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PubMed2001

In ovo chronic neurosteroid treatment affects the function and allosteric interactions of GABA(A) receptor modulatory sites.

We investigated the effects of in ovo chronic administration of the endogenous neurosteroid epipregnanolone (5beta-pregnan-3beta-ol-20-one) on the GABA(A) receptor complex present in chick optic lobe synaptic membranes. Chronic epipregnanolone treatment failed to exert any effect on the chick optic lobe total protein content and wet weight at the different doses tested. [3H]Flunitrazepam control binding remained unaltered after neurosteroid exposure, however, the positive allosteric modulation of this ligand by 4 microM allopregnanolone was reduced in a dose-dependent manner by neurosteroid treatment. Embryo exposure to 30 microM epipregnanolone decreased allopregnanolone EC(50) and E(max) values. Analyses of saturation binding isotherms disclosed that such administration had no effect on K(d) and B(max) values for [3H]flunitrazepam and [3H]GABA binding. [3H]GABA binding modulation disclosed an increase in allopregnanolone EC(50) value with a decrease in its E(max) value. Diazepam EC(50) and E(max) values were enhanced, while low affinity sodium pentobarbital EC(50) value was reduced by epipregnanolone treatment. The investigation of the GABA(A) receptor function revealed that administration of this neurosteroid reduces the efficacy of GABA to induce 36Cl(-) influx into microsacs prepared from chick optic lobe. These results indicate that endogenous neurosteroid epipregnanolone chronically administered in ovo produces homologous uncoupling between steroid modulatory sites, and those corresponding to benzodiazepine and GABA receptors. Thus epipregnanolone is able to induce heterologous changes in the allosteric linkage between benzodiazepine and barbiturate modulatory sites, and the GABA receptor site. Taken jointly with results on epipregnanolone enhancing effects on [3H]flunitrazepam and [3H]GABA binding, in the context of its endogenous synthesis, our present findings support this neurosteroid as the endogenous modulator of GABA(A) receptor sites and function during chick optic lobe development.

Allosteric Regulation↗
PubMed1998

Chronic ethanol administration alters the modulatory effect of 5alpha-pregnan-3alpha-ol-20-one on the binding characteristics of various radioligands of GABAA receptors.

In this study, we investigated the modulatory effect of 5alpha-pregnan-3alpha-ol-20-one, a neurosteroid, on the binding characteristics of [3H]flunitrazepam (2 nM), [3H]muscimol (5 nM), and 4 nM [35S]t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5alpha-Pregnan-3alpha-ol-20-one potentiated the binding of [3H]flunitrazepam and [3H]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [3H]flunitrazepam as well as [3H]muscimol binding (Emax) in the ethanol-dependent rat cerebellum as compared to control group (p<0. 025). Furthermore, 5alpha-pregnan-3alpha-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [35S]TBPS at lower concentrations (<=100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [35S]TBPS binding (-Emax) by 5alpha-pregnan-3alpha-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats (p<0.025). These observations suggest that the neurosteroid binding site associated with the gamma-aminobutyric acidA (GABAA) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.

Animals↗
PubMed1999

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices.

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [3H]muscimol, [3H]flunitrazepam and [35S]TBPS in rat brain slices. The density of [3H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [3H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [3H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [3H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu [35S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg2+. Elevated [35S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg2+. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.

Animals↗
PubMed2001

Evaluation of native GABA(A) receptors containing an alpha 5 subunit.

The type A receptor for gamma-aminobutyric acid (GABA), or GABA(A) receptor, is a pentamer of highly variable quaternary structure. It includes two alpha subunits, drawn from a pool of six genes, which largely determine benzodiazepine pharmacology of the receptor. In brain sections, both [(3)H]RY-80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate) and [(3)H]L-655,708 (ethyl (S)-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate), which are selective for the benzodiazepine site of alpha 5 subunit-containing receptors, showed high-affinity, specific binding, but to fewer regions than did the nonselective benzodiazepine, [(3)H]flunitrazepam. The pattern mirrored alpha 5 mRNA distribution, and was similar to that previously reported for [(3)H]L-655,708 binding. Displacement of [(3)H]RY-80 bound to hippocampal homogenates, and of [(3)H]flunitrazepam bound to cerebellar and hippocampal homogenates showed comparable displacement by flumazenil (K(i)'s 5--7 nM). However, the K(i)'s for diazepam and for clobazam to displace [(3)H]RY-80 binding in hippocampus were about fourfold higher than for [(3)H]flunitrazepam, and the K(i) for clonazepam was sixfold larger, suggesting that these benzodiazepine receptor agonists bind with relatively lower affinity at hippocampal alpha 5-containing receptors.

