Like diazepam,CL 218,872, a selective ligand for the benzodiazepine ω1 receptor subtype,impairs place learning in the Morris water maze

The sedative, anxiolytic, and amnesic effects of diazepam were compared to those of CL 218,872, a triazolopyridazine that has a preferential affinity for the benzodiazepine ₁ receptor subtype. Spontaneous locomotion was assessed using a running wheel, anxiety was assessed using an open-field divided into central and peripheral areas (thigmotaxis), and amnesia was assessed using the Morris water maze. It was found that CL 218,872, like diazepam, depressed spontaneous locomotion, reduced anxiety, and impaired place learning in a dose-dependent manner. Flumazenil, a benzodiazepine receptor antagonist with a similar affinity for both ₁ and ₂ subtypes, reversed all of the effects of diazepam and antagonized the anxiolytic and amnesic effects, and some but not all of the sedative effects of CL 218,872. These results suggest that the selective activation of the ₁ receptor subtype by CL 218,872 is sufficient to produce sedation, anxiolysis, and amnesia in a manner similar to that produced by the coactivation of both the ₁ and ₂ receptor subtypes with diazepam.     

Is the mitochondrial benzodiazepine receptor involved in the control of airway smooth muscle tone?

• 1.1. In addition to binding to GABA_A receptors in the central nervous system, benzodiazepines have also been reported to recognize high affinity binding sites in several different peripheral tissues.
• 2.2. These peripheral benzodiazepine receptors likely consist of distinct integral membrane proteins, which are predominantly localized in the outer mitochondrial membrane and may be associated to form a heteropolymeric receptor complex. One such protein, identified for its ability to bind a class of benzodiazepines and isoquinolines, has been purified and the corresponding complementary DNA (cDNA) has been cloned and characterized. Furthermore, the structure of the rat gene encoding this protein has been clarified, thus potentially opening new insights into the molecular mechanisms responsible for receptor regulation.
• 3.3. Although the exact physiologic and/or pharmacologic role of peripheral benzodiazepine receptors is still unknown, their wide tissue distribution suggests an involvement in many cellular phenomena.
• 4.4. In particular, several lines of investigation indicate that these receptors, densely expressed on airway smooth muscle of various species, may contribute to the modulation of bronchomotor tone and perhaps to the pathogenesis of asthma and airway hyperresponsiveness.

     

Autoradiographic localization of “peripheral-type” benzodiazepine binding sites in the rat brain,heart and kidney

Summary The localization of peripheral-type benzodiazepine binding sites has been accomplished using quantitative receptor autoradiography after labeling slidemounted tissue sections with [³H]-RO 5-4864. Specific [³H]-RO 5-4864 binding sites were observed in the choroid plexus and ependyma cells within the brain. Lower levels of specific binding were seen in areas corresponding to the glomerular layer of the olfactory bulb. Stereotaxic administration of the excitotoxin, ibotenic acid, into the piriform cortex produced a dramatic increase in binding to the peripheral-type site in this brain region. Binding in the kidney was associated with the ascending limb of the loop of Henle and the distal convoluted tubule, while in the heart a more diffuse binding was found throughout the ventricle wall. The localization of peripheral-type benzodiazepine binding sites to areas involved in ion transport gives additional support to previously suggested physiological roles for these sites.     

β-Carboline binding indicates the presence of benzodiazepine receptor subclasses in the bovine central nervous system

Summary Receptor binding studies were performed with tritiated propyl -carboline-3-carboxylate ([³H]PrCC), tritiated ethyl -carboline-3-carboxylate ([³H]ECC), and tritiated flunitrazepam ([³H]FNT) in membrane preparations from different regions of the bovine brain and retina. Specific binding in all regions investigated was associated with benzodiazepine receptor sites. However, not all benzodiazepine receptor sites. However, not all benzodiazepine receptors in the regions investigated as determined by the specific binding of tritiated flunitrazepam ([³H]FNT) are available for [³H]PrCC suggesting that specific [³H]PrCC binding labels only one subclass or subpopulation of the benzodiazepine receptor. This benzodiazepine receptor subclass is sensitive to GABAergic modulation and amounts for about 60% of the benzodiazepine receptors in bovine cortex, hippocampus, and retina but for about 80% of the benzodiazepine receptors in the bovine cerebellum. By contrast, specific [³H]ECC binding in the cerebellum and the hippocampus labeled the same number of benzodiazepine receptors as [³H]FNT, giving no evidence for a benzodiazepine receptor subclass specificity of this compound in the bovine CNS.Professor Dr. U. Wollert died on February 26, 1981     

