PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Anesthesiology. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
PMCID: PMC2739411
NIHMSID: NIHMS125743

Methoxycarbonyl-etomidate: A Novel Rapidly Metabolized and Ultra-Short Acting Etomidate Analogue That Does Not Produce Prolonged Adrenocortical Suppression

Joseph F. Cotten, M.D., Ph.D,* S. Shaukat Husain, D.Phil., Stuart A. Forman, M.D., Ph.D, Keith W. Miller, D.Phil.,§ Elizabeth W. Kelly, B.A.,|| Hieu H. Nguyen, B.A.,|| and Douglas E. Raines, M.D.

Abstract

Background

Etomidate is a rapidly-acting sedative-hypnotic that provides hemodynamic stability. However because it causes prolonged suppression of adrenocortical steroid synthesis, its clinical utility and safety are limited. We 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 γ-aminobutyric acid type A receptor activities of MOC-etomidate and etomidate were compared using electrophysiological techniques in human α1β2γ2L receptors. MOC-etomidate’s hypnotic concentration was determined in tadpoles using a loss of righting reflex assay. Its in-vitro metabolic half-life was measured in human liver S9 fraction and the resulting metabolite provisionally identified 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 γ-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 > 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 minutes 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 following bolus administration.

Introduction

Etomidate is a rapidly acting imidazole-based intravenous (IV) sedative-hypnotic that is used to induce general anesthesia. In common with other IV induction agents, etomidate’s hypnotic action in humans terminates following bolus delivery as it redistributes from the brain to other tissues and ultimately undergoes elimination by the liver with a half-life of several hours.1,2 However, etomidate is distinguished from other induction agents by its ability to maintain hemodynamic stability even in the setting of cardiovascular compromise.36 Consequently, it has emerged as an agent of choice for use in critically ill patients.

Etomidate also potently inhibits 11β-hydroxylase, an enzyme in the biosynthetic pathway leading to adrenocortical steroid synthesis.710 Etomidate’s potency for inhibiting 11β-hydroxylase is ≥100-fold greater than its hypnotic potency.11 Therefore, inhibition of steroid synthesis occurs even with sub-hypnotic doses of etomidate. At the doses necessary to produce hypnosis, etomidate causes adrenocortical suppression that can persist for more than 4 days after discontinuing a prolonged infusion, resulting in significantly increased mortality in critically ill patients.7,8,10 Recent studies and reports of critically ill patients show that adrenocortical suppression following even a single induction dose of etomidate can last for 24 hrs or longer and several suggest that it increases morbidity and/or mortality.1221

Based on our previous studies of etomidate analogues, 22,23 we hypothesized that analogues of etomidate could be designed that are metabolized quickly, providing etomidate’s favorable pharmacological properties (e.g. rapid onset of action, high hypnotic potency, and hemodynamic stability) but also ultra-rapid recovery from both hypnosis and adrenal suppression. In this report, we describe the results of studies characterizing (R)-3-methoxy-3-oxopropyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MOC-etomidate), the first etomidate analogue designed to undergo ultra-rapid metabolism by esterases.

Materials and Methods

Animals

All animal studies were conducted in accordance with rules and regulations of the Subcommittee on Research Animal Care at the Massachusetts General Hospital, Boston, Massachusetts. Early prelimb-bud stage Xenopus laevis tadpoles and adult female Xenopus laevis frogs were purchased from Xenopus One (Ann Arbor, MI) and maintained in our laboratory (tadpoles) or in the Massachusetts General Hospital Center for Comparative Medicine animal care facility (frogs). Adult male Sprague-Dawley rats (300–450 gm) were purchased from Charles River Laboratories (Wilmington, MA) and housed in the Massachusetts General Hospital Center for Comparative Medicine animal care facility.

All blood draws and all IV drug administrations used a lateral tail vein IV catheter (24 gauge, 19 mm) placed under brief (approximately 1–5 min) sevoflurane anesthesia delivered using an agent specific variable bypass vaporizer with continuous gas monitoring. Animals were weighed immediately prior to IV catheter placement and were allowed to fully recover from sevoflurane exposure (at least 15 to 30 min) prior to any study. IV catheters were secured with tape, and the tail was further secured with tape to a 1 inch by 6 inch rigid, acrylic support to prevent catheter dislodgement.

During all studies, rats were placed on a warming stage (Kent Scientific, Torrington, CT). Rectal temperatures were maintained between 36–38 °C (BAT-12, Kent Scientific) as confirmed immediately upon recovery of righting reflexes and/or completion of measurements.

Synthesis of MOC-etomidate

Synthesis of (R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid (1)

A solution of (R)-ethyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate ((R)-etomidate), 281 mg, 1.2 mmol) in methanol (5 ml) and 10 % aqueous NaOH (1.7 ml) was refluxed for 30 min. After cooling, the solution was neutralized with 12 M HCl (0.351 ml). The mixture was dried by rotary evaporation, the residue suspended in methanol-dichloromethane 1:4 v/v, and the sodium chloride removed by filtration. (R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid 1 was obtained by chromatography on a silica gel column, equilibrated with methanol-dichloromethane 1:4 V/V to give 220 mg (85% yield) of the product (figure 1). 1HNMR spectrum: (CD3OD) δ 9.30(d, 1H, imidazole CH), 8.23 (d, 1H, imidazole CH), 7.37 (m, 5H, phenyl), 6.64 (q, 1H, methine), 1.97 (d, 3H, methyl).

Figure 1
(A) Synthesis of methoxycarbonyl-etomidate (MOC-etomidate). (B) Structure of etomidate.

Synthesis of methyl-3-hydroxypropanoate (2)

The compound was prepared essentially as described by Bartlett and Rylander.24 β-Propriolactone (4.36 g, 60.5 mmol) was added drop-wise to a stirred solution of sodium methoxide (121 mg, 2.24 mmol) in anhydrous methanol (15 ml) at −78°C. The mixture was neutralized by adding an equivalent amount of HCl (2.24 ml 1M HCl). The mixture was filtered, rotary evaporated to remove methanol, and the oily residue distilled at reduced pressure to obtain methyl-3-hydroxypropanoate 2 (2.72 g, 43% yield). 1HNMR spectrum: (CDCl3) δ 3.88 (t, 2H, methylene), 3.73 (s, 3H, methyl), 2.59 (d, 2H, methylene).

