Changes of blood endocannabinoids during anaesthesia: a special case for fatty acid amide hydrolase inhibition by propofol?

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1 British Journal of Clinical Pharmacology DOI: /j x Changes of blood endocannabinoids during anaesthesia: a special case for fatty acid amide hydrolase inhibition by propofol? Carina Jarzimski, 1 Matthias Karst, 2 Alexander A. Zoerner, 1 Christin Rakers, 1 Marcus May, 1 Maria T. Suchy, 1 Dimitrios Tsikas, 1 Joachim K. Krauss, 3 Dirk Scheinichen, 2 Jens Jordan 1 & Stefan Engeli 1 Correspondence Dr Stefan Engeli, Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, Hannover, Germany. Tel.: Fax: engeli.stefan@mh-hannover.de Keywords anaesthesia, anandamide, endocannabinoids, fatty acid amide hydrolase, propofol Received 25 August 2011 Accepted 25 December 2011 Accepted Article Published Online 13 January Institute of Clinical Pharmacology, 2 Department of Anaesthesiology and 3 Department of Neurosurgery, Hannover Medical School, Hannover, Germany WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Available data from animal studies suggest that the narcotic drug propofol interacts with the endocannabinoid system. Inhibition of enzymatic degradation of anandamide could explain some of the characteristics of propofol. Direct measurements have not been reported yet in humans. WHAT THIS STUDY ADDS Propofol does not change the time course of anandamide plasma concentrations during anaesthesia. Furthermore, propofol does not inhibit fatty acid amide hydrolase activity ex vivo or in vitro. Thus, specific characteristics of the narcotic drug propofol cannot be explained by peripheral inhibition of anandamide degradation in humans. AIMS The aim of our study was to describe the time course of endocannabinoids during different anaesthesia protocols in more detail, and to challenge the hypothesis that propofol acts as a FAAH inhibitor. METHODS Endocannabinoids were measured during the first hour of anaesthesia in 14 women and 14 men undergoing general anaesthesia with propofol and in 14 women and 14 men receiving thiopental/sevoflurane. We also incubated whole human blood samples ex vivo with propofol and the known FAAH inhibitor oloxa and determined FAAH enzyme kinetics. RESULTS Plasma anandamide decreased similarly with propofol and thiopental/sevoflurane anaesthesia, and reached a nadir after 10 min. Areas under the curve for anandamide (mean and 95% CI) were 53.3 (47.4, 59.2) nmol l min with propofol and 48.5 (43.1, 53.8) nmol l min with thiopental/sevoflurane (P = NS). Anandamide and propofol plasma concentrations were not correlated at any time point. Ex vivo FAAH activity was not inhibited by propofol. Enzyme kinetics (mean SD) of recombinant human FAAH were K m = mmol l -1 and V max = nmol mg 1 min 1 FAAH without, and K m = mmol l -1 and V max = nmol mg 1 min -1 FAAH with 50 mmol l -1 propofol (P = NS for both). CONCLUSIONS Our findings challenge the idea that propofol anaesthesia and also propofol addiction are directly mediated by FAAH inhibition, but we cannot exclude other indirect actions on cannabinoid receptors. 54 / Br J Clin Pharmacol / 74:1 / The Authors British Journal of Clinical Pharmacology 2012 The British Pharmacological Society

2 Propofol anaesthesia and endocannabinoids Introduction Endocannabinoids are arachidonic acid derivatives activating cannabinoid receptors (CB1 and CB2). Anandamide (arachidonoyl ethanolamide) and 2-arachidonoyl glycerol (2-AG) are among the best characterized endocannabinoids [1]. Peripheral CB1 receptors may modulate pain sensation and transmission [2, 3]. Ongoing studies are evaluating endocannabinoid-modulating drugs for the treatment of pain syndromes [4, 5]. Moreover, endocannabinoids may be affected by anaesthesia. General anaesthesia with a combination of midazolam, sufentanil, pancuronium and isoflurane rapidly decreased anandamide in plasma early after induction. During cardiopulmonary bypass surgery, 2-AG plasma concentrations initially remained stable and strongly increased later on [6]. Circulating anandamide showed a differential response to general anaesthesia with either etomidate/sevoflurane or propofol. Anandamide decreased during the first 10 min in the etomidate/sevoflurane group, but remained unchanged during total intravenous anaesthesia (TIVA) with propofol [7]. The authors suggested that propofol inhibited anandamide degradation through fatty acid amide hydrolase (FAAH), thereby maintaining anandamide blood concentrations during propofol-tiva. Indeed, in