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Zolpidem, a nonbenzodiazepine hypnotic, binds to the benzodiazepine binding site on the gammaaminobutyric acid type A (GABA-A) receptors. Many studies have reported efficacy and safety of zolpidem in treatment of insomnia, low abuse, and dependence capability. We present a case of zolpidem dependence in a 33-year-old male to emphasize that clinicians should pay close attention to the potential of zolpidem tolerance, abuse and dependence. Key Word : Zolpidem, Dependence

Data on the basal (constitutive or spontaneous) activities of receptors, as well as their neurochemical and electrophysiological correlates, are presented. Inverse receptor agonists are a group of pharmacological drugs that can suppress constitutive activities. We describe the neurochemical mechanisms of the pharmacological activities of benzodiazepine binding site inverse agonists. These compounds inhibit chloride currents caused by gamma-aminobutyric acid at low non-physiological concentrations. This activity is the basis of the sobering action during the action of ethanol and the ability to suppress the addictive potential of psychoactive substances. In addition, the benzodiazepine binding site inverse agonists are able to activate memory formation processes and improve learning. The possible uses of drugs from this group in modern medicine are discussed. They may be used to treat a number of diseases in the fields of narcology, therapy, neurology, and psychiatry.

The synthesis, in vitro ligand binding study and in vivo Elevated Plus Maze test (EPM) of a series of pyrazolo[4,3-c]quinolin-3-ones (PQs) are reported. Multistep synthesis of PQs started from anilines and diethyl 2-(ethoxymethylene)malonate to give the quinolin-4-one nucleus, via the Gould-Jacobs reaction. These quinolinones were transformed to 4-chloroquinolines, which react with aryl-hydrazines affording the final compounds. PQs exhibited different potency in displacing specific [3H]Flunitrazepam binding from the benzodiazepine binding site at the γ-aminobutyric acid receptor (GABAA-R) depending on the substitution of the pyrazoloquinolone nucleus. PCA helped determine how different substituents contributed to the differential behavior of the PQs studied. Compounds with high affinity for the GABAA-R were tested regarding their anxiolytic properties in Wistar adult male rats using the Elevated Plus Maze (EPM). Thus, PQs with a p-methoxy phenyl group at N-1 (7b-ii and 7c-ii) displayed a remarkable anxiolytic activity at low doses (0.5–1.0 mg/kg). Meanwhile, PQs featuring an unsubstituted phenyl (7b-i) or p-fluoro phenyl group (7b-iii) at the N-1 showed anxiogenic effects in the EPM test. Fil: Lopez Rivilli, Marisa Juana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Turina, Anahi del Valle. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina Fil: Bignante, Elena Anahi. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina Fil: Cena Molina, Victor Hugo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina Fil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; Argentina Fil: Briñon, Margarita Cristina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina Fil: Moyano, Elizabeth Laura. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina

The structural resolution of a bound ligand-receptor complex is a key asset to efficiently drive lead optimization in drug design. However, structural resolution of many drug targets still remains a challenging endeavor. In the absence of structural knowledge, scientists resort to structure-activity relationships (SARs) to promote compound development. In this study, we incorporated ligand-based knowledge to formulate a docking scoring function that evaluates binding poses for their agreement with a known SAR. We showcased this protocol by identifying the binding mode of the pyrazoloquinolinone (PQ) CGS-8216 at the benzodiazepine binding site of the GABAA receptor. Further evaluation of the final pose by molecular dynamics and free energy simulations revealed a close proximity between the pendent phenyl ring of the PQ and γ2D56, congruent with the low potency of carboxyphenyl analogues. Ultimately, we introduced the γ2D56A mutation and in fact observed a 10-fold potency increase in the carboxyphenyl analogue, providing experimental evidence in favor of our binding hypothesis.

