ARTICLE IN PRESS. Prostaglandins & other Lipid Mediators xxx (2014) xxx xxx. Contents lists available at ScienceDirect

Transcription

1 Prostaglandins & other Lipid Mediators xxx (2014) xxx xxx Contents lists available at ScienceDirect Prostaglandins and Other Lipid Mediators Q Review Endocannabinoid metabolism by cytochrome P450 monooxygenases Susan Zelasko a, William R. Arnold b, Aditi Das a,b,c,d, a Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, IL 61802, United States b Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61802, United States c Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61802, United States d Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL 61802, United States a r t i c l e i n f o Article history: Available online xxx Keywords: Endocannabinoids Cytochrome P450s Anandamide 2-Arachidonylglycerol Eicosanoids CYP2J2 a b s t r a c t The endogenous cannabinoid system was first uncovered following studies of the recreational drug Cannabis sativa. It is now recognized as a vital network of signaling pathways that regulate several physiological processes. Following the initial discovery of the cannabinoid receptors 1 (CB1) and 2 (CB2), activated by Cannabis-derived analogs, many endogenous fatty acids termed endocannabinoids are now known to be partial agonists of the CB receptors. At present, the most thoroughly studied endocannabinoid signaling molecules are anandamide (AEA) and 2-arachidonylglycerol (2-AG), which are both derived from arachidonic acid. Both AEA and 2-AG are also substrates for the eicosanoid-synthesizing pathways, namely, certain cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes. In the past, research in the endocannabinoid field focused on the interaction of AEA and 2-AG with the COX and LOX enzymes. Yet, accumulating evidence also points to the involvement of CYPs in modulating endocannabinoid signaling. The focus of this review is to explore the current understanding of CYP-mediated metabolism of endocannabinoids Published by Elsevier Inc. 22 Contents Introduction Discovery of the endocannabinoid signaling system Endocannabinoid metabolism by eicosanoid-synthesizing enzymes Cytochrome P450 enzymes Overview of human CYP enzymes Cytochrome P450 mediated metabolism of AA to form epoxyeicosatrienoic acid (EET) and hydroxyeicosatrienoic acid (HETE) Anandamide Anandamide (AEA) Overview of CYP involvement in AEA metabolism Anandamide and family 2 CYP epoxygenases Anandamide and family 4 CYPs Anandamide and polymorphic CYPs (CYP3A4, 2D6, and 2B6) Interaction of the EET-EA and HETE-EAs with the CB receptors Abbreviations: 2-AG, 2-arachidonylglycerol; AEA, anandamide; AA, arachidonic acid; CB 1, cannabinoid receptor 1; CB 2, cannabinoid receptor 2; CNS, central nervous system; COX, cyclooxygenase; CYP, cytochrome P450; DAG, diacylglycerol; DHET, dihydroxyeicosatrienoic acid; EGFR, epidermal growth factor receptor; EET, epoxyeicosatrienoic acid; EA, ethanolamine; ERK, extracellular signal-regulated protein kinase; FAAH, fatty acid amide hydrolase; GPCR, G protein coupled receptor; EG, glycerol; HETE, hydroxyeicosatrienoic acid; HEET, hydroxyepoxyeicosatrienoic acid; LOX, lipoxygenase; MAGL, monoacylglycerol lipase; MI, myocardial infarction; NAPE, N-Arachidonylphosphatidylethanolamine; PPAR, peroxisome proliferator-activated receptor; PGH 2, prostaglandin H 2; TRPV1, vanniloid receptor 1; THC, 9 -tetrahydrocannabinol. Corresponding author at: Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 3813 Veterinary Medicine Basic Sciences Building, 2001 South Lincoln Avenue, Urbana, IL 61802, United States. Tel.: address: aditidas@illinois.edu (A. Das) / 2014 Published by Elsevier Inc.

2 2 S. Zelasko et al. / Prostaglandins & other Lipid Mediators xxx (2014) xxx xxx 4. 2-Arachidonylglycerol Arachidonylglycerol (2-AG) CYP-mediated metabolism of 2-AG Interaction of the 2-EET-EGs with the CB receptors Endocannabinoid hydrolysis Concluding remarks Uncited references Acknowledgements References Q Introduction The endogenous cannabinoid signaling system was first uncovered following studies of the common recreational drug, Cannabis sativa, or marijuana. The psychoactive component of C. sativa, 9 -tetrahydrocanabinol (THC), elicits its activity by binding two cannabinoid receptors: cannabinoid receptor 1 (CB 1 ) and 2 (CB 2 ) [1 5]. Besides Cannabis-derived analogs, a number of endogenous cannabinoids (endocannabinoids) produced by the body bind to these cannabinoid receptors and elicit similar effects to those typically associated with Cannabis use. The two most thoroughly characterized endocannabinoids are anandamide (AEA) and 2-arachidonylglycerol (2-AG) [6,7]. AEA and 2-AG exhibit neuroprotective, anti-nociceptive, and anti-inflammatory properties that are mediated primarily by CB-receptor-dependent pathways [8,9]. The dysregulation of the endocannabinoid system has been implicated in a wide range of pathologies that include nociception [10,11], emotional disorders [12,13] energy imbalance [14], neurodegenerative diseases [15 19], cancer [20,21], and cardiovascular disease [22 27]. Taken together, these observations provide convincing evidence for the importance of the endocannabinoids in maintaining homeostasis. Therefore, a systematic understanding of all the potential pathways that control endocannabinoid metabolism in the body is crucial for drug development targeted toward this signaling system. In this review, we discuss the cytochrome P450 (CYP) mediated metabolism of endocannabinoids 1.1. Discovery of the endocannabinoid signaling system The discovery of the endocannabinoid system occurred in 1988 following the observation that Cannabis-derived THC inhibits adenylate cyclase activity in neuroblastoma cells. From this came the discovery of a novel G protein-coupled receptor (GPCR) in these cells, which was termed CB 1 [28,29]. CB 1 is the most abundant GPCR in the central nervous system (CNS) [28 32] with expression in the pre-frontal cortex, hippocampus, substantia nigra, cerebellum, and spinal cord. It is also detectable in cardiac [33], pulmonary [33], intestinal [34], hepatic [35,36], pancreatic [37], and reproductive tissues [33,38,39]. The identification of CB 2 followed in 1990, shortly after the discovery of CB 1 [40]. CB 2 is also a GPCR, but it is expressed primarily in the tonsils, spleen, and immune cells, with the highest levels of expression occurring in B lymphocytes, natural killer cells, and macrophages [33]. There is no clear consensus on the expression of CB 2 in the nervous system, but CB 2 mrna and protein has been detected in the brainstem [41]. The role of CB 2 in the CNS includes mediation of immunoregulation via microglial cells, which are phenotypically and functionally similar to macrophages [42]. Activation of either CB receptor leads to a myriad of responses that are accompanied by inhibition of adenyl cyclase and attenuation of subsequent pathways [43]. Given the variety of tissue types that express the CB 1 and CB 2 receptors, the endocannabinoid signaling system has a wide range of physiological implications. These initial studies of CB-receptor activation by the nonendogenous, Cannabis-derived THC molecule piqued the interest in researching the endogenous role of the cannabinoid receptors. Following the identification of CB 1 and CB 2, the interest in finding the respective endogenous ligands for these receptors grew. Within a few years, the fatty-acid-derived ligands AEA and 2-AG were found to interact with the CB receptors, and upon binding were shown to trigger responses analogous to those observed using non-endogenous THC [44 46]. Overall, the endocannabinoid system is presently known to consist of signaling molecules such as AEA, 2-AG, and other fatty acid compounds that regulate several physiological and cognitive processes in humans via CB 1 and CB 2. Besides the cannabinoid receptors, the endocannabinoids AEA and 2-AG also bind to other receptors including certain nuclear peroxisome proliferator-activated receptors (PPAR) [47,48] and, in the case of AEA, the heat-sensing vanniloid type-1 receptor (TRPV1) [49] Endocannabinoid metabolism by eicosanoid-synthesizing enzymes Currently, AEA and 2-AG are the most well studied endocannabinoids. Due to the structural similarity of AEA and 2-AG to the parent molecule arachidonic acid (AA) (Fig. 1) these molecules may also serve as substrates for the eicosanoid-synthesizing enzymes involved in AA metabolism. Eicosanoids are involved in inflammation, cardiovascular diseases, and cancer [50 52]. The generation of eicosanoids from AA occurs through three enzymatic pathways: cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 epoxygenase (EPOX). These eicosanoid-generating pathways have been reviewed more thoroughly elsewhere [53]. Both AEA and 2- AG are known substrates for certain COX and LOX enzymes and initial studies have demonstrated that these endocannabinoids are also metabolized by specific CYPs. Recently, there has been advances in determining the role of CYPs in endocannabinoid oxidation, particularly with respect to 2-AG metabolism. Additionally, there possibly exists a large degree of cross-talk between the three endocannabinoid-metabolizing pathways therefore novel fattyacid derivatives with presently unknown structures and functions may yet to be discovered [6]. Of the above mentioned eicosanoid-synthesizing pathways, the COX and LOX systems have been thoroughly studied and have been reviewed previously [6,54,55]. Briefly, two COX isoforms are known to oxidize AA to form prostaglandin H 2 (PGH 2 ), which serves as a precursor for many subsequent pathways. With respect to endocannabinoid metabolism, only the COX-2 isoform converts 2-AG to PGH 2 -glycerol (-EG) and converts AEA to PGH 2 -ethanolamine (-EA) and PGE 2 -EA [55]. COX-2 metabolizes AA, 2-AG, and AEA with similar enzymatic efficiencies [56]. The LOX enzymes typically add an oxygen to AA at C5, C12, and C15 positions [57,58] and various LOX isoforms also metabolize AEA and 2-AG at regioselective positions C12 and C15, with varying degrees of efficiency [59,60,50]. The third route of endocannabinoid metabolism is the CYP pathway, which is an important emerging field within endocannabinoid research. As a whole, the CYPs are a superfamily of hemoproteins that typically monooxygenate a

3 S. Zelasko et al. / Prostaglandins & other Lipid Mediators xxx (2014) xxx xxx 3 Fig. 1. The formation of AEA and 2-AG and subsequent oxidation by the CYP-mediated pathway. Anandamide (AEA, purple/solid pathway) formation is initiated upon phospholipase A2 (PLA2) releasing arachidonic acid (AA) from the plasma membrane. AA is combined with phophatidylethanolamine (PEA) by N-acyltransferase (NAT) to form N-arachidonyl-phophatidylethanolamine (NAPE), which is finally converted to AEA by N-arachidonyl-phosphatidylethanolamine phospholipase D (NAPE-PLD). In the case of 2-arachidonylglycerol (2-AG, green/dashed pathway), formation is initiated upon, (1) phospholipase C (PLC) producing diacylglycerol (DAG) that is converted to 2-AG by DAG lipase (DAGL), or (2) phospholipase A1 (PLA1) producing 2-AA lysophosphatidic acid (2-AA-LPA) that is converted to 2-AG by lysophospholipase C (lyso-plc). The cytochrome P450s (CYPs) then oxidize AEA and 2-AG. Single epoxidation reactions with AEA may form any of the four epoxyeicosatrienoic acid ethanolamides (EET-EAs) and the hydroxylation leads to two hydroxyeicosatetraenoic acid ethanolamides (HETE-EAs). Double oxidation reactions with AEA produces hydroxylated epoxyeicosatrienoic acid ethanolamides (HEET-EAs). Hydroxylation of the 5,6- and 14,15-EETs can occur at olefin positions 16, 17, 18, 19 and 20. Oxidation of 2-AG produces 2-epoxyeicosatrienoic acid glycerols (2-EET-EGs). Presently, oxidation has only been shown to occur at the 11,12- and 14,15- positions. Finally, CYP2J2 and CYP2C8 also hydrolyze the ester group in 2-AG and the 2-EET-EGs to either AA or the corresponding EET, respectively, and glycerol variety of endogenous and xenobiotic compounds [51 53]. Evidence supporting the metabolism of AEA and 2-AG by members of the CYP2, -3, and -4 subfamilies continues to accumulate, although further examinations both in vitro and in vivo are needed to understand the full scope of CYP involvement in the endocannabinoid system. This review will focus on the recent advances in the area of CYP-mediated oxidation of AEA and 2-AG as well as the potential physiological implications of the CYP-derived metabolites in this signaling system. The significance of 2-AG is emphasized in this review as recent work has demonstrated it is both a more abundant and potent CB activator [54,61]. Finally, we provide insights on the future directions this research field ought to consider and pose questions to address the remaining gaps in the present understanding of CYP-mediated endocannabinoid metabolism. 