Specific Benzodiazepine Antagonist Research Articles

Toxicity of intravenous anaesthetics

Intravenous anaesthetic agents are generally remarkably safe. However, it is clear that propofol infusion syndrome is a real, albeit rare, entity. This often lethal syndrome of metabolic acidosis, acute cardiomyopathy and skeletal myopathy is strongly associated with infusions of propofol at rates of 5mg/kg/hour and greater for more than 48 hours. There is evidence to support the hypothesis that the syndrome is caused by the failure of free fatty acid metabolism due to inhibition of free fatty acid entry into the mitochondria and also specific sites in the mitochondrial respiratory chain. The syndrome therefore mimics the mitochondrial myopathies. Midazolam causes seizure-like activity in very-low-birthweight premature infants requiring the drug prior to tracheal intubation or during prolonged positive pressure ventilation. This can be successfully reversed with the specific benzodiazepine antagonist flumazenil. Midazolam can also cause paradoxical reactions, including increased agitation, poor co-operation and aggressive or violent behaviour, which has been successfully managed with flumazenil.

In vivo imaging of anaesthetic action in humans: approaches with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI)

In vivo imaging of anaesthetic action in humans: approaches with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI)

Sedation and Analgesia for Procedures in Children

The management of acute pain and anxiety in children undergoing therapeutic and diagnostic procedures outside the operating room has developed substantially in the past 15 years. The widespread availability of noninvasive monitoring, short-acting opioids and sedatives, and specific opioid and benzodiazepine antagonists has enabled clinicians to administer sedation safely for procedures in diverse settings. The goal of procedural sedation is the safe and effective control of pain, anxiety, and motion so as to allow a necessary procedure to be performed and to provide an appropriate degree of memory loss or decreased awareness. This article reviews the current status of sedation . . .

Sedation and analgesia for procedures in children.

The management of acute pain and anxiety in children undergoing therapeutic and diagnostic procedures outside the operating room has developed substantially in the past 15 years. The widespread availability of noninvasive monitoring, short-acting opioids and sedatives, and specific opioid and benzodiazepine antagonists has enabled clinicians to administer sedation safely for procedures in diverse settings. The goal of procedural sedation is the safe and effective control of pain, anxiety, and motion so as to allow a necessary procedure to be performed and to provide an appropriate degree of memory loss or decreased awareness. This article reviews the current status of sedation . . .

Modulation of GABA(A) receptors and inhibitory synaptic currents by the endogenous CNS sleep regulator cis-9,10-octadecenoamide (cOA).

1. Cis-9,10-octadecenoamide (cOA) accumulates in the CSF of sleep-deprived cats and may represent a novel signalling molecule. Synthetic cOA has been shown to induce physiological sleep when injected into laboratory rats. Here we assess the cellular mode of action of cOA in vitro. 2. In all rat cultured cortical neurones (pyramidal cells) examined, the synthetic brain lipid (3.2-64 microM) enhanced the responses to subsaturating GABA concentrations (up to circa 2x) in a concentration-dependent manner (EC50, circa 15 microM). 3. (20 microM) cOA significantly enhanced the affinity of exogenous GABA for its receptor without changing the Hill slope or the maximal response. These effects were not voltage-dependent or secondary to shifts in E(Cl). 4. In the absence of GABA, cOA directly evoked small inhibitory currents in a subpopulation (<7%) of sensitive cells. 5. 20 microM cOA reversibly enhanced the duration of spontaneous inhibitory post synaptic currents (circa 2 fold) without significantly altering their amplitude. 6. At 32-64 microM, cOA reversibly reduced the incidence and amplitude of both inhibitory post synaptic currents (i.p.s.cs) and excitatory post synaptic currents (e.p.s.cs) in the cultured neuronal circuits in common with other depressant drugs acting at the GABA(A) receptor. 7. 32 microM Oleic acid did not modulate exogenous GABA currents or synaptic activity suggesting that cOAs actions are mediated through a specific receptor. 8. A specific, protein-dependent interaction with GABA(A) receptors was confirmed in Xenopus oocytes. Recombinant human receptors were modulated by 10 microM cOA (and diazepam) only when a gamma2 subunit was co-expressed with alpha1beta2: the cOA response was not sensitive to the specific benzodiazepine antagonist flumazenil (1 microM). 9. cOA may represent an endogenous ligand for allosteric modulatory sites on isoforms of GABA(A) receptors which are crucial for the regulation of arousal and have recently been implicated in the circadian control of physiological sleep.

