Flumazenil-insensitive benzodiazepine binding sites in GABAA receptors contribute to benzodiazepine-induced immobility in zebrafish larvae.

AIMS: Benzodiazepines (BZDs) produce various pharmacological actions by binding to and allosterically regulating GABAA receptors. Several in vitro studies have demonstrated diazepam, the prototypic BZD, produces a high-dose action that cannot be countered with the classical BZD-binding site antagonist flumazenil. Here, we investigate the existence and behavioral relevance of non-classical BZD binding sites in zebrafish larvae. MAIN METHODS: Zebrafish larvae were treated with a series of BZDs alone or combined with flumazenil, bicuculline (a non-selective GABAA receptor antagonist), or RO 15-4513 (a general BZD antagonist and a proposed antagonist interacting with α+/ß- interfaces in α4/6/ß3δ receptors), and their locomotor activities and behavioral phenotypes were recorded. KEY FINDINGS: Diazepam-induced hypolocomotion (sedation-like state) at low doses (10 and 20 mg L-1) was effectively antagonized by flumazenil or bicuculline, while diazepam-induced immobility (anesthesia-like state) at higher dose (30 mg L-1) was prevented by bicuculline (3 mg L-1) but not flumazenil, even at doses up to 150 mg L-1. Ro 15-4513 also failed to efficiently antagonize diazepam-induced immobility. Immobility induced by high dose of another 1,4-BZD, clonazepam, was also resistant to flumazenil. SIGNIFICANCE: These results provide direct in vivo evidence for non-classical BZD-binding sites, which may be located at the second transmembrane domain of GABAA receptors and contribute to BZD-induced anesthesia.

Individual differences in aggressive responding to intravenous flumazenil administration in adult male parolees.

Nonhuman and human studies have shown that benzodiazepine (BZD) receptor agonists can modify aggressive behaviour. However, it is unknown whether flumazenil, a BZD receptor antagonist, enhances or inhibits aggressive behaviour. The present study was designed to investigate the effects of acute administrations of flumazenil on aggressive responding in adult humans. Six adult males with histories of childhood conduct disorder (DSM IV R) participated in experimental sessions. Aggression was measured using the Point Subtraction Aggression Paradigm (PSAP; Cherek 1992), which provided subjects with aggressive and monetary-reinforced response options. Acute doses of flumazenil (2 and 3mg) did not produce statistically significant changes in either monetary-reinforced responding or aggressive responding. The analysis of individual subjects data revealed that aggressive responses varied across subjects. The results are discussed in terms of individual differences based on the previous history of BZD abuse. Additional laboratory research is needed to better clarify the behavioural mechanisms by which BZD receptor antagonists modify human aggressive responding.

Flumazenil--treatment or toxin.

Flumazenil is frequently administered to the poisoned patient. Seizures may be precipitated and resedation may occur in patients who awakened following flumazenil administration. Seizures may increase morbidity and mortality of the overdose. Benefit:Risk ratio of administering flumazenil should be determined in each overdose patient. Indications for flumazenil are limited.

Comparative pharmacokinetics of submucosal vs. intravenous flumazenil (Romazicon) in an animal model.

PURPOSE: This study was performed to determine the bioavailability and local tissue toxicological safety of flumazenil (Romazicon) when administered by oral submucosal (SM) as opposed to intravenous (i.v.) injection. METHODS: Six dogs each received SM flumazenil (0.2 mg), and their serum was collected at predetermined time intervals (0-2 h) and frozen (-70 degrees C). Seven days later, the dogs received an identical dose of i.v. flumazenil, and serum samples were again collected, as above. Comparative quantitation of flumazenil levels (i.v. vs. SM) was made using a sensitive HPLC assay (UV detection). Direct/local drug toxicity was visually scored by unbiased raters of color photographs (test and control mucosa) taken at 1, 2, and 7 days following SM flumazenil injection. An oral pathologist examined slides processed from control and treatment tissues (hematoxylin and eosin staining) taken (punch biopsy) 1 week following SM injection to compare with direct clinical scores. RESULTS: Serum flumazenil levels reached a plateau (8.5 +/- 1.5 ng/mL, mean +/- SD) within 4 min of SM drug injection and declined thereafter to -2 ng/mL by 2 h. Bioavailability of SM flumazenil was 101 +/- 14%, based upon measuring the area under the serum concentration-time curves over 1.5 h (AUC 0-1.5 h, SM vs. i.v. drug). Thus, serum drug levels following SM drug administration were broadly comparable to those obtained during the elimination phase of corresponding i.v. drug delivery. Regarding drug tissue toxicity, no evidence of direct drug toxicity was observed by unbiased raters of color photographs (test and control mucosa) taken at 1, 2, and 7 days following SM flumazenil injection. Following pathologic review, no difference was seen in the degree of inflammation between treatment and control tissue. CONCLUSION: At the quantity and concentration used, SM drug flumazenil administration appears to be both a safe and a viable alternative to bolus i.v. drug delivery and worthy of further investigation.

