Abstract

History

On October 30-31, 1986, the National Institute on Drug Abuse organized a “Technical Review” event in Building 1 of the National Institutes of Health (NIH) campus in Bethesda, MD, whose purpose was to bring together scientists involved in the field of cannabinoids to discuss the most current research efforts. I had just received my first cannabinoid grant and was invited by its organizer, Dr. Rao Rapaka, who had recently joined the extramural program of the National Institute on Drug Abuse, to help put together such an event. It was generally felt that scientific activity in this important aspect of drug abuse was at a low point and such an event may help energize the field.

By all accounts, this effort by the National Institute on Drug Abuse (NIDA) was very successful in achieving its goals. Publication of the conference proceedings2, 17 was followed by a number of key discoveries, including the identification, cloning, characterization and imaging of the CB1 receptor; the discovery of the key endocannabinoids, anandamide and 2-arachidonylglycerol, and the design and synthesis of novel ligands that enabled the elucidation of the cannabinoid biochemical system and established its major role in mammalian physiology. It is now universally recognized that cannabinoids are a very active research area. Also, because of its pleiotropic nature and its drug-friendly targets, the endocannabinoid system has excellent prospects in serving as a basis for drug discovery.

The three decades preceding this conference had witnessed a great deal of exciting work aimed at accessing the therapeutic properties of cannabis and its ingredients and developing novel therapeutic medications. This involved the development of new chemistries for the synthesis of terpenoid analogs with structural similarities to cannabis’ endogenous constituents. The effort was led by Alexander Todd from University of Manchester35 and Roger Adams from the Noyes Chemical Laboratory at the University of Illinois, Urbana-Champaign,37 and produced new molecules with pronounced physiological effects when tested in different animal species. A major boost to the field was the isolation of the key bioactive constituent of cannabis and its subsequent synthesis by Raphael Mechoulam in Israel.38, 39 This gummy non-crystallizable compound, which was identified as a tricyclic terpene encompassing a middle pyran ring, a phenolic hydroxyl, and a linear 3-pentyl side chain attached to the aromatic ring, was named (-)-Δ1-tetrahydrocannabinol and later renamed (-)-Δ9-tetrahydrocannabinol (Δ9-THC). Its structure served as a prototype for additional synthetic efforts by a number of academic laboratories, including the Mechoulam, as well as the Razdan and Pars laboratories in Cambridge, MA.40

The above efforts were paralleled by significant programs within the pharmaceutical industry to develop cannabinoid-based medications principally as non-opioid effective analgesic agents. Notably, Lilly’s efforts had led to the synthesis and development of the drug Nabilone,41 which has been used by patients receiving cancer chemotherapy. Also, companies such as Abbott and Arthur D. Little Inc. were developing nitrogen containing analogs (eg. Nabitan) that were deemed to have more drug-like properties.40 One of the major programs was undertaken at Pfizer in Groton, Connecticut. The effort for the discovery of cannabinoid analgesics was led by two talented medicinal chemists, Larry Melvin and Ross Johnson, whose work led to their first clinical candidate, Levonantradol,42 a compound that was less lipophilic than the key phytocannabinoid Δ9-THC, and also was 10- to 100-fold more potent in analgesia tests. In their systematic SAR, they had developed a series of analogs lacking the middle ring of the tricyclic terpenoid structure which they named non-classical cannabinoids,17 the most prominent of which was CP-55940,17 a 3β-hydroxycyclohexane phenol in which the 3-pentyl chain of Δ9-THC was substituted with a 1’,1’-dimethylheptyl chain.