Modulation of γ-aminobutyric acid (GABA) receptors and the feeding response by neurosteroids in Hydra vulgaris

γ-aminobutyric acid (GABA) receptors are present in membrane preparations from Hydra vulgaris, one of the most primitive organisms with a nervous system. These receptors are sensitive to muscimol and benzodiazepines and appear to be important in the regulation of the feeding response. The effects of neurosteroids, general anaesthetics, and GABA antagonists on GABA A receptors in membranes prepared from hydra and on the feeding response have now been investigated. The neurosteroids tetrahydroprogesterone and tetrahydrodeoxycorticosterone increased [ 3H]GABA binding to hydra membranes with nanomolar potency (EC 50, 141±11 and 623±36 nM, respectively) and high efficacy (maximal increase 79±6.5 and 62±4%, respectively), whereas the 3 β-hydroxy epimer of tetrahydroprogesterone was ineffective. The benzodiazepine receptor ligands diazepam (100 μM), clonazepam (100 μM) and abecarnil (30 μM) enhanced [ 3H]GABA binding to Hydra membranes by 22, 20 and 24%, respectively; effects abolished by the specific benzodiazepine antagonist flumazenil (100 μM). On the contrary, the peripheral benzodiazepine receptor ligand 4′chlorodiazepam failed to affect [ 3H]GABA binding to Hydra membranes. The general anaesthetics propofol and alphaxalone similarly increased (+38% and +30%, respectively) [ 3H]GABA binding. Moreover, [ 3H]GABA binding to Hydra membranes was completely inhibited by the GABA A receptor antagonist SR 95531, whereas bicuculline was without effect. The modulation of GABA A receptors in vitro by these various drugs correlated with their effects on the glutathione-induced feeding response in the living animals. Tetrahydroprogesterone and tetrahydrodeoxycorticosterone (1 to 10 μM) prolonged, in a dose-dependent manner, the duration of mouth opening induced by 10 μM glutathione, with maximal effects of +33 and +29%, respectively, apparent at 10 μM neurosteroid. Alphaxalone (10 μM) similarly increased (+33%) the effect of glutathione. The effects of steroids on the feeding response were inhibited by SR 95531 in a dose-dependent manner; t-butylbyclophosphorothyonate (1 μM), a specific Cl − channel blocker, which per se, like picrotoxin but not bicuculline, shortened the duration of the response, also counteracted the steroids effects at 1 μM. These results suggest that the modulation of GABA A receptors by steroids is an ancient characteristic of the animal kingdom and that the pharmacological properties of these receptors have been highly conserved through evolution.

Yohimbine and flumazenil: effect on nitrous oxide-induced suppression of dorsal horn neurons in cats.

The purpose of this study was to determine the mechanisms of nitrous oxide (N2O) antinociception at the spinal level with yohimbine (an α2-adrenergic antagonist) and flumazenil (a specific benzodiazepine antagonist) using chemonociceptive stimuli in spinal dorsal horn neurons in the cat. A lumbar laminectomy extending from L4 to L6 was performed to allow insertion of a extracellular recording device via a microelectrode. Additional laminectomy was performed at the T12 level to transect the spinal cord. As a noxious stimulus, bradykinin (BK) was injected via the cannula inserted into the femoral artery. Animals were divided into four treatment groups for subsequent experiments: N2O+flumazenil, N2O+yohimbine, flumazenil (alone), and yohimbine (alone). N2O suppressed BK-induced nociceptive responses in transected feline spinal cords. The BK-induced neuronal firing rates were significantly suppressed: to 69.2%, 61.8%, and 52.2% of the baseline firing rate at 10, 20, and 30 min, respectively, after N2O administration. The 47.8% suppression on BK-induced neuronal responses at 30 min after N2O administration was reversed 5 min after administration of yohimbine (25.2% suppression). Similarly, N2O suppression (42.5%) on chemically induced neuronal responses was reversed by flumazenil (24.9% suppression) at identical postadministration intervals. These data imply that N2O suppresses the nociceptive responses in part probably through its agonistic binding activity to the α2-adrenergic, γ-aminobutyric acid (GABA)-benzodiazepine, or both receptor systems in dorsal born neurons of the feline spinal cord.

