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Methoxycarbonyl etomidate is the prototypical soft etomidate analog. Because it has relatively low potency and is extremely rapidly metabolized, large quantities must be infused to maintain hypnosis. Consequently with prolonged infusion, metabolite reaches sufficient concentrations to delay recovery. Dimethyl-methoxycarbonyl metomidate (DMMM) and cyclopropyl-methoxycarbonyl metomidate (CPMM) are methoxycarbonyl etomidate analogs with higher potencies and slower clearance. Because of these properties, we hypothesized that dosing would be lower and electroencephalographic and hypnotic recoveries would be faster - and less context-sensitive - with DMMM or CPMM versus methoxycarbonyl etomidate or etomidate.
Etomidate, DMMM, and CPMM where infused into rats (n=6 per group) for either 5 min or 120 min. After infusion termination, electroencephalographic and hypnotic recovery times were measured. The immobilizing ED50 infusion rates were determined using a tail clamp assay.
Upon terminating 5-minute infusions, electroencephalographic and hypnotic recovery times were not different among hypnotics. However upon terminating 120-minute infusions, recovery times varied significantly with respective values (mean ± SD) 48 ± 13 min and 31 ± 6.5 min (etomidate), 17 ± 7.0 min and 14 ± 3.4 min (DMMM), and 4.5 ± 1.1 min and 4.2 ± 1.6 min (CPMM). The immobilizing ED50 infusion rates were (mean ± SD) 0.19 ± 0.03 mg/kg·min (etomidate), 0.60 ± 0.12 mg/kg·min (DMMM), and 0.89 ± 0.18 mg/kg·min (CPMM).
Electroencephalographic and hypnotic recoveries following prolonged infusions of DMMM and CPMM are faster than those following methoxycarbonyl etomidate or etomidate. In the case of CPMM infusion, recovery times are 4 min and context-insensitive.
Methoxycarbonyl etomidate is the prototypical ultra-rapidly metabolized etomidate analog (figure 1). 1 Similar to remifentanil, methoxycarbonyl etomidate is hydrolyzed by esterases to a carboxylic acid metabolite whose potency is orders of magnitude lower than that of the parent compound. 1-3 However unlike remifentanil, methoxycarbonyl etomidate has a duration of action that is markedly context-sensitive as encephalographic and hypnotic recoveries following prolonged (e.g. 30-minute) continuous infusions are 1-2 orders of magnitude slower than those following brief (e.g. 5-minute) infusions or single boluses. 4,5 This context sensitivity likely reflects the accumulation of methoxycarbonyl etomidate's carboxylic acid metabolite in the central nervous system during prolonged continuous methoxycarbonyl etomidate infusion. 4
In the accompanying manuscript, we described the development of a family of methoxycarbonyl etomidate analogs whose members exhibit widely varying pharmacokinetic and pharmacodynamics properties. 6 Among the fourteen new compounds, dimethylmethoxycarbonyl metomidate (DMMM) and cyclopropyl-methoxycarbonyl metomidate (CPMM) had the highest hypnotic potencies and clearances that were intermediate between those of methoxycarbonyl etomidate and etomidate. Based on these properties, we hypothesized that hypnosis could be maintained for prolonged periods of time with DMMM and CPMM using substantially lower infusion rates than with methoxycarbonyl etomidate and that post-infusion recovery would be relatively rapid and less context-sensitive because less metabolite would be produced.
To test these hypotheses in a rat model, we used a closed-loop infusion system with electroencephalographic feedback to induce and maintain approximately equivalent hypnotic depths for either 5 minutes or 120 minutes using etomidate, DMMM, and CPMM. We defined the infusion rates necessary to maintain an electroencephalographic burst suppression ratio (BSR) of 80% (in a background of 1% isoflurane) and the times required for the electroencephalogram to recover upon infusion termination. We also measured the times required for rats to regain their righting reflexes following 5 minute or 120 minute infusions of etomidate, DMMM, and CPMM (in the absence of isoflurane) as a behavioral measure of recovery. Finally, we determined ED50 infusion rates of etomidate, DMMM, and CPMM infusion that maintain immobility in the rat to define the relative anesthetic potencies of these agents and assess the anesthetic depths achieved during our experiments.
