Both propofol and volatile anesthetics have been reported to interact with the endocannabinoid system. The purpose of this study was to evaluate the effect of selective agonists for cannabinoid receptor types 1 and 2 on etomidate-induced sedation.
A controlled, blinded, experimental study was performed in 20 mice that received intraperitoneal injections of etomidate, the cannabinoid1 receptor agonist arachidonyl-2-chloroethylamide (ACEA), the cannabinoid2 receptor agonist JWH 133 alone, and both ACEA and JWH 133 combined with etomidate. The cannabinoid1 receptor antagonist AM 251 and the cannabinoid2 receptor antagonist AM 630 were administered 10 min before the delivery of ACEA and JWH 133, respectively. Each drug combination was applied to 6-8 mice of these 20 study animals. Sedation was monitored by a Rota-Rod (Ugo Basile, Comerio, Italy). Isobolographic analysis was used for evaluation of pharmacologic interaction.
Single drug administration of etomidate and ACEA produced dose- and time-dependent decreased time on the Rota-Rod (P < 0.05). No sedative effect was seen after JWH 133. Etomidate-induced sedation was significantly increased and prolonged with ACEA (P < 0.05), but not with JWH 133. Isobolographic analysis revealed an additive interaction between ACEA and etomidate that was antagonized by the cannabinoid1 receptor antagonist AM 251. The cannabinoid1 receptor antagonist had no effect on etomidate alone.
Etomidate-induced sedation was increased and prolonged by activation of the cannabinoid1 receptor, but not of the cannabinoid2 receptor, in mice. However, this interaction was only additive.
ENDOCANNABINOIDS have been demonstrated to play an important role in the physiologic control of sleep, sedation, anxiety, pain processing, and emesis, suggesting a possible role as adjuvants during anesthesia.1The endocannabinoid system includes two identified cannabinoid receptors: type 1, which mainly exists in the central nervous system, and type 2, which is absent from the brain but is enriched in peripheral neuronal and immune tissues.2It has recently been proposed that the anesthetic drug propofol induces an increase in the brain content of the endocannabinoid anandamide and that this may contribute to the sedative effects of propofol.3Furthermore, volatile anesthetic–evoked sleep duration has been reported to be prolonged by different exogenously administered cannabinoids.4
Etomidate (R -(+)ethyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate) is a widely used potent hypnotic drug whose major advantage has been described as hemodynamic stability. This pharmacologic profile renders etomidate particularly suitable for induction of anesthesia in critically ill patients and patients with cardiovascular disease.5The anesthetic effect is thought to be mediated primarily through an action on γ-aminobutyric acid receptors.6In addition, interactions of etomidate with α2adrenoreceptors7and the nitric oxide metabolism8have been suggested.
To elucidate the role of cannabinoid receptors in the anesthetic action of etomidate, we studied the interaction of etomidate with selective agonists and antagonists for cannabinoid1/2receptors in vivo in mice. We hypothesized that the activation of the cannabinoid1receptor increases etomidate-induced sedation, but not activation of the cannabinoid2receptor.
Materials and Methods
This project was approved by the Animal Investigation Committee of the University Schleswig-Holstein, Campus Kiel, Germany, and the animals were managed in accordance with institutional guidelines. This was a controlled, blinded, randomized, experimental study in 20 mice (129S2/SVHsd) of either sex, weighing 25–35 g. Mice were housed 4 animals per cage and maintained on a 12-h light–dark cycle with free access to water and food. All experiments were conducted between 08:00 and 18:00 h.
A total of 20 mice were used in this study. Each following drug combination was applied to 6–8 mice of these 20 study animals. Thus, each animal was repeatedly exposed to different drug combinations. To avoid any interference with drug remnants from the previous regimen, a washout period of at least 20 days was chosen.
Lipid emulsion (Lipofundin® 20%; B. Braun, Melsungen, Germany) was used as the solvent for etomidate (Etomidate®-Lipuro; B. Braun) and as an inactive control. Arachidonyl-2-chloroethylamide (ACEA) and JWH 133 (Tocris Bioscience, Ellisville, MO) are cannabinoid1and cannabinoid2receptor agonists with 1,400-fold9and 200-fold10selectivity for binding to the cannabinoid1and cannabinoid2receptor in vitro , respectively. AM 251 and AM 630 (Tocris Bioscience) are cannabinoid1and cannabinoid2receptor antagonists with 306-fold11and 165-fold12selectivity for binding to the cannabinoid1and cannabinoid2receptors in vitro , respectively. ACEA, JWH 133, AM 251, and AM 630 were dissolved in ethanol, Cremophor (Sigma-Chemie, Deisenhofen, Germany), and saline in a 1:1:18 ratio. Solvents were also used as vehicle control for cannabinoid1/2receptor agonists and antagonists, respectively. All drugs were administered intraperitoneally in a volume of 10 ml/kg body weight, and animals were weighed on the day of the experiment for calculation.
