Competitive Antagonism of Etomidate Action by Diazepam: In Vitro GABAA Receptor and In Vivo Zebrafish Studies.

BACKGROUND: Recent cryo-electron microscopic imaging studies have shown that in addition to binding to the classical extracellular benzodiazepine binding site of the α1ß3γ2L γ-aminobutyric acid type A (GABAA) receptor, diazepam also binds to etomidate binding sites located in the transmembrane receptor domain. Because such binding is characterized by low modulatory efficacy, the authors hypothesized that diazepam would act in vitro and in vivo as a competitive etomidate antagonist. METHODS: The concentration-dependent actions of diazepam on 20 µM etomidate-activated and 6 µM GABA-activated currents were defined (in the absence and presence of flumazenil) in oocyte-expressed α1ß3γ2L GABAA receptors using voltage clamp electrophysiology. The ability of diazepam to inhibit receptor labeling of purified α1ß3γ2L GABAA receptors by [H]azietomidate was assessed in photoaffinity labeling protection studies. The impact of diazepam (in the absence and presence of flumazenil) on the anesthetic potencies of etomidate and ketamine was compared in a zebrafish model. RESULTS: At nanomolar concentrations, diazepam comparably potentiated etomidate-activated and GABA-activated GABAA receptor peak current amplitudes in a flumazenil-reversible manner. The half-maximal potentiating concentrations were 39 nM (95% CI, 27 to 55 nM) and 26 nM (95% CI, 16 to 41 nM), respectively. However, at micromolar concentrations, diazepam reduced etomidate-activated, but not GABA-activated, GABAA receptor peak current amplitudes in a concentration-dependent manner with a half-maximal inhibitory concentration of 9.6 µM (95% CI, 7.6 to 12 µM). Diazepam (12.5 to 50 µM) also right-shifted the etomidate-concentration response curve for direct activation without reducing the maximal response and inhibited receptor photoaffinity labeling by [H]azietomidate. When administered with flumazenil, 50 µM diazepam shifted the etomidate (but not the ketamine) concentration-response curve for anesthesia rightward, increasing the etomidate EC50 by 18-fold. CONCLUSIONS: At micromolar concentrations and in the presence of flumazenil to inhibit allosteric modulation via the classical benzodiazepine binding site of the GABAA receptor, diazepam acts as an in vitro and in vivo competitive etomidate antagonist.

Using dezocine to prevent etomidate-induced myoclonus: a meta-analysis of randomized trials.

OBJECTIVE: This study was designed to evaluate the efficacy and safety of preinjection of dezocine in preventing etomidate-induced myoclonus. METHODS: PubMed, Embase, The Cochrane Library, and China National Knowledge Infrastructure (CNKI) were searched to collect relevant randomized controlled trials (RCTs) from inception to July 2016 on the preinjection of dezocine in preventing etomidate-induced myoclonus. Two researchers independently screened literature, extracted data, and evaluated bias risks in accordance with inclusion and exclusion criteria, and then used RevMan 5.2 to perform the meta-analysis. RESULTS: A total of six RCTs were included in this study. The meta-analysis showed that 1) the preinjection of dezocine can reduce the incidence of etomidate-induced myoclonus (relative risk [RR] =0.25, 95% CI [0.13, 0.50], P<0.0001), which is consistent with the result of subgroup analysis; 2) the preinjection of dezocine can reduce the incidence of mild, moderate, and severe myoclonus; 3) dezocine was not related to an increasing incidence of etomidate-induced dizziness and nausea (RR =2.83, 95% CI [0.66, 12.08], P=0.6); and 4) dezocine did not reduce heart rates after the administration of etomidate (mean difference =1.06, 95% CI [-4.08, 6.19], P=0.69). CONCLUSION: The preinjection of dezocine has the effect of both lowering the incidence of etomidate-induced myoclonus and easing the severity of myoclonus, but without increasing dizziness and nausea or affecting the heart rate.

A Novel Strategy to Reverse General Anesthesia by Scavenging with the Acyclic Cucurbit[n]uril-type Molecular Container Calabadion 2.