Melvin and Johnson were among the participants of this historic 1986 event, where they described the detailed SAR obtained from testing the non-classical cannabinoids for their analgesic effects.17 Their results underscored the remarkable correlation between analgesic potency with their respective absolute and relative stereochemistries,17 as well as subtle structural modifications. The results argued for the existence of a specific site of interaction through which the new cannabinoids were producing their effects. Coincidentally, an interesting presentation during the meeting was from a young investigator, Allyn Howlett,17who had been testing some of the phytocannabinoids for their effects on modulating the levels of cAMP. She showed that the most biologically-active of these compounds, including Δ9-THC, dose-dependently reduced the levels of cAMP, suggesting an inhibitory effect on the enzyme adenylyl cyclase. During that meeting, the Pfizer scientists offered to develop a tritiated analog from their non-classical cannabinoid series, which they provided to Howlett’s laboratory. She and her graduate student William Devane tested a variety of synthetic cannabinoid analogs, in a heterologous displacement assay, using the non-classical Pfizer analog CP55940 as the radioligand, and obtained high quality sigmoid curves that accurately reflected the in vivo potencies of the compounds.43

These successful results were immediately followed by the autoradiographic imaging of the putative receptor in rat brain by Miles Herkenham at the NIH, using the same radioligand,28and its cloning by Lisa Matsuda in Tom Bonner’s laboratory,44 which was next to that of Herkenham’s, thus allowing for a great deal of communication between the two laboratories.

My own involvement with cannabinoid chemistry and biochemistry had started in the early eighties, when the prevalent theory for cannabinoid activity was one invoking drug-induced specific membrane perturbations that modulated the properties of functional proteins within the cell membrane. Based on this approach, I was awarded a NIDA grant, which involved detailed structural and conformational studies aimed at developing correlations between ligand structure and function with their effects on model and cellular membrane systems. A turning point in my approach came when, in 1985, I heard a presentation by a Pfizer scientist describing their very extensive medicinal chemistry with their new cannabinoid-like ligands and the striking differences in the pharmacological properties based on subtle structural modifications. The results strongly argued for the presence of a yet to be identified functional protein, in all likelihood a G-protein coupled receptor.

With accumulating evidence for the existence of one or more cannabinoid receptors, I started a research program whose goals were to design and develop new biochemical and pharmacological tools that would allow us to characterize these new GPCRs and also decipher the circuitries of what was anticipated to be an intriguing and extensive biochemical system. An early effort in my laboratory to identify this putative receptor was through the design and synthesis of novel photoaffinity and electrophilic probes. This initial work by my student Avgui Charalambous led to a family of photoactivatable Δ8-tetrahydrocannabinol analogs designed to covalently attach to the receptor either through a carbene or an aliphatic nitrene mechanism.1 One of these, 5’-azido-Δ8-THC1, was subsequently radioiodinated and served as the first covalent probe for the CB1 receptor.2 This work was being carried out in collaboration with my colleague Sumner Burnstein, contemporaneously with the characterization of the CB1 receptor in the Howlett and Bonner laboratories. When rat brain membranes were allowed to photoreact with our 125I-labeled azido probe, the gel, when developed, showed two bands reflecting the presence of the corresponding proteins. This radiolabeling was prevented when the preparation was pretreated with Δ9-THC. The experiment provided the first chemical evidence for the presence of a ligand-cannabinoid receptor complex. Other information obtained from this early simple labeling experiment was the identification of a second, fainter band of lower molecular weight which allowed us to speculate on the presence of another cannabinoid receptor. This first photoaffinity labeling opened the door for future experiments in my laboratory to use covalent chemistry to characterize the structural, biochemical and biophysical properties of the cannabinoid receptors.

The story of the cannabinoid biochemical system was further expanded by the identification of the second cannabinoid receptor, designated as CB2, and initially described as peripheral because of its presumed absence in mammalian brain.45 More recently evidence was obtained for its CNS presence, albeit in low concentrations.46, 47 We also know now that CB2 levels can be very significantly enhanced in non-homeostatic conditions such as inflammation, neurodegeneration and cancer.