Synthesis of (R)-3-methoxy-3-oxopropyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MOC-etomidate, 3)

To a mixture of (R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid 1 (220 mg, 1 mmol) and methyl-3-hydroxypropanoate 2 (115 mg, 1.1 mmol) in anhydrous dichloromethane (3.5 ml) was added dicyclohexylcarbodiimide (139 mg, 1.1 mmol) and p-dimethylaminopyridine (134 mg, 1.1 mmol). The solution was stirred at room temperature for 48 h. The precipitate was removed by filtration and the clear solution applied to a silica gel column equilibrated with dichloromethane. Elution with 10% ether in dichloromethane gave the product, which was further purified by preparative thin layer chromatography with hexane-ethyl acetate 1:1 v/v on 1 mm thick silica gel plate. The oily product was treated with HCl in anhydrous ether to obtain white, crystalline (R)-3 -methoxy-3-oxopropyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate.HCl (MOC-etomidate.hydrochloride; 198 mg, 59% yield). This product was pure as judged by high performance liquid chromatography. 1HNMR spectrum: (CDCl3) δ 8.92 (d, 1H, imidazole CH), 7.76 (d, 1H, imidazole CH), 7.36 (m, 5H, phenyl), 6.49 (q, 1H, methine), 4.60 (m, 2H, methylene), 3.73 (s, 3H, methyl), 2.76 (t, 2H, methylene), 2.01 (d, 3H, methyl).

Tadpole Loss of Righting Reflex (LORR)

Groups of 5 early prelimb-bud stage Xenopuslaevis tadpoles were placed in 100ml of oxygenated water buffered with 2.5 mM Tris HCl buffer (pH = 7.4) and containing a concentration of MOC-etomidate ranging from 0.1–128 μM. Tadpoles were manually tipped every 5 min with a flame polished pipette until the response stabilized. Tadpoles were judged to have LORR if they failed to right themselves within 5 seconds after being turned supine. At the end of each study, tadpoles were returned to fresh water to assure reversibility of hypnotic action. The EC50 for LORR was determined from the MOC-etomidate concentration-dependence of LORR using the method of Waud.25

γ-aminobutyric acid type A (GABAA) Receptor Electrophysiology

Adult female Xenopus laevis frogs were anesthetized with 0.2% tricaine (ethyl-m-aminobenzoate) and hypothermia. Ovary lobes were then excised through a small laparotomy incision and placed in OR-2 solution (82 mM NaCl, 2 mM KCl, 2 mM MgCl2, 5 mM HEPES, pH 7.5) containing collagenase 1A (1 mg/ml) for 3 hrs to separate oocytes from connective tissue.

Stage 4 and 5 oocytes were injected with messenger RNA encoding the α1, β2, and γ2L subunits of the human GABAA receptor (~ 40 ng of messenger RNA total at a subunit ratio of 1:1:2). This messenger RNA was transcribed from complementary DNA encoding for GABAA receptor α1, β2, and γ2L subunits using the mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit (Ambion, Austin, Tx). Injected oocytes were incubated in ND-96 buffer solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 0.8 mM MgCl2, 10 mM HEPES, pH 7.5) containing 50 U/ml of penicillin and 50 μg/ml of streptomycin at 17°C for at least 18 hours before electrophysiological experiments.

All electrophysiological recordings were performed using the whole cell two-electrode voltage-clamp technique. Oocytes were placed in a 0.04 ml recording chamber and impaled with capillary glass electrodes filled with 3 M KCl and possessing open tip resistances less than 5 MΩ. Oocytes were then voltage-clamped at −50 mV using a GeneClamp 500B amplifier (Axon Instruments, Union City, CA), and constantly perfused with ND-96 buffer at a rate of 4–6 ml/min. Buffer perfusion was controlled using a six-channel valve controller (Warner Instruments, Hamden, CT) interfaced with a Digidata 1322A data acquisition system (Axon Instruments), and driven by a Dell personal computer (Round Rock, TX). Current responses were recorded using Clampex 9.2 software (Axon Instruments), and processed using a Bessel (8-pole) low pass filter with a cutoff at 50 Hz using Clampfit 9.2 software (Axon Instruments).

For each oocyte, the concentration of γ-aminobutyric acid (GABA) that produces 5–10% of the maximal current response (EC5–10 GABA) was determined by measuring the peak current responses evoked by a range of GABA concentrations (in ND-96 buffer) and comparing them to the maximal peak current response evoked by 1 mM GABA. The effect of each hypnotic (i.e. MOC-etomidate or etomidate) on EC5–10 GABA-evoked currents was then assessed by first perfusing the oocyte with EC5–10 GABA for 90 s and measuring the control peak evoked current. After a 5 min recovery period, the oocyte was perfused with drug for 90 s and then with EC5–10 GABA plus hypnotic for 90 s and the peak evoked current was measured again. After another 5 min recovery period, the control experiment (i.e. no drug) was repeated to assure reversibility. The peak current response in the presence of drug was then normalized to the average peak current response of the two control experiments. Drug-induced potentiation was quantified from the normalized current responses in the presence versus absence of hypnotic.

The effects of hypnotics on the GABA EC50 for peak current activation were determined as described above, except that a wide range of GABA concentrations was used to evoke GABAA receptor current responses. All currents were normalized to that evoked by 1 mM GABA in the same oocyte in the absence of hypnotic. The EC50 for peak current activation was then calculated from the GABA concentration-dependence of the normalized peak current response using a Hill equation.