2. Cytochrome P450 enzymes 2.1. Overview of human CYP enzymes At present, there are 57 known human CYPs, which are expressed in the liver [56] and in several extrahepatic tissues [57,58] such as the lungs [59], kidneys [60], brain [62], cardiovascular system [63 65], intestinal tissues [66], and platelets [67,68]. In addition, individuals may have polymorphic variants of certain

4 4 S. Zelasko et al. / Prostaglandins & other Lipid Mediators xxx (2014) xxx xxx CYP isoforms that significantly alter catalytic efficiency of the enzyme. Polymorphisms in CYPs 2D6, 2C19, and 2C9 account for the most frequent variations in phase I drug metabolism in humans [69]. CYPs typically catalyze the epoxidation, hydroxylation, or, less commonly, the isomerization of endogenous and/or xenobiotic compounds [70]. Many CYPs, especially those concerned with xenobiotic metabolism, promiscuously bind several substrates, allowing for a wide variety of side reactions to occur, which can result in drug-drug interactions [71 73]. A number of CYPs are known to primarily metabolize AA to produce eicosanoid signaling molecules [74 80]. Finally, while the various roles of many CYPs in humans have been elucidated, a handful of orphan CYPs with unknown functions remain, including CYP20A1 of the CNS and members of the CYP4A and 4F subfamilies [81]. Given the variety of CYP functions, the understanding of how each CYP isoform influences the metabolism of endocannabinoids is critical to understand Cytochrome P450 mediated metabolism of AA to form epoxyeicosatrienoic acid (EET) and hydroxyeicosatrienoic acid (HETE) Endocannabinoids (AEA and 2-AG) are derivatives of AA. Therefore, it is useful to consider CYP-mediated metabolism of the endocannabinoids as typically CYPs metabolizes AA to form epoxyeicosatrienoic acids (EETs) [82,83] and hydroxyeicosatrienoic acid (HETE) [83]. Collectively, the EETs mediate inflammation, vascular tone, and angiogenesis that is important in mitigating ischemia and myocardial reperfusion injury [84]. HETEs produced by CYPs are also involved in vasoconstriction and vasodilation [85 87]. Thus far, enzymes in the CYP1, -2, -3, and -4 families have been shown to produce AA metabolites, including CYP2J, 2D, 3A, 4A, and 4F subfamilies in different mammalian species that have been implicated in endocannabinoid metabolism as well [74 80,88]. Epoxygenation reactions are primarily carried out by the CYP2C and 2J families in humans, while CYP4 members have been shown to carry out -20 hydroxylation to form HETEs [89 91]. CYP epoxygenases may form several EET regioisomers that contain R/S enantiomeric forms in varying proportions [92 94] where each possesses specific physiological functions [74,95]. The predominant regioisomers formed by CYPs include 5,6-; 8,9-; 11,12-; and 14,15-EETs, and 12-, 19-, and 20-HETEs [74,96]. Enzymes in the CYP2J subfamily form all four regioisomeric EETs as the major product, as well as 19-HETE [97]. However, the propensity to form these various regio- and stereoisomers is not the same among all CYP isoforms. For instance, CYP2C8 produces only the 14,15- and 11,12-EETs in appreciable quantities [98] [99] and 2C9 only produces 14,15-; 11,12-; and 8,9-EET [99]. The CYP4 subfamily, which includes CYP4A11, CYP4A22, and CYP4F isoforms, predominantly produce 20-HETE [78,100]. CYP2D18 from rat only forms the 14,15-; 11,12-; and 8,9-EETs [77]. The eicosanoids formed by CYP metabolism maintain homeostasis in part by regulating blood pressure, inflammation, renal function, immunity, and hemostasis [101]. When formed in excess under pathological conditions, these molecules can contribute to the onset of many acute and chronic diseases. The EET class of molecules is of particular importance in vasculature, renal, and respiratory systems [74,102,103]. The HETE-producing enzymes CYPs 4F and 4A are upregulated in a number of human cancers [100], along with having roles similar to the EET metabolites, e.g. concerning vasculature and hypertension [74]. Additionally, the regioisomers of EETs and HETEs may have different effects even among themselves. The 14,15- and 11,12-EETs promote cell survival of cultured human coronary artery endothelial cells and cultured human lung microvascular endothelial cells, while the 8,9- and 5,6-EET do not [104]. Furthermore, the EETs have been shown to induce vasodilation in vascular smooth muscle cells [74], although in other cell types certain EETs mediate vasoconstriction by various mechanisms [ ]. As aforesaid, HETEs can be either vasoconstrictive or vasodilatory depending on the site of release and action [85,86]. Therefore, the EETs and HETEs play an important and opposing role in cell signaling. 