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Several new benzodiazepines selectively interact with a benzodiazepine receptor subtype
Abstract
The potency of several new benzodiazepines as inhibitors of [3H]flunitrazepam binding was investigated in membranes from rat cerebellum or hippocampus. It was found that quazepam and two of its metabolites have a higher affinity for benzodiazepine receptors in cerebellum than for those in hippocampus, indicating a preferential interaction of these compounds with BZ1-receptors. Other experiments indicated that these compounds have benzodiazepines agonistic properties similar to diazepam or flunitrazepam. Thus for the first time, benzodiazepines have been identified with differentially interact with different benzodiazepine receptors.
... Recently, quazepam [7-chloro-1-(2,2,2-trifluoroethyl)-5-(o-fluorophenyl)-1,3-dihydro-2H-1,4benzodiazepine-2-thione] and its derivatives con- (Sieghart, 1983;Iorio et al., 1984;Barnett et al., 1985;Wamsley e t al., 1985). ...
... In the present study we have shown that quazepam and its active metabolite SCH 15725, two trifluoroethyl benzodiazepines with preferential affinity for type 1 benzodiazepine recognition sites (Sieghart, 1983;Wamsley et al., 1985), produce a concentration-dependent increase in the density of low-affinity GABA receptors in membrane preparations from rat cerebral cortex. This finding is in agreement with several biochemical and pharmacological studies showing that benzodiazepines facilitate GABAergic transmission (for review, see Costa and Guidotti, 1979). ...
... Our present results, however, do not allow us to conclude that type I benzodiazepine recognition sites are solely responsible for the increase in l3H1GABA binding induced by quazepam and SCH 15725. Thus, the ICso values of these two compounds for type I benzodiazepine recognition sites are in the low nanomolar range (Sieghart, 1983). At the latter concentrations, however, these compounds failed to increase [3H]GABA binding, whereas at the concentrations required to enhance the density of GABA receptors (lop6 M ) , the occupancy of both type I and type I1 sites is >99%. ...
We evaluated the effect of the two N-trifluoroethyl benzodiazepines, quazepam and its 2-oxo metabolite SCH 15725, which possess preferential affinity for type I benzodiazepine recognition sites, on the binding of [3H] gamma-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. The study also included compounds such as diazepam and N-desalkyl-2-oxoquazepam (SCH 17514), which have equal affinity for the type I and type II receptor subtypes. Binding of [3H]GABA was studied in frozen-thawed and repeatedly washed cortical membranes incubated in 20 mM KH2PO4 plus 50 mM KCl, pH 7.4, at 4 degrees C in the absence and presence of quazepam or its metabolites. Addition of 10(-6) M quazepam increased by 30% specific [3H]GABA binding; as revealed by Scatchard plot analysis, the effect was due to an increase in the total number of GABA receptors. The effect of quazepam was concentration dependent, and it was shared by its active metabolite SCH 15725. The potency of quazepam and SCH 15725 in enhancing [3H]GABA binding was similar to that of diazepam, whereas CL 218872 and SCH 17514 were less active. Moreover, the [3H]GABA binding-enhancing effect of quazepam was mediated by an occupancy of benzodiazepine receptors, because it was specifically antagonized by 5 X 10(-6) M Ro15-1788.
... Most BZDs bind to the central BZD receptors with similar affinities throughout the brain, but the binding properties of several compounds, most notably CL 218872 (Squires et al., 1979;Sieghart, 1983) and β-carboline-3-carboxylic acid ethyl ester (βCCE) demonstrate the heterogeneity of BZD receptors. ...
... Two types of BZD receptor were originally proposed based on their pharmacology and distribution. The BZD1 subtype found throughout the brain but predominant in the cerebellum scarcely in the hippocampus (Faull et al., 1987;Faull and Villiger, 1988;Olsen et al, 1990;Lüddens et al., 1995), show high affinity to the triazolopyridazine CL 218872, and βCCE (β-carboline-3-carboxylic acid ethyl ester) (Squires et al., 1979;Nielsen and Braestrup, 1980;Sieghart, 1983). The BZD2 ...
... Regional heterogeneity in the binding of certair ligands of the benzodiazepine receptor suggest that there art two subtypes of receptor with difIerent regional distribution BZ I is predominant in the cerebellum, while hippocampu! contains an equal density of BZ I and BZ 2 [1, 25,[54][55]63). Among the Iigands that bind preferentially to the BZ! recep· tor are the benzodiazepines quazepam [43][44]54] an( lorazepam, the ,B-carbolines, ,B-CCM, ,B-CCE, ,B-CCP ane ,B-CCtB [l, 15, 34, 53-55], and the triazolopyridazine, Cl 218 872 (see [25,33]). ...
... contains an equal density of BZ I and BZ 2 [1, 25,[54][55]63). Among the Iigands that bind preferentially to the BZ! recep· tor are the benzodiazepines quazepam [43][44]54] an( lorazepam, the ,B-carbolines, ,B-CCM, ,B-CCE, ,B-CCP ane ,B-CCtB [l, 15, 34, 53-55], and the triazolopyridazine, Cl 218 872 (see [25,33]). ...
Derivatives of ethyl-beta-carboline-3-carboxylate, ZK 91296, ZK 93423 and ZK 95962 have potent anticonvulsant activity against sound-induced seizures in audiogenic DBA/2 mice and against photically-induced seizures in the baboon, Papio papio. The convulsant beta-carbolines, DMCM and beta-CCM, have proconvulsant and convulsant activity in the same animal models. DMCM and beta-CCM are similar in potency as convulsants in DBA/2 mice (ED50 value for DMCM: 1.3 mg/kg; ED50 value for beta-CCM; 0.8 mg/kg), but differ with respect to their profiles for protection by anticonvulsant drugs. The anticonvulsant potencies of diazepam and clobazam are similar against both types of beta-carboline-induced seizures, whereas quazepam protects better against beta-CCM seizures (4 fold elevation in ED50 value at 1 mg/kg quazepam IP) than against DMCM seizures (1.7 fold elevation in ED50 value), supporting a preferential action of beta-CCM on BZ1 receptors. Valproate (400 mg/kg) and gamma-vinyl-GABA (1.5 g/kg) protect better against beta-CCM seizures (9.5 and 5.9 fold elevations in ED50 values respectively) than against DMCM seizures (1.8 and 2.7 fold elevations in ED50 values respectively). The excitatory amino acid antagonist, 2-amino-7-phosphonoheptanoic acid, has significant anticonvulsant activity against DMCM seizures. The elevated regional GABA levels in brains of DBA/2 mice observed during beta-CCM seizures are eliminated by the pretreatment with Ro 15-1788, which also blocks the seizure activity.
... Metabolism results in the replacement of the =S to =O, the 2oxoquazepam metabolite (Fig. 56). This metabolite has 2-to 3-fold greater potency than the parent (Sieghart, 1983) and is present in circulation at about two-thirds of the parent (Chung et al., 1984). As such, it can be estimated that 2-oxoquazepam contributes substantially to the total activity. ...
Metabolism represents the most prevalent mechanism for drug clearance. Many drugs are converted to metabolites that can retain the intrinsic affinity of the parent drug for the pharmacological target. Drug metabolism redox reactions such as heteroatom dealkylations, hydroxylations, heteroatom oxygenations, reductions, and dehydrogenations can yield active metabolites, and in rare cases even conjugation reactions can yield an active metabolite. To understand the contribution of an active metabolite to efficacy relative to the contribution of the parent drug, the target affinity, functional activity, plasma protein binding, membrane permeability, and pharmacokinetics of the active metabolite and parent drug must be known. Underlying pharmacokinetic principles and clearance concepts are used to describe the dispositional behavior of metabolites in vivo. A method to rapidly identify active metabolites in drug research is described. Finally, over 100 examples of drugs with active metabolites are discussed with regard to the importance of the metabolite(s) in efficacy and safety.