All studies were conducted in accordance with rules and regulations of the Subcommittee on Research Animal Care at the Massachusetts General Hospital, Boston, Massachusetts. Adult male Sprague-Dawley rats (250-500 gm) were purchased from Charles River Laboratories (Wilmington, MA) and housed in the Massachusetts General Hospital Center for Comparative Medicine animal care facility. All intravenous drugs were administered through a femoral venous catheter pre-implanted by the vendor prior to animal delivery to our animal care facility.
Etomidate (2 mg/ml in 35% propylene glycol/water) was from Hospira (Lake Forest, IL). DMMM and CPMM were synthesized (>97% purity) by Aberjona Laboratories (Beverly, MA) using the previously described approach and solubilized at 5 mg/ml in 10% propylene glycol/normal saline. 6 Isoflurane was purchased from Baxter (Deerfield, IL). Bupivicaine and heparin were from APP Pharmaceuticals (Schaumburg, IL).
Electroencephalographic electrodes were placed in each rat's skull under 2-3% isoflurane anesthesia as previously described. 7 The electrodes were connected via a cable to a P511 AC preamplifier (Grass Technologies, West Warwick, RI). The electroencephalographic signal was amplified 5000-fold, filtered (low frequency pass: 0.3 Hz, high frequency pass 0.03 kHz), digitized at 128 Hz using a USB-6009 data acquisition board (National Instruments, Austin, TX), and the BSR measured in real time with LabView Software (version 8.5 for Macintosh OS X; National Instruments) to provide feedback for a closed-loop infusion system and to monitor BSR recovery after infusion termination.
We used the approach described in Rampil and Laster, Vijn and Sneyd, and Cotten et al. to estimate the BSR during each 6 s time epoch. 7-9 Suppression was defined as an interval during which the time-differentiated electroencephalographic signal amplitude stayed within a suppression voltage window for at least 100 ms. This suppression voltage window was defined individually in each rat as previously described. 7 Rats were then equilibrated with 1% inhaled isoflurane delivered through a tight-fitting nose cone for at least 45 minutes until the BSR stabilized prior to study. Closed-loop hypnotic infusion studies were done in a background of 1% inhaled isoflurane.
A KDS Model 200 Series infusion pump (KD Scientific, Holliston, MA) was used for continuous hypnotic infusion. The pump was controlled remotely via its RS 232 serial port by a Macintosh computer using a Keyspan USB-Serial port adapter (Tripp Lite, Chicago, IL). A LabView 8.5 instrument driver using Virtual Instrument Software Architecture protocols provided computer-to-pump communication. For closed-loop infusions, we used the algorithm described by Vijn & Sneyd in which the hypnotic infusion rate is increased or decreased every 6 seconds depending upon whether the BSR is below or above, respectively, the target value. 9 The magnitude of the change in the infusion rate is dependent upon the difference between the current BSR measured in the rat and the BSR target. For all closed-loop experiments, we used a BSR target of 80%. To prevent overdosing, the algorithm was modified with a maximum infusion rate of 2 mg/kg·min for etomidate and 3 mg/kg·min for DMMM and CPMM. For each hypnotic, these maximal rates deliver a cumulative dose equivalent to 4X the ED50 for loss of righting reflexes (as determined in single bolus studies) every minute. 6 We also employed a minimum infusion rate of 0.1 mg/kg·min for all hypnotics. The BSR was measured for 5 minutes prior to beginning closed-loop hypnotic infusion and then until the BSR recovered to the baseline value after infusion termination.
Because the BSR increased from a pre-infusion baseline to the 80% target in an approximately sigmoidal manner when hypnotic infusions were initiated and then decreased to a post-infusion baseline in a sigmoidal manner once hypnotic infusions were terminated, we analyzed the BSR data by fitting this entire infusion time-dependent change in the BSR to a biphasic sigmoidal equation using the analysis software Igor Pro 6.1 (Wavemetrics, Lake Oswego, OR). This biphasic equation was formed by the combination of two monotonic relationships: 10
where t is the time during the infusion experiment, h and h1 approximate the respective midpoints as the BSR rises and falls upon hypnotic infusion initiation and termination, r and r1 are the respective slopes of the rising and falling phases of the BSR, and M, B, and B1 together define the maximal and pre- and post-infusion baseline BSR values. From each fit, we calculated the 90% BSR recovery time, which was defined as the time from infusion termination until the time when the BSR fell 90% towards the post-infusion baseline value.
