New Approaches and Challenges to Targeting the Endocannabinoid

Home , ABHD12, Ajulemic acid, Arachidonoyl serotonin, Caryophyllene, Diacylglycerol lipase, Hemopressin

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G protein-coupled receptors (GPCRs) — OPINION cannabinoid receptor 1 (CB1) and CB2 (REFS7,8) — and that CB1 is responsible New approaches and challenges for the psychoactive effects of marijuana5,6,9. However, to date, no specific receptor for CBD has been identified. Several different to targeting the endocannabinoid molecular targets have been suggested to mediate distinct pharmacological effects of system this cannabinoid. The identification of CB1 and CB2 led Vincenzo Di Marzo to the isolation and characterization of endogenous ligands for these proteins, Abstract | The endocannabinoid signalling system was discovered because N‑arachidonoyl-ethanolamine (AEA) receptors in this system are the targets of compounds present in psychotropic and 2‑arachidonoylglycerol (2‑AG)10–12 preparations of Cannabis sativa. The search for new therapeutics that target (FIG. 1), which were named the endo endocannabinoid signalling is both challenging and potentially rewarding, as cannabinoids13, and of five main enzymes endocannabinoids are implicated in numerous physiological and pathological for their biosynthesis and inactivation: N‑acyl-phosphatidylethanolamine- processes. Hundreds of mediators chemically related to the endocannabinoids, hydrolysing phospholipase D (NAPE-PLD), often with similar metabolic pathways but different targets, have complicated the sn‑1‑specific diacylglycerol lipase-α (DGLα), development of inhibitors of endocannabinoid metabolic enzymes but have also DGLβ, fatty acid amide hydrolase 1 (FAAH) stimulated the rational design of multi-target drugs. Meanwhile, drugs based on and monoacylglycerol lipase (MAGL; also 14–17 botanical cannabinoids have come to the clinical forefront, synthetic agonists known as MGL) . This system of two designed to bind cannabinoid receptor 1 with very high affinity have become a signalling lipids, their two receptors and their metabolic enzymes became known as societal threat and the gut microbiome has been found to signal in part through the endocannabinoid system and was soon the endocannabinoid network. The current development of drugs that alter assigned a wealth of physiological roles that endocannabinoid signalling and how this complex system could be went far beyond what could be predicted pharmacologically manipulated in the future are described in this Opinion article. from the pharmacological actions of THC18. Later, alterations in endocannabinoid signalling, owing to changes in the expression and function of cannabinoid receptors and Along with its controversial recreational Despite the fact that CBD and THC were endocannabinoid metabolic enzymes, as use, the recent history of psychotropic identified at almost the same time, most of well as modified endocannabinoid tissue preparations of Cannabis sativa has the pharmacological efforts were dedicated concentrations, were found to be associated been marked by their ever increasing, to understanding the mechanism of action with diverse pathological conditions. although mostly anecdotal, therapeutic of THC, and, initially, relatively few studies Therapies that exploit or correct such applications for a wide variety of ailments1. investigated the effects of CBD. This was alterations might therefore be developed When, in the mid‑1950s and 1960s2–4, the possibly for two reasons: the need to fully from agonists or antagonists of CB1 or CB2 first chemical constituents of cannabis appreciate the potential toxicological and or from inhibitors of endocannabinoid were identified, it was imagined that the addictive effects of the principal psychoactive degradation or biosynthesis19. medicinal activity of this plant could be component of what had become (and still is) The road to the clinical development of teased out from its psychotropic effects. the most widely used illicit drug in Western synthetic endocannabinoid system-based The first cannabinoids to be chemically societies; and the fact that the varieties of drugs (TABLE 1) has been paved with characterized, cannabidiol (CBD)3 and cannabis used recreationally, in which THC great hopes and bitter disappointments. Δ9-tetrahydrocannabinol (THC)4, were the was most abundant, were perceived as the In the meantime, plant cannabinoids, most abundant members of this class of ones with the most promising therapeutic particularly CBD, either alone or in natural products in the dried and heated effects. However, there was really no reason combination with THC, have come back flowers of C. sativa varieties that are used to believe that such effects were exclusively into the limelight as efficacious and for the production of hemp and marijuana, caused by THC nor that preparations from moderately safe therapeutic drugs20,21. respectively. Accordingly, CBD was found non-psychotropic varieties of cannabis, Several endocannabinoid-based drugs and to be non-psychotropic, whereas THC is in which CBD was often most abundant, their therapeutic applications have been responsible for the psychoactive effects of would not have therapeutic value. We thoroughly reviewed in this journal19. Here, marijuana5,6. now understand that THC acts via two I provide some explanation of what has gone

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wrong so far with these molecules and Targeting cannabinoid receptors anecdotal data had previously suggested why. I also discuss various possible ways CB1 and CB2. CB1 is possibly the most that marijuana is effective. Subsequent out of this impasse and highlight the role abundant and widespread GPCR in the preclinical studies confirmed that the of the gut microbiota as a potential source mammalian brain. This has made the activation of CB1 in peripheral and central of information for new endocannabinoid- therapeutic use of THC very difficult nervous system (CNS) cells (including based therapies. in pathological conditions for which but not exclusively neurons) might be beneficial in neuropathic and inflammatory OA and other primary fatty acid amides pain; neuropsychiatric disorders including anxiety, depression and post-traumatic stress disorder; neurological diseases NAEs Non speciﬁc targets (including CB1) 2-AcGs such as multiple sclerosis and Huntington disease chorea; and inflammatory bowel PPAR γ disorders22–25. By contrast, the use of CB1 PPARα antagonists or inverse agonists for conditions in which CB1 overactivation may contribute DHEA to the progression or symptoms of a disease — such as obesity, type 2 diabetes, GPR110 hepatic or kidney disorders and even some neurological conditions such as Alzheimer PEA disease and schizophrenia24,26,27 — has been GPR55 proposed. However, the endocannabinoid system is often activated on demand and in a cell-specific and time-specific manner during pathological states to exert a homeostatic GPR119 function19. Hence, the use of systemic direct or inverse agonists, which indiscriminately activate or inhibit the function of all CB1 molecules, can interfere with normal CB1 CB2 function in non-target cells. Typical examples of unwanted effects of such compounds are OEA LEA AEA 2-AG 2-OG 2-LG the intoxication associated with CB1 agonists (which might also interfere with cognitive CB1 functions)28 and the anxiety and depression caused by CB1 antagonists or inverse agonists29. Thus, rimonabant (Acomplia), a CB1 inverse agonist marketed in Europe TRPV1 N-acyl- N-acyl- serotonins dopamines in 2006 for the treatment of obesity and the metabolic syndrome, had to be withdrawn in 2008 because it induced depression and Ca 3 N-acyl- N-acyl- v suicidal ideation in a subset of patients29. taurines glycines For CB1 activators, marinol (botanical THC) and nabilone (a synthetic THC GPR18 analogue) are employed for the treatment of cachexia in cancer and AIDS and for chemotherapy-induced nausea, but their very narrow therapeutic window prevents their TRPV4 widespread use30 (TABLE 1). N-acyl- Peripherally restricted agonists and ? serines antagonists for CB1 (compounds with very Lipoamino acids Acyl neurotransmitters low penetration through the blood–brain barrier) were next designed and found to be Figure 1 | Endocannabinoidome mediators and receptors. The main endocannabinoids, devoid of CNS side effects31. Efforts have also N-arachidonoyl-ethanolamine (AEA) and 2‑arachidonoylglycerol (2Nature‑AG), are Reviews part of |larger Drug familiesDiscovery of been dedicated to develop indirect agonists: lipids, the N-acylethanolamines (NAEs) and the 2‑acylglycerols (2‑AcGs), respectively. Numerous other drugs that act on only the populations members of these families signal through other G protein-coupled receptors (GPCRs), ion channels of receptors that are subject to defective and nuclear receptors, as shown. In addition, long-chain primary fatty acid amides, lipoamino acids and acyl neurotransmitters signal through some of the receptors used by NAEs and 2‑AcGs. 2‑LG, activation by endocannabinoids. These 2+ indirect agonists inhibit the inactivation of 2‑linoleoyl glycerol; 2‑OG, 2‑oleoyl glycerol; Cav3, T-type Ca channel; CB, cannabinoid receptor; DHEA, N‑docosahexaenoyl ethanolamine; GPR18, G protein-coupled receptor 18; LEA, N‑linoleoyl endocannabinoids by catabolic enzymes ethanolamine; OA, oleamide; OEA, N-oleoylethanolamine; PEA, N-palmitoylethanolamine; PPAR, (see section below on endocannabinoid peroxisome proliferator-activated receptor; TRPV1, transient receptor potential cation channel sub- metabolism)32–36 or enhance their activity family V member 1. through positive allosteric modulation37–39.

