Ratiometric determination of etomidate based on an albumin-based indicator displacement assay (IDA).

The first fluorescent sensor based on the indicator displacement assay (IDA) for on-site determination of etomidate.

Substituted Cysteine Modification and Protection with n-Alkyl-MTS Reagents Quantifies Steric Changes Induced by a Mutation in Anesthetic Binding Sites on GABA Type A Receptors.

Multiple approaches, including cryogenic electron microscopy (cryo-EM), indicate that the anesthetics etomidate and propofol modulate α1ß2/3γ2 GABAA receptors by binding in overlapping transmembrane inter-subunit sites near ßM286 and αL232 sidechains. High-precision approaches in functional receptors are needed for comparisons with cryo-EM. We previously used substituted cysteine modification and protection (SCAMP) with n-alkyl-methanethiosulfonate (MTS) reagents and electrophysiology in α1ß3M286Cγ2L receptors to estimate the distance from etomidate to ß3M286 with precision near 1.3 Å. Here, we address three more aims using this approach: (i) SCAMP with etomidate was tested in α1L232Cß3γ2L receptors; (ii) studies in α1L232Wß3M286Cγ2L receptors assessed whether α1L232W displaces etomidate relative to ß3M286C; and (iii) results with propofol were compared with those with etomidate. Voltage-clamp electrophysiology in Xenopus oocytes was used to assess persistent functional changes after exposing cysteine-substituted receptors to methyl-MTS through n-decyl-MTS. Overlap of modified cysteine sidechains with bound anesthetic was inferred when anesthetic co-application with alkyl-MTS reagent blocked the development of persistent effects. In α1L232Cß3γ2L receptors, only pentyl-MTS and hexyl-MTS induced persistent effects that were unaltered by etomidate co-application, precluding a direct estimate of intermolecular distance. In α1L232Wß3M286Cγ2L receptors, sidechain overlap with bound etomidate was inferred for modifications with ethyl-MTS through n-pentyl-MTS, with unambiguous cut-on and cut-off. Comparison with results in α1ß3M286Cγ2L reveals that α1L232W, which increases maximal sidechain length by 2.1 Å, displaces etomidate closer to ß3M286C by about 1.3 Å. Propofol results largely mirrored those with etomidate. These findings indicate that both etomidate and propofol bind within 1 Å of α1L232, consistent with cryo-EM structures. SIGNIFICANCE STATEMENT: We combined electrophysiology, cysteine substitutions, and n-alkyl-methanethiosulfonate modifiers in functional GABAA receptors to enable precise estimates of the distance between ß3M286C sidechains and anesthetics (etomidate and propofol) bound in transmembrane ß+/α- inter-subunit pockets. Comparing results in α1ß3M286Cγ2L and α1L232Wß3M286Cγ2L receptors reveals that α1L232W mutations displace both anesthetics toward ß3M286C, indicating that these anesthetics bind within 1 Å of the α1L232 sidechain in functional receptors, consistent with cryogenic electron microscopy structures derived under nonphysiologic conditions.

(+)-Catharanthine potentiates the GABAA receptor by binding to a transmembrane site at the ß(+)/α(-) interface near the TM2-TM3 loop.

(+)-Catharanthine, a coronaridine congener, potentiates the γ-aminobutyric acid type A receptor (GABAAR) and induces sedation through a non-benzodiazepine mechanism, but the specific site of action and intrinsic mechanism have not beendefined. Here, we describe GABAAR subtype selectivity and location of the putative binding site for (+)-catharanthine using electrophysiological, site-directed mutagenesis, functional competition, and molecular docking experiments. Electrophysiological and in silico experiments showed that (+)-catharanthine potentiates the responses to low, subsaturating GABA at ß2/3-containing GABAARs 2.4-3.5 times more efficaciously than at ß1-containing GABAARs. The activity of (+)-catharanthine is reduced by the ß2(N265S) mutation that decreases GABAAR potentiation by loreclezole, but not by the ß3(M286C) or α1(Q241L) mutations that reduce receptor potentiation by R(+)-etomidate or neurosteroids, respectively. Competitive functional experiments indicated that the binding site for (+)-catharanthine overlaps that for loreclezole, but not those for R(+)-etomidate or potentiating neurosteroids. Molecular docking experiments suggested that (+)-catharanthine binds at the ß(+)/α(-) intersubunit interface near the TM2-TM3 loop, where it forms H-bonds with ß2-D282 (TM3), ß2-K279 (TM2-TM3 loop), and ß2-N265 and ß2-R269 (TM2). Site-directed mutagenesis experiments supported the in silico results, demonstrating that the K279A and D282A substitutions, that lead to a loss of H-bonding ability of the mutated residue, and the N265S mutation, impair the gating efficacy of (+)-catharanthine. We infer that (+)-catharanthine potentiates the GABAAR through several H-bond interactions with a binding site located in the ß(+)/α(-) interface in the transmembrane domain, near the TM2-TM3 loop, where it overlaps with loreclezole binding site.