... Several drugs that interact with brain BDZ binding sites differ in their affinity in different brain regions. For example quazepam, halazepam, CL 218,872 and fiCCE are more potent at displacing [3H]BDZ ligands in the rat cerebellum than the rat hippocampus (Braestrup & Nielsen, 1981;Stapleton et al., 1982;Sieghart, 1983;Iorio et al., 1984;Sieghart & Schuster, 1984). The proposed explanation is that the above drugs have a higher affinity for the so-called BDZ, subset of binding sites, which is the predominant (or only) type in the cerebellum whereas the hippocampus contains both BDZ1 and BDZ2 subtypes (Braestrup & Nielsen, 1981) The present results indicate that the binding of substituted benzamides to BDZ receptors is highly structure specific, and the nature of aromatic substituents, the basic side-chain and N-substituents all seem to play a critical role. ...
The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [ ³ H]‐flunitrazepam ([ ³ H]‐FN m ) from specific binding sites in bovine cortical membranes in vitro .
Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration‐dependent displacement of [ ³ H]‐FNM with IC 50 values of 73 nM, 132 nM, 7.7 μ m and 5.9 μ m , respectively. Other substituted benzamides including metoclopramide, sulpiride, tiapride, sultopride and cisapride were inactive at 10 ⁻⁵ m .
Inhibition by clebopride and Delagrange 2674 of [ ³ H]‐FNM binding was apparently competitive and readily reversible.
In the presence of γ‐aminobutyric acid (GABA), the ability of diazepam and Delagrange 2674 to displace [ ³ H]‐Ro 15–1788 binding was increased 3.6 and 1.6 fold respectively, compared to the absence of GABA, while ethyl β‐carboline‐3‐carboxylate (βCCE) and clebopride were less potent in the presence of GABA.
Diazepam was 30 fold less potent at displacing [ ³ H]‐Ro 15–1788 in membranes that had been photoaffinity labelled with FNM than in control membranes, whereas the potency of βCCE did not differ. Clebopride and Delagrange 2674 showed a less than two fold loss of potency in photoaffinity labelled membranes.
The pattern of binding of clebopride and Delagrange 2674 in these in vitro tests is similar to that found previously with partial agonists or antagonists at BDZ binding sites.
Clebopride and Delagrange 2674 inhibited [ ³ H]‐FNM binding with similar potency in rat cerebellar and hippocampal membranes, suggesting they have no selectivity for BDZ 1 and BDZ 2 binding sites.
Clebopride and Delagrange 2674 are structurally dissimilar to other BDZ ligands and represent another chemical structure to probe brain BDZ binding sites.
DN-2327, 2-(7-chloro-1,8-naphthyridin-2-yl)-3-[(1,4-dioxa-8-azaspiro-[4.5]dec-8-yl)carbonylmethyl]isoindolin-1 -one, produced anxiolytic, taming and anti-convulsive effects when administered orally to several species of animals. DN-2327 produced few of the sedative-hypnotic and muscle-relaxant effects observed with diazepam. The durations of the anxiolytic and anti-convulsive activities of DN-2327 were much longer than those of diazepam. Tolerance to DN-2327 did not develop when it was administered daily for 14 days in an anti-conflict test (Vogel conflict test). DN-2327 showed potent displacement activity against [³H]diazepam binding. The binding affinity of DN-2327 for benzodiazepine receptors was about twenty times that of diazepam. Furthermore, the affinity of DN-2327 for benzodiazepine receptors was not enhanced by the presence of GABA. There is a wide margin between the doses of DN-2327 that cause the anxiolytic effects and its sedative-hypnotic/muscle-relaxant effects. These results suggest that DN-2327 has more marked anxioselective properties compared with the benzodiazepines.
Insomnia is a prevalent disorder that affects over one-third of the U.S. population to varying degrees and is highly disruptive towards quality of life. Pharmacological treatments for insomnia include benzodiazepines (BZs) and the non-BZ 'Z-drugs' (zolpidem, zaleplon, eszopiclone, zopiclone), which are amongst the most widely prescribed medications. Yet, these agents can produce adverse effects such as tolerance to the hypnotic effect, rebound insomnia, next-day residual drowsiness, as well as amnesia and complex behaviours such as sleep-walking, sleep-eating and sleep-driving. Quazepam, one of the five BZ approved for treatment of insomnia, was recently relaunched to the U.S. market in 2016 and is distinguished amongst hypnotic BZ by unique pharmacological characteristics including selectivity for sleep-promoting α1-subunit containing γ-aminobutyric acid (GABA-A) receptors and a significantly lower relative receptor binding affinity. These features likely drive the decreased rate of adverse events seen clinically with quazepam, such as tolerance, rebound insomnia and amnesic behaviours, compared with other BZ. Given the recent reintroduction of quazepam as a pharmacotherapeutic option, and the lack of head-to-head comparative trials against newer agents, the purpose of this review is to provide an update on distinguishing features of quazepam with regard to its pharmacology, pharmacokinetics, sleep efficacy and potential adverse effects compared to other agents used for insomnia.
Nach der Entdeckung der „zentralen“ Benzodiazepinrezeptoren im Jahre 1977 (Braestrup u. Squires 1977; Möhler u. Okada 1977) wurden diese Rezeptoren durch eine Vielzahl an pharmakologischen Untersuchungen charakterisiert. Es konnte gezeigt werden, daß die Benzodiazepinrezeptoren eng mit einem GABA-Rezeptor, einem Chloridionenkanal und Bindungsstellen für Pikrotoxinin, Avermektin und andere Substanzen assoziiert sind (Tallman et al. 1980; Olsen 1982). Alle diese Bindungsstellen beeinflussen einander auf komplizierte Art und Weise, und es ist heute klar, daß der GABA-Benzodiazepinrezeptor eine hoch regulierbare, komplexe molekulare Struktur besitzt.
Im Laufe der Zeit wurden viele verschiedene Substanzen zur Bekämpfung der Angst verwendet, so z. B. Äthanol, in Form von alkoholischen Getränken, aber auch Opiate, Barbiturate, andere Sedativa, β-Blocker, Neuroleptika und Antide-pressiva. 1951 wurde Meprobamat erstmals ausgetestet und galt damals bald als das relativ potenteste Anxiolytikum (Berger 1963). 1957 wurde dann die Tranquilizerwirkung des von Sternbach synthetisierten Benzodiazepins Chlordiazepoxid entdeckt, und bereits 2½ Jahre später wurde diese Substanz unter dem Namen Librium in die Klinik eingeführt (Sternbach 1980). Weitere intensive Forschungsarbeiten führten zur Synthese einer Vielzahl von verschiedenen Benzodiazepinen (BZD), die sich von Librium v. a. durch ihre wesentlich höhere anxiolytische Potenz unterscheiden (Sternbach 1980). Da BZD von allen Medikamenten die stärkste anxiolytische Wirksamkeit aufwiesen, bestand von allem Anfang an die Hoffnung, daß die genaue Kenntnis ihres Wirkungsmechanismus möglicherweise auch Aufschluß über die physiologischen und biologischen Mechanismen der Angst geben würde. Neben ihrer anxiolytischen Wirkung besitzen BZD aber auch antikonvulsive, muskelrelaxierende und hypnotische Eigenschaften, und da alle bekannten BZD diese verschiedenen Wirkungen in mehr oder weniger ähnlichem Maße ausüben, wurde ursprünglich angenommen, daß die einzelnen BZD-Wirkungen nicht voneinander zu trennen sind.
The challenging basic science of anxiolytic drug action and the potential for compounds with profiles different from the traditional anti-anxiety drugs has focused attention upon more selective structure activity relationships. Insights into the gamma-aminobutyric acid (GABA)ergic effects of these drugs and their anti-convulsant properties continue to grow. The effects of drugs upon deprivation-induced drinking should be considered to avoid false-positive indications. The susceptibility of compounds anti-conflict effects to blockade by BZ antagonists appears to distinguish BZ receptor binders from nondisplacers. Midazolam continues to receive substantial support as an effective hypnotic with neither tolerance effects nor rebound insomnia on drug withdrawal. There is no distinguishable difference between the nocturnal effects of triazolam or flurazepam, however, the “hangover” effects of these two drugs are still under evaluation. The hypnotic and anti-convulsant profile of etomidate have been discussed in the chapter. Based on the comparative clinical effects on the photopalpebral reflex, zopiclone was slightly more potent than nitrazepam with more rapid onset, shorter duration of action, and fewer side effects. The effects of several marketed anti-epileptic drugs including carbamazepine, phenytoin, and sodium valproate, upon cognitive function and behavior, have been clinically assessed. Adenosine receptor binding studies of carbamazepine indicate that its anticonvulsant action may be due in part to A1 receptor agonist and A2 receptor antagonist activities. Potential anti-convulsant activity of α-adrenergic agonists was indicated by their in vitro inhibition of interictal discharge in rat hippocampal slices; β-adrenergic agonists showed proconvulsant actions.