Non-GABAergic effects of midazolam, diazepam and flumazenil on voltage-dependent ion currents in NG108-15 cells.

Non-gamma-Aminobutyric acid (GABA)-mediated effects of benzodiazepines (BZs) have not been widely investigated. However, there is significant evidence in the literature to suggest that several experimental and clinical observations are inconsistent with the commonly accepted GABAergic mechanisms of action for these drugs. The purpose of the present study was to explore electrophysiological effects of midazolam, diazepam and a specific BZ antagonist, flumazenil, using patch-clamp techniques in NG108-15 cells which do not express the GABAA receptor. Midazolam and diazepam decreased Na+, K+ and Ca2+ currents in a dose-related manner. Ca2+ currents were reduced more significantly by diazepam than by midazolam. Flumazenil showed no effects on voltage-dependent ion currents. GABA by itself showed neither effects on the membrane potential nor these ion currents. Midazolam and diazepam, but not flumazenil, exhibited effects on voltage-dependent ion currents in cultured neurons.

Role of GABA-A and mitochondrial diazepam binding inhibitor receptors in the anti-stress activity of neurosteroids in mice.

Neuroactive steroidal modulation of immobilization-stress and possible involvement of GABA-A and mitochondrial diazepam binding inhibitor (DBI) receptors (MDR) has been investigated in mice. Immobilization of mice for 2 h induced intense antinociception, anxiety state, and associated with a fall in adrenal ascorbic acid levels. Pretreatment with high dose of progesterone (10 mg/kg), a precursor of neurosteroids, significantly decreased the stress-induced antinociception, anxiety and fall in adrenal ascorbic acid, while low doses (1 and 5 mg/kg) or hydrocortisone (10 and 100 mg/kg) were ineffective. In contrast, progesterone (1 mg/kg, for 9 days) produced a significant antistress effect, which was blocked by GABA-A antagonists picrotoxin (1 mg/kg) and bicuculline (1 mg/kg), but not by flumazenil (2 mg/kg), a specific benzodiazepine (BZD) antagonist. 4'-chlordiazepam (0.1 and 0.25 mg/kg), a specific high affinity MDR agonist, produced significant anti-stress effect in a flumazenil-insensitive manner, but was blocked by pretreatment with PK11195 (1.5 mg/kg), a selective partial agonist of MDR, and with bicuculline (1 mg/kg), a potent GABA-A receptor antagonist. At higher doses, progesterone and 4'-chlordiazepam which are effective in immobilization stress also reduced locomotion. However, lower doses of progesterone (6.5 mg/kg) neither affected locomotion, nor produced any motor toxicity on rota-rod test. At the lower doses, the MDR ligand 4'-chlordiazepam (50 micrograms/kg) decreased locomotor activity, without altering motor toxicity on rota-rod test. Further, the per se effects of these treatments on unstressed mice were not significantly different from those of untreated controls, except for plus-maze test. The antistress profile of progesterone may be attributed to the in vivo production of neurosteroid allopregnanolone, thus resembled that of BZDs. Furthermore, the antistress actions are flumazenil-resistant, reaffirming that there may be an increase in the levels of pregnane neurosteroids in vivo, which may act on a specific allosteric site on GABA-A receptors distinct from BZD site. Because 4'-chlordiazepam binds to MDRs and stimulate mitochondrial neurosteroidogenesis, the anti-stress effects of 4'-chlordiazepam may be imputed to its MDR-induced neurosteroids, which then act on GABA-A receptors. These data suggest a pivotal role for GABA-A and mitochondrial DBI receptors in the antistress actions of neurosteroids and reinforces their ameliorative effect in physiological stress.

The role of flumazenil in the critically ill.

Flumazenil is a specific benzodiazepine antagonist. It competitively inhibits the action of benzodiazepines at gamma aminobutyric acid (GABA) receptors in the central nervous system and thus reverses the sedative effects of benzodiazepines. Usually, it is given intravenously as a bolus dose or as an infusion. It has a short duration of action. Flumazenil is extensively metabolized to compounds that have minimal benzodiazepine antagonistic properties. Reversal of sedation can be useful in many conditions that are often encountered in the critically ill. The adverse effects of its use are usually predictable and, with sufficient clinical monitoring, are usually avoidable. These properties make it a useful and safe drug when used appropriately.