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Table 1 | Selected clinically tested, synthetic and botanical endocannabinoid system-based drugs Drug Mechanism of Indications Current status Reasons for limited use Refs and action or failure ClinicalTrials. gov identifiers Synthetic CB1 or CB2 modulators Nabilone (Cesamet) CB1 and CB2 agonism Nausea and emesis in Marketed in the USA Narrow therapeutic 242 patients with cancer and elsewhere window Rimonabant CB1 inverse agonism Obesity, type 2 diabetes Withdrawn Psychiatric side effects 243 (Acomplia) and/or antagonism and dyslipidaemia (depression, anxiety and suicidal ideation) in target patient population GW842166 CB2 agonism Pain Terminated No efficacy in clinical 244 trials S-777469 CB2 agonism Atopic dermatitis Phase II trial recently NA 245 completed NCT00703573 JBT‑101 CB2 agonism Systemic lupus Phase III trial ongoing NA 246 erythematosus, (systemic scleroderma) NCT03398837, scleroderma, and phase II trial NCT03451045, dermatomyositis and ongoing or completed NCT02466243, cystic fibrosis for other indications NCT03093402 and NCT02465437 APD371 CB2 agonism Abdominal pain in Phase IIa trial recruiting NA NCT03155945 Crohn’s disease Namacizumab CB1 negative Nonalcoholic Phase I trial planned NA 247 allosteric modulation steatohepatitis SAB378 CB1 and CB2 HIV-associated Terminated NA 248 agonism (peripherally neuropathy restricted) NEO1940 CB1 and CB2 Cancer and anorexia or Phase I trial NA 249 agonism (peripherally weight loss associated completed and restricted) with cancer further development announced Synthetic endocannabinoid metabolism modulators PF‑04457845 FAAH inhibition Osteoarthritic pain Phase II trial completed No substantial effect on 34 primary end point URB597 FAAH inhibition Symptoms of Phase I trial not yet NA NCT00916201 schizophrenia recruiting V158866 FAAH inhibition Spinal cord Completed Failed to meet primary NCT01748695 injury-induced end point neuropathic pain JNJ‑42165279 FAAH inhibition Social anxiety disorders, Phase II trial recruiting NA NCT02432703 major depressive and disorder with anxious NCT02498392 distress BIA 10‑2474 FAAH inhibition with Anxiety, Parkinson Phase II trial One death and mild to 35,36 other targets disease, chronic pain, discontinued severe injury in four other cancer and hypertension subjects in a phase II clinical trial PF‑06818883 MAGL inhibition Cerebral haemorrhage Phase I trial Safety issues NCT03020784 discontinued ABX‑1431 MAGL inhibition Tourette syndrome, Phase Ib trial completed; NA 250 neuromyelitis optica, encouraging results NCT03138421 neuralgia, myelitis, communicated by the and neuropathies and developers NCT03447756 multiple sclerosis Phytocannabinoids THC (dronabinol; CB1 agonism and CB2 Nausea and emesis in Marketed in the USA Narrow therapeutic 251 Marinol) partial agonism patients with cancer and elsewhere window and wasting syndrome in patients with AIDS

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Table 1 (cont.) | Selected clinically tested, synthetic and botanical endocannabinoid system-based drugs Drug Mechanism of Indications Current status Reasons for limited use Refs and action or failure ClinicalTrials. gov identifiers Phytocannabinoids (cont.) Botanical CB1 agonism and Spasticity and pain Marketed in over 30 NA 21,179,183,184 THC + CBD CB2 partial agonism in multiple sclerosis, countries for spasticity NCT01812603 (nabiximols; Sativex (see TABLE 3 for other glioblastoma and cancer in multiple sclerosis and in the USA) targets of CBD) pain NCT01812616 Botanical CBD Multi-target (see Refractory paediatric Approved by the FDA NA 20,195–197,252 (for example, TABLE 3) epilepsies (Dravet for the treatment of Epidiolex and other and Lennox–Gastaut convulsions in Dravet formulations) syndromes) and and Lennox–Gastaut schizophrenia syndromes THCV Multi-target (see Type 2 diabetes and Two phase II trials Primary end points were 194 TABLE 3) dyslipidaemia completed not met CB, cannabinoid receptor; CBD, cannabidiol; FAAH, fatty acid amide hydrolase 1; MAGL, monoacylglycerol lipase; NA, not applicable; THC, Δ9-tetrahydrocannabinol; THCV, Δ9-tetrahydrocannabivarin.

These strategies would restore the Other endocannabinoid receptors. The channels for neurotransmitters, including, for site-specificity and time-specificity of discovery of the first endocannabinoid, example, serotonin, glycine and the GABAA 19 10 REF.62 endocannabinoid activity . Inhibitors of AEA , in 1992 was soon accompanied receptor (GABAAR) (see for a recent endocannabinoid biosynthesis (see section by the realization that this compound review), although the physiopathological below on endocannabinoid metabolism)40–42 had numerous molecular targets. AEA relevance of these interactions is yet to and CB1 negative allosteric modulators43 are and other endocannabinoids bind to be clarified. in the early stages of development; inhibitors L‑type Ca2+ channels50,51, produce other From these studies, evidence for the of endocannabinoid biosynthesis have non‑CB1‑mediated and non‑CB2‑ existence of ionotropic receptors that interact been investigated in preclinical studies of mediated effects in vivo52 and induce with both endocannabinoids and plant obesity and inflammatory and neuropathic GTP-mediated signalling in brain membranes cannabinoids has increased. Numerous pain44–46. Recently, the first biologic drug in from Cnr1‑knockout mice53. In the following reports have shown that AEA (and more the endocannabinoid field, namacizumab, decades, pharmacological evidence emerged recently 2‑AG) can bind to and activate a negative allosteric antibody that stabilizes for the existence of other receptors that could transient receptor potential cation channel CB1 in an inactive conformation, was mediate some of the effects of AEA, THC and subfamily V member 1 (TRPV1) both submitted for approval to initiate a phase 1a/b synthetic THC-mimetic aminoalkylindoles — in vitro and in vivo (see REF.63 for a recent clinical trial for nonalcoholic steatohepatitis such as WIN 55,212 (REFS53,54) — in the brain, review). These channels were previously (see Related links), a frequent comorbidity and the effects of a CBD isomer — abnormal considered orphan and believed to be of abdominal obesity. Importantly, cannabidiol — in vascular endothelial cells55. activated by only heat (>42 °C), protons namacizumab, like other biologics, is likely to Although these receptors have not yet been and the hot chilli pepper component be peripherally restricted. characterized, the activity of some orphan capsaicin. They were later found to also CB2 is predominantly expressed in GPCRs, such as GPCR 55 (GPR55)56,57, be activated by CBD and other non-THC immune tissues and cells but is also present GPR18 (REF.58) and GPR110 (also known as cannabinoids, such as cannabigerol (CBG) at low levels in neuronal and non-neuronal ADGRF1)59, can be modulated by THC, CBD and Δ9-tetrahydrocannabivarin (THCV) (see (for example, activated microglia) cells of the and/or AEA and/or some of its congener the section below on phytocannabinoids), as brain47. Since its discovery in 1993 (REF.8), it N‑acylethanolamines (NAEs), although this well as by other long-chain fatty-acid amides has been seen as a promising target for the evidence is not without some controversy60,61. (denoted as N‑acyl-amides) and esters64–66. treatment of inflammatory and autoimmune Most of these interactions, even when they AEA and several plant cannabinoids, diseases and more recently for liver and occur in vitro at concentrations similar to including THC, also antagonize TRP cation kidney fibrosis48–49. Numerous CB2 agonists those necessary for AEA to activate CB1 or channel subfamily M member 8 (TRPM8), have been designed, and several clinical CB2, have not been demonstrated to occur which is activated by menthol and low trials have been initiated (TABLE 1), but few in vivo and validated through the use of temperatures63,67. Finally, whereas high outcomes have been reported48. Nevertheless, knockout mice. Furthermore, the lack of micromolar concentrations of AEA are particularly because it is often overexpressed high-affinity radiolabelled ligands for most of required to activate TRPA1 (the mustard oil during pathological conditions in selected these orphan GPCRs has thus far prevented receptor) and TRPV2 (also activated by heat) cells, CB2 is still considered a potential target the experimental demonstration of their in vitro, CBD and other plant cannabinoids for specific, and hence safe, therapeutic direct interaction with AEA or cannabinoids, can produce these effects at submicromolar drugs48. The involvement of this receptor in and this interaction has not been suggested concentrations (discussed in more detail in neurogenesis as well as in neurodegenera‑ by other structural biology approaches. the phytocannabinoids section)67–69. tive, neuroinflammatory and, albeit more Other reports have suggested that AEA, and In summary, endocannabinoids interact controversially, neuropsychiatric disorders47 more recently 2‑AG, are similar to some with multiple receptors. Therefore, altering indicates that future clinical development of non-THC cannabinoids in their capability the levels of endocannabinoids with the CB2 agonists could include these indications. to bind to GPCRs and ligand-activated ion purpose of indirectly manipulating CB1