We report herein the design and optimization of a novel series of sulfamides and sulfamates derived from amino esters with anticonvulsant properties. The structures were designed based on the pharmacophoric pattern previously proposed, with the aim of improving the anticonvulsant action. The compounds were obtained by a new synthetic procedure with microwave assisted heating and the use of adsorbents in the isolation process. All the derivatives showed protection against the maximal electroshock seizure test (MES test) in mice at the lowest dose tested (30 mg/kg) but they did not show significant protection against the chemical induced convulsion by pentylenetetrazole. These results verify the ability of the computational model for designing new anticonvulsants structures with anti-MES activity. Additionally, we evaluated the capacity of the synthesized structures to bind to the benzodiazepine binding site (BDZ-bs) of the γ-aminobutiric acid receptor (GABAA receptor). Some of them showed medium to low affinity for the BDZ-bs. Fil: Villalba, Maria Luisa. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Enrique, Andrea Verónica. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Higgs, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Castaño, Rocío A.. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina Fil: Goicoechea, Sofia. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Taborda, Facundo. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina Fil: Gavernet, Luciana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lick, Ileana Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Química Inorgánica "Dr. Pedro J. Aymonino". Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Química Inorgánica "Dr. Pedro J. Aymonino"; Argentina Fil: Marder, Nora Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Bruno Blanch, Luis Enrique. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Indolylglyoxylamides are a class of distinctive benzodiazepine receptor ligands, proposed in the mid-eighties as open analogues of -carbolines. Thorough and long-lasting studies of their structure-activity relationships led to the development of a great deal of derivatives, to satisfy increasingly structural and pharmacophoric requirements of the benzodiazepine binding site in the central nervous system. Efforts to pre-organize their flexible structure in the three-dimensional shape adopted when bound to the receptor led to the identification of two novel classes of rigid ligands, characterized by planar tricyclic heteroaromatic cores: the [1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one and the [1,2,3]triazolo[1,2-a][1,2,4]benzotriazin-1,5(6H)-dione. The present review focuses on these selected classes of ligands, whose rational development, in terms of chemical structures and structure-activity relationships, will be fully discussed.

A set of sulfamides designed, synthesized and evaluated against maximal electroshock seizure (MES) and pentilenetetrazol (PTZ) tests with promising results, were tested for their affinity for the benzodiazepine binding site of the GABAA receptor. The most active compounds, N,N′-dicyclohexylsulfamide (7) and N,N′-diphenethylsulfamide (10), competitively inhibited the binding of [3H]-flunitrazepam to the benzodiazepine binding site with Ki ± SEM values of 27.7 ± 4.5 μM (n = 3) and 6.0 ± 1.2 μM (n = 3), respectively. The behavioral actions of these sulfamides, i.p. administered in mice, were examined in the plus-maze, hole-board and locomotor activity assays. Compound 7 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the hole-board test (at 1 and 3 mg/kg) and the plus-maze assay (at 1 and 3 mg/kg). Compound 10 evidenced anxiolytic activity in the plus-maze and the hole-board tests at 1 mg/kg. Locomotor activity of mice was not modified by compound 7 or 10 at the doses tested. Flumazenil, a non selective benzodiazepine binding site antagonist, was able to completely reverse the anxiolytic-like effects of these sulfamides, proving that the GABAA receptor is implicated in this action. Anxiety represents a major problem for people with epilepsy. The use of anxiolytic and anticonvulsant sulfamides would be beneficial to individuals who suffer from both disorders. Fil: Wasowski, Cristina Lucia N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Gavernet, Luciana. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Barrios, Ivana Analia. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Villalba, Maria Luisa. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pastore, Valentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Samaja, Gisela Anabel. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Enrique, Andrea Verónica. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bruno Blanch, Luis Enrique. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Química Medicinal; Argentina Fil: Marder, Nora Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Using the guide of a competitive assay for the benzodiazepine binding site in the γ-aminobutyric acid type A receptor (GABAA), 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 [3H]-FNZ to the benzodiazepine binding site with Ki ± 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. Fil: Wasowski, Cristina Lucia N.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marder, Nora Mariel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

A novel series of agonists at the benzodiazepine binding site of the GABAA receptor was prepared by functionalizing a known template. Adding substituents to the pyrazolone-oxygen of CGS-9896 led to a number of compounds with selectivities for either α2- or α1-containing GABAA receptor subtypes offering an entry into indications such as anxiety and insomnia. In this communication, structure–activity relationship and efforts to increase in vitro stabilities are discussed.