3. Anandamide 3.1. Anandamide (AEA) Anandamide (N-arachidonoylethanolamine or AEA) is an endocannabinoid that derives its name from the Sanskrit word ananda meaning joy. AEA is presently the most thoroughly examined endocannabinoid with respect to metabolism by CYPs and other enzymes, although there are other important endocannabinoid signaling molecules beyond AEA, such as 2-AG. AEA is a highly lipophilic signaling molecule that has a relatively short half-life [93] and is present at low levels (<3.5 pmol/g of tissue) in fresh rat brain, liver, and heart under basal conditions [ ]. It is believed that AEA primarily forms in a stimulusdependent manner and acts on local receptors in a paracrine and/or autocrine manner, which may explain the low basal levels [93]. AEA is produced from the precursor C20:4-N-Arachidonylphosphatidylethanolamine (NAPE). This precursor forms via the transfer of an arachidonyl fatty acid to the amino group of phosphatidylethanolamine by N-acyltransferase (NAT) [93,112] (Fig. 1). It appears that AEA regulates physiological processes primarily through CB 1 -mediated signaling, as well as through TRPV1 and PPAR- and - signaling pathways [47,49,113] Overview of CYP involvement in AEA metabolism Prior to inactivation, AEA may undergo oxidative metabolism by various enzymes within the COX, LOX, and CYP pathways [56,59,114]. The metabolites produced by these pathways alter the physiological fate of AEA by either enhancing or diminishing AEA signaling. The first characterization of AEA metabolism by CYPs was demonstrated in mouse liver microsomes, which formed 20 AEAderived products, and in mouse brain microsomes, which formed two products [115]. The production of these metabolites could be inhibited by antibodies against hepatic CYP3A, 2B, and 1A and CYP3A in the brain. Further evidence for the participation of hepatic and nervous system CYPs in AEA metabolism was provided by other groups. It is also worth noting that in human liver microsomes, inhibitory antibodies against CYP 1A1, 1A2, 2A6, 2B6, 2D6, 2C8, 2C9, and 2E1 showed no appreciable decrease in AEA metabolite formation so these CYPs could not be attributed to AEA metabolism. In the same experiment, CYP3A4 and CYP2C19 antibody based inhibition resulted in a decrease in dioxygenated AEA metabolite (HEET-EA) formation [114]. In human kidney microsomes, none of the above listed CYPs showed decrease in AEA metabolism upon inhibition. The specific CYP proteins that oxidize AEA are listed in Table 1, though this list continues to expand. Several isoforms have not yet been evaluated for AEA metabolism, therefore the full scope of CYP involvement in the endocannabinoid system remains unknown. In particular, the CYPs that mediate AA oxidation may prove to be involved in the metabolism of AEA. Based on the present literature, certain CYPs metabolize AEA into four EET-EAs and two HETE-EAs, similar to AA metabolism. It also remains possible that additional oxidized products may be formed that have not yet been identified. The EET-EA and HETE-EA turnover rates have been determined in human liver microsomes, with the rate of formation for the 11,12- and 14,15-EET-EAs being approximately 40 pmol/min/mg

5 S. Zelasko et al. / Prostaglandins & other Lipid Mediators xxx (2014) xxx xxx Table 1 CYPs involved in AEA and 2-AG oxidation. CYP enzymes Compounds References CYP2J2 2-11,12-EET-EG [1] 2-14,15-EET-EG 5,6-EET-EA; 8,9-EET-EA; 11,12-EET-EA; 14,15-EET-EA (all four EET-EAs) 19-HETE-EA 20-HETE-EA CYP2D6 All four EET-EAs [2,3] 20-HETE-EA 16-, 17-, 18-, 19-, and 20-5,6-HEET-EA 16-, 17-, 18-, 19-, and 20-14,15-HEET-EA CYP2D6.34 Dec. rates compared to WT: [3] 14,15-EET-EA 20-HETE-EA 11,12-EET-EA 8,9-EET-EA 16-, 17-, 18-, 19-, and 20-5,6-HEET-EA 16-, 17-, 18-, 19-, and 20-14,15-HEET-EA CYP2B6 All four EET-EAs [3] CYP2B6.4 11,12-EET-EA [3] 5,6-EET-EA 8,9-EET-EA 14,15-EET-EA 20-HETE-EA CYP2B6.6 11,12-EET-EA (dec. rate compared to WT) [3] 14,15-EET-EA 8,9-EET-EA (dec. rate compared to WT) CYP2B6.9 11,12-EET-EA [3] CYP3A4 All four EET-EAs [4] CYP3A4.4 All four EET-EAs [5] 19-HETE-EA CYP4F2 20-HETE-EA [4,6] 16-, 17-, 18-, 19-, and 20-5,6-HEET-EA 16-, 17-, 18-, 19-, and 20-14,15-HEET-EA CYP4F3B 20-HETE-EA [6] CYP4A11 20-HETE-EA [6] CYP4X1 14,15-EET-EA [7] microsomal protein; 5,6-EET-EA being 184 ± 24 pmol/min/mg; 8,9- EET-EA being 480 ± 56 pmol/min/mg; and the 20-HETE-EA being 266 ± 26 pmol/min/mg [114]. In human kidney microsomes, the 20-HETE-EA rate was 122 ± 7.4 pmol/min/mg microsomal protein [114]. The involvement of the CYP enzymes in AEA metabolism has been previously reviewed by Snider and coworkers [112], though a number of advances in the field have since been made that we discuss in the next section that explore the details of CYPs implicated in AEA metabolism Anandamide and family 2 CYP epoxygenases The family 2 CYPs include several epoxygenases that oxidize AA, some of which have recently been demonstrated to metabolize AEA. CYP2J2 is an important epoxygenase in humans that is expressed in cardiovascular structures including the myocardium and aorta [117]. CYP2J2 produces all four EET regioisomers from AA and 19-HETE [97]. We also showed that CYP2J2 also forms all four EET regioisomers of AEA, as well as 19- and 20-HETE-EA. The production of 14,15-EET-EA by CYP2J2 was 4-fold faster than that of 14,15-EET from AA. Furthermore, spectral titrations, molecular modeling, and steady-state cyanide binding studies revealed that AEA binds differently than AA [61]. To date, the metabolism of AEA by classical epoxygenases besides CYP2J2, such as CYP2C subfamily members, has not been reported. We hypothesize that this is due to the larger active site of CYP2J2 as compared to another epoxygenase CYP2C8. Other family 2 CYPs that metabolize AEA, but are traditionally considered hydroxylases include CYP2D6 and 2B6. Unlike CYP2J2, the isoforms CYP2D6 and 2B6 are highly polymorphic in nature and the most recent insights in to the metabolism of AEA by variants of these CYPs are discussed in Section 3.5 [118]. At present, very few family 2 CYPs have been evaluated for AEA metabolism though it is likely that other isoforms metabolize AEA, particularly AA-metabolizing CYPs. Further structure-function studies are need to clarify how CYPs associated typically with hydroxylation can epoxygenate AEA Anandamide and family 4 CYPs As with family 2 CYPs, a number of family 4 CYPs that were previously known to metabolize AA have recently been shown to oxidize AEA as well. In terms of AA oxidation, the CYP4A subfamily produces 19- and 20-HETE from AA, while CYP4F enzymes are only known to form the 20-HETE regioisomer [119,120]. Previous work has established that human CYP4F2, CYP4F3B, and CYP4A11 hydroxylate xenobiotic molecules and endogenous fatty acids, including AA [53, ]. Human 4A11, CYP4F2, 4F3B, and the previously orphaned 4X1 are now known to display AEA oxidation activity as well [114,125,126]. The expression profiles of the family 4F and 4A CYPs include hepatic, renal, and immune. CYP4F2 and CYP4F3B are expressed in the liver and kidneys, with CYP4F2 representing approximately 15% of all hepatic CYPs [ ]. Additionally, CYP4F3B is expressed in leukocytes [131]. In terms of AEA oxidation, CYP4F2 is more active than CYP4A11, since the ratio of 20-HETE-EA formed by 4F2:4F3B:4A11 in kidney microsomes was 58:7:1 [114]. This ratio may, however, be altered by external factors in vivo, so further investigations are needed to confirm the predominance of CYP4F2-mediated AEA metabolism. It appears that AEA also has a higher affinity for CYP4F2 (0.7 M) than AA (24 M), potentially making this isoform more relevant in endocannabinoid signaling [114,121]. Furthermore, AEA displays lower affinity for both COX-2 and 12-LOX compared to CYP4F2 [112]. Finally, CYP4F2 was also shown to produce the dioxygenated hydroxyepoxyeicosatrienoic acids (HEETs) that have unknown physiological functions (Fig. 1) [114,125]. Given the high abundance of CYP4F isoforms in the liver and efficient production of 20-HETE-EA, the role of endocannabinoid oxidation by CYP4F members should be investigated in vivo as all the currently known studies are in microsomes [11 15]. As for CYP4A11, it is expressed in the liver and kidneys [130,132]. It is now known that CYP4X1 is expressed mostly in the amygdala and skin, but it is also in the cerebellum, basal ganglia, heart, liver, breast, and prostate, which gives it close proximity to the CB 1 receptor [126]. Recently, the orphan CYP4X1 was shown to be involved in AEA oxidation, producing 14,15-EET-EA; however, it does not appear to metabolize the typical family 4 substrates, only metabolizing AA in the presence of cytochrome b 5 and not metabolizing any other fatty acids included in the study [126]. Binding and kinetic experiments will help in comparing the contribution of this previously orphaned CYP to endocannabinoid oxidation by other family 4 members. In summary, the family 4 CYPs, particularly CYP4F2 and CYP4X1 are known metabolize AEA. Additional family 4 isoforms in AEA metabolism have yet to be determined Anandamide and polymorphic CYPs (CYP3A4, 2D6, and 2B6) CYP3A4, CYP2D6, and CYP2B6 are three polymorphic CYPs that are known to metabolize xenobiotic substances and several endogenous ligands such as polyunsaturated fatty acids [ ]. There is also recent evidence of AEA metabolism by these polymorphic CYPs [114,118,138]. Expression of these CYPs occurs in the liver as well as in the CNS, showing regio-specific expression profiles in these organs [55, ]. CYP3A4 is also expressed in the intestines [142]. Concerning wild type activity, both CYP3A4 and 2B6 metabolize AA to form EETs [136,143]. Contrariwise, CYP2D6 is unique in that it does not metabolize AA, but does show highaffinity binding to and metabolism of AEA [138]. Structure-function

6 6 S. Zelasko et al. / Prostaglandins & other Lipid Mediators xxx (2014) xxx xxx Table 2 Metabolites of AEA and 2-AG Oxidation by CYPs. Compound Detection in vivo Receptor binding CB1 CB2 AEA Yes a 155 nm [162] 11,400 nm [162] 5,6-EET-EA Unknown 3,200 nm Degraded slowly and binds with a lower affinity than AEA [162] 8.9 nm Degraded slowly and binds with a higher affinity than AEA [162] 8,9-EET-EA Unknown Unknown Unknown 11,12-EET-EA Unknown Unknown Unknown 14,15-EET-EA Unknown 1,560 nm Unknown Binds with a lower affinity than AEA [129] 20-HETE-EA Unknown 985 nm Unknown Binds with a lower affinity than AEA [129] 19-HETE-EA Unknown Unknown Unknown 16-, 17-, 18-, 19-, and 20-5,6-HEET-EA Unknown Unknown Unknown 16-, 17-, 18-, 19-, and 20-14,15-HEET-EA Unknown Unknown Unknown 2-AG Yes a 45 nm 251 nm 2-8,9-EET-EG Undetected in vivo and in Unknown Unknown CYP2J2 incubations [66,67]. 2-5,6-EET-EG Undetected in vivo and in CYP2J2 incubations [66,67]. Unknown Unknown 2-11,12-EET-EG Detected in vivo and in CYP2J2 incubations [66,67]. 2-14,15-EET-EG Detected in vivo and in CYP2J2 incubations [66,67]. 23 nm Binds with higher affinity than 2-AG [66]. 40 nm Binds with comparable affinity compared to 2-AG [66]. 75 nm Binds with higher affinity than 2-AG [66]. 138 nm Binds with higher affinity than 2-AG [66]. a Zoerner AA, Batkai S, Suchy M-T, Gutzki F-M, Engeli S, Jordan J, et al. Simultaneous UPLC MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction. J Chromatogr B, 2012; : studies ought to be conducted to elucidate the apparent ability of CYP2D6 to metabolize AEA and not AA. CYP2D6 converts AEA into four EET-EA regioisomers, 20-HETE-EA, and diooxygenated HEETs [138]. The relative abundance of these products from wild type CYP2D6 are 20-HETE-EA > 14,15-EET-EA > 8,9-EET-EA > 11,12-EET- EA > 5,6-EET-EA. Wild type CYP2B6 also produces these four EET-EA metabolites; however, in contrast to CYP2D6 it does not produce significant quantities of 20-HETE-EA. Furthermore, CYP2B6 produces more 11,12-EET-EA and less 14,15-EET-EA than CYP2D6 [118]. Both the biochemical mechanisms and the physiological implications of the CYP-specific metabolite profiles by these, and other CYPs, remain unknown. It should be noted that the observations of CYP2B6 and CYP2D6 metabolizing AEA contradict the kidney and liver studies that show that there is no metabolism of AEA by these enzymes (see Section 3.2) [114]. However, these studies were done using direct assays in vitro with brain samples [118,138] as opposed to the inhibitory antibody screening performed in earlier studies, which may help to explain the discrepancy [114]. Finally, the metabolism of AEA by CYP3A4 also results in the formation of the four EET-EA regioisomers. Based on antibody inhibition in human liver microsomes, it was proposed that a large proportion of hepatic EET-EAs are produced by CYP3A4 though this must be confirmed in vivo [118]. The polymorphic variants of CYP3A4, 2D6, and 2B6 display a range of functionality with respect to AEA metabolism. This may impact the ability of different