The AM Compounds

Through the use of target-based design and focused medicinal chemistry, over the past three decades, we have developed a wide variety of structurally-distinct analogs, the most interesting of which have found applications in pharmacology, biochemistry, and biophysics, as well as serving as early leads or advanced preclinical candidates for drug development. Under the AM acronym, many of these compounds are universally used by research laboratories. As pharmacological probes, AM compounds have played a substantial role in elucidating the physiological role of the endocannabinoid system. In this medicinal chemistry effort, we were assisted by an excellent collaboration with Marcus Tius at the University of Hawaii. Initial in vivo testing of key AM compounds were provided by a number of very productive collaborations with the laboratories of Toby Järbe (Northeastern University), John Salamone (University of Connecticut), Philip Malan (University of Arizona), Frank Porreca (University of Arizona), Andrea Hohmann (University of Indiana), Linda Parker (University of Guelph), and Keith Sharkey (University of Calgary).

Endocannabinoids

What are the endogenous molecules that engage and activate the two receptors? The first endocannabinoid anandamide (AEA) was isolated from pig brain in Mechoulam’s laboratory.48 This was followed by the characterization of 2-arachidonylglycerol (2AG) as the second endogenous cannabinoid.49, 50 We now know that the endocannabinoid system is modulated by a large family of structurally-related lipid mediators belonging to either the amide or ester families, all of which are involved in the activation of the cannabinoid receptors. Additionally, there is evidence that these same lipid molecules may also modulate other systems such as the vanilloid receptor 1 (VR1) and other transient receptor potential vanilloid (TRPV) channels, as well as the largely unexplored GPR55, GPR35, GPR18 and GPR119 receptors. This property of pleiotropic action by lipid modulators is an important feature of the cannabinoid system, and distinguishes it from other non-lipid neurotransmitters. Arguably, the levels of individual modulators, as an aggregate within a human or animal biological sample, may serve as a unique descriptor of the organism’s physiological state. This realization prompted us to develop an accurate LC/MS/MS-focused lipidomic assay for the measurement of the larger family of these endocannabinoid lipids, which we named the “endocannabinoid metabolome.”51–54 This assay expands the family of endocannabinoid lipid modulators beyond its two major players AEA and 2AG, and affords us a more accurate description of the endocannabinoid system by providing quantitative information on its individual components. Such measurements can find utility in identifying endocannabinoid fluctuations that are drug-related or the result of cannabinoid-associated pathologies, such as depression and schizophrenia, where they could serve as important biomarkers.

As mentioned above, the endocannabinoid family comprises a variety of ethanolamides and glycerol long chain fatty acid esters. However, among those, AEA,, also known as anandamide, and 2AG are the most potent and best studied. AEA and 2AG are substrates for the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL), respectively, as well as other hydrolytic enzymes, and consequently have relatively short in vivo half lives, a property that limits their usefulness as probes for the in vivopharmacological characterization of the endocannabinoid system. An early goal in my cannabinoid journey was to develop more stable analogs, encompassing the key structural features of the endogenous ligands. Design of the metabolically-stable anandamides was based on introducing substituents in the vicinity of the sessile amide bond to impede access of the ligand to the enzyme’s catalytic site. Alternatively, AEA can be modified in a manner which maintains all features required for the recognition of CB1 and CB2 receptors while impeding its fit within the enzyme’s catalytic domain. These approaches led to the design of ligands with significantly enhanced stability toward the key deactivating enzyme FAAH. The first of these, AM356, an anandamide analog substituted at the headgroup with a chiral methyl group, and also known as R-methanandamide, was a successful probe with significantly higher metabolic stability and substantially improved CB1 potency.55–57AM356 has gained popularity among pharmacologists and is universally used as a surrogate for the endogenous AEA. A second generation of such probes was inspired by our early observation that the inverse amides or retroamide analogs of anandamide were significantly more resistant to enzymatic hydrolysis.57 Experimentation around that concept resulted in the development of AM1346,58, 59 a very potent and metabolically-stable anandamide, which we named “mahanandamide” or superanandamide, a designation contributed by Dr. Rapaka. Ongoing efforts in our laboratory toward the development of hydrolytically-stable 2AG have led to the dimethylated headgroup analog AM10336 (K. Vadivel and A. Makriyannis, unpublished).