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and CB2 activity may not be safer or more Targeting endocannabinoid metabolism on the resulting diacylglycerols (DAGs); selective than directly targeting these AEA and 2‑AG belong to two large, hence, 2‑AcGs are derived ultimately from receptors. In particular, the capability of distinct families of lipids, the NAEs and the lipid present at the sn‑2 position of endocannabinoids to activate TRPV1, which, the 2‑acylglycerols (2‑AcGs), respectively. phospholipids, which is most commonly contrary to CB1 and CB2, exacerbates pain AEA is one of the least abundant NAEs, arachidonic acid (FIG. 2). DGLα and DGLβ and neurogenic inflammation63, may explain with levels of N‑stearoylethanolamine inhibitors40–44 are in the early stages of in part why inhibitors of FAAH, the enzyme (SEA), N‑palmitoylethanolamine (PEA) development and have been suggested to be mostly responsible for AEA hydrolysis, have and N‑oleoylethanolamine (OEA) usually useful in preclinical studies of obesity and not been successful in clinical trials (discussed at least tenfold higher in most of the tissues inflammatory pain, respectively44–46. below). In fact, AEA has been suggested to and cells analysed thus far76. Conversely, By contrast, NAEs are derived be more efficacious at activating human than 2‑AG is among the most abundant 2‑AcGs. from the processing of N‑acyl- rat recombinant TRPV1 (REFS70,71), and in phosphatidylethanolamines (NAPEs), which preclinical settings, FAAH inhibition often Endocannabinoid biosynthesis. The in turn are formed by the transfer of an acyl unmasks the TRPV1‑mediated effects (for 2‑AcGs are synthesized through the chain from the sn‑1 position of phospholipids

example, proalgesic) of AEA and its NAE sequential action of phospholipase C (PLC) to the NH2 group of phosphatidylethanola‑ congeners72–75. on phospholipids and DGLα or DGLβ mines. Arachidonic acid is one of the least

NAEs 2-AcGs

GPR55 NATs Phospholipids (using Phospholipids (namely, Phosphatidylethanolamines Phosphatidic acids the sn-1-acyl chain) phosphatidylinositols)

Acyl- PA phospho- PLCβ PLA1A NAPEs CoA hydrolase

ABHD4 DAGs sn-1-lysophospholipids PLC sPLA2 NAPE-PLD (x2) (eliminating the (namely, sn-1-lyso- sn-1-acyl chain) phosphatidylinositols) 2-lyso-N-acyl- Glycero- Phosphoacyl- phosphatidyl- phosphoacyl- ethanolamides ethanolamines ethanolamides sn-1-lysophosphatidic acids DGLα or Lyso-PLC DGLβ LPA PTN22 Lyso-PLD GDE1 phosphatase

NAEs 2-AcGs LPA1–LPA3

Amino acids Serotonin Dopamine Acyl- Acyl- CoA CoA ? ? FA GLYATL3 AANATL2 AANATL2 ?

N-acyl- N-acyl- N-acyl- N-acyl- N-acyl- taurines serines glycines serotonins dopamines

5-HT receptors D1–D3 FAAH

Lipoamino acids Acyl neurotransmitters

Figure 2 | Synthesis of the endocannabinoidome mediators. Although N‑acyl-glycines, for which the anabolic processes and their role as biosyn- N‑acyl-phosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) thetic precursors of primary amides are wellNature understood. Reviews |5 Drug‑HT, 5 Discovery‑hydroxy- and sn‑1‑specific diacylglycerol lipase-α (DGLα) or DGLβ are considered to tryptamine; AANATL2, arylalkylamine N‑acyltransferase-like 2, isoform A;

be the main enzymes involved in the biosynthesis of the N‑acylethanolamines ABHD4, α/β‑hydrolase 4; D1, dopamine receptor 1; DAGs, diacylglycerols; (NAEs) and 2‑acylglycerols (2‑AcGs), respectively, numerous other potential DHEA, N‑docosahexaenoyl ethanolamine; FA, fatty acid; FAAH, fatty acid biosynthetic pathways exist. In addition, some of the precursors are signalling amide hydrolase 1; GDE1, glycerophosphodiester phosphodiesterase 1; lipids themselves. Among the sn‑1‑lysophospholipids, only lysophosphatidy- GLYATL3, glycine N‑acyltransferase-like protein 3; LPA, lysophosphatidic acid; linositols have been shown to activate G protein-coupled receptor 55 LPA1, LPA receptor 1; NAPEs, N‑acyl-phosphatidylethanolamines; NATs, (GPR55)60. Limited knowledge is available regarding the biosynthesis of N‑acyl N‑acyltransferases (including phospholipase A2 group IVE and phospholipase neurotransmitters, and this has only been studied in Drosophila melano‑ A/acyltransferase 1); PA, phosphatidic acid; PLA1A, phospholipase A1 mem- gaster, in which the corresponding neurotransmitters play important roles. ber A; PLC, phospholipase C; PTPN22, tyrosine-protein phosphatase non- Similarly, little is known about the biosynthesis of lipoamino acids, except for receptor type 22; sPLA2, soluble phospholipase A2.

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abundant esterified fatty acids at the sn‑1 and no biosynthetic pathway specific to In summary, it may be difficult to position, explaining the relatively low levels the generation of polyunsaturated NAEs, manipulate, either pharmacologically or of AEA compared with other NAEs. NAPEs such as AEA and N‑docosahexanoyl- genetically, the biosynthetic pathways of AEA can be produced by both Ca2+-dependent ethanolamine (also known as and 2‑AG without also interfering with the and Ca2+-independent acyltransferases, and synaptamide)59, has been identified so far. biosynthesis of related NAEs and 2‑AcGs. several pathways can convert them to NAEs As a consequence, AEA is thought to be (FIG. 2). Although Ca2+-independent NAPE- biosynthesized using the same pathway Endocannabinoid catabolism. synthesizing acyltransferases seem to be as other, usually more abundant, NAEs. Similar considerations apply to most predominantly involved in the formation of Likewise, 2‑AG may be released together endocannabinoid inactivation pathways bioactive saturated and monounsaturated with its congener 2‑AcGs, although these (FIG. 3). All long-chain NAEs are substrates NAEs, such as OEA, PEA and N‑linoleoyl- are usually less abundant. NAEs and 2‑AcGs for FAAH, which hydrolyses AEA to ethanolamine77, Ca2+-dependent acyltrans‑ other than AEA and 2‑AG have receptors arachidonic acid and ethanolamine16, ferases, as well as distinct NAPE-converting and biological effects of their own (FIG. 1), although this enzyme has less affinity enzymes, have not been selectively assigned and thus, they should not be considered for saturated NAEs than for AEA. to the biosynthesis of any specific NAE78, endocannabinoids (see below). N‑Acylethanolamine-hydrolysing acid

FPs, EPs, DPs, leukotriene CB1 CB2 FP FPAlt4 P2Y6 LPA1–LPA3 receptors, etc.

Hydroperoxy-N- Prostaglandin arachidonoyl- glycine dopamine endoperoxide Hydroxy-eCBs Prostamide F2α PGE2-G and epoxy-eCBs 2-oxo- Hydroperoxy-N- arachidonoyl- arachidonoyl- Prostaglandin serotonin glycine synthases sn-1-lysophos- COX2 p450s Endoperoxides phatidic acids LOX12 p450 LOX12 and LOX15 COX2 2U1 and LOX15

Only the arachidonoyl homologues MAGK

PAM

N-acyl- N-acyl- N-acyl- N-acyl- Primary amides serotonins taurines serines glycines (e.g. oleamide) OEA, PEA and LEA AEA 2-AG 2-OG, 2-PG and 2-LG

N-acyl- ? dopamines

FAAH (also FAAH2 in NAAA ABHD6 ABHD12 MAGL humans for OEA and LEA) (for PEA) COMT