individuals to produce different levels of endocannabinoid metabolites. Polymorphisms, therefore, ought to be considered in future endocannabinoid research and pharmacologic therapies [140,141,144]. For instance, recent investigation of the common CYP2B6.4 variant indicates it displays higher production of 20-HETE-EA, whereas 2B6.9 only produces the 11,12-EET-EA regioisomer [118]. CYP2D6 exhibits an even higher amount of polymorphism [144] and the CYP2D6.34 variant produces the same AEA metabolites as the wild type isoform, except 5,6-EET-EA that is not formed [118]. The common polymorphism CYP3A4.4 shows 60% lower production of the four EET-EAs compared to wild-type, but unlike wild type CYP3A4, it uniquely forms the 19-HETE-EA [116]. Hence, certain polymorphic CYPs appear to mediate endocannabinoid signaling through the oxidation of AEA and may account for variation in susceptibility to endocannabinoidrelated pathologies. Additional in vivo investigations are required to better understand the impact of these polymorphisms on endocannabinoid signaling and associated pathologies Interaction of the EET-EA and HETE-EAs with the CB receptors The significance of CYP-mediated metabolism of endocannabinoids such as AEA lies in the resulting physiological consequences of these novel endocannabinoid derivatives. The independent function of AEA signaling at the CB receptors has been reviewed previously [145,146]. It is known, for instance, that prior to oxidative metabolism, AEA mediates analgesia, hypothermia, appetite, and anti-proliferation [147,148]. In regard to CYP expression, AEA has been observed to enhance the expression of CYP3A and CYP2B in rat liver and brain tissue [149,150]. Nevertheless, the precise function of AEA oxidation by the CYP enzymes remains a novel area for detailed study and only a handful of in vivo studies have been performed. Presently, it is known that the EET-EAs and HETE- EAs bind to the CB 1 and CB 2 receptors with different affinities than AEA (Table 2). For instance, the propensity of 5,6-EET-EA binding to CB 2 is 1000-fold stronger than AEA [151]. This EET-EA is also slower to degrade than AEA, giving it additional potential to interact with the receptor. The 5,6-EET-EA displays a 300-fold higher binding to the CB 2 receptor versus the CB 1 receptor, indicating that CYP oxidation may serve as a pathway for CB 2 -selective activation that is more robust than AEA [151]. CB 2 has been implicated in some of the downstream effects concerning the liver and CNS [112]. For example, CB 2, although not found in normal human liver, was found to be expressed in cirrhotic, hepatic fibrogenic cells and aided in anti-fibrogenesis [152]. The CB 2 receptor is also

7 S. Zelasko et al. / Prostaglandins & other Lipid Mediators xxx (2014) xxx xxx upregulated in microglia cells during experimental autoimmune encephalomyelitis in mice [153]. In contrast to the 5,6-EET-EA, the 14,15-EET-EA and 20-HETE-EA appear to bind the CB 1 receptor, but with a lower affinity than AEA [118]. Stability assays using rat brain homogenate indicate the 14,15-EET-EA and 20-HETE-EA metabolites also display shorter half-lives compared to AEA, implying that these metabolites are less potent than AEA [118]. Together, these studies suggest that CYP action may alter the physiological fate of AEA as a means for fine-tuned homeostatic regulation. One of many important, unanswered questions is whether the remaining regioisomeric products of CYP-mediated AEA metabolism interact with the CB 1, CB 2, or other receptors. Given the wide and important role AEA plays in maintaining homeostasis, it is critical to better discern how the CYP pathway enhances or diminishes such signals, especially if the endocannabinoid system is to be considered a target of clinical intervention Arachidonylglycerol Arachidonylglycerol (2-AG) Currently, the other well-studied endocannabinoid is 2-AG. 2- AG is derived from the commonly distributed pre-cursor molecule diacylglycerol (DAG) that bears an AA at the 2-position [7]. It has also been proposed that 2-AG can be synthesized by dephosphorylation of arachidonoyl-lysophosphatic acid (LPA) by lysophospholipase C [154,155] (Fig. 1). A more detailed review of 2-AG synthesis and degradation has been previously published [156]. The role of CYP-mediated metabolism of 2-AG has not been a topic of discussion in previous reviews, though accumulating evidence suggests that 2-AG plays a significant role in the CNS signaling. 2-AG and AEA follow different routes of synthesis and degradation that one could speculate would partly account for the specificities of each molecule. Accordingly, the level of 2-AG in the brain was found to be 800 times higher than that of AEA [44,108]. The relative CB 1 and CB 2 binding capacities of these two endocannabinoids are also different, where 2-AG preferentially binds the most abundant CNS GPCR, namely CB 1 [28,29], while AEA is unique in that it activates TRPV1 in addition to CB 1 and CB 2 [49]. Finally, altering 2-AG metabolism exerts dramatic changes on central synaptic transmission via CB 1 with concurrent 2-AG accumulation in CB 1 expression regions, whereas alteration of AEA levels does not [15, ]. Therefore, while it was once believed that 2-AG and AEA exhibit nearly identical pharmacological properties due to their close structural similarities and potential to activate the same receptors, this notion ought to be reevaluated [1, ] CYP-mediated metabolism of 2-AG Within the last few years, CYP-mediated metabolism of 2-AG that enhances binding to CB 1 has been demonstrated. With respect to the CYP branch, it is conceivable that CYPs would produce four regioisomeric EET-EGs due to the similarity in structure of 2-AG to AA. However, at present only two regioisomers (2-11,12- and 2-14,15-EET-EG) have been isolated from renal epithelial cells, while the 2-8,9- and 2-5,6-EET-EGs were not detected in these tissues [54]. Surprisingly, early studies showed the 2-AG is not metabolized by rat liver and kidney microsomes that express the highest levels of CYPs, nor is it oxidized by recombinant