Additive Interaction of the Cannabinoid Receptor I Agonist Arachidonyl-2-chloroethylamide with Etomidate in a Sedation Model in Mice

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 cannabinoid^1/2receptor 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.

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.

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 cannabinoid^1/2receptor 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 (Z^add= (f)ED^{50, drug A}+ (1 − f)ED^{50, 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 (Z^mix, 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 (Z^mix) and theoretical (Z^add) 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 cannabinoid^1/2receptor 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 cannabinoid^1/2receptor 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, Z^mix, and the theoretical one, Z^add, 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 cannabinoid^1/2receptors 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.