We are all familiar with the term benzodiazepine, as are a significant proportion of the general public. In the public domain it is a term synonymous with diazepam that connotes dependence and withdrawal. The scientific community also has its misconceptions about the term benzodiazepine, which is perhaps not surprising given the speed of evolution of our knowledge about these drugs, their receptors and the uniqueness of their pharmacology. In no other field of CNS pharmacology has such a short time elapsed between the discovery of a receptor and its cloning and expression. It is at times hard to believe that the first report of brain-specific benzodiazepine receptors was made only 12 years ago (Squires and Braestrup, 1977; Mohler and Okada, 1978). Not surprisingly, some of the more recent and novel aspects of benzodiazepine pharmacology have not yet been fully assimilated.
Cell-to-cell communication in the nervous system is brought about mainly by the exchange of chemical signals — the neurotransmitters or neuromodulators. A biological response is elicited by the interaction of these signals with highly selective receptors on target cells which discriminate between various neurotransmitters. Psychoactive drugs interfere in various ways with chemical signalling in the central nervous system, frequently producing their specific effects by interacting with distinct proteins or glycoproteins in the target ceil membrane — the pharmacological receptors (Ariëns, 1983; Snyder, 1984). The benzodiazepines are no exception.
In der vorliegenden Studie wurde die Pharmakokinetik von Prazepam, Quazepam und Temazepam mit Hilfe einer Rezeptorbindungsmethode untersucht. Die dabei erhaltenen Ergebnisse stimmen in allen Punkten mit anderen Untersuchungen überein, die die Pharmakokinetik dieser Substanzen mit Hilfe von chromatographischen Methoden bestimmt haben. Auf Grund der Anwendbarkeit der Rezeptorbindungsmethode für alle Benzodiazepine bzw. Benzodiazepin-ähnliche Substanzen und auf Grund der Einfachheit dieser Bestimmungen wird vorgeschlagen, diese Methode für pharmakokinetische Untersuchungen und Routineuntersuchungen der Serumspiegel von Benzodiazepinen zu verwenden. Für die Beantwortung spezieller Fragestellungen müssen aber nach wie vor Chromatographische Methoden zur Bestimmung von Benzodiazepinen und ihren aktiven Metaboliten verwendet werden.
2-Oxoquazepam (2oxoquaz) is a novel benzodiazepine which shows preferential affinity for type I benzodiazepine recognition sites. In the present study, we analyzed the effect of γ-aminobutyric acid (GABA), pentobarbital, and chloride ions on [3H]2oxoquaz and [3H]flunitrazepam ([3H]FNT) binding to membrane preparations from rat and human brain. GABA stimulated [3H]-2oxoquaz and [3H]FNT binding in a concentration-dependent manner. The maximal enhancement produced by GABA on [3H]2oxoquaz binding was higher than that produced on [3H]FNT binding in both rat and human tissues. In the rat brain, the effect of GABA on [3H]2oxoquaz was similar throughout different brain areas, whereas the effect on [3H]FNT binding was lower in the cerebral cortex and hippocampus than in the cerebellum. Moreover, both [3H]2oxoquaz and [3H]FNT binding were stimulated by chloride ions and pentobarbital. The results are consistent with the hypothesis that type I benzodiazepine recognition sites are linked functionally to the GABA recognition site and the chloride ionophore.
: We evaluated the effect of the two N-trifluoroethyl benzodiazepines, quazepam and its 2-oxo metabolite SCH 15725, which possess preferential affinity for type I benzodiazepine recognition sites, on the binding of [3H]γ-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. The study also included compounds such as diazepam and N-desalkyl-2-oxoquazepam (SCH 17514), which have equal affinity for the type I and type II receptor subtypes. Binding of [3H]GABA was studied in frozen-thawed and repeatedly washed cortical membranes incubated in 20 mM KH2PO4 plus 50 mM KCl, pH 7.4, at 4°C in the absence and presence of quazepam or its metabolites. Addition of 10−-6M quazepam increased by 30% specific [3H]GABA binding; as revealed by Scatchard plot analysis, the effect was due to an increase in the total number of GABA receptors. The effect of quazepam was concentration dependent, and it was shared by its active metabolite SCH 15725. The potency of quazepam and SCH 15725 in enhancing [3H]GABA binding was similar to that of diazepam, whereas CL 218872 and SCH 17514 were less active. Moreover, the [3H]GABA binding-enhancing effect of quazepam was mediated by an occupancy of benzodiazepine receptors, because it was specifically antagonized by 5 × 10−-6M Ro15–1788.
Starting with a short overview on the pharmacology of Benzodiazepine (BZ) receptor ligands that includes aspects of their therapeutic actions, development of tolerance and dependence to BZ and their metabolism this review pays special attention to the interaction and mode of action of BZ with the various γ‐aminobutyric acid type A (GABA A ) receptors.
We discuss the diversity of GABA A receptors with respect to the subunits, receptor subtypes, brain distribution and function. The functional domains on GABA A receptors needed for the recognition and action of GABA and BZ as well as those needed for assembly are described. The structure activity relations of benzodiazepines are discussed with an emphasis on GABA A receptor subtypes.
Tranquilizers, and specifically benzodiazepines (BZD), have become one of the most widely used group of drugs in the world; some have suggested too widely used. Interest in these drugs in still extremely high, both from the scientific and sociological point of view. This is best documented by the fact that the present review is based on more than 700 articles published between 1981 and 1984 covering various aspects of clinical pharmacology. Pharmacodynamic studies are mostly concerned with effects of the drug on the central nervous system (CNS), as well as on behavious and neuroendocrine functions. The former include computer-assisted quantitative analyses of the scalp-recorded wake electroencephalogram (EEG), based on which it seems possible to determine if, how, when and at what dose a newly developed compound affects the human brain. Sleep investigations are now concerned not only with the effect of the drug on the all-night sleep, but also on the quality of awakening and early morning behaviour as well as with residual effects during daytime, affecting amnestic and cognitive functions, psychomotor performance (specifically in regard to driving ability), as well as mood and affectivity. Finally, for almost a decade increasing concern has been expressed about the widespread use of tranquillizers in society. Scientific evidence concerning thiss issue will be reviewed so that an appropriate medical decision may be reached for the treatment of the individual patients after consideration of the beneficial effects as well as undesirable side-effects.
Benzodiazepine (BDZ) hypnotics bind at a specific receptor located on postsynaptic neurons. Some data support specificity of binding for several hypnotics to receptor subtypes. We evaluated BDZ receptor binding in cerebral cortical membranes using agonist, antagonist, and subtype-specific ligands for commonly used hypnotics and their metabolites. All hypnotics competed similarly at BDZ1 and BDZ2 receptor subtypes except quazepam and its metabolite 2-oxo-quazepam and to a lesser extent hydroxyethyl flurazepam (EtOH) flurazepam. These compounds had relative specificity for the BDZ1 site. Triazolam, estazolam, and flurazepam bound equally to sites labeled by agonists and antagonists but desalkylflurazepam, EtOH flurazepam, temazepam, quazepam, and 2-oxo-quazepam did not; in addition, these four compounds did not bind to the "peripheral" BDZ site labeled by Ro 5-4864. BDZ hypnotics differ in their receptor subtype and ligand binding characteristics.
Electroencephalogram (EEG) effect parameters may be useful in pharmacokinetic-pharmacodynamic modelling studies of drug effects on the central nervous system (CNS). Effect parameters derived from a quantitative analysis of the EEG appear to be perfectly suited to characterise the relationships between pharmacokinetics and pharmacodynamics of benzodiazepines and intravenous anaesthetics. EEG parameters represent many of the characteristics of ideal pharmacodynamic measures, being continuous, objective, sensitive and reproducible. These features provide the opportunity to derive concentration-effect relationships for these drugs in individuals, which yield important quantitative information on the potency and intrinsic efficacy of these drugs. The EEG techniques presented can be used to study the influences of factors such as age, disease, chronic drug use and drug interactions on the concentration-effect relationships of psychotropic drugs.