Bz-423 is an inhibitor of the mitochondrial F1F0-ATPase, with therapeutic properties in murine models of immune diseases. Here, we study the binding of a water-soluble Bz-423 analog (5-(3-(aminomethyl)phenyl)-7-chloro- 1-methyl-3-(naphthalen-2-ylmethyl)-1H-benzo][e][1,4]diazepin-2(3H)-one); (1) to its target subunit on the enzyme, the oligomycin sensitivity conferring protein (OSCP), by NMR spectroscopy using chemical shift perturbation and cross-relaxation experiments. Titration experiments with constructs representing residues 1–120 or 1–145 of the OSCP reveals that (a) 1 binds to a region of the protein, at the minimum, comprising residues M51, L56, K65, V66, K75, K77, and N92, and (b) binding of 1 induces conformational changes in the OSCP. Control experiments employing a variant of 1 in which a key binding element on the small molecule was deleted; it had no perturbational effect on the spectra of the OSCP, which indicates that the observed changes with 1 represent specific binding interactions. Collectively, these data suggest that 1 might inhibit the enzyme through an allosteric mechanism where binding results in conformational changes that perturb the OSCP-F1 interface resulting in disrupted communication between the peripheral stalk and the F1-domain of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 29: 85–92, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Polygala sabulosa, a folk medicine, presents dihydrostyryl-2-pyrones (DST) and styryl-2-pyrones (STY), compounds structurally similar to kavalactones. Our previous study showed that the ethyl acetate fraction (EA) and these constituents present anxiolytic-like, hypno-sedative, and anticonvulsant effects in mice.This study investigated the role of benzodiazepine binding site (BDZ-bs) in the central effects of either EA or three DST (1, 2, and 3) and three STY (4, 5, and 7), using in vivo and in vitro assays.In the elevated plus-maze (EPM), flumazenil (FMZ), a BDZ antagonist, partially blocked the anxiolytic-like effect of DST-3 or STY-4 and STY-7, but not DST-1. Using electroencephalogram (EEG), EA protected against pentylenetetrazole (PTZ)-induced convulsion in rats, an effect partially blocked by FMZ, suggesting the participation of the BDZ-bs in this action. EA also protected against the maximal electroshock (MES)-induced convulsions in mice, a profile distinct from diazepam (DZP). DST and STY compounds inhibited the [(3)H]-flunitrazepam ([(3)H]-FNZ) binding to BDZ-bs in rat cortical synaptosomes with K (i) higher than 100 microM (DST-1), 41.7 microM (DST-2), 35.8 microM (DST-3), 90.3 microM (STY-4), 31.0 microM (STY-5) and 70.0 microM (STY-7). In the saturation assay, DST-3 and STY-7 competitively inhibited the binding of [(3)H]-FNZ to BDZ-bs with a significant decrease in apparent affinity (K (d)) and no change in maximal binding (B (max)).The present data support a partial BDZ-bs mediation of the anxiolytic-like and anticonvulsant effects of EA of P. sabulosa and its main isolated constituents, DST and STY.

Ganaxolone (3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one) (GNX) is the 3beta-methylated synthetic analog of allopregnanolone; it belongs to a class of compounds referred to as neurosteroids. GNX is an allosteric modulator of GABA(A) receptors acting through binding sites which are distinct from the benzodiazepine binding site. It has activity in a broad range of animal models of epilepsy. GNX has been shown to be well tolerated in adults and children. In early phase II studies, GNX has been shown to have activity in adult patients with partial-onset seizures and epileptic children with history of infantile spasms. It is currently undergoing further development in infants with newly diagnosed infantile spasms, in women with catamenial epilepsy, and in adults with refractory partial-onset seizures.

