Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.     

Behavioral effects of GABA agonists in relation to anxiety and benzodiazepine action

A considerable body of biochemical and neurophysiological evidence implicates GABA in anxiety and in benzodiazepine action. The present article surveys the behavioral effects of GABA agonists and their interactions with drugs acting at the benzodiazepine receptor in animal anxiety paradigms. Certain GABA agonists, notably valproate, simulate many behavioral actions of benzodiazepines. Moreover, several behavioral studies of the interaction of GABA agonists with benzodiazepines support the hypothesis of a benzodiazepine receptor complex with one or more GABA, benzodiazepine and probably other binding sites. However, there are also a number of anomalous findings of GABA agonist action alone and in combination with benzodiazepines. It is argued that these paradoxical results can better be accounted for in terms of the receptor complex and the distribution of the drugs, rather than by suggesting that the anxiolytic actions of benzodiazepines are not mediated by GABA systems. The potential clinical usefulness of GABA agonists in anxiety is commented upon.     

Characterization of benzodiazepine receptors with a fluorescence-quenching ligand

A conjugate of the high affinity benzodiazepine receptor ligand Ro 15-1788 and the fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) moiety was synthesized. This novel compound (BD 623) exhibited excitation and emission maxima at 486 and 542 nm, respectively, and possessed fluorescent properties that are dependent upon the polarity of its environment. BD 623 bound reversibly to benzodiazepine receptors in the central nervous system with an apparent affinity (K(i) 5.7 nM) comparable to the parent imidazobenzodiazepine (K(d) 2.8 nM). Addition of BD 623 to a suspension of brain membranes resulted in a time-dependent quenching of its fluorescence. Fluorescence quenching of this compound was readily reversed by specific benzodiazepine receptor ligands but not by a variety of other substances. Moreover, inactivation of benzodiazepine receptors by photoaffinity labeling with Ro 15-4513 resulted in a reduction in the fluorescence quenching of BD 623 consistent with the reduction in density of benzodiazepine receptors measured using a radioreceptor assay. Monitoring of fluorescence/dequenching of BD 623 in real time permitted a quantitative characterization of the ligand-receptor interaction, with both the K(d) of BD 623 (13.9 nM) and K(i) of Ro 15-1788 (5.7 nM) comparable with the estimates obtained using radioreceptor techniques. These results indicate that application of fluorescence quenching techniques with BD 623 could prove a useful adjunct for the study of benzodiazepine receptors. BD 623 may serve as a prototype for the development of other fluorescent ligands to study ligand-receptor interactions.     

Metabolism of Anxiolytics and Hypnotics: Benzodiazepines, Buspirone, Zoplicone, and Zolpidem

1. The benzodiazepines are among the most frequently prescribed of all drugs and have been used for their anxiolytic, anticonvulsant, and sedative/hypnotic properties. Since absorption rates, volumes of distribution, and elimination rates differ greatly among the benzodiazepine derivatives, each benzodiazepine has a unique plasma concentration curve. Although the time to peak plasma levels provides a rough guide, it is not equivalent to the time to clinical onset of effect. The importance of and half-lives in the actions of benzodiazepines is discussed.2. The role of cytochrome P450 isozymes in the metabolism of benzodiazepines and in potential pharmacokinetic interactions between the benzodiazepines and other coadministered drugs is discussed.3. Buspirone, an anxiolytic with minimal sedative effects, undergoes extensive metabolism, with hydroxylation and dealkylation being the major pathways. Pharmacokinetic interactions of buspirone with other coadministered drugs seem to be minimal.4. Zopiclone and zolpidem are used primarily as hypnotics. Both are extensively metabolized; N-demethylation, N-oxidation, and decarboxylation of zopiclone occur, and zolpidem undergoes oxidation of methyl groups and hydroxylation of a position on the imidazolepyridine ring system. Zopiclone has a chiral centre, and demonstrates stereoselective pharmacokinetics. Metabolic drug–drug interactions have been reported with zopiclone and erythromycin, trimipramine, and carbamazepine. Reports to date indicate minimal interactions of zolpidem with coadministered drugs; however, it has been reported to affect the C _max and clearance of chlorpromazepine and to decrease metabolism of the antiviral agent ritonavin. Since CYP3A4 has been reported to play an important role in metabolism of zolpidem, possible interactions with drugs which are substrates and/or inhibitors of that CYP isozyme should be considered.     