Evaluation of perinatal flumazenil effects on the behavior of female RLA/Verh rats in anxiety tests and shuttle box avoidance.

The present study investigated the effects of perinatal flumazenil (Ro 15-1788, a benzodiazepine receptor antagonist), given from gestational day 15 to the 14th day after giving birth, at two doses (3.5 and 6.3 mg/kg/ day) on the behavior of female RLA/Verh rats. This rat line has been selectively bred for non-acquisition of two-way active avoidance. Offspring of treated/non-treated dams were tested when adults for two-way active (shuttle box) avoidance acquisition. For comparison reasons additional offspring coming from the same treatments were used in an hyponeophagia test and in both an open field and a plus maze tests. The results show that perinatal flumazenil, specially the lower dose (and also the highest dose, although only in the final phases of the shuttle box training) was able to improve two-way active avoidance acquisition, as it has been previously found with males, whereas it failed to show any effect in the hyponeophagia task, the open field and the plus-maze tests. As two-way active avoidance acquisition can be enhanced both by reducing emotionality/anxiety or by improving memory, it is suggested that flumazenil treatment could have affected either one or both processes.

Gender differences in diazepam withdrawal syndrome in mice.

The present work was designed to study the influence of testosterone and oestrogens on the benzodiazepine withdrawal syndrome in mice. Several withdrawal signs were induced by 15 mg/kg intraperitoneally of flumazenil in diazepam-treated mice. The most noticeable were jerks, usually accompanied by tail lifts, and seizures. The intensity of the diazepam withdrawal syndrome was significantly lower in male than in female mice, especially in relation to the incidence of seizures. Castrated male mice showed a significant increase in the intensity of withdrawal syndrome. In addition, diazepam produced a significant increase of body weight in males but not in females. The principal finding of the present work is that the incidence of seizures produced by the administration of flumazenil was significantly lower in male than in female diazepam-treated mice. This fact suggests that the mechanism of action of benzodiazepines is modulated by the action of sexual hormones, and that testosterone plays a relevant role.

Antídotos de uso reciente

Los antídotos son sustancias cuya función es contrarrestar el efecto farmacológico y tóxico de otras sustancias, teniendo en cuenta la importancia de las medidas generales en el manejo del intoxicado (baño general, emesis, lavado gástrico, carbón activado, catárticos). Cada día aparecen sustancias nuevas con dichas características. En el presente artículo se pretende dar información breve y detallada sobre las propiedades farmacológicas, indicaciones, dosificación, efectos secundarios y contraindicaciones de algunos de uso general (carbón activado, soluciones electrolíticas con polietilenglycol) y principalmente de algunos específicos de uso reciente: flumazenil, fragmentos Fab-antidigoxina, glucagón, naloxona, clonidina, N-acetil-cisteína, azul de metileno, nitrito y tiosulfato de sodio, ácido-2-3-dimercaptosuccínico, penicilina benzatínica, glicopirrolato y S-adenosil-metionina.

Flumazenil and lignocaine-induced toxicity: is the inverse agonist type activity circadian-time dependent?

We have investigated the possible influence of time of day on the activity of flumazenil on lignocaine-induced toxicity in mice. The circadian pattern of the period of latency for convulsions and the induced mortality for lignocaine alone and for lignocaine plus flumazenil was not statistically significant (cosinor or analysis of variance) but the influence of flumazenil on lignocaine-induced toxicity was circadian-time dependent (F = 27.8, P = 0.0001).

Relative anticonvulsant effects of GABAmimetic and GABA modulatory agents.

