Clinical and Molecular Pharmacology of Etomidate

Abstract

This review focuses on the unique clinical and molecular pharmacology of etomidate. Among general anesthesia induction drugs, etomidate is the only imidazole, and it has the most favorable therapeutic index for single bolus administration. It also produces a unique toxicity among anesthetic drugs-- inhibition of adrenal steroid synthesis that far outlasts its hypnotic action and that may reduce survival of critically ill patients. The major molecular targets mediating anesthetic effects of etomidate in the central nervous system are specific γ-aminobutyric acid type A receptor subtypes. Amino acids forming etomidate binding sites have been identified in transmembrane domains of these proteins. Etomidate binding site structure models for the main enzyme mediating etomidate adrenotoxicity have also been developed. Based on this deepening understanding of molecular targets and actions, new etomidate derivatives are being investigated as potentially improved sedative-hypnotics or for use as highly selective inhibitors of adrenal steroid synthesis.

A Brief History of Etomidate

Etomidate [R-1-(1-ethylphenyl)imidazole-5-ethyl ester] (Figure 1) is a unique drug used for induction of general anesthesia and sedation. The first report on etomidate was published in 1965 as one of several dozen arylalkyl imidazole-5-carboxylate esters¹ synthesized by Janssen Pharmaceuticals (a division of Ortho-McNeil-Jannsen Pharmaceuticals, Titusville, New Jersey, USA). Initially developed as anti-fungal agents, the potent hypnotic activity of several compounds was observed during animal testing, and several compounds, including etomidate, appeared significantly safer than barbiturates.

Figure 1. Chemical Structure of Etomidate.

Critical structural features for anesthetic activity include a single methylene group between the imidazole and the phenyl group and the R(+) configuration at the chiral center (labeled with an asterix).

Etomidate contains a chiral carbon (Figure 1). Initial studies of racemic etomidate in rats demonstrated lethality at about 12 times its effective hypnotic dose (LD50/ED50 ≈ 12), in comparison to barbiturates with LD50/ED50 ratios (therapeutic indices) of 3–5.¹ Subsequent studies found that the isolated R(+) enantiomer of etomidate has ten to twenty-fold greater hypnotic potency than S(−)-etomidate ^2,3. The LD50/ED50 ratio for R(+)-etomidate is 26 in rats,⁴ significantly higher than therapeutic indices for other general anesthetics (Table 1). Preclinical experiments in mammals also demonstrated that etomidate injection was associated with minimal hemodynamic changes or respiratory depression, features that were presumed to result in its unusually favorable safety profile.⁵

Table 1.

Acute Toxicity Ratios of Intravenous Anesthetic Induction Drugs.

Anesthetic Induction Drug	Acute Toxicity Ratio * (LD50/ED50)
R(+)-Etomidate	26 5
Althesin	17.3 159
(alphaxalone/alphadolone) Ketamine (racemic) †	6.3 160
Methohexital	4.8 – 9.5 5,159
Thiopental	3.6– 4.6 5,159,161
Pentobarbital	3.4 161
Propofol	3.4 159

Abbreviations: ED50 = dose at which 50% of animals lose righting reflexes. LD50 = dose resulting in 50% mortality within 24 hours.

^*Data are from therapeutic index studies in mice and rats using intravenous injection.

^†The therapeutic index of (+)-ketamine is 10, while that of the (−) enantiomer is 4.0.

Etomidate was introduced into clinical practice in 1972 and initial reports of its use in humans emerged in the clinical literature soon afterward.^6,7 Academic publications focusing on etomidate rose steadily until 1983, when the number of reports rapidly doubled following discovery of its adrenal toxicity (Figure 2). Subsequently, the number of yearly published papers focusing on etomidate diminished (apparently in parallel with its use in operating rooms), but this rate has resurged in the last decade. Renewed interest in etomidate parallels its widening use during intubations in emergency rooms and intensive care units, as well as new concerns about the impact of etomidate-induced adrenal toxicity in critically ill patients. The recent increase in publications on etomidate also reflects scientific progress in understanding this drug’s molecular pharmacology.

Figure 2. Etomidate Publications in PubMed.

The graph displays numbers of publications within a calendar year, based on PubMed searches with “etomidate” as a MESH term (gray + white bars), or the subset of these publications with humans as the subjects (white bars). Data are inclusive through December, 2009.