The protective effects of etomidate against interleukin-1ß (IL-1ß)-induced oxidative stress, extracellular matrix alteration and cellular senescence in chondrocytes.

Osteoarthritis (OA) is a common chronic inflammatory disease associated with aging. Etomidate is an intravenous anesthetic with profound antioxidant and anti-inflammatory effects. We speculated that etomidate might exert a beneficial effect on OA. Herein, we explored the effects of etomidate on interleukin-1ß (IL-1ß)- induced chondrocytes. Our results prove that etomidate ameliorated the IL-1ß-induced oxidative stress in C28/12 chondrocytes by decreasing and increasing the reactive oxygen species (ROS) and glutathione peroxidase (GPx) levels, respectively. Etomidate prevented the IL-1ß-induced increase in the expressions of matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-13 (MMP-13) in C28/I2 chondrocytes at both mRNA and protein levels. It also caused a significant reduction in the percentage of senescence-associated-ß-galactosidase (SA-ß-Gal)-stained chondrocytes, while inducing elevated telomerase activity in IL-1ß-treated C28/I2 chondrocytes. The expression levels of senescence regulators, plasminogen activator inhibitor-1 (PAI-1) and p16, were also inhibited by etomidate in IL-1ß-treated C28/I2 chondrocytes. In addition, etomidate caused the activation of Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), along with upregulated expression levels of phosphorylated AMPKα and phosphorylated acetyl-Co A carboxylase (ACC). Moreover, blockage of AMPK using compound C abolished the protective effects of etomidate on IL-1ß-challenged C28/I2 chondrocytes. Taken together, these results demonstrate that etomidate protected C28/I2 chondrocytes from IL-1ß-induced oxidative stress, ECM degradation, and cellular senescence via activating AMPK signaling.

Physical and Chemical Compatibility of Etomidate and Propofol Injectable Emulsions.

INTRODUCTION: The mixture of etomidate and propofol is widely used in clinical practice to improve efficacy of general anesthesia and to minimize side effects. As a thermodynamically unstable system, emulsion is prone to destabilization through mechanisms including coalescence, flocculation, and creaming. Such unwanted phenomenon can induce fat embolism after intravenous administration. This study was aimed to investigate the physical and chemical stability of the mixture of etomidate and propofol in the dosage form of emulsion. METHODS: This compatibility study focused on the critical quality attributes (CQAs) of drug-containing emulsions, such as appearance, pH, particle size and distribution, zeta potential, the observation under centrifugation, and drug content and impurity. RESULTS: As the results, there were no significant changes in the CQAs of the mixed emulsions up to 24 h after mixing at refrigeration temperature (4°C), room temperature (25°C), and body temperature (37°C). CONCLUSIONS: These results demonstrate that etomidate emulsion is physically and chemically compatible with propofol emulsions up to 24 h at 4°C, 25°C, and 37°C, suggesting that etomidate and propofol can be administrated in mixture without adversely affecting product characteristics, at least in vitro.

The general anesthetic etomidate and fenamate mefenamic acid oppositely affect GABAAR and GlyR: a structural explanation.