Glycerol + Fatty acid

Prostanoids O-methylated Taurine, glycine, NH , Fatty Ethanol- Palmitic Arachidonic acid COXs and 2 + + Glycerol + N-acyl-dopamines ethanolamine or glycerol acid amine acid or other fatty acids LOXs leukotrienes

| 239 Figure 3 Catabolism of the endocannabinoidome mediators. Oxidizing prostaglandin F2α receptor (FP) and its splicing variant 4 (FPAlt4) . Several enzymes (indicated in medium blue) and more pathway-specific serine hydro- NAEs, including AEA, as well as 2‑AG have also been suggested to have non- lases are primarily responsible for the catabolism of endocannabinoidome specific intracellular carriers (not shown in the figure) such as fatty-acid- signalling molecules. Fatty acid amide hydrolase 1 (FAAH) and mono binding proteins240 and heat shock 70 kDaNature protein Reviews (HSP70) | Drug (REF. Discovery241), which acylglycerol lipase (MAGL) are thought to be the primary enzymes responsible seem to be important for their inactivation. 2‑LG, 2‑linoleoyl glycerol; 2‑OG, for the catabolism of N-arachidonoyl-ethanolamine (AEA) and 2‑arachi- 2‑oleoyl glycerol; 2‑PG, 2‑palmitoyl glycerol; ABHD, α/β‑hydrolase; CB, can- donoylglycerol (2‑AG) respectively. Products of oxidation (indicated in red) nabinoid receptor; COMT, catechol-O‑methyltransferase; COX, cyclo often signal through G protein-coupled receptors (GPCRs), as shown. The oxygenase; DPs, prostaglandin D2 receptors; eCBs, endocannabinoids; EPs, thickness of the arrows indicates the importance of the pathway in metabo- prostaglandin E2 receptors; LEA, N‑linoleoyl ethanolamine; LOX, arachido- lite degradation. N-Acylethanolamines (NAEs) are shown in light blue, nate lipoxygenase; LPA1, lysophosphatidic acid receptor 1; MAGK, mono 2‑acylglycerols (2‑AcGs) are shown in grey, lipoamino acids are shown in light acylglycerol kinase; NAAA, N-acylethanolamine-hydrolyzing acid amidase; green, and the acyl neurotransmitters are shown in pink. FAAH (dark blue) OEA, N-oleoylethanolamine; P2Y6, P2Y purinoceptor 6; p450 2U1, catabolizes multiple endocannabinoidome signalling molecules. The recep- cytochrome p450 2U1; PAM, peptidyl-glycine α‑amidating monooxygenase; tor for prostamide F2α has been suggested to be a heterodimer between the PEA, N-palmitoylethanolamine; PGE2-G, prostaglandin E2-glycerol.

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amidase (NAAA), which has a tissue A strong impetus seems to have been put on and prostaglandin glycerol esters (PG‑Gs), and cell distribution quite different from producing and testing several chemotypes of which may act on their own receptors that of FAAH, predominantly hydrolyses inhibitors of MAGL87 and determining the and often produce pronociceptive and saturated NAEs, particularly PEA79. preclinical efficacy of those that are selective pro-inflammatory effects100–104. Thus, Furthermore, FAAH also hydrolyses other for this enzyme versus those that also inhibit manipulation of endocannabinoid metabolic long-chain N‑acyl-amides, such as the FAAH88. This push towards MAGL inhibitors enzymes could affect not only the levels N‑acyl-taurines and the primary amides may be the result of the present pause in the of endocannabinoid congeners but also (such as the sleep-inducing factor oleamide), clinical development of FAAH inhibitors. those of other biochemically related, but which preferably modulate non‑CB1 and The pharmacological and genetic functionally unrelated, lipid mediators. In non‑CB2 receptors16,80,81. MAGL is not manipulation of endocannabinoid catabolic theory (often supported by experimental selective for 2‑AG over other 2‑AcGs or enzymes is likely to affect other NAEs and evidence), inhibition of DGLs reduces even other monoacylglycerols82; other 2‑AcGs and hence interfere with signalling levels of 2‑AG, as well as prostanoid and esterases, such α/β‑hydrolase 6 (ABHD6) pathways other than CB1 and CB2. Indeed, LPA signalling downstream of 2‑AG105, and ABHD12, which have been suggested treatment of mice or humans with FAAH but increases DAG and protein kinase C to catalyse the hydrolysis of 2-AcGs, are inhibitors increases the levels of AEA as well signalling. Inhibition of NAPE-PLD, the likewise non-selective83. Oxidation of endo‑ as OEA and PEA36,89 and often unmasks enzyme that catalyses the direct conversion cannabinoids occurs via the arachidonate effects mediated by peroxisome proliferator- of NAPEs into NAEs14, reduces AEA cascade, which includes cyclooxygenase 2 activated receptor-α (PPARα) or TRPV1, signalling at cannabinoid receptors and (COX2; also known as PTGS2)84, 12- and for which OEA and PEA also act as TRPV1, as well as NAE signalling at PPARα, 15-lipoxygenases85, and some cytochrome agonists72–75,90,91,92. Likewise, inhibition but will also reduce the levels of the other p450 oxygenases86. NAEs and 2‑AcGs of MAGL elevates the tissue levels of bioactive product of the hydrolytic reaction, with saturated acyl chains are less likely numerous monoacylglycerols, and this may phosphatidic acid. Levels of NAPE-PLD to be metabolized through oxidation, be accompanied by a beneficial metabolic substrates, which have been shown to affect whereas those with monounsaturated and phenotype that is more reminiscent of metabolism, are also increased106. Finally, polyunsaturated chains can be recognized by indirect activation of some of the targets of inhibition (or genetic inactivation) of FAAH some of these enzymes. non-endocannabinoid 2‑AcGs (such as the or MAGL increases the levels of AEA or Several different chemotypes of inhibitors orphan receptor GPR119)93 than of CB1. 2‑AG, respectively, but may also increase of FAAH have been developed32,33. At least Clearly, as shown in most of the studies their oxidation by COX2 and hence enhance four of them, PF‑04457845, BIA 10–2474, on selective FAAH and MAGL inhibitors prostamide and PG‑G signalling102,107. For V158866 (ClinicalTrials.gov identifier reviewed here, these compounds generally MAGL, this metabolic shift may also reduce NCT01748695) and JNJ‑42165279 elicit effects that can be antagonized in prostanoid98,99 and increase LPA105 receptor (NCT02432703 and NCT02498392), have rodents by CB1 or CB2 antagonists94,95. activity. been administered to humans, and a fifth, However, a consequence of the redundancy In summary, the above considerations URB597, is about to be tested in patients and promiscuity of endocannabinoid suggest that, as for numerous target with schizophrenia (NCT00916201). catabolic enzymes is that the effects of their pathways, great care must be taken when PF‑04457845 was tested in a clinical trial inhibition can be unpredictable, although interpreting studies that pharmaco‑ for inflammatory (osteoarthritic) pain, and the premise that endocannabinoids are logically manipulate endocannabinoid although it was safe and elevated plasma produced and released on demand might anabolic and catabolic enzymes. In most NAE levels, it was inefficacious34. The mitigate some of this unpredictability. cases, generalized knockout of the major clinical development of BIA 10–2474 for endocannabinoid hydrolysing enzymes the treatment of anxiety, Parkinson disease, Other mediators involved in is accompanied by phenotypes that chronic pain, multiple sclerosis, cancer and endocannabinoid metabolism. In addition are sensitive to cannabinoid receptor hypertension had to be interrupted following to being biosynthesized and inactivated antagonism (for example, see REF.108). These a disastrous trial in which one volunteer together with bioactive congeners, AEA and phenotypes are therefore due to elevation of was killed and another seriously harmed for 2‑AG can be produced from, and/or endocannabinoid levels in tissues, although reasons that are yet to be fully established35 catabolized to, other lipid mediators. MAGL genetic inactivation can also produce but are likely due to an interaction of the A particular species of lysophosphatidic CB1 desensitization owing to the subsequent drug with critical targets other than FAAH acid (LPA), sn‑1‑lyso‑2‑arachidonoyl- elevation of levels of 2‑AG, which functions in the brain36. Other FAAH inhibitors phosphatidic acid, which preferentially as a CB1 agonist 109,110. However, cell-specific tested in humans, such as PF‑04457845 and activates the GPCRs LPA receptor 2 (LPA2), and time-specific roles for each of these V158866, were well tolerated even following LPA1 and LPA3 (REF.96), is both a biosynthetic enzymes may exist, possibly during different extensive testing. precursor and a metabolic product of 2‑AG97 physiological and pathological conditions. More recently, the development of MAGL (FIG. 3). Hydrolysis of 2‑AG can also generate Thus, studies using inducible conditional inhibitors has also proceeded steadily, and arachidonic acid, which is a precursor of knockouts of the corresponding genes or at least two such compounds, ABX‑1431 prostanoids, although this occurs in an their mRNA transcripts might cast light on (Abide Therapeutics) and PF‑06818883 organ-dependent manner (in the brain which enzyme needs to be inhibited, and (Pfizer), have entered phase I clinical trials. but not in the gastrointestinal tract)98,99. when and where it needs to be inhibited, Although promising results were announced Endocannabinoids are also oxidized by to selectively affect the levels of particular for ABX‑1431 (see Related links), the COX2, and the ensuing endoperoxides are endocannabinoids and specifically modulate trial with PF‑06818883 was interrupted converted by prostaglandin synthases to the activity of one or more endocannabinoid because of safety reasons (NCT03020784). prostaglandin ethanolamides (prostamides) receptor. Such studies will need to include