For each 120-minute closed-loop infusion experiment, we defined the average infusion rate required to maintain an 80% BSR during each 5-minute epoch by recording the infusion rate every six seconds and binning this data into twenty-four 5-minute periods. For each study group (etomidate, DMMM, and CPMM), we then calculated the within group average infusion rate during each 5-minute period and fit the time-dependent change in infusion rate to an exponential equation using Igor Pro 6.1 in the form:
where y is the infusion rate at time t, A + y0 is the initial infusion rate, y0 is the steady-state infusion rate after long infusion times, and invTau is the inverse time constant that defines the change in infusion rate over time.
In individual rats, loss of righting was produced using a continuous infusion of the desired hypnotic (etomidate, DMMM, or CPMM) for either 5 minutes or 120 minutes. To achieve approximately equivalent hypnotic depths, we used the infusion protocols defined by equation 2 using values of A, yo, and invTau determined for each hypnotic in the closed-loop experiments. Approximately 3 minutes after the hypnotic infusion was begun, rats were turned supine. After the infusion was terminated, the recovery time (spontaneous righting onto all four legs) was measured with a stopwatch.
The immobilizing ED50 for each hypnotic was determined as generally described by Zhang et al. 11 We chose an initial infusion rate for each hypnotic that was 30% of the steady-state infusion rate value determined in closed-loop studies (y0 in equation 2). This initial infusion rate was maintained for 40 min and then the tail was clamped with an alligator clip and the clip rotated 180° at 1-2 Hz for 1 min or until the rat made a purposeful response. If the rat responded, the infusion rate was increased by 20% and after another 40 min equilibration period, the tail was clamped as before. This procedure was repeated every 40 min with escalating hypnotic infusion rates until the rat failed to respond. The ED50 infusion rate for immobility was then defined in that rat as the average of the highest rate that produced a response and the subsequent rate that did not.
All data are reported as mean +/- SD. Statistical analyses were done using Prism v5.0 for the Macintosh (GraphPad Software, Inc., LaJolla, CA) or Igor Pro 6.1. Statistical comparisons among the 6 groups of rats receiving different hypnotics for different durations of time were made using a one-way ANOVA followed by a Tukey post-test. P < 0.05 was considered statistically significant.
In closed-loop experiments using rats pre-equilibrated with 1% isoflurane, the BSR reached the target value of 80% 3-4 minutes after initiating the infusion. Once the infusion was terminated, the BSR decreased to a post-infusion value that was similar to the pre-infusion value. Figures 2A and B show the results of typical experiments in which a rat was infused with etomidate for either 5 minutes or 120 minutes, respectively. The respective 90% BSR recovery times for these experiments, calculated from the biphasic sigmoidal fit, were 9.0 min and 50.3 min. Figures 2C and D show the results of analogous experiments in which DMMM was infused. In this case, the calculated 90% BSR recovery times were 8.8 min and 15.7 min for infusion durations of 5 minutes and 120 minutes, respectively. Figures 2E and F show typical results of experiments in which CPMM was infused. The calculated 90% BSR recovery times following the 5-minute and 120-minute CPMM infusions were 6.3 min and 3.3 min, respectively.
Figure 3 summarizes the results of all closed-loop experiments to define the 90% BSR recovery times after infusing etomidate, DMMM, or CPMM. Upon terminating 5-minute infusions, the 90% BSR recovery times did not vary significantly with the identity of the administered hypnotic and averaged 11.1 ± 3.9 min (etomidate), 10.0 ± 3.9 min (DMMM), and 6.2 ± 1.7 min (CPMM). However upon terminating 120-minute infusions, recovery times varied significantly with average values of 48 ± 13 min (etomidate) 17 ± 7.0 min (DMMM), and 4.5 ± 1.1 (CPMM). For etomidate, recovery times after 120-minute infusions were significantly longer than that after 5-minute infusions (p<0.001) whereas for both DMMM and CPMM, recovery times did not vary significantly with infusion duration.
Figure 4 shows the cumulative hypnotic doses received by rats during closed-loop infusions of the three hypnotics. With 5-minute infusions, the cumulative doses administered by the closed-loop system were not significantly different among the three hypnotics and averaged 6.2 ± 2.3 mg/kg (etomidate), 8.6 ± 1.2 mg/kg (DMMM), and 7.7 ± 3.4 mg/kg (CPMM). However with 120-minute infusions, the cumulative doses varied significantly with average values of 36 ± 12 mg/kg (etomidate), 107 ± 18 mg/kg (DMMM), and 143 ± 38 mg/kg (CPMM).