GABA and glycine act as inhibitory neurotransmitters in the CNS. Inhibitory neurotransmission is mediated via activation of ionotropic GABAA and glycine receptors. We used a modeling approach to explain the opposite effects of the general anesthetic etomidate (ETM) and fenamate mefenamic acid (MFA) on GABA- and glycine-activated currents recorded in isolated cerebellar Purkinje cells and hippocampal pyramidal neurons, respectively. These drugs potentiated GABAARs but blocked GlyRs. We built a homology model of α1ß GlyR based on the cryo-EM structure of open α1 GlyR, used the α1ß3γ2 GABAAR structure from the PDB, and applied Monte-Carlo energy minimization to optimize models of receptors and ligand-receptor complexes. In silico docking suggests that ETM/MFA bind at the transmembrane ß( +)/α( -) intersubunit interface in GABAAR. Our models predict that the bulky side chain of the highly conserved Arg19' residue at the plus interface side wedges the interface and maintains the conducting receptor state. We hypothesized that MFA/ETM binding at the ß( +)/α( -) interface leads to prolongation of receptor life-time in the open state. Having analyzed different GABAAR and GlyR structures available in the PDB, we found that mutual arrangement of the Arg19' and Gln-26' side chains at the plus and minus interface sides, respectively, plays an important role when the receptor switches from the open to closed state. We show that this process is accompanied by narrowing of the intersubunit interfaces, leading to extrusion of the Arg19' side chain from the interface. Our models allow us to explain the lack of GlyR potentiation in our electrophysiological experiments.

Shared structural mechanisms of general anaesthetics and benzodiazepines.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

Behavioral and steroidogenic pharmacology of phenyl ring substituted etomidate analogs in rats.

BACKGROUND: Cushing's syndrome is an endocrine disorder characterized by the overproduction of adrenocortical steroids. Steroidogenesis enzyme inhibitors are the mainstays of pharmacological treatment. Unfortunately, they produce significant side effects. Among the most potent inhibitors is the general anesthetic etomidate whose GABAA receptor-mediated sedative-hypnotic actions restrict use. In this study, we defined the sedative-hypnotic and steroidogenesis inhibiting actions of etomidate and four phenyl-ring substituted etomidate analogs (dimethoxy-etomidate, isopropoxy-etomidate, naphthalene-etomidate, and naphthalene (2)-etomidate) that possess negligible GABAA receptor modulatory activities. METHODS: In the first set of experiments, male Sprague-Dawley rats were assessed for loss of righting reflexes (LoRR) after receiving intravenous boluses of either etomidate (1 mg/kg) or an etomidate analog (40 mg/kg). In the second set of experiments, rats were assessed for LoRR and their abilities to produce adrenocortical and androgenic steroids after receiving 2-h infusions (0.5 mg kg- 1 min- 1) of either etomidate or an etomidate analog. RESULTS: All rats that received etomidate boluses or infusions had LoRR that persisted for minutes or hours, respectively. In contrast, no rat that received an etomidate analog had LoRR. Compared to rats in the vehicle control group, rats that received etomidate analog infusions had plasma corticosterone and aldosterone concentrations that were reduced by 80-84% and 68-94%, respectively. Rats that received etomidate infusions had plasma corticosterone and aldosterone concentrations that were also significantly reduced (by 92 and 96%, respectively). Rats that received etomidate or isopropoxy-etomidate had significant reductions (90 and 57%, respectively) in plasma testosterone concentrations whereas those that received naphthalene-etomidate had significant increases (1400%) in plasma dehydroepiandrosterone concentrations. Neither etomidate nor any etomidate analog significantly affected plasma androstenedione and dihydrotestosterone concentrations. CONCLUSIONS: Our studies demonstrate that the four phenyl-ring substituted etomidate analogs form a novel class of compounds that are devoid of sedative-hypnotic activities and suppress plasma concentrations of adrenocortical steroids but vary in their effects on plasma concentrations of androgenic steroids.

A newly developed anesthetic based on a unique chemical core.