Clinical Pharmacology

Formulation and Dosing

Etomidate formulations for clinical use contain the purified R(+) enantiomer. Etomidate has a pKa of 4.2 and is very hydrophobic at physiologic pH. To increase solubility, it is formulated as a 0.2% solution either in 35% propylene glycol (Amidate; Hospira, Inc., Lake Forest, Illinois, USA) or in lipid emulsion (Etomidate-Lipuro; B. Braun, Melsungen, Germany)⁸. Formulations in cyclodextrins have also been developed.^9,10

Early clinical studies determined that intravenous bolus doses of 0.2–0.4 mg/kg provided hypnosis for 5–10 minutes. Following a bolus, maintenance of general anesthesia can be achieved by continuous infusion of etomidate at 30–100 µg/kg/min.^11–13 Oral transmucosal etomidate has been used to induce sedation¹⁴ and rectal administration has been used to induce general anesthesia in pediatric patients.¹⁵

Systemic effects

Etomidate does not inhibit sympathetic tone or myocardial function,^16,17 and at typical anesthetic induction doses produces minimal blood pressure and heart rate changes in patients, including those with valvular or ischemic heart disease.^12,18–20 For the same reason, etomidate does not block sympathetic responses to laryngoscopy and intubation, and these are often blunted by premedication with opioids.^21,22 Etomidate produces less apnea than barbiturates or propofol, no histamine release, and very rare allergic reactions. Because of its remarkably benign hemodynamic effects, etomidate has proven useful for general anesthetic induction in patients undergoing cardiac surgery and those with poor cardiac function.²³

Etomidate also provides advantages for induction of anesthesia in the setting of hemorrhagic shock. In a pig model of hemorrhagic shock, the pharmacodynamic and pharmacokinetics of etomidate are minimally altered,²⁴ in contrast to other anesthetic drugs.^25,26 As a result of its favorable profile for anesthetic induction in a variety of critically ill patients, etomidate has been adopted by many emergency medicine physicians as the hypnotic drug of choice for rapid sequence induction and intubation.^27–29

Hepatic blood flow is modestly reduced following induction of general anesthesia with etomidate, but this has minimal impact on pharmacokinetics and metabolism of anesthetic agents.^30,31 Cerebral blood flow is reduced along with cerebral metabolic rate and intracranial pressure, while cerebral perfusion pressure is maintained or increased during etomidate-induced anesthesia.^32–34 Electroencephalographic changes during hypnosis with etomidate are similar to those seen with barbiturates.³⁵ Bispectral index monitor values drop following etomidate bolus administration and return to baseline during recovery of consciousness.³⁶ During brief etomidate infusions, bispectral index values correlate well with sedation scores.³⁷ Etomidate increases latency and decreases amplitude of auditory evoked potentials.³⁸ The duration of epileptiform activity following electroconvulsive therapy is longer after anesthetic induction with etomidate versus methohexital or propofol.³⁹ Somatosensory evoked potential amplitudes are enhanced by etomidate,⁴⁰ and motor evoked potential amplitudes are suppressed less by etomidate than propofol, thiopental, or methohexital.⁴¹

Pharmacokinetics and metabolism

In healthy patients, etomidate is approximately 75% protein bound.⁴² Etomidate is characterized by a large central volume of distribution, 4.5 litres/kg, and a very large peripheral volume of distribution, 74.9 litres/kg, due to its high solubility in fat.^30,37,43 The single bolus pharmacokinetic profile of plasma etomidate concentration is described by a three compartment model (Figure 3).³⁰ The fast, intermediate, and slow declines in plasma etomidate are thought to correspond to, respectively, distribution into highly perfused tissues, redistribution into peripheral tissues (mostly muscle), and terminal metabolism. The hypnotic effect of an IV bolus of 3 mg/kg etomidate terminates as redistribution into the peripheral compartment starts to dominate the plasma concentration profile. Etomidate metabolism in laboratory animals and humans depends on hepatic esterase activity, which hydrolyzes the drug to a carboxylic acid and an ethanol leaving group.⁴⁴ The carboxylate metabolite is excreted mostly in urine, and to a lesser degree in bile. Total plasma clearance is 15–20 ml/kg/min and the terminal metabolic half-life of etomidate in humans ranges from 2–5 hours. Elderly or ill patients often require lower etomidate doses because of reduced protein binding and reduced clearance.^42,45,46 The pharmacokinetic parameters for etomidate indicate its suitability for use as a continuous infusion, with a context-sensitive half time shorter than that of propofol.⁴⁷ Prolonged etomidate infusion for anesthesia and sedation was practiced during the first decade of clinical availability.^32,48–52 Other considerations (adrenal toxicity) now preclude this application (see below, Adrenal toxicity, sepsis, and exogenous steroids).

Figure 3. Single Intravenous Bolus Pharmacokinetics of Etomidate.

The etomidate plasma concentration following a single intravenous bolus (3 mg/kg) is depicted on a semi-logarithmic plot with the early decline period expanded. This concentration versus time profile is based on pharmacokinetic parameters determined by Van Hamme et al,³⁰ showing three distinct decline phases with half-times of 2 minutes, 21 minutes, and 3.9 hours. Graded colored areas indicate etomidate plasma concentration ranges associated with hypnosis (blue) and adrenocortical suppression (red). Together, these data illustrate why the duration of hypnosis (~ 8 minutes) is much shorter than the duration of adrenocortical suppression (~ 8 hours) following a single etomidate dose.