Novel syntheses of 2,3,8-trisubstituted pyrido[2,3- d ]pyridazines and 2,3,5-trisubstituted pyrazino[2,3- d ]pyridazines are described. Two complementary routes to pyrido[2,3- d ]pyridazines were developed, the first of which began by constructing the pyridine ring, and the second of which started by constructing the pyridazine ring. Pyrazino[2,3- d ]pyridazines were prepared in a route employing an aza-Wadsworth–Emmons cyclization as the key step. The resulting compounds were found to be high affinity ligands for the GABA A receptor benzodiazepine binding site.

Imidazo[1,2-a]pyrimidines are GABA(A) receptor benzodiazepine binding site ligands which can exhibit functional selectivity for the alpha(3) subtype over the alpha(1) subtype. SAR studies to optimize this functional selectivity are described.

Recently, reference tissue methods have been proposed to estimate binding potential from PET data. A reference region without specifically bound ligand is used as an indirect input function to enable the expression of the time-concentration curve of a region of interest using a compartment model. However, PET dopaminergic and serotoninergic studies have shown differences between binding potential (BP) values obtained with reference tissue methods and those obtained with conventional kinetic modeling using an arterial input function. In this study, we measured the BP values for the benzodiazepine receptors in seven subjects using PET [(11)C]flumazenil and SPECT [(123)I]iomazenil radioligands. We compared the BP values obtained using the reference tissue methods with those obtained using the conventional kinetic method. These values were also compared with the absolute value of receptor density, B'(max). For the PET studies, a multi-injection approach employing labeled and unlabeled flumazenil was used to estimate the main binding parameters, BP and B'(max). For SPECT studies, a single injection protocol of [(123)I]iomazenil was used to estimate BP values. The BP values were estimated using one- and two-tissue compartment models for the target region. Similar BP values were obtained using either the one- or two-tissue compartment model. This is probably due to the rapid equilibrium between tissue compartments reached with these radioligands. For PET and SPECT, these BP values were highly correlated (r > 0.960) to the BP values obtained using the arterial input function. We also found high correlations between the BP values obtained using the simplified reference tissue method and the receptor density parameter B'(max) (r > 0.884). However, the reference tissue methods yielded lower BP values than those obtained using the conventional approach. Moreover, there was a bias on BP values that was not a simple scaling. It seems that the physiological values found in gray matter structures using these radioligands give acceptable BP values. We conclude that the reference tissue methods should be carefully evaluated for each radioligand.

6,3′-Dibromoflavone and 6-nitro-3′-bromoflavone inhibited [3H]flunitrazepam binding to the benzodiazepine binding site of the gamma amino butyric acid receptor complex with Ki values between 17 and 36 nM in different brain regions. Their gamma amino butyric acid ratio for [3H]flunitrazepam binding to cerebral cortex membranes indicated partial agonistic properties. Both compounds had similar pharmacological effects: they produced anxiolytic-like effects at low doses but did not alter locomotor activity or muscle tonicity; sedation was caused only at doses higher than 30 mg/kg in mice. These synthetic flavone derivatives join an existing family of 6,3′-disubstituted flavone compounds with high affinity for the benzodiazepine binding site and partial agonistic profiles.

1. We reviewed studies performed to characterize central benzodiazepine binding sites. 2. An overview of the different radioligands used to characterize BZ1 and BZ2 binding sites and a mapping of these central benzodiazepine sites are described. 3. Saturation studies carried out by autoradiogram quantification also are reviewed. 4. The specific use of the autoradiographic technique to carry out studies on ontogeny, development, and phylogeny is discussed, as well as studies performed using this technique on some diseases and experimental conditions, such as drug treatments or chemical and mechanical lesions.