Evaluation of the β-carboline ZK 93 426 as a benzodiazepine receptor antagonist

We describe here biochemical and pharmacological effects of the -carboline ZK 93426, a new and potent benzodiazepine (BZ) receptor antagonist. ZK 93426 was compared with Ro 15-1788 and CGS 8216, two compounds previously described as BZ receptor antagonists. Certain effects of ZK 93426, Ro 15-1788 and CGS 8216 were quite similar (e.g., ³H-FNM displacement, GABA ratio, photo-shift). In most pharmacological tests ZK 93426 and Ro 15-1788 lacked overt effects; Ro 15-1788 was a weak agonist in some paradigms, while ZK 93426 exhibited a potent proconflict effect but also a weak anticonvulsant effect. This interesting finding with ZK 93426 suggests that BZ receptor ligands may possess differential efficacy at BZ receptor subtypes. In contrast, CGS 8216 exhibited potent proconvulsant effects in several paradigms in addition to proconflict and pentylenetetrazol generalizing effects. ZK 93426, Ro 15-1788 and CGS 8216 were almost equally potent as antagonists of the effects of BZ receptor agonists, such as diazepam and lorazepam. However, ZK 93426 was the most potent inhibitor of the convulsions produced by the BZ receptor inverse agonist DMCM.     

GABAA receptor subtypes: any clues to the mechanism of benzodiazepine dependence?

Chronic use of benzodiazepines for the treatment of anxiety has revealed that these drugs can lead to dependence as indicated by withdrawal symptoms following cessation and tolerance to the drugs effects. Together with their reinforcing properties, this has led to them being labelled as scheduled drugs. Our new knowledge regarding the molecular structure of the benzodiazepine binding site and the growing ability to differentiate GABA(A) receptor subtypes, either by genetic manipulation or subtype selective compounds, have begun to facilitate our understanding of what underlies the mechanism of benzodiazepine dependence. In addition, the involvement of GABA(A) receptors in this phenomenon is leading to a greater understanding of other drugs such as alcohol and opiates.     

The plasma–occupancy relationship of the novel GABAA receptor benzodiazepine site ligand, α5IA,is similar in rats and primates

Background and purpose:

α5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is a triazolophthalazine with subnanomolar affinity for α1-, α2-, α3- and α5-containing GABA_A receptors. Here we have evaluated the relationship between plasma α5IA concentrations and benzodiazepine binding site occupancy in rodents and primates (rhesus monkey).

Experimental approach:

In awake rats, occupancy was measured at various times after oral dosing with α5IA (0.03–30 mg·kg⁻¹) using an in vivo {[³H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} binding assay. In anaesthetized rhesus monkeys, occupancy was measured using {[¹²³I]iomazenil (ethyl 5,6-dihydro-7-iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} γ-scintigraphy and a bolus/infusion paradigm. In both rat and rhesus monkey, the plasma drug concentration corresponding to 50% occupancy (EC₅₀) was calculated.

Key results:

In rats, α5IA occupancy was dose- and time-dependent with maximum occupancy occurring within the first 2 h. However, rat plasma EC₅₀ was time-independent, ranging from 42 to 67 ng·mL⁻¹ over a 24 h time course with the average being 52 ng·mL⁻¹ (i.e. occupancy decreased as plasma drug concentrations fell). In rhesus monkeys, the EC₅₀ for α5IA displacing steady-state [¹²³I]iomazenil binding was 57 ng·mL⁻¹.

Conclusions and implications:

Rat plasma EC₅₀ values did not vary as a function of time indicating that α5IA dissociates readily for the GABA_A receptor in vivo. These data also suggest that despite the different assays used (terminal assays of [³H]flumazenil in vivo binding in rats and [¹²³I]iomazenil γ-scintigraphy in anaesthetized rhesus monkeys), these techniques produced similar plasma α5IA EC₅₀ values (52 and 57 ng·mL⁻¹ respectively) and that the plasma–occupancy relationship for α5IA translates across these two species.     