Side effects

Several unfavorable side effects associated with etomidate were noted in early studies, including pain on injection and myoclonic movements during induction of general anesthesia.^53–55 Pain on injection was found to be worse with etomidate in aqueous solutions in comparison to the formulation in 35% propylene glycol.⁵⁶ Formulation into medium chain-length lipids or cyclodextrins appears to further decrease the incidence of injection pain and hemolysis.^9,57 Myoclonus has been shown to increase with etomidate dose and can be attenuated by split-dose induction⁵⁸ or premedication with benzodiazepines,⁵⁹ thiopental, dexmedetomidine⁶⁰, and/or opioids.^22,61,62

Post-operative nausea and vomiting is cited as a frequent side-effect of etomidate, but very few studies have formally compared post-operative nausea and vomiting following etomidate versus other agents used for induction of general anesthesia. Early investigators reported that post-operative nausea and vomiting incidence after induction with etomidate is around 40%,^50,55 comparable to that following barbiturates,^43,56 and higher than that following propofol.⁶³ More recently, the incidence of nausea after induction with etomidate in lipid emulsion was reported to be similar to that associated with propofol,^64,65 while the incidence of vomiting was higher with etomidate.⁶⁵

Adrenal toxicity, sepsis, and exogenous steroids

Adrenal cortical inhibition by etomidate has received a great deal of attention and significantly limits its use as both an anesthetic and sedative. Nonetheless, the effect of etomidate on clinical outcomes has never been carefully studied in large numbers of surgical or intensive care patients.

In 1983, a decade after its introduction into clinical use, Ledingham and Watt^66,67 reported retrospective data showing increased mortality among intensive care patients receiving prolonged etomidate infusions for sedation, in comparison to patients receiving benzodiazepines (69% with etomidate vs. 25% with benzodiazepines). Soon afterward, McKee and Finlay⁶⁸ reported that cortisol replacement therapy could reduce the mortality in a similar group of critically ill patients receiving etomidate infusions. At that time, there was emerging preclinical evidence that etomidate suppressed adrenocortical function in rats,⁶⁹ and clinical investigators rapidly confirmed this toxicity in patients.^70,71 Etomidate was found to suppress normal cortisol and aldosterone increases following surgery as well as adrenal responses to corticotrophin. Adrenal suppression was found to last 6–8 hours in patients following a single induction dose of etomidate^72,73 and more than 24 hours following etomidate infusion.⁷⁴

The clinical community reacted to revelations about adrenal toxicity by ceasing the use of etomidate for long-term infusions. Some editorials^75,76 recommended halting its use altogether, while others suggested that etomidate had value as a single-dose induction drug for selected patients.⁷⁷ The drug package insert was amended to state that etomidate usage is approved for induction of general anesthesia and anesthetic maintenance for short operative procedures. It specifically warns against administration by prolonged infusion. Subsequent research showed that etomidate is far more potent as an inhibitor of steroid synthesis than as a sedative-hypnotic.⁷⁸ Etomidate plasma concentrations associated with hypnosis in patients are above 200 ng/ml (1 µM), while concentrations lower than 10 ng/ml are associated with adrenal cortical suppression.⁷² The in vitro IC₅₀ for etomidate inhibition of cortisol synthesis in cultured adrenal cells is 1 nanomolar (nM), which closely matches the apparent dissociation constant (K_D) for etomidate binding to membranes of these cells.⁷⁹ Together, the disparate etomidate concentration-dependences for hypnosis versus adrenotoxicity and multi-phase pharmacokinetics account for the dramatic difference in the durations of these two actions following a single intravenous bolus (Figure 3).⁷²

Recently, concern about etomidate-induced adrenal toxicity in critically ill patients and the use of corticosteroids to treat this effect has re-emerged. Exposure to single dose etomidate was found to be a confounding variable in a large multicenter trial evaluating the use of supplemental corticosteroids in septic patients with and without adrenal insufficiency.⁸⁰ Enrollment in this study ran from September, 1995 until March, 1999, and in July, 1996 inclusion criteria were altered to exclude patients who had received etomidate within 6 hours. At that point 72 enrollees had received etomidate and 68 of these were non-responders to corticotrophin.⁸¹ Thus, at least 30 percent of the non-responders in this study (229 in total) had received etomidate, and it is likely that additional patients received etomidate between 6 and 24 hours before enrollment. In a 500 patient follow-up study of low-dose corticosteroid therapy in septic shock (CORTICUS),⁸² etomidate was administered to 20% of patients before enrollment and 8% of patients after enrollment. Even though etomidate was given on average 14 hours before testing for adrenal insufficiency, it was associated with a 60% non-response rate to corticotrophin, significantly higher than that of enrollees who did not receive etomidate. Similar results have been reported by others.⁸³ The CORTICUS study⁸² concluded that supplemental steroids did not improve the long-term outcome of patients found to have adrenal insufficiency. Retrospective analyses of the CORTICUS cohort suggest that patients receiving etomidate before enrollment had 28 day mortality significantly higher than other patients in the trial, and that steroids provided no benefit to those who received etomidate.^82,84,85