Metabolic Stability and Metabolite Identification

The metabolic stabilities of MOC-etomidate and etomidate were assessed in-vitro by adding each (20 μM from a 10 mM ethanolic stock solution) to a 1 ml incubation mixture containing 0.3 mg of pooled liver S9 fraction with 1 mM nicotinamide adenine dinucleotide phosphate in phosphate buffer. After the desired time interval (5–40 min) at 37 °C, 100 μL aliquots of the mixture were withdrawn and metabolism stopped by vortexing with 200 μL of acetonitrile. After centrifugation (10,000g for 15 min), the supernatant was removed and evaporated to dryness under vacuum. The drug was then reconstituted in H2O/acetonitrile (98/2%) and its relative levels determined by liquid chromatography (LC)/mass spectrometry (MS) using a Thermo Finnigan TSQ 7000 mass spectrometer (Thermo Finnigan, San Jose, CA) operating in electrospray ionization mode and interfaced with a Michrom BioResources Paradigm MS4 high performance liquid chromatograph (Michrom Bioresources, Auburn, CA). The spectrometer was operating under the following conditions: polarity positive, capillary temperature 375°C, spray voltage 4.5 V, sheath gas (nitrogen) pressure 70 psi, and auxillary gas flow 5 l/m. Argon was used as the collision gas at 2.0 mT. The MS was operated in the selected reaction monitoring mode using a MS/MS transition unique to the parent compound. The LC method used water (A) and acetonitrile (B) as the mobile phase, 0.1% formic acid as a mobile phase modifier, a rapid linear gradient from 2% A to 100% B in 3min at 0.7μl/min, and a 2.1mm X 20mm MAGIC C18-AQ Bullet column from Michrom BioResources. The retention times of MOC-etomidate and its metabolite were 11.5 min and 9.4 min, respectively. In-vitro half-life was calculated by curve fitting a plot of percent parent drug remaining values. Each percent parent drug remaining value was calculated from the ratio of parent compound signal (MS peak area) observed at each time point versus the time zero sample. Although this rapid in vitro approach is not useful for determining absolute concentrations in a given sample, it is satisfactory for estimating and comparing metabolic stability among related analogues. For metabolite identification, an aliquot obtained after 40 min incubation with liver S9 fraction (same as above) was analyzed using high performance liquid chromatography/ion trap mass spectrometry. The same ion source and LC devices were used for the profiling analysis. The LC method was modified for detailed analysis using a longer linear gradient (75% B in 24min at 300μl/min) and longer column (150mm). The ion trap MS (Thermo Finnegan LTQ equipped with an Ion Max Source) was operated in data-dependent MS/MS mode, whereby, full and product ion spectra were obtained on all major components/ions observed throughout the LC run. The spectrometer was operating under the following conditions: polarity positive, capillary temperature 350°C, capillary voltage 40 V, spray voltage 4.00 kV, and tube lens voltage 100 V. Proposed structures were generated by comparing a given metabolite’s product ion spectra with the product ion spectrum of the known parent compound. Additionally, MS/MS spectra were observed on several source generated fragments from each metabolite. These source fragments matched expected fragment ions produced in the corresponding product ion spectra of each parent compound, and were used for elucidative purposes as well.

Rat LORR

Rats were briefly restrained in a 3 inch diameter, 9 inch long acrylic chamber with a tail exit port. The desired dose of hypnotic was injected through a lateral tail vein catheter followed by an approximately 1 ml normal saline flush. Immediately after injection, rats were removed from the restraint device and turned supine. A rat was judged to have LORR if it failed to right itself (onto all four paws) within 5 sec of drug administration. A stop-watch was used to measure the duration of LORR, which was defined as the time from drug injection until the animal spontaneously righted itself. The ED50 for LORR upon bolus administration was determined from the dose-dependence of LORR using the method of Waud.25

Rat Hemodynamics

Femoral arterial catheters, tunneled between the scapulas, were preimplanted by the vendor (Charles River Laboratories). Animals were fully recovered from the placement procedure upon arrival. During housing and between studies, catheter patency was maintained with a heparin (500 U/ml) and hypertonic (25%) dextrose locking solution, which was withdrawn prior to each use and replaced just after.

On the day of study and following weighing and lateral tail vein IV catheter placement, rats were restrained in the acrylic tube with a tail exit port and allowed to acclimate for approximately 15 to 20 min prior to data collection. The signal from the pressure transducer (TruWave, Edwards Lifesciences, Irvine, CA) was amplified using a custom built amplifier (AD620 operational amplifier, Jameco Electronics, Belmont, CA) and digitized (1 kHz) using a USB-6009 data acquisition board (National Instruments, Austin, TX) without additional filtering. All data were acquired and analyzed using LabView Software (version 8.5 for Macintosh OS X; National Instruments).

Data used for heart rate and blood pressure analysis were recorded for 5 min immediately prior to drug administration and for 15 min thereafter. All drugs were administered through the tail vein catheter followed by approximately 1 ml normal saline flush.

Rat Adrenocortical Suppression

Methods for study of rat adrenal function were adapted and optimized from several previously published reports.2628 Immediately following weighing and IV catheter placement, dexamethasone (0.2 mg/kg IV; American Regent, Shirley, NY) was administered to each rat to inhibit endogenous adrenocorticotropic hormone (ACTH) release, to suppress baseline corticosterone production, and to inhibit the variable stress response to restraint and handling. The IV tail vein catheter, used for both drug administration and blood draws, was heparin-locked after each use with 10 U/ml heparin to maintain patency; the heparin locking solution was “wicked” out of the catheter prior to drug administration and blood draws to minimize rat and sample heparinization. All blood draws were approximately 0.3 mls in volume. All drugs administrations were followed by a 1 ml normal saline flush to assure complete drug delivery.

Two hours following dexamethasone treatment, blood was drawn (for baseline measurement of serum corticosterone concentration) and a second dose of dexamethasome (0.2 mg/kg) was administered along with either IV MOC-etomidate, etomidate, propofol, or vehicle (35% propylene glycol v/v in water) as a control. Fifteen minutes later, ACTH1–24 (25 μg/kg; Sigma-Aldrich Chemical Co, St. Louis, MO) was given intravenously to stimulate corticosterone production. Fifteen minutes after ACTH1–24 administration (i.e., 30 min after drug or vehicle administration), a second blood sample was drawn to measure the ACTH1–24-stimulated serum corticosterone concentration. ACTH1–24 was dissolved in 1 mg/ml in deoxygenated water as stock, aliquoted, and frozen (−20 °C); a fresh aliquot was thawed just prior to each use. Rats in all three groups (vehicle, etomidate, and MOC-etomidate) received the same volume of propylene glycol.

