Concentrations Of Etomidate Research Articles

Characterization of etomidate and emerging analogs in human hair using UHPLC-MS/MS and confirmation in real forensic cases

The regulation of etomidate, a widely used drug for anesthesia induction and short surgical procedures, has led to the emergence of etomidate analogs such as metomidate, propoxate, and isopropoxate. This study introduces and validates a simple, rapid, and cost-effective ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination and quantification of etomidate, etomidate acid, metomidate, propoxate, and isopropoxate in human hair. Using a five-minute gradient elution on a Phenomenex Kinetex Biphenyl column, the method achieves low limits of quantification (LOQs), ranging from 5 to 20 pg/mg. Method validation confirms robust linear calibration curves for the target substances in hair samples within the range of LOQs to 1000 pg/mg, with average correlation coefficients (r) all exceeding 0.999. The method also achieves acceptable intra-day and inter-day precision (<15 %) and trueness(bias, −10.9 % to 14.4 %). The matrix effect ranges from 46.9 % to 94.2 %, and the extraction recovery rate ranges from 84.5 % to 105 %. When applied to authentic cases, this method successfully identified 56 positive samples. The concentrations of etomidate, etomidate acid, metomidate, propoxate, and isopropoxate in positive samples ranged from 27 to 1.5 × 105 pg/mg, 21 to 1.5 × 103 pg/mg, 18 to 8.7 × 104 pg/mg, 25 to 1.6 × 104 pg/mg, and 22 to 5.0 × 105 pg/mg, respectively.

Detection of "smoke powder" etomidate and its metabolite etomidate acid in blood and urine by UHPLC-MS/MS: Application in authentic cases.

Recently, etomidate has been widely used as an alternative in illicit drug market. It is usually added to regular cigarette tobacco (commonly known as "cigarette powder") or mixed in e-cigarette oil sold through the Internet, retail stores, or entertainment outlets and other channels. An ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify etomidate and etomidate acid in human blood and urine. The limit of detection (LOD) of etomidate and etomidate acid in blood are 0.5 ng/mL and 2 ng/mL, respectively, and the lower limit of quantification (LLOQ) are 1 ng/mL and 5 ng/mL, respectively. The LOD of etomidate and etomidate acid in urine are1 ng/mL and 2 ng/mL, respectively, and the LLOQ are 2 ng/mL and 5 ng/mL, respectively. The precision, accuracy, recoveries and matrix effects of etomidate and etomidate acid determinations in blood and urine met the requirements for methodological validation. The method was successfully applied to the identification and quantification of etomidate and etomidate acid in blood and urine of 62 forensic cases. The concentration of etomidate ranged from 1.52 to 8.41 ng/mL (positive cases, n=5) and the concentration of etomidate acid ranged from 2.76 to 112 ng/mL (positive cases, n=5) in blood. The concentrations of etomidate and etomidate acid in urine samples were 2.64-79,300 ng/mL (positive cases, n=59) and 6.11-518,000 ng/mL (positive cases, n=60), respectively. Therefore, the concentration of etomidate in blood and urine is mostly higher than that of etomidate.

Etomidate protects retinal ganglion cells from hydrogen peroxide-induced injury via Nrf2/HO-1 pathway.