Does high-dose benzodiazepine abuse really produce liver toxicity? Results from a series of 201 benzodiazepine monoabusers

Background: Several side-effects related to prolonged benzodiazepines (BZD) use have been reported. Given the primary role of liver in BZD metabolism, toxicity related to prolonged high-dose BZD use could be conceivable. No data are available on the long-term impact of high-dose BZD use on liver.

Research design and methods: A total of 201 BZD mono-abusers admitted to an Addiction Unit for detoxification were evaluated. Liver enzymes were evaluated at admission, before starting any treatment. An elevation of more than five times the upper limit of normal range (ULN) in serum ALT or conjugated bilirubin, or a combined elevation of AST, alkaline phosphatase and total bilirubin, one of which exceeding >2 the ULN, was considered diagnostic for drug-induced liver injury.

Results: None of the evaluated subjects showed significant alterations of liver enzymes. Those with the highest transaminase levels were showing high body mass index. Twenty patients (10%) showed elevated gamma-glutamyl-transferase. No alteration of alkaline phosphatase, nor bilirubin was found in any patient. The average dosage of BZD was 307 mg of diazepam-equivalents for 7 years.

Conclusions: Present data suggest that prolonged use of high-dose BZD, although very dangerous for several reasons, does not seem to produce a significant drug-induced liver injury.     

A β-carboline antagonizes benzodiazepine actions but does not precipitate the abstinence syndrome in cats

The -carbolines, which are potent ligands for benzodiazepine receptors, antagonize the pharmacological actions of benzodiazepines. In the cat, the stable -carboline derivative methylamide--carboline-3-carboxylate, FG 7142, and the specific benzodiazepine antagonist Ro 15-1788 reversed behavioral and electroencephalographic (EEG) changes produced by a single dose of diazepam. Surprisingly, the -carboline did not elicit signs of withdrawal when given after 22 days of a daily dose regimen of diazepam, while Ro 15-1788 precipitated an acute abstinence syndrome largely characterized by tremors, increased muscle tone, back arching, myoclonic jerks and pupil dilatation. Unlike Ro 15-1788, the -carboline produced effects of its own such as behavioral states of arousal and fearfulness. These findings indicate that the -carboline functionally interacts with benzodiazepine receptors in a manner unlike that seen with typical agonists and antagonists.     

Scopolamine-induced amnesia in humans: lack of effects of the benzodiazepine receptor antagonist β-carboline ZK 93426

It has been suggested from pharmacological studies in animals that ZK 93426 may improve memory and other cognitive processes in humans. Scopolamine has been used to model aspects of memory impairment. To test the effects of ZK 93426 alone and in combination with scopolamine, ZK 93426 (0.04 mg/kg) or vehicle (Intralipid R) was administered intravenously (i.v.) to normal controls, pre-treated with either scopolamine 0.5 mg administered subcutaneously (s.c.) or the same volume of saline. A visual (presentation of pictures) and a verbal (words list) memory test were applied. Both drugs on their own and in combination were found to be safe and well tolerated. ZK 93426 did not antagonize the scopolamine-induced impairment of acquisition of the words list. Scopolamine did not impair delayed recall of visual or verbal material. ZK 93426 alone improved performance in delayed recall of visual material presented after drug application, whereas it impaired performance in delayed recall of visual material presented before drug administration.     

High affinity ligands for ‘diazepam-insensitive’ benzodiazepine receptors

Structurally diverse compounds have been shown to possess high affinities for benzodiazepine receptors in their ‘diazepam-sensitive’ (DS) conformations. In contrast, only the imidazobenzodiazepinone Ro 15-4513 has been shown to exhibit a high affinity for the ‘diazepam-insensitive’ (DI) conformation of benzodiazepine receptors. We examined a series of 1,4-diazepines containing one or more annelated ring systems for their affinities at DI and DS benzodiazepine receptors, several 1,4-diazepinone carboxylates including Ro 19-4603, Ro 16-6028 and Ro 15-3505 were found to possess high affinities (K_i ∼ 2.6–20 nM) for DI. Nonetheless, among the ligands examined, Ro 15-4513 was the only substance with a DI/DS potency ratio ∼ I: other substances had ratios ranging from 13 to > 1000. Ligands with high to moderate affinities at Di were previously classified as partial agonists, antagonists, or partial inverse agonists at DS benzodiazepine receptors, but behaved as ‘GABA neutral’ (antagonist) substances at DI. The identification of several additional high affinity ligands at DI benzodiazepine receptors may be helpful in elucidating the pharmacological and physiological importance of these sites.     

