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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

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

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

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.

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.

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.

A Catalyst pharmacophore model has been developed for the benzodiazepine site within the GABA(A) receptor complex. The model is based on a pharmacophore model originally proposed by Cook and co-workers (Drug Des. Discovery 1995, 12, 193-248) and further developed by Kahnberg et al. (J. Med. Chem. 2002, 45, 4188-4201). The Catalyst pharmacophore model has been validated by using a series of flavonoids with varying affinities for the benzodiazepine receptor and has then been used as a search query in database searching with the aim of finding novel structures which have the possibility to be modified into novel lead compounds. Five of the hits from the database searching were purchased and their affinities for the benzodiazepine site of the GABA(A) receptor were determined. Two of the compounds displayed K(i) values below 10 microM. The substance showing highest potency in-vitro displayed an affinity of 121 nM making it an interesting compound for optimization. The false positive compounds (K(i) values10 microM affinities) have been analysed in terms of conformational energy penalties and possibilities for hydrogen bond interactions. The analysis clearly demonstrates the need for post processing of Catalyst hits.

Mercury compounds are neurotoxic compounds with a great specificity for cerebellar granule cells. The interaction of mercury compounds with proteins in the central nervous system may underlie some of their effects on neurotransmission. In this work we study the interaction of mercuric chloride (HgCl2) and methylmercury (MeHg) with the GABAA receptor in primary cultures of cerebellar granule cells. Both compounds increased, dose dependently, the binding of flunitrazepam to the benzodiazepine recognition site. EC50 values for this effect were 3.56 and 15.24 μM for HgCl2 and MeHg, respectively, after 30 min exposure of intact cultured cerebellar granule cells. The increase of flunitrazepam binding by mercury compounds was completely inhibited by the GABAA receptor antagonists bicuculline and picrotoxinin, and by the organochlorine pesticide α-endosulfan. It was also partially inhibited by the anion transporter blocker DIDS, however this effect could be due to a possible chelation of mercury by DIDS. Intracellular events, like intracellular calcium, kinase activation/inactivation or antioxidant conditions did not affect flunitrazepam binding or its increase induced by mercury compounds. The sulfhydryl alkylating agent N-ethylmaleimide mimicked the effect of mercury compounds on flunitrazepam binding suggesting a common mechanism. We conclude that mercury compounds interact with the GABAA receptor by the way of alkylation of SH groups of cysteinyl residues found in GABAA receptor subunit sequences., This research was supported by Grant number 00/1094 and 01/1318 from FIS (Spanish Fondo de Investigaciones Sanitarias de la Seguridad Social) and Grant numbers 1999SGR00210 and 1999SGR00212 from CIRIT (Generalitat de Catalunya, Spain). E. Fonfrı́a is recipient of a fellowship from the Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS).

Summation analysis strategies are recognized throughout diverse scientific fields as powerful means of differentially enhancing experimental signals over random fluctuations (noise). Such techniques, applied to emission tomographic cerebral blood flow scans, reveal subtle alterations in neuronal activity during specific behavioral states. In the present work, we extend the principles of intersubject image summation analysis to the evaluation of emission tomographic ligand-binding studies. A general methodology is presented that may be applied to a wide variety of binding site determinations. The procedure consists of anatomic standardization of individual brains to a common stereotaxic orientation, followed by statistical analyses of group versus group or individual versus group differences. We develop and evaluate performance of our technique with the use of positron emission tomographic [11C]flumazenil scans from normal volunteers, depicting the regional cerebral distribution of benzodiazepine binding sites.

