Binding activity of Valeriana fauriei root extract on GABAA receptor flunitrazepam sites and distribution of its active ingredients in the brain of mice - A comparison with that of V. officinalis root.

ETHNOPHARMACOLOGICAL RELEVANCE: Valeriana fauriei root (VF) is a crude drug registered in the Japanese Pharmacopeia 17th Edition and a known substitute for V. officinalis (VO). Although VO has been pharmacologically evaluated for its sedative effects and mechanism of action, data regarding VF remain scarce. AIM OF THE STUDY: We compared the binding affinity of VF and VO extracts, as well as examined the active ingredients in the VF extract, on flunitrazepam sites of γ-aminobutyric acid receptor type A (GABAA receptor). Furthermore, we confirmed whether these active ingredients were distributed in the brain of mice orally administered the VF extract. MATERIALS AND METHODS: We prepared the assay system to evaluate the binding activity of flunitrazepam sites of GABAA receptor using a 96-well plate and assessed the activities of VF and VO extracts. We then analyzed their constituents using HPLC with principal component analysis (PCA) and evaluated active ingredients correlated with their activities. The distribution of active ingredients in the plasma and brain of mice orally administered the VF extract prepared with different emulsifiers were analyzed by LC-MS/MS. RESULTS: The ethanol extract of VF exhibited significantly higher activity on flunitrazepam sites of GABAA receptor than VO. For the VF extract, kessyl glycol diacetate (KGD) was markedly associated with the binding activities; however, active ingredients included KGD, kessyl glycol 8-acetate (KG8), α-kessyl acetate (α-KA), and coniferyl isovalerate (CI). For VO, valerenic acid and five other compounds were associated with the binding affinity on flunitrazepam sites of GABAA receptor. On emulsifying the VF extract with a fat-soluble glycerin fatty acid ester, the plasma and brain distributions of KGD tended to be higher, those of KG8 were significantly more than 10-times higher, and those of α-KA was lower than those of the VF extract emulsified with water-soluble gum arabic, after oral administration in mice. CONCLUSIONS: Based on the binding activity on flunitrazepam sites of GABAA receptor and brain distribution, KGD, KG8, and α-KA can be considered active ingredients of VF. The addition of a fat-soluble emulsifier promoted the absorption of KGD, the main active ingredient, and KGD was metabolized to KG8 in the body. The present results suggest a possible mechanism underlying the sedative effect for VF, and these three compounds can be used as marker compounds to evaluate the quality of VF products.

Hops compounds modulatory effects and 6-prenylnaringenin dual mode of action on GABAA receptors.

Hops (Humulus lupulus L.), a major component of beer, contain potentially neuroactive compounds that made it useful in traditional medicine as a sleeping aid. The present study aims to investigate the individual components in hops acting as allosteric modulators in GABAA receptors and bring further insight into the mode of action behind the sedative properties of hops. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native GABAA receptors. Flumazenil sensitivity of GABA-potentiating effects, [3H]Ro 15-4513, and [3H]flunitrazepam binding assays were used to examine the binding to the classical benzodiazepines site. Humulone (alpha acid) and 6-prenylnaringenin (prenylflavonoid) were the most potent compounds displaying a modulatory activity at low micromolar concentrations. Humulone and 6-prenylnaringenin potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner where the IC50 values for this potentiation in native GABAA receptors were 3.2 µM and 3.7 µM, respectively. Flumazenil had no significant effects on humulone- or 6-prenylnaringenin-induced displacement of [3H]EBOB binding. [3H]Ro 15-4513 and [3H]flunitrazepam displacements were only minor with humulone but surprisingly prominent with 6-prenylnaringenin despite its flumazenil-insensitive modulatory activity. Thus, we applied molecular docking methods to investigate putative binding sites and poses of 6-prenylnaringenin at the GABAA receptor α1ß2γ2 isoform. Radioligand binding and docking results suggest a dual mode of action by 6-prenylnaringenin on GABAA receptors where it may act as a positive allosteric modulator at α+ß- binding interface as well as a null modulator at the flumazenil-sensitive α+γ2- binding interface.

Anxiolytic-like effect of lavender essential oil inhalation in mice: participation of serotonergic but not GABAA/benzodiazepine neurotransmission.