Blood samples were allowed to clot at room temperature (10 to 60 min) before centrifugation at 3500g for 5 min. Serum was carefully expressed from any resulting superficial fibrin clot using a clean pipette tip prior to a second centrifugation at 3500g for 5 min. Following the second centrifugation, the resultant straw colored, clot-free serum layer was transferred to a fresh vial for a final, high-speed centrifugation (16000g, for 5 min) to pellet any contaminating red blood cells or particulates. The serum was transferred to a clean vial and promptly frozen (−20 °C) pending corticosterone measurement within 1 to 2 days. Following thawing and heat inactivation of corticosterone binding globulins (65 °C for 20 min), serum baseline and ACTH1–24 stimulated corticosterone concentrations were quantified using an Enzyme-Linked ImmunoSorbent Assay (ELISA) (Diagnostic Systems Laboratories, Webster, TX) and a 96-well plate reader (Molecular Devices, Sunnyvale, CA).

Statistical Analysis

All data are reported as mean +/− SD. Statistical analysis and curve fitting (using linear or nonlinear least squares regression) were done using either Prism v4.0 for Macintosh (GraphPad Software, Inc., LaJolla, CA) or Igor Pro 4.01 (Wavemetrics, Lake Oswego, OR). Metabolic half-life data were linearized by log transformation prior to analysis. Statistical significance indicates a P-value < 0.05 unless otherwise indicated. For multiple comparisons of physiological data derived from rats, we performed a one-way analysis of variance (ANOVA) followed by either a Newman-Keuls or a Bonferroni post test (which relies on an unpaired t-test with a Bonferroni correction).

Results

Loss of Righting Reflexes in Tadpoles by MOC-etomidate

The MOC-etomidate concentration-response relationship for LORR in Xenopus laevis tadpoles (n = 100) is shown in figure 2. The fraction of tadpoles that had LORR increased with MOC-etomidate concentration and at the highest MOC-etomidate concentrations studied (48–128 μM), all tadpoles had LORR. All tadpoles that had LORR recovered their righting reflexes when removed from MOC-etomidate and returned to fresh water. MOC-etomidate’s EC50 for LORR was 8 ± 2 μM (mean ± SD), a value that is 3.5-fold higher than etomidate’s previously reported EC50 for LORR from this laboratory.22

Figure 2
Methoxycarbonyl-etomidate (MOC-etomidate) concentration response curve for loss of righting reflex (LORR) in tadpoles. Each data point represents the results from a single tadpole. The curve is a fit of the data set yielding an EC50 of 8 ± 2 μM. ...

GABAA Receptor Modulation by MOC-etomidate and Etomidate

At the molecular level, there is compelling evidence that etomidate produces hypnosis by modulating the function of GABAA receptors containing β2 or β3 subunits.2931 To test whether MOC-etomidate acts by a similar mechanism, we defined its effects on human α1β2γ2L GABAA receptors. Figure 3A shows representative current traces recorded upon perfusing an oocyte expressing α1β2γ2L GABAA receptors with EC5–10 GABA alone or along with either MOC-etomidate or etomidate at their respective EC50s for producing LORR in tadpoles. Both MOC-etomidate and etomidate significantly potentiated GABA-evoked currents. MOC-etomidate enhanced the peak current amplitudes of GABA-evoked currents by 450 ± 130% (n=6 oocytes). In the same six oocytes, etomidate enhanced peak currents by 660 ± 240%, a value that was not significantly different from that produced by MOC-etomidate (Student’s t-test).

Figure 3
Methoxycarbonyl-etomidate (MOC-etomidate) and etomidate modulation of human α1β2γ2Lγ-aminobutyric acid type A receptor function. (A) Representative traces showing enhancement of currents evoked by EC5–10γ-aminobutyric ...

Although both drugs enhanced currents evoked by low GABA concentrations, they had relatively little effect on currents evoked by high GABA concentrations (figure 3B). This produced a statistically significant leftward shift in the GABA concentration-response curves, reducing the GABA EC50 from 12.7 ± 0.4 μM in the absence of drug to 3.3 ± 0.1 μM and 1.6 ± 0.1 μM in the presence of 8 μM MOC-etomidate and 2 μM etomidate, respectively.

In-Vitro Metabolism of MOC-etomidate and Etomidate

As the first step towards evaluating MOC-etomidate metabolism, we defined its stability in pooled human liver S9 fraction. Figure 4 plots the percentage of unmetabolized drug remaining as a function of incubation time in pooled human liver S9 fraction (+ nicotinamide adenine dinucleotide phosphate) on a semi-logarithmic scale. Even after 40 min, we could detect no reduction in the concentration of etomidate, indicating that its metabolic half-life was much longer than 40 min. In contrast, MOC-etomidate was rapidly metabolized in human liver S9 fraction. The concentration of MOC-etomidate decreased as a first-order process reaching <1% of the original concentration by 40 min. From this data, the metabolic half-life of MOC-etomidate was determined to be approximately 4.4 min. In these studies, buspirone was used as an internal standard to confirm metabolic activity in the liver fraction. Its metabolic half-life was approximately 15.4 min (data not shown).

Figure 4
Metabolic stability of methoxycarbonyl-etomidate (MOC-etomidate) vs. etomidate in pooled human liver S9 fraction. The metabolic half-life of methoxycarbonyl-etomidate was approximately 4.4 min. There was no detectable metabolism of etomidate during the ...

The structure of the metabolite formed after 40 min of incubation in pooled human liver S9 fraction (+ nicotinamide adenine dinucleotide phosphate) was analyzed using high performance liquid chromatography/ion trap mass spectrometry. The ion chromatogram detected the presence of only one major metabolite. It had an m/z of 289.2, which is consistent with the carboxylic acid formed upon hydrolysis of MOC-etomidate’s distal ester moiety. Figure 5A shows the MS/MS spectra of the major metabolite (main spectrum) and its major fragment ion (left inset spectrum). The right inset shows a possible fragmentation pathway supporting the proposed metabolite structure. Based on these results, we conclude that rapid metabolism of MOC-etomidate likely occurs exclusively via the designed pathway shown in figure 5B in which the distal ester moiety of MOC-etomidate is hydrolyzed to form the corresponding carboxylic acid along with methanol as the leaving group.

