Sources of Drugs and Chemicals
ACTH, human chorionic gonadotropin hormone (hCG), and Glycine-HCl were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Isoflurane was purchased from Patterson Veterinary (Saint Paul, MN, USA). Heparin and saline were purchased from Hospira (Lake Forest, IL, USA). EDTA-containing vacutainer tubes were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Etomidate was purchased from Bachem (Torrance, CA, USA). Enzyme-linked immunosorbent assay kits for corticosterone were purchased from IDS (Gaithersburg, MD, USA), whereas those for aldosterone, dehydroepiandrosterone (DHEA), and testosterone were purchased from Enzo Life Sciences (Farmingdale, NY, USA), and those for androstenedione and dihydrotestosterone were purchased from MyBioSource (Cambridge, MA, USA). Dimethoxy-etomidate, (R)-ethyl 1-(1-(3-isopropoxyphenyl)ethyl)-1H-imidazole-5-carboxylate (isopropoxy-etomidate), (R)-ethyl 1-(1-(naphthalen-2-yl)ethyl)-1H-imidazole-5-carboxylate (naphthalene-etomidate), and (R)-ethyl 1-(1-(naphthalen-1-yl)ethyl)-1H-imidazole-5-carboxylate (naphthalene(2)-etomidate) were custom synthesized by Aberjona Laboratories (Beverly, MA, USA) and their structures confirmed by nuclear magnetic resonance spectroscopy.
Animals
All studies were conducted with the approval of and in accordance with rules and regulations of the Institutional Animal Care and Use Committee at the Massachusetts General Hospital, Boston, Massachusetts (Protocol # 2013N000130) and the principles outlined in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Adult male Sprague-Dawley rats (300–500 gm) were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed with a 12-12 light-dark cycle with light switched on at 7AM. Rats were caged in our animal care facility, which is maintained at 22.5° C, and allowed free access to food and drinking water until the day of study. Rats were housed socially (i.e. with other rats) whenever possible with access to enrichment materials (wooden block and plastic tube). Each rat was weighed on the day of study. All studies were performed in our laboratory at the Massachusetts General Hospital and carried out at room temperature. During infusions, animal core temperature was monitored and maintained at a target temperature of 36-37° C using a heat lamp. At the end of study, all animals were euthanized by our veterinary staff in the animal care facility by carbon dioxide asphyxiation or in our laboratory under propofol anesthesia using intravenous KCl as recommended under American Veterinary Medical Association guidelines.
Etomidate and Etomidate Analog Formulation, Dosing and Administration
For experiment 1, our goal was to test all etomidate analogs for sedative-hypnotic activity at the highest attainable intravenous bolus dose that was reliably non-lethal. That dose was determined in pilot studies to be 40 mg kg-1. An etomidate intravenous bolus dose of 1 mg kg-1 was chosen for context with the expectation that it would produce loss of righting reflexes in all rats [25]. On the day of study, rats were blocked randomized into 6 rats per treatment group (i.e. etomidate group, dimethoxy-etomidate group, naphthalene-etomidate group, naphthalene(2)-etomidate group). To allow intravenous dosing, a 24-gauage catheter was placed in a tail vein under brief isoflurane (2 – 3%) anesthesia. This method of anesthesia was chosen to allow rapid reversibility. Catheter patency was maintained with a heparin lock flush. Animals were then allowed to recover for at least two hours prior to study. Etomidate and etomidate analogs were formulated for these bolus studies using Captisol (Ligand, San Diego, CA) at a 1:1 molar ratio in distilled water at concentrations of 2 mg/ml and 50 mg/ml, respectively.
For experiment 2, our primary goal was to assess the impact of 2-hr etomidate and etomidate analog infusions on steroidogenesis. Our secondary goal was to determine whether such infusions produce sedation/hypnosis. For these studies, we used the highest attainable dose (0.5 mg kg-1min-1) given the limitations in etomidate and analog aqueous solubility and infusion volume constraints in rats. On the day of study, rats were blocked randomized into 6 rats per treatment group (i.e. vehicle control group, etomidate group, dimethoxy-etomidate group, naphthalene-etomidate group, naphthalene(2)-etomidate group). Two 24-gauge intravenous catheters were placed in each rat (one in each lateral tail vein) under brief isoflurane (2 – 3%) anesthesia to allow the reliable intravenous administration of drugs and test compounds and to draw blood. Catheter patency was maintained with a heparin lock flush. Animals were then allowed to recover for at least two hours prior to study. To avoid potential interference with our steroid assays by Captisol [26, 27], etomidate and etomidate analogs were formulated for these infusion studies on the study day in 10 mM glycine-HCl buffer (pH 2 – 2.2) at a concentration of 3 mg/ml.
Loss of Righting Reflex by Etomidate and Etomidate Analogs
Immediately after receiving an etomidate or etomidate analog bolus (Experiment 1) or at the end of a 2-hr etomidate or etomidate analog infusion (Experiment 2), each rat was assessed for sedation/hypnosis using a loss of righting reflex assay [28]. In this assay, a rat is determined to have lost its righting reflexes if (after being turned supine) it fails to turn itself back onto all four paws within 5 seconds. Each rat that has loss of righting reflexes is then monitored and the duration of loss of righting reflexes defined as the time when etomidate or etomidate analog administration was complete until the animal returned onto all four paws.
Steroidogenesis inhibition by Etomidate and Etomidate Analogs
In Experiment 2, the in vivo steroidogenic actions of etomidate and etomidate analogs were assessed in male rats (six per group) using a hormone stimulation test (Fig 3). For the two days immediately prior to study, rats were restrained in a broome style rodent restrainer from 10AM until 4PM to accommodate them to this environment and reduce stress-induced fluctuations in steroid biosynthesis. On the study day, each rat was placed in a restrainer at 10AM and intravenous catheters were placed as described above. At 1PM, a 2-hour infusion of 0.5mg/kg/min etomidate, etomidate analog, or an equivalent volume of 10mM Glycine-HCl, pH 2.2 vehicle was started. At the timepoints indicated in figure 3, adrenocortical and androgenic steroid biosynthesis was stimulated in rats by administering intravenous ACTH (25μg/kg) and hCG (10U/kg), respectively. At the end of the infusion, a blood sample was drawn and rats were assessed for loss of righting reflexes as described in the following section. Stimulated plasma steroid concentrations were determined using enzyme-linked immunosorbent assays and a 96-well plate reader (Molecular Devices, Sunnyvale, CA).
Statistical Analysis
All individual data points are expressed as the mean ± the S.E.M. of six independent measurements. Group size was chosen based on previous experience [23]. Statistical analyses to assess differences in plasma steroid concentrations between etomidate or etomidate analog groups versus the vehicle control groups were carried out with a Mann-Whitney U test using Prism 5 for Mac OS X. A Bonferroni correction was used to account for multiple (i.e. five) comparisons. Thus, statistical significance was assumed for a p < 0.01