Intravenous anesthetic agents are associated with cardiovascular instability and poorly tolerated in patients with cardiovascular disease, trauma, or acute systemic illness. We hypothesized that a new class of intravenous (IV) anesthetic molecules that is highly selective for the slow type of γ-aminobutyric acid type A receptor (GABAAR) could have potent anesthetic efficacy with limited cardiovascular effects. Through in silico screening using our GABAAR model, we identified a class of lead compounds that are N-arylpyrrole derivatives. Electrophysiological analyses using both an in vitro expression system and intact rodent hippocampal brain slice recordings demonstrate a GABAAR-mediated mechanism. In vivo experiments also demonstrate overt anesthetic activity in both tadpoles and rats with a potency slightly greater than that of propofol. Unlike the clinically approved GABAergic anesthetic etomidate, the chemical structure of our N-arylpyrrole derivative is devoid of the chemical moieties producing adrenal suppression. Our class of compounds also shows minimal to no suppression of blood pressure, in marked contrast to the hemodynamic effects of propofol. These compounds are derived from chemical structures not previously associated with anesthesia and demonstrate that selective targeting of GABAAR-slow subtypes may eliminate the hemodynamic side effects associated with conventional IV anesthetics.

Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the α1ß3γ2 GABAAR Transmembrane Domain.

GABAA receptors (GABAARs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([3H]azietomidate) and mephobarbital [[3H]1-methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid ([3H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the α1ß3γ2 GABAAR transmembrane domain at ß +-α - (ß + sites) and α +-ß -/γ +-ß - (ß - sites) subunit interfaces. We now use competition photolabeling with [3H]azietomidate and [3H]R-mTFD-MPAB to identify para-substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to ß +, while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to ß - sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the α +-ß - and γ +-ß - sites. However, we discovered four compounds that bind with different affinities to the two ß - interface sites. Two of these bind with higher affinity to one of the ß - sites than to the ß + sites. We deduce that 4-benzoyl-propofol binds with >100-fold higher affinity to the γ +-ß - site than to the α +-ß - or ß +-α - sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the α +-ß - site than to the ß + and γ +-ß - sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABAAR transmembrane domain, a property that may facilitate the development of subtype selective GABAAR PAMs.

GABA allosteric modulators: An overview of recent developments in non-benzodiazepine modulators.

γ-Aminobutyric acid (GABA) is the major inhibitory transmitter controlling synaptic transmission and neuronal excitability. It is present in a high percentage of neurons in the central nervous system (CNS) and also present in the peripheral nervous system, and acts to maintain a balance between excitation and inhibition. GABA acts via three subclasses of receptors termed GABAA, GABAB, and GABAC. GABAA and GABAC receptors are ligand-gated ion channels, while GABAB receptors are G-protein coupled receptors. Each class of GABA receptor has distinct pharmacology and physiology. GABAA receptors are heteropentameric transmembrane protein complexes made up of α1-6, ß1-3, γ1-3, δ, ε, θ, π subunits, giving rise to numerous allosteric binding sites and have thus attracted much attention targets for the treatment of conditions such as epilepsy, anxiety and sleep disorders. The development of ligands for these binding sites has also led to an improved understanding of the different physiological functions and pathological processes and offers the opportunity for the development of novel therapeutics. This review focuses on the medicinal chemistry aspects including drug design, structure-activity relationships (SAR), and mechanism of actions of GABA modulators, including non-benzodiazepine ligands at the benzodiazepine binding site and modulators acting at sites other than the high-affinity benzodiazepine binding site. Recent advances in this area their future applications and potential therapeutic effects are also highlighted.

A simple and efficient automated cGMP-compliant radiosynthesis of [11 C]metomidate using solid phase extraction cartridge purification.

[11 C]metomidate ([11 C]MTO) is a radiotracer widely used to detect disorders of adrenocortical origin by positron emission tomography (PET) imaging. [11 C]MTO PET/computed tomography (PET/CT) is considered a sensitive and specific noninvasive alternative to adrenal vein sampling (AVS) in the management of primary hyperaldosteronism (PHA). Herein, we report a reliable automated procedure for the routine manufacturing of [11 C]MTO in current good manufacturing practice (cGMP) conditions on the commercial Synthra MeIPlus Loop Vessel synthesizer. The method is based on a combination of the captive-solvent 11 C-methylation of the carboxylate salt 1b of the MTO precursor 1a followed by solid phase extraction (SPE) cartridge purification methodology, which substitutes HPLC purification of the crude reaction mixture. Starting from 45 GBq [11 C]CO2 at the end of bombardment (EOB), 3 GBq of pure [11 C]MTO was produced in 18 minutes with 12% decay corrected radiochemical yield (RCY) at the end of synthesis (EOS) and with the modest molar activity of 13 GBq/µmol at the time of application. Each dose produced met all established quality control (QC) criteria. The method can easily be implemented into other commercial automated radiosynthesizers for manufacturing carbon-11 labeled radiotracers.

Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission tomography (PET) radiotracer for imaging CYP11B1 and CYP11B2 enzymes in adrenal glands.

INTRODUCTION: Primary aldosteronism accounts for 6-15% of hypertension cases, the single biggest contributor to global morbidity and mortality. Whilst ~50% of these patients have unilateral aldosterone-producing adenomas, only a minority of these have curative surgery as the current diagnosis of unilateral disease is poor. Carbon-11 radiolabelled metomidate ([11C]MTO) is a positron emission tomography (PET) radiotracer able to selectively identify CYP11B1/2 expressing adrenocortical lesions of the adrenal gland. However, the use of [11C]MTO is limited to PET centres equipped with on-site cyclotrons due to its short half-life of 20.4 min. Radiolabelling a fluorometomidate derivative with fluorine-18 (radioactive half life 109.8 min) in the para-aromatic position ([18F]FAMTO) has the potential to overcome this disadvantage and allow it to be transported to non-cyclotron-based imaging centres. METHODS: Two strategies for the one-step radio-synthesis of [18F]FAMTO were developed. [18F]FAMTO was obtained via radiofluorination via use of sulfonium salt (1) and boronic ester (2) precursors. [18F]FAMTO was evaluated in vitro by autoradiography of pig adrenal tissues and in vivo by determining its biodistribution in rodents. Rat plasma and urine were analysed to determine [18F]FAMTO metabolites. RESULTS: [18F]FAMTO is obtained from sulfonium salt (1) and boronic ester (2) precursors in 7% and 32% non-isolated radiochemical yield (RCY), respectively. Formulated [18F]FAMTO was obtained with >99% radiochemical and enantiomeric purity with a synthesis time of 140 min from the trapping of [18F]fluoride ion on an anion-exchange resin (QMA cartridge). In vitro autoradiography of [18F]FAMTO demonstrated exquisite specific binding in CYP11B-rich pig adrenal glands. In vivo [18F]FAMTO rapidly accumulates in adrenal glands. Liver uptake was about 34% of that in the adrenals and all other organs were <12% of the adrenal uptake at 60 min post-injection. Metabolite analysis showed 13% unchanged [18F]FAMTO in blood at 10 min post-administration and rapid urinary excretion. In vitro assays in human blood showed a free fraction of 37.5%. CONCLUSIONS: [18F]FAMTO, a new 18F-labelled analogue of metomidate, was successfully synthesised. In vitro and in vivo characterization demonstrated high selectivity towards aldosterone-producing enzymes (CYP11B1 and CYP11B2), supporting the potential of this radiotracer for human investigation.

Insights Into Receptor-Based Anesthetic Pharmacophores and Anesthetic-Protein Interactions.

General anesthetics are thought to allosterically bind and potentiate the inhibitory currents of the GABAA receptor through drug-specific binding sites. The physiologically relevant isoform of the GABAA receptor is a transmembrane ligand-gated ion channel consisting of five subunits (γ-α-ß-α-ß linkage) symmetrically arranged around a central chloride-conducting pore. Although the exact molecular structure of this heteropentameric GABAA receptor remains unknown, molecular modeling has allowed significant advancements in understanding anesthetic binding and action. Using the open-channel conformations of the homologous glycine and glutamate-gated chloride receptors as templates, a homology model of the GABAA receptor was constructed using the Discovery Studio computational chemistry software suite. Consensus structural alignment of the homology templates allowed for the construction of a three-dimensional heteropentameric GABAA receptor model with (γ2-ß3-α1-ß3-α1) subunit linkage. An anesthetic binding site was identified within the transmembrane α/ß intersubunit space by the convergence of three residues shown to be essential for anesthetic activity in previous studies with mutant mice (ß3-N265, ß3-M286, α1-L232). Propofol derivatives docked into this binding site showed log-linear correlation with experimentally derived GABAA receptor potentiation (EC50) values, suggesting this binding site may be important for receptor activation. The receptor-based pharmacophore was analyzed with surface maps displaying the predominant anesthetic-protein interactions, revealing an amphiphilic binding cavity incorporating the three residues involved in anesthetic modulation. Quantum mechanics calculations of the bonding patterns found in complementary high-resolution receptor systems further elucidated the complex nature of anesthetic-protein interactions.