Sedation was determined by placing mice on a rotating wheel (Rota-Rod; Ugo Basile, Comerio, Italy), and measuring the duration of time they remained on the rod as described previously.7Mice were initially trained until they could stay on the Rota-Rod for at least 60 s at a speed of 28 revolutions per minute. Time on the Rota-Rod was recorded 1, 2.5, 5, 7.5, 10, 15, 20, 30, 45, 60, 75, and 90 min after drug administration. The observer was blinded with respect to the drugs applied.
Agonists and Etomidate
For evaluation of the single drug dose response of the sedative action of the cannabinoid1receptor agonist (ACEA, 2.5, 3, 4, 5, 6, 8, 10, and 15 mg/kg), cannabinoid2receptor agonist (JWH 133, 2.5, 5, 10, and 15 mg/kg), and hypnotic drug (etomidate, 0.5, 1, 2, 4, 5, 6, 8, and 10 mg/kg), each agent was intraperitoneally administered. ED50values of ACEA and etomidate were separately calculated representing the effective dose that produced a reduction in time on the Rota-Rod to an average of 30 s in the six to eight mice tested. Injection time of each single drug experiment was defined as t = 0.
Role of Cannabinoid Receptor Subtypes
To further determine whether the effects of ACEA and JWH 133 were mediated through certain subtypes of cannabinoid receptors, the cannabinoid1receptor antagonist (AM 251, 5 mg/kg) and cannabinoid2receptor antagonist (AM 630, 5 mg/kg) were administered 10 min before the delivery of ACEA (ED50) and JWH 133 (5 mg/kg), respectively. Then, we investigated the drug combination of 5 mg/kg AM 251, ED50ACEA, and ED50etomidate. Furthermore, we evaluated the role of the endocannabinoid system in etomidate-induced sedation. Therefore, we examined the effect of the cannabinoid1/2receptor antagonists (AM 251, 5 mg/kg; and AM 630, 5 mg/kg) combined with both lipid emulsion (0.2 mg/kg) and etomidate (ED50) on Rota-Rod performance, respectively.
An isobolographic analysis13was used to determine the nature of pharmacologic interaction between ACEA and etomidate. This method is based on comparisons of doses that are determined to be equieffective. First, each ED50value was determined from the single drug dose–response curves. Next, ACEA was coadministered with etomidate or 0.2 mg/kg lipid emulsion (t = 0) in a fixed 1:1 ratio of their respective agonist ED50values (0.1, 0.25, 0.33, 0.4, 0.5, 0.6, and 1.0). From the dose–response curve of the combined drugs, the ED50value of the mixture was calculated. The isobologram was constructed by plotting the ED50values of the single agents on the x- and y-axes, respectively. The theoretical additive dose combination was calculated.
Statistics were performed using commercially available statistics software (GraphPad Prism version 4.03 for Windows; GraphPad Software, San Diego, CA). A Kolmogorov-Smirnov test was used to test for gaussian distribution. Data were analyzed using two-way repeated-measures analysis of variance factoring for time and drug effects with post hoc Bonferroni correction. Data are expressed as mean ± SEM. The dose–response lines were fitted using least-squares linear regression and ED50. Drug combinations were analyzed for additive interactions using a “fixed ratio design” isobologram whereby combinations of two drugs in known ratios were administered as fractions of their respective ED50, as outlined above.13The isobologram consists of an additivity line that connects the ED50of ACEA on the vertical axis to the ED50of etomidate on the horizontal axis. The theoretical dose required for a purely additive interaction (Zadd= (f)ED50, drug A+ (1 − f)ED50, drug B, where f is the fraction of drug A used) was calculated and compared via an unpaired Student t test to the actual dose (Zmix, determined from the ED50of the combination dose–response curve) required to achieve the same effect experimentally. Statistical significance was considered at a two-sided P value of less than 0.05.
Single drug administration of etomidate and ACEA to conscious mice produced dose- and time-dependent decreased time on the Rota-Rod (figs. 1A and B; P < 0.05). JWH 133 in different dosages from 2.5 to 15 mg/kg did not affect ability to remain on the Rota-Rod. Rota-Rod values at the time points at which the greatest sedative responses were observed for each respective drug were used to plot the agonist log dose–response curves displayed in figure 1C. The mean ED50values (±SEM) of etomidate and ACEA were 4.84 (±0.35) and 6.23 (±0.40) mg/kg, respectively. Comparison of curve fits revealed that a sigmoidal dose–response model with variable slope provided the best fit for etomidate (goodness of fit, R 2= 0.7034) and ACEA (R 2= 0.7779).