Alkynes↗
PubMed1997

Size-exclusion chromatographic reconstitution of the bovine brain benzodiazepine receptor. Effects of lipid environment on the binding characteristics.

The benzodiazepine receptor from calf brain was solubilized with sodium deoxycholate (2 mg/ml) in the presence of 0.5 M KCl and protease inhibitors, and bound flunitrazepam with an equilibrium dissociation constant (Kd) of 2.7 +/- 1.2 nM and with 0.40 +/- 0.04 pmol binding sites per mg protein (Bmax). Up to 60% of the benzodiazepine binding sites (average 25%) could be reconstituted in lipid vesicles, upon size-exclusion chromatography of protein-detergent-lipid mixtures on Sephadex G-50 Medium for detergent depletion. The flunitrazepam affinity for the reconstituted receptor varied with the lipid composition (Kd 1.4-4 nM). Freezing and thawing increased the size of the small proteoliposomes obtained by chromatographic reconstitution and, on the average, doubled the number of operative flunitrazepam binding sites. When the proteoliposomes were stored at -20 degrees C or -80 degrees C or in lyophilized state, the receptor retained its benzodiazepine binding affinity and Bmax over a period of 2 months.

Animals↗
PubMed1997

Modulation of long-term potentiation in CA1 region of mouse hippocampal brain slices by GABAA receptor benzodiazepine site ligands.

Enhancement of GABAA receptor function with benzodiazepine (BZ) site agonists can disrupt memory formation and hippocampal synaptic plasticity. To investigate this further the effects of the agonist, flunitrazepam, were contrasted with that of the inverse agonist, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), on NMDA-dependent LTP induction in the CA1 region of mouse hippocampus. Under control conditions, a priming stimulus (10 stimuli at 100 Hz) potentiated e.p.s.p. slopes by 198%, and subsequent burst stimuli (4 x 10 events at 100 Hz every 20 sec) by 306%. This potentiation was blocked by the non-competitive NMDA receptor antagonist MK-801 and the glycine site antagonist L-701,324. Flunitrazepam (1 microM) alone caused a slight but significant reduction in e.p.s.p.s to 83% of control, suppressed LTP induced by priming stimuli (133%) and burst stimuli (188%), but not that induced by sustained high-frequency stimulation (2 x 100 events at 100 Hz, 20 sec apart). The suppression of LTP induction by flunitrazepam was blocked by the benzodiazepine site antagonist flumazenil. In contrast, the inverse agonist DMCM (100 nM) potentiated LTP formed by both priming (to 283%) and burst stimuli (to 477%). This was associated with an enhancement of paired pulse facilitation during the induction phase and the subsequent appearance of paroxysmal burst discharges. Therefore, in addition to improvements in learning and memory as a result of improved vigilance, benzodiazepine inverse agonists can have direct effects on synaptic processes thought to contribute to memory formation.

Animals↗
PubMed2002

GABA(A) receptor sites in the developing human foetus.

GABA(A) receptor sites were characterised in cerebral cortex tissue samples from deceased neurologically normal infants who had come to autopsy during the third trimester of pregnancy. Pharmacological parameters were obtained from homogenate binding studies which utilised the 'central-type' benzodiazepine ligands [3H]diazepam and [3H]flunitrazepam, and from the GABA activation of [3H]diazepam binding. It was found that the two radioligands behaved differently during development. The affinity of [3H]flunitrazepam for its binding site did not vary significantly between preparations, whereas the [3H]diazepam K(D) showed marked regional and developmental variations: infant tissues showed a distinctly lower affinity than adults for this ligand. The density of [3H]flunitrazepam binding sites increased approximately 35% during the third trimester to reach adult levels by term, whereas [3H]diazepam binding capacity declined slightly but steadily throughout development. The GABA activation of [3H]diazepam binding was less efficient early in the trimester, in that the affinity of the agonist was significantly lower, though it rose to adult levels by term. The strength of the enhancement response increased to adult levels over the same time-frame. The results strongly suggest that the subunit composition of cortical GABA(A) sites changes significantly during this important developmental stage.

Adult↗
PubMed1997

Developmental expression of chick cortical GABA(A) receptor alpha1 subunits in vivo and in vitro.