Dimethoxy-etomidate: A Nonhypnotic Etomidate Analog that Potently Inhibits Steroidogenesis.

Cushing's syndrome is characterized by the overproduction of adrenocortical steroids. Steroidogenesis inhibitors are mainstays of medical therapy for Cushing's syndrome; unfortunately, adverse side effects and treatment failures are common with currently available drugs. The general anesthetic induction agent etomidate is among the most potent inhibitors of adrenocortical steroidogenesis. However, its use as a treatment of Cushing's syndrome is complicated by its sedative-hypnotic activity and ability to produce myoclonus, central nervous system actions thought to be mediated by the GABAA receptor. Here, we describe the pharmacology of the novel etomidate analog (R)-ethyl 1-(1-(3,5-dimethoxyphenyl)ethyl)-1H-imidazole-5-carboxylate (dimethoxy-etomidate). In contrast to etomidate, dimethoxy-etomidate minimally enhanced GABA-evoked GABAA receptor-mediated currents even at a near-saturating aqueous concentration. In Sprague-Dawley rats, dimethoxy-etomidate's potency for producing loss of righting reflexes-an animal model of sedation/hypnosis-was 2 orders of magnitude lower than that of etomidate, and it did not produce myoclonus. However, similar to etomidate, dimethoxy-etomidate potently suppressed adrenocortical steroid synthesis primarily by inhibiting 11ß-hydroxylase. [3H]etomidate binding to rat adrenocortical membranes was inhibited by dimethoxy-etomidate in a biphasic manner with IC50 values of 8.2 and 3970 nM, whereas that by etomidate was monophasic with an IC50 of 22 nM. Our results demonstrate that, similar to etomidate, dimethoxy-etomidate potently and dose-dependently suppresses adrenocortical steroid synthesis by inhibiting 11ß-hydroxylase. However, it is essentially devoid of etomidate's GABAA receptor positive modulatory and sedative-hypnotic activities and produces no myoclonus, providing proof of concept for the design of etomidate analogs without important central nervous system actions for the pharmacologic treatment of Cushing's syndrome.

The role of etomidate as an anaesthetic induction agent for critically ill patients.

Etomidate is an anaesthetic induction agent which may be favoured in critically ill patients because of its cardiovascular stability. However, etomidate causes adrenocortical suppression which may be particularly harmful in these patients. This article reviews current evidence and debates the use of etomidate in critical illness.

Biophysical study on the interaction of etomidate and the carrier protein in vitro.

Etomidate is a unique drug used for induction of general anesthesia and sedation, and is usually used through intravenous injection clinically. Before targeting to the receptor, etomidate binds proteins in blood when it comes into veins. Thus to study the interaction of etomidate and serum albumin would be of great toxicological and pharmacological importance. In this study, the interaction between etomidate and human serum albumin (HSA) was studied using fluorescence spectroscopy, UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) spectroscopy, site maker displacement and molecular modeling methods. Investigations of the binding constant (K = 3.55 × 10(5 )M(-1), 295 K), the number of binding sites (n = 1.16), thermodynamic parameters (ΔG = 3.13 × 10(4 )J·mol(-1), ΔS = 364 J·mol(-1)·K(-1) and ΔH = -6.85 × 10(5 )J·mol(-1)) for the reaction and changes to the binding sites and conformation in HSA in response to etomidate were presented. Results show that etomidate can bind HSA tightly through electrostatic forces, and the protein skeleton conformation and secondary structure changes thereby. This is the first spectroscopic report for etomidate-HSA interactions which illustrates the complex nature of this subject.