Other studies of sepsis and trauma patients have examined the duration of adrenal insufficiency after single-dose etomidate and its effect on outcomes. In this population, the duration of adrenal suppression following a single dose of etomidate is more than 24 hours^86–88 and may last up to 72 hours.⁸⁶ However, the impact of single dose etomidate on outcomes in critically ill patients remains unclear. Hildreth et al⁸⁹ reported that trauma patients randomized to intubation using etomidate had longer hospital and intensive care unit length of stay than a group intubated using fentanyl and midazolam. In contrast to these and the CORTICUS study results, a non-randomized study by Tekwani et al⁹⁰ found no difference in mortality among septic patients who received etomidate for intubation in the emergency department versus those who received other agents. Ray and McKeown⁹¹ also found no evidence of excess mortality associated with etomidate in a retrospective study. A recent randomized controlled trial comparing etomidate with ketamine for intubation of critically ill, mostly non-septic, patients also found no difference in mortality.⁹² Clearly, large well-designed trials are needed to define the clinical impact of single-dose etomidate in critically ill patients. In the meantime, a vigorous debate about the use of etomidate for intubation of these patients continues.^93,94

Molecular Pharmacology

Clinical studies focusing on etomidate are less numerous than those focusing on either propofol or isoflurane^*, yet the molecular pharmacology of etomidate is currently understood far better than other intravenous or inhaled general anesthetics. Etomidate appears to produce hypnosis, amnesia, and inhibition of nociceptive responses almost exclusively via actions at one class of neuronal ion channels, γ-aminobutyric acid type A (GABA_A) receptors.^95,96 Molecular targets mediating adrenal steroid inhibition and pain on injection have also been identified.

GABA_A receptors: Mediators of Etomidate Anesthesia

Soon after etomidate became available for clinical use, it was noted to have effects similar to the endogenous neurotransmitter γ-aminobutyric acid (GABA) in the nervous system.⁹⁷ Indeed, it is now firmly established that the molecular targets underlying the anesthetic actions of etomidate are GABA_A receptors, which are the major inhibitory neurotransmitter receptors in mammalian brains.⁹⁸ GABA_A receptors are neurotransmitter-activated ion channels that selectively conduct chloride ions. Under normal conditions their activation stabilizes neuronal membrane voltage near the chloride Nernst potential of −70 mV. GABA_A receptors are members of the superfamily of cys-loop ligand-gated ion channels that includes nicotinic acetylcholine receptors from muscle and nerve, glycine receptors, and serotonin type A receptors. All of these receptors are structurally similar, formed from five polypeptide subunits surrounding an ion-conductive transmembrane channel. All cys-loop receptor subunits consist of a large amino-terminal extracellular domain, four hydrophobic transmembrane domains (M1 through M4), and a large intracellular domain between M3 and M4. Structural models of GABA_A receptors (Figure 4) are based upon high resolution studies of crystallized acetylcholine binding protein from snail synapses, homologous to extracellular domains,⁹⁹ Torpedo nicotinic acetylcholine receptors¹⁰⁰, and crystallized pentameric prokaryotic channels.^101–103

Figure 4. Molecular Structure of GABA_A Receptors.

A GABA_A receptor homology model, based on the structure of Torpedo nicotinic acetylcholine receptors, is shown in two views. The subunits are color-coded: α, yellow; β, blue; γ, green. A) The receptor is depicted in a membrane cross-sectional view, showing the extracellular domains containing GABA binding sites (purple), and the transmembrane domains forming the etomidate sites (red) between α and β subunits. Two amino acid residues, αM236 (blue) and βM286 (yellow) are shown adjacent to the etomidate binding site. The intracellular domains between M3 and M4 are not shown; their structures remain undefined. B) The pentameric model is depicted as viewed from the extracellular space with subunits labeled. The ion channel is formed by the M2 domains at the center of the subunits. C) The transmembrane domains are depicted with the extracellular domains removed. Transmembrane domains of one α subunit are labeled. (This figure was kindly provided by David Chiara, Harvard Medical School, Boston, MA).

