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.1
The 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.2
It 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.3
Furthermore, volatile anesthetic–evoked sleep duration has been reported to be prolonged by different exogenously administered cannabinoids.4
-(+)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.5
The anesthetic effect is thought to be mediated primarily through an action on γ-aminobutyric acid receptors.6
In addition, interactions of etomidate with α2
and the nitric oxide metabolism8
have 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 cannabinoid1/2 receptors in vivo in mice. We hypothesized that the activation of the cannabinoid1 receptor increases etomidate-induced sedation, but not activation of the cannabinoid2 receptor.
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 cannabinoid1
receptor agonists with 1,400-fold9
selectivity for binding to the cannabinoid1
receptor in vitro
, respectively. AM 251 and AM 630 (Tocris Bioscience) are cannabinoid1
receptor antagonists with 306-fold11
selectivity for binding to the cannabinoid1
receptors 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 cannabinoid1/2
receptor 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.7
Mice 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 cannabinoid1 receptor agonist (ACEA, 2.5, 3, 4, 5, 6, 8, 10, and 15 mg/kg), cannabinoid2 receptor 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. ED50 values 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 cannabinoid1 receptor antagonist (AM 251, 5 mg/kg) and cannabinoid2 receptor 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, ED50 ACEA, and ED50 etomidate. Furthermore, we evaluated the role of the endocannabinoid system in etomidate-induced sedation. Therefore, we examined the effect of the cannabinoid1/2 receptor 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 analysis13
was 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 ED50
value 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 ED50
values (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 ED50
value of the mixture was calculated. The isobologram was constructed by plotting the ED50
values 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.13
The isobologram consists of an additivity line that connects the ED50
of ACEA on the vertical axis to the ED50
of 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 ED50
of 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
< 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 ED50
values (±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, R2
= 0.7034) and ACEA (R2
Dose- and time-dependent sedative effects of the cannabinoid1
receptor 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 ED50
value coadministered in a fixed 1:1 ratio of the ED50
of 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) ED50
values were determined and converted to absolute dose values for isobolographic analysis. The ED50
value (±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 (R2
= 0.7467). The cannabinoid2
receptor agonist JWH 133 combined with ED50
etomidate did not change time on the Rota-Rod compared with ED50
Accordingly, isobolographic analysis revealed an additive interaction between intraperitoneal ACEA and etomidate. The experimental ED50
value (A) did not significantly differ from the theoretical ED50
value (B) (P
= 0.5787; fig. 3
). Experimentally obtained (Zmix
) and theoretical (Zadd
) additive doses of ED50
, and ED20
are presented in table 1
receptor antagonist AM 251 reversed the sedative effect of single drug administration of ED50
< 0.01), and the sedative component of ACEA when ED50
ACEA was combined with ED50
< 0.01; fig. 4A
). In contrast, the cannabinoid1/2
receptor antagonists AM 251 and AM 630 combined with ED50
etomidate did not significantly differ from ED50
etomidate 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.5
In agreement with previous experimental studies,14,15
intraperitoneal 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 cannabinoid1 receptor agonist ACEA alone reduced time on the Rota-Rod in a dose-dependent manner, indicating increased sedation, whereas the cannabinoid2 receptor 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.16
In our experimental study, the synthetic cannabinoid1
receptor agonist ACEA altered the Rota-Rod performance by decreasing time on the Rota-Rod in a dose-dependent manner, whereas the cannabinoid2
receptor agonist JWH 133 had no effect. Cannabinoid1
receptors are located throughout the central nervous system, including the neocortex, hippocampus, basal ganglia, and brainstem,17
regions that have been associated with sedation.18
In 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 cannabinoid1
Delta-9-Tetrahydrocannabinol enhanced thiopental-induced loss of righting reflex, too.19
In addition, propofol-evoked loss of righting reflex was increased by coadministration of a nonselective cannabinoid1
These 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 cannabinoid1/2
receptor 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 cannabinoid1
receptor 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.21
In addition, as etomidate is not used for repetitive administration and long-term sedation because of its detrimental effect on adrenal function,22
enhanced 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 ED50 levels 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 etomidate3
is 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.23
Moreover, 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 cannabinoid1
receptor antagonist AM 251 that did not significantly change etomidate-induced sedation, sedative properties of etomidate may not depend on activation of cannabinoid1/2
receptors by endocannabinoids per se
, whereas an endogenous cannabinoid tone mediated by cannabinoid1
receptors has been suggested to contribute to sedative–hypnotic effects of propofol.3
To further determine whether the effects of cannabinoids were mediated through certain subtypes of cannabinoid receptors, the cannabinoid1
receptor antagonist AM 251 and cannabinoid2
receptor 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 cannabinoid1
receptor activation, the cannabinoid2
receptor agonist JWH 133 did not affect etomidate-induced Rota-Rod performance. This difference is not astonishing, because cannabinoid2
receptors have been demonstrated to be predominantly expressed in peripheral neuronal tissue and in the immune system,2
and single cannabinoid2
receptor 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 cannabinoid1 receptors 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 cannabinoid1 receptor 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 cannabinoid1 receptor 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 cannabinoid1 receptor, but not of the cannabinoid2 receptor, resulted in increased and prolonged etomidate-evoked sedation based on an additive interaction. Therefore, these data suggest that selective cannabinoid1 receptor agonists could be novel targets for anesthetic drug development.