Although there were no differences in response to an acute injection of pentylenetetrazole (PTZ), there were strain differences in the development of kindled seizures to repeated injections (PTZ; 30 mg/kg 3 times weekly for 13 injections), with Wistar rats reaching stage 4 or 5 of clonic-tonic seizures, but hooded Lister rats reaching only stage 2 or 3 of convulsive waves axially through the body. The strains also reacted differently to a test dose of PTZ (20 mg/kg) one week after the end of kindling, with the Wistar strain showing stage 3 and the Lister strain stage 2 seizures. When the rats were tested 24 h after the end of the kindling injections there was an anxiolytic effect in the social interaction test, in both the low light, familiar and the low light, unfamiliar test conditions that reached significance in the Wistar strain. The Wistar kindled rats showed an anxiolytic effect in the elevated plus-maze test of anxiety when they were tested 24 h after the end of kindling. The anxiolytic effects found 24 h after kindling could not be due to the seizure 24 h earlier, since no changes were found in rats tested 24 h after a single seizure from PTZ (60 mg/kg). When the rats were tested 1 week after the end of kindling there were no changes, compared with vehicle-injected controls, in either test of anxiety. There was no change in benzodiazepine binding in platelets of the kindled Lister rats but there was a significant increase in the kindled Wistar rats 1 week after the end of kindling and also 24 h after a single PTZ seizure. The pattern of increased platelet benzodiazepine binding did not correspond with the time course of rebound anxiolytic effects. However, after kindling it seems that there are long-lasting changes in benzodiazepine binding that are similar to the short-term increases that are found following a single seizure.

[3H]PK 11195 binding to the peripheral benzodiazepine binding site was investigated in the brain and liver of mice treated with ethanol (4 g/kg, p.o.) daily for 5 days. In the brain stem, Bmax was decreased by 78% in the ethanol-treated group with unaltered Kd (2 nM). The ethanol-withdrawn group did not differ from the control group in both parameters. In the cerebellum, Bmax was decreased by 74% but the binding affinity increased 5-fold as the Kd decreased from 10 to 2 nM. The ethanol-withdrawn group did not differ significantly from the ethanol-treated group. No changes were observed in the cerebrum and liver. These results further support the idea that [3H]PK 11195 binding may be a useful marker for ethanol consumption. The observed changes in these binding sites may represent a functional adaptive response to the ethanol insult and/or a role in the mediation of the effects of ethanol.

GABA(A) ion channel is a validated drug target, implicated in the pathophysiology of various neurological and psychiatric disorders. Structural investigations on GABA(A) are currently precluded in the absence of experimentally resolved structure. Pharmacophore modeling circumvents such issues and proves to be a powerful and successful method in drug discovery.The present reviews encompass pharmacophoric models available in the literature for the orthosteric GABA and the allosteric benzodiazepine binding site. Success stories from these simplistic pharmacophore models in scaffold hopping and strategic lead optimization have been highlighted. Recent advances in pharmacophore modeling that can leverage CNS drug discovery programs and deliver astounding results have been reviewed.Readers are bound to gain a comprehensive insight on different computational techniques used by different groups to arrive at simple, yet sophisticated pharmacophore models. In the absence of experimentally unresolved active site geometry of GABA(A), these models will provide the reader an opportunity to translate these pharmacophoric features to the microscopic phenomenon of supramolecular ligand interaction.Pharmacophore modeling has now evolved as a mainstay approach for lead generation and optimization in drug discovery programs. Of late, many advances in pharmacophore perception have emerged. Such advancements should be used to confront activity profiling and early stage risk assessment in a high-throughput fashion. Extending such technologies has the potential not only to reduce time and cost, but also to prevent late stage attrition in drug discovery.