To determine whether etomidate (ET) has a protective effect on retinal ganglion cells (RGCs) injured with hydrogen peroxide (H2O2) and to explore the potential mechanism underlying the antioxidative stress effect of ET. Cultured RGCs were identified by double immunofluorescent labeling of microtubule-associated protein 2 and Thy1.1. An injury model of H2O2-induced RGCs oxidative stress was established in vitro. Cells were pretreated with different concentrations of ET (1, 5, and 10 µmol/L) for 4h, followed by further exposure to H2O2 at 1000 µmol/L. Cell counting kit 8 and Annexin V/propidium iodide assays were applied to detect the viabilities and apoptosis rates of the RGCs at 12, 24, and 48h after H2O2 stimulation. The levels of nitric oxide, malondialdehyde, and glutathione in culture media were measured at these time points. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were performed to observe the effects of ET on the messenger RNA and protein expression of inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione peroxidase 1 and the level of conjugated acrolein in RGCs at 12, 24, and 48h after H2O2 stimulation and in the retina at 12h after optic nerve transection (ONT). The applications of 5 and 10 µmol/L of ET significantly increased the viability of RGCs. Results from qRT-PCR indicated a decrease in the expression of iNOS and an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs at 12, 24 and 48h after H2O2 stimulation, as well as in ET-treated retinas at 12h after ONT. Western blot analysis revealed a decrease in the expression of iNOS and levels of conjugated acrolein, along with an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs in vitro and ET-treated retinas in vivo. ET is a neuroprotective agent in primary cultured RGCs injured by H2O2. The effect of ET is dose-dependent with the greatest effect being at 10 µmol/L. ET plays an antioxidant role by inhibiting iNOS, up-regulating Nrf2/HO-1, decreasing the production of acrolein, and increasing the scavenge of acrolein.

Etomidate as an anesthetic in Colossoma macropomum: Behavioral and electrophysiological data complement each other as a tool to assess anesthetic safety.

The use of anesthetic agents in the management of fish in fish farming or ornamental fish breeding aims to minimize stress and promote animal welfare. Therefore, this study aims to investigate behavioral, electrocardiographic, and ventilatory characteristics of tambaquis exposed to anesthetic baths with etomidate. The study was conducted with juvenile tambaquis (27.38 ± 3.5g) n = 99, at etomidate concentrations of 2-4 mg.L -1, analyzing induction and anesthetic recovery behavior (experiment I), electrocardiogram (experiment II), and opercular movement (experiment III). Fish exposed to high concentrations of etomidate reached the stage of general anesthesia faster, however, the recovery time was longer, characterizing a dose-dependent relationship. Cardiorespiratory analyzes demonstrated a reduction in heart rate (69.19%) and respiratory rate (40.70%) depending on the concentration of etomidate used during anesthetic induction. During the recovery period, there was cardiorespiratory reversibility to normality. Therefore, etomidate proved to be safe as an anesthetic agent for this species at concentrations of 2 to 3 mg.L -1 for short-term anesthesia, but at higher doses the animals showed slow reversibility of anesthesia in a gradual manner and without excitability. The hemodynamic effect due to the rapid decrease in heart rate includes a negative factor of using higher concentrations of etomidate for Colossome macropomum anesthesia.

Direct injection ultra-performance liquid chromatography-tandem mass spectrometry for the high-throughput determination of etomidate and etomidate acid in wastewater.

Etomidate (ET), a hypnotic agent used for the induction of anesthesia, is rapidly metabolized to etomidate acid (ETA) in the liver. Recently, ET has become one of the most serious alternative drugs of abuse in China. Therefore, an urgent need exists to develop a fast and convenient analysis method for monitoring ET. The current work presents a simple, fast, and sensitive direct injection method for the determination of ET and ETA in wastewater. After the optimization of the ultra-performance liquid chromatography-tandem mass spectrometry and sample filtration conditions, the method exhibited satisfactory limits of detection (1 ng/L) and good filtration loss. The validated method was successfully applied to determine the concentrations of ET and ETA in wastewater samples (n = 245) from several wastewater treatment plants in China. The concentrations of the targets in positive samples ranged from less than the lower limits of quantitation to 47.71 ng/L. The method can meet ET monitoring and high-throughput analysis requirements.

Pharmacokinetic and pharmacodynamic evaluation study of etomidate: a randomized, open-label, 2-period crossover study in healthy Chinese subjects