The Role of Cell Membrane in Cannabinoid Activity

Prior to the discovery of the cannabinoid receptors, the dominant postulate was that these compounds act by perturbing the cell membrane, thus, indirectly affecting the function of key membrane proteins. This concept coincided with my first entry into the cannabinoid field, during which time we adapted or developed a variety of biophysical methods to study the effects of cannabinoids on model and natural membranes. The methods included solution60,61 and deuterium solid state NMR,29, 62 as well as X-ray31, 62, 63 and neutron small angle diffraction.64, 65 These approaches, which were initially used to study the molecular mechanism of cannabinoid action, were subsequently directed to a more general program aimed at understanding the interaction of lipophilic and amphipathic ligands, including cannabinoids and endocannabinoids with cell membranes.

Because of their lipophilic properties, cannabinergic compounds have very limited aqueous solubility, raising the question of how they can access their sites of action within the respective membrane-bound proteins? Our studies provided evidence that cannabinoids and amphipathic ligands in general, assume a preferred orientation within the membrane bilayer.9,33, 66, 67 In early works, we introduced the concept that amphipathic cannabinoids favorably partition into the membrane and, through fast lateral diffusion, interact with the receptor.9, 66,67 We postulated that the ligand aligns itself within the bilayer, in a favorable orientation and conformation which optimizes its ability for a productive interaction with the respective functional protein.9, 67 To provide experimental evidence for our postulate, we used high-resolution NMR to study the conformational properties of select cannabinergic ligands.60, 61,68 We also developed 2H-solid state NMR,33, 69, 70 small angle x-ray31, 62, 71, 72 and neutron diffraction approaches65 described in a series of publications that allowed us to identify the manner by which the ligand aligns itself within the membrane.63, 72–75 Our studies demonstrated that because of their amphipathic properties, cannabinergic ligands align themselves in an orientation in which the polar components of the molecule interact with the respective membrane headgroups. Such an orientation enhances their ability to engage in fast lateral diffusion within the bilayer and allows for a productive collision at the receptor site.9, 33 We, thus, demonstrated that Δ9-THC aligns itself in an “awkward” orientation, in which its phenolic hydroxyl interacts with the polar headgroup within the bilayer, while its hydrophobic tricyclic component orients with its access orthogonal to those of phospholipid chains.29, 31, 72 Cannabinoids with two or more hydroxyl groups such as the Pfizer analog CP55940 align in a manner in which all three OHs interact with the polar membrane components.9 We also showed that this “amphipathic reciprocal alignment” of amphipathic ligands within a membrane bilayer has general applicability with endogenous and exogenous lipid-friendly ligands such as steroids, including neurosteroids63, 74, 76 and estrogens. Conversely, hydrophobic ligands with no polar groups capable of hydrogen bonding (e.g. OH, NH2), such as O-methyl-Δ9-THC, align with their long axis parallel to the membrane bilayer chains.71, 72, 74 The endocannabinoid anandamide aligns with its extended arachidonyl chain parallel with the membrane chains and its polar ethanolamide group interacting with the phospholipid headgroups.33

In the above series of biophysical experiments, we provided substantial experimental evidence supporting our postulate that hydrophobic and amphipathic ligands access their sites of action by fast lateral diffusion after preferentially partitioning in the cellular membrane bilayer.9, 33 This “through membrane” access of ligands to their respective GPCR binding domains is now generally well-accepted within the GPCR scientific community.