An important issue is the choice of the EEG parameter to characterise the CNS effects of the compounds. More attention must be paid to evaluating the relevance of EEG parameters to the pharmacological effects of the drugs. Knowledge of the relationship between EEG effect parameters and clinical effects of drugs under different physiological and pathophysiological conditions is crucial to determining the value of EEG parameters in drug effect monitoring.
Pharmacodynamic parameters derived from the concentration-EEG effect relationship may be correlated to pharmacodynamic parameters obtained from other in vitro and in vivo effect measurements. These comparisons revealed that changes in the amplitudes in the β frequency band of EEG signals is a relevant measure of pharmacological effect intensity of benzodiazepines, which reflects their affinity and intrinsic efficacy at the central γ-aminobutyric acid (GABA) benzodiazepine receptor complex. The exact EEG correlates of the anxiolytic, anticonvulsant, sedative and hypnotic actions of benzodiazepines have not yet clearly been elucidated. For intravenous anaesthetics, close correlations between the potency determined with EEG measurements and clinical measures of anaesthetic depth have been established, suggesting that, in principle, EEG parameters can adequately reflect depth of anaesthesia. However, more study is required to further substantiate these findings.
In order to characterize the pharmacologic profile of DN-2327, an isoindoline benzodiazepine (BZD) receptor ligand, its interactions with Ro15-1788 and diazepam were analyzed in rodents. The anti-conflict action of DN-2327 in two conflict tests using rats, the punished water-lick conflict (Vogel conflict) and the punished bar-pressing conflict test, was completely attenuated by treatment with Ro15-1788. The anti-convulsive (pentylenetetrazol [PTZ] induced convulsion) effect of DN-2327 was also reduced by Ro15-1788. These results suggest that the anti-conflict and anti-convulsive actions of DN-2327 may be mediated via BZD receptors. On the other hand, DN-2327 only slightly affected the motor coordination in mice and rats, as estimated by the inclined screen test and the climbing test, respectively; however, the compound attenuated the motor incoordination produced by diazepam. Furthermore, the pentobarbital potentiating effect of diazepam was reduced by pretreatment with DN-2327 in mice. In the Vogel conflict test, additive effects were observed upon the conflict test, additive effects were observed upon the concomitant administration of subeffective doses (5 mg/kg, PO) of DN-2327 and diazepam. DN-2327 at 20 mg/kg, PO, did not reduce but slightly potentiated the anti-conflict effect of the maximum effective dose of diazepam. For PTZ-induced convulsions, DN-2327, 0.5 and 20 mg/kg, PO, doses which produced partial and complete anti-convulsive effects, respectively, in rats did not reduce but increased additively the effects of diazepam. DN-2327 at 10 and 20 mg/kg, PO, doses which both produced partial anti-convulsive effects in mice, showed an additive effect with the partial effects of diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)
Recombinantly expressed gamma-aminobutyric acid type A (GABAA) receptors consisting of alpha 1, beta 2, and gamma 2 subunits contain a binding site for benzodiazepines that differs in its properties from that of alpha 3 beta 2 gamma 2 receptors. Amino acid substitutions between the GABAA receptor alpha subunits were analyzed for their effect on the binding of compounds to the benzodiazepine site. By converting ever smaller regions of the alpha 3 subunit sequence to that of the alpha 1 subunit, we show that a single substitution (glycine for glutamic acid) increases the affinity for several compounds approximately 10-fold without changing the affinity for nonselective compounds. Hence, the identified amino acids may interact directly with the ligand and define part of the benzodiazepine binding sites in these receptors.
The hypnotics, quazepam (a benzodiazepine), brotizolam (a thienotriazolodiazepine), zopiclone (a cyclopyrrolone) and zolpidem (an imidazopyridine) have a common ability to bind to the benzodiazepine recognition site (omega receptor) within the GABAA receptor. For this reason we compared their pharmacological profiles in mice. All compounds shared anticonvulsant and central depressant effects. However, the sedative activity of zolpidem appeared at much lower doses than did the anticonvulsant and myorelaxant effects but the opposite was observed with the other hypnotics. In contrast to brotizolam, quazepam and zopiclone, zolpidem did not increase food intake in mice placed in a novel environment, indicating that this drug lacks disinhibitory activity. Moreover the efficacy of zolpidem at the GABAA receptor, as indicated by its activity against convulsions induced by the GABA synthesis inhibitor, isoniazid, was much greater than that of other hypnotics. These results suggest that the hypnoselective properties observed with zolpidem might be related to a high selectivity for the omega 1 recognition site of the GABAA receptor coupled with a very high intrinsic activity.
The effects of a single oral dose of alprazolam (1 mg), quazepam (15 mg) and diazepam (10 mg) on the peak saccadic velocity (PSV) of saccadic eye movements (SEM), the Sternberg memory scanning and choice reaction time (SMS-CRT), critical flicker fusion frequency (CFFF), spectral analysis of the EEG and a mood scale were assessed in 9 healthy volunteers in a double-blind, placebo-controlled cross-over study. Alprazolam revealed greater sedative effects than diazepam in the above-mentioned tests. Quazepam had the least sedative effect of the 3 drugs tested, showed a time lag at the onset of its effects and a more prolonged effect on psychomotor impairment than reported previously.
GABAA (gamma-aminobutyric acid A)-benzodiazepine receptors expressed in mammalian cells and assembled from one of three different alpha subunit variants (alpha 1, alpha 2, or alpha 3) in combination with a beta 1 and a gamma 2 subunit display the pharmacological properties of either type I or type II receptor subtypes. These receptors contain high-affinity binding sites for benzodiazepines. However, CL 218 872, 2-oxoquazepam, and methyl beta-carboline-3-carboxylate (beta-CCM) show a temperature-modulated selectivity for alpha 1 subunit-containing receptors. There were no significant differences in the binding of clonazepam, diazepam, Ro 15-1788, or dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) to all three recombinant receptors. Receptors containing the alpha 3 subunit show greater GABA potentiation of benzodiazepine binding than receptors containing the alpha 1 or alpha 2 subunit, indicating that there are subtypes within the type II class. Thus, diversity in benzodiazepine pharmacology is generated by heterogeneity of the alpha subunit of the GABAA receptor.
The interaction between GABAA receptors and benzodiazepine (BZD) recognition site subtypes in the spinal cord of the rat was investigated. Computer analysis of displacement curves for [3H]flunitrazepam [( 3H]FNT) binding by 2-oxo-quazepam (2OXOQ) indicated the presence of two subtypes of BZD recognition sites in this region. Type I sites accounted for approximately 25% of the total number of BZD recognition sites, the remainder being Type II sites. A similar proportion of Type I and Type II sites was obtained by Scatchard analysis of the saturation curves for [3H]FNT, [3H]2OXOQ and [3H]ethyl-beta-carboline-3-carboxylate [( 3H]beta CCE) binding. The in vitro addition of GABA (10(-8)-10(-4) M) to spinal cord membrane preparations produced an increase in the binding of [3H]FNT and [3H]2OXOQ. The maximal enhancement produced by GABA was 50 and 82% above control values for [3H]FNT and [3H]2OXOQ, respectively. In contrast, GABA stimulated both [3H]FNT and [3H]2OXOQ binding in the cerebellum to a similar extent. We also evaluated the effects of different ligands for BZD recognition sites on the binding of [3H]GABA to spinal cord membranes, as compared with brain areas containing a higher proportion ( greater than 30%) of Type I sites. Diazepam, quazepam and the beta-carboline, ZK 93423, enhanced the specific binding of [3H]GABA in a concentration-dependent manner (10(-7)-10(-5) M) in the cerebral cortex and hippocampus but not in the spinal cord and cerebellum. These results indicate that there is a regional variation in the interaction between GABA and BZD recognition sites in the central nervous system.