Eighteen distinct GABA_A receptor subunits are encoded in the human genome,¹⁰⁴ but only about a dozen subunit combinations have been shown to form neuronal channels. Most of these consist of two α subunits, two β subunits, and one γ subunit arranged γ-β-α-β-α counterclockwise when viewed from the extracellular space.¹⁰⁵ Heterologously expressed receptors containing α1, β2, and γ2 subunits display GABA sensitivity, drug sensitivity, and open-closed transition rates similar to synaptic GABA_A receptors in brain.¹⁰⁶ Synaptic GABA concentrations are thought to briefly reach several millimolar, and decay within milliseconds due to uptake via GABA transporters. Postsynaptic GABA_A receptor channels open within a millisecond, generating an inhibitory postsynaptic current, which deactivates over tens of milliseconds, far longer than GABA remains in the synapse.¹⁰⁷ During an inhibitory postsynaptic current, action potential generation is impaired in the postsynaptic neuron, so current deactivation is thought to be a factor in regulating the frequency response of neuronal circuits.^108,109 Some GABA_A receptors, formed from α and β subunits in combination with δ or ε subunits, are expressed on neuronal cell bodies and axons.¹¹⁰ These extrasynaptic receptors produce small tonic chloride “leak” currents in response to low micromolar concentrations of GABA in the extrasynaptic space.^111,112

Etomidate actions at GABA_A receptors

Two effects on GABA_A receptors, produced by different concentrations of etomidate, have been described. At concentrations associated with clinical doses, etomidate positively modulates GABA_A receptor activation by agonists.⁹⁸ In other words, when etomidate is present, GABA_A receptors are activated by lower concentrations of GABA than required under normal conditions.^2,113,114 Clinical concentrations of etomidate also slow the inhibitory postsynaptic current decay mediated by synaptic GABA_A receptors^115,116, prolonging postsynaptic inhibition and reducing the frequency response of neuronal circuits. Enhanced activation of extrasynaptic receptors is also observed at clinical etomidate concentrations, increasing the tonic inhibitory “leak” current, and reducing neuronal excitability. Yang¹¹⁵ noted that etomidate effects on tonic currents mediated by extrasynaptic GABA_A receptors may be more important than effects on synaptic currents. Etomidate at supra-clinical concentrations also directly activates synaptic GABA_A receptor channels in the absence of GABA, an action variously termed direct activation, GABA-mimetic activity, or allosteric agonism.^{2,114,115,117}

Both positive modulation of GABA-mediated activity and direct activation of GABA_A receptors display parallel dependences on drug and receptor structures. For both etomidate actions, stereoselectivity for the R(+) enantiomer is of the same magnitude (10 to 20-fold) seen in animal studies of hypnotic and anti-nociceptive activity.^{2,114,118,119} Both etomidate actions also show similar dependence on GABA_A receptor subunit makeup. Receptors containing β2 and/or β3 subunits are modulated and activated by etomidate, while those containing β1 are much less sensitive to both etomidate actions.^113,117,120 Etomidate sensitivity is also affected by the presence of a γ subunit¹¹³ and weakly by the α subtype.¹¹⁷

These parallels suggest that a single class of etomidate sites on GABA_A receptors mediate both modulation of GABA activation and direct activation. Indeed, both of these effects in α1β2γ2L receptors can be quantitatively modeled with an equilibrium Monod-Wyman-Changeux allosteric co-agonist mechanism, wherein etomidate binding to its sites is determined by whether the receptor is in one of two canonical states: open versus closed (Figure 5).¹¹⁴ In essence, etomidate binds weakly (K_E ≈ 35 µM) to closed receptors, but tightly (K_E* ≈ 0.27 µM) to open receptors, so the drug stabilizes open states whether or not GABA is bound. This class of model was found to be optimal with two equivalent etomidate sites.

Figure 5. A Monod-Wyman-Changeux Two-State Equilibrium Model for Etomidate and GABA Activation of GABA_A Receptors.

The scheme depicts allosteric co-agonism for GABA_A receptors with two equivalent GABA (G; orthosteric agonist) sites and two equivalent etomidate (E; allosteric agonist) sites. The L₀ parameter describes the basal equilibrium between the two canonical states: inactive (R) and active (O). K_G is the dissociation constant for GABA interactions with R-state receptors and K_G* is the dissociation constant for GABA interactions with O-state receptors. The GABA efficacy factor, c, is defined as K_G*/K_G. K_E is the dissociation constant for etomidate interactions with R-state receptors and K_E* is the dissociation constant for etomidate interactions with O-state receptors. The etomidate efficacy factor, d, is defined as K_E*/K_E. The different size arrows illustrate how equilibria shift as ligands bind and functional state changes.

Mutations that alter etomidate sensitivity of GABA_A receptors

A β subunit region containing the M2 domain was found to underlie the differential etomidate sensitivity of GABA_A receptors containing β1 versus β2 subunits.¹²¹ The only amino acid in M2 that differs between β1 and β2 is at position 265 of the mature protein. β265 is a serine (S) in β1 and an asparagine (N) in β2 and β3. A point mutation replacing β1S265 with N (β1S265N) increases etomidate sensitivity, while replacing β2 or β3N265 with S (β2/3N265S) dramatically reduces etomidate sensitivity.¹²¹ Similarly, an anesthetic-insensitive mutant drosophila melangaster (fruit fly) line contains a methionine (M) at the homologous amino acid in M2, instead of the N found in wild-type. A mutation from N265 to M in the β2 or β3 subunit of mammalian GABA_A receptors also confers insensitivity to etomidate.^122–124 Mutations at βN265 produce parallel changes in etomidate modulation of GABA-activated receptor-mediated currents and direct activation of channels. Quantitative electrophysiological analysis of GABA_A receptors containing both β2N265S and β2N265M mutations show little impact on basal or GABA-mediated activation, and different degrees of reduced etomidate sensitivity.¹²⁵ The β2N265M mutation totally eliminates etomidate sensitivity, while the β2N265S mutation reduces etomidate-induced shifts in GABA EC₅₀ (EC₅₀ ratio) over eight-fold relative to wild-type (Table 2).

