vascular smooth muscle cells.

Several studies indicate that oxytocin and vasopressin receptors in the human uterus are heterogeneous. We have investigated whether oxytocin and vasopressin bind to separate receptors or one class of receptors in human uterine smooth muscle cells. [3H]d(CH2)5Tyr(Me)AVP, the vasopressin V1A receptor selective radioligand, was used for comparison of vasopressin binding sites in human uterine and vascular smooth muscle cell membranes. Both membrane preparations exhibited one class of high-affinity binding sites with Kd values of 6.44 and 0.47 nM, Bmax values of 166 and 34.8 fmol/mg protein for uterine and vascular smooth muscle cells, respectively. In vascular preparations, the selective vasopressin V1A receptor antagonist, SR 49059 ((2S) 1-[(2R 3S)-(5-chloro-3-(2-chlorophenyl)- -(3.4-dimethoxybenzenesulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2- carbonyl]-pyrrolidine-2-carboxamide), showed high affinity with Ki value of 0.98 nM, confirming that these receptors belong to the vasopressin V1A receptor subtype. On the contrary, in uterine preparations, binding of [3H]d(CH2)5Tyr(Me)AVP was more effectively displaced by oxytocin and the oxytocin receptor selective antagonist, L-371257, (1-[1-[4-[ N-Acetyl-4-piperidinyl)oxy]2-methoxybenzoyl]piperidin-4-yl]- 4H-3,1-benzoxazin-2(1H)-one), than vasopressin and SR 49059, suggesting that binding may be due to cross-reaction with the oxytocin receptors. These results suggest that human uterine smooth muscle cells express only a high density of oxytocin receptors.

Binding Sites↗
PubMedDate not supplied

4-quinazolinones: synthesis and reduction of prostaglandin E2 production.

We synthesized and evaluated the anti-inflammatory activity of a series of 4-quinazolinone derivatives. Two approaches were used to yield the title compounds. A first group of quinazolinone derivatives was obtained by the appropriate substituted anthranilates. A second group of quinazolinone compounds was prepared through the benzoxazin-4-ones intermediate. The pharmacological results reveal that the synthesized derivatives exhibit a significant anti-inflammatory effect in an experimental ocular inflammation model. In fact, all the tested compounds lowered the prostaglandin E2 (PGE2) production with respect to the control group (P < 0.05). The 3-cyclohexyl-6-chloro-quinazolin-4(3H)-one and 3-cyclohexyl-quinazolin-4(3H)-one derivatives were the most active compounds. These compounds significantly reduced PGE2 levels even more than the reference drug tolmetin and significantly lower protein concentration and polymorphonuclear leukocytes number compared to the control group (P < 0.05). Therefore, these compounds may be useful to prevent ocular inflammatory reactions.

Animals↗
PubMed1998

Trace determination of methanol in water-ethanol solution by derivatization and high-performance liquid chromatography.

A simple and sensitive high-performance liquid chromatographic method has been established for the determination of methanol in water-ethanol solution. The method is based on the transfer of the methoxide anion, which is formed from methanol under strong alkaline treatment in aqueous solution, by benzalkonium chloride into the dichloromethane organic phase for derivatization with 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one. The derivative obtained was separated on a LiChrospher diol column with n-hexane-dichloromethane (9:1, v/v) as the mobile phase. Several parameters affecting the partition/derivatization of methanol were investigated. The linear range for the determination of methanol was 2-20 mumol/ml; the detection limit (signal-to-noise ratio = 5; sample size, 10 microliters) of methanol was about 0.10 mumol/ml (R.S.D. = 16%, n = 3). The method has been satisfactorily applied to the assay of methanol in spiked commercial liquors.

Chromatography, High Pressure Liquid↗
PubMed1998

Effect of DIMBOA on growth and digestive physiology of Sesamia nonagrioides (Lepidoptera: noctuidae) larvae.