ETHNOPHARMACOLOGICAL RELEVANCE: Lavandula angustifolia (lavender) inhalation has been used in folk medicine for the treatment of anxiety, and clinical and animal studies have corroborated its anxiolytic effect, although its mechanism of action is still not fully understood. AIMS OF THE STUDY: The objective of the present study was to determine whether the GABAA/benzodiazepine complex or serotonin neurotransmission mediates the anxiolytic-like effect of lavender essential oil. MATERIALS AND METHODS: Male Swiss mice were subjected to the marble-burying test after being exposed to the aroma of lavender essential oil (1-5%), amyl acetate (5%; used as a behaviorally neutral odor), or distilled water for 15 min via inhalation. Additionally, the effect of 5% lavender essential oil was also evaluated in mice subjected to the elevated plus maze. GABAA/benzodiazepine mediation was evaluated by pretreating the mice with the GABAA receptor antagonist picrotoxin before the marble burying test and [(3)H]flunitrazepam binding to the benzodiazepine site on the GABAA receptor. Serotonergic mediation was studied by pretreating the mice with O-methyl-[3H]-N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY100635), a serotonin 5-HT1A receptor antagonist before the marble burying test. We also evaluated changes in the pharmacologically induced serotonin syndrome and the effects of combined administration of subeffective doses of lavender essential oil and the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). RESULTS: Lavender essential oil (1-5%) decreased the number of marbles buried compared with the control and amyl acetate groups. In the elevated plus maze, 5% lavender essential oil inhalation increased the percentage of time spent on and number of entries into the open arms compared with controls. No effect was seen in the number of closed arm entries or number of beam interruptions in the automated activity chamber. Pretreatment with the GABAA receptor antagonist picrotoxin (0.5mg/kg) did not modify the behavioral effect of 5% lavender essential oil in the marble-burying test. Lavender essential oil also did not alter [(3)H]flunitrazepam binding to the benzodiazepine site on the GABAA receptor. Pretreatment with the serotonin 5-HT1A receptor antagonist WAY100635 (3mg/kg) blocked the anxiolytic-like effect of lavender essential oil and the 5-HT1A receptor agonist 8-OH-DPAT (3mg/kg). A combination of ineffective doses of 8-OH-DPAT (0.5mg/kg) and lavender essential oil (0.1%) reduced the number of marbles buried. Finally, 5% lavender essential oil attenuated the serotonin syndrome induced by 40 mg/kg fluoxetine plus 80 mg/kg 5-hydroxytryptophan. CONCLUSIONS: These results indicate an important role for the serotonergic system in the anxiolytic-like effect of lavender essential oil.

Structure-based development of bacterial nitroreductase against nitrobenzodiazepine-induced hypnosis.

Nitrobenzodiazepine (NBDZ) is an addictive drug of the abused substances that causes severe neurological effects and even death. Bacterial type I nitroreductase NfsB (EC 1.5.1.34) has been reported to catalyze NBDZ into inactive metabolite 7-amino-benzodiazepine (7ABDZ) with promising activity, so as to become an attractive candidate for treatment of NBDZ overdose and addiction. Here, we investigate the nitroreduction of an NBDZ, flunitrazepam (FZ), by various mutants of NfsB designed from the solved crystal structure and characterize their in vitro and in vivo potency. Conformational changes occurred in the active site of N71S/F124W in contrast to the wild-type, including the flipping on the aromatic rings of W124 and F70 as well as the extension on the hydrogen bond network between flavin mononucleotide (FMN) and S71, which allow the significant enlargement in the active site pocket. In the complex structure of N71S/F124W and nicotinamide (NIA), stacking sandwich attractions of W124-FMN-NIA were also found, implying the importance of W124 in substrate accessibility. Consequently, N71S/F124W exhibited increased 7AFZ production in vitro with nearly no toxicity and reduced 50% sleeping time (hypnosis) in vivo. Taken together, we demonstrate for the first time that N71S/F124W can serve as an effective antidote for NBDZ-induced hypnosis and provide the molecular basis for designing NfsB and the like in the future.

Central nervous system activities of two diterpenes isolated from Aloysia virgata.