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not only the observation of the ensuing the name ‘endocannabinoidome’ was to make sense of its overall physiological phenotypes but also quantitative analysis proposed129–131. The endocannabinoidome and pathological role. In particular, two of the tissue (and, when possible, cellular) includes these lipid mediators, as well as the questions will need to be addressed. First, levels of both endocannabinoids and their aforementioned endocannabinoid congeners given the commonalities among the related mediators, preferably at different (the NAEs and 2‑AcGs), the N‑acyl-taurines regulatory and signalling mechanisms of ages and physiopathological settings. Only and the fatty-acid primary amides, bioactive distinct endocannabinoidome mediators, through the assessment of which lipid endocannabinoid metabolites (such as do these act in concert, with different mediators are affected and to what extent prostamides, PG‑Gs, lipoxygenase or or redundant roles, to turn on and off they are affected can one surmise what type cytochrome p450 oxygenase products cellular responses to external homeostasis- of receptors are modulated by the genetic and sn‑1-lyso-2‑arachidonoyl‑LPA) and perturbing stimuli, thus resulting in fine (or pharmacological) manipulation of their biosynthetic precursors (NAPEs and physiological tuning or the emergence of endocannabinoid anabolic and catabolic DAGs), and the many molecular targets and pathological states? Second, do specific enzymes. metabolic enzymes (several of which are yet profiles for these hundreds of mediators exist unidentified) of all these molecules. Thus, for different individuals or distinct diseases, The endocannabinoidome the endocannabinoidome is a considerable and will deciphering these signatures lead to In the past 15 years, numerous bioactive expansion from the initial two mediators, new personalized diagnoses and treatments? N‑acyl-amides have been identified, five enzymes and two receptors of the and their biosynthesis, inactivation and endocannabinoid system129–131. Multi-target drugs function have been investigated. These Predicting the effects of the manipulation On the basis of the considerations described putative lipid mediators (FIG. 1) often share of endocannabinoidome enzymes, such as in the previous sections, manipulating receptors and/or catabolic enzymes with MAGL, FAAH, the DGLs and NAPE-PLD, endocannabinoid levels without endocannabinoids and their congeners. is particularly difficult because several affecting other mediators that are related Examples include the N‑acyl-amino endocannabinoidome receptors do not biochemically, although not necessarily acids (or lipoamino acids), such as the necessarily play similar roles in pathological functionally, to the endocannabinoids is N‑acyl-glycines, which are inactivated conditions. For example, TRPV1 and often difficult. Directly interfering with the by FAAH and can activate the orphan prostamide F2α receptors have opposite activity of CB1, and even CB2, with agonists GPCR GPR18 and, like AEA, can inhibit effects to CB1 and CB2 on pain, and insulin and antagonists has also been problematic. 2+ 58,111–114 T‑type Ca channels (Cav3s) , and sensitivity is increased by GPR119, a target One way to deal with endocannabinoid the N‑acyl-serines, which have important for some 2‑AcGs and NAEs, and inhibited target promiscuity and metabolic enzyme biological effects in vivo and for which by CB1, as mentioned above72,73,90,100–103,132. redundancy is to develop drugs with anabolic and catabolic pathways, as well Furthermore, GPR55, a proposed (and still multiple but specific groups of targets. For 114 as direct targets (other than Cav3s) , are controversial) target for endocannabinoids, example, one of the possible consequences not yet identified114–116. Examples of acyl seems to exacerbate cancer growth, whereas of inhibiting endocannabinoid catabolic neurotransmitters include the N‑acyl- CB1 and CB2 have the opposite effect133,134. enzymes is to activate receptors other dopamines, most of which activate TRPV1 LPA1–LPA3, again contrary to the effects than CB1 and CB2, which could obstruct 114,117–121 and inhibit FAAH and Cav3s , and of CB1 and CB2, contribute to pain and any beneficial effects or cause unwanted the N‑acyl-serotonins122, which inhibit cancer growth135,136. GPR55 also seems to side effects. Therefore, new compounds FAAH and often antagonize TRPV1 but activate pain pathways but, contrary to containing one pharmacophore that 137,138 also inhibit Cav3s and whose biosynthesis CB1, ameliorates glucose tolerance . inhibits FAAH or MAGL and another so far has been investigated in only PPARα, for which some NAEs are agonists, that antagonizes these non-cannabinoid Drosophila melanogaster123–126. Furthermore, and CB1 have opposing roles in regulating receptors could be therapeutically useful. other long-chain N‑acyl-amides, lipid accumulation in the liver or in nicotine Several multi-target drugs that modulate such as N‑arachidonoyl-tyrosine and addiction (inhibitory and stimulatory, endocannabinoidome receptors and 91,139,140 N‑arachidonoyl-tyramine, N‑acyl-GABAs, respectively) , and prostamide F2α enzymes, either obtained by rational design N-acyl-alanines, N-acyl-methionines, receptors and CB1 have anti-adipogenic or found in a serendipitous manner, already N-acyl-prolines, N-acyl-valines, N-acyl- and pro-adipogenic effects, respectively, in exist (TABLE 2). For example, olvanil, a leucines, N-acyl-isoleucines and N-acyl- preadipocytes141. non-pungent TRPV1 agonist, was originally phenylalanines, have been identified66,127,128, To further complicate matters, several developed (but never marketed) as an although their metabolism and/or biological endogenous and environmental stimuli analgesic, as it could rapidly desensitize effects have not yet been investigated. Any (such as the relative amounts of different TRPV1 and was later found to also inhibit bioactive or biogenic amine might be able fatty acids in the diet) are predicted to endocannabinoid transport across the cell to form an amide with any long-chain affect the tissue concentrations of several membrane144. This as-yet uncharacterized fatty acid, thereby generating hundreds endocannabinoidome mediators, which mechanism for the cellular reuptake of of novel bioactive lipids, which could may act either in concert or in competition endocannabinoids is necessary for their constitute specific signalling signatures with endocannabinoids via non‑CB1, degradation by intracellular catabolic responsible for physiological and non‑CB2 targets (including Ca2+ channels, enzymes145,146. Conversely, AM404, which pathological phenotypes. Because these TRP channels, PPARs and orphan GPCRs). was initially designed as an endocannabinoid novel mediators may have numerous Therefore, ever more sophisticated, targeted membrane transport inhibitor, was later chemical features in common with the omics (transcriptomics, proteomics and found to also activate and desensitize endocannabinoids, such as inactivating lipidomics) methodologies142,143 are required TRPV1 (REF.147) and, more importantly, to enzymes and molecular targets (FIGS. 1,2,3), to study the endocannabinoidome and be a metabolic product of acetaminophen

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Table 2 | Selected marketeda and preclinical drugs with multiple therapeutically relevant endocannabinoidome targets Drug Cannabinoid receptor and Other endocannabinoidome Potential therapeutic use Refs endocannabinoid metabolic receptor and metabolic enzyme enzyme targets targets Acetaminophen (via • FAAH (–) • TRPV1 (++) Pain and fever 147–152,253 AM404 and other • eCB transport across the • TRPA1 (++) metabolites)a membrane (– –) • COX2 (–; ns and nss) Dipyrone (via its • CB1 (+) • TRPV1 (+) Pain and fever 254 AM404‑like metabolites)a • CB2 (+) • COX2 (–; ns and nss) ARN2508 FAAH (– – –) COX2 (– – –; ns and nss) Pain without gastric mucosal 171 damage and IBDs OMDM-198 FAAH (–) TRPV1 (– –) Inflammatory pain and anxiety 158,159 OMDM-202 FAAH (–) TRPA1 (++) Inflammatory pain 158 AGN211335 and FAAH (– –) Heterodimer between FP and FPAlt4 (– –) Inflammatory pain 163 AGN211336 (R)-2- FAAH (–) COX2 (–; ns and ss) Inflammatory pain and anxiety 169 (2‑fluorobiphenyl‑4‑yl)- N-(3‑methylpyridin‑2‑yl) propanamide Compound 11e • FAAH (–) COX2 (–; ss) Inflammatory pain and anxiety 170 • eCB transport across the membrane (–) Compound 11f • CB2 (+) COX2 (–; ss) Inflammatory pain and anxiety 170 • eCB transport across the membrane (–) AGN220653 FAAH (– –) Various prostanoid receptors (DP1, DP2, Inflammatory pain 172 EP1, EP4, FPs and TP) (– –) Compound 2 and CB1 (– –) PPARα Metabolic syndrome and 173 compound 3 hepatosteatosis JZL195 • FAAH (– –) None Neuropathic and visceral pain, 88,174–178 • MAGL (– – –) nausea, pruritus and depression (but not anxiety disorders)

The strength of the activation (+) or inhibition (–) is indicated by the number of + or –. CB, cannabinoid receptor; COX, cyclooxygenase; DP, prostaglandin D2 receptor; eCB, endocannabinoid; EP, prostaglandin E2 receptor; FAAH, fatty acid amide hydrolase 1; FP, prostaglandin F2 receptor; FPAlt4, splicing variant 4 of FP; IBDs, inflammatory bowel disorders; MAGL, monoacylglycerol lipase; ns, non-selective for COX2 over COX1 (also known as PTGS1); nss, non-substrate-selective for inhibition of oxidation of 2‑arachidonoylglycerol (2‑AG) over arachidonic acid; PPARα, peroxisome proliferator-activated receptor-α; ss, substrate selective for inhibition of oxidation of 2‑AG over arachidonic acid; TP, thromboxane receptor; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPV1, transient receptor potential cation channel subfamily V member 1. aLists drugs that have been tested in clinical trials and are currently marketed.