Summary: Distribution of the enzyme monoamine oxidase B (MAO-B) and the peripheral benzodiazepine binding site (ψ3 site) was studied by quantitative autoradiography using [3H]L-deprenyl and [3H]PK 11195, two tentative glial markers, as ligands. Sclerotic hippocampus from seven patients who had had anterotemporal lobe resection because of intractable complex partial epilepsy were investigated and compared with postmortem hippocampus from three nonepileptic controls. A significantly higher degree of L-deprenyl and PK 11195 binding was observed in the epileptic cases. The increased binding of both ligands correlated to extent of neuronal loss, but only PK 11195 showed correlation to degree of gliosis. We conclude that both ligands could provide useful markers for quantitating the degree of gliosis in pathologic states such as epilepsy. They may be applicable in future in vivo studies with positron emission tomography (PET). RESUME La distribution de la monoamine oxydase B (MAO-B) et du site de liaison peripherique benzodiazepine (site ω3) a eteetudiee parautoradiographie quantitative au moyen de deux ligands, le H3-L-deprenyl et le H3-PK 11 195, deux marqueurs probables de la glie. L'hippocampe sclerose de sept patients qui avaient beneficie d'une lobectomie temporale anterieure pour epilepsie partielle complexe rebelle a eteetudie et comparea I'hippocampe recueilli a l'autopsie de trois controles non-epileptiques. Une liaison significativement elevee de L-deprenyl et du PK 11 195 a ete observee chez les epileptiques. L'augmentation de liaisons pour les deux ligands etait correlee a I'etendue des pertes neuronales, mais seul le PK 11195 a montre une correlation au degre de gliose. Les auteurs concluent que les deux ligands pourraient fournir des marqueurs utiles pour la quantification du degre de gliose dans les etats pathologiques comme l'epilepsie. IIs pourraient etre utilises dans des etudes in vivo futures utilisant la tomographie aemissions de positons (TEP). RESUMEN Utilizando el [3H]-L-deprenil y el [3H]-PK 1 1195, dos marcadores gliales tentativos usados como ligandos en autorradiografia, se ha estudiado la distribucion de la enzima monoamino oxidasa B (MAO-B) y el lugar periferico de acoplamiento de la benzodiacepina. Se ha investigado el hipocampo-esclerotico de 7 enfermos que habian sido sometidos a una reseccion anterior del loulo temporal, debido a una epilepsia parcial compleja e incontrolable y los resultados se han comparado con hipocampo obtenido en post-mortem de 3 casos control no-epilepticos. Se ha observado un grado significativamente mas elevado de acoplamiento del L-deprenil y el PK 111195 en los casos de epilepsia. El incremento del acoplamiento de los dos ligandos mostro una aceptable coordinacion con la perdida neuronal pero solo el PK 111195 mostro correlacion con el grado de gliosis. Los autores concluyen que ambos ligandos puden proporcionar marcadores utiles para cuantificar el grado de gliosis en estados patologicos tales como la epilepsia, que pueden aplicarse en estudios futuros in vivo con Tomografia por Emision de Positrones (PET). ZUSAMMENFASSUNG Die Verteilung des Enzyms Monoaminooxidase B (MAO-B) und die peripheren Benzodiazepinbindungslotic (ω3-sites) wurden durch quantitative Auradiographie untersucht unter Verwendung von (3H)-L-depoprenyl und (3H-) 11195, 2 gliale Marker, als Liganden. Bei 7 Patienten mit therapieresistenten komplex-partialen Anfallen wurde eine vordere Temporallap- pen-Resektion vorgenommen. Sklerotisches Hippokampus- gewebe wurde untersucht und mit post-mortem Hippokampus von 3 nicht epileptischen Kontrollen verglichen. Bei den Epilep- siekranken fanden sich ein signifikant erhohter Grad an L-De- prenyl und PK 11 195-Bindungen. Die erhohte Bindung beider Llganden korrelierte mit dem Ausmap von neuronalem Unter- gang; aber nur PK I 1195 zeigte eine Korrelation zum Grad der Gliose. Daraus schliβen wir, daβ beide Liganden nutzliche Marker darstellen, zur Quantifizierung der Gliose bei pathologischem Zustanden wie der Epilepsie. Sie konnten in Zukunft bei in vivo Untersuchungen, wie dem PET, eingesetzt werden.

We demonstrate direct visualization of benzodiazepine binding parameters collected in a 3H-flunitrazepam saturation study. In this way the relationship between anatomy and specific binding, Kd and Bmax, can be appreciated. These experiments have resulted in image representations of specific flunitrazepam binding, Kd and Bmax. The images retain the anatomic localization inherent in the original autoradiogram and hence make these parameters amenable to visual survey. Binding constants were consistent with data presented elsewhere in the literature. The image of Bmax showed regional heterogeneity. Kd, on the other hand, was relatively homogeneous. These results demonstrate a means by which autoradiographic binding experiments can be extended to saturation studies without significant loss of data.

The specific photoaffinity labeling of membrane-bound and detergent-solubilized benzodiazepine binding sites has been investigated using UV irradiated [3H] flunitrazepam as a photochemical probe. The time course and the regional and pharmacological specificity of the photolabeling reaction has been determined for "brain-specific" benzodiazepine binding sites; "peripheral-type" binding sites treated in an identical manner were not specifically labeled. Comparison of the number of sites labeled and blocked by [3H]flunitrazepam photolabeling of detergent-solubilized preparations indicated that about one site was blocked and unavailable for reversible binding for each site photolabeled. In contrast, when membrane-bound sites were photolabeled, about four sites were inactivated for each site photolabeled. Examination of photolabeled binding sites from various brain regions including cortex, striatum, and hippocampus using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave only a single labeled band of apparent Mr = 48,000.