Conditioned Suppression and the Effects of Pentobarbital with Picrotoxin, Flumazenil, and R05-3663

In two experiments, rats trained to lever press for food were maintained under a conditioned suppression procedure and then given an acute regime of drug administration involving pentobarbital (10 mg/kg). In both experiments, pentobarbital reduced conditioned suppression. In the first experiment (4 rats), this effect was increased by the combination of the specific benzodiazepine antagonist flumazenil (10 mg/kg) with pentobarbital but unaffected by the combination of picrotoxin (1.5 mg/kg) with pentobarbital. In the second experiment (3 rats), this effect was attenuated when picrotoxin (2.0 mg/kg) or Ro5-3663 (5.0 mg/kg) were also administered. These findings add to the data on the effects of anxiolytics and related compounds on conditioned suppression, they provide more information on the possible nature of the GABA/benzodiazepine receptor complex and suggest that flumazenil can have anxiolytic as well as anxiogenic effects.

An assessment of resedation following flumazenil-induced antagonism of intravenous midazolam: comparison of psychomotor and amnesic recovery with a non-sedated reference group

The specific benzodiazepine antagonist flumazenil can enhance patient recovery following local anaesthetic day-case surgery performed under sedation. However, in view of its short elimination half-life, concerns have been expressed about the risk of resedation following its use. An open, randomised, parallel group study was designed to explore this question. Eighty-five patients were studied. Group A (n=43) patients underwent local anaesthetic cystoscopy with intravenous (i.v.) midazolam sedation. Following cystoscopy, and 30 min after the injection of midazolam, a bolus dose of flumazenil (0.5 mg i.v.) was given. Group B (n=42) patients underwent no operation and received no drugs but, in all other respects, were treated in an identical fashion to patients in group A. Tests of psychomotor function and memory were administered at baseline and again at 0.5, 1, 2, 3 and 4 h (or equivalent times for group B patients) following the injection of flumazenil. The test results showed no evidence of resedation, but there was evidence of incomplete reversal, as shown by significant differences in critical flicker fusion and delayed word recall at the 0.5-h test point. Group B patients showed no evidence of practice effects but did demonstrate an impairment in test performance possibly related to motivational factors. In conclusion, this study provides no evidence of resedation when using flumazenil to reverse the acute effects of midazolam. Incomplete reversal of amnesia need not delay patient discharge but has important implications with respect to the timing and nature of information imparted to patients prior to their release from hospital.

Astroglia‐released factor shows similar effects as benzodiazepine inverse agonists

Media conditioned by cultured neonatal cerebral cortex microexplants (CCM) or astrocytes (ACM) contain low molecular weight (< 1,000 Da) substance(s) which inhibits the gamma aminobutyric acid (GABA)-induced inward current recorded in cerebellar granule cells and hippocampal neurons in culture using the whole-cell patch-clamp technique. This effect is specific for CCM and ACM, as medium conditioned by PC12 cells (PC12CM) does not affect the GABA response of these cells. It is also specific for GABA-induced currents because glutamate-induced currents do not change either in amplitude or in shape in the presence of CCM or ACM. The inhibitory effect on the GABA response in cerebellar granule cells of both ACM and CCM could be suppressed by flumazenil, a specific benzodiazepine (BZD) antagonist and could be mimicked by two BZD inverse agonists. These data thus demonstrate the presence of a BZD inverse agonist-like activity in CCM and ACM. This effect of ACM on different neuronal cell types was heterogenous since no detectable effect could be observed on the GABA-induced current in GABA-responsive dorsal root ganglion (DRG) neurons, presumably reflecting a functional heterogeneity of the GABAA receptors present in these different neuronal subsets. By the release of such an endogenous BZD inverse agonist-like activity, glia cells could possibly modulate GABAA receptor-mediated responses.