Figure 5
Methoxycarbonyl-etomidate (MOC-etomidate) metabolite identification. (A) Mass spectrometry spectra of the major metabolite (main spectrum; m/z 289.2) and its major fragment ion (left inset spectrum; m/z 185.2). The metabolite produced a single major fragment ...

Loss of Righting Reflex in Rats by Propofol, Etomidate, and MOC-etomidate

The hypnotic potencies and durations of hypnotic action of MOC-etomidate were compared with those of etomidate and propofol in a rat model. Figure 6A shows the propofol, etomidate, and MOC-etomidate dose-response relationships for LORR in rats. The fraction of rats that had LORR increased with the dose. At the highest doses, all rats had LORR and there was no obvious toxicity. From these data, the ED50s for LORR following bolus administration of etomidate, propofol, and MOC-etomidate were determined to be 1.00 ± 0.03 mg/kg (n=18), 4.1 ± 0.3 mg/kg (n=20), and 5.2 ± 1 mg/kg (n=20), respectively.

Figure 6
Dose-response curves for loss of righting reflex (LORR) and duration of loss of righting reflex in rats. Each data point represents the results from a single rat. (A) Etomidate, propofol, and methoxycarbonyl-etomidate (MOC-etomidate) produced loss of ...

At doses sufficient to produce LORR in rats, all three drugs produced LORR within several seconds of IV bolus administration. The duration of LORR increased approximately linearly with the logarithm of the dose (figure 6B); however, the slope of this relationship, which depends upon the drug’s half-life in the brain32,33, was an order of magnitude lower for MOC-etomidate (2.8 ± 0.4) than for etomidate (27 ± 7) or propofol (22 ± 4). The slopes for etomidate and propofol were not significantly different from one another.

Hemodynamic Actions of Propofol, Etomidate, and MOC-etomidate in Rats

Etomidate is often chosen for induction over other agents in the critically ill patient because it better preserves hemodynamic stability. To determine whether MOC-etomidate similarly preserves hemodynamic stability, we measured and compared the actions of propofol, etomidate, MOC-etomidate, and vehicle (35% v/v propylene glycol in water) on heart rate and blood pressure in rats. To compare these drugs at equi-hypnotic doses, each was administered intravenously at twice its ED50 for LORR (i.e., 2 mg/kg etomidate, 10 mg/kg MOC-etomidate, and 8 mg/kg propofol). The volume of propylene glycol administered was the same for vehicle, etomidate, and MOC-etomidate groups. Following animal acclimatization, data were recorded for 5 min prior to (baseline) and for 15 min after drug/vehicle injection (figure 7). Rats in each group had similar mean heart rates and blood pressure at baseline over the first 5 minutes (391 ± 49 beats/min, 118 ± 9 mmHg). Vehicle caused no significant change in mean blood pressure relative to baseline (5 ± 11 mmHg, n = 3, at 90 sec); data not shown in figure 7 for clarity. However, MOC-etomidate, etomidate, and propofol (n =3 animals each) each caused a significant decrease in mean blood pressure relative to baseline and to each other in this rank order for both maximum magnitude (−11 ± 15 mmHg, −36 ± 11 mmHg, and −51 ± 19 mmHg, respectively) and duration of significant effect (30 sec, 6.5 min, and 7 min, respectively). For all groups, vehicle (36 ± 14 beats/min), MOC-etomidate (24 ± 33 beats/min), etomidate (49 ± 67 beats/min), and propofol (64 ± 56 beats/min), there was a small, transient and variable increase in heart rate shortly after injection.

Figure 7
The effects of methoxycarbonyl-etomidate (MOC-etomidate), propofol, and etomidate on mean blood pressure in rats. Drugs were given at doses equal to twice their respective ED50s for loss of righting reflex. Drug was injected at time 0. Each data point ...

Adrenocortical Suppression Following Administration of Etomidate and MOC-etomidate

To test whether MOC-etomidate produced prolonged adrenocortical suppression, we measured ACTH1–24-stimulated serum corticosterone concentrations in dexamethasone pre-treated rats that had received MOC-etomidate, etomidate, or vehicle. Baseline serum corticosterone concentrations in rats (n=12) after dexamethasone administration averaged 29 ± 39 ng/ml and were not significantly different among the three groups (vehicle, etomidate, and MOC-etomidate). Injection of ACTH1–24 stimulated adrenocortical steroid production as all rats had significantly higher serum corticosterone concentrations fifteen minutes after ACTH1–24 administration. However figure 8 shows that rats that had received etomidate fifteen minutes prior to ACTH1–24 stimulation had significantly lower serum corticosterone concentrations than those that had received either vehicle or an equi-hypnotic dose of MOC-etomidate. In contrast, rats that had received MOC-etomidate had serum corticosterone concentrations that were not different from those that had received only vehicle.

Figure 8
Adrenocorticotropic hormone1–24-stimulated serum corticosterone concentrations in rats 30 min following administration of vehicle, etomidate, or methoxycarbonyl-etomidate (MOC-etomidate). Drugs were given at doses equal to twice their respective ...

Discussion

MOC-etomidate is a well-tolerated etomidate analogue that retains etomidate’s important favorable pharmacological properties including rapid onset of action, high hypnotic potency, and hemodynamic stability. Like etomidate, it potently enhances GABAA receptor function. However in contrast to etomidate, MOC-etomidate is very rapidly metabolized, ultra-short acting, and does not produce prolonged adrenocortical suppression following single IV bolus administration.