Table 2.

GABA_A Receptor Mutant Effects on GABA and Etomidate Sensitivity ^*

Receptor	Spontaneous Activation †	GABA EC50‡ (µM)	GABA Efficacy §	Etomidate EC50‡ (µM)	Etomidate Efficacy **	Left-Shift Ratio (CNTL/ETO) ††
α1β2γ2L	<0.001	26	0.9	36	0.4	20
α1M236Wβ2γ2L	0.16	2.0	0.99	12	0.97	1.7
α1β2M286Wγ2L	0.04	6.6	1.0	NA	<0.001	1.1
α1β2N265Sγ2L	<0.001	27	0.93	78	0.03	2.3
α1β2 N265Mγ2L	<0.001	32	0.84	NA	<0.001	0.95

Abbreviations: CNTL, control; EC₅₀, half-maximal effect concentration; ETO, etomidate; GABA, γ-aminobutyric acid; GABA_A, γ-aminobutyric acid type A;

^*All functional effects are estimated from voltage-clamp electrophysiological experiments on receptors expressed in Xenopus oocytes.

^†Spontaneous activation is a measure of the propensity of channels to open in the absence of agonist and other ligands. It is estimated using a potent channel blocker (picrotoxin) that inhibits the spontaneously active receptors. The picrotoxin-sensitive current is reported as a fraction of maximum GABA current.

^‡GABA EC₅₀ is the GABA concentration eliciting half maximal activation of receptors. Etomidate EC₅₀ is defined similarly for etomidate’s direct activating (agonist) activity.

^§GABA efficacy is an estimate of the fraction of receptors activated when all agonist sites are occupied by GABA. It is estimated using positive allosteric modulators to enhance the maximum current elicited by high GABA concentrations. We assume that the combination of high GABA plus allosteric enhancer activates all receptors.

^**Etomidate efficacy is the maximum current elicited by etomidate, normalized to the maximum current elicited with GABA.

^††The left-shift ratio is a measure of etomidate modulation of GABA responses. It is calculated as the ratio of GABA EC₅₀s in the absence of etomidate to that in the presence of 3.2 µM etomidate. A large ratio indicates sensitivity to etomidate modulation, while a ratio of 1.0 or smaller indicates no positive modulation.

All GABA_A receptor β subunits contain a methionine at position 286 in their M3 domains and βM286 mutations also influence etomidate sensitivity. The βM286W mutation eliminates etomidate modulation of receptors, whereas the homologous αA291W mutation has no effect on etomidate actions.^123,124,126 Quantitative electrophysiological analysis demonstrates that GABA_A receptors containing the β2M286W mutation display both enhanced sensitivity to GABA and spontaneous activity, effects that mimic the actions of etomidate on wild-type channels (Table 2).¹²⁷

Etomidate anesthesia in transgenic animals

Mutations at β2N265 and β3N265 have been incorporated into transgenic “knock-in” mice to test the role of these subunits in anesthetic actions. Jurd et al¹²⁸ reported that β3N265M knock-in animals have grossly normal morphological and behavioral phenotypes, but are resistant to both loss of righting reflexes and anti-nociceptive (immobilizing) actions of etomidate and propofol at doses higher than those affecting 100% of wild-type animals. Reynolds et al¹²⁹ developed β2N265S knock-in mice and reported that they also have normal morphology and behavior, including sleep and electroencephalographic activity. The β2N265S knock-in mice show normal sensitivity to etomidate for loss of righting reflexes and anti-nociceptive actions, but these mice are resistant to sedative and hypothermic actions of etomidate.^129,130 Etomidate enhancement of tonic currents associated with extrasynaptic receptors is lost in neurons from β2N265S transgenic mice.¹³¹ Further evidence implicating extrasynaptic receptors comes from knock-out mice lacking GABA_A receptor α5 subunits, which are insensitive to the amnestic effects of etomidate.¹³² However, sedative-hypnotic actions in α5^−/− animals are similar to those in wild-type littermates. Similarly, δ^−/− knock-out animals show normal sensitivity to etomidate hypnosis.¹³³ Transgenic animal studies like these confirm that etomidate acts via GABA_A receptors, and that different clinical actions of etomidate are mediated by specific receptor subtypes containing different subunits. Hypnotic and immobilizing actions are mediated by receptors containing β3 subunits, while sedation is linked to receptors containing β2. Extrasynaptic receptors, which often contain α5 and δ subunits appear to be linked to etomidate-induced amnesia, but not to hypnosis and immobility.