Human studies on the benzodiazepine receptor antagonist β-carboline ZK 93 426: antagonism of lormetazepam's psychotropic effects

The effects of lormetazepam (0.03 mg/kg IV) a benzodiazepine (BZ) derivative in combination with ZK 93 426 (0.04 mg/kg IV) a -carboline, benzodiazepine receptor antagonist were evaluated in humans. Independently, the effects of ZK 93 426 on its own were investigated. A psychometric test battery to evaluate sedation (visual analog scales (VAS), anxiolysis (state-trait-anxiety inventory scale (STAIG X₁) and cognitive functions [logical reasoning test (LR), letter detection test (LD)] was applied before and several hours after initiation of treatment. Multiple sleep latency test (MSLT), which measures day time sleepiness, was also applied. Vigilosomnograms analysed from standard EEG recordings were evaluated shortly before and for 1 h after treatment. Treatment started with an intravenous injection of either lormetazepam (LMZ) or placebo (PLA), which was followed 30 min later by administration of either ZK 93 426 or placebo; thus four treatment groups were created (PLA + PLA, LMZ + PLA, LMZ + ZK 93 426 and PLA + ZK 93 426). ZK 93 426 antagonized the sedative and hypnotic effect of LMZ as estimated by MSLT and vigilosomnograms, respectively. Impairment of cognitive functions (LR and LD) induced by LMZ was also antagonized by ZK 93 426. ZK 93 426 had no effect on the changes in the time estimation seen in the LMZ group. Furthermore, ZK 93 426 on its own increased vigilance (alertness) as measured by the vigilosomnogram. A competitive antagonism at the benzodiazepine binding site between ZK 93 426 and LMZ is suggested by their combination effects; the intrinsic activity of ZK 93 426 seems to be due to its weak partial inverse agonist component.     

Testing for benzodiazepine inebriation—relationship between benzodiazepine concentration and simple clinical tests for impairment in a sample of drugged drivers

Objective To study how the various 25 subtests and observations of the Norwegian clinical test for impairment related to the blood benzodiazepine concentrations of apprehended drivers suspected of driving under the influence of benzodiazepines. The impact of single-dose intake in non-daily users of benzodiazepines on the clinical picture of inebriation was also studied.Methods Included in the study were 818 drivers suspected of driving under the influence of non-alcoholic drugs with blood samples containing only one benzodiazepine. We determined which of the 25 subtests and observations of the clinical test for impairment related significantly to the blood benzodiazepine concentrations.Results Significantly related to blood benzodiazepine concentrations were 13 subtests and observations. Of these, 9 withstood adjustment for a variety of background variables. Singledose intake in non-daily users only influenced 3 subtests and observations after adjustment for blood benzodiazepine concentration and background variables. Romberg's test, 1 observation concerning alertness (oriented for time and place), 4 tests on motor and coordination (walk and turn on line, finger-to-nose and finger-to-finger tests), 2 observations on speech (articulation and content) and 1 observation regarding appearance (general conduct) were related to blood benzodiazepine concentrations.Conclusion Many of these simple clinical tests are included in the standardized field sobriety test and are of value in revealing benzodiazepine impairment. The present study offered some possible additions. Combinations of these robust tests can also be used to reveal benzodiazepine inebriation in other contexts.     

Human studies on the benzodiazepine receptor antagonist β-carboline ZK 93 426: preliminary observations on psychotropic activity

The -carboline ZK 93 426, a benzodiazepine receptor antagonist, was administered intravenously to human volunteers at two different doses (0.01 mg/kg, 0.04 mg/kg) according to a double-blind, placebo controlled design. Vital functions (i.e. blood pressure, heart rate, ECG, EEG), peripheral (finger) skin temperature and performance in psychometric tests for psychotropic and cognitive effects were evaluated. Blood samples were collected in addition and certain pharmacokinetic parameters were estimated. ZK 93 426 in both doses was well tolerated and exhibited no side effects. A decrease in peripheral skin temperature and heart rate was observed. In a general estimation of behavioural changes, volunteers experienced a stimulant and activating effect of the drug. An improvement in performance was observed in two cognitive tasks, the logical reasoning task and pictures differences task which estimated concentration and attention, respectively. No effects were found in time estimation. Plasma levels 5 min after intravenous administration of ZK 93 426 were 16±10 ng/ml and 52±31 ng/ml for 0.01 mg/kg and 0.04 mg/kg, respectively. Total clearance was calculated as 46±22 ml/min/kg (0.04 mg/kg).     