In the present study, the distribution of benzodiazepine recognition site subtypes in the brain of the cat was investigated. To this aim, the binding properties of [3H]2-oxo-quazepam ([3H]2OXOQ) and [3H]beta-CCE, two ligands with preferential affinity for Type I benzodiazepine recognition sites, were compared to binding parameters for [3H]flunitrazepam ([3H]FNT) in different areas of the cat brain. The ratio of [3H]2OXOQ to [3H]FNT binding sites indicated that, in the cerebellum, Type I sites accounted for 90% of the total number of benzodiazepine recognition sites. The cerebral cortex, thalamus and mesencephalic reticular formation had also a high proportion of Type I sites (73-78%), whilst the two subtypes were almost equally distributed in the hippocampus, amygdala and bulbar reticular formation. A similar distribution of subtypes of benzodiazepine recognition sites was indicated by the ratio of [3H]beta CCE to [3H]FNT binding sites for different areas of the brain. These results demonstrate the existence of heterogeneity of recognition sites for benzodiazepines in the brain of the cat and support the view that [3H]2OXOQ preferentially labels Type I sites.
2-Oxoquazepam (2oxoquaz) is a novel benzodiazepine which shows preferential affinity for type I benzodiazepine recognition sites. In the present study, we analyzed the effect of gamma-aminobutyric acid (GABA), pentobarbital, and chloride ions on [3H]2oxoquaz and [3H]flunitrazepam ( [3H]FNT) binding to membrane preparations from rat and human brain. GABA stimulated [3H]-2oxoquaz and [3H]FNT binding in a concentration-dependent manner. The maximal enhancement produced by GABA on [3H]2oxoquaz binding was higher than that produced on [3H]FNT binding in both rat and human tissues. In the rat brain, the effect of GABA on [3H]2oxoquaz was similar throughout different brain areas, whereas the effect on [3H]FNT binding was lower in the cerebral cortex and hippocampus than in the cerebellum. Moreover, both [3H]2oxoquaz and [3H]FNT binding were stimulated by chloride ions and pentobarbital. The results are consistent with the hypothesis that type I benzodiazepine recognition sites are linked functionally to the GABA recognition site and the chloride ionophore.
Receptor autoradiography was used to localize and quantify the distribution of benzodiazepine receptor sites in human post mortem materials using [³H]flunitrazepam. The distribution and density of these sites was analysed in the brains of 21 patients dying without reported neurological disease. The distribution of benzodiazepine receptors in the human brain was found to be comparable from case to case although differences in the density occurred among the brains examined. No influence of the post mortem delay, age, gender or pre mortem drug treatment on the distribution and densities was observed in our series. The highest densities of benzodiazepine receptors in human brain were localized in cortical and hippocampal areas, nucleus accumbens, amygdala and mammillary bodies. Intermediate densities were found in the basal ganglia and thalamic and hypothalamic nuclei. [³H]Flunitrazepam binding was low in the brainstem nuclei and very low in white matter.
The displacement of [3H]flunitrazepam binding activity by ethyl-beta-carboline-3-carboxylate (beta CCE) was studied in both membrane-bound and purified GABAA receptors from adult bovine cerebral cortex, hippocampus and cerebellum. It was found that the best fit for the displacement of benzodiazepine binding in the cerebellar membranes was a single site with IC50 = 0.55 +/- 0.21 nM, whereas the best fit for cortical and hippocampal membranes was a two-site model with respective values of IC50 = 0.2 +/- 0.09 nM (high affinity), IC50 = 21 +/- 6 nM (low affinity) (cortex) and IC50 = 0.25 +/- 0.05 nM, IC50 = 20 +/- 2 nM (hippocampus). These same properties were retained in the purified GABAA receptor from the three brain regions. Thus, we have demonstrated that binding site heterogeneity as defined by the displacement of beta CCE is preserved in purified GABAA receptors and we suggest that this provides evidence for the existence of GABAA receptor isoforms.
This paper reviews selected aspects of benzodiazepine binding site heterogeneity. These include receptor heterogeneity revealed by biochemical determinations of receptor numbers, autoradiographic localization in histological sections of brain, lesion studies, solubilization of receptors, and photoaffinity labelling. The data summarized support the concept of benzodiazepine receptor multiplicity. In addition, we have reviewed recent work on peripheral-type benzodiazepine binding sites and suggest that further study of these sites may increase our understanding of both the central and peripheral actions of benzodiazepines and other ligands.
Receptor autoradiographic techniques have been used to demonstrate the selectivity of two trifluoroethyl-containing benzodiazepines for one of the subtypes of benzodiazepine receptor. Indirect localization of the binding sites for quazepam and halazepam was accomplished by using the ability of these compounds to displace [3H]-flunitrazepam binding. The appropriate binding parameters were selected on the basis of initial studies aimed at identifying the binding characteristics of several benzodiazepine compounds in comparison with the triazolopyridine CL218,872. Autoradiographic analysis of the benzodiazepine sites displaceable with quazepam and halazepam revealed the two benzodiazepine compounds preferentially labeled receptor sites in regions of the brain dominated by the type 1 benzodiazepine receptor subtype. Thus, quazepam and halazepam preferentially bind to benzodiazepine type-1 receptors in lamina IV of the cerebral cortex, the zona incerta, substantia nigra and the cerebellum.
We have previously described the synthesis of a novel compound, 3-(methoxycarbonyl)-amino-beta-carboline (beta-CMC), which has a high in vitro affinity for the benzodiazepine receptor. In vivo testing showed that this compound had a restricted pharmacological profile. beta-CMC lacked intrinsic activity but it antagonized the convulsions induced by the methyl ester of beta-carboline-3-carboxylic acid, an inverse agonist of the benzodiazepine receptor. Moreover, beta-CMC selectively antagonized the sedative but not the anxiolytic or anticonvulsant effects of benzodiazepines. The possible mechanisms involved in the selective antagonism of the sedative effects of benzodiazepines by beta-CMC are discussed.
Dispositional pharmacokinetics have been shown to have little relationship to drug effects after acute doses of certain benzodiazepines (Ellinwood et al. 1983, 1985; Lader 1979; Ziegler et al. 1983). For diazepam the rapid onset of acute tolerance follows the peak behavioral effect; that is, impairment of performance in cognitive-neuromotor tests declines considerably faster than the corresponding serum drug concentration (Ellinwood et al. 1983, 1985). Other studies have described a relatively short period of impairment for high single doses of diazepam (George and Dundee 1977) and a more prolonged period of impairment for lorazepam (Seppala et al. 1976), although the elimination half-lives (t1/2) of diazepam and its active metabolite N-desmethyldiazepam (Mandelli et al. 1978) are over twice as long as that of lorazepam, which has no active metabolites.
The pharmacological activity of quazepam, a BZ1 specific benzodiazepine, was compared to the effects of triazolam, a BZ1, BZ2 nonspecific benzodiazepine. Using a double-blind procedure, single oral doses of quazepam (15 or 30 mg), triazolam (0.5 or 1.0 mg) and placebo were administered to 21 healthy young men according to a random Latin square design balanced for order of drug administration. The drug effects on the performance of motor coordination and cognitive tasks were monitored for 7 h following drug ingestion. The results did not indicate any differential effects on cognitive-neuromotor performance for the BZ1 specific quazepam and 2-oxoquazepam compared with the BZ1, BZ2 nonspecific N-desalkylflurazepam metabolite. The impairment magnitude for 30 mg quazepam was closer to that of 0.5 mg triazolam. The onset of the initial drug effect was considerably slower for quazepam than for triazolam. The time course of the impairment profiles for the tasks was compared to pharmacokinetic data from previous studies and suggested that published pharmacokinetic rate constants explain only a limited portion of the impairment time course. In particular, the performance scores were already showing recovery from peak impairment 2 h post-drug ingestion, although quazepam's potent N-desalkylflurazepam metabolite has been found to maintain a maximum plateau level from 2 to 24 h.