Development of a high-throughput screening system for the compounds that inhibit collagen–protein interactions

Collagen-binding proteins (CBPs) play important roles in various physiological events. Some CBPs are regarded as targets for drug development; for example, platelet glycoprotein VI (GPVI) and heat shock protein 47 (HSP47) are promising targets for the development of novel antiplatelet and antifibrotic drugs, respectively. However, no systematic screening method to search compounds that inhibit collagen–CBP interactions have been developed, and only a few CBP inhibitors have been reported to date. In this study, a facile turbidimetric multiwell plate assay was developed to evaluate inhibitors of CBPs. The assay is based on the finding that CBPs retard spontaneous collagen fibril formation in vitro and that fibril formation is restored in the presence of compounds that interfere with the collagen–CBP interactions. Using the same platform, the assay was performed in various combinations of fibril-forming collagen types and CBPs. This homogeneous assay is simple, convenient, and suitable as an automated high-throughput screening system.     

Characteristics of Flunitrazepam Binding to Intact Primary Cultured Spinal Cord Neurons and Its Modulation by GABAergic Drugs

The interaction of [3H]flunitrazepam and its modulation by various drugs was studied in intact primary cultured spinal cord neurons. In the intact cells, the [3H]-flunitrazepam binding was rapid and saturable. The benzodiazepine binding sites exhibited high affinity and saturability, with an apparent KD of 6.1 +/- 1.6 nM and Bmax of 822 +/- 194 fmol/mg protein. The association and dissociation of [3H]flunitrazepam binding exhibited monoexponential kinetics. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by benzodiazepines like flunitrazepam, clonazepam, diazepam, Ro 15-1788, and beta-carbolines like methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3'-carboxylate. Specific [3H]flunitrazepam binding to intact cells was enhanced in a concentration-dependent manner by gamma-aminobutyric acid (GABA) agonists and drugs which facilitate GABAergic transmission like etazolate, (+)-etomidate, and pentobarbital. The enhancing effect of GABA agonists was antagonized by bicuculline and picrotoxinin. These results suggest that the intact cultured spinal cord neurons exhibit the properties of benzodiazepine GABA receptor-ionophore complex. Since these cells can also be studied in parallel for characterizing GABA-induced 36Cl-influx, they provide an ideal in vitro assay preparation to study GABA synaptic pharmacology.     

Benzodiazepine drugs in general medical patients.

Data from a hospital-based drug surveillance programme were used to determine how often benzodiazepine drugs were used in general medical wards. Benzodiazepines were the drugs most commonly used as hypnotics and were given to 32% of these patients. Concomitant use of more than one benzodiazepine drug or of benzodiazepines with other psychoactive drugs was common and often irrational. A series of double-blind patient-preference studies comparing various benzodiazepines and a benzodiazepine with an antihistamine showed that for short-term hypnotic effect there were no differences between three common benzodiazepines but elderly patients preferred benzodiazepines to the antihistamine, which produced more undesired effects. These results suggest that currently diazepam is the hypnotic of choice for medical ward inpatients.     

Microanalysis for MDR1 ATPase by high-performance liquid chromatography with a titanium dioxide column

MDR1 is clinically important because it is involved in multidrug resistance of cancer cells and affects the pharmacokinetics of various drugs. Because MDR1 harnesses adenosine 5'-triphosphate (ATP) hydrolysis for transporting drugs, examining the effect on ATPase activity is imperative for understanding the interactions between drugs and MDR1. However, conventional assay systems for ATPase activity are not sensitive enough for screening drugs using purified MDR1. Here we report a novel method to measure ATPase activity of MDR1 using high-performance liquid chromatography equipped with a titanium dioxide column. The amount of adenosine 5'-diphosphate (ADP) produced by the ATPase reaction was determined within 2 min with a titanium dioxide column (4.6 mm ID x 100 mm). The relationship between ADP amount and chromatogram peak area was linear from 5 pmol to 10 nmol. This method made it possible to reduce the amount of purified MDR1 required for a reaction to 0.5 ng, about 1/20th of the conventional colorimetric inorganic phosphate detection assay. This method is sensitive enough to detect any subtle changes in ATPase activity of MDR1 induced by drugs and can be applied to measure ATPase activity of any protein.     