Using the guide of a competitive assay for the benzodiazepine binding site in the γ-aminobutyric acid type A receptor (GABA(A)), two active diterpenes were isolated from the aerial parts of Aloysia virgata (Ruíz & Pavón) A.L. Jussieu var. platyphylla (Briquet) Moldenke. These compounds, identified as (16R)-16,17,18-trihydroxyphyllocladan-3-one (1) and (16R)-16,17-dihydroxyphyllocladan-3-one (2) on the basis of spectral data, competitively inhibited the binding of [(3)H]-FNZ to the benzodiazepine binding site with K(i)±S.E.M. values of 56±19 µM and 111±13 µM, respectively. The behavioral actions of these diterpenes, intraperitoneally (i.p.) administered in mice, were examined in the plus-maze, holeboard, locomotor activity and light/dark tests. Compound 1 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the holeboard test (the number of head dips at 0.3 mg/kg and 3 mg/kg, the rears at 1 mg/kg and the time spent head-dipping at 3 mg/kg), in the plus-maze assay (the percentage of open arm entries at 1 mg/kg) and in the light/dark test (the time in light and the number of transitions at 1 mg/kg). Compound 2 augmented the number of rearings in the holeboard apparatus (at 0.3 mg/kg and 1 mg/kg) and the locomotor activity (at 1 mg/kg). These results reveal the presence of neuroactive compounds in Aloysia virgata.

Betulin binds to gamma-aminobutyric acid receptors and exerts anticonvulsant action in mice.

The lupane type pentacyclic triterpenes: lupeol, betulin, and betulinic acid are widely distributed natural compounds. Recently, pharmaceutical compositions from plant extracts (family Marcgraviaceae) containing betulinic acid, have been patented as anxiolytic remedies. To extend our knowledge of the CNS effects of the triterpenes, we suggest here that the chemically related lupeol, betulin and betulinic acid may interact with the brain neurotransmitter gamma-aminobutyric acid (GABA) receptors in vitro and in vivo. Using radioligand receptor-binding assay, we showed that only betulin bound to the GABA(A)-receptor sites in mice brain in vitro and antagonised the GABA(A)-receptor antagonist bicuculline-induced seizures in mice after intracisternal and intraperitoneal administration. Neither betulinic acid nor lupeol bound to GABA(A) receptor nor did they inhibit bicuculline-induced seizures in vivo. These findings demonstrate for the first time the CNS effects of betulin in vivo, and they also show distinct GABA(A)-receptor-related properties of lupane type triterpenes. These findings may open new avenues in understanding the central effects of betulin, and they also indicate possibilities for novel drug design on the basis of betulin structure.

Antidepressant activity of standardised extract of Marsilea minuta Linn.

AIM OF THE STUDY: Marsilea minuta Linn. (Marsileaceae) has been referred in Indian traditional medicine system (Ayurveda) for the treatment of insomnia and other mental disorders. Marsiline isolated from Marsilea minuta was reported to have sedative and anticonvulsant property. The ethanol extract of Marsilea minuta was standardised for marsiline (1.15%, w/w) and studied for its antidepressant activity. MATERIALS AND METHODS: Antidepressant activity was studied using forced swimming test (FST), tail suspension test (TST), learned helplessness test (LHT) and 5-hydroxytryptophan (5-HTP) induced head twitches response in rodents. Standardised extract of Marsilea minuta in doses of 100, 200 and 400 mg/kg/day were administered orally for three consecutive days and evaluated on day 3, 1h after the last dose treatment. Imipramine (15 mg/kg/day, i.p.) was used as the standard drug. Neurochemical mechanism of antidepressant activity was elucidated by using radioligand receptor binding assays for 5-HT2A and benzodiazepine receptors in rat frontal cortex. RESULTS: Immobility time in FST and TST was significantly (P<0.05) reduced by ethanol extract of Marsilea minuta treated animals. A decrease in number of escape failures in LHT was also observed in Marsilea minuta treated rats. Head twitch response induced by 5-HTP was significantly attenuated by Marsilea minuta (400 mg/kg, p.o.) and imipramine showing the involvement of serotonergic system. This effect was corroborated with radioligand receptor binding study where Marsilea minuta (400 mg/kg, p.o.) significantly (P<0.05) down regulated 5-HT2A receptor in frontal cortex, whereas, no marked effect was observed for benzodiazepine receptor. CONCLUSION: The antidepressant effect exhibited by Marsilea minuta extract may be due to its effect on 5-HT2A density in rat frontal cortex.

