Stuart A. Forman

Clinical and Molecular Pharmacology of Etomidate

This review focuses on the unique clinical and molecular pharmacologic features 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 &ggr;-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.

Methoxycarbonyl-etomidate: a novel rapidly metabolized and ultra-short-acting etomidate analogue that does not produce prolonged adrenocortical suppression.

Background:Etomidate is a rapidly acting sedative-hypnotic that provides hemodynamic stability. It causes prolonged suppression of adrenocortical steroid synthesis; therefore, its clinical utility and safety are limited. The authors describe the results of studies to define the pharmacology of (R)-3-methoxy-3-oxopropyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MOC-etomidate), the first etomidate analogue designed to be susceptible to ultra-rapid metabolism. Methods:The &ggr;-aminobutyric acid type A receptor activities of MOC-etomidate and etomidate were compared by using electrophysiological techniques in human &agr;1&bgr;2&ggr;2l receptors. MOC-etomidate’s hypnotic concentration was determined in tadpoles by using a loss of righting reflex assay. Its in vitro metabolic half-life was measured in human liver S9 fraction, and the resulting metabolite was provisionally identified by using high-performance liquid chromatography/mass spectrometry techniques. The hypnotic and hemodynamic actions of MOC-etomidate, etomidate, and propofol were defined in rats. The abilities of MOC-etomidate and etomidate to inhibit corticosterone production were assessed in rats. Results:MOC-etomidate potently enhanced &ggr;-aminobutyric acid type A receptor function and produced loss of righting reflex in tadpoles. Metabolism in human liver S9 fraction was first-order, with an in vitro half-life of 4.4 min versus more than 40 min for etomidate. MOC-etomidate’s only detectable metabolite was a carboxylic acid. In rats, MOC-etomidate produced rapid loss of righting reflex that was extremely brief and caused minimal hemodynamic changes. Unlike etomidate, MOC-etomidate produced no adrenocortical suppression 30 min after administration. Conclusions:MOC-etomidate is an etomidate analogue that retains etomidate’s important favorable pharmacological properties. However, it is rapidly metabolized, ultra–short-acting, and does not produce prolonged adrenocortical suppression after bolus administration.

Azo-propofols: photochromic potentiators of GABA(A) receptors.

Shine and rise! GABA(A) receptors are ligand-gated chloride ion channels that respond to γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter of the mammalian central nervous system. Azobenzene derivatives of propofol, such as compound 1 (see scheme), increase GABA-induced currents in the dark form and lose this property upon light exposure and thus function as photochromic potentiators. Compound 1 can be employed as a light-dependent general anesthetic in translucent tadpoles.

Ethanol increases agonist affinity for nicotinic receptors from Torpedo

The presence of ethanol increases the apparent affinity with which acetylcholine and carbamylcholine elicit 86Rb+ flux from Torpedo nicotinic acetylcholine receptor-rich vesicles at 4 degrees C. Affinity increased exponentially with ethanol concentration, reaching nearly 200-fold by 3.0 M ethanol without sign of saturation. At submaximal agonist concentrations 50-100 mM ethanol enhanced flux by 15-35%, but the maximum agonist-induced flux was unaffected in quenched-flow assays. The effect was independent of the agonist and of the time over which flux was measured (5 ms to 10 s), indicating that ethanol acts before agonist-induced desensitization occurs. Ethanol also caused an increase in the apparent affinity with which acetylcholine caused fast desensitization. This affinity increase was equal to that for flux-response curves, but the maximum fast desensitization rate was increased 50% at 0.5 M ethanol. This was the most pronounced of ethanols actions and has not been reported before. Prolonged preincubation with 1.0 M ethanol alone reduced agonist-induced flux activity by only 25%. The rate of agonist-induced slow desensitization was also increased, but neither of these effects was as marked as those on fast desensitization and cation flux.

Tryptophan Mutations at Azi-Etomidate Photo-Incorporation Sites on α1 or β2 Subunits Enhance GABAA Receptor Gating and Reduce Etomidate Modulation