Laboratory rats exhibit innate behavioural and corticosterone responses when exposed to cat odour. However, not all are responsive and differences in benzodiazepine receptor binding between responders and non-responders were explored. Rats were exposed to cat odour for 5 min and based on time spent sheltering were divided into responders (n = 21; mean ± SEM = 244 ± 8.2 sec) or non-responders (n = 20; 43.9 ± 4.8 sec). Four days later, both groups were randomly allocated among 3 experimental conditions: home-cage, neutral or cat odour, and killed 30 min after exposure. [3H]flunitrazepam binding was performed at two ligand concentrations (2 and 10 nM); where significant differences in single point binding were found, Scatchard analysis was performed on pooled samples. In hippocampus and frontal cortex responders had significantly lower binding than non-responders. In hippocampus this was most apparent when the rats were exposed to the novel test situation, i.e. neutral odour and was due to a reduction in affinity (Kd = 0.4 and 1.2 nM non-responders and responders). In frontal cortex, differences were significant only following exposure to cat odour (Bmax = 2663 and 1501 fmol/mg protein in non-responders and responders). The changes in amygdala were not significant.

The central benzodiazepine binding site mediates the therapeutically relevant pharmacologic actions of benzodiazepine agonists, including reducing anxiety, sedation, muscle relaxation, and antagonism of seizure production. Benzodiazepines potentiate the ability of gamma-aminobutyric acid (GABA) to promote membrane chloride ion conductance by binding reversibly to a distinct site on the GABAA receptor complex. Although the role of this central benzodiazepine binding site in mediating the pharmacologic actions of benzodiazepine agonists has been shown conclusively, its participation in an animal's response to stress is less certain. Data are reviewed consistent with an environmental stress-induced modification of the structure and function of the central benzodiazepine binding site. Modifications show brain anatomic regional selectivity. The mechanisms of these modifications include rapid enzyme-mediated phosphorylations of the GABAA receptor complex and the selective transcription of individual polypeptide subunits. The potential relevance of the data on environmental stress-induced functional modifications of the central benzodiazepine binding site to the development of newer medications and improved understanding of the pathophysiology of stress-related neuropsychiatric disorders is discussed.

The present study used saturation binding analyses to test the hypothesis that pentobarbital would differentially increase the affinity of benzodiazepine binding across brain regions. The results show

The status of the radiochemical development and biological evaluation of radioligands for PET studies of central benzodiazepine (BZ) receptors and the so-called peripheral benzodiazepine binding sites, here discriminated and referred to as PK binding sites, is reviewed against current pharmacological knowledge, indicating those agents with present value and those with future potential. Practical recommendations are given for the preparation of two useful radioligands for PET studies, [N-methyl-11C]flumazenil for central BZ receptors, and [N-methyl-11C]PK 11195 for PK binding sites. Quality assurance and plasma metabolite analysis are also reviewed for these radioligands and practical recommendations are given on methodology for their performance.

Benzodiazepines are widely used in clinical anesthesia as premedication, but also to induce general anesthesia. Recent in vitro studies suggest that γ-aminobutyric acid type A receptors, harboring a classical high-affinity benzodiazepine binding site, possess another "nonclassical" binding site for benzodiazepines. At present, it is unclear if, and to what extent, this novel nonclassical binding site is of relevance for the actions of benzodiazepines in the central nervous system.Because neocortex is involved in mediating the sedative and hypnotic properties of general anesthetics, we quantified the actions of diazepam over a wide range of concentrations (from 10 nM up to 100 μM) in organotypic slice cultures using extracellular multiunit recordings of spontaneous action potential activity.Up to a concentration of 6.25 μM, diazepam reduced the activity of neocortical neurons, approaching a maximum of approximately 20%. This action was nullified by the benzodiazepine antagonist flumazenil. At concentrations12.5 μM, diazepam evoked a second concentration-dependent dampening of network activity. Unlike the low concentration effect, this high concentration component was resistant to flumazenil.Diazepam induced a biphasic attenuation of spontaneous action potential firing of neocortical neurons. Low to moderate concentrations caused a monotonic, mild depression that is mediated via the classical binding site as it is antagonized by flumazenil. However, the effects of diazepam observed at high concentrations were not affected by flumazenil. Hence, these findings support the concept of at least 2 different binding sites for benzodiazepines on γ-aminobutyric acid type A receptors. Furthermore, our results are consistent with the hypothesis that the classical high-affinity binding site mediates low-dose diazepam actions, such as amnesia, anxiolysis, and sedation, while a second, nonclassical and independent site contributes to the anesthetic effects of diazepam, such as hypnosis and immobility.