Antipsychotic drug administration differentially affects [3H]muscimol and [3H]flunitrazepam GABA(A) receptor binding sites.

Post-mortem studies of the human brain indicate that certain GABA(A) receptor subtypes may be differentially altered in schizophrenia. Increased binding to the total population of GABA(A) receptors using [3H]muscimol is observed in the post-mortem schizophrenic brain, yet a proportion of these receptors which bind benzodiazepines and are labelled with [3H]flunitrazepam, show decreased or unaltered expression. Data from animal studies suggest that antipsychotic drugs alter GABA(A) receptor expression in a subtype selective manner, but in the opposite direction to that observed in schizophrenia. To broaden our understanding of the effects of antipsychotic drugs on GABA(A) receptors, we examined the saturation binding maximum (B(max)) and binding affinity (K(D)) of [3H]muscimol and [3H]flunitrazepam in the prefrontal cortex (PFC), hippocampus and thalamus of male SD rats that received a sucrose solution containing either haloperidol (1.5 mg/kg), olanzapine (6.5 mg/kg) or no drug daily for up to 28 days using quantitative receptor autoradiography. [3H]Muscimol binding density was increased most prominently in the PFC after 7 days, with larger and more prolonged effects being induced by the atypical antipsychotic drug olanzapine in subcortical regions. While no changes were observed in [3H]muscimol binding in any region after 28 days of drug administration, [3H]flunitrazepam binding density (B(max)) was increased for both antipsychotic treatments in the PFC only. These findings confirm that the subset of GABA(A) receptors sensitive to benzodiazepines are regulated differently from other GABA(A) receptor subtypes following antipsychotic drug administration, in a time- and region-dependent manner.

Anxiolytic-like effects of obovatol isolated from Magnolia obovata: involvement of GABA/benzodiazepine receptors complex.

This experiment was performed to investigate whether obovatol isolated from the leaves of Magnolia obovata has anxiolytic-like effects through GABA-benzodiazepine-receptors Cl(-) channel activation. The anxiolytic-like effects of obovatol in mice were examined using the elevated plus-maze and the automatic hole-board apparatus. Oral administration of obovatol (0.2, 0.5 and 1.0 mg/kg) significantly increased the number of open arm entries and the spent time on open arm in the elevated plus-maze test, compared with those of saline. Obovatol (0.2, 0.5 and 1.0 mg/kg) also produced anxiolytic-like effects, as reflected by an increase in head-dipping behaviors. These effects were comparable to those of diazepam (1.0 mg/kg), a well known anxiolytic drug. On the other hand, the anxiolytic-like effects of obovatol and diazepam were reversed by flumazenil, a benzodiazepine receptor antagonist, suggesting that the anxiolytic-like effects of obovatol were involved in GABA-benzodiazepine receptors complex. Obovatol was muscle relaxant by rota-rod test, but its effect was weaker than diazepam. Spontaneous locomotor activity also was inhibited by obovatol. Obovatol selectively increased the GABA(A) receptors alpha(1) subunit expression in amygdala of mouse brain. Obovatol also showed to bind to benzodiazepine receptors competitively in experiments using [(3)H]flunitrazepam in the cerebral cortex of mouse brain. Moreover, obovatol (10, 20 and 50 microM) increased Cl(-) influx and the increased Cl(-) influx was inhibited by flumazenil, in primary cultured neuronal cells and IMR-32 human neuroblastoma cells. These results suggest that obovatol has anxiolytic-like effects, and these pharmacological effects may be mediated by GABA-benzodiazepine receptors-activated Cl(-) channel opening.

The anxiolytic-like effects of Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) in mice.