The hydroxamic acid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) was assessed for its effect on growth and digestive physiology of larvae of the stalk corn borer Sesamia nonagrioides Lef. Nutritional indices and activities of some digestive and detoxification enzymes were determined for larvae feeding on a DIMBOA-containing diet for the first two days of the third instar (short-term feeding assays), and from neonates to third instar (long-term feeding assays). DIMBOA reduced the relative growth rate and the efficiency of conversion of ingested food without affecting the relative consumption rate in long-term feeding assays, but it had no effect in short-term assays. Moreover, elastase-like activity was significantly increased by DIMBOA in short-term feeding assays, whereas microsomal oxidase activity was increased and esterase activity was reduced in long-term feeding assays. In vitro, DIMBOA inhibited the activities of carboxypeptidases, aminopeptidase, glutathione S-transferase and esterase, but it had no effect on trypsin, chymotrypsin and elastase. The implications of the altered levels of proteases and detoxification enzyme activities on the digestive physiology of larvae feeding on DIMBOA-containing diets are discussed.

Journal Article↗
PubMed2003

The modulatory effects of the anxiolytic etifoxine on GABA(A) receptors are mediated by the beta subunit.

The anxiolytic compound etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride) potentiates GABA(A) receptor function in cultured neurons (Neuropharmacology 39 (2000) 1523). However, the molecular mechanisms underlying these effects are not known. In this study, we have determined the influence of GABA(A) receptor subunit composition on the effects of etifoxine, using recombinant murine GABA(A) receptors expressed in Xenopus oocytes. Basal chloride currents mediated by homomeric beta receptors were reduced by micromolar concentrations of etifoxine, showing that beta subunits possess a binding site for this modulator. In oocytes expressing alpha(1)beta(x) GABA(A) receptors (x=1, 2 or 3), etifoxine evoked a chloride current in the absence of GABA and enhanced GABA (EC10)-activated currents, in a dose-dependent manner. Potentiating effects were also observed with alpha(2)beta(x), beta(x)gamma(2s) or alpha(1)beta(x)gamma(2s) combinations. The extent of potentiation was clearly beta-subunit-dependent, being more pronounced at receptors containing a beta(2) or a beta(3) subunit than at receptors incorporating a beta(1) subunit. The mutation of Asn 289 in the channel domain of beta(2) to a serine (the homologous residue in beta(1)) did not significantly depress the effects of etifoxine at alpha(1)beta(2) receptors. This specific pattern of inhibition/potentiation was compared with that of other known modulators of GABA(A) receptor function like benzodiazepines, neurosteroids, barbiturates or loreclezole.

Animals↗
PubMed2000

Modulation of GABAergic synaptic transmission by the non-benzodiazepine anxiolytic etifoxine.

We have investigated the effects of 2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride (etifoxine) on GABA(A) receptor function. Etifoxine displaced [(35)S]TBPS (t-butylbicyclophosphorothionate) from GABA(A) receptors of rat cortical membranes with an IC(50) of 6.7+/-0.8 microM and [(3)H]PK11195 from peripheral (mitochondrial)-type benzodiazepine receptors (PBRs) of rat heart homogenates with an IC(50) of 27.3+/-1.0 microM. Etifoxine displayed anxiolytic properties in an anticonflict test in rats, and potentiated GABA(A) receptor-mediated membrane currents elicited by submaximal (5-10 microM) but not saturating (0.5 mM) concentrations of GABA in cultured rat hypothalamic and spinal cord dorsal horn neurones. In hypothalamic cultures, etifoxine induced a dose-dependent inward current for concentrations >1 microM which reflected the post-synaptic potentiation of a small ( approximately 20 pA) tonic and bicuculline-sensitive GABA(A) receptor-gated Cl(-) current. Etifoxine also increased the frequency of spontaneous and miniature GABAergic inhibitory post-synaptic currents without changing their amplitude and kinetic characteristics. Both effects of etifoxine were insensitive to flumazenil (10 microM), an antagonist of central-type benzodiazepine sites present at GABA(A) receptors, but were partly inhibited by PK11195 (10 microM) an antagonist of PBRs which control the synthesis of neurosteroids. Our results indicate that etifoxine potentiates GABA(A) receptor-function by a direct allosteric effect and by an indirect mechanism involving the activation of PBRs.

Animals↗