In order to examine the expression of the GABA(A) receptor alpha1 subunit during chick cortical development in vivo and in vitro, we have utilized a polyclonal antibody (RP4) directed against an alpha1(331-381) fusion protein. This antibody exhibits a high titer for precipitation of [3H]flunitrazepam binding sites in chick cortical extracts, no significant cross-reactivity with GABA(A) receptor beta2- or beta4-subunit fusion proteins, and a robust reaction with a single 51-kDa polypeptide on immunoblots of cortical membranes. This indicates monospecificity of the RP4 antiserum for the GABA(A) receptor alpha1 subunit. The alpha1-subunit antibody also showed strong immunocytochemical reactions with neurons in the embryonic mediodorsal cortex and Purkinje cells of the chick cerebellum. The ontogeny of the alpha1 subunit in chick cortex and in derived neuronal cultures was examined by quantitative Western blotting. The level of the alpha1 polypeptide increased from day 2 to day 6 in culture, acquiring 50% of the maximum expression at day 4. Expression of the cortical GABA(A) receptor alpha1 subunit increased in vivo from embryonic day 8 (E8) to day 7 post-hatching, reaching 50% of adult levels at E16. Levels of the corresponding alpha1-subunit mRNA, analyzed from E8 to E20 by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), showed a corresponding incline. These findings correlated well with previous developmental studies of GABA(A) receptor ligand binding sites both in vivo and in vitro. The parallel increase of the alpha1 subunit transcript and polypeptide with [3H]flunitrazepam binding sites suggests that this subunit may be an important component of GABA(A) receptors early in cortical ontogeny. This was investigated further by quantitative immunoprecipitation. At saturation, the RP4 antiserum consistently precipitated 50-65% of the central [3H]flunitrazepam binding sites in the developing cortex from E12 through P7, despite a 5-fold increase in the binding level. The data suggest that during cortical development the fraction of GABA(A) receptors containing alpha1 subunits remains relatively constant. Furthermore, the alpha1 polypeptide appears to be a major component of GABA(A) receptor oligomers at all stages of cortical maturation.

Animals↗
PubMed1999

Chronic D1 and D2 dopaminomimetic treatment of MPTP-denervated monkeys: effects on basal ganglia GABA(A)/benzodiazepine receptor complex and GABA content.

The effect of various chronic dopaminergic treatments in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys on the brain gamma-aminobutyric acid type A (GABA(A)) /benzodiazepine receptor complex and GABA content was investigated in order to assess the GABAergic involvement in dopaminomimetic-induced dyskinesia. Three MPTP monkeys received for one month pulsatile administrations of the D1 dopamine (DA) receptor agonist SKF 82958 whereas three others received the same dose of SKF 82958 by continuous infusion. A long acting D2 DA receptor agonist, cabergoline, was given to another three animals. Untreated MPTP as well as naive control animals were also included. Pulsatile SKF 82958 relieved parkinsonian symptoms but was also associated with dyskinesia in two of the three animals whereas animals treated continuously with SKF 82958 remained as untreated MPTP monkeys. Chronic cabergoline administration improved motor response with no persistent dyskinesia. MPTP treatment induced a decrease of 3H-flunitrazepam binding in the medial anterior part of caudate-putamen and an increase in the internal segment of globus pallidus (GPi) which was in general unchanged by pulsatile or continuous SKF 82958 administration. Throughout the striatum, binding of 3H-flunitrazepam remained reduced in MPTP monkeys treated with cabergoline but was not significantly lower than untreated MPTP monkeys. Moreover, cabergoline treatment reversed the MPTP-induced increase in 3H-flunitrazepam binding in the GPi. GABA concentrations remained unchanged in the striatum, external segment of globus pallidus and GPi following MPTP denervation. Pulsatile but not continuous SKF 82958 administration decreased putamen GABA content whereas cabergoline treatment decreased caudate GABA. No alteration in GABA levels were observed in the GPe and GPi following the experimental treatments. These results suggest that: (1) D2-like receptor stimulation with cabergoline modulates GABA(A) receptor density in striatal subregions anatomically related to associative cortical afferent and (2) the absence of dyskinesia in dopaminomimetic-treated monkeys might be associated with the reversal of the MPTP-induced upregulation of the GABA(A)/benzodiazepine receptor complex in the Gpi.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1997

Detection of benzodiazepines and other psychotropic drugs in human hair by GC/MS.