MOC-etomidate is a “soft analogue” of etomidate. A soft analogue is a derivative of a parent compound that is specifically designed to undergo rapid and predictable metabolism after exerting its therapeutic actions.34 Commonly used soft analogues include the opioid remifentanil and the β-blocker esmolol. Both of these compounds contain labile carboxylate ester moieties that are rapidly hydrolyzed to carboxylic acids by esterases found in various organs and/or blood. The elimination half-life of these two drugs in humans is 1–2 orders of magnitude shorter than their non-ester containing analogues fentanyl and propranolol.3540 Etomidate also contains a carboxylate ester moiety that is hydrolyzed by liver esterases to a carboxylic acid, but it is a poor substrate for these esterases as reflected by its several hour elimination half-life. Comparison of the structures of remifentanil and esmolol with that of etomidate suggests two reasons for etomidate’s slow rate of ester hydrolysis. First, the ester moiety in etomidate is attached directly to its imidazole ring whereas the labile ester moieties in remifentanil and esmolol are attached to ring structures via a spacer composed of two CH2 groups. This spacer may be critical because it reduces steric hindrance, allowing esterases freer access to the carbonyl group. In support of this, as esmolol’s spacer is decreased in length, its rate of ester hydrolysis decreases.34 Second, the electrons in etomidate’s carbonyl group contribute to a π electron system that extends into the imidazole ring. This reduces the carbonyl carbon’s partial positive charge, making it a poorer substrate for nucleophilic attack by esterases.

Based on this reasoning, we developed the strategy of adding a new ester moiety to etomidate that is both sterically unhindered and electronically isolated from the π electron systems in the imidazole ring to produce an etomidate analogue that would be rapidly metabolized. We expected that this ester moiety, like those in remifentanil and esmolol, would be rapidly hydrolyzed by esterases present in various tissues and/or blood. This was confirmed by our in-vitro metabolic studies of MOC-etomidate that showed that this moiety was rapidly metabolized to a carboxylic acid in S9 liver fraction, a commonly used in vitro drug biotransformation assay. Future work will need to define the specific in-vivo site (e.g., blood, plasma, and/or liver) and to confirm the identity of in-vivo metabolites. In addition, a more complete understanding of MOC-etomidate metabolism may suggest methods by which the duration of action of future related drugs might be further optimized through changes in drug structure (e.g., changes in spacer length or leaving group).

Our studies demonstrated that MOC-etomidate is a hypnotic in two species. It has a potency that is 1/4th–1/5th of etomidate’s potency and likely produces hypnosis via the same receptor mechanism. Our rat studies further demonstrated that MOC-etomidate is an ultra-short acting hypnotic even when given at large multiples of its ED50 for LORR. For example when given at dose that is 4X its ED50 for LORR (20 mg/kg), MOC-etomidate produced LORR in rats for only 55 ± 11 sec. In comparison, propofol and etomidate produced LORR for 9.7 ± 3.5 min and 24 ± 7 min, respectively at approximately equi-hypnotic doses.

Recovery from IV bolus administration of propofol and etomidate is considered to reflect redistribution of drug from the brain to other tissues rather than metabolism. Therefore, the similar slopes in the relationship between the duration of LORR and the logarithm of the drug dose (figure 6B) suggests that propofol and etomidate redistribute from the brain at similar rates. The much faster recovery of righting reflexes and shallower slope of this relationship with MOC-etomidate suggests that ultra-rapid metabolism contributes significantly to the termination of MOC-etomidate’s hypnotic action.

MOC-etomidate produced a correspondingly brief (30 s) reduction in blood pressure, suggesting that MOC-etomidate’s hemodynamic effects also terminate upon metabolism. In addition, we found that the maximum magnitude of this reduction was significantly less following administration of MOC-etomidate than following administration of equi-hypnotic doses of etomidate or propofol. Thus, it is possible to modify etomidate’s chemical structure while retaining its favorable hemodynamic effects.

In common with other hydrophobic imidazole-containing compounds, etomidate suppresses adrenocortical steroid production.9,10,41,42 The primary mechanism underlying this suppression is inhibition of 11β-hydroxylase, a critical enzyme in the biosynthetic pathway leading to adrenocortical synthesis of cortisol, corticosterone, and aldosterone.43 It has been hypothesized that etomidate inhibits 11β-hydroxylase by competing with steroid precursors at the enzyme’s presumably hydrophobic catalytic site.44 Because MOC-etomidate was designed to be rapidly metabolized by esterases to a highly polar carboxylic acid, we expected that MOC-etomidate would not produce prolonged adrenocortical suppression following administration. This expectation was realized as thirty minutes after administration, MOC-etomidate produced no reduction in the ACTH1–24-stimulated serum corticosterone concentration whereas an equi-hypnotic dose of etomidate significantly reduced it. Our results also imply that any effect of MOC-etomidate’s rapidly formed metabolite(s) on corticosterone synthesis is negligible following administration of a single IV dose; however, additional studies will be necessary to determine whether the metabolite could reach sufficiently high levels after repeat dosing or a continuous infusion to produce significant adrenocortical suppression. We also acknowledge the possibility that MOC-etomidate may spare adrenal function, at least in part, by binding to 11β-hydroxylase with lower affinity than etomidate. This would open the possibility that MOC-etomidate or other etomidate analogues might be developed for continuous infusion, regardless of mode of metabolism, without suppressing adrenal function.

In our studies, we utilized rats as our experimental model to assess duration of action. Rats and other small animals usually metabolize drugs significantly faster than humans. For example, the elimination half-life of remifentanil is less than one minute in Sprague Dawley rats as compared to 10 minutes (or longer) in humans.35,4547 Therefore, the duration of hypnosis produced by MOC-etomidate will almost certainly be longer in humans than in rats and probably be similar to those of remifentanil and esmolol (5–10 min), the prototypical esterase-metabolized drugs after which MOC-etomidate was modeled.

Acknowledgments

The authors thank Jeffrey Whitney B.S., Vice President of Novatia LLC, Monmouth Junction, New Jersey for analytical assistance.

Supported by grant P01-GM58448 from the National Institutes of Health, Bethesda, MD; the Foundation for Anesthesia Education and Research, Rochester, Minnesota; and the Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, Massachusetts.

Footnotes

Received from the Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, Massachusetts.

Summary Statement: Methoxycarbonyl-etomidate is an etomidate analogue designed to be highly susceptible to ester metabolism. In rats, it is a potent, ultra-short acting hypnotic that maintains hemodynamic stability and does not cause prolonged suppression of adrenocortical function.