Etomidate is a sedative and hypnotic drug through intravenous administration that act on the central nervous system through GABA (Gamma-Amino Butyric Acid) receptors, which is widely used in anesthesia induction and maintenance and long-term sedation in severe patients. The study aimed to evaluate the pharmacokinetic and pharmacodynamic properties of two etomidate fat emulsions after administration through the intravenous infusion pump in healthy Chinese subjects. A randomized, open-label, 2-period crossover study was performed in 52 healthy subjects. The wash-out period was 7 days. Blood samples and pharmacodynamic index values were collected at the specified time points. Etomidate concentrations were measured using validated liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were analyzed using a non-compartment model method. Pharmacodynamic parameters were calculated using pharmacodynamic index values. The study also evaluated the safety of the etomidate. Both the pharmacokinetic parameters and pharmacodynamic parameters result of the test and reference formulation were very similar. The 90% confidence intervals (CI) of the geometric least-squares mean (GLSM) ratios of the test to reference formulation were 91.33–104.96% for the maximum plasma concentration (Cmax), 97.21–102.03% for the area under the plasma concentration time curve from time 0 to the time of the last measurable concentration (AUC0–t), and 97.22–102.33% for the area under the plasma concentration time curve from time 0 to infinity (AUC0–∞). Meanwhile, the 90% CI of the GLSM ratios of the test to reference formulation were 102.28–110.69% for the minimal BIS value (BISmin), 99.23–101.17% for the area under the BIS time curve from time 0–60 min after administration (BISAUC0–60 min), respectively. The 90% CI of these pharmacokinetic and pharmacodynamic parameters all fall in the accepted bioequivalence range of 80.00–125.00%. No serious adverse events occurred during the study. This study has shown that the etomidate fat emulsion test and reference formulation had similar pharmacokinetic and pharmacodynamic characteristics in vivo. The two formulations exhibited good safety and well-tolerance.Clinical trials registration number:http://www.chinadrugtrials.org.cn/index.html. # CTR20191836.

Detection and quantification of etomidate and metomidate in human hairs by ultraperformance liquid chromatography with triple quadrupole mass spectrometry (UPLC-MS/MS).

Intravenous narcotic agents, such as etomidate and metomidate, has been widely spread and abused in the world, including in Korea and China; thus, it is important to establish validated and sensitive analytical method for these compounds. Human hair as a biological sample has various advantages, including a wide detection window of drugs, compared to other typical samples, such as urine and blood in investigation. The purpose of this communication is to develop a reliable and useful method for the simultaneous detection and quantification of etomidate and metomidate in human hair samples by ultraperformance liquid chromatography combined with triple quadrupole mass spectrometry (UPLC-MS/MS), and to apply it for authentic samples in abuse cases. The hair samples were washed with a detergent solution, followed by with water and acetone. After drying, they were cut into approximately 2mm sections and then ground to powder by a low-temperature grinder. The 20mg of hair powder plus internal standard in 1mL of methanol was vortexed and then centrifuged to obtain the supernatant layer, followed by subjecting to analysis. The coefficient of determination (r2) values of the calibration curves of etomidate and metomidate in the hair samples were both more than 0.99 in the range of 1-500ng/mg and 1-500pg/mg, respectively. The limits of detection and lower limits of quantification were 0.5 and 1pg/mg, respectively, for the both target compounds. Other tested validation data were all satisfactory. Etomidate and metomidate could be detected in the all hair samples and cigarette oil, which were seized by the police. The concentrations of etomidate and metomidate obtained from 10 samples from suspects were 5.48-45.7ng/mg and 3.60-377pg/mg, respectively. The concentrations of etomidate and metomidate in the cigarette oil were 95.8μg/mg and 2.8μg/mg, respectively. In this study, a simple and reliable analytical method for etomidate and metomidate in the human hair has been established. To the best of our knowledge, this is the first report to establish a method for the simultaneous detection and quantification of etomidate and metomidate in the human hair, and to apply it to authentic samples seized in authentic cases.

A highly efficient liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for etomidate and etomidate acid in urine, liver and kidney