Analogues of etomidate: modifications around etomidate's chiral carbon and the impact on in vitro and in vivo pharmacology.

BACKGROUND: R-etomidate possesses unique desirable properties but potently suppresses adrenocortical function. Consequently, efforts are being made to define structure-activity relationships with the goal of designing analogues with reduced adrenocortical toxicity. The authors explored the pharmacological impact of modifying etomidate's chiral center using R-etomidate, S-etomidate, and two achiral etomidate analogues (cyclopropyl etomidate and dihydrogen etomidate). METHODS: The γ-aminobutyric acid type A receptor modulatory potencies of drugs were assessed in oocyte-expressed α1(L264T)ß3γ2L and α1(L264T)ß1γ2L γ-aminobutyric acid type A receptors (for each drug, n = 6 oocytes per subtype). In rats, hypnotic potencies and durations of action were measured using a righting reflex assay (n = 26 to 30 doses per drug), and adrenocortical potencies were quantified by using an adrenocorticotropic hormone stimulation test (n = 20 experiments per drug). RESULTS: All four drugs activated both γ-aminobutyric acid type A receptor subtypes in vitro and produced hypnosis and suppressed adrenocortical function in rats. However, drug potencies in each model ranged by 1 to 2 orders of magnitude. R-etomidate had the highest γ-aminobutyric acid type A receptor modulatory, hypnotic, and adrenocortical inhibitory potencies. Respectively, R-etomidate, S-etomidate, and cyclopropyl etomidate were 27.4-, 18.9-, and 23.5-fold more potent activators of receptors containing ß3 subunits than ß1 subunits; however, dihydrogen etomidate's subunit selectivity was only 2.48-fold and similar to that of propofol (2.08-fold). S-etomidate was 1/23rd as potent an adrenocortical inhibitor as R-etomidate. CONCLUSION: The linkage between the structure of etomidate's chiral center and its pharmacology suggests that altering etomidate's chiral center may be used as part of a strategy to design analogues with more desirable adrenocortical activities and/or subunit selectivities.

Etomidate produces similar allosteric modulation in α1ß3δ and α1ß3γ2L GABA(A) receptors.

BACKGROUND AND PURPOSE: Neuronal GABA(A) receptors are pentameric chloride ion channels, which include synaptic αßγ and extrasynaptic αßδ isoforms, mediating phasic and tonic inhibition respectively. Although the subunit arrangement of αßγ receptors is established as ß-α-γ-ß-α, that of αßδ receptors is uncertain and possibly variable. We compared receptors formed from free α1, ß3 and δ or γ2L subunits and concatenated ß3-α1-δ and ß3-α1 subunit assemblies (placing δ in the established γ position) by investigating the effects of R-(+)-etomidate (ETO), an allosteric modulator that selectively binds to transmembrane interfacial sites between ß3 and α1. EXPERIMENTAL APPROACH: GABA-activated receptor-mediated currents in Xenopus oocytes were measured electrophysiologically, and ETO-induced allosteric shifts were quantified using an established model. KEY RESULTS: ETO (3.2 µM) similarly enhanced maximal GABA (1 mM)-evoked currents in oocytes injected with 5 ng total mRNA and varying subunit ratios, for α1ß3(1:1), α1ß3δ(1:1:1) and α1ß3δ(1:1:3), but this potentiation by ETO was significantly greater for ß3-α1-δ/ß3-α1(1:1) receptors. Reducing the amount of α1ß3δ(1:1:3) mRNA mixture injected (0.5 ng) increased the modulatory effect of ETO, matching that seen with ß3-α1-δ/ß3-α1(1:1, 1 ng). ETO similarly reduced EC50s and enhanced maxima of GABA concentration-response curves for both α1ß3δ and ß3-α1-δ/ß3-α1 receptors. Allosteric shift parameters derived from these data depended on estimates of maximal GABA efficacy, and the calculated ranges overlap with allosteric shift values for α1ß3γ2L receptors. CONCLUSION AND IMPLICATIONS: Reducing total mRNA unexpectedly increased δ subunit incorporation into receptors on oocyte plasma membranes. Our results favour homologous locations for δ and γ2L subunits in α1ß3γ2/δ GABA(A) receptors.