mice, propofol increased brain anandamide concentrations and attenuated forebrain FAAH activity [8]. Another substrate of FAAH is the sleep inducing fatty acid amide, oleamide [9]. Propofol may also act on oleamide during anaesthesia. Furthermore, FAAH deficiency in animals [10] and a polymorphism in the human FAAH gene leading to decreased FAAH activity predispose to drug abuse [11]. Thus, FAAH inhibition could contribute to the recently discussed addiction potential of propofol [12] as well as to anaesthetic and anti-emetic actions of the drug. We hypothesized that during propofol-tiva, plasma anandamide is correlated with propofol blood concentrations and that this correlation is explained by FAAH inhibition by propofol. Methods Study design Patients aged 18 years undergoing elective spinal surgery in the Department of Neurosurgery at Hannover Medical School were eligible for participation. Exclusion criteria were any oncological diseases, immunosuppression due to drugs or chronic diseases, drug and alcohol abuse, heavy smoking (>20 cigarettes day -1 ) and a history of psychiatric disease. Written informed consent was obtained from all patients independently of the preoperative information about surgery and anaesthesia. Blood sampling in these patients was approved by the Ethics Committee of Hannover Medical School and all procedures were in accordance with the Declaration of Helsinki (2008). The study was not randomized, not interventional by design, not blinded for blood sampling, but blinded for laboratory and data analysis. Choice of the anaesthesia protocol was at the discretion of the anaesthesiologist and was not influenced by the study protocol. After losing consciousness, patients receiving propofol- TIVA had a second venous catheter inserted on the contralateral arm to the propofol infusion line for blood sampling. Informed consent included this information. Blood sampling All patients received standard doses of midazolam, atracurium and fentanyl/remifentanil. Anaesthesia induction was achieved with either propofol or with thiopental plus sevoflurane. Blood samples were collected in EDTAcontaining tubes before injection of propofol or thiopental (0 min) and 10, 30 and 60 min after induction.in about 50% of the patients in each group, surgery had already begun when the 60 min sample was obtained. To avoid contamination of the blood samples with endocannabinoids from blood cells [13], tubes were immediately cooled and centrifuged at 4 C (10 min, 3500 g). Plasma was then quickly separated from blood cells and kept on ice until storage at -80 C. Propofol and endocannabinoid analysis Propofol plasma concentrations were determined by HPLC using thymol (Sigma-Aldrich, Munich, Germany) as internal standard for quantification (Gynkotec RF1002 fluorescence detector, Germering, Germany, and Dionex Ultimate 3000 HPLC pump, Idstein, Germany). Chromatographic separation was achieved on a Sphinx C18 Gravity column (2.1 mm 250 mm, 3.5 mm particle size, Macherey-Nagel, Düren, Germany) by isocratic elution with water : methanol (65 : 35, v/v). Proteins were removed before injection by adding ice-cold methanol and centrifugation (4 C, 4500 g, 5 min). The limit of detection for propofol (LOD) was <2ng (on column), the coefficient of variation (CV) was <10% and the linearity of the method was r 2 = Anandamide was determined by GC-MS/MS as described in detail previously using d 4-anandamide as internal standard [14].The LOD for anandamide was pg (on column), the CV was <10% and the linearity of the method was r 2 = AG was determined by LC-MS/MS using the internal standard d 5-2-AG as previously described [15].The LOD for 2-AG was 6.4 pg (on column), the CV was <15% and the linearity of the method was r 2 = All deuterated internal standards for endocannabinoid and FAAH analysis were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Determination of FAAH activity We determined ex vivo FAAH activity in 1 ml whole blood aliquots of six healthy volunteers by adding 1 mmol l -1 d 4-anandamide and measuring generated d 4-ethanolamine. At 30 min, samples were centrifuged (4 C, 1000 g, 5 min) and d 4-ethanolamine concentrations in Br J Clin Pharmacol / 74:1 / 55