Etomidate (ETO) is a highly-efficient drug that can induce anesthesia with increasing doses, thus subject to strict regulation. However, an accurate and efficient method for ETO intake detection is currently lacking. Therefore, this study developed a straightforward sample preparation method using LC-MS/MS to analyze ETO and its primary metabolite, etomidate acid (ETA), in urine, liver, and kidney samples. Snap frozen pig liver and kidney samples were ground into a fine powder. Then, all the biological samples, including human urine, pig liver and kidney tissues, were deproteinized using acetonitrile and filtered for analysis. The separation was achieved in 9.01 min with gradient elution. The calibration curves ranged from 0.5 to 50 ng/mL for ETO in urine and 0.5 to 50 ng/g in liver and kidney, while the curves ranged from 1 to 100 ng/mL for ETA in urine and 1 to 100 ng/g in liver and kidney. The correlation coefficients (R2) were greater than 0.9957. The Limit of detection (LOD) and limit of quantitation (LOQ) for ETO were 0.2 and 0.5 ng/mL in urine samples and 0.2 and 0.5 ng/g in liver and kidney samples, respectively. For ETA, the LOD and LOQ were 0.5 and 1 ng/mL in urine samples and 0.5 and 1 ng/g in liver and kidney samples. This method was assessed by validation parameters, including selectivity, intra- and inter-day precision and accuracy, recovery, matrix effect, dilution integrity and stability. It was successfully applied to a practical case, revealing ETO and ETA concentrations in urine of 1.01 and 5.58 μg/mL, in liver samples of 12.30 and 1.13 μg/g, and in kidney samples of 6.95 and 4.23 μg/g. This suggests that the method is suitable for routine forensic detection of illicit ETO abuse.

Anesthetic drug concentrations and placental transfer rate in fetus between term and preterm infants, twins, and singletons.

Objective: This study aims to determine the drug concentration of etomidate, remifentanil, and rocuronium bromide for general anesthesia in fetus as well as the placental transport rate between term and preterm delivery, twins, and singleton. Study design: Sixty parturients with 72 fetuses undergoing cesarean section under general anesthesia were included. According to whether the fetus was a twin or premature, parturients were divided into Group I (term singleton), Group II (premature singleton), Group III (term twins), and Group IV (premature twins). The preoperative demographic characteristics and laboratory examination of parturients, hemodynamic indicators, the Apgar score of neonates at 1, 5, and 10min after delivery and at specific assigned values, umbilical artery blood gas analysis results, neonatal weight, and resuscitative measures were recorded. Anesthetic drug concentrations in maternal arterial (MA), umbilical arterial (UA), and umbilical venous (UV) blood were detected by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS). Result: No significant differences were observed in the concentrations of etomidate, remifentanil, and rocuronium bromide in MA, UV, and UA blood, or in the UV/MA and UA/UV ratios between term and preterm infants, twins, and singletons. Moreover, there was no variation in the anesthetic drug concentration among each pair of twins. Additionally, no correlation was found between the neonatal weight and the plasma concentrations of anesthetic drugs in UV and UA blood, except for remifentanil in UA blood. Conclusion: Preterm or twin deliveries do not affect the neonatal concentration of etomidate, remifentanil, and rocuronium bromide used in general anesthesia for cesarean sections. Clinical Trial Registration: www.chictr.org.cn, identifier ChiCTR2100046547.

Prolonged Anesthesia Induction to Delivery Time Did Not Influence Plasma Remifentanil Concentration in Neonates.

Remifentanil, in combination with etomidate and sevoflurane, is commonly used in clinics for general anesthesia induction in cesarean section (CS). This study aimed to evaluate the correlation between the induction to delivery (I-D) time and neonatal plasma drug concentration and anesthesia, as well as its effects on neonates. Fifty-two parturients in whom general anesthesia was induced for CS were divided into group A (I-D<8 min) and group B (I-D≥8 min). Maternal arterial (MA), umbilical venous (UV), and umbilical arterial (UA) blood samples were collected at delivery to analyze the remifentanil and etomidate concentrations using liquid chromatography-tandem mass spectrometry. There were no statistically significant differences between the two groups in terms of plasma concentrations of remifentanil in the MA, UA, and UV blood (P > 0.05). The plasma concentration of etomidate in MA and UV was higher in group A than that in group B (P<0.05), whereas the UA/UV ratio of etomidate was higher in group B than that in group A (P<0.05). The Spearman rank correlation test showed no correlation between the I-D time and plasma remifentanil concentration in the MA, UA, and UV plasma (P>0.05). The concentrations of etomidate in the MA and UV were negatively correlated with the I-D time (P < 0.05). Prolonged I-D time did not significantly influence the maternal or neonatal plasma concentration of remifentanil. It is safe to administer remifentanil target-controlled infusion in combination with etomidate and sevoflurane for general anesthesia induction during CS.