The aim of the present work is to demonstrate the putative sedative and anxiolytic-like effects of a hydro-ethanolic extract obtained from the aerial parts of Aloysia polystachya (Verbenaceae) in male mice using several behavioural assays. Groups of male mice orally treated with doses of 1.0, 10.0 and 100.0 mg/kg of the extract did not show any significant alteration of their locomotor activity, body temperature or motor coordination. The same treatment increased the duration of the sleeping time induced by 30.0 mg/kg i.p. of sodium pentobarbital. However, the sleeping time induced by ethyl ether was not modified by the oral administration of the extract, not confirming the putative sedative effect of the plant. The ethanolic extract also significantly increased the percentage of both entries (1.0 and 100.0 mg/kg) and the time spent (10.0 and 100.0 mg/kg) into the open arms of the elevated plus maze (EPM). Nevertheless, the binding of (3)H-flunitrazepam ((3)H-FNZ) to the benzodiazepine binding site (BDZ-bs), in washed crude synaptosomal membranes from rat cerebral cortex, was not affected by the semi-purified components from Aloysia polystachya. These results indicate an anxiolytic-like profile of action for the extract of Aloysia polystachya without sedative side effect, being this activity probably mediated by other mechanism than BDZ-bs modulation at the GABA(A) receptors.

Novel plant substances acting as beta subunit isoform-selective positive allosteric modulators of GABAA receptors.

GABAA receptors are modulated by a large variety of compounds. A common chemical characteristic of most of these modulators is that they contain a cyclic entity. Three linear molecules of a polyacetylene structure were isolated from the East African medicinal plant Cussonia zimmermannii Harms and shown to allosterically stimulate GABAA receptors. Stimulation was not abolished by the absence of the gamma2 subunit, the benzodiazepine antagonist Ro15-1788 (8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester), or the point mutation beta2N265S that abolishes effects by loreclezole. At a concentration of 30 microM, the substances by themselves elicited only tiny currents. Maximal stimulation at alpha1beta2gamma2 amounted to 110 to 450% for the three substances, and half-maximal stimulation was observed at concentrations of 1 to 2 muM. Stimulation was subunit composition-dependent and was for the substance MS-1, alpha1beta2gamma2 approximately alpha1beta2 approximately alpha3beta2gamma2 > alpha2beta2gamma2 > alpha5beta2gamma2 approximately alpha1beta3gamma2 approximately alpha6beta2gamma2 > alpha1beta1gamma2, for MS-2 alpha1beta2gamma2 approximately alpha3beta2gamma2 approximately alpha1beta2 > alpha2beta2gamma2 approximately alpha6beta2gamma2 approximately alpha5beta2gamma2 > alpha1beta1gamma2, and for MS-4, alpha1beta2gamma2 approximately alpha1beta2 approximately alpha5beta2gamma2 approximately alpha3beta2gamma2 approximately alpha2beta2gamma2 > alpha6beta2gamma2 >> alpha1beta1gamma2. Maximal stimulation by MS-1 was 450% at alpha1beta2gamma2, 80% at alpha1beta1gamma2, and 150% at alpha1beta3gamma2. MS-1 was thus specific for receptors containing the beta2 subunit. The reversal potential was unaffected by 10 microM MS-1, whereas apparent picrotoxin affinity for current inhibition was increased approximately 3-fold. In summary, these positive allosteric modulators of GABAA receptors of plant origin have a novel unusual chemical structure and act at a site independent of that of benzodiazepines and loreclezole.

Brain neurotransmitter receptor binding and nootropic studies on Indian Hypericum perforatum Linn.