The hypnotic drug quazepam and its active metabolite 2-oxo-quazepam (2-oxo-quaz) are two benzodiazepines (BZ) containing a trifluoroethyl moiety on the ring nitrogen at position 1, characterized by their preferential affinity for Type I BZ recognition sites. In the present study we characterized the binding of 3H-2-oxo-quaz in discrete areas of the human brain. Saturation analysis demonstrated specific and saturable binding of 3H-2-oxo-quaz to membrane preparations from human cerebellum. Hill plot analysis of displacement curves of 3H-flunitrazepam (3H-FNT) binding by 2-oxo-quaz yielded Hill coefficients of approximately 1 in the cerebellum and significantly less than 1 in the cerebral cortex, hippocampus, caudate nucleus, thalamus and pons. Self and cross displacement curves for 3H-FNT and 3H-2-oxo-quaz binding in these brain areas indicated that 2-oxo-quaz binds with different affinities to two populations of binding sites. High affinity binding sites were more abundant in the cerebellum (95% of total sites), cerebral cortex, hippocampus and thalamus, whereas low affinity sites were predominant in the caudate nucleus and pons. Competition studies of 3H-2-oxo-quaz (2 nM) and 3H-FNT (0.5 nM) using unlabelled ligands indicated that compounds which preferentially bind to Type I sites are more potent at displacing 3H-2-oxo-quaz than 3H-FNT from cerebral cortex membrane preparations. The results suggest that 3H-2-oxo-quaz may be used for selectively studying Type I BZ recognition sites in the human brain.
Quazepam and 2-oxo-quazepam are novel benzodiazepines containing a trifluoroethyl substituent on the ring nitrogen at position #1. Detailed competition binding experiments (25 to 30 concs.) at 4 degrees C were undertaken with these compounds versus 3H-flunitrazepam using synaptic membranes from rat cortex or cerebellum. Unlike other benzodiazepines, both quazepam and 2-oxo-quazepam distinguished two populations of 3H-flunitrazepam binding sites in rat cortex which were present in roughly equal proportions and for which the compounds displayed a greater than 20-fold difference in affinity. In cerebellum, no such discrimination of sites was noted for 2-oxo-quazepam, but quazepam did distinguish a small, low affinity (15% of total) population of sites. 3H-2-oxo-quazepam was prepared and used in competition studies to substantiate the conclusion that these compounds discriminate two populations of benzodiazepine sites in rat cortex. This new radioligand was shown to specifically label BZ binding sites with high affinity in a saturable manner. The competition experiments were then conducted using 3H-2-oxo-quazepam at a radioligand concentration sufficiently low (0.5 nM) to ensure that only the higher affinity binding sites which 2-oxo-quazepam discriminates would be occupied. Competition experiments in both cortex and cerebellum under these conditions indicated single site binding for unlabelled quazepam and 2-oxo-quazepam in every instance. This suggests that 3H-2-oxo-quazepam should be a useful new tool for selectively labeling and studying the BZ1 population of benzodiazepine binding sites.
Quazepam is a trifluoroethyl benzodiazepine hypnotic with a half-life of 27 to 41 hours, which has been shown to induce and maintain sleep in the short to long term (up to 4 weeks) treatment of patients with chronic or transient insomnia. Although its hypnotic efficacy has been well characterised against placebo, there are few clinical studies in comparison with established hypnotics, particularly over long term administration. However, preliminary evidence suggests that quazepam 15 to 30 mg is as effective as flurazepam and triazolam in usual therapeutic doses, and causes minimal rebound insomnia following its withdrawal, unlike rapidly eliminated benzodiazepines such as triazolam. The lack of rebound phenomena is likely to be attributable to the 'carryover' effects occurring after discontinuation of quazepam, which has pharmacologically active metabolites with half-lives of elimination similar to or longer than that of the parent drug. Probably because of the long half-lives of quazepam's metabolites, daytime sedation, fatigue and lethargy are the most frequently reported side effects. These side effects are most intense with the 30 mg dose and least with the 7.5mg dose, which has not been studied extensively. Hence, quazepam is an effective hypnotic which may be particularly suitable for short or medium term use in patients in whom withdrawal effects or rebound insomnia may be especially bothersome. Further definition of certain characteristics of its profile--such as its long term use and potential for development of tolerance or dependence, effects on psychomotor skills, efficacy of the 7.5mg dose, and suitability in elderly patients and patients with chronic organic diseases--will assist in more clearly defining its ultimate place in therapy.
Receptor autoradiographic techniques have been used to localize benzodiazepine-1 (BZ-1) receptor sites by employing a tritiated form of 2-oxo-quazepam (SCH 15-725), a metabolite of the benzodiazepine quazepam (SCH 16-134). Labeling of the receptors was quantitatively determined to be most abundant in areas of the rat brain known through previous autoradiographic studies to bind preferentially with BZ-1 selective ligands. These areas include lamina IV of the parietal cortex, substantia innominata, anterior amygdaloid nucleus, substantia nigra, zona incerta, and molecular layer of the cerebellum. Autoradiographic localization of binding sites for [3H]2-oxo-quazepam provides additional support for the hypothesis that this compound identifies the BZ-1 receptor subtype and demonstrates the utility of this ligand for quantitation of the distribution and density of these sites.
1. 2-oxo-quazepam (2oxoquaz) is a novel benzodiazepine (BZD) hypnotic containing a trifluoethyl substituent on the ring nitrogen at position 1, which, unlike other BZDs, distinguishes two populations of BZD binding sites. In the present study we characterized the binding of 3H-2oxoquaz to human brain membrane preparations. 2. Self and cross displacement curves for 3H-FNT and 3H-2oxoquaz binding in different brain areas indicate that 2oxoquaz binds with different affinities to two populations of binding sites in the human brain. 3. Competition studies of 3H-2oxoquaz (2 nM) and 3H-FNT (0.5 nM) binding with a series of unlabelled ligands indicate that compounds which preferentially bind to Type I sites are more potent at displacing 3H-2oxoquaz than 3H-FNT from cerebral cortex membrane preparations. 4. The binding of 3H-2oxoquaz is stimulated by gamma-aminobutyric acid (GABA) and pentobarbital in a concentration-dependent manner. 5. The results suggest that in the human brain 3H-2oxoquaz binds with high affinity to a subpopulation of BZD recognition sites (Type I sites) which are functionally linked to the GABA receptor and the chloride ionophore.
The postnatal development of several proteins irreversibly labeled by [3H]flunitrazepam in membranes from rat cerebral cortex was investigated. It was demonstrated that in the early postnatal days proteins with apparent molecular weights 55,000 and 59,000 were predominantly labeled whereas irreversible labeling of a protein with apparent molecular weight 51,000 started to predominate only in the second postnatal week. Irreversible labeling of another protein with apparent molecular weight 62,000 was weak throughout development. All these proteins seem to be associated with central benzodiazepine receptors. Irreversible labeling at various time points after birth seems to parallel the postnatal development of these proteins, and the different time course of development and different binding properties of the individual proteins support the hypothesis that these proteins are associated with separate and distinct benzodiazepine receptor subtypes. The pharmacological properties of the individual receptor subtypes seem to be fully developed in the early postnatal days, and therefore newborn animals seem to be a good model system for the investigation of properties and function of these various benzodiazepine receptor subtypes.
Several lines of evidence from reversible binding studies seem to indicate there are at least two “central” benzodiazepine receptor subtypes, the BZ1 and BZ2 receptors. Irreversible binding studies, using3H-flunitrazepam as a photoaffinity label for benzodiazepine receptors, not only are in perfect agreement with the data from reversible binding studies but extend these studies by identifying P51, a protein with apparent molecular weight 51,000, as a protein associated with the BZ1 receptor and by suggesting that the BZ2 receptor might actually consist of several different benzodiazepine receptors associated with different and distinct proteins irreversibly labeled by3H-flunitrazepam.
Other reversible binding studies have accumulated indicating the existence of several different conformations of benzodiazepine receptors. Irreversible binding studies support this conclusion and in addition suggest the existence of four different benzodiazepine binding sites within the GABA-benzodiazepine receptor complex. It is therefore hypothesized that there are several different GABA-benzodiazepine receptor subtypes all of which have four distinct benzodiazepine binding sites which can exist in at least three different but freely interconvertible conformations. This hypothesis can account for all experimental observations obtained so far and might partially explain the distinct clinical effects of structurally similar benzodiazepines.
Reversible and irreversible binding of [3H]flunitrazepam was investigated in membranes from cerebellum and inferior colliculus of young and adult rats. Results indicate that in adult animals predominantly BZ1 receptors are enriched in both brain regions. In the brains of newborn animals, however, additional benzodiazepine receptor subtypes seem to exist in cerebellum as well as in inferior colliculus.