Location of etomidate sites on GABA_A receptors

Etomidate, with its high potency and stereoselectivity, proved an excellent candidate for creating photo-reactive derivatives that covalently modify target channels. Husain and colleagues synthesized a diaziryl derivative, azi-etomidate³, while Bright et al¹³⁴ produced an azide. These photolabels display stereoselectivity and pharmacological activity almost identical to that of etomidate in both animals and GABA_A receptors.^3,134,135 In the presence of ultraviolet light, azi-etomidate effects on GABA_A receptors become irreversible.¹¹⁶ Radiolabeled azi-etomidate was used to photolabel affinity-purified bovine GABA_A receptor protein, leading to the identification of two photo-modified amino acids: M236 in M1 on α subunits and M286 in M3 on β subunits.¹³⁶ Addition of etomidate blocked photo-incorporation at both positions in parallel, suggesting that they contribute to the same binding pockets formed where α subunits abut β subunits (Figure 4A). Two such interfacial sites are predicted to be formed by most GABA_A receptors, consistent with the predictions from functional analysis.¹¹⁴

More evidence that αM236 and βM286 are involved in etomidate binding comes from recent molecular studies of mutations at these residues. GABA_A receptors with tryptophan mutations at either α1M236 or β2M286 display functional characteristics that mimic the reversible effects of etomidate on wild-type receptors.¹²⁷ Both α1M236W and β2M286W also reduce receptor sensitivity to etomidate, perhaps because the large tryptophan side-chains occupy the space where etomidate binds. Cysteine mutations have been used to introduce free sulfhydryls at α1M236 and β2M286, which are accessible to modification by selective reagents.¹³⁷ Sulfhydryl modification of α1M236C or β2M286C is blocked by etomidate (Deirdre Stewart, Ph.D, Boston, MA, USA, unpublished research findings), confirming that the drug binds close to both residues. The hypothesis that etomidate binds between transmembrane helices on two adjacent GABA_A receptor subunits differs from earlier proposals that anesthetics bind within a single subunit.¹³⁸ Recently, Bali et al¹³⁷ provided further evidence that αM236 and βM286 residues of GABA_A receptors are on nearby helical domains and oriented toward interfacial clefts between subunits. Their experiments showed that β2M286C forms inter-subunit cross-linking disulfide bonds with cysteines substituted at two α subunit M1 domain loci on the same helical face as α1M236.

Etomidate interactions with adrenal steroidogenesis enzymes

During etomidate infusion, plasma levels of cortisol, cortisone, and aldosterone drop, while those of 11-deoxycorticosterone, 11-deoxycortisol, progesterone, and 17-hydroxyprogesterone become elevated.¹³⁹ These clinical results, and related in vitro studies¹⁴⁰, indicate that etomidate inhibits adrenal steroid synthesis primarily by blocking the activity of CYP11B1, also known as 11β-hydroxylase or P450c11. This mitochondrial cytochrome enzyme converts 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone and is 95% homologous to the CYP11B2 (aldolase) enzyme in the pathway leading to aldosterone.¹⁴¹

The imidazole ring of etomidate is likely to be a major determinant of its binding to adrenal cytochrome enzymes. Many other imidazole compounds inhibit CYP11B enzymes¹⁴², and a variety of crystal structure studies confirm that imidazole nitrogens coordinate (form dipolar bonds with) heme irons located at the active sites of prokaryotic and eukaryotic cytochromes.^143–145 High level production of purified human CYP11B1 has recently been reported,¹⁴⁶ and high resolution structural data for the molecule may be available in the near future. Homology models based on crystal structures of related enzymes have been developed and used for in silico ligand etomidate docking studies (Figure 6).¹⁴⁷

Figure 6. Homology model for etomidate binding to CYP11B1.

The binding pocket of CYP11B1 is depicted, based on high resolution crystal structures of related cytochromes.¹⁴⁷ Etomidate is shown bound within the binding pocket, oriented to form a strong coordinate bond between its free imidazole nitrogen and the heme iron of the enzyme. (This figure was kindly provided by Keith Miller and Shunmugasundararaj Sivananthaperumal, Massachusetts General Hospital, Boston, MA).

Adrenergic receptors and cardiovascular stability with etomidate

Alpha-2 adrenergic receptors are activated by etomidate, but this action is unrelated to its hypnotic effects in mice.¹⁴⁸ However, the transient hypertension produced by etomidate in wild-type mice is absent in knock-out mice lacking either α2B or α2A adrenergic receptor subtypes. This result indicates that α2 adrenergic receptors may contribute to the hemodynamic effects of etomidate.