BACKGROUND: Calabadion 2 is a new drug-encapsulating agent. In this study, the authors aim to assess its utility as an agent to reverse general anesthesia with etomidate and ketamine and facilitate recovery. METHODS: To evaluate the effect of calabadion 2 on anesthesia recovery, the authors studied the response of rats to calabadion 2 after continuous and bolus intravenous etomidate or ketamine and bolus intramuscular ketamine administration. The authors measured electroencephalographic predictors of depth of anesthesia (burst suppression ratio and total electroencephalographic power), functional mobility impairment, blood pressure, and toxicity. RESULTS: Calabadion 2 dose-dependently reverses the effects of ketamine and etomidate on electroencephalographic predictors of depth of anesthesia, as well as drug-induced hypotension, and shortens the time to recovery of righting reflex and functional mobility. Calabadion 2 displayed low cytotoxicity in MTS-3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-based cell viability and adenylate kinase release cell necrosis assays, did not inhibit the human ether-à-go-go-related channel, and was not mutagenic (Ames test). On the basis of maximum tolerable dose and acceleration of righting reflex recovery, the authors calculated the therapeutic index of calabadion 2 in recovery as 16:1 (95% CI, 10 to 26:1) for the reversal of ketamine and 3:1 (95% CI, 2 to 5:1) for the reversal of etomidate. CONCLUSIONS: Calabadion 2 reverses etomidate and ketamine anesthesia in rats by chemical encapsulation at nontoxic concentrations.

Effects of etomidate on complications related to intubation and on mortality in septic shock patients treated with hydrocortisone: a propensity score analysis.

INTRODUCTION: Endotracheal intubation in the ICU is associated with a high incidence of complications. Etomidate use is debated in septic shock because it increases the risk of critical illness-related corticosteroid insufficiency, which may impact outcome. We hypothesized that hydrocortisone, administered in all septic shock cases in our ICU, may counteract some negative effects of etomidate. METHODS: A single-center observational study was carried out in septic shock patients, treated with hydrocortisone and intubated within the first 48 hours of septic shock. Co-primary end points were life-threatening complications incidence occurring within the first hour after intubation and mortality during the ICU stay. Statistical analyses included unmatched and matched cohorts using a propensity score analysis. P < 0.05 was considered significant. RESULTS: Sixty patients in the etomidate cohort and 42 patients in the non-etomidate cohort were included. Critical illness-related corticosteroid insufficiency was 79% in the etomidate cohort and 52% in the non-etomidate cohort (P = 0.01). After intubation, life-threatening complications occurred in 36% of the patients whatever the cohort. After adjustment with propensity score analysis, etomidate was a protective factor for death in the ICU both in unmatched (hazard ratio, 0.33 (0.15 to 0.75); P < 0.01)) and matched cohorts (hazard ratio, 0.33 (0.112 to 0.988); P = 0.04). CONCLUSION: In septic shock patients treated with hydrocortisone, etomidate did not decrease life-threatening complications following intubation, but when associated with hydrocortisone it also did not impair outcome.

Investigating paradoxical hysteresis effects in the mouse neocortical slice model.

Clinically, anesthetic drugs show hysteresis in the plasma drug concentrations at induction versus emergence from anesthesia induced unconsciousness. This is assumed to be the result of pharmacokinetic lag between the plasma and brain effect-site and vice versa. However, recent mathematical and experimental studies demonstrate that anesthetic hysteresis might be due in part to lag in the brain physiology, independent of drug transport delay - so-called "neural inertia". The aim of this study was to investigate neural inertia in the reduced neocortical mouse slice model. Seizure-like event (SLE) activity was generated by exposing cortical slices to no-magnesium artificial cerebrospinal fluid (aCSF). Concentration-effect loops were generated by manipulating SLE frequency, using the general anesthetic drug etomidate and by altering the aCSF magnesium concentration. The etomidate (24 µM) concentration-effect relationship showed a clear hysteresis, consistent with the slow diffusion of etomidate into slice tissue. Manipulation of tissue excitability, using either carbachol (50 µM) or elevated potassium (5mM vs 2.5mM) did not significantly alter the size of etomidate hysteresis loops. Hysteresis in the magnesium concentration-effect relationship was evident, but only when the starting condition was magnesium-containing "normal" aCSF. The in vitro cortical slice manifests pathway-dependent "neural inertia" and may be a valuable model for future investigations into the mechanisms of neural inertia in the cerebral cortex.