For the detection of psychotropic drugs in human hair we collected hair obtained from 21 corpses that died from an overdose of legal or illicit drugs. These persons were known to have taken psychotropic drugs prior to their death as determined by post-mortem toxicological analysis in blood. After washing, cutting hair into segments of 3 cm, pulverization, enzymatic hydrolysis with beta-glucuronidase/arylsulfatase and solid phase extraction, drugs were identified by GC/MS. For quantification of flunitrazepam we used its metabolite amino-flunitrazepam; for oxazepam and lorazepam we used the hydrolysed forms of the corresponding drugs. In the hair of 21 subjects tested we found in 20 cases nordazepam, in 15 cases diazepam, in 15 cases oxazepam and in eight cases flunitrazepam with maximal concentrations of 1.8, 2.2, 3.4 and 9.5 ng/mg hair respectively. In addition to these compounds, in subject 11 to 21 we also analyzed for and detected amitriptyline (seven positive), carbamazepine (eight positive), lormetazepam (three positive) and lorazepam (one positive) and found maximal concentrations of 106.0, 13.5, 29.0 and 4.9 ng/mg hair respectively. The comparison of hair analysis versus post-mortem blood and tissues analysis of all the drugs studied shows that in 40 cases, where a positive result was found in blood, the corresponding drug could also be detected in hair in 37 cases. Our results show that hair testing is complementary to classical post-mortem analysis in forensic toxicology.

Adult↗
PubMed2003

High-dose midazolam and the attenuation of the contractile response to vasoconstrictors in coronary artery segments.

BACKGROUND AND OBJECTIVE: Benzodiazepines may cause hypotension and are reported to interfere with smooth vascular muscle activity. The aim was to elucidate the influence of three different benzodiazepines on the vascular reactivity of coronary arteries. METHODS: Using the model of isolated vessels, we studied the impact of midazolam (0.15, 1.5, 15 microg mL(-1)), diazepam (0.1, 1.0, 10 microg mL(-1)) and flunitrazepam (0.01, 0.1, 1.0 microg mL(-1)) on the contractile responses to histamine (2 x 10(-5) mol L(-1)) and serotonin (3 x 10(-5) mol L(-1)) in isolated intact and denuded coronary arteries. RESULTS: Midazolam significantly attenuated the contractile response when administered in high concentrations (15 microg mL(-1)). This effect was more pronounced in intact than in denuded preparations (histamine: -22.7 versus -7.3%, P = 0.0079; serotonin: -47.1 versus -15.9%, P < 0.0001). Diazepam and flunitrazepam exerted no significant effects on the vascular tone of coronary arteries. CONCLUSIONS: Midazolam, but not diazepam or flunitrazepam, attenuates the contractile responses to vasoconstrictors in concentrations beyond those used in clinical practice. This effect is in part mediated by endothelial factors.

Animals↗
PubMed1989

Functional reconstitution of the bovine brain GABAA receptor from solubilized components.

The GABAA/benzodiazepine receptor has been solubilized from membrane preparations of bovine cerebral cortex and has been reconstituted, in a functionally active form, into phospholipid vesicles. In preliminary experiments, the receptor was labeled with the photoactive benzodiazepine [3H]flunitrazepam prior to solubilization. A peptide of apparent molecular weight 53,500 was specifically labeled by this method, and this was used as a marker for the receptor during the reconstitution procedures. The labeled protein was solubilized with approximately 40% efficiency by 1% beta-octyl glucoside. Reconstitution was achieved by mixing the solubilized proteins with a 4:1 mixture of soybean asolectin and bovine brain phospholipids, followed by chromatography on Sephadex G-50-80 to remove detergent. The incorporation of the GABAA receptor into membrane vesicles has been verified by sucrose gradient centrifugation in which the [3H]-flunitrazepam-labeled peptide comigrated with [14C]phosphatidylcholine used as a lipid marker. Vesicles prepared without labeled markers retained the ability to bind both [3H]flunitrazepam and the GABA analogue [3H]muscimol. Furthermore, the binding parameters were very similar to those measured using native membrane preparations. A novel fluorescence technique has been used to measure chloride transport mediated by the GABAA receptor in reconstituted vesicles. Chloride influx was rapidly stimulated in the presence of micromolar concentrations of muscimol and was blocked by preincubation of the membranes with muscimol (desensitization). Flux was also blocked by pretreatment with the competitive GABAA receptor blocker bicuculline or with the noncompetitive GABAA receptor antagonist picrotoxin.