Effects of the benzodiazepine GABAA α1-preferring ligand, 3-propoxy-β-carboline hydrochloride (3-PBC), on alcohol seeking and self-administration in baboons

Rationale

The various α subtypes of GABA_A receptors have been strongly implicated in alcohol reinforcement and consumption.

Objectives

The effects of the GABA_A α1-preferring ligand, 3-propoxy-β-carboline hydrochloride (3-PBC), on seeking and self-administration responses were evaluated in two groups of baboons trained under a 3-component chained schedule of reinforcement (CSR).

Methods

Alcohol (4 %?w/v; n?=?5; alcohol group) or a preferred nonalcoholic beverage (n?=?4; control group) was available for self-administration only in component 3 of the CSR. Responses in component 2 provided indices of motivation to drink (seeking). 3-PBC (1.0–30.0 mg/kg) and saline were administered before drinking sessions under both acute and 5-day dosing conditions.

Results

Repeated, and not acute, doses of 3-PBC significantly decreased total self-administration responses (p?<?0.05), volume consumed (p?<?0.05), and gram per kilogram of alcohol (p?<?0.05) in the alcohol group. In the control group, 5-day administration of 3-PBC significantly decreased total self-administration responses (p?<?0.05) but produced nonsignificant decreases in volume consumed. Within-session pattern of drinking was characterized by a high level of drinking in the first 20 min of the session for both groups, which was significantly (p?<?0.05) decreased by all doses of 3-PBC (1.0–18.0 mg/kg) only in the alcohol group. In contrast, the first drinking bout in the control group was only reduced at the highest doses of 3-PBC (10.0 and 18.0 mg/kg).

Conclusions

The results support the involvement of the GABA_A α1 subtype receptor in alcohol reinforcement and consumption.     

Adaptation and validation of the revised Patients’ Attitudes towards Deprescribing (rPATD) questionnaire for benzodiazepine receptor agonists

BackgroundThe revised Patients' Attitudes Towards Deprescribing (rPATD) questionnaire explores older adults’ views on deprescribing in general. Those views may differ, however, when the target is a specific drug such as benzodiazepine receptor agonists (BZRA).ObjectiveThis study aimed to adapt the 22-item French rPATD questionnaire to create a BZRA-specific instrument and to assess the psychometric properties of this new tool.MethodsThe adaptation of the questionnaire comprised 3 steps: 1) item transformation during group discussions with 8 healthcare providers and 8 BZRA users (aged ≥65 years), 2) pre-test of the questionnaire with 12 other older adults to ensure items understanding, 3) evaluation of the psychometric properties of the new questionnaire with 221 older BZRA users recruited in Belgium, France, and Switzerland. Construct validity was assessed using exploratory factor analysis (EFA), internal consistency with Cronbach's alpha, and test-retest reliability with intraclass correlation coefficient (ICC).ResultsAfter the pre-test, the questionnaire had 24 items (19 adapted from the French rPATD, 3 removed, and 5 added). The EFA, however, found that several items performed poorly. Eleven items were consequently removed, based on statistical performance and clinical relevance. Three factors were extracted from the EFA performed on the 11 retained items and were named “Concerns about stopping BZRA”, “BZRA inappropriateness”, and “Dependence on BZRA”. The questionnaire also includes two global questions about willingness to reduce BZRA dosage and willingness to discontinue BZRA. All factors showed acceptable internal consistency (0.68 ≤ Cronbach's alpha ≤0.74). Two factors showed acceptable test-retest reliability. The “Concerns about stopping BZRA” factor was found to vary over time (ICC [95%CI]: 0.35[-0.02; 0.64]).ConclusionsWe developed and validated a 13-item questionnaire to evaluate the attitudes of older people towards BZRA deprescribing. Despite some limitations, this questionnaire appears to be a useful tool for facilitating shared decision-making on BZRA deprescribing.