Cholinergic modulation of sevoflurane potency in cortical and spinal networks in vitro.

BACKGROUND: Victims of organophosphate intoxication with cholinergic crisis may have need for sedation and anesthesia, but little is known about how anesthetics work in these patients. Recent studies suggest that cholinergic stimulation impairs gamma-aminobutyric acid type A (GABAA) receptor function. Because GABAA receptors are major targets of general anesthetics, the authors investigated interactions between acetylcholine and sevoflurane in spinal and cortical networks. METHODS: Cultured spinal and cortical tissue slices were obtained from embryonic and newborn mice. Drug effects were assessed by extracellular voltage recordings of spontaneous action potential activity. RESULTS: Sevoflurane caused a concentration-dependent decrease in spontaneous action potential firing in spinal (EC50=0.17+/-0.02 mM) and cortical (EC50=0.29+/-0.01 mM) slices. Acetylcholine elevated neuronal excitation in both preparations and diminished the potency of sevoflurane in reducing action potential firing in cortical but not in spinal slices. This brain region-specific decrease in sevoflurane potency was mimicked by the specific GABAA receptor antagonist bicuculline, suggesting that (1) GABAA receptors are major molecular targets for sevoflurane in the cortex but not in the spinal cord and (2) acetylcholine impairs the efficacy of GABAA receptor-mediated inhibition. The latter hypothesis was supported by the finding that acetylcholine reduced the potency of etomidate in depressing cortical and spinal neurons. CONCLUSIONS: The authors raise the question whether cholinergic overstimulation decreases the efficacy of GABAA receptor function in patients with organophosphate intoxication, thereby compromising anesthetic effects that are mediated predominantly via these receptors such as sedation and hypnosis.

Gamma-aminobutyric acid type A receptor beta 2 subunit mediates the hypothermic effect of etomidate in mice.

BACKGROUND: The authors have previously described that the gamma-aminobutyric acid type A (GABAA) receptor beta 2N265S mutation results in a knock-in mouse with reduced sensitivity to etomidate. After recovery from etomidate anesthesia, these mice have improved motor performance and less slow wave sleep. Because most clinically used anesthetics produce hypothermia, the effect of this mutation on core body temperature was investigated. METHODS: The effect of etomidate and propofol on core body temperature were measured using radiotelemetry in freely moving GABAA receptor beta 2N265S mutant mice and wild-type controls. RESULTS: beta 2N265S mutant mice have a reduced hypothermic response to anesthetic doses of etomidate compared with wild-type controls and after a transient loss of righting reflex regain normothermia more rapidly compared with wild-type controls. Subanesthetic doses of etomidate produce hypothermia, which was not observed in the mutant mice. Vehicle administration resulted in a stress-induced hyperthermic response in both genotypes. Propofol produced a hypothermic response that was similar in both genotypes. CONCLUSIONS: The GABAA receptor beta 2 subunit mediates a significant proportion of the hypothermic effects of etomidate. As the beta 2 subunit mediates postrecovery ataxia and sedation, anesthetic agents that do not have in vivo potency at beta 2 subunit-containing receptors offer the potential for surgical anesthesia with improved recovery characteristics.

Midazolam pretreatment reduces etomidate-induced myoclonic movements.

During induction of anaesthesia with etomidate, myoclonic muscle movements are frequent. In this study, pretreatment with a small dosage of etomidate or midazolam was compared with placebo for the prevention of myoclonic muscle movements. Sixty patients, premedicated with oral midazolam, were pretreated in a randomized double-blinded fashion with etomidate 0.05 mg/kg i.v., midazolam 0.015 mg/kg i.v. or normal saline i.v. (placebo) in three groups of 20 patients each. The pretreatment was followed after 90 seconds by etomidate 0.3 mg/kg i.v. One minute after onset of hypnosis, induction of anaesthesia was completed with sufentanil and rocuronium. From the time of pretreatment to completion of anaesthesia, patients were observed for myoclonic muscle movements by a single physician, blinded to group allocation. Myoclonic movements were graded on a scale of 0 to 3. The incidence of myoclonic movements was significantly lower in patients pretreated with midazolam (4 of 20) compared with placebo (18/20) (P < 0.01). Midazolam 0.015 mg/kg i.v., administered 90 seconds before induction of anaesthesia with etomidate, is effective in reducing etomidate-induced myoclonic muscle movements.