Animals↗
PubMed1996

Synthesis and pharmacological properties of novel 8-substituted imidazobenzodiazepines: high-affinity, selective probes for alpha 5-containing GABAA receptors.

The synthesis and pharmacological properties of imidazobenzodiazepines with both high affinity and selectivity for alpha 5-containing GABAA receptors are described. Four of these compounds (5, 6, 8, and 9) inhibited [3H]flunitrazepam binding to recombinant alpha 5 beta 2 gamma 2 GABAA receptors with IC50 values between approximately 0.4 and 5 nM. These compounds were > or = 24-75-fold more selective for recombinant receptors containing alpha 5 subunits compared to other, "diazepam-sensitive" GABAA receptors containing either alpha 1, alpha 2, or alpha 3 subunits. Imidazobenzodiazepine 9 (used as the prototypical alpha 5 selective ligand) inhibited [3H]flunitrazepam binding to hippocampal membranes with high- and low-affinity components (IC50 0.6 +/- 0.2 and 85.6 +/- 13.1 nM, respectively), representing approximately 16% and approximately 84% of the receptor pool. Inhibition of [3H]flunitrazepam binding to cerebellar membranes with imidazobenzodiazepine 9 was best fitted to a single population of sites with an IC50 of 79.8 +/- 18.3 nM. These imidazobenzodiazepines behaved as GABA negative ligands in recombinant GABAA receptors expressed in Xenopus oocytes and were convulsant in mice after parenteral administration. The relative potencies of flumazenil and zolpidem in blocking convulsions induced by 9 and DMCM, respectively, indicated that occupation of alpha 5-containing GABAA receptors substantially contributed to the convulsant properties of acetylene analog 9. These 8-substituted imidazobenzodiazepines (5, 6, 8 and 9) should prove useful in examining the physiological roles of GABAA receptors bearing an alpha 5 subunit and may also lead to the development of novel, subtype selective therapeutic agents.

Animals↗
PubMed1998

Structural requirements for ligand interactions at the benzodiazepine recognition site of the GABA(A) receptor.

His101 of the GABA(A) receptor alpha1 subunit is an important determinant of benzodiazepine recognition and a major site of photolabeling by [3H]flunitrazepam. To investigate further the chemical specificity of the residue in this position, we substituted it with phenylalanine, tyrosine, lysine, glutamate, glutamine, or cysteine. The mutant alpha subunits were coexpressed with the rat beta2 and gamma2 subunits in TSA201 cells, and the effects of the substitutions on the binding of benzodiazepine site ligands were examined. [3H]Ro 15-4513 bound to all mutant receptors with equal or greater affinity than to the wild-type receptor. However, flunitrazepam and ZK93423 recognition was adversely affected by substitutions of the amino acid in this position. The binding of the antagonists, Ro 15-1788 and ZK93426, was also sensitive to the mutations, with the largest decreases in affinity occurring with the tyrosine, lysine, and glutamate substitutions. In all mutants that recognized flunitrazepam, GABA potentiated the binding of this ligand to a similar extent, suggesting that it is a full agonist at these receptors. The effects of GABA on the binding of Ro 15-1788 and Ro 15-4513 suggest that their efficacies may have been changed by some of the substitutions. This study further emphasizes the importance of the residue at position 101 in both ligand recognition and pharmacological effect.

Allosteric Site↗
PubMed2003

Chronic ethanol consumption enhances internalization of alpha1 subunit-containing GABAA receptors in cerebral cortex.

The molecular mechanisms that underlie ethanol dependence involve alterations in the functional properties and subunit expression of GABAA receptors. Chronic ethanol exposure decreases GABAA receptor alpha1 subunits and increases alpha4 subunit levels in cerebral cortical membranes. This study explored the effect of chronic ethanol exposure on internalization of GABAA/benzodiazepine receptors. Chronic ethanol exposure increased alpha1 subunit levels by 46 +/- 12% and [3H]flunitrazepam binding by 35 +/- 9% in the clathrin-coated vesicle (CCV) fraction. There was a corresponding 34 +/- 8% decrease in alpha1 peptide expression and 37 +/- 6% decrease in [3H]flunitrazepam binding in the synaptic fraction. Chronic ethanol consumption also increased the alpha1 subunit immunoprecipitate in the cytosolic fraction (77 +/- 22%), measured by western blot analysis. Moreover, co-immunoprecipitation of both clathrin and adaptin-alpha with alpha1 subunits was increased in the cytosolic fraction, suggesting that alpha1 subunit endocytosis is enhanced by chronic ethanol consumption. In contrast, alpha4 subunit peptide levels were not altered in the CCV fraction despite a 39 +/- 13% increase in peptide levels in the synaptic fraction of cortex. Moreover, acute ethanol exposure did not alter alpha1 subunit peptide expression or [3H]flunitrazepam binding in the synaptic or CCV fractions. These results suggest that chronic ethanol consumption selectively increases internalization of alpha1 subunit-containing GABAA receptors in cerebral cortex.