Behavioural Aversion and Cortisol Level Assessment When Adult Zebrafish Are Exposed to Different Anaesthetics.

Simple SummaryThe increased popularity of zebrafish as a research model calls for appropriate refinement of procedures, such as finding the best anaesthetic protocol to use in adult zebrafish in a laboratory setting. The scarce literature available regarding aversion described the most used anaesthetic (MS222) as aversive to adult zebrafish; therefore, the study of alternatives is urgent. Thus, we studied the impact of three anaesthetic alternatives (a combination of propofol with lidocaine, clove oil, and etomidate), and the standard anaesthetic (MS222) on zebrafish aversion and on cortisol levels. Contrary to what was expected, MS222 did not result in clear signs of aversion, while only etomidate generated a similar profile to the aversive substance (hydrochloric acid). Our results suggested that all the anaesthetic protocols except for etomidate were valid candidates for use in a laboratory setting, although none were innocuous.The use of zebrafish (Danio rerio) as an animal model is growing and occurs in a wide range of scientific areas. Therefore, researchers need better and more appropriate anaesthetics for stressful and/or painful procedures to prevent unpleasant experiences. Thus, we aimed to study if adult zebrafish displayed aversion-associated behaviours (conditioned place aversion) and alterations in cortisol levels when exposed to equipotent concentrations of MS222, propofol/lidocaine, clove oil, or etomidate. Adult AB zebrafish (mixed-sex, N = 177) were randomly assigned to MS222 (150 mg/L), Propofol/Lidocaine (5 mg/L propofol + 150 mg/L lidocaine), Clove Oil (45 mg/L), or Etomidate (2 mg/L) groups. The conditioned place aversion test was used to assess behavioural aversion. Only etomidate resulted in a similar aversion to the positive control group (HCl; pH = 3). Cortisol levels were measured 5 and 15 min after loss of equilibrium. Etomidate induced low levels of cortisol by impairing its synthesis, whereas all the other groups had similar cortisol levels. Based on our data, etomidate was ruled out as an alternative to MS222, as it showed an aversive profile. The remaining protocols were not innocuous, displaying a weak aversive profile when compared to the positive control. In conclusion, a combination of propofol with lidocaine, clove oil, and MS222 were valid candidates for use as anaesthetic protocols.

Determination of etomidate and etomidate acid in hair using liquid chromatography-tandem mass spectrometry.

Etomidate, with efficacy similar to that of propofol, has been used as a propofol substitute because propofol is a designated narcotic drug, and an increase in the frequency of illegal distribution and misuse has been reported in Korea. Previous analytical studies on etomidate used blood and urine. For long-term use and timing estimation, a method for etomidate analysis using hair should be developed. Therefore, in this study, an analytical method using LC-MS/MS was developed to determine etomidate and its major metabolite in hair. Human hair samples were segmented after washing to eliminate possible contaminants on the hair and stirred with methanol. The LC-MS/MS conditions were optimized, and the chromatographic separation time was 10min. Selectivity, linearity, limit of detection, limit of quantification, precision, accuracy, recovery, process efficiency, matrix effect, and stability were evaluated to validate the analytical method. The calibration curves ranged from 0.25 to 50 pg/mg for etomidate and 2-250 pg/mg for etomidate acid; the coefficients of determination were higher than 0.997. The intra- and inter-assay precision results for all the compounds were <15% and satisfied at recovery, process efficiency, matrix effect, and stability. In addition, this method was applied to the hair of 4 rats which are administered with etomidate to evaluate. The etomidate concentrations in the rat hair ranged from 2.60 to 8.50 pg/mg, and the etomidate acid concentrations were 2.06-7.13 pg/mg. Thus, this method can be used as basic data for monitoring etomidate in hair.