Effect of thiopental, propofol, and etomidate on vincristine toxicity in PC12 cells.

Neurotoxicity is the dose-limiting side-effect of vincristine in cancer therapy. Using the nerve growth factor (NGF)-dependent neurite outgrowth and cell proliferation of the PC12 pheochromocytoma cell line as an in vitro assay, the protective effect of different intravenous anesthetics was assessed. Vincristine (1 nmol/L) significantly decreased the percentage of neurite-forming cells from 68% +/- 9% to 27% +/- 7% within a 3-day incubation period. The longer neurites (> 2 x cell body) in particular proved to be extremely sensitive to vincristine (from 17% +/- 4% to 0% of total neurite-expressing cells). Flow cytometry results revealed an S-phase percentage of 15.85% +/- 3.25% after NGF induction, with vincristine reducing this percentage to 0.68% +/- 0.38%. Reversal of the inhibitory effect of vincristine was noted in the cells treated with thiopental or propofol but not etomidate. Bicuculline partially antagonized the protective effect of thiopental and propofol in both studies. We conclude that thiopental and propofol, but not etomidate, have a protective effect in vincristine-induced neurotoxicity. The protective effect produced by thiopental and propofol is probably secondary to activation of GABAA receptors.

[Does flumazenil antagonize the anesthetic effect of ketamine, etomidate or thiopental?].

The effect of flumazenil, a benzodiazepine antagonist, was assessed in a random, double-blind clinical study in which each of the four groups of surgical outpatients comprising 20 in each was given either ketamine 100 mg (K), etomidate 20 mg (E), thiopental 300 mg (T) or flunitrazepam 4 mg (F) for induction of anesthesia. On emergence, patients in each group were randomly given 2cc of either 2 coded solutions, one of which contained 0.2 mg flumazenil and the other of which was normal saline. Following injection of coded solution, all patients were assessed at 0, 5, 15, 30, 60 and 120 min for wakefulness. All 10 patients of group F who received flumazenil were alert and able to recall at 5 min, whereas in group T this was noted from 15 to 30 min. Patients of group E and K responded alike in a manner as of those who received normal saline placebo with onset of wakefulness at 30 and 60 min respectively. These results confirm that flumazenil antagonizes flunitrazepam (within 5 min) and also indicate that the antagonizing effect occurs 30 min following injection for thiopental, suggestive of some cross-reactivity between these two drugs.

Diazepam, pentobarbital and D-etomidate produced increases in bicuculline seizure threshold; selective antagonism by RO15-1788, picrotoxin and (+/-)-DMBB.

The seizure threshold for different seizure components was measured after slow intravenous infusion of bicuculline in the rat. Clear differences were seen in the seizure threshold for tremor (TRE) and clonic-forepaw (CLOF) as compared to clonic-hindpaw (CLOH) and tonic-forepaw (TONF). Seizure threshold was measured after treatment with different doses of diazepam, pentobarbital, D-etomidate, picrotoxin, RO15-1788 and (+/-)-5-(1,3,-dimethylbutyl)-5-barbituric acid (DMBB). Direct and indirect antagonism between the agonists and antagonists was examined. The interactions between the drugs for TRE and CLOF resemble those described in in vitro receptor-binding assays using the GABA-benzodiazepine-chloride ionophore complex (GBCI). The interactions for CLOH and TONF do not show this resemblance, suggesting less involvement of GABA in these phenomena. Diazepam was selectively antagonized by RO15-1788. D-Etomidate and pentobarbital were directly antagonized by DMBB, suggesting shared activity at the barbiturate site. No evidence was found for an interaction between compounds acting at different sites within the GBCI.

GABA-mimetic action of etomidate.

A comparison of antagonism by bicuculline or strychnine of the effects of GABA or etomidate on rat isolated superior cervical ganglia, frog isolated hemisected spinal cords and rat central neurones in vivo indicates that etomidate has GABA-mimetic actions.