CB2 Selective Ligands

The initial concept that cannabinoid-induced analgesia was an exclusively CB1 associated response was challenged in early work published by Porreca and Makriyannis.82Subsequently, the analgesic effects of CB2 agonists were established with the synthesis of CB2 selective ligands including HU308 from the Mechoulam laboratory83 and our own more potent and in vivo more efficacious AM1241.84–86 In a series of detailed publications involving collaborative work with Phil Malan at the University of Arizona and Andrea Hohmann at the University of Indiana, we documented the role of CB2 activation in peripheral and chronic analgesia, as well as inflammation.87–94 More recently, in collaboration with Andrea Hohmann, using three different CB2 selective chemotypes, including AM1241, AM1710, and AM1714, we demonstrated that CB2 activation effectively relieves chemotherapy-induced neuropathy.95–98 We also showed that the protective and therapeutic effects of these compounds were superior to those of gabapentin (Neurontin), the most commonly used medication for the different neuropathies. Our early success demonstrating the analgesic and anti-inflammatory effects obtained through activation of the CB2 receptor motivated a number of pharmaceutical companies to establish active research programs for the development of therapeutically-useful CB2 agonists. Unfortunately, the limited data obtained from testing CB2-selective agonists in humans did not fully validate the animal-based results. It can be argued that the limited clinical trials in which CB2 agonists were tested for their analgesic properties involved suboptimal therapeutic indications. I propose that more successful clinical results may be expected by choosing better targeted therapies.

Moreover, the development of novel CB2-selective agonists within academic and industrial laboratories opened the door for understanding the role of CB2 activation as a potential target for other therapeutic indications, such as bone disorders.99 It has also become clear that, contrary to earlier findings, CB2 is expressed in brain.46 Although brain expression under homeostatic conditions is limited, it becomes much more pronounced in pathological conditions, such as inflammation, neurodegeneration and cancer, where the CB2 is highly overexpressed both in brain and the periphery, suggesting that CB2 is a suitable target for such conditions as amyotrophic lateral sclerosis100 and colitis.101

Very recent evidence has identified a role for CB2 activation in addiction,102 which, arguably, can be considered a drug-induced inflammatory syndrome, and offers opportunities for additional therapeutic intervention.

Ligand Assisted Protein Structure

Following their discovery, the structure and function of GPCRs has been a major goal of biologists and pharmacologists. Although these proteins were cloned and expressed over three decades ago, their structural and functional characterization remained elusive and relied mostly on indirect biophysical approaches. Based on crystal structure determination, first of bacterial rhodopsin, and later of rhodopsin,103 computational methods were developed to obtain homology models of individual GPCRs, and their respective ligand receptor complexes based on the rhodopsin structure. Only recently were the crystal structures of pharmacologically-interesting GPCRs achieved. First among these was the beta adrenergic receptor, an important accomplishment through collaborative efforts between the Brian Kobilka and Ray Stevens laboratories, which required significant protein engineering of the GPCR protein.104 In parallel, Chris Tate and his collaborators at MRC, UK,105 used extensive receptor mutations to obtain crystallizable GPCR samples.

In my laboratory over the past ten years, we have been developing a novel approach aimed at obtaining experimental information on the binding motifs and functional characteristics of cannabinergic ligands with their respective receptors. This approach, which we named “Ligand Assisted Protein Structure” (LAPS), relies on the availability of covalent CB1 and CB2 probes. To identify the binding motif of individual ligands within CB1 and CB2, we use a dual methodology which includes the development of targeted CB1 and CB2 mutants and a focused proteomic characterization of the complex using LC/MS/MS.

CB1 Receptor Antagonists

The development of rimonabant (SR141716A, Acomplia), a selective CB1 antagonist, by Sanofi as a potential antiobesity medication was initially greeted enthusiastically by cannabinoid researchers. When tested in humans, the drug was shown to reduce body weight and to ameliorate dyslipidemias, diabetes and metabolic syndrome. However, this enthusiasm was dampened by later observations of undesirable side effects, which included nausea, anxiety, depression, and in some cases, suicidal tendencies, which led to its withdrawal from the market in October 2008.117, 118

In my laboratory, we have been developing CB1 receptor antagonists since the late nineties,119–123 among which, AM251 and AM281 received special attention as useful pharmacological probes with inverse agonist pharmacological profiles.119, 120, 122 AM281 was later used to demonstrate the first in vivo imaging of the receptor in non-human primates using SPECT technology.119–121 In tandem, and anticipating potential CNS side effects resulting from CB1 antagonism, we also made serious efforts to develop novel compounds with no or reduced side effect profiles. We followed two distinct approaches. The first was to develop effective CB1 antagonists with no inverse