(paracetamol) in both rodents and counteract one of the aforementioned COX inhibitors, the R‑profens, were shown humans148,149. The analgesic activity of consequences of FAAH inhibition — the to inhibit the COX2‑mediated peroxidation acetaminophen in rodents was indeed indirect activation of TRPV1, which not of endocannabinoids selectively over the shown to be mediated by indirect activation only exacerbates pain but also has been peroxidation of arachidonic acid167,168, of CB1 and activation or desensitization of implicated in anxiety and depression160,161, and some derivatives of R‑flurbiprofen, TRPV1150–152. N‑Arachidonoyl-serotonin, two other proposed indications for selective such as (R)-2-(2‑fluorobiphenyl‑4‑yl)- initially designed as an FAAH inhibitor123 FAAH inhibitors. N-(3‑methylpyridin‑2‑yl)propanamide, and later shown to be an endogenous As mentioned above, inhibition of AEA were found to also inhibit FAAH169. lipid122, was found to also antagonize TRPV1 hydrolysis can trigger AEA catabolism Functionalization of β‑caryophyllene, a (REF.124) and to have CB1‑mediated and by COX2 and the subsequent production naturally occurring CB2 agonist, generated TRPV1‑mediated analgesic, anxiolytic and of prostamides that, in some cases, have an FAAH-specific and endocannabinoid antidepressant activity in rodents124,153–156. been shown to induce inflammatory and substrate-specific COX2 inhibitor170. Adding This compound can potentially also be pronociceptive responses, thus counteracting a COX-blocking moiety to FAAH inhibitors used for inflammatory or irritable bowel one of the most theoretically promising might improve the analgesic efficacy of disorders157. In the attempt to mimic the therapeutic effects of FAAH inhibitors162. FAAH blockade in humans, and notably,

pharmacophores and improve the stability In this case, dual FAAH–prostamide F2α inserting an FAAH-blocking feature in COX of N‑arachidonoyl-serotonin, synthetic receptor inhibitors, or dual FAAH–COX inhibitors was shown to counteract their piperazinyl carbamates were designed as inhibitors, might be more efficacious at damaging effects on the gastric mucosa171. dual inhibitors of FAAH and TRPV1, and inhibiting inflammatory pain than selective Finally, a new generation of pan-prostanoid one of them, OMDM‑198 (REF.158), was FAAH inhibitors, as suggested for some receptor antagonists with FAAH inhibitory shown to effectively reduce osteoarthritic synthetic prototypes of such compounds163–166. activity produced analgesic activity via 159 pain in rats . These compounds can Interestingly, some previously developed mixed antagonism of prostaglandin E2

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receptors (EPs), prostaglandin F2 receptors of THC and CBD have had positive and safely with the multi-factorial and 180–182 (FPs) and prostaglandin D2 receptors (DPs) outcomes . In the clinic, nabiximols has multi-receptor nature of several diseases, and indirect activation of CB1 (REF.172). been tested for the treatment of cancer pain, including pain, spasticity, epilepsy and Stronger efficacy can be obtained by not with mixed but promising results183,184; the inflammatory bowel disorders. This only inhibiting targets that are activated progression of glioblastoma after recurrence model enables the prediction of whether as a consequence of inhibition of FAAH, of the tumour, with a seemingly positive a ‘therapeutic handshake’ occurs between but also by altering two or more endo outcome (see Related links); and the main the aetiopathological handprint of these cannabinoidome proteins involved in a symptoms of Huntington disease, with disorders and the polypharmacological given pathological condition. For example, disappointing results in terms of efficacy but handprint of some phytocannabinoids dual CB1 antagonists–PPARα agonists with not safety185. Of note, for pain studies, the and can be applied in principle to other nanomolar affinity could potentially be used numeric rating scale used as a primary end non-selective natural and synthetic in metabolic disorders173. Furthermore, dual point is strongly affected by an individual’s drugs200. If supported by mathematical FAAH–MAGL inhibitors, such as JZL195, expected effects and greatly varies among and other bioinformatics approaches, and have been designed and exert stronger individuals, which complicates clinical trials. together with systems biology-guided effects in several animal models of disease Previous epidemiological and preclinical methodologies, the therapeutic handshake than inhibitors selective for either enzyme studies suggest that CBD, although inactive model might predict that existing alone, at least for some indications (TABLE 2). in animal models of spasticity in multiple multi-target molecules are safe and They may be used at lower doses than sclerosis186, can substantially reduce many effective in unexpected conditions — thus single MAGL inhibitors, thereby potentially of the psychotropic side effects of THC, leading to their repositioning — and help minimizing the induction of drug tolerance thus widening its therapeutic window and to rationally design new multi-target and their addictive potential174–178. allowing administration at higher and drugs200. In summary, while the search for possibly more effective doses187–190. This The clinical success of nabiximols and synthetic ‘magic bullets’ has recently reduction in the intoxicating effects of THC CBD (Epidiolex was recently approved by waned, there are already several examples was not due, as initially hypothesized, to the FDA following successful clinical trials of molecules that affect more than one direct antagonism of CB1 (see REF.191 for for the treatment of orphan paediatric endocannabinoidome target (TABLE 2). review), but was rather due to a combination epilepsies20,197) might further boost the This is not surprising if one remembers of effects on other targets, such as adenosine development of other phytocannabinoids, that endocannabinoids and other receptor A2a and 5‑hydroxytryptamine as more than 100 such metabolites are 189,191 pro-homeostatic endocannabinoidome receptor 1A (5‑HT1A) . found in the flowers of different varieties mediators modulate the activity of multiple Another non-psychotropic phytocannab‑ of C. sativa. These metabolites interact targets (FIG. 1). However, whether the endo‑ inoid, THCV, reduced disease in preclinical with receptors and enzymes of the endo cannabinoidome-based multi-target drugs models of obesity and insulin resistance192,193, cannabinoidome (TABLE 3) (see REF.201 mentioned in this section exhibit improved which was partly confirmed in a phase II for a recent review). For example, as efficacy and safety over the corresponding clinical trial194. These effects were suggested already mentioned, phytocannabinoids, selective compounds still needs to be to occur via interaction with several targets particularly CBD, CBG and THCV, activate confirmed in further preclinical studies and rather than, as initially hypothesized, and desensitize TRPV1 much in the same demonstrated in humans. through neutral antagonism of CB1. The way as NAEs, N‑acyl-taurines, N‑acyl- antipsychotic effects of CBD in patients with dopamines and other N‑acyl-amides do. Phytocannabinoids schizophrenia may occur through non‑CB1 CBD was reported to antagonize GPR55, While several pharmaceutical companies targets195,196, and the anti-convulsant which was suggested to be activated by were struggling to make CB1 antagonists, effect of CBD in phase III clinical trials in endocannabinoids and PEA (although, as CB2 agonists and FAAH inhibitors, paediatric patients with Dravet or Lennox– discussed above, this is still controversial). active pharmacological research on the Gastaut syndrome20,197 is also probably not THC was recently shown to activate GPR18, neglected cousins of THC, such as CBD, CB1‑mediated, as pro-convulsant activity an effect also suggested for N‑arachidonoyl- but also THCV, CBG, cannabidivarin has occasionally been observed with glycine. Finally, both phytocannabinoids (CBDV), cannabidiolic acid (CBDA) and synthetic CB1 agonists and antagonists198. and N‑acyl-amides may interact with PPARs cannabichromene (CBC), started again at the Indeed, unlike THC, the pharmacology (see REF.202 for a recent review). Although turn of the century. Nabiximols (marketed of non-psychotropic phytocannabinoids phytocannabinoids also interact with non- in the USA as Sativex), the first botanical seems to be driven by interactions with endocannabinoidome proteins, the partial drug based on plant cannabinoids, consists more than one receptor or enzyme199. This overlap between the phytocannabinoids and of a 1:1 ratio of standardized extracts from combination of molecular mechanisms the endocannabinoidome might facilitate two different cultivars of C. sativa enriched, of action might explain why CBD is quite the rational design of new synthetic endo‑ respectively, in CBD and THC and delivers safe in humans (doses of up to 800 mg cannabinoidome-based drugs, which approximately equal doses of THC and per day were administered in one of the could begin with the chemical structures CBD together with other minor cannabis two schizophrenia clinical trials195) and of multi-target phytocannabinoids and components as an oromucosal spray. It has efficacious in most of the preclinical endocannabinoidome mediators. This been marketed since 2007 as an efficacious studies in which this compound has could be especially useful when phyto‑ and safe treatment for neuropathic pain (only been tested. Recently, a model has been cannabinoids and endocannabinoidome in Canada) or spasticity (in 30 countries so developed that attempts to explain how the mediators cannot be clinically developed far) in patients with multiple sclerosis21,179. multi-target nature of non-psychotropic because of stability, pharmacokinetics or Preclinical studies with various combinations phytocannabinoids fits successfully marketing issues.