3 C. Jarzimski et al. plasma were measured by LC-MS/MS according to a recently published method [16]. In brief, 13 C 2-ethanolamine (1 mmol l -1 ) was spiked into the plasma samples as internal standard. Samples were treated with acetonitrile (1:1, v/v) to precipitate proteins and 1 ml aliquots of the supernatant were injected into the Waters Acquity/XEVO TQ MS UPLC- MS/MS system (Waters, Milford, MA, USA). Chromatographic separation was achieved with a ZIC-Hilic column (2.1 mm 100 mm, 3.5 mm particle size, Merck, Darmstadt, Germany), and isocratic elution with aqueous ammonium acetate (25 mmol l -1, ph 3.0)/acetonitrile (1:1, v/v) at a flow rate of 0.2 ml min -1. The following mass transitions were monitored for quantitative analysis: m/z 66 m/z 48 for d 4-ethanolamine, 13 m/z 64 m/z 46 for C 2- ethanolamine. The LOD for 13 C 2-ethanolamine was 128 fg and for d 4-ethanolamine <1 pg (on column), the CV was <2% and the linearity of the method was r 2 = The effects of drugs on d 4-ethanolamine production were studied by incubating whole blood samples for 30 min with propofol (2 mg l -1 ) or thiopental (25 mg l -1 ) at anaesthetic concentrations. The highly specific FAAH inhibitor oloxa (500 nmol l -1, Cayman Chemicals, Ann Arbor, MI, USA) served as positive control [17]. FAAH activity was also studied by incubating 0.1 units ml -1 human recombinant FAAH (Cayman Chemicals, Ann Arbor, MI, USA) in 10 mmol l -1 phosphate buffered saline with 0.25, 0.5, 1, 5 and 10 mmol l -1 d 4-anandamide for 5 min in the absence and in the presence of 50 mmol l -1 propofol. The reaction was stopped with 1.2 mol l -1 hydrochloric acid and the concentration of generated d 4-ethanolamine was measured by LC-MS/MS as described above. Statistical analysis SPSS 17.0 (SPSS Inc. Chicago, IL, USA) was used for statistical analysis. Clinical data are given as mean and 95% confidence interval (95% CI) unless otherwise stated and in vitro data as mean SD. Endocannabinoids during anaesthesia were analyzed by two-way ANOVA for repeated measures over time and type of anaesthesia as betweengroup factor. Areas under the curve were calculated over 60 min by GraphPad Prism 5.0 (GraphPad Software, Inc. La Jolla, CA, USA) and compared by unpaired t-test. Correlations between endocannabinoids and propofol concentrations were determined by Pearson s coefficient of correlation. Ex vivo and in vitro FAAH assays were analyzed by one-way ANOVA and Bonferroni s post-hoc test (whole blood) or unpaired t-test (recombinant FAAH). A P value <0.05 was considered statistically significant. Results Patient characteristics Fifty-six patients undergoing general anaesthesia were analyzed in total. Twenty-eight patients underwent propofol-tiva and 28 patients received a thiopental/ sevoflurane combination. Each anaesthesia protocol group consisted of 14 women and 14 men. Both groups were matched for age [propofol group: 55 (50 60) years; thiopental/sevoflurane group: 54 (49, 59) years], and body mass index [propofol group: 27.4 (25.7, 29.0) kg m -2 ; thiopental/sevoflurane group: 28.2 (26.4, 30.0) kg m -2 ].Two diabetics and seven smokers (propofol) vs. three diabetics and six smokers (thiopental/sevoflurane) were included. C-reactive protein was similar in both groups [propofol group: 3.0 (1.8, 4.3) mg l -1 ; thiopental/sevoflurane group: 2.9 (1.8, 3.9) mg l -1 ]. Endocannabinoids and propofol during anaesthesia In both groups, plasma anandamide concentrations rapidly decreased after anaesthesia induction reaching a minimum concentration at 10 min. At 30 min, anandamide concentrations were still significantly decreased compared with baseline and then recovered towards baseline concentrations at 60 min. The responses at each time point were similar with propofol and thiopental/sevoflurane (Figure 1). Areas under the curve for anandamide were 53.3 (47.4, 59.2) nmol l min with propofol and 48.5 (43.1, 53.8) nmol l min with thiopental/sevoflurane (P = NS). Plasma 2-AG concentrations were stable in the propofol group over 1 h whereas a small, but significant decrease occurred in the thiopental/sevoflurane group at 10 min (Figure 1). Areas under the curve for 2-AG were (125.1, 179.3) nmol l min with propofol and (98.6, 167.8) nmol l min with thiopental/sevoflurane (P = NS). According to our hypothesis, we analyzed the correlation between plasma concentrations of both endocannabinoids and propofol at 10, 30 and 60 min separately and found no correlation at all. Figure 2 illustrates pooled data of the three time points. Propofol plasma concentrations [2.7 (2.4, 3.1) mg l -1 ] were within narcotic concentration ranges reported by other authors [17]. Ex vivo and in vitro FAAH activity assays We incubated whole blood samples ex vivo with narcotic propofol or thiopental concentrations, or with the potent FAAH inhibitor oleoyl oxazolopyridine (oloxa) [17]. After adding deuterated