Pulse purge thermal desorption ion mobility spectrometer for rapid and sensitive determination of intravenous anesthetic etomidate in blood

Rapid and sensitive determination of etomidate in blood is of great significance for anesthesiologists to adjust the depth of anesthesia, ensure the safety of operation and reduce the incidence of adverse reactions in patients, especially those with unstable hemodynamics. Herein, a positive photoionization ion mobility spectrometry method with acetone doped into the drift gas was developed, increasing the reaction efficiency of ion-molecule reactions and improving the detection sensitivity of etomidate in blood. Furthermore, the proposed injection method of pulse purge thermal desorption in bidirectional flow mode not only successfully improved the analysis performance of etomidate by increasing the effective injection amount of gaseous etomidate molecules, but also significantly reduced the impact of pressure fluctuation caused by the pulse on the quantitative accuracy. In view of the effective blood concentration range of etomidate from 1.25 to 3.75 ng μL−1, the proposed method with the quantitative range of 0.1–10 ng μL−1 was suitable for etomidate determination throughout the perioperative period. As a demonstration, the proposed method has been applied clinically to determine the concentration of etomidate in blood of patients undergoing tonsillectomy or nasal endoscopic functional sinus surgery.

Etomidate inhibits cell proliferation and induces apoptosis in A549 non-small cell lung cancer cells via downregulating WWP2.

Etomidate (ETO) is a commonly used intravenous anesthetic that has been reported to exert a tumor suppressive effect in several types of cancer. The present study aimed to investigate the effect of ETO on cell proliferation and apoptosis in non-small cell lung cancer (NSCLC) cells and elucidate its potential mechanism of action. Therefore, Cell Counting Kit-8 assay was performed to evaluate the effect of different concentrations of ETO (0, 1, 2 or 3 µg/ml) on A549 cell viability. In addition, the possible interaction between ETO and WW domain containing E3 ubiquitin protein ligase 2 (WWP2) was predicted using the STITCH database. Additionally, a stable WWP2-overexpressing A549 cell line was constructed by transfecting A549 cells with the pcDNA3.1-WWP2 plasmid. Cell proliferation and apoptosis were assessed using colony formation and TUNEL assays, respectively. The mRNA and protein expression levels of the apoptosis-related proteins Bcl-2, Bax, caspase 3 and cleaved-caspase 3 were determined by reverse transcription-quantitative PCR and western blotting. In addition, the expression and phosphorylation levels of proliferation-associated genes (PCNA and Ki-67) and proteins in the PI3K/Akt pathway were analyzed by western blotting. The results showed that treatment with ETO attenuated the cell viability and proliferation of A549 cells. ETO also promoted cell apoptosis and decreased the expression of the anti-apoptotic protein Bcl-2, whilst increasing that of pro-apoptotic proteins Bax and cleaved caspase 3 in a dose-dependent manner. Furthermore, ETO was found to negatively regulate the expression of WWP2, such that WWP2 overexpression reversed the potentiating effects of ETO on cell apoptosis. In addition, ETO promoted the expression of PTEN and reduced the phosphorylation levels of the PI3K/AKT pathway-related proteins. These effects aforementioned could also be reversed by WWP2 overexpression. Therefore, data from the present study suggest that ETO can attenuate the progression of NSCLC through by the PI3K/AKT pathway, specifically by targeting WWP2. These findings may provide a novel target for the treatment of NSCLC.

Optimization, and in vitro and in vivo evaluation of etomidate intravenous lipid emulsion

The aim of this investigation was to develop an etomidate intravenous lipid emulsion (ETM-ILE) and evaluate its properties in vitro and in vivo. Etomidate (ETM) is a hydrophobic drug, and organic solvents must be added to an etomidate injectable solution (ETM-SOL) to aid dissolution, that causes various adverse reactions on injection. Lipid emulsions are a novel drug formulation that can improve drug loading and reduce adverse reactions. ETM-ILE was prepared using high-pressure homogenization. Univariate experiments were performed to select key conditions and variables. The proportion of oil, egg lecithin, and poloxamer 188 (F68) served as variables for the optimization of the ETM-ILE formulation by central composite design response surface methodology. The optimized formulation had the following characteristics: particle size, 168.0 ± 0.3 nm; polydispersity index, 0.108 ± 0.028; zeta potential, −36.4 ± 0.2 mV; drug loading, 2.00 ± 0.01 mg/mL; encapsulation efficiency, 97.65% ± 0.16%; osmotic pressure, 292 ± 2 mOsmol/kg and pH value, 7.63 ± 0.07. Transmission electron microscopy images showed that the particles were spherical or spheroidal, with a diameter of approximately 200 nm. The stability study suggested that ETM-ILE could store at 4 ± 2 °C or 25 ± 2 °C for 12 months. Safety tests showed that ETM-ILE did not cause hemolysis or serious vascular irritation. The results of the pharmacokinetic study found that ETM-ILE was bioequivalent to ETM-SOL. However, a higher concentration of ETM was attained in the liver, spleen, and lungs after administration of ETM-ILE than after administration of ETM-SOL. This study found that ETM-ILE had great potential for clinical applications.