Inosine, 2-deoxyinosine, and 2-deoxyguanosine completely reversed the increase in exploratory activity elicited in mice by diazepam. The inhibition of exploratory behavior by purines occurred at doses that when given alone have no effect on exploratory behavior. 7-Methylinosine, which does not bind to the brain benzodiazepine binding site in vitro, had no effect on the diazepam-induced increase in exploratory behavior. Behavioral effects produced by various combinations of inosine and diazepam indicate that the interaction between purine and benzodiazepine is antagonistic and support the hypothesis that the naturally occurring purines function in anxiety-related behaviors that respond to benzodiazepine treatment.

Benzodiazepines are widely used anxiolytic and anticonvulsant drugs, and brain receptors for these drugs have been characterized by Möhler and Okada and Squires and Braestrup. Recently, substances that antagonize benzodiazepine binding to brain receptors have been discovered. These benzodiazepine antagonists were shown to block the central effects of benzodiazepines and particularly their anticonvulsive properties. Two such antagonists, Ro 15-1788 (an imidazodiazepine) and methyl beta-carboline-3-carboxylate (beta-CCM), have recently been shown to have different intrinsic pharmacological properties. beta-CCM, injected into baboons, cats, mice and rats, is a convulsant, whereas Ro 15-1788 lacks such an activity. Thus, the separation of convulsant and non-convulsant antagonists has been proposed. We suggest here that a subclassification of antagonists is also valid at the behavioural level, based on a conflict model in mice. We show that Ro 15-1788 and beta-CCM antagonize the anxiolytic effect of benzodiazepines. In addition, we find that, when injected alone, Ro 15-1788 has no anxiogenic effects while beta-CCM has anxiogenic properties. We therefore propose that beta-CCM is an anxiogenic convulsant benzodiazepine antagonist and that Ro 15-1788 is a non-anxiogenic non-convulsant benzodiazepine antagonist.

A number of studies have suggested the existence of multiple benzodiazepine binding sites in the brain1–20. We have recently reported the physical separation of two apparent benzodiazepine binding site subtypes21, the pharmacological properties, and distribution in tissue sections of which correspond to the putative type I and type II sites20,22. Benzodiazepine and γ-aminobutyric acid (GABA) receptors have been shown to interact23, and lesions of the GABAergic striatonigral pathway, which lead to GABA supersensitivity24,25, both increase the numbers of GABA binding sites24,26 and enhance GABA-stimulated benzodiazepine binding27. We demonstrate here that degeneration of striatonigral fibres increases the density of putative type I benzodiazepine binding sites in the substantia nigra and decreases the density of the putative type II sites. This suggests that type I sites that increase after denervation are postsynaptic, whereas the type II sites reduced by the lesion may be localized to axons or terminals of the striatonigral pathways.

Experimental hyperthermic seizures in 15-day-old rats resulted in both an alteration in the benzodiazepine (BDZ) binding site in the hippocampal region and morphologic changes in hippocampal neurons. These alterations were not apparent until adulthood. Experimental adult animals also showed a reduced anticonvulsant response to a selective BDZ ligand when challenged with pentylenetetrazol. Developmental hyperthermic seizures may initiate dynamic changes in the hippocampus that contribute to adult functional impairment.

Although the benzodiazepines have been extensively investigated since their discovery about 20 years ago1, only recently have antagonists of the classical actions of benzodiazepines been described2. We have now discovered a class of benzodiazepines which have lost all affinity for benzodiazepine binding sites but which have moderate to high affinity for opiate receptors. Depending on the substituents introduced, it is possible to obtain compounds with selective actions on μ, δ and κ subpopulations of opiate receptors. The compounds are effective analgesics in various animal models of pain, exhibit an opioid activity spectrum in various test systems, and show none of the typical effects of minor tranquillizers. Here we report that 1-methyl-2(3-thienylcarbonyl)-aminomethyl-5-(2-fluorophenyl)-H-2,3-dihydro-l,4-benzodiazepine (KC5103; tifluadom), acts selectively on opiate κ-receptors.

The benzodiazepines are a class of drugs used to alleviate anxiety. As such they constitute one of the most commonly prescribed compounds1, due in part to their efficacy and safety. The physiological effect of these drugs is probably through interactions with a low affinity benzodiazepine binding site2 and two (types 1 and 2) higher affinity sites3–8 . The ontogenesis of these latter two binding sites in the rat differs, with the type 2 binding site being predominant at birth and the type 1 binding site increasing in number after the second week after birth9. The differential development of these two receptor types is important because the immature organism may have different physiological and behavioural responses from the adult. Here we demonstrate an important difference: that a prototypic benzodiazepine, chlordiazepoxide, and a water-soluble benzodiazepine, flurazepam, produce behavioural convulsions in the preweanling rat. The convulsions are antagonized by the benzodiazepine blocker Ro-15-1788. The triazolopyridizine CL-218872, specific to the type 1 receptor, does not share this action. We suggest that this paradoxical convulsant effect of chlordiazepoxide and flurazepam is due to activation of the type 2 receptor in the absence of the type 1 receptor in the immature rat.