The high affinity binding sites for serotonin and benzodiazepine in the frontal cortex, for dopamine in the striatum and muscarinic cholinergic receptors in the hippocampus were investigated in the brains of Charles Foster rats treated for 3 days. Transfer latency on elevated plus maze (TL), passive and active avoidance behaviour (PA and AA) and electroconvulsive shock (ECS) induced amnesia were also studied. Pilot studies indicated that single dose administration of Indian Hypericum perforatum (IHp) had little or no acute behavioural effects and hence the extract of IHp was administered orally at two dose levels (100 and 200 mg/kg, p.o.) once daily for 3 consecutive days, while piracetam (500 mg/kg, i.p.), a clinically used nootropic agent, was administered acutely to rats as the standard nootropic agent. Control rats were treated with an equal volume of vehicle (0.3% carboxymethyl cellulose). The results indicate that IHp treatment caused a significant decrease in the binding of [3H] spiroperone (DA-D2 receptor) to the striatum and an increase in the binding of [3H] ketanserin (5-HT2A receptor) and [3H] flunitrazepam (BDZ receptor) to the frontal cortex in rats. Preliminary pharmacological studies with IHp extract indicate the presence of two major behavioural actions, namely, antidepressant and anxiolytic. The present findings tend to elucidate the mechanism of earlier observations, the downregulation of the dopamine D2 receptor being consonant with anxiolytic and the upregulation of 5-HT2A and BDZ receptors being consonant with antidepressant activity. Piracetam when given alone, shortened the TL on days 1, 2 and 9 day and also antagonized the amnesic effects of ECS on the TL significantly, whereas IHp antagonized the amnesia produced by ECS. IHp had no significant effect per se on the retention of the PA in rats but produced a significant reversal of ECS induced PA retention deficit. Piracetam showed a significant facilitatory effect per se on PA retention and also reversed the ECS induced impaired PA retention. In the AA test, piracetam facilitated the acquisition and retention of AA in rats but IHp had no effect per se. Both the doses of IHp and piracetam significantly attenuated the ECS induced impaired retention of AA. These results indicate a possible nootropic action of IHp in amnesic animals, which was comparable qualitatively to piracetam.

Modulation of radioligand binding to the GABA(A)-benzodiazepine receptor complex by a new component from Cyperus rotundus.

Four sesquiterpenes, beta-selinene, isocurcumenol, nootkatone and aristolone and one triterpene, oleanolic acid were isolated from the ethylacetate fraction of the rhizomes of Cyperus rotundus and tested for their ability to modulate gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor function by radioligand binding assays using rat cerebrocortical membranes. Among these compounds, only isocurcumenol, one of the newly identified constituents of this plant, was found to inhibit [3H]Ro15-1788 binding and enhance [3H]flunitrazepam binding in the presence of GABA. These results suggest that isocurcumenol may serve as a benzodiazepine receptor agonist and allosterically modulate GABAergic neurotransmission via enhancement of endogenous receptor ligand binding.

Honokiol and magnolol selectively interact with GABAA receptor subtypes in vitro.

Honokiol and magnolol have been identified as modulators of the GABAA receptors in vitro. Our previous study suggested a possible selectivity of honokiol and magnolol on GABAA receptor subtypes. This possibility was examined in the current study by 3H-muscimol and 3H-flunitrazepam binding assays on various rat brain membrane preparations and human recombinant GABA(A) receptor subunit combinations expressed by the Sf-9/baculovirus system. Generally, honokiol and magnolol have a similar enhancing effect on (3)H-muscimol binding to various membrane preparations in nonsaturation binding assays. Honokiol and magnolol preferentially increased (3)H-muscimol binding to hippocampus compared to cortex and cerebellum (with a maximum enhancement of 400% of control). As for subunit combinations, honokiol and magnolol have a more potent enhancing effect on alpha2 subunit containing combinations (with a maximum enhancement of 400-450% of control). This action was independent of the gamma subunit. In saturation binding assays, magnolol affected either the number of binding sites (ca. 4-fold on alpha2 containing combinations) or the binding affinity (on alpha1 containing combinations) of (3)H-muscimol binding to various GABAA receptor subunit combinations. In contrast, honokiol increased only binding sites on alpha2beta3gamma2s and alpha2beta3 combinations, but both the number of binding sites and the binding affinity on alpha1beta2gamma2S and alpha(1)beta2 combinations. These results indicate that honokiol and magnolol have some selectivity on different GABAA receptor subtypes. The property of interacting with GABAA receptors and their selectivity could be responsible for the reported in vivo effects of these two compounds.

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.

Synthesis and preliminary pharmacological evaluation of coumestans with different patterns of oxygenation.

Five coumestans with different patterns of oxygenation in rings A and D were synthesized from resorcinol and aromatic aldehydes, and screened for their antimyotoxic activity. The most potent compound (2b, IC50 = 1 microM) was selected for study of its pharmacological profile.

Lack of interaction of buprenorphine with flunitrazepam metabolism.