Electrophoretic methods for studying protein-protein interactions.

Protein-protein interactions are involved in many biological processes ranging from DNA replication, to signal transduction, to metabolism control, to viral assembly. The understanding of those interactions would allow the effective design of new drugs and further manipulation of those interactions. Several useful analytical methods are available for the study of protein-protein binding, and among them, electrophoresis is commonly used. We describe two types of electrophoresis: gel electrophoresis and capillary electrophoresis. Gel electrophoresis is a well-established method used to study protein-protein interactions and includes overlay gel electrophoresis, charge shift method, band shift assay, countermigration electrophoresis, affinophoresis, affinity electrophoresis, rocket immunoelectrophoresis, and crossed immunoelectrophoresis. These techniques are briefly described along with their advantages and limitations. Capillary electrophoresis, on the other hand, is a relatively new method and affinity capillary electrophoresis has demonstrated its value in the measurement of binding constants, the estimation of kinetic rate constants, and the determination of stoichiometry of biomolecular interactions. It offers short analysis time, requires minute amounts of protein samples, usually involves no radiolabeled compounds, and, most importantly, is carried out in solution. We summarize the principles of affinity capillary electrophoresis for studying protein-protein interactions along with current limitations and describe in depth its application to the determination of stoichiometries of tight and weak binding protein-protein interactions. The protocol presented in the experimental section details the use of affinity capillary electrophoresis for the determination of stoichiometry of protein complexes.     

Putative endogenous ligands for the benzodiazepine receptor.

The recent discovery of pharmacologically relevant, high affinity, stereospecific binding sites for the benzodiazepines in the central nervous system (CNS) has rekindled investigations concerning the mechanism of action of these drugs. It has become increasingly clear that elucidation of benzodiazepine action will provide new and important insights into the neurochemical substances of seizure activity, centrally mediated muscle relaxation and anxiety, three major actions of this class of drugs.The existence of a functional receptor for the benzodiazepines, compounds not present $\text{in vivo}$, suggests that endogenous substances exist that serve as natural substrates for this receptor. Furthermore, the characterization of endogenous benzodiazepine receptor ligands affords an opportunity to determine the neurochemical mechanisms underlying the pharmacologic and behavioral effects manifested by the benzodiazepines.Using receptor binding methodology to assay tissue extracts for [³H] diazepam binding inhibitory activity, putative endogenous ligands for the benzodiazepine receptor have been isolated and identified as the purine nucleosides. Compounds such as inosine and hypoxanthine exhibit competitive inhibition of [³H] diazepam binding. The low affinity purinergic inhibition of diazepam binding is consistent with their $\text{in vivo}$ concentrations. Distinct structure-activity relationships exist for the purines with subtle structural alterations having marked effects on diazepam binding inhibitory potency. The methylxanthine stimulants, caffeine, theophylline, and theobromine, also competitively inhibit diazepam binding, suggesting that some of their actions may be mediated by the benzodiazepine receptor.The purines also have “benzodiazepine-like” pharmacologic properties, since they have been shown to antagonize pentylenetetrazol induced seizures in mice in a dose dependent manner. Neurophysiologic studies have also shown that iontophoresis of inosine on cultured mouse primary neurons produce neurotransmitter like effects. Furthermore, these effects are similar to those observed with flurazepam, a finding that provides additional evidence for the “benzodiazepine-like” properties of the purines.The preliminary studies outlined below indicate that the purines are good candidates as putative endogenous ligands for the benzodiazepine receptor and provide a foundation for future studies that concern the homeostatic mediation of seizure activity and anxiety.     