Dose- and time-dependent sedative effects of the cannabinoid1receptor agonist ACEA combined with etomidate are shown in figure 2A, and combinations were of equal fractions (0.1, 0.25, 0.33, 0.4, 0.5, 0.6, and 1.0) of each paired drug’s respective ED50value coadministered in a fixed 1:1 ratio of the ED50of ACEA:ED50of etomidate. Rota-Rod values at the time points at which the greatest sedative responses were observed for each respective combination were used to plot the dose– response curve shown in figure 2B. Paired combinations of ACEA and etomidate produced a dose-dependent decrease of time on the Rota-Rod (P < 0.05). Dose fraction (an arbitrary value) ED50values were determined and converted to absolute dose values for isobolographic analysis. The ED50value (±SEM) of the fixed-ratio combination ACEA and etomidate was 0.47 (±0.04). Comparison of curve fits revealed that a sigmoidal dose–response model with variable slope provided the best fit for the combination of ACEA and etomidate (R 2= 0.7467). The cannabinoid2receptor agonist JWH 133 combined with ED50etomidate did not change time on the Rota-Rod compared with ED50etomidate alone.
Accordingly, isobolographic analysis revealed an additive interaction between intraperitoneal ACEA and etomidate. The experimental ED50value (A) did not significantly differ from the theoretical ED50value (B) (P = 0.5787; fig. 3). Experimentally obtained (Zmix) and theoretical (Zadd) additive doses of ED50, ED30, ED25, and ED20are presented in table 1.
The cannabinoid1receptor antagonist AM 251 reversed the sedative effect of single drug administration of ED50ACEA (P < 0.01), and the sedative component of ACEA when ED50ACEA was combined with ED50etomidate (P < 0.01; fig. 4A). In contrast, the cannabinoid1/2receptor antagonists AM 251 and AM 630 combined with ED50etomidate did not significantly differ from ED50etomidate alone. Further, AM 251 and AM 630 combined with 0.2 mg/kg lipid emulsion did not affect baseline Rota-Rod performance (fig. 4B).
Etomidate is widely used for induction of anesthesia, particularly in critically ill patients, because of its beneficial properties, including rapid, predictable onset of action, cardiovascular stability, and short half-life.5In agreement with previous experimental studies,14,15intraperitoneal injection of etomidate reduced time on the Rota-Rod, an index of the sedative action of general anesthetics in mice, in a dose-dependent manner.
Main findings of our experimental study in mice are as follows. First, etomidate and the cannabinoid1receptor agonist ACEA alone reduced time on the Rota-Rod in a dose-dependent manner, indicating increased sedation, whereas the cannabinoid2receptor agonist JWH 133 had no effect, irrespective of the dosage used. Second, etomidate-induced sedation was significantly increased and prolonged with ACEA, but not with JWH 133. However, isobolographic analysis revealed that this interaction is based on simple additivity. Third, the anesthetic action of etomidate is not mediated via cannabinoid receptors.
With regard to natural cannabinoids, their analgesic and sedative properties have historically been used during surgical procedures more than three centuries ago.16In our experimental study, the synthetic cannabinoid1receptor agonist ACEA altered the Rota-Rod performance by decreasing time on the Rota-Rod in a dose-dependent manner, whereas the cannabinoid2receptor agonist JWH 133 had no effect. Cannabinoid1receptors are located throughout the central nervous system, including the neocortex, hippocampus, basal ganglia, and brainstem,17regions that have been associated with sedation.18In this respect, sleep duration of volatile anesthetics such as halothane or isoflurane has been reported to be prolonged when combined with both nonselective and selective cannabinoid1receptor agonists.4Delta-9-Tetrahydrocannabinol enhanced thiopental-induced loss of righting reflex, too.19In addition, propofol-evoked loss of righting reflex was increased by coadministration of a nonselective cannabinoid1receptor agonist.3These authors have further suggested that propofol induces an inhibition of the anandamide-degrading enzyme, the fatty acid amide hydrolase that leads to elevated concentration of anandamide, an endogenous nonselective cannabinoid1/2receptor ligand, which in turn may contribute to the sedative effects of propofol. More recently, even a reduced anandamide concentration has been reported after etomidate administration in patients, suggesting counteracting effects of etomidate and fatty acid amide hydrolase.20
The current study indicates that activation of the cannabinoid1receptor by ACEA increased and prolonged significantly etomidate-induced sedation, suggesting a potentially anesthetic-sparing effect. Furthermore, isobolographic analysis of this study revealed that our results for the combination of ACEA and etomidate represent a simple additive interaction, suggesting that activation of both cannabinoid receptors and γ-aminobutyric acid receptors cause sedation by independent mechanisms or sites of action. However, the fact that lower doses of sedative drugs may be administered in combination to cause effective sedation may have potential clinical benefit. Additive drug combinations may enhance the pharmacodynamic safety margin because the lower clinical dose requirements for each agent will minimize drug-specific adverse effects.21In addition, as etomidate is not used for repetitive administration and long-term sedation because of its detrimental effect on adrenal function,22enhanced and prolonged sedative effects after a single etomidate injection might be advantageous under special circumstances.