CYP2C8 (human), CYP2C11 (rat), and CYP2C23 (rat), which are known to epoxygenate AA [54]. However, our recent work shows that upon incubation of 2-AG with CYP2J2, the 2-11,12- and 2-14,15-EET-EG regioisomers are produced in vitro [61]. These 2-EET-EGs display tighter binding to CB 1 as compared to 2-AG [54]. Consequentially, the cardiovascular epoxygenase CYP2J2 remains the only CYP shown to metabolize 2-AG (Table 1). Structure-function studies may help determine why the traditional CYP2C epoxygenases do not oxidize 2-AG, while CYP2J2 does. Considering that AEA is metabolized by CYPs not typically considered as epoxygenases (e.g. CYP2D6), it may be worth evaluating such CYPs in 2-AG metabolism as well. Finally, if not by CYPs, these EET-EGs may form by other means. As Chen and coworkers speculate, the formation of the 2-EET-EGs may also be possible by cleaving EET glycerophospholipids via PLC and diacylglycerol lipase and/or by hydrolysis of EET phospholipids via PLA1 and lysophospholipase C [54] Interaction of the 2-EET-EGs with the CB receptors The compound 2-AG and its physiological consequences have been studied and reviewed previously [146,147,165], but the implications of the CYP-mediated metabolites have only recently begun to be elucidated. The two aforementioned metabolites of 2-AG (2-11,12-EET-EG and 2-14,15-EET-EG) have been isolated directly from physiological sources, which corroborates the observed formation of these metabolites by either CYP-mediated or other alternate pathways [54]. Yet, the precise role of these oxidized products remains unclear [54]. Initial insights into the metabolite functions show that both the 2-11,12- and 2-14,15-EET-EGs bind rat brain extracts overexpressing CB 1 with K i values of 23 ± 3 nm and 40 ± 5 nm, respectively [54] (Table 2). Both metabolites also bind rat spleen extracts overexpressing CB 2 receptors with K i values of 75 ± 6 nm and 138 ± 9 nm for the 2-11,12-, and 2-14,15- EET-EGs, respectively [54] (Table 2). From these results, it would appear that the 2-11,12-EET-EG is a more potent agonist for the two CB receptors overall and that 2-AG metabolites favor CB 1 binding. Additionally, the affinities of the metabolites for the CB receptors appear to be higher than 2-AG itself for either receptor [54]. Therefore, the CYP oxidized metabolites of 2-AG would be expected to constitute a bioactivation of the CB 1 receptor. This appears to contrast with AEA derived 5,6-EET-EA that preferentially binds the CB 2 receptor with higher affinity than AEA. Note that 2-AG itself shows a higher affinity for the CB receptors than AEA. This evidence suggests a hierarchy of binding capacities among 2-AG, AEA, and their oxidized metabolites where the EET-EGs bind the tightest among these three. Our studies indicate CYP2J2, which is primarily present in cardiovascular structures, metabolizes 2-AG to form 2-11,12-EET- EG and 2-14,15-EET-EG (Table 1). Furthermore, 2-AG levels are present at reasonable levels in the heart (3.25 ± 1.15 nmol/g tissue [169], compared to 3.36 ± 1.34 in the brain from rat models [168]) and platelets [171], suggesting a need for 2-AG signaling in this organ. A potential role for 2-AG and its oxidized metabolites is cardiovascular CB 1 signaling is following acute tissue damage. In experimental rat models of acute myocardial infarction (MI), blocking CB 1 with the specific antagonist SR141716A increased endothelial dysfunction, decreased post-mi hypotension, and led to overall worse mortality rates [171]. Blocking the shock-related hypotension attributed to CB 1 signaling had a detrimental effect on early rat survival. This is in agreement with the finding that post-mi restoration of blood pressure alone is not beneficial, as illustrated by the persistent high mortality rate in patients given high doses of vasoconstricting catecholamines [171]. The ability of 2-AG and/or the 2-EET-EGs to act as vasodilators via CB 1 suggests a role in minimizing acute tissue damage by mediating vascular tone and endothelial function [171]. Additionally, there have been initial studies with hydrolysisresistant ether analogs of the two 2-EET-EGs that were shown to activate p44/p42 extracellular signal-regulated kinase pathways in Chinese hamster ovary cell lines expressing both of the human CB receptors [54]. The 2-EET-EGs were also shown to activate

8 8 S. Zelasko et al. / Prostaglandins & other Lipid Mediators xxx (2014) xxx xxx N18TG2 neuroblastoma cells that only express CB 1 and promyelocytic leukemia HL-60 cells that only express CB 2 receptors [54]. The interaction of the 2-14,15-EET-EG has been further characterized using a renal proximal tubule epithelial cell line and was shown to activate epidermal growth factor receptor (EGFR) signaling that which aids renal cell recovery, whereas 2-AG alone exerts no effect [166]. Interestingly, the AA-derived 14,15-EET also activates EGFR signaling via heparin-binding EGF-like growth factor, while 2-14,15-EET-EG does so using transforming growth factor (TGF ) [166,167]. Both eicosanoids are, therefore, implicated in the regulation of various stages of renal cell injury recovery and do so using separate pathways. Together, this demonstrates a possible physiological activation pathway for the oxidized 2-AG metabolites. Further evidence from mouse model studies show that both 2-11,12- and 2-14,15-EET-EG analogs induce a significant decrease in locomotor activity as well as a hypothermic response, which corroborates the physiological role of endocannabinoid activity [54]. The analogs have also been shown to induce a CB 1 -dependent vasorelaxing response in renal glomerular afferent arterioles and a CB 2 -dependent neutrophil-like chemotaxis of HL-60 cells [54]. Together, these results highlight the importance CYP-mediated metabolism of 2-AG and the physiological roles of these metabolites; however, further studies are needed to clearly elucidate other unknown physiological effects particularly in the CNS and cardiovasculature. 