Conflict of Interest Statement: The Massachusetts General Hospital has submitted a patent application for MOC-etomidate and related analogues. Five authors (Raines, Cotten, Forman, Husain, and Miller), their laboratories, the Dept. of Anesthesia and Critical Care at the Massachusetts General Hospital, and the Massachusetts General Hospital could receive royalties relating to the development or sale of these drugs.

References

1. de Ruiter G, Popescu DT, de Boer AG, Smeekens JB, Breimer DD. Pharmacokinetics of etomidate in surgical patients. Arch Int Pharmacodyn Ther. 1981;249:180–8. [PubMed]
2. Hebron BS, Edbrooke DL, Newby DM, Mather SJ. Pharmacokinetics of etomidate associated with prolonged i.v. infusion. Br J Anaesth. 1983;55:281–7. [PubMed]
3. McCollum JS, Dundee JW. Comparison of induction characteristics of four intravenous anaesthetic agents. Anaesthesia. 1986;41:995–1000. [PubMed]
4. Gooding JM, Weng JT, Smith RA, Berninger GT, Kirby RR. Cardiovascular and pulmonary responses following etomidate induction of anesthesia in patients with demonstrated cardiac disease. Anesth Analg. 1979;58:40–1. [PubMed]
5. Gooding JM, Corssen G. Effect of etomidate on the cardiovascular system. Anesth Analg. 1977;56:717–9. [PubMed]
6. Ebert TJ, Muzi M, Berens R, Goff D, Kampine JP. Sympathetic responses to induction of anesthesia in humans with propofol or etomidate. Anesthesiology. 1992;76:725–33. [PubMed]
7. Watt I, Ledingham IM. Mortality amongst multiple trauma patients admitted to an intensive therapy unit. Anaesthesia. 1984;39:973–81. [PubMed]
8. Ledingham IM, Watt I. Influence of sedation on mortality in critically ill multiple trauma patients. Lancet. 1983;1:1270. [PubMed]
9. Wagner RL, White PF. Etomidate inhibits adrenocortical function in surgical patients. Anesthesiology. 1984;61:647–51. [PubMed]
10. Wagner RL, White PF, Kan PB, Rosenthal MH, Feldman D. Inhibition of adrenal steroidogenesis by the anesthetic etomidate. N Engl J Med. 1984;310:1415–21. [PubMed]
11. Zolle IM, Berger ML, Hammerschmidt F, Hahner S, Schirbel A, Peric-Simov B. New selective inhibitors of steroid 11beta-hydroxylation in the adrenal cortex. Synthesis and structure-activity relationship of potent etomidate analogues. J Med Chem. 2008;51:2244–53. [PubMed]
12. Absalom A, Pledger D, Kong A. Adrenocortical function in critically ill patients 24 h after a single dose of etomidate. Anaesthesia. 1999;54:861–7. [PubMed]
13. Malerba G, Romano-Girard F, Cravoisy A, Dousset B, Nace L, Levy B, Bollaert PE. Risk factors of relative adrenocortical deficiency in intensive care patients needing mechanical ventilation. Intensive Care Med. 2005;31:388–92. [PubMed]
14. den Brinker M, Joosten KF, Liem O, de Jong FH, Hop WC, Hazelzet JA, van Dijk M, Hokken-Koelega AC. Adrenal insufficiency in meningococcal sepsis: bioavailable cortisol levels and impact of interleukin-6 levels and intubation with etomidate on adrenal function and mortality. J Clin Endocrinol Metab. 2005;90:5110–7. [PubMed]
15. den Brinker M, Hokken-Koelega AC, Hazelzet JA, de Jong FH, Hop WC, Joosten KF. One single dose of etomidate negatively influences adrenocortical performance for at least 24h in children with meningococcal sepsis. Intensive Care Med. 2007;34:163–8. [PMC free article] [PubMed]
16. Lundy JB, Slane ML, Frizzi JD. Acute adrenal insufficiency after a single dose of etomidate. J Intensive Care Med. 2007;22:111–7. [PubMed]
17. Lipiner-Friedman D, Sprung CL, Laterre PF, Weiss Y, Goodman SV, Vogeser M, Briegel J, Keh D, Singer M, Moreno R, Bellissant E, Annane D. Adrenal function in sepsis: The retrospective Corticus cohort study. Crit Care Med. 2007;35:1012–8. [PubMed]
18. Vinclair M, Broux C, Faure P, Brun J, Genty C, Jacquot C, Chabre O, Payen JF. Duration of adrenal inhibition following a single dose of etomidate in critically ill patients. Intensive Care Med. 2008;34:714–9. [PubMed]
19. Sprung CL, Annane D, Keh D, Moreno R, Singer M, Freivogel K, Weiss YG, Benbenishty J, Kalenka A, Forst H, Laterre PF, Reinhart K, Cuthbertson BH, Payen D, Briegel J. Hydrocortisone therapy for patients with septic shock. N Engl J Med. 2008;358:111–24. [PubMed]
20. Hildreth AN, Mejia VA, Maxwell RA, Smith PW, Dart BW, Barker DE. Adrenal suppression following a single dose of etomidate for rapid sequence induction: A prospective randomized study. J Trauma. 2008;65:573–9. [PubMed]
21. Cotton BA, Guillamondegui OD, Fleming SB, Carpenter RO, Patel SH, Morris JA, Jr, Arbogast PG. Increased risk of adrenal insufficiency following etomidate exposure in critically injured patients. Arch Surg. 2008;143:62–7. [PubMed]
22. Husain SS, Ziebell MR, Ruesch D, Hong F, Arevalo E, Kosterlitz JA, Olsen RW, Forman SA, Cohen JB, Miller KW. 2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate: a derivative of the stereoselective general anesthetic etomidate for photolabeling ligand-gated ion channels. J Med Chem. 2003;46:1257–65. [PubMed]