Etomidate is devoid of genotoxicty and mutagenicity in human lymphocytes and in the Salmonella typhimurium/microsomal activation test

No carcinogenesis or mutagenesis studies have been carried out with etomidate. The current study showed that etomidate has weak cytotoxic potential after 48 h exposure in human lymphocytes and has no hemolytic activity. The weak cytotoxicity seems to be related with redox imbalance of etomidate (40.9 and 81.9 μM) treated lymphocytes. At both etomidate concentrations, a slight decrease of the levels of GSH intracellular content and a significant increase in the amount of carbonylated proteins were observed after 48 h. The contribution of oxidative stress to genetic toxicity was only perceived when the enzyme Fpg was applied in the comet assay. Etomidate (40.9 and 81.9 μM) is a weak generator of oxidative DNA damage in lymphocytes. These damages to DNA probably were repaired, since no DNA strand breaks were detected in the standard alkaline comet assay (in the presence or absence of hepatic S9 microsomal fraction) without Fpg. Also, no micronucleated lymphocytes or carrying chromosomal aberrations were observed. Finally, etomidate (2046.8 and 4093.5 μM) was not mutagenic in the Salmonella/microsome mutagenicity assay, which used four Salmonella typhimurium strains (TA97a, TA98, TA100, and TA102) to detect frameshift and base-substitution mutations. In summary, etomidate is a weak oxidative DNA damaging anesthetic and is devoid of mutagenic properties in eukaryotic and prokaryotic models.

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

Recent cryo-electron microscopic imaging studies have shown that in addition to binding to the classical extracellular benzodiazepine binding site of the α1β3γ2L 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. 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 α1β3γ2L GABAA receptors using voltage clamp electrophysiology. The ability of diazepam to inhibit receptor labeling of purified α1β3γ2L 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. 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. 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.

Etomidate reduces excitability of the neurons and suppresses the function of nAChR ventral horn in the spinal cord of neonatal rats

To investigate the effects of etomidate on electrophysiological properties and nicotinic acetylcholine receptors (nAChRs) of ventral horn neurons in the spinal cord. The spinal cord containing lumbosacral enlargement was isolated from 19 neonatal SD rats aged 7-12 days. The spinal cord were sliced and digested with papain (0.18 g/30 mL artificial cerebrospinal fluid) and incubated for 40 min. At the ventral horn, acute mechanical separation of neurons was performed with fire-polished Pasteur pipettes, and perforated patch-clamp recordings combined with pharmacological methods were employed on the adherent healthy neurons. In current-clamp mode, the spontaneous action potential (AP) of the ventral horn neurons in the spinal cord was recorded. The effects of pretreatment with different concentrations of etomidate on AP recorded in the ventral horn neurons were examined. In the voltage-clamp mode, nicotine was applied to induce inward currents in the ventral horn neurons, and the effect of pretreatment with etomidate on the inward currents induced by nicotine were examined with different etomidate concentrations, different holding potentials and different use time. The isolated ventral horn neurons were in good condition with large diverse somata and intact processes. The isolated spinal ventral horn neurons (n=21) had spontaneous action potentials, and were continuously perfused for 2 min with 0.3, 3.0 and 30.0 μmol/L etomidate. Compared with those before administration, the AP amplitude, spike potential amplitude and overshoot were concentration-dependently suppressed (P &lt; 0.01), and spontaneous discharge frequency was obviously reduced (P &lt; 0.01, n=12). The APs of the other 9 neurons were completely abolished by etomidate at 3.0 or 30 μmol/L. At the same holding potential (VH=-70 mV), pretreatment with 0.3, 3.0 or 30.0 μmol/L etomidate for 2 min concentration-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P &lt; 0.01, n=7). At the holding potentials of - 30, - 50, and - 70 mV, pretreatment with 30.0 μmol/L etomidate for 2 min voltage-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine (P &lt; 0.01, n=6 for each holding potential). During the 6 min of 30.0 μmol/L etomidate pretreatment, the clamped cells were exposed to 0.4 mmol/L nicotine for 4 times at 0, 2, 4, and 6 min (each exposure time was 2 s), and the nicotinic current amplitude decreased gradually as the number of exposures increased. But at the same concentration, two nicotine exposures (one at the beginning and the other at the end of the 6 min pretreatment) resulted in a significantly lower inhibition rate compared with 4 nicotine exposures (P &lt; 0.01, n=6). etomidate reduces the excitability of the spinal ventral neurons in a concentration-dependent manner and suppresses the function of nAChR in a concentration-, voltage-, and use-dependent manner.