Antagonism of RO 15-1788 with benzodiazepines in the effect on motivated aggression and the action of analgesics

The influence of Ro 15-1788 and bicuculline on the action of GABA-positive drugs (muscimol), GABA cethyl ester, piracetam and depakine and benzodiazepine tranquilizers (diazepam, phenazepam) on motivated aggression has been studied. It has been shown that Ro 15-1788 which has a weak antiaggressive effect selectively antagonizes the anti-aggressive effect of tranquilizers but not that of GABA-positive drugs. Bicuculline antagonizes antiaggressive activity of the drugs of both types. The action of these antagonists on the effect of the drugs under study as regards the analgetic activity of morphine was also studied. It has been shown that Ro 15-1788 antagonizes the potentiation of morphine analgesia caused by diazepam. At the same time Ro 15-1788 does not influence morphine analgesia potentiated by muscimol. Bicuculline removes the potentiation of morphine analgesia caused both by diazepam and muscimol it is concluded that bicuculline-sensitive GABA receptors modulate the antiaggressive effect of benzodiazepines and their influence on the analgetic action of opiates.     

Some insights into the binding mechanism of the GABAA receptor: a combined docking and MM-GBSA study

Gamma-aminobutyric type A receptor (GABA_AR) is a member of the Cys-loop family of pentameric ligand gated ion channels (pLGICs). It has been identified as a key target for many clinical drugs. In the present study, we construct the structure of human 2α₁2β₂γ₂ GABA_AR using a homology modeling method. The structures of ten benzodiazepine type drugs and two non-benzodiazepine type drugs were then docked into the potential benzodiazepine binding site on the GABA_AR. By analyzing the docking results, the critical residues His102 (α₁), Phe77 (γ₂) and Phe100 (α₁) were identified in the binding site. To gain insight into the binding affinity, molecular dynamics (MD) simulations were performed for all the receptor–ligand complexes. We also examined single mutant GABA_AR (His102A) in complexes with the three drugs (flurazepam, eszopiclone and zolpidem) to elucidate receptor–ligand interactions. For each receptor–ligand complex (with flurazepam, eszopiclone and zolpidem), we calculated the average distance between the C_α of the mutant residue His102A (α₁) to the center of mass of the ligands. The results reveal that the distance between the C_α of the mutant residue His102A (α₁) to the center of flurazepam is larger than that between His102 (α₁) to flurazepam in the WT type complex. Molecular mechanic-generalized Born surface area (MM-GBSA)-based binding free energy calculations were performed. The binding free energy was decomposed into ligand-residue pairs to create a ligand-residue interaction spectrum. The predicted binding free energies correlated well (R ²?=?0.87) with the experimental binding free energies. Overall, the major interaction comes from a few groups around His102 (α₁), Phe77 (γ₂) and Phe100 (α₁). These groups of interaction consist of at least of 12 residues in total with a binding energy of more than 1 kcal mol^?1. The simulation study disclosed herein provides a meaningful insight into GABA_AR–ligand interactions and helps to arrive at a binding mode hypothesis with implications for drug design.     

In vivo quantification of protein-protein interactions in Saccharomyces cerevisiae using bimolecular fluorescence complementation assay

Most of the biological processes are carried out and regulated by dynamic networks of protein-protein interactions. In this study, we demonstrate the feasibility of the bimolecular fluorescence complementation (BiFC) assay for in vivo quantitative analysis of protein-protein interactions in Saccharomyces cerevisiae. We show that the BiFC assay can be used to quantify not only the amount but also the cell-to-cell variation of protein-protein interactions in S. cerevisiae. In addition, we show that protein sumoylation and condition-specific protein-protein interactions can be quantitatively analyzed by using the BiFC assay. Taken together, our results validate that the BiFC assay is a very effective method for quantitative analysis of protein-protein interactions in living yeast cells and has a great potential as a versatile tool for the study of protein function.     

In vitro and in vivo effects of some benzodiazepine drugs on human and rabbit erythrocyte carbonic anhydrase enzymes

Carbonic anhydrase inhibitors (CAIs) are a class of pharmaceuticals used as antiglaucoma agents, diuretics and antiepileptics. Thus, discovery of novel CAIs has become of great importance in the recent years. In the current study, in vitro and in vivo inhibition effects of benzodiazepine drugs, diazepam and midazolam, on human erythrocytes carbonic anhydrase I and II isozymes were investigated. After purification of the isoenzymes, in vitro inhibition assays were performed and K(i) values were determined to be of 141.5 μM and 40.7 μM for hCA I and of 5.11 μM and 0.58 μM against hCA II by the esterase activity assay, respectively. The drugs showed strong inhibitory effects on hCA II, in the same range as the clinically used sulphonamide acetazolamide. For in vivo studies, five adult male New Zealand White rabbits (3-4.2 kg) were selected for intravenous administrations of the drugs (2 mg/kg and 0.2 mg/kg body weight, respectively). The enzyme was significantly inhibited by 2 mg/kg diazepam (p < 0.05), and 0.2 mg/kg midazolam (p < 0.05) for up to 30 min following intravenous administration.     