The potent general anesthetic etomidate produces its effects by enhancing GABAA receptor activation. Its photolabel analog [3H]azi-etomidate labels residues within transmembrane domains on α and β subunits: αMet236 and βMet286. We hypothesized that these methionines contribute to etomidate sites formed at α-β subunit interfaces and that increasing side-chain bulk and hydrophobicity at either locus would mimic etomidate binding and block etomidate effects. Channel activity was electrophysiologically quantified in α1β2γ2L receptors with α1M236W or β2M286W mutations, in both the absence and the presence of etomidate. Measurements included spontaneous activation, GABA EC50, etomidate agonist potentiation, etomidate direct activation, and rapid macrocurrent kinetics. Both α1M236W and β2M286W mutations induced spontaneous channel opening, lowered GABA EC50, increased maximal GABA efficacy, and slowed current deactivation, mimicking effects of etomidate on α1β2γ2L channels. These changes were larger with α1M236W than with β2M286W. Etomidate (3.2 μM) reduced GABA EC50 much less in α1M236Wβ2γ2L receptors (2-fold) than in wild type (23-fold). However, etomidate was more potent and efficacious in directly activating α1M236Wβ2γ2L compared with wild type. In α1β2M286Wγ2L receptors, etomidate induced neither agonist-potentiation nor direct channel activation. These results support the hypothesis that α1Met236 and β2Met286 are within etomidate sites that allosterically link to channel gating. Although α1M236W produced the larger impact on channel gating, β2M286W produced more profound changes in etomidate sensitivity, suggesting a dominant role in drug binding. Furthermore, quantitative mechanistic analysis demonstrated that wild-type and mutant results are consistent with the presence of only one class of etomidate sites mediating both agonist potentiation and direct activation.

Carboetomidate: a pyrrole analog of etomidate designed not to suppress adrenocortical function.

Background:Etomidate is a sedative hypnotic that is often used in critically ill patients because it provides superior hemodynamic stability. However, it also binds with high affinity to 11&bgr;-hydroxylase, potently suppressing the synthesis of steroids by the adrenal gland that are necessary for survival. The authors report the results of studies to define the pharmacology of (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate), a pyrrole analog of etomidate specifically designed not to bind with high affinity to 11&bgr;-hydroxylase. Methods:The hypnotic potency of carboetomidate was defined in tadpoles and rats using loss of righting reflex assays. Its ability to enhance wild-type &agr;1&bgr;2&ggr;2l and etomidate-insensitive mutant &agr;1&bgr;2M286W&ggr;2l human &ggr;-aminobutyric acid type A receptor activities was assessed using electrophysiologic techniques. Its potency for inhibiting in vitro cortisol synthesis was defined using a human adrenocortical cell assay. Its effects on in vivo hemodynamic and adrenocortical function were defined in rats. Results:Carboetomidate was a potent hypnotic in tadpoles and rats. It increased currents mediated by wild-type but not etomidate-insensitive mutant &ggr;-aminobutyric acid type A receptors. Carboetomidate was a three orders of magnitude less-potent inhibitor of in vitro cortisol synthesis by adrenocortical cells than was etomidate. In rats, carboetomidate caused minimal hemodynamic changes and did not suppress adrenocortical function at hypnotic doses. Conclusions:Carboetomidate is an etomidate analog that retains many beneficial properties of etomidate, but it is dramatically less potent as an inhibitor of adrenocortical steroid synthesis. Carboetomidate is a promising new sedative hypnotic for potential use in critically ill patients in whom adrenocortical suppression is undesirable.

A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc

The present study describes two novel compound heterozygous mutations, c.410C>T(p.T137M) (T137M) on the maternal and g.4225_50del on the paternal allele of SLC34A3, in a previously reported male with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and recurrent kidney stones (Chen C, Carpenter T, Steg N, Baron R, Anast C. Pediatrics 84: 276-280, 1989). For functional analysis in vitro, we generated expression plasmids encoding enhanced green fluorescence protein (EGFP) concatenated to the NH2 terminus of wild-type or mutant human type IIc Na-Pi cotransporter (NaPi-IIc), i.e., EGFP-hNaPi-IIc, EGFP-[M137]hNaPi-IIc, or EGFP-[Stop446]hNaPi-IIc. The V446Stop mutant showed complete loss of expression and function when assayed for apical patch expression in opossum kidney (OK) cells and sodium-dependent 33P uptake into Xenopus laevis oocytes. Conversely, EGFP-[M137]hNaPi-IIc was inserted into apical patches of OK cells and into oocyte membranes. However, when quantified by confocal microscopy, surface fluorescence was reduced to 40% compared with wild-type. After correction for surface expression, the rate of 33P uptake by oocytes mediated by EGFP-[M137]hNaPi-IIc was decreased by an additional 60%. The resulting overall reduction of function of this NaPi-IIc mutant to 16%, taken together with complete loss of expression and function of g.4225_50del(V446Stop), thus appears to be sufficient to explain the phenotype in our patient. Furthermore, the stoichiometric ratio of 22Na and 33P uptake was increased to 7.1 +/- 3.65 for EGFP-[M137]hNaPi-IIc compared with wild-type. Two-electrode studies indicate that EGFP-[M137]hNaPi-IIc is nonelectrogenic but displayed a significant phosphate-independent inward-rectified sodium current, which appears to be insensitive to phosphonoformic acid. M137 thus may uncouple sodium-phosphate cotransport, suggesting that this amino acid residue has an important functional role in human NaPi-IIc.