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Table 3 | Proposed key molecular targets for the most studied non-psychotropic phytocannabinoids Phytocannabinoid Effect on Effect Effect on Effect on enzymes Effect on Potential Refs CB1 and on TRP PPARs and and transporters neurotransmitter therapeutic uses CB2 channels orphan receptors and GPCRs voltage-dependent ion channels 64,67,68,199, CBD Negative • TRPV1 (+) • PPARγ (+) • FAAH (–) • 5‑HT1A (+) Chronic and allosteric • TRPV2 (+) • GPR55 (–) • ENT (–) • Glycine receptors (+) inflammatory pain, 202,255–260 a a a modulator • TRPV3 (+) • GPR3 (–) • eCB transport across • GABAAR (+) epilepsy, IBDs, a a for CB1 • TRPA1 (+) • GPR6 (–) the membrane (–) • CaV3s (–) schizophrenia, a • TRPM8 (–) • GPR12 (–) • CaV1s (–) cancer and a • Nav1.6 (–) neuroinflammatory • VDAC1 (–)a diseases CBDV None • TRPV1 (+) None • DGLα (–)a None Epilepsy 67,68,199,256 • TRPA1 (+) • eCB transport across • TRPM8 (–) the membrane (–)a CBDA None None PPARγ (+) • DGLα (–)a Positive allosteric Nausea and cancer 199,202,256, a a 261,262 • NAAA (–) modulator for 5‑HT1A

a 67,199,256, THCV • CB1 (–) • TRPV1 (+) None None 5‑HT1A (+) Obesity, metabolic • CB2 (+) • TRPV2 (+) syndrome, insulin 257,263 • TRPV3 (+) resistance, • TRPA1 (+)a steatosis, • TRPM8 (–) schizophrenia and inflammatory pain

CBG Weak CB2 • TRPV1 (+) PPARγ (+) eCB transport across • ADRA2 (+)a Cancer, 67,202,256, a a 257,261,264 agonist • TRPV2 (+) the membrane (–) • 5‑HT1A (–) neurodegenerative • TRPA1 (+) diseases and IBDs • TRPM8 (–) CBC None TRPA1 (+) None • ENT (–)a,b None Pain and gliosis 67,199, • eCB transport across 256,260 the membrane (–)a THCA None None PPARγ (+) • DGLα (–)a None Neurodegenerative 67,256,261 • MAGL (–)a diseases 2+ + indicates activation, and – indicates inhibition. 5‑HT1A, 5‑hydroxytryptamine receptor 1A; ADRA2, α2-adrenergic receptor; Cav1s, L-type Ca channels; Cav3s, T-type Ca2+ channels; CBC, cannabichromene; CBD, cannabidiol; CBDA, cannabidiolic acid; CBDV, cannabidivarin; CBG, cannabigerol; DGLα, sn‑1‑specific diacylglycerol lipase-α; GABAAR, GABAA receptor; eCB, endocannabinoid; ENT, equilibrative nucleoside transporter; FAAH, fatty acid amide hydrolase 1; GPCRs, G protein-coupled receptors; GPR3, GPCR 3; IBDs, inflammatory bowel disorders; MAGL, monoacylglycerol lipase; NAAA, N-acylethanolamine-hydrolysing acid

amidase; Nav1.6, voltage-gated sodium channel type 1.6 (also known as SCN8A); PPAR, peroxisome proliferator-activated receptor; THCA, Δ9-tetrahydrocannabinolic acid; THCV, Δ9-tetrahydrocannabivarin; TRP, transient receptor potential; TRPA1, transient receptor potential cation channel subfamily A member 1; TRPM8, transient receptor potential cation channel subfamily M member 8; TRPV1, transient receptor potential cation channel subfamily V member 1; VDAC1, voltage-dependent anion-selective channel protein 1. aOn the basis of only one study; bOn the basis of only in vivo pharmacological data.

Synthetic cannabinoids preparations, does not exhibit strong a very serious problem because of their Whereas plant cannabinoids, either addictive properties187. On the other hand, often dangerous side effects, which range as purified compounds (CBD) or some synthetic cannabinoids or chemically from agitated delirium, hallucinations, as standardized botanical extracts unrelated cannabimimetic compounds, tachycardia and hypertension to psychosis, (nabiximols), are achieving success in such as the aminoalkylindoles, exhibit up seizures, lethargy and coma208,209; the clinic, synthetic cannabinoids with to fivefold higher efficacy in most in vitro these effects also occasionally lead to ultra-potent agonist activity at CB1 have functional assays for CB1 activity204,205. As fatalities, which may also be the result of started obtaining a different type of such, these compounds can produce much interactions with targets other than CB1 fame. THC is a relatively high affinity but stronger effects, especially in adolescents, (REF.210). Compounds detected in eight inefficacious agonist for both CB1 and CB2, in view of the important role played by CB1 synthetic cannabinoid-associated deaths even compared with endocannabinoids in neural development, synaptic plasticity in adolescents revealed that five of the like 2‑AG, and can therefore be considered and cognition but also in cardiovascular deaths had no other discernible cause on a partial agonist203. Indeed, the partial function (see REFS206,207 for recent reviews). autopsy, and the autopsies of these five agonist activity of THC, combined with Several chemical variations of these deaths uncovered detectable plasma levels the presence of CBD and other non- synthetic drugs have further increased their of synthetic CB1 agonists such as PB‑22 psychotropic, multi-target phytocannabi‑ potency. These ultra-potent CB1 agonists, (1.1 ng per ml), JWH‑210 (12.0 ng per ml), noids, likely explains why this compound, which can be made in large amounts with XLR‑11 (1.3 ng per ml), AB‑CHMINACA at the relatively low concentrations usually inexpensive starting materials and fairly (8.2 ng per ml), UR‑144 (12.3 ng per ml) found in traditional, 1960s‑style cannabis simple organic reactions, are becoming and JWH‑022 (3.0 ng per ml)210. Such levels

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may yield concentrations from ~2 to 20 nM, study, the protective effects of capsaicin, NAPE-PLD-knockout mice did not have which, for these compounds, would be the prototypical TRPV1 agonist and a reduced endocannabinoid tone in the sufficient to fully activate CB1. hot chilli pepper component, against white adipose tissue but rather had reduced The scale and rapidity of the evolution obesity-induced and dysbiosis-mediated levels of NAEs (namely SEA, OEA and of these new recreational drugs (more low-grade inflammation in mice were found PEA but not AEA)221; hence, the observed than 150 chemical structures are known to be accompanied by downregulation of phenotype was possibly due to impaired to date) make their legal control and colonic CB1 expression215. Activation of signalling from receptors (that is, GPR119, analytical detection in biological samples CB1 with a synthetic agonist eliminated TRPV1 and PPARα) other than CB1. This difficult, if not impossible208. Therefore, the beneficial effects of capsaicin on body is the first study to suggest an important their consumption is on the rise, and it weight, food intake, glucose tolerance and role for some non-endocannabinoid lipid is estimated that between 3% and 10% of fat accumulation215. These data suggest that mediators of the endocannabinoidome in high school students in the USA have used CB1 signalling by endocannabinoids acts as inhibiting the deleterious effects of dysbiosis these compounds210. New efforts are thus one of the intermediate actors between high- on gut permeability and metabolic control. urgently required to increase the analytical fat-diet-induced dysbiosis and its low-grade- Furthermore, the study exemplifies how capabilities of forensic institutions in order inflammation-mediated negative effects on experiments with conditional knockout of to allow the identification of new chemical metabolism. endocannabinoidome metabolic enzymes entities as soon as they appear on the market The relationship between the must always be accompanied by the to understand their potential dangerous side endocannabinoid system and the gut understanding of which endocannabinoi‑ effects and find potential pharmacological microbiota seems to be bi‑directional. dome mediator is affected and where these treatments for serious cases of intoxication Blockade of CB1 was recently suggested effects occur. from these compounds. For example, a single to ameliorate diet-induced obesity and An intestine-specific knockout of administration of dismissed CB1 antagonists accompanying dysmetabolism by increasing myeloid differentiation primary response such as rimonabant and taranabant, which the relative abundance of Akkermansia protein (MYD88) also linked intestinal were administered with relative overall muciniphila, a commensal Gram-negative inflammation with the endocannabi‑ safety to thousands of healthy volunteers bacterium with beneficial effects on noidome following the quantification of and patients with obesity in clinical trials26,29, metabolism216, and subsequently reducing metabolites. MYD88 is a central adaptor might block the acute effects of synthetic inflammation217. These data indicate that molecule for most of the Toll-like receptors cannabinoids211 if their consumption can the increased peripheral CB1 signalling through which microbiota-derived be deemed responsible for life-threatening usually associated with obesity and related inflammatory molecules (such as LPS) signal effects in hospitalized individuals. By disorders (see REFS31,132,218 for recent to the immune system. MYD88 has been contrast, ultra-potent synthetic CB1 reviews) may result in aberrant metabolism suggested to mediate the function of the agonists are unlikely to lead to clinically by facilitating dysbiosis and suggest gut microbiota in energy homeostasis. Mice useful compounds, given the difficulties the existence of a vicious cycle between lacking MYD88 in epithelial intestinal cells of harnessing THC for therapeutic use. In increased CB1 signalling and dysbiosis — were protected from diet-induced obesity fact, their ever increasing abuse may cast a this cycle is induced by a high-fat diet and and insulin resistance and had reduced shadow on the future therapeutic use of plant amplifies low-grade inflammation and the hepatic steatosis, fat mass and inflammation, cannabinoids, thus prolonging the partly metabolic syndrome. Consistent with this and had increased energy expenditure, unjustified stigma that has affected this class hypothesis, chronic administration of THC, improved glucose homeostasis and an of natural compounds since the mid‑1960s. which may lead to CB1 internalization and increased number of regulatory T cells in desensitization219, counteracts diet-induced the intestine222. Protection was transferred Endocannabinoids and gut microbiota obesity and concomitantly alters the gut to wild-type germ-free mice following gut The gut microbiota is increasingly known microbiota, increasing the relative amount microbiota transplantation — indicating to be involved in a plethora of physio‑ of A. muciniphila220. Interestingly, however, that the beneficial effects of MYD88 deletion pathological conditions (see REF.212 for a in another report, selective depletion of were mediated by alterations in the intestinal recent review). Interestingly, a probiotic, NAPE-PLD in white adipose tissue induced microbiome — and was accompanied by Lactobacillus acidophilus, was shown to obesity, glucose intolerance, adipose tissue decreased levels of AEA and increased levels produce antinociceptive effects against inflammation and dyslipidaemia221. This of 2‑AcGs (including 2‑AG) and increased visceral pain by increasing the expression phenotype was caused by inflammation of expression of GPR119 in the intestines of of CB2 and μ-type opioid receptor (MOR1) white adipocytes, which then became less mice fed a high-fat diet222. Increased 2‑AG in intestinal epithelial cells in rats with responsive to cold-induced browning and levels seem contradictory to the role of colonic hypersensitivity213. However, from hence to cold-induced energy expenditure. CB1 in promoting the dysmetabolic and this report, it was not clear whether the These alterations were mediated by a shift in pro-inflammatory effects of dysbiosis. effects of the probiotic were direct or through gut microbiota composition (and resulting However, decreased CB1 tone, via lower alterations in the gut microbiota, as are many increased gut permeability) and hence AEA levels, and increased GPR119 and of its other effects. Later, several papers could be partially transferred to wild-type CB2 tone, via higher levels of 2‑oleoyl - showed that increased gut permeability germ-free mice221. These data are counter‑ glyerol or 2‑palmitoyl glycerol and 2‑AG, (similar to that induced by obesity), which intuitive, as NAPE-PLD is the biosynthetic respectively, may explain the beneficial causes intestinal dysbiosis and thus leads enzyme for AEA, and its deletion might be effects on insulin resistance and the to lipopolysaccharide (LPS)-induced expected to reduce CB1 activity and produce reduction in inflammation. Accordingly, inflammation, can occur through activation beneficial metabolic effects. However, the another study showed that the beneficial of CB1 (REF.214). Accordingly, in a subsequent authors also showed that adipocyte-specific effects of A. muciniphila on metabolism,