5. Endocannabinoid hydrolysis The endocannabinoids undergo inactivation through hydrolysis by specific enzymes. Fatty acid amide hydrolase (FAAH) carries out the hydrolytic cleavage of AEA to form AA and ethanolamine. This integral membrane enzyme is expressed mostly in the liver and brain, as well as the intestines, spleen, and lungs [173]. Evidence for the role of FAAH in inactivating AEA stems from in vivo experiments that demonstrate, in the absence or inhibition of FAAH, cells rapidly succumb to necrosis after AEA treatment [174]. N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is also capable of inactivating AEA [175]. The hydrolysis of 2-AG to AA and glycerol is carried out primarily by monoacylglycerol lipase (MAGL), although a minor percentage of 2-AG is also hydrolyzed by FAAH [176] and serine lipases hydrolase domains 6 and 12 (ABHD6, ABHD12) [165,177]. Expression of MAGL occurs in the brain, adipose tissue, intestines, and pancreatic islet cells [165, ]. The inactivation of AEA and 2-AG plays a necessary role in attenuating endocannabinoid signaling. In addition to FAAH- and MAGL-mediated hydrolysis, alternative routes of endocannabinoid inactivation have been elucidated. For instance, it has been shown that the cleavage of the ester bond in 2-AG into free AA and glycerol occurs in vitro using incubations of either CYP2J2 or CYP2C8 with NADPH and cytochrome P450 reductase [61]. The AA formed in this process can then be converted into signaling molecules EETs and HETEs that are essential to mediating cardiovascular homeostasis. This was the first evidence of CYP-mediated inactivation of 2-AG, although previously CYPs have been shown to cleave ester bonds [70]. In tissues where CYP concentrations are higher, this may become a predominant mechanism of 2-AG inactivation and could also explain why 2-AG epoxides were not detected in liver microsomes incubations. These findings provide a groundwork by which further studies may be stimulated. In regards to the CYP-generated metabolites of AEA and 2- AG, the epoxygenated EET-EGs and EET-EAs undergo secondary metabolism that involves epoxide ring opening. Epoxide hydrolysis has been previously demonstrated in EETs derived from AA using microsomal and soluble epoxide hydrolases that catalyze the addition of water to epoxides, forming the corresponding dihydroxyeicosatrienoic acids (DHETs) [181,182]. The inhibition of epoxide hydrolase has been shown to elevate levels of EETs [181]. In a similar fashion, the four EET-EAs have been shown to form DHET-EAs via secondary metabolism by epoxide hydrolase [114]. For instance, the 5,6-EET-EA is degraded by epoxide hydrolase in mouse brain homogenate with a half-life of 32 min (though it does not completely disappear) [151], whereas AEA is entirely hydrolyzed (presumably by FAAH) in 14 min. The physiological relevance of the produced DHET-EAs and putative DHET-EGs remains unclear. Furthermore, the degradation of AEA and 2-AG by additional enzymatic routes remains to be investigated. 6. Concluding remarks Since the initial discovery of the endogenous cannabinoid receptors, CB 1 and CB 2, substantial progress has been made in characterizing the biochemical and physiological properties of the endocannabinoid system. Thus far, the most thoroughly studied components of this signaling network remain the two cannabinoid receptors, CB 1 and CB 2, as well as the lipid signaling molecules AEA and 2-AG. The biological activity of AEA and 2-AG may be altered by eicosanoid synthesizing enzymes in the COX, LOX, and CYP pathway. The involvement of certain CYP enzymes in oxidizing AEA and 2-AG was demonstrated within the last few years. The CYPs add a level of fine-tuned modulation to the endocannabinoid system that attenuates or enhances the capacity of AEA and 2-AG to act as signaling molecules. Several CYPs have been shown to form four AEA and two 2-AG epoxide derivatives. A number of CYPs are also known to produce hydroxylated AEA and 2-AG derivatives. Each modification of the endocannabinoid molecules represents a potential alteration in binding capacity to the target receptors, which has been observed for certain metabolites as mentioned in Sections 3.6 and 4.3. Additionally, CYP2J2 and CYP2C8 are involved in the inactivation of 2-AG that may influence the bioavailability of this key non-classical neurotransmitter. While it is clear that CYP enzymes play a significant role in the metabolism of AEA and 2-AG, there remain many potential avenues of study that will provide a more complete understanding of how CYPs influence the endocannabinoid system, or vice versa. For instance, the physiological role of the EET-EAs, HETE-EAs, and 2-EET-EGs in activating or attenuating endocannabinoid signaling requires further attention. The inactivation mechanisms of the endocannabinoid molecules by the remaining human CYPs beyond those enzymes described in Sections 3 and 4 should be evaluated. There are several CYPs isoforms that metabolize AA in the CYP1A, 2B, 2C, 2E, 2J, 2D, 2N, 4A, and 4F families, which have not yet been evaluated for endocannabinoid metabolism. Furthermore, the CYPs previously evaluated for endocannabinoid oxidation ought to be revisited in light of the new discovery that CYP2J2 and 2C8 to induce 2-AG cleavage. Another potentially useful route of study may be the orphan CYPs, considering that the previously orphaned CYP4X1 was shown to metabolize AEA [81,126]. The remaining orphan CYPs include CYP2A7, 2S1, 2U1, 2W1, 3A43, 4A22, 4F11, 4F22, 4V2, 4Z1, 20A1, and 27C1. Additionally, several endocannabinoids beyond AEA and 2-AG have been recognized, which include 1-arachidonylglycerol, noladin ether [186], virodhamine [187], N-arachidonyl dopamine [188], N-arachidonylglycine [189], and oleamide [190]. The metabolism of these endogenous molecules by CYP isoforms should be considered to better understand their role within the endocannabinoid system. Overall, CYP involvement in the endocannabinoid system remains a relatively new field that requires further study to fully elucidate and understand the physiological and pharmacological relevance. As CYPs perform numerous functions on a myriad of substrates, including xenobiotic