Monod-Wyman-Changeux Allosteric Shift Analysis in Mutant α1β3γ2L GABAA Receptors Indicates Selectivity and Crosstalk among Intersubunit Transmembrane Anesthetic Sites.

Propofol, etomidate, and barbiturate anesthetics are allosteric coagonists at pentameric α1β3γ2 GABAA receptors, modulating channel activation via four biochemically established intersubunit transmembrane pockets. Etomidate selectively occupies the two β +/α - pockets, the barbiturate photolabel R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB) occupies homologous α +/β - and γ +/β - pockets, and propofol occupies all four. Functional studies of mutations at M2-15' or M3-36' loci abutting these pockets provide conflicting results regarding their relative contributions to propofol modulation. We electrophysiologically measured GABA-dependent channel activation in α1β3γ2L or receptors with single M2-15' (α1S270I, β3N265M, and γ2S280W) or M3-36' (α1A291W, β3M286W, and γ2S301W) mutations, in the absence and presence of equipotent clinical range concentrations of etomidate, R-mTFD-MPAB, and propofol. Estimated open probabilities were calculated and analyzed using global two-state Monod-Wyman-Changeux models to derive log(d) parameters proportional to anesthetic-induced channel modulating energies (where d is the allosteric anesthetic shift factor). All mutations reduced the log(d) values for anesthetics occupying both abutting and nonabutting pockets. The Δlog(d) values [log(d, mutant) - log(d, wild type)] for M2-15' mutations abutting an anesthetic's biochemically established binding sites were consistently larger than the Δlog(d) values for nonabutting mutations, although this was not true for the M3-36' mutant Δlog(d) values. The sums of the anesthetic-associated Δlog(d) values for sets of M2-15' or M3-36' mutations were all much larger than the wild-type log(d) values. Mutant Δlog(d) values qualitatively reflect anesthetic site occupancy patterns. However, the lack of Δlog(d) additivity undermines quantitative comparisons of distinct site contributions to anesthetic modulation because the mutations impaired both abutting anesthetic binding effects and positive cooperativity between anesthetic binding sites.

Trapping of Syntaxin1a in Presynaptic Nanoclusters by a Clinically Relevant General Anesthetic

Propofol is the most commonly used general anesthetic in humans. Our understanding of its mechanism of action has focused on its capacity topotentiate inhibitory systems in the brain. However,it is unknown whether other neural mechanismsareinvolved in general anesthesia. Here, we demonstrate that the synaptic release machinery isalso a target. Using single-particle tracking photoactivationlocalization microscopy, we show that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility.Removing the interaction with the t-SNARE partnerSNAP-25 abolishes propofol-induced syntaxin1A confinement, indicating that syntaxin1A and SNAP-25 together form an emergent drug target. Impaired syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated syntaxin1A construct that interacts with SNAP-25. Our results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional syntaxin1A nanoclusters.