OBJECTIVE: The authors' goal was to determine if the reported clinical adverse interaction of flunitrazepam and buprenorphine was caused by inhibition of drug metabolism. METHOD: Inhibition of flunitrazepam metabolism by buprenorphine and norbuprenorphine were determined in three human liver microsome preparations carrying the CYP2C19*1/*1 allele. Omeprazole metabolism mediated by CYP2C19 and CYP3A4 was used as a control reaction. Apparent K(i) values were determined. RESULTS: Norbuprenorphine did not inhibit the metabolism of flunitrazepam or omeprazole. Buprenorphine inhibited the formation of CYP3A4-mediated pathways of 3-hydroxyflunitrazepam and omeprazole sulfone formation (K(i) 118 and 16 microM) in human liver microsomes. Corresponding values were 38 and 90 microM in cDNA-expressed CYP3A4 microsomes. Projected in vivo inhibition of CYP3A4-mediated metabolism of flunitrazepam by buprenorphine is 0. 1%-2.5%. Estimated inhibition of buprenorphine N-dealkylation by flunitrazepam in vivo is 0.08%. CONCLUSIONS: The clinical interaction of flunitrazepam and buprenorphine is likely based on a pharmacodynamic mechanism.

Effects of Valeriana officinalis extracts on [3H]flunitrazepam binding, synaptosomal [3H]GABA uptake, and hippocampal [3H]GABA release.

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with gamma-aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [3H]flunitrazepam binding (EC50 4.13 x 10(-10) mg/ml). However, this increased [3H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC50 of 4.82 x 10(-1) mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [3H]flunitrazepam binding sites. Valerian extracts also potentiate K+ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [3H]GABA. Finally, inhibition of synaptosomal [3H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA(A) receptors, but can also interact at other presynaptic components of GABAergic neurons.

Influence of plasma dialysate from normal and renal dysfunction rats on the electroencephalogram and gamma-aminobutyric acid A receptor complex modulation of thiopental.

We have previously reported that brain sensitivity to thiopental with respect to electroencephalogram (EEG) is enhanced in uranyl acetate pretreated renal dysfunction rats. The results were attributed to pharmacodynamic factors. In this study, in vivo EEG and in vitro binding studies for gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex were performed to investigate the mechanism of the enhanced effect of thiopental. The receptor binding properties in the brain membrane from normal and renal dysfunction rats were examined using [3H]tertbutylbicycloorthobenzoate (TBOB), [3H]flunitrazepam and [3H]muscimol. The effect of plasma dialysate from normal (ND) and renal dysfunction rats (RDD) on the thiopental induced EEG and receptor binding were also examined to confirm the role of endogenous compounds. The intrinsic receptor binding characteristics of various sites and their allosteric interaction with thiopental was similar in membrane preparations from normal and renal dysfunction rats. However, RDD, when compared to ND, enhanced the EEG induced by thiopental. At the receptor level, RDD significantly enhanced the thiopental induced inhibition of TBOB. No difference was found between the influence of ND and RDD on the interaction between thiopental and flunitrazepam or muscimol binding. These results showed that the thiopental induced allosteric inhibition of TBOB binding was potentiated by some endogenous compounds in RDD and suggests that this action might be the mechanism, at least in part, for the increased sensitivity of thiopental in renal dysfunction rats.

Benzodiazepine binding sites in the human hypothalamus. Autoradiographic study.

Using in vitro labelling and autoradiographic techniques, we have analyzed the fine and the detailed distribution of benzodiazepine binding sites in the post-mortem human hypothalamus. Binding sites were labelled in mounted tissue sections from adult brains, using the selective high affinity ligand [3H]-Flunitrazepam. A heterogeneous distribution of benzodiazepine binding sites was found throughout the rostrocaudal extent of human hypothalamus. The autoradiographic labelling was shown in the three hypothalamic parts, i.e., anterior, mediobasal and posterior levels. At the anterior level, the highest densities were present in the diagonal band of Broca, the preoptic area (medial and lateral parts) and the septohypothalamic nucleus. At the mediobasal hypothalamic level, the highest densities were mainly localized in the ventromedial nucleus, whereas the other structures were moderately labelled with [3H]-Flunitrazepam. The mammillary complex as well as the posterior hypothalamic area represented the most heavily labelled structures in the posterior hypothalamus. The results obtained in this study, indicate the presence of a large and heterogeneous distribution of benzodiazepine binding sites in human adult hypothalamus. This could support their implication in the control of distinct neural functions (like neuroendocrine role).