Specific high affinity binding of [3H]flunitrazepam to membranes from human brain was stimulated by gamma-aminobutyric acid (GABA), pentobarbital, 1-ethyl-4-(isopropylidene-hydrazino)-1H-pyrazolo[3,4b]pyridine-5-carboxy lic acid ethyl ester hydrochloride (SQ 20009) and avermectin B1a and was unaffected by 2 microM 4'-chlorodiazepam (Ro 5-4864) indicating that [3H]flunitrazepam in human brain as well as in rat brain predominantly binds to benzodiazepine receptors specific to brain, which was associated with a GABA receptor and several modulatory binding sites for drugs. The potency of several selective and non-selective ligands for benzodiazepine receptors for inhibition of the binding of [3H]flunitrazepam was compared in membranes from human or rat brain cerebellum, hippocampus and cerebral cortex. It was demonstrated that all these compounds, derived from different chemical structures, had a remarkably similar potency for inhibition of the binding of [3H]flunitrazepam in the corresponding regions of the human or rat brain. However, irreversible labelling of benzodiazepine binding sites with [3H]flunitrazepam and subsequent SDS-polyacrylamide gel electrophoresis and fluorography revealed more photolabelled protein bands in human than in rat cerebellum and hippocampus. The results seem to indicate that, although the pharmacological properties of reversible binding of [3H]flunitrazepam are remarkably similar in membranes from rat or human brain, the molecular heterogeneity of benzodiazepine binding sites is even greater in human than in rat brain.
The relationships between the pharmacological activities of quazepam and flurazepam and the concentrations of each drug and its major active metabolites in brain and plasma following single oral doses of either drug to mice were investigated. At various time points after either quazepam or flurazepam administration, pharmacological activity was measured by the inhibition of electroconvulsive shock (ECS)-induced seizures. After quazepam, the plasma and brain samples obtained at the same time points were assayed for concentrations of quazepam, 2-oxoquazepam and N-desalkyl-2-oxoquazepam by specific GLC methods. After flurazepam, the plasma and brain samples were assayed for flurazepam, hydroxyethyl-flurazepam, and N-desalkyl-2-oxoquazepam, also by specific GLC methods. The results showed that both quazepam and flurazepam were rapidly metabolized and that parent drugs and metabolites were rapidly distributed to the brain. The brain levels of all the benzodiazepines analyzed in this study paralleled plasma levels. After quazepam, pharmacological activity most closely paralleled the combined brain concentrations of quazepam and 2-oxoquazepam rather than N-desalkyl-2-oxoquazepam levels. In contrast, following the flurazepam dose, activity most closely paralleled N-desalkyl-flurazepam concentrations. From these data, it can be concluded quazepam is distinctly different from flurazepam, and that, in the presence of quazepam and 2-oxoquazepam, N-desalkyl-2-oxoquazepam does not contribute extensively to the observed pharmacological activity.
The triazolobenzodiazepine triazolam (0.1-1.0 mg/kg i.p.) and quazepam (0.3-30.0 mg/kg i.p.) were administered to non-food-deprived rats which had been partially-satiated on a palatable diet. In a subsequent 30 min feeding test, both compounds produced a significant increase in the level of food consumption. While triazolam had a dose-related effect and produced a 151.5% increase in the level of food intake, quazepam exerted only a partial effect, achieving a 73.9% increase in food intake at 3.0 mg/kg but no additional increase in food intake at higher doses. The two beta-carbolines, ZK 93423 (0.1-3.0 mg/kg i.p.) and ZK 91296 (1.0-30.0 mg/kg i.p.), a full agonist and a partial agonist at benzodiazepine receptors respectively, also produced significant increases in food consumption under the same experimental conditions. ZK 93423 had effects which were similar to those of triazolam, ZK 91296 had effects similar to quazepam. The beta-carboline benzodiazepine inverse agonist FG 7142 (10.0 mg/kg i.p.) had an anorectic effect in non-food-deprived rats given 30 min access to the highly palatable diet. This effect was reversed by the beta-carboline benzodiazepine receptor antagonist ZK 93426 in a dose-dependent manner. These results emphasize that within the series of beta-carboline ligands for benzodiazepine receptors, their characterization in terms of agonists, antagonists and inverse agonists has validity with respect to the behavioural response of palatable food consumption in non-food-deprived rats.
Investigation of the actions of the benzodiazepines has provided insights into the neurochemical mechanisms underlying anxiety,
seizures, muscle relaxation, and sedation. Behavioral, electrophysical, pharmacological, and biochemical evidence indicates
that the benzodiazepines exert their therapeutic effects by interacting with a high-affinity binding site (receptor) in the
brain. The benzodiazepine receptor interacts with a receptor for gamma-aminobutyric acid, a major inhibitory neurotransmitter,
and enhances its inhibitory effects. The benzodiazepine receptor may also interact with endogenous substances and several
naturally occurring compounds, including the purines and nicotinamide, are candidates for this role. Both the purines and
nicotinamide possess some benzodiazepine-like properties in vivo, although further work will be required to confirm their
possible roles as endogenous benzodiazepines.
In this chapter, I would like to discuss the discovery and development of the centrally acting 1,4-benzodiazepines which started with the synthesis and pharmacological evaluation of a compound which is the active ingredient of Librium and is now known by the generic name chlordiazepoxide.
Ethyl β-carboline-β-carboxylate (β-CCE) is a mixed-type inhibitor of [3H]flunitrazepam ([3H]FNM) binding to benzodiazepine receptors in noncerebellar regions of rat brain. These findings may represent the presence of either receptor multiplicity or negative cooperativity among benzodiazepine receptors. [3H]Propyl β-carboline-3-carboxylate ([3H]PrCC) has previously been shown to bind specifically to benzodiazepine receptors of rat cerebellum. In the present study we found no indication of the presence of true negative cooperativity among benzodiazepine receptors when [3H]PrCC was used as radioligand. However, we observed that [3H]PrCC labelled only 57% of [3H]FNM binding sites in rat hippocampus (Bmax values) and 71% in rat cerebral cortex, whereas the number of receptors labelled by both ligands was equal in the cerebellum. Hofstee analyses of the shallow inhibition curves seen in hippocampus and cerebral cortex when [3H]FNM binding was inhibited by β-CCE indicate that β-CCE and some other β-carboline-3-carboxylate derivatives interact preferentially with a subclass of receptors, and that the percentage of this subclass is equivalent to the number of receptors labelled by [3H]PrCC. We conclude that [3H]PrCC at low concentration (0.3–0.4 × 10-9 M) labels a subclass of benzodiazepine receptors, BZ1, while another class, BZ2 receptors, are not labelled by [3H]PrCC when filtration assays are used. By parallel determinations of the proportion between [3H]FNM and [3H]PrCC binding we calculated the percentage of BZ1 receptors in several regions of rat, guinea pig and calf brain and in mouse forebrain. The values ranged from approximately 50% in hippocampus to 90% in the guinea pig pons.
Brain-specific binding sites have been isolated on synaptosomal membrane fragments which recognize pharmacologically active benzodiazepines (BDZ's) and triazolopyridazines (TPZ's). While early evidence indicated the existence of a single homogeneous class of BDZ binding sites, more recent biological and pharmacological studies support the notion of BDZ receptor multiplicity. We now propose that two biochemically distinct BDZ receptors exist in brain which are responsible for the mediation of different pharmacological activities. Type I BDZ receptors display a high affinity for both BDZ's and TPZ's, are not coupled to GABA receptors or to chloride ionophores, and are the sites which mediate anxiolytic actions. Type II BDZ receptors display a high affinity for BDZ's, display a low affinity for TPZ's, are coupled to GABA receptors and/or chloride ionophores, and are the sites which mediate pharmacological effects other than anxiolytic activity.
When membranes of rat cerebellum are exposed to UV light in the presence of flunitrazepam this ligand can be incorporated into one of the assumed 4 benzodiazepine binding sites of the GABA-benzodiazepine receptor complex. This irreversible incorporation of flunitrazepam, in contrast to reversible binding of this substance, leads to conformational changes of the remaining 3 benzodiazepine binding sites which result in a decreased affinity of benzodiazepine agonists, but not of benzodiazepine antagonists. The investigation of the affinity of drugs for [3H]benzodiazepine antagonist binding before and after photoaffinity labelling of benzodiazepine receptors with flunitrazepam can therefore be used as a sensitive and simple test to distinguish between agonists and antagonists in vitro.