Subcellular localization of two neurotropic drugs in three varieties of central nervous system cells by secondary ion mass spectroscopy (SIMS) microscopy.

The intracellular localization of two neurotropic drugs, flunitrazepam (benzodiazepine) and triflupromazine (phenothiazine), was studied by secondary ion mass spectrometry microscopy (SIMS) in three varieties of cells. The images of the intracellular distributions of the two drugs are easily obtained by selecting the fluorine atom of the molecules. These images show that the drug from the benzodiazepine group is mainly located in the nuclei, whereas the phenothiazine is exclusively located inside the cytoplasm.     

Effects of diazepam and muscimol on GABA-mediated neurotransmission: interactions with inosine and nicotinamide.

Inosine and nicotinamide have been proposed as endogenous ligands for the brain benzodiazepine receptor. An $\text{in vivo}$ method for detecting drugs with GABA-mimetic properties was used to examine the effects of inosine, nicotinamide and diazepam in the rat globus pallidus. Inosine and nicotinamide completely prevented the GABA-mimetic action of diazepam but neither compound alone had any GABA-like activity. These findings suggest that inosine and nicotinamide are able to antagonize but are not able to mimic the GABA-like actions of diazepam at the benzodiazepine receptor.     

Prevalence,frequency, and duration of hypnotic drug use among the elderly living at home

Details of consumption of hypnotic drugs derived from a nationally representative sample of elderly people were analysed in terms of the prevalence, duration, and likely frequency of use. Of 1020 randomly selected subjects aged 65 and over 16% (166) reported using (mainly benzodiazepine) hypnotic drugs, and of these 89% reported having taken such a drug the night before the interview. Most of these users (73%) had been taking hypnotic drugs for more than one year, with 25% reporting drug use for more than 10 years.These results suggest that for most elderly users of hypnotic drugs, patterns of consumption encourage the development of cumulative effects and benzodiazepine dependence.     

Effect of ergot alkaloids on 3H-flunitrazepam binding to mouse brain GABAA receptors

In vitro effects of dihydroergotoxine, dihydroergosine, dihydroergotamine, alpha-dihydroergocriptine (ergot alkaloids), diazepam, methyl-beta-Carboline-3-carboxilate (beta-CCM), flumazenil (benzodiazepines), gamma-amino butyric acid (GABA) and thiopental (barbiturate) were studied on mouse brain (cerebrum minus cerebral cortex) benzodiazepine binding sites labeled with 3H-flunitrazepam. Specific, high affinity (affinity constant, Kd = 57.7 8.6 nM) binding sites for 3H-flunitrazepam on mouse brain membranes were identified. All benzodiazepine drugs inhibited 3H-flunitrazepam binding with nanomolar potencies. In contrast to benzodiazepines, all ergot drugs, GABA and thiopental produced an enhancement of 3H-flunitrazepam binding to its binding site at the GABAA receptor of the mouse brain. The rank order of potency was: neurotransmitter (GABA) > dihydroergotoxine > thiopental > alpha-dihydroergocriptine > dihydroergosine > dihydroergotamine. The results suggest that dihydrogenated ergot derivatives do not bind to the brain benzodiazepine binding sites labeled with 3H-flunitrazepam. However, an enhancement of 3H-flunitrazepam binding by all ergot drugs tested, clearly identifies an allosteric interaction with the benzodiazepine binding sites of GABAA receptors.     

Specific Adsorbents for Affinity Chromatography of Benzodiazepine Binding Proteins

The use of affinity chromatography as means of isolating/ purifying proteins which have an affinity for benzodiazepine is described. Three such drugs are employed: chlorazepate, clonazepam and delorazepam. The results presented in this paper indicate that the proposed technique only works for chlorazepate and delorazepam. In fact these benzodiazepine–Sepharose derivatives are able to retain specifically proteins from human serum and rat kidney, lung, skeletal muscle and brain.