Management of Benzodiazepine Overdose

Today, benzodiazepines are the drugs most commonly involved in cases of self-poisoning. Fortunately, compared with several other drugs they are relatively safe in overdose, and symptoms of severe poisoning are rarely observed in young adults with pure benzodiazepine overdosage. However, respiratory depression, hypotension and prolonged coma are often seen in other patient groups with benzodiazepine overdosage, such as the elderly, children and patients with chronic pulmonary disease. Flumazenil, a specific benzodiazepine antagonist, is a valuable and safe diagnostic and therapeutic tool in the short term management of drug poisoning with involvement of benzodiazepines. Its use should therefore be considered in all unconscious patients admitted because of benzodiazepine overdosage. In self-poisoning, the drugs ingested are frequently a mixture of several compounds and their identity is often unknown. Flumazenil can also be used diagnostically in cases of unclear multiple drug poisoning or coma of unknown aetiology. During use of flumazenil, consideration should be paid to a few contraindications, including patients with epilepsy who are receiving long term benzodiazepine medication. Moreover, some precautions should be taken during administration, such as individual titration of the dosage to minimise the risk of inducing benzodiazepine withdrawal syndrome or unmasking toxic effects of other concurrently ingested drugs. A normal electrocardiogram recording before the antidote is given will imply a minimal risk of any adverse reactions in cases of combined intake of benzodiazepine and antidepressants. Further, the short half-life of flumazenil necessitates a period of careful supervision after its administration. Supportive care in a patient with benzodiazepine overdose includes close monitoring, preferably in the drainage position. If hypotension or prolonged eNS depression occurs, intravenous fluids should be administered. Occasionally, gastric lavage and administration of activated charcoal is indicated, but only if the patient is awake and potentially sensitive to benzodiazepines and if a large dose has been ingested within the last 1 to 2 hours.

Endogenous benzodiazepine system and regulation of respiration in the cat

Benzodiazepines, a class of drugs widely used as anxiolytics, can induce a depression of respiration. This study was designed to determine if endogenous benzodiazepine ligands could act in a similar fashion and exert a tonic inhibitory influence on respiration. Administration of a benzodiazepine antagonist should then facilitate respiration. This might be especially visible in hypoxia, the condition characterized by both central respiratory depression and potentially enhanced benzodiazepine expression. We addressed this issue by comparing the effects on the phrenic neurogram of the specific benzodiazepine antagonist flumazenil (200 μg i.v. boluses) in the contrasting conditions of hypoxia and hyperoxia in anesthetized, both spontaneously breathing and paralyzed ventilated cats. Contrary to our hypothesis, flumazenil showed a modest but definite inhibitory effect on respiration. Flumazenil also lengthened the duration of the Hering-Breuer inspiratory inhibition. The respiratory depression was neither related to chemical drive nor to the GABA receptor complex, for it was sustained after antagonism of GABA with picrotoxin and bicuculline. We conclude that the endogenous benzodiazepine system is unlikely to play an inhibitory role in the regulation of respiration. The physiologic role of this system remains to be established.

The effect of oral midazolam and diazepam on respiration in normal subjects

Benzodiazepine have been shown to suppress ventilatory responses to hyperoxic hypercapnia (HCVR) and isocapnic HVR when taken parenterally. Most patients would, however, prefer to take an oral rather than parenteral preparation but the effect of oral benzodiazepine on these ventilatory responses has not been well studied. We therefore studied the effect of oral midazolam (7.5 mg) and diazepam (5 mg) both given orally on resting ventilation and respiratory drive, as assessed by HCVR and HVR. Flumazenil, a specific benzodiazepine antagonist, was administered intravenously to reverse the effect. A mental alertness-drowsiness index in five grades, from 1 (awake and alert) to 5 (asleep), was used to assess the sedation effect. Six normal male subjects, (aged 31 +/- 1.6 yrs) (mean +/- SD), participated in the study. Mean resting ventilation, and ventilatory response to HCVR and HVR were not significantly altered by these drugs when taken orally. Flumazenil also had not significant effect on HCVR and HVR. However the mental alertness-drowsiness index rose from 1 to 2.83 with oral midazolam and reversed to 1.25 with flumazenil. Similarly, this index increased from 1 to 2.25 after oral diazepam and reversed to 1.42 after flumazenil. In conclusion, we found that even though oral midazolam and diazepam produced a significant sedation effect, which was reversed with flumazenil, the drugs had no effect on ventilation at rest and the ventilatory responses to hypoxia and hypercapnia.