7-Chloro-1-(2,2,2-trifluoroethyl)-5-(o-fluorophenyl)-1, 3-dihydro-2H-1,4-benzodiazepine-2-thione (Sch 16134, quazepam) demonstrated high safety and effective hypnotic properties. Toxicity of quazepam in mice was extremely low at the largest doses administered of 5000 mg/kg p.o. and 1370 mg/kg i.p. as compared to LD50 values for flurazepam of 756 (635-889) mg/kg p.o. and 254 (234-280) mg/kg i.p. In cats, quazepam did not produce overt toxicity even at the highest dose administered, 1000 mg/kg p.o., whereas death occurred after the administration of flurazepam at a median dose of 250 mg/kg p.o. In addition, flurazepam produced central excitation and convulsions. Quazepam did not affect hemodynamic parameters in conscious dogs and anesthetized cats. Autonomic functions were virtually unaffected by the drug in anesthetized cats. In interaction studies in mice, quazepam did not interact with cimetidine while it potentiated the sedative activity of alpha-methyldopa, ethanol and propranolol. The CNS depressant properties of quazepam were further investigated by studying its effects on spontaneous motor activity in mice and EEG pattern in immobilized cats. In mice, quazepam reduced locomotor activity at doses which did not impair motor coordination thus differing from flurazepam which did. In the EEG study, quazepam produced a slow wave EEG pattern comparable to that of physiological sleep, while flurazepam induced a similar EEG activity which alternated with a fast frequency pattern of moderate amplitude. Quazepam also showed potent anticonvulsant activity. These results suggest that quazepam is likely to be a safe and effective sleep-promoting agent.
Benzodiazepines exhibit reversible, stereospecific high affinity binding to mammalian brain membranes, and the respective binding sites for 3H-flunitrazepam represent pharmacologically and clinically relevant receptors for benzodiazepines. Recently it has been demonstrated that reversibly bound 3H-flunitrazepam becomes irreversibly attached to a specific membrane protein with apparent molecular weight of 50,000 when incubations are performed in the presence of UV light. Irreversible binding of 3H-flunitrazepam to this protein had pharmacological properties similar to reversible benzodiazepine receptor binding, indicating that 3H-flunitrazepam is a photoaffinity label for the benzodiazepine receptor. Using irreversible binding of 3H-flunitrazepam and subsequent electrophoretic separation of the labelled proteins in SDS-gels followed by fluorography, we found that in hippocampus and several other brain regions at least two different types of benzodiazepine receptors exist. Each seems to be associated with a gamma-aminobutyric acid (GABA) receptor.
Ethyl beta-carboline-3-carboxylate (beta-CCE) is a mixed-type inhibitor of [3H]flunitrazepam ([3H]FNM) binding to benzodiazepine receptors in noncerebellar regions of rat brain. These findings may represent the presence of either receptor multiplicity or negative cooperativity among benzodiazepine receptors. [3H]Propyl beta-carboline-3-carboxylate ([3H]PrCC) has previously been shown to bind specifically to benzodiazepine receptors of rat cerebellum. In the present study we found no indication of the presence of true negative cooperativity among benzodiazepine receptors when [3H]PrCC was used as radioligand. However, we observed that [3H]PrCC labelled only 57% of [3H]FNM binding sites in rat hippocampus (Bmax values) and 71% in rat cerebral cortex, whereas the number of receptors labelled by both ligands was equal in the cerebellum. Hofstee analyses of the shallow inhibition curves seen in hippocampus and cerebral cortex when [3H]FNM binding was inhibited by beta-CCE indicate that beta-CCE and some other beta-carboline-3-carboxylate derivatives interact preferentially with a subclass of receptors, and that the percentage of this subclass is equivalent to the number of receptors labelled by [3H]PrCC. We conclude that [3H]PrCC at low concentration (0.3-0.4 X 10(-9) M) labels a subclass of benzodiazepine receptors, BZ1, while another class, BZ2 receptors, are not labelled by [3H]PrCC when filtration assays are used. By parallel determinations of the proportion between [3H]FNM and [3H]PrCC binding we calculated the percentage of BZ1 receptors in several regions of rat, guinea pig and calf brain and in mouse forebrain. The values ranged from approximately 50% in hippocampus to 90% in the guinea pig pons.
Benzodiazepines are thought to exert their pharmacological and clinical effects by interacting with specific receptors on neurones in the central nervous system1-3. Originally, only benzodiazepinoid compounds were known to interact with these receptors, but recently other classes of agents have been discovered which have high affinity for benzodiazepine receptors4. A representative from one of these classes, ethyl beta-carboline-3-carboxylate (beta-CCE), was obtained from human urine by virtue of its high affinity for benzodiazepine receptors. It was hypothesized that derivatives or congeners of this beta-carboline could be related to presumed endogenous ligands, the exact nature of which are unknown3,5. Another ester of beta-carboline-3-carboxylic acid, the propyl ester, PrCC, has recently been used as a radioligand for labelling benzodiazepine receptors6: in particular, 3H-PrCC has been observed selectively to label a BZ1 receptor subclass7. Binding of PrCC to benzodiazepine receptors, however, was less enhanced by gamma-aminobutyric acid (GABA) than expected6,8. The affinity of benzodiazepines for benzodiazepine receptors is enhanced two to threefold by GABA9-11, probably reflecting the functional coupling of benzodiazepine receptors and GABA receptors at the molecular level. Here we have investigated binding of the methyl ester of beta-carboline-3-carboxylic acid (beta-CCM), which by itself is a convulsant, in contrast to beta-CCE and PrCC. We report that 3H-beta-CCM binds to brain benzodiazepine receptors and that, in contrast to binding of 3H-diazepam, 3H-beta-CCM binding is reduced by GABA in a bicuculline-sensitive manner.
A representative of a novel series of imidazodiazepines, Ro 15-1788, selectively antagonizes all major central actions of benzodiazepines by competitive, high-affinity interactions with benzodiazepine receptors (BR) in the central nervous system (CNS)1-3. Doses of Ro 15-1788 sufficient to antagonize benzodiazepine actions have been shown per se to lack phamacological action in animals and man1-4. Thus Ro 15-1788 provides a highly selective tool for experimental investigations of BR-mediated events and is of therapeutic value in all cases where a rapid termination of benzodiazepine actions is indicated. Using 3H-labelled Ro 15-1788 as radioligand in equilibrium binding studies in vitro, we show here that 3H-Ro 15-1788 interacts with the same number of BR sites as the agonist 3H-clonazepam in various brain regions. However, their mode of receptor interaction is different. In conditions which alter receptor affinity for 3H-clonazepam binding, such as addition of gamma-aminobutyric acid (GABA) or certain ions, no change is seen in 3H-Ro 15-1788 binding. This effect can be used to distinguish between benzodiazepine receptor agonists and antagonists in vitro. Furthermore, the thermodynamics of agonist and antagonist receptor interaction are different, but only at temperatures above 21 °C.
The gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex, which is composed of distinct proteins embedded in the
neuronal plasma membrane, is important for several effects of benzodiazepines, including protection afforded against convulsions.
During structural modification of ethyl beta-carboline-3-carboxylate an agent was discovered which has high affinity for brain
benzodiazepine receptors but which is a potent convulsant. Also in contrast to benzodiazepines, this type of benzodiazepine
receptor ligand favors benzodiazepine receptors in the non-GABA-stimulated conformation, which may explain the convulsive
properties.
GABA reduces bi,~ding of 13H]melhyI-B-carboline-3-carboxylate to brain benzodiazepine receptors
- C Braeslrup
- M J Nielsen
BraeSlrup, C. and Nielsen, M.J., GABA reduces bi,~ding of 13H]melhyI-B-carboline-3-carboxylate to
brain benzodiazepine receptors, Nature (Lond.), 294 (1981) 472-474.
Distinction of benzodiazepine agonists from antagonists by photoaffinity labeling of benzodiazepine receptors in vitro
- Mo Karobath
- P Supavjlai
Karobath, Mo and SupavJlai, P., Distinction of benzodiazepine agonists from antagonists by photoaffinity labeling of benzodiazepine receptors in vitro, Neurosci. Lett., 31 (1982) 65-69.