Figure 5 shows the average infusion rate for each 5-minute period during 120-minute closed-loop infusions of etomidate, DMMM, and CPMM. For all hypnotics, the infusion rates determined by the closed-loop system were highest during the first 5-minute epoch before declining to steady-state values of 0.25 ± 0.01 mg/kg·min (etomidate), 0.86 ± 0.01 mg/kg·min (DMMM), and 1.14 ± 0.02 mg/kg·min (CPMM). For each hypnotic, the curve in figure 5 is an exponential fit of the data set. This fit defined the infusion protocol used subsequently to assess the rate of hypnotic recovery (i.e. recovery of righting reflexes) in the absence of isoflurane (see next section).
To assess hypnotic recovery rates using a behavioral endpoint, we infused each hypnotic for either 5 minutes or 120 minutes in the absence of isoflurane. We used the infusion rates that we had previously defined during the closed-loop studies (i.e. the exponential fit of the data shown in figure 5) to induce and maintain approximately equivalent hypnotic depths. We then measured the time required for rats to recover their righting reflexes after the infusion was stopped (figure 6). We found that all rats lost their righting reflexes approximately 3 minutes after initiating the hypnotic infusion. Upon terminating 5-minute infusions, the recovery times did not vary significantly with the identity of the administered hypnotic and averaged 4.0 ± 0.8 min (etomidate), 3.3 ± 0.7 min (DMMM), and 4.2 ± 1.3 min (CPMM). However upon terminating 120-minute infusions, these recovery times varied significantly (p<0.001) with average values of 31 ± 6 min (etomidate), 14 ± 3 min (DMMM), and 4.2 ± 1.6 min (CPMM). For etomidate and DMMM, recovery times after 120-minute infusions were significantly (p<0.001) longer than after 5-minute infusions. However for CPMM, recovery times following 5-minute and 120-minute infusions were identical.
Continuous infusion of all three hypnotics produced immobilization at sufficiently high infusion rates. The average immobilizing ED50 (n=5 rats/hypnotic) were 0.19 ± 0.03 mg/kg·min (etomidate), 0.60 ± 0.12 (DMMM), and 0.89 ± 0.18 (CPMMM).
Together with our previous studies, the current studies show that DMMM and CPMM maintain hypnosis with doses that are 1-2 orders of magnitude lower than methoxycarbonyl etomidate. 4 They also show that following prolonged hypnotic infusion, encephalographic and hypnotic recovery times range from several minutes to several hours with CPMM < DMMM < etomidate < methoxycarbonyl etomidate. In the case of CPMM, recovery times were independent of infusion duration.
Methoxycarbonyl etomidate, DMMM, and CPMM are members of a structurally related family of hypnotics that we have termed “spacer-linked etomidate esters” because each has a metabolically labile ester moiety that is linked to the etomidate backbone via a carbon spacer. 6 Methoxycarbonyl etomidate is the prototypical member of this family and its metabolically labile ester is linked to the etomidate backbone via a simple two-carbon spacer. When designing methoxycarbonyl etomidate, our objective was to minimize any steric hindrance that might inhibit esterase-catalyzed hydrolysis of the labile ester and thus slow recovery. 1In vitro studies demonstrated that methoxycarbonyl etomidate's half-life in rat blood is very short (20 s) and its in vivo duration of hypnotic action in rats following single bolus administration is extremely brief (1-2 min) even when given at several multiples of its hypnotic ED50 dose. 1,12
In subsequent infusion studies, it became apparent that methoxycarbonyl etomidate dosing requirements were high and electroencephalographic and hypnotic recoveries upon infusion termination were remarkably context-sensitive. 4 For example, electroencephalographic recovery following a single methoxycarbonyl etomidate bolus occurred within several minutes whereas recovery following a 30-minute methoxycarbonyl etomidate infusion occurred on the time scale of hours. 4,7 Analysis of metabolite levels in the cerebrospinal fluid revealed that with prolonged methoxycarbonyl etomidate infusion, methoxycarbonyl etomidate's carboxylic acid metabolite reached millimolar concentrations. 4 These are concentrations that produce significant hypnotic effects, which strongly suggested that the high context-sensitivity was the result of accumulated metabolite in the brain. 4,13 Such high metabolite concentrations can logically be attributed to the large quantity of methoxycarbonyl etomidate that must be infused to maintain hypnosis for prolonged periods of time.