d 4-anandamide, we measured the amount of produced d 4-ethanolamine after 30 min. d 4-ethanolamine formation with propofol or thiopental was identical to control samples without additives. In contrast, oloxa significantly inhibited d 4-ethanolamine formation in whole human blood samples (Figure 3, P < ). Finally, in vitro studies with recombinant human FAAH and d 4-anandamide as substrate revealed identical enzyme kinetics with and without propofol: K M = mmol l -1, V max = nmol mg -1 min 1 FAAH in control experiments; K M = mmol l -1, V max = nmol mg -1 min 1 FAAH with 50 mmol l -1 propofol. K M and V max with and without propofol were not significantly different as determined by unpaired t-test. 56 / 74:1 / Br J Clin Pharmacol

4 Propofol anaesthesia and endocannabinoids Plasma anandamidt (nm) Baseline Time (min) propofol AUC = 53.3 (47.4, 59.2) nmol l 1 60min thio/sevo AUC = 48.5 (43.1, 53.8) nmol l 1 60min Plasma Z-AG (nm) propofol AUC = (125.1,179.3) nmol l 1 60min thio/sevo AUC = (98.6, 167.8) nmol l 1 60min Baseline Time (min) Figure 1 Anandamide and 2-AG plasma concentrations during the first hour of general anaesthesia with either propofol ( ) or thiopental/sevoflurane ( ) (n = 28 in each group). Data are shown as mean SEM and were analyzed by two-way ANOVA for repeated measures over time and type of anaesthesia as between-group factor. Anandamide: P < 0.05 vs. baseline for both anaesthesia groups. 2-AG: P < 0.05 for interaction between propofol and thiopental/ sevoflurane at 10 min. Inserts: calculated anandamide and 2-AG areas under the curve (AUC, mean and 95% CI) over 60 min. AUCs were compared by unpaired t-test (NS for both anaesthesia groups) Plasma anandamide (nm) Plasma 2-AG (nm) Plasma propofol (mg ml -1 ) Plasma propofol (mg ml -1 ) Figure 2 Correlation between anandamide (Pearson sr = 0.07), or 2-AG (Pearson s r = 0.20) plasma concentrations and propofol plasma concentrations in patients from the propofol group were both not statistically significant. Data from 10, 30 and 60 min were pooled (n = 79 measurements for each endocannabinoid) Discussion We hypothesized that the narcotic drug propofol acts as an inhibitor of FAAH, thereby stabilizing anandamide plasma concentrations during anaesthesia, as proposed by previous studies in mice and patients [7, 8]. Our findings challenge this idea. First, anandamide plasma concentrations during the first hour of general anaesthesia were similar in patients on either propofol or thiopental/sevoflurane. Second, propofol plasma concentrations did not correlate with anandamide plasma concentrations. Third, ex vivo d 4-ethanolamine formation from the added precursor d 4-anandamide, a reaction catalyzed by the FAAH enzyme, was not inhibited by propofol, but was by the well characterized FAAH inhibitor oloxa. Fourth, in vitro activity of human recombinant FAAH was identical whether propofol was present or not. FAAH inhibition by propofol was previously described in isolated cell membranes from mouse brain [8]. The reported IC 50 for propofol in that study was 52 mmol l -1 (= 9.2 mg l -1 ), a concentration in the range of narcotic concentrations in humans of 2 6 mg l -1 that we (Figure 2) and others have measured [18]. Based on these and our findings, we cannot exclude species or organ-specific responses of FAAH activity to the presence of propofol.the use of thiopental as a comparator in our study is justified by our finding that thiopental does not interfere with ethanolamine formation. Also, a very high IC 50 for thiopental of 2 mm toward FAAH inhibition was reported by others [8]. The rapid anandamide decline during the first 10 min after anaesthesia induction could be explained by stress mediators acting on anandamide [19], fear responses accompanying anaesthesia induction [20] or sleep induction itself [21]. Stable 2-AG concentrations during anaesthesia induction and an acute anandamide reduction have been previously described, albeit in smaller patient numbers [6, 7]. Schelling et al. suggested that propofol may have inhibited FAAH activity based on their observation that anandamide did not change with propofol-tiva whereas anandamide Br J Clin Pharmacol / 74:1 / 57