Flumazenil

Flumazenil is a specific benzodiazepine antagonist which is indicated when the central effects of a benzodiazepine need to be attenuated or terminated. Following intravenous administration of up to 1 mg, flumazenil effectively reverses sedation and improves psychomotor performance following administration of short and longer acting benzodiazepines used for sedation, or general anaesthesia supplemented with benzodiazepines. The duration of action is short at generally 30 to 60 minutes and supplemental doses of flumazenil may be needed to maintain the desired level of consciousness in some patients. After poisoning with high dosages of benzodiazepines alone or combined with other drugs, the initial single dose of flumazenil will require supplementing with repeated low intravenous doses or an infusion to maintain wakefulness. In such patients, flumazenil also facilitates differential diagnosis and reduces the necessity for interventions. Flumazenil thus enhances recovery and allows more rapid discharge of patients sedated with benzodiazepines for diagnostic procedures and facilitates management of patients during the initial recovery period following general anaesthesia supplemented with benzodiazepines, but does not preclude normal monitoring during the recovery period. Flumazenil is clearly very useful in treating drug poisoning when benzodiazepines are a major component. By virtue of its specific benzodiazepine antagonist effects, flumazenil provides an innovative and well tolerated approach in clinical situations requiring rapid reversal of benzodiazepine-induced central nervous system depressant effects.

Cardiorespiratory Effects of Antagonism of Diazepam Sedation With Flumazenil in Patients With Cardiac Disease

The specific benzodiazepine antagonist flumazenil is currently under intense study. Despite much clinical experience, no detailed invasive hemodynamic studies of its use in cardiac patients have been published. In the present study, hemodynamic and respiratory variables were measured in 10 cardiac patients undergoing catheterization of the right and left sides of the heart, before and after sedation with intravenous diazepam, and after reversal of sedation with flumazenil. A sleep dose of diazepam (12.2 +/- 5.1 mg, mean +/- SD) caused only slight decreases in mean arterial pressure (103 +/- 12 to 98 +/- 14 mm Hg; P less than 0.05), pulmonary capillary wedge pressure (13.2 +/- 6.3 to 11.7 +/- 6.6 mm Hg; P less than 0.05), and left ventricular end-diastolic pressure (20.8 +/- 7.5 to 17.3 +/- 10.0 mm Hg; P less than 0.05), with no significant changes in respiratory gas homeostasis. Intravenous flumazenil (0.22 +/- 0.07 mg) resulted in spontaneous awakening and return to full orientation, yet caused no significant alteration in either hemodynamic or respiratory variables measured. Reversal of diazepam-induced sedation by flumazenil in cardiac patients appears safe and effective.

HPLC Quantification of Zolpidem and Prothipendyl in a Voluntary Intoxication

Zolpidem, a recently developed sleep inducer, and prothipendyl, a neuroleptic azaphenothiazine, were involved in a voluntary intoxication along with ethanol. After administration of flumazenil, a specific benzodiazepines antagonist, respiratory depression was corrected. HPLC with UV detection methods after selective extraction were developed to measure simultaneously prothipendyl and zolpidem without flumazenil interaction. These methods could be applied in drug monitoring and in emergency toxicology.

Protection against ethanol-induced gastric damage by drugs acting at the GABA-benzodiazepine receptor complex

The protective effects of drugs acting at the GABA-benzodiazepine receptor complex against ethanol-induced gastric damage in rats were investigated. Gastric lesions were induced by administration of 1 ml absolute ethanol orally to rats. Administration of clonazepam (0.625-2.5 mg/kg, IP), which binds with high affinity to the benzodiazepine binding site of the GABA-benzodiazepine receptor complex, or Ro 5-3663 (2.5 or 5 mg/kg), which binds to the piorotoxinin site of the receptor complex, protected against ethanol-induced gastric damage. The protective effect of clonazepam (1.25 mg/kg, IP) against ethanol-induced gastric damage was reversed, dose dependently, by the specific benzodiazepine antagonist, flumazenil (5-20 mg/kg, IP). This protective effect of clonazepam or Ro 5-3663 seems to be specific to ethanol-induced gastric damage, since neither drug protected against indomethacin-induced gastric damage. These results present for the first time evidence of the involvement of drugs acting at GABA-benzodiazepine receptors in protection against ethanol-induced gastric damage.