Specific binding of (3)H-flunitrazepam with synaptosomal membranes after exposure to open field test and "contact with predator" test was measured in C57Bl/6 and BALB/c mice. Stress-induced decrease in benzodiazepine binding after open field test was observed only in BALB/c mice and after contact with predator in both animal strains.

Benzodiazepine binding sites were studied by autoradiography in several hippocampic layers in brains of drug-free violent suicide victims (hanging) and matched controls. Kd was increased in suicides, and when brain sections from control subjects were incubated in the bath fluid that had previously served to incubate sections from suicides, Kd was increased in the same way. Results are discussed in terms of possible modulators of benzodiazepine binding sites, mainly tissue GABA and amino acid concentrations.

This work describes the distribution of the central benzodiazepine-binding sites (BZD-BS) in several structures recently described as involved in memory processes. The study was performed on 4 postmortem human brains by in vitro quantitative autoradiography using 3H-flunitrazepam as a ligand. Very high densities of BZD-BS (1,500–2,000 fmol/mg protein) were observed in the anterior and posterior cingulate cortex, in the indusium griseum and in the accessory basal nucleus of the amygdala, and moderate concentrations (500–700 fmol/mg) in the magnocellular region of the mediodorsal thalamic nucleus. In the classical cerebral memory circuits, high densities of BZD-BS (1,000 to 1,500) were also found, except in the septum where the density of BZD-BS (400 to 600) was not significantly different from that in control structures not involved in memory. No evidence for lateralization of the sites was observed.

The presence of benzodiazepine binding inhibitory activity (B.B.I.A.) in sera from 44 psychiatric patients and from 14 healthy volunteers, prompted us to investigate whether or not this activity underwent changes in stressful situations. We measured the inhibitory units (IU) of deproteinized sera of 12 subjects, immediately before and 2 weeks after sitting for a difficult university exam. Our results showed significantly higher IU values (i.e., higher B.B.I.A. concentrations) in the samples taken just before the exam. This preliminary finding clearly suggests the involvement of B.B.I.A. in anxiety mechanisms.

Benzodiazepine binding was measured in amygdala and hippocampus from 19 suicides in whom a firm diagnosis of depression was established retrospectively, and 19 well-matched controls. The number and affinity of benzodiazepine binding sites did not differ significantly between the drug-free or drug-treated suicides and controls.

It is demonstrated that benzodiazepine binding sites on human peripheral blood lymphocytes are the peripheral type receptors. The binding sites for the selective agonist3H-Ro 5-4864, but not for the antagonist3H-PK 11195, are completely inactivated by freezing-thawing of lymphocytes. The binding of3H-Ro 5-4864 to intact lymphocytes is activated by GABA and (+)baclofen. It is shown that the selective ligands Ro 5-4864 and PK 11195 bind to different subtypes of benzodiazepine receptors on human peripheral blood lymphocytes.

Novel synthetic routes have been devised for the preparation of previously inaccessible 2,3,7-trisubstituted pyrazolo[1,5-d][1,2,4]triazines 2. These compounds are high affinity ligands for the GABAA benzodiazepine binding site and some analogues show functional selectivity for agonism at α3-containing receptors over α1-containing receptors with the lead compound being 32.

Novel syntheses of 2,3,8-trisubstituted pyrido[2,3- d ]pyridazines and 2,3,5-trisubstituted pyrazino[2,3- d ]pyridazines are described. Two complementary routes to pyrido[2,3- d ]pyridazines were developed, the first of which began by constructing the pyridine ring, and the second of which started by constructing the pyridazine ring. Pyrazino[2,3- d ]pyridazines were prepared in a route employing an aza-Wadsworth–Emmons cyclization as the key step. The resulting compounds were found to be high affinity ligands for the GABA A receptor benzodiazepine binding site.