With respect to an appropriate effect size for the difference between the actually measured additive dose, Zmix, and the theoretical one, Zadd, we considered a difference of 10% or greater between the observed and expected absolute dose in mg/kg of etomidate or ACEA to be clinically meaningful. At none of the four different fractional ED50levels did we obtain any such difference. Hence, not only did the Student t test give a nonsignificant result, but also the mean data differed by less than the clinically relevant effect size. Therefore, it can reasonably concluded that the interaction is simply additive.
Furthermore, a pharmacokinetic alteration of the endocannabinoid system by etomidate3is unlikely because an inhibition of fatty acid amide hydrolase by etomidate has not yet been demonstrated, and ACEA metabolism is independent of fatty acid amide hydrolase.23Moreover, an interaction between ACEA and the lipid solvent contained in the etomidate emulsion also seems highly improbable, because the combination of both drugs did not affect Rota-Rod performance. In addition, although etomidate is indeed known as an inhibitor of cytochrome P450 3A4, ACEA has, to the best of our knowledge, not been reported as a substrate, inhibitor, or inducer of any CYP isoenzyme including CYP3A4. Hence, CYP-mediated drug–drug interactions are also unlikely. However, it remains speculative whether other interactions between ACEA and etomidate, especially given by the intraperitoneal route, may have influenced the results obtained.
In terms of pretreatment of mice with the cannabinoid1receptor antagonist AM 251 that did not significantly change etomidate-induced sedation, sedative properties of etomidate may not depend on activation of cannabinoid1/2receptors by endocannabinoids per se , whereas an endogenous cannabinoid tone mediated by cannabinoid1receptors has been suggested to contribute to sedative–hypnotic effects of propofol.3To further determine whether the effects of cannabinoids were mediated through certain subtypes of cannabinoid receptors, the cannabinoid1receptor antagonist AM 251 and cannabinoid2receptor antagonist AM 630 were administered 10 min before the delivery of ACEA and JWH 133, respectively. Therefore, AM 251 reversed the sedative component of ACEA when ACEA was administered both alone and in combination with etomidate. In contrast to cannabinoid1receptor activation, the cannabinoid2receptor agonist JWH 133 did not affect etomidate-induced Rota-Rod performance. This difference is not astonishing, because cannabinoid2receptors have been demonstrated to be predominantly expressed in peripheral neuronal tissue and in the immune system,2and single cannabinoid2receptor activation did not induce impairment in motor coordination in our study.
Several limitations to this study should be noted. First, the use of the intraperitoneal route enables hepatic metabolism, and we did not determine serum concentration or brain content of the drugs applied and their active metabolites. Second, we did not perform any ligand-binding studies to elucidate a direct activation of cannabinoid1receptors by etomidate. Third, effects of drugs given throughout the study on systemic hemodynamic and respiratory variables were not evaluated. Further, both the timing and the dose of the cannabinoid1receptor antagonist used may be responsible for the negative effect on etomidate-induced sedation. However, 5 mg/kg AM 251 reversed the sedative effect of single drug administration of ACEA completely. Therefore, the dose range of AM 251 used in our study may provide sufficient antagonistic properties at the cannabinoid1receptor when combined with etomidate. With respect to effect site concentrations, in the clinical context, dosing of anesthetic drugs is usually accomplished irrespective of plasma concentrations. Hence, our results are particularly meaningful because they translate from dose to response as opposed to concentration to response. Finally, data from animals should be extrapolated to humans with caution.
In conclusion, activation of the cannabinoid1receptor, but not of the cannabinoid2receptor, resulted in increased and prolonged etomidate-evoked sedation based on an additive interaction. Therefore, these data suggest that selective cannabinoid1receptor agonists could be novel targets for anesthetic drug development.