Anticonvulsant properties of compounds that enhance GABA-mediated inhibition through modulatory sites on the GABAA receptor [phenobarbital (PB), clonazepam (CZP), alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone (alpha-EMTBL)] were compared with anticonvulsant effects of compounds believed to be antagonists at these modulatory sites (Ro15-1788 and alpha-isopropyl-alpha-methyl-gamma-butyrolactone gamma-IMGBL)] and to 4,5,6,7-tetrahydroisoxazolo-[4,5-c]-pyridin-3-ol (THIP, GABAA receptor agonist), (+/-) baclofen (GABAB receptor agonist), and gamma-vinyl GABA, a compound that increases endogenous GABA. The compounds were tested for their ability to block experimental seizures caused by maximal electroshock, pentylenetetrazol, picrotoxin, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), bicuculline (BIC), aminophylline, strychnine, and t-butyl-bicyclophosphorothionate (TBPS) in mice. CZP blocked all but strychnine seizures. PB was also highly effective, blocking all but TBPS seizures. alpha-EMTBL, representing a new class of experimental anticonvulsant drugs, prevented all seizures except strychnine (STR)- and aminophylline-induced seizures. The antagonists are effective only against one convulsant stimulus. Ro15-1788 and alpha-IMGBL prevented only DMCM- and pentylenetetrazol (PTZ)-induced seizures, respectively. THIP and gamma-vinyl GABA both blocked only BIC and picrotoxin seizures. Baclofen had no anticonvulsant activity. These data demonstrate that compounds that increase neuronal inhibition by potentiating the action of GABA have a broader spectrum of anticonvulsant action than either antagonists or GABAmimetic agents or compounds that increase endogenous GABA.

Effects of prenatal exposure to benzodiazepine-related drugs on early development and adult social behaviour in Swiss mice--II. Antagonists.

1. The present experiment examined, using a battery of tests, the effects of in utero exposure of Swiss mice to the benzodiazepine antagonists Ro 15-1788 and CGS 8216 upon early development and adult behaviour. 2. Early development was facilitated by treatment of Ro 15-1788. CGS 8216 suppressed during later development. Effects on the righting reflex were seen on different postnatal days following exposure to different treatments. The other factors studied remained unaffected. 3. Effects on adult social behaviour were examined using the resident-intruder paradigm, by determining the time allocated to broad behavioural categories. Increases in attack and threat in male offspring of dams treated with Ro 15-1788 and CGS 8216 were found. Female offspring differed from controls in digging and defensive/submissive behaviour.

Toxicological investigations with the benzodiazepine antagonist flumazenil.

The benzodiazepine antagonist ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4] benzodiazepine-3-carboxylate (flumazenil, Ro 15-1788, Anexate) was investigated in a series of toxicological studies. In a single intravenous injection study in male and female mice and rats, the highest non-lethal (maximum tolerated) doses were found to be between 62.5 and 125 mg/kg (the amounts of flumazenil present in the aqueous ampoules available for intravenous injection in man are 0.5 and 1.0 mg). In intravenous studies of 4 weeks duration, 10 mg/kg/d were systemically well tolerated in dogs and rats. In 13-week oral studies, 80 mg/kg/d were very well tolerated in dogs (capsule administration) and, after 125 mg/kg/d (by feed-admix) in rats, no untoward compound-related findings apart from a 10-15% increase of the liver weights in females were made. In reproductive toxicity studies, flumazenil revealed no drug-related embryotoxic or teratogenic effect and no adverse effects upon fertility of dosed animals themselves or on the peri- and postnatal development of their offspring. There was no indication for mutagenic potential of flumazenil in vitro concerning induction of gene mutations or clastogenic effects. An in vivo DNA repair test using germ cells of male mice did not yield DNA-damaging activities. From all these toxicological investigations it can be concluded that the risk in man given therapeutic doses or even intentional or accidental overdoses of flumazenil is extremely small.

Protection against the lethal effects of pentobarbital in mice by a benzodiazepine receptor inverse agonist, 6,7-dimethoxy-4-ethyl-3-carbomethoxy-beta-carboline.

The benzodiazepine receptor inverse agonist 6,7-dimethoxy-4-ethyl-3-carbomethoxy-beta-carboline (DMCM) (1.5-15 mg/kg) was administered to mice 5 min after a lethal (LD94) injection of pentobarbital. DMCM (1.5-5 mg/kg) increased short-term (1 h) survival in a dose-dependent fashion, with an optimum survival rate more than five times greater than mice receiving pentobarbital alone. Statistically significant increases in long-term (24 h) survival were also observed after both 5 and 10 mg/kg of DMCM (34 and 33%, respectively) compared with animals receiving pentobarbital alone (6%). Two doses of DMCM (5 and 2.5 mg/kg, respectively) administered 55 min apart produced an even greater increase (58%) in 24-h survival rates. Doses of DMCM that increased 1- and 24-h survival were not lethal when administered alone, and were below the dose that produced convulsions in 50% (CD50) of the animals. The protective effects of DMCM were blocked by pretreatment with the benzodiazepine receptor agonist ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo- 4H-imidazo[1,5a][1,4]benzodiazodiazepine-3-carboxylate (Ro 15-1788), which suggests the effects of DMCM are mediated through the benzodiazepine receptor. These findings suggest that DMCM or another benzodiazepine receptor ligand with full inverse agonist qualities could prove effective as an antidote for barbiturate intoxication in man.