DMMM and CPMM are analogs of methoxycarbonyl etomidate that contain aliphatic groups (two methyl groups and a cyclopropyl group, respectively) designed to sterically protect the labile ester from enzymatic attack and increase metabolic stability relative to methoxycarbonyl etomidate. We hypothesized that this increased stability would reduce the DMMM and CPMM infusion rates necessary to maintain hypnosis (and thus the quantity of metabolite generated), resulting in less context-sensitivity than we had observed with methoxycarbonyl etomidate. 4 During the initial characterization of DMMM and CPMM, we unexpectedly found that these agents also had hypnotic potencies that were nearly 8-fold higher than methoxycarbonyl etomidate, a property that would further reduce dosing requirements.
The current studies confirm our hypothesis as hypnotic dosing requirements are significantly lower for DMMM and CPMM than previously shown for methoxycarbonyl etomidate; the total (cumulative) doses of DMMM and CPMM required to maintain an 80% BSR for 120 minutes (107 ± 18 mg/kg and 143 ± 38 mg/kg, respectively) approximated the total dose of methoxycarbonyl etomidate that maintained an 80% BSR for just 2-3 minutes (all in a background of 1% isoflurane). 4 In addition, electroencephalographic recovery upon terminating DMMM or CPMM infusions did not show the marked context-sensitivity previously observed with methoxycarbonyl etomidate. 4 With DMMM and CPMM (and etomidate), we also failed to see the slow phase of electroencephalographic recovery that was apparent after even brief methoxycarbonyl etomidate infusions and which was attributed to the slow clearance of metabolite from the brain. 4 Although we did not measure metabolite concentrations in the present study, our results imply that metabolite failed to reach concentrations sufficient to significantly affect the BSR even after 120 minutes as electroencephalographic recovery occurred over minutes, not hours.
The results of the righting reflexes studies closely paralleled those of the electroencephalographic studies. However in the former studies, the difference in the recovery times following 120-minute versus 5-minute DMMM infusions reached statistical significance.
Our electroencephalographic studies utilized a high (80%) target BSR to more easily quantify recovery upon termination of hypnotic infusions. Although this degree of burst suppression is indicative of a relatively deep level of anesthesia, it was achieved in a background of 1% isoflurane. 14,15 This background allowed us to measure the full time course of burst suppression induction and recovery without motion artifact. It was also expected to reduce the hypnotic infusion rates necessary to achieve such burst suppression towards a more clinically relevant range. This expectation was met as our immobility studies indicate that the steady-state infusion rates used in the electroencephalographic and hypnotic studies correspond to immobilizing ED50 multiples of only 1.3, 1.4, and 1.3 for etomidate, DMMM, and CPMM, respectively.
In conclusion, DMMM and CPMM are sterically hindered analogs of methoxycarbonyl etomidate that maintain equivalent levels of hypnosis with significantly lower infusion rates than methoxycarbonyl etomidate. Encephalographic and hypnotic recoveries following DMMM and CPMM infusion occur without the marked context-sensitivity characteristic of methoxycarbonyl etomidate, which is attributed to metabolite accumulation in the brain. In the case of CPMM, encephalographic and hypnotic recoveries occur in approximately 4 minutes independent of infusion duration.
Electroencephalographic and hypnotic recoveries following prolonged infusions of dimethyl-methoxycarbonyl metomidate and cyclopropyl-methoxycarbonyl metomidate are significantly faster than following prolonged infusions of methoxycarbonyl etomidate and etomidate and, in the case of cyclopropyl-methoxycarbonyl metomidate, context-insensitive.
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Supported by grants R01-GM087316 and R21-DA029253 from the National Institutes of Health, Bethesda, MD and the Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts.
Received from the Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts.
Conflict of Interest Statement: The Massachusetts General Hospital has submitted patent applications for methoxycarbonyl etomidate and related analogues. Three authors (Husain, Cotten, and Raines), and their respective laboratories, departments, and institutions could receive compensation related to the development or sale of the technology presented in this report. Dr. Raines is a consultant for and holds an equity interest in Annovation BioPharma Inc. (Cambridge, MA), which has licensed this technology.
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