Administration, Oral↗
PubMed2001

Radioligand binding assays in the drug discovery process: potential pitfalls of high throughput screenings.

Radioligand binding assays evaluating directly the ability of a drug to interact with a defined molecular target is part of the drug discovery process. The need for a high throughput rate in screening drugs is actually leading to simplified experimental schemes that increase the probability of false negative results. Special concern involves voltage-gated ion channel drug discovery where a great care is required in designing assays because of frequent multiplicity of (interacting) binding sites. To clearly illustrate this situation, three different assays used in the academic drug discovery program of the authors were selected because they are rich of intrinsic artifacts: (I) (20 mmol/l caffeine almost duplicated [3H]ryanodine binding (89% higher than control) to rat heart microsomes at 0.3 mumol/l free calcium but did not exert any effect when using a high (107 mumol/l) free calcium, as mostly used in ryanodine binding assays; (II) An agonist for the ionotropic glutamate receptor of the kainate type can distinctly affect [3H]kainate binding to chicken cerebellum membranes depending on its concentration: unlabelled kainic acid per se either stimulated about 30% (at 50-100 nmol/l), had no effect (at 200 nmol/l) or even progressively decreased (at 0.3-2 mumol/l) the binding of 5 nmol/l [3H]kainate, emphasizing the risk of using a single concentration for screening a drug; (III) in a classical [3H]flunitrazepam binding assay, the stimulatory effect of a GABA (gamma-aminobutyric acid) agonist was only observed when using extensively washed rat brain synaptosomes (10 mumol/l GABA increased flunitrazepam binding by 90%). On the other hand, the inhibitory effect of a GABA antagonist was only observed when using crude synaptosomes (10 mumol/l bicuculine reduced [3H]flunitrazepam binding by 40%). It can be concluded that carefully designed radioligand assays which can be performed in an academic laboratory are appropriate for screening a small number of drugs, especially if these are potential hits because of their rational design. Therefore, the low throughput rate could be partially balanced by a higher performance when compared to what is done in a robotic high throughput screening where simplification of assay conditions can lead to false negative results.

Animals↗
PubMedDate not supplied

Solid-phase microextraction and GC-ECD of benzophenones for detection of benzodiazepines in urine.

Benzodiazepines are common drugs that cause intoxication. Benzodiazepines and their metabolites can be converted by hydrolysis in acid to the corresponding benzophenones, which are easier to be separated from matrices because of their hydrophobic properties. In this study, a new separation technique called solid-phase microextraction (SPME), which can integrate extraction, concentration, sampling and sample introduction into one single procedure, has been employed to extract the products of benzodiazepines from urine after acid hydrolysis. The extracts were determined by gas chromatography with electron-capture detection (GC-ECD). The hydrolysis conditions were optimized by a statistic orthogonal design. Factors influencing direct-immersion (DI)-SPME process were also checked and chosen experimentally. The method was evaluated with spiked human urine samples. The recoveries of nine benzodiazepines ranged from 1 to 25%, with the highest for oxazolam and the lowest for bromazepam. The calibration curves were linear from 10 to 500 ng/mL for oxazolam, haloxazolam, flunitrazepam, nimetazepam, and clonazepam and from 20 to 1000 ng/mL for the others except bromazepam. The detection limits were 2-20 ng/mL for most drugs tested. The intraday and interday coefficients of variation of the developed method were within 10 and 17%, respectively. In addition, the utility of the method was confirmed by determining two ingested benzodiazepines (flunitrazepam and oxazolam) in a volunteer's urine; urine flunitrazepam was still detectable 32 h after a therapeutic dose (1.2 mg) of the drug. Finally, the DI-SPME was compared with the conventional liquid-liquid extraction with regard to detection limits and extraction efficiency of the analytes. By DI-SPME, more amounts of analytes could be introduced into GC column than by conventional liquid-liquid extraction, and thus lower detection limits of the analytes were reached, although benzophenone recoveries by DI-SPME were rather low.

Adult↗