23. Husain SS, Nirthanan S, Ruesch D, Solt K, Cheng Q, Li GD, Arevalo E, Olsen RW, Raines DE, Forman SA, Cohen JB, Miller KW. Synthesis of trifluoromethylaryl diazirine and benzophenone derivatives of etomidate that are potent general anesthetics and effective photolabels for probing sites on ligand-gated ion channels. J Med Chem. 2006;49:4818–25. [PubMed]
24. Bartlett PD, Rylander PN. β-Propriolactone. XII Mechanisms involved in the reaction of β-propriolactone with acids and bases. J Amer Chem Soc. 1951;73:4273–4.
25. Waud DR. On biological assays involving quantal responses. J Pharmacol Exp Ther. 1972;183:577–607. [PubMed]
26. Fish KJ, Rice SA, Margary J. Contrasting effects of etomidate and propylene glycol upon enflurane metabolism and adrenal steroidogenesis in Fischer 344 rats. Anesthesiology. 1988;68:189–93. [PubMed]
27. Cole MA, Kim PJ, Kalman BA, Spencer RL. Dexamethasone suppression of corticosteroid secretion: Evaluation of the site of action by receptor measures and functional studies. Psychoneuroendocrinology. 2000;25:151–67. [PubMed]
28. Johnson EO, Kamilaris TC, Calogero AE, Gold PW, Chrousos GP. Experimentally-induced hyperthyroidism is associated with activation of the rat hypothalamic-pituitary-adrenal axis. Eur J Endocrinol. 2005;153:177–85. [PubMed]
29. Belelli D, Muntoni AL, Merrywest SD, Gentet LJ, Casula A, Callachan H, Madau P, Gemmell DK, Hamilton NM, Lambert JJ, Sillar KT, Peters JA. The in vitro and in vivo enantioselectivity of etomidate implicates the GABAA receptor in general anaesthesia. Neuropharmacology. 2003;45:57–71. [PubMed]
30. Rusch D, Zhong H, Forman SA. Gating allosterism at a single class of etomidate sites on alpha1beta2gamma2L GABA A receptors accounts for both direct activation and agonist modulation. J Biol Chem. 2004;279:20982–92. [PubMed]
31. Jurd R, Arras M, Lambert S, Drexler B, Siegwart R, Crestani F, Zaugg M, Vogt KE, Ledermann B, Antkowiak B, Rudolph U. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. Faseb J. 2003;17:250–2. [PubMed]
32. Shafer SL. In: Principles of Pharmacokinetics and Pharmacodynamics, Anesthesiology. Longnecker DE, Tinker JH, Morgan GE, editors. St. Louis: Mosby Publishers; 1998. pp. 1159–210.
33. Liao M, Sonner JM, Husain SS, Miller KW, Jurd R, Rudolph U, Eger EI., 2nd R (+) etomidate and the photoactivable R (+) azietomidate have comparable anesthetic activity in wild-type mice and comparably decreased activity in mice with a N265M point mutation in the gamma-aminobutyric acid receptor beta3 subunit. Anesth Analg. 2005;101:131–5. [PubMed]
34. Bodor N, Buchwald P. Soft drug design: General principles and recent applications. Med Res Rev. 2000;20:58–101. [PubMed]
35. Westmoreland CL, Hoke JF, Sebel PS, Hug CC, Jr, Muir KT. Pharmacokinetics of remifentanil (GI87084B) and its major metabolite (GI90291) in patients undergoing elective inpatient surgery. Anesthesiology. 1993;79:893–903. [PubMed]
36. Sum CY, Yacobi A, Kartzinel R, Stampfli H, Davis CS, Lai CM. Kinetics of esmolol, an ultra-short-acting beta blocker, and of its major metabolite. Clin Pharmacol Ther. 1983;34:427–34. [PubMed]
37. Wiest DB, Trippel DL, Gillette PC, Garner SS. Pharmacokinetics of esmolol in children. Clin Pharmacol Ther. 1991;49:618–23. [PubMed]
38. Schleimer R, Benjamini E, Eisele J, Henderson G. Pharmacokinetics of fentanyl as determined by radioimmunoassay. Clin Pharmacol Ther. 1978;23:188–94. [PubMed]
39. Bentley JB, Borel JD, Nenad RE, Jr, Gillespie TJ. Age and fentanyl pharmacokinetics. Anesth Analg. 1982;61:968–71. [PubMed]
40. Williams FM, Leeser JE, Rawlins MD. Pharmacodynamics and pharmacokinetics of single doses of ketanserin and propranolol alone and in combination in healthy volunteers. Br J Clin Pharmacol. 1986;22:301–8. [PubMed]
41. Como JA, Dismukes WE. Oral azole drugs as systemic antifungal therapy. N Engl J Med. 1994;330:263–72. [PubMed]
42. Pont A, Williams PL, Loose DS, Feldman D, Reitz RE, Bochra C, Stevens DA. Ketoconazole blocks adrenal steroid synthesis. Ann Intern Med. 1982;97:370–2. [PubMed]
43. de Jong FH, Mallios C, Jansen C, Scheck PA, Lamberts SW. Etomidate suppresses adrenocortical function by inhibition of 11 beta-hydroxylation. J Clin Endocrinol Metab. 1984;59:1143–7. [PubMed]
44. Roumen L, Sanders MP, Pieterse K, Hilbers PA, Plate R, Custers E, de Gooyer M, Smits JF, Beugels I, Emmen J, Ottenheijm HC, Leysen D, Hermans JJ. Construction of 3D models of the CYP11B family as a tool to predict ligand binding characteristics. J Comput Aided Mol Des. 2007;21:455–71. [PMC free article] [PubMed]
45. Haidar SH, Moreton JE, Liang Z, Hoke JF, Muir KT, Eddington ND. Evaluating a possible pharmacokinetic interaction between remifentanil and esmolol in the rat. J Pharm Sci. 1997;86:1278–82. [PubMed]
46. Egan TD, Minto CF, Hermann DJ, Barr J, Muir KT, Shafer SL. Remifentanil versus alfentanil: Comparative pharmacokinetics and pharmacodynamics in healthy adult male volunteers. Anesthesiology. 1996;84:821–33. [PubMed]
47. Egan TD, Lemmens HJ, Fiset P, Hermann DJ, Muir KT, Stanski DR, Shafer SL. The pharmacokinetics of the new short-acting opioid remifentanil (GI87084B) in healthy adult male volunteers. Anesthesiology. 1993;79:881–92. [PubMed]