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gut permeability and inflammation are In summary, components of the endo‑ be dedicated to other strategies, such as accompanied by increased intestinal levels cannabinoidome likely modulate some the clinical development of some of the of 2‑AG and 2‑AcG223. of the effects of the gut microbiome, over 100 C. sativa cannabinoids whose The altered gut microbiome has not only particularly in the context of host– pharmacology is now being investigated. been implicated in metabolic disorders. microorganism interactions that alter Endocannabinoidome receptors that The excessive use of antibiotics and the immune and intestinal function, recognize fewer endogenous ligands, such subsequent perturbation of the intestinal metabolic control and behaviour212,224. as CB2 (for which even AEA is only a partial flora have been suggested to play a role in Understanding such interactions is likely agonist)232, and that are selectively expressed neuropsychiatric disorders such as autism, to suggest new therapeutic drugs for in tissues or cells during pathophysiological psychoses, anxiety and depression224. A dysbiosis-related diseases, for example, conditions also need to be studied more recent study suggested that alterations in through the identification of bioactive thoroughly. While waiting for effective and some endocannabinoidome mediators are metabolites that are produced by commensal safe non-brain-penetrant analogues, CB1 partly responsible for some of these effects microorganisms alone or in collaboration antagonists could be repositioned for use in mice225. Perturbation of the microbiota with the host (that is, ‘post-biotics’). New in patients with a low risk of developing by prolonged treatment with antibiotics endocannabinoidome-based medicines may depression relative to individuals who are was accompanied by an inflammatory come from these efforts. obese (the population in which rimonabant state and strong behavioural changes in was unsafe) or for orphan and otherwise two experimental tests of depression. The Conclusions and outlook untreatable diseases. CB1 antagonists could authors also observed altered brain-derived Drugs targeting the endocannabinoid be useful in treating muscular dystrophies, neurotrophic factor (BDNF)–tropomyosin- system, particularly if viewed as part for example, given the anti-differentiating related kinase B (TRKB; also known as of a larger signalling system — the activity of CB1 in muscle cells233. Existing NTRK2) signalling and neuronal firing and endocannabinoidome — already exist, and new CB2 agonists or, possibly even increased microglial inflammatory activity in and new ones are being developed. Several better, dual CB1 antagonists–CB2 agonists234 the hippocampus. Simultaneously, the levels widely used therapeutic interventions have might be extremely useful to treat fibrosis of two N‑acyl-serotonins that inhibit FAAH been found to affect the endocannabinoidome of the liver, lung, heart and kidney48,49. and TRPV1 were substantially reduced in and likely act partly through it (reviewed Exploiting the positive and negative the small intestine. This finding, given the in REF.229): for example, acetaminophen, allosteric sites in CB1 and CB2 (REFS37-39) suggested role of these two endocannabino one of the most popular antipyretic and could be useful in diseases such as chronic idome proteins in depression (see above) anti-inflammatory drugs on the market, is pain, cancer, anxiety, depression and and the capability of N‑acyl-serotonins to increasingly suggested to act in part through schizophrenia and in metabolic and neuro cross the blood–brain barrier226, suggested the endocannabinoidome; the R‑profens may inflammatory disorders. In this respect, that the observed depression-like signs owe part of their analgesic effects to reduced recently identified endogenous negative were due in part to disinhibited central COX2‑mediated peroxidation of endocan‑ allosteric modulators of cannabinoid FAAH and TRPV1 activity. In support nabinoids; and ketamine may also act partly receptors, like some hemopressin analogues of this hypothesis, administration of a through these mediators230. and pregnenolone235–238, could provide probiotic counteracted the antibiotic- The increasing success of botanical starting points for new treatments for induced behavioural and CNS functional drugs based on non-psychotropic phyto addiction disorders and cannabis and alterations and concomitantly restored cannabinoids is likely due, at least in synthetic cannabinoid intoxication. Finally, near-physiological levels of intestinal N‑acyl- part, to their simultaneous modulation of the thorough biomolecular investigation serotonins225. The mechanism through several endocannabinoidome proteins200. of gut microbiome–endocannabinoidome which the antibiotics reduced the levels of Indeed, most endocannabinoidome lipids interactions will likely lead to new drugs, these compounds remains to be established, modulate the activity of multiple targets which could include synthetic analogues of and the contribution of microbial species (FIG. 1), and formulations of at least one of multi-target endocannabinoid-like mediators to the biosynthesis of N‑acyl-serotonins them, PEA, have been efficacious in clinical designed to have drug-like properties. should be explored. Indeed, another trials of chronic and inflammatory pain, We now understand that the recent study suggested that gut bacterial dermatological conditions, intraocular endocannabinoid system is complex. species can produce small molecules that pressure in glaucoma and other disorders231. However, the plethora of potential are chemically very similar to some endo All this should not come as a surprise, as therapeutic opportunities that it offers cannabinoidome mediators and can interact several successful therapeutic drugs that is precisely because of this complexity. with the same receptors, thus potentially have been on the market for years (such as Multiple methodologies and technologies, affecting metabolic disorders227. Additionally, many nonsteroidal anti-inflammatory drugs, such as the omics, bioinformatics and endocannabinoidome mediators that are tyrosine kinase inhibitors and metformin) systems biology approaches suitable to deal typically produced by the host, such as OEA are now known to be promiscuous in with and make sense of ‘big data’, are now and PEA, could also be produced by the their mechanism of action, and poly available and will allow us to cope with microbiome and might modulate the effects pharmacology is increasingly used to treat most of the complications described in the of dysbiosis. For example, production of several multi-factorial disorders in a more present article, much in the same way that these lipids could increase in response to efficacious and safer manner. these tools are now proposed to be used for dysbiosis and subsequently decrease gut Therefore, there is a strong rationale precision and personalized medicine. This barrier permeability by stimulating PPARα for developing synthetic or natural should push drug developers to look at this and TRPV1, thereby counteracting multi-target drugs from the endocannabino field as a challenging and exciting goldmine inflammation228. idome. However, efforts will also need to rather than a ‘no‑go’ area.

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