5 C. Jarzimski et al. d4-ethanolamine (mm) Figure 3 Control Propofol Thiopental Oloxa d 4-ethanolamine formation from 1 mmol l -1 of the precursor d 4-anandamide was measured in 1 ml human whole blood aliquots after 30 min incubation. Control sample contained no further additives; other samples contained 2 mg l -1 propofol, 25 mg l -1 thiopental or 500 nm of the FAAH inhibitor oloxa. Data are mean SEM. Group comparison by one-way ANOVA and Bonferroni s post hoc test (n = 6 donors in each group). P < 0.05 vs. control decreased with another anaesthetic regimen. Yet, anandamide plasma concentrations were not related to the administered cumulative propofol dose in this study. Furthermore, neither propofol plasma concentrations nor FAAH activity were actually measured by these authors [7]. When we measured propofol plasma concentrations, they were not correlated with plasma anandamide. However, we cannot exclude that propofol affected anandamide turnover in the brain or interacted indirectly with brain cannabinoid receptors. The latter was suggested in an animal model of inflammatory pain [22]. In the light of our findings, the recently reported effects of propofol on memory formation in rats must be interpreted more carefully, because these authors also referred to FAAH inhibition by propofol as the most likely mechanism [23]. Based on a previous study [7], we assumed that the difference between anandamide concentrations in the propofol and the thiopental/sevoflurane group at 10 min should have been at least 50%, starting at identical baseline values. Given the reduction of anandamide by nmol l -1 between baseline and 10 min, a group size of six patients would have been sufficient to detect this expected difference in our study with a=0.05 and a statistical power of 80% using a two-sided t-test. Taking also into account the reliability of our validated anandamide, 2-AG, and FAAH assays we are confident that our methodologies and the actual sample size were sufficient to detect clinically relevant influences of propofol on circulating endocannabinoids and FAAH activity. These propofol effects were not verified in our study. A potential limitation of our study is that we obtained baseline blood samples within a few minutes before narcotic drugs were injected. Thus, baseline anandamide and 2-AG concentrations may have already been affected by psychological stress or premedication with midazolam, which was given to all patients. Yet, baseline anandamide concentrations of 1.1 (0.9, 1.3) nmol l -1 in the propofol group and 1.0 (0.8, 1.1) nmol l -1 in the thiopental/ sevoflurane group in the present study are close to previously determined reference values in plasma samples from resting healthy subjects on no medications [14]. Propofol is often used in patients with a history of post-operative nausea and vomiting. Decreased circulating anandamide concentrations were associated with nausea and vomiting in patients with motion sickness [24]. However, anandamide concentrations at baseline were slightly increased in patients receiving propofol in our study. Thus, we can exclude a premedication and selection bias influencing our endocannabinoid measurements. In summary, we did not detect any effects of propofol on FAAH activity or anandamide plasma concentrations in a sufficiently powered study employing validated analytical methods. Therefore, the anaesthetic and anti-emetic actions of propofol as well as its addictive potential in human subjects cannot be explained by peripheral FAAH inhibition. However, our findings do not exclude direct or indirect generated central cannabinoid receptor activation by propofol. Competing Interests There are no competing interests to declare. The expert technical assistance of B. Beckmann and F.M. Gutzki is gratefully acknowledged. REFERENCES 1 Pacher P, Batkai S, Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacol Rev 2006; 58: Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin CE, Brenner GJ, Rubino T, Michalski CW, Marsicano G, Monory K, Mackie K, Marian C, Batkai S, Parolaro D, Fischer MJ, Reeh P, Kunos G, Kress M, Lutz B, Woolf CJ, Kuner R. Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors. Nat Neurosci 2007; 10: Pernia-Andrade AJ, Kato A, Witschi R, Nyilas R, Katona I, Freund TF, Watanabe M, Filitz J, Koppert W, Schuttler J, Ji G, Neugebauer V, Marsicano G, Lutz B, Vanegas H, Zeilhofer HU. Spinal endocannabinoids and CB1 receptors mediate C-fiber-induced heterosynaptic pain sensitization. Science 2009; 325: Karst M, Wippermann S, Ahrens J. Role of cannabinoids in the treatment of pain and (painful) spasticity. Drugs 2010; 70: Pillarisetti S, Alexander CW, Khanna I. Pain and beyond: fatty acid amides and fatty acid amide hydrolase inhibitors in cardiovascular and metabolic diseases. Drug Discov Today 2009; 14: / 74:1 / Br J Clin Pharmacol

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