Etomidate’s remarkably benign cardiovascular and pulmonary effects are also likely due to its selectivity for a small number of molecular targets. In comparison, clinically relevant concentrations of barbiturates, propofol, and volatile anesthetics modulate a broader array of GABA_A receptor subtypes together with multiple other etomidate-insensitive ion channels found in both neurons and cardiovascular structures.¹⁴⁹

Channels that mediate etomidate injection pain

Transient receptor potential type A1 (TRPA1) cation channels are involved in inflammation and pain sensation. Like propofol and other general anesthetics, etomidate at high concentrations activates TRPA1 channels, a mechanism that may underlie pain during injection.¹⁵⁰

New drugs based on etomidate

Selective adrenal steroid inhibitors

Because of its unequaled potency as an inhibitor of cortisol and aldosterone synthesis, etomidate derivatives have been explored as selective biomarkers and inhibitors for diseases associated with excess adrenocortical activity. Positron-emitting derivatives of etomidate have been developed for localization of adrenal tumors¹⁵¹, and infusion of etomidate is gaining popularity as an acute treatment for poorly controlled Cushing’s disease.¹⁵² Subhypnotic doses of etomidate effectively reduce the high systemic cortisol and aldosterone concentrations associated with this disease with mild sedation as a side-effect.¹⁵³ In addition to inhibiting steroid synthesis, etomidate inhibits proliferation of adrenal cortical cells, making it particularly useful in the treatment of metastatic adrenocortical tumors.¹⁵⁴ In a recent report on several dozen synthetic etomidate derivatives, none demonstrated greater potency than etomidate for inhibition of cortisol synthesis by cultured adrenal cells.¹⁵⁵ Several of these compounds show high potency for CYP11B binding, but weak interactions with GABA_A receptors, suggesting that treatment for excess cortisol or aldosterone synthesis may be achieved without sedating side effects.¹⁵⁶

Novel anesthetic agents

Recent research has also aimed at modifying etomidate to improve its clinical utility as an anesthetic and sedative. Two molecular strategies have been described to maintain the favorable clinical features of etomidate, while reducing the activity that most limits its clinical use: prolonged inhibition of adrenal steroidogenesis.

Methoxycarbonyl-etomidate (MOC-etomidate) is a “soft” analog that contains a second ester bond distal to the existing etomidate ester linkage (Figure 7A).¹⁵⁷ MOC-etomidate modulates GABA_A receptors with potency near that of etomidate, but is rapidly (with a half-life of a few minutes) metabolized by non-specific esterase enzymes in blood and tissue, and converted to a carboxylic acid metabolite. The MOC-etomidate metabolite is inactive both as an anesthetic and as an inhibitor of adrenal steroid synthesis (verbal communication, Douglas Raines, M.D., Associate Professor, Dept. of Anesthesia Critical Care & Pain Medicine, Massachusetts General Hospital, Boston, MA, USA; April, 2010). In rats, MOC-etomidate bolus administration produced anesthesia lasting only a few minutes, while an equipotent bolus of etomidate produced loss of righting reflexes for nearly an hour. Thirty minutes after MOC-etomidate bolus administration, no adrenal suppression is found, whereas significant adrenal suppression is associated with etomidate bolus administration. MOC-etomidate is currently in pre-clinical development. Its potential use includes anesthesia induction and maintenance for periods up to several hours. Adrenal suppression may be present during anesthesia with MOC-etomidate, but adrenal function is predicted to recover rapidly after cessation of drug infusion.

Figure 7. Structures of MOC-etomidate and Carboetomidate.

Panel A shows the structure of methoxycarbonyl etomidate (MOC-etomidate), a rapidly metabolized “soft analog” of etomidate. The dashed box outlines the parent molecule, which is depicted in Figure 1. Panel B shows the structure of carboetomidate, a molecule that retains the molecular shape of etomidate, while replacing the imidazole ring with a pyrrole ring that is unable to form coordinate bonds with heme iron. (The structures were kindly provided by Douglas Raines, Massachusetts General Hospital, Boston, MA).

Carboetomidate is an etomidate “look alike” drug which contains a 5-membered pyrrole ring instead of an imidazole (Figure 7B).¹⁵⁸ The loss of the free imidazole nitrogen eliminates coordination interactions with heme irons, reducing adrenal suppression potency by three orders of magnitude (IC₅₀ ≈ 1 µM vs. etomidate IC₅₀ = 1 nM), based on adrenal cell cortisol synthesis assays. Carboetomidate retains the ability to modulate and directly activate GABA_A receptors and is a potent sedative-hypnotic with systemic effects and duration of action similar to those of etomidate in laboratory animals.

Summary Statement.

Etomidate is a potent hypnotic drug with unique clinical characteristics, and the molecular mechanisms underlying its actions are understood better than those of other anesthetics. This knowledge has engendered several promising translational drug developments.