Classic Benzodiazepines Modulate the Open–close Equilibrium in α1β2γ2l γ-aminobutyric Acid Type A Receptors

Background: Classic benzodiazepine agonists induce their clinical effects by binding to a site on &ggr;-aminobutyric acid type A (GABAA) receptors and enhancing receptor activity. There are conflicting data regarding whether the benzodiazepine site is allosterically coupled to &ggr;-aminobutyric acid binding versus the channel open–close (gating) equilibrium. The authors tested the hypothesis that benzodiazepine site ligands modulate &agr;1&bgr;2&ggr;2L GABAA receptor gating both in the absence of orthosteric agonists and when the orthosteric sites are occupied. Methods: GABAA receptors were recombinantly expressed in Xenopus oocytes and studied using two-microelectrode voltage clamp electrophysiology. To test gating effects in the absence of orthosteric agonist, the authors used spontaneously active GABAA receptors containing a leucine-to-threonine mutation at residue 264 on the &agr;1 subunit. To examine effects on gating when orthosteric sites were fully occupied, they activated wild-type receptors with high concentrations of a partial agonist, piperidine-4-sulfonic acid. Results: In the absence of orthosteric agonists, the channel activity of &agr;1L264T&bgr;2&ggr;2L receptors was increased by diazepam and midazolam and reduced by the inverse benzodiazepine agonist FG7142. Flumazenil displayed very weak agonism and blocked midazolam from further activating mutant channels. In wild-type receptors activated with saturating concentrations of piperidine-4-sulfonic acid, midazolam increased maximal efficacy. Conclusions: Independent of orthosteric site occupancy, classic benzodiazepines modulate the gating equilibrium in &agr;1&bgr;2&ggr;2L GABAA receptors and are therefore allosteric coagonists. A Monod-Wyman-Changeux coagonist gating model quantitatively predicts these effects, suggesting that benzodiazepines minimally alter orthosteric ligand binding.

Nonhalogenated Alkane Anesthetics Fail to Potentiate Agonist Actions on Two Ligand-gated Ion Channels

Background Although ether, alcohol, and halogenated alkane anesthetics potentiate agonist actions or increase the apparent agonist affinity of ligand-gated ion channels at clinically relevant concentrations, the effects of nonhalogenated alkane anesthetics on ligand-gated ion channels have not been studied. The current study assessed the abilities of two representative nonhalogenated alkane anesthetics (cyclopropane and butane) to potentiate agonist actions or increase the apparent agonist affinity of two representative ligand-gated ion channels: the nicotinic acetylcholine receptor and &ggr;-aminobutyric acid type A (GABAA) receptor. Methods Nicotinic acetylcholine receptors were obtained from the electroplax organ of Torpedo nobiliana, and human GABAA receptors (&agr;1&bgr;2&ggr;2L) were expressed in human embryonic kidney 293 cells. The Torpedo nicotinic acetylcholine receptors apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the apparent rates of desensitization induced by a range of acetylcholine concentrations. The GABAA receptor’s apparent agonist affinity in the presence and absence of anesthetic was assessed by measuring the peak currents induced by a range of GABA concentrations. Results Neither cyclopropane nor butane potentiated agonist actions or increased the apparent agonist affinity (reduced the apparent agonist dissociation constant) of the Torpedo nicotinic acetylcholine receptor or GABAA receptor. At clinically relevant concentrations, cyclopropane and butane reduced the apparent rate of Torpedo nicotinic acetylcholine receptor desensitization induced by low concentrations of agonist. Conclusions Our results suggest that the in vivo central nervous system depressant effects of nonhalogenated alkane anesthetics do not result from their abilities to potentiate agonist actions on ligand-gated ion channels. Other targets or mechanisms more likely account for the anesthetic activities of nonhalogenated alkane anesthetics.

Molecular sites of anestheticaction in postsynaptic nicotinic membranes

Theories of general anesthesia have traditionally been based on correlations between potency and the properties of simple models such as apolar solvents, lipid bilayers and soluble proteins. However, mechanisms can now be determined directly by studying excitable proteins in their membrane environment. Stuart Forman and Keith Miller describe the physiological, biophysical and molecular biological evidence pointing to the location of a discrete allosteric site on the nicotinic acetylcholine receptor at which local anesthetics act. General anesthetics, while superficially resembling local anesthetics in their actions on the receptor, do not appear to act upon such a site.