1. Wilson RI, Nicoll RA: Endocannabinoid signaling in the brain. Science 2002; 296:678–82
2. Cravatt BF, Lichtman AH: The endogenous cannabinoid system and its role in nociceptive behavior. J Neurobiol 2004; 61:149–60
3. Patel S, Wohlfeil ER, Rademacher DJ, Carrier EJ, Perry LJ, Kundu A, Falck JR, Nithipatikom K, Campbell WB, Hillard CJ: The general anesthetic propofol increases brain N-arachidonylethanolamine (anandamide) content and inhibits fatty acid amide hydrolase. Br J Pharmacol 2003; 139:1005–13
4. Schuster J, Ates M, Brune K, Guhring H: The cannabinoids R(-)-7-hydroxy-delta-6-tetra-hydrocannabinol-dimethylheptyl (HU-210), 2-O-arachidonoylglycerylether (HU-310) and arachidonyl-2-chloroethylamide (ACEA) increase isoflurane provoked sleep duration by activation of cannabinoids 1 (CB1)-receptors in mice. Neurosci Lett 2002; 326:196–200
5. Bergen JM, Smith DC: A review of etomidate for rapid sequence intubation in the emergency department. J Emerg Med 1997; 15:221–30
6. Tomlin SL, Jenkins A, Lieb WR, Franks NP: Stereoselective effects of etomidate optical isomers on γ-aminobutyric acid type A receptors and animals. Anesthesiology 1998; 88:708–17
7. Paris A, Philipp M, Tonner PH, Steinfath M, Lohse M, Scholz J, Hein L: Activation of α2B-adrenoceptors mediates the cardiovascular effects of etomidate. Anesthesiology 2003; 99:889–95
8. Tonner PH, Scholz J, Suppe E, Schulte am Esch J: L-nitroargininemethylester (L-NAME), a nitric oxide synthase inhibitor, increases the anesthetic potency of etomidate [in German]. Anasthesiol Intensivmed Notfallmed Schmerzther 1999; 34:136–9
9. Hillard CJ, Manna S, Greenberg MJ, DiCamelli R, Ross RA, Stevenson LA, Murphy V, Pertwee RG, Campbell WB: Synthesis and characterization of potent and selective agonists of the neuronal cannabinoid receptor (CB1). J Pharmacol Exp Ther 1999; 289:1427–33
10. Pertwee RG: Pharmacology of cannabinoid receptor ligands. Curr Med Chem 1999; 6:635–64
11. Gatley SJ, Gifford AN, Volkow ND, Lan R, Makriyannis A: 123I-labeled AM251: A radioiodinated ligand which binds in vivo to mouse brain cannabinoid CB1 receptors. Eur J Pharmacol 1996; 307:331–8
12. Ross RA, Brockie HC, Stevenson LA, Murphy VL, Templeton F, Makriyannis A, Pertwee RG: Agonist-inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656, and AM630. Br J Pharmacol 1999; 126:665–72
13. Tallarida RJ: Drug synergism: Its detection and applications. J Pharmacol Exp Ther 2001; 298:865–72
14. Gomwalk NE, Healing TD: Etomidate: A valuable anaesthetic for mice. Lab Anim 1981; 15:151–2
15. Zeller A, Arras M, Lazaris A, Jurd R, Rudolph U: Distinct molecular targets for the central respiratory and cardiac actions of the general anesthetics etomidate and propofol. FASEB J 2005; 19:1677–9
16. Piomelli D: The molecular logic of endocannabinoid signalling. Nat Rev Neurosci 2003; 4:873–84
17. Herkenham M, Lynn AB, Little MD, Johnson MR, Melvin LS, de Costa BR, Rice KC: Cannabinoid receptor localization in brain. Proc Natl Acad Sci U S A 1990; 87:1932–6
18. Nelson LE, Guo TZ, Lu J, Saper CB, Franks NP, Maze M: The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway. Nat Neurosci 2002; 5:979–84
19. Sofia RD, Knobloch LC: The effect of delta9-tetrahydrocannabinol pretreatment on ketamine thiopental or CT-1341–induced loss of righting reflex in mice. Arch Int Pharmacodyn Ther 1974; 207:270–81
20. Schelling G, Hauer D, Azad SC, Schmoelz M, Chouker A, Schmidt M, Hornuss C, Rippberger M, Briegel J, Thiel M, Vogeser M: Effects of general anesthesia on anandamide blood levels in humans. Anesthesiology 2006; 104:273–7
21. Raffa RB, Clark-Vetri R, Tallarida RJ, Wertheimer AI: Combination strategies for pain management. Expert Opin Pharmacother 2003; 4:1697–708
22. 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
23. Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, Felder CC, Herkenham M, Mackie K, Martin BR, Mechoulam R, Pertwee RG: International Union of Pharmacology: XXVII. Classification of cannabinoid receptors. Pharmacol Rev 2002; 54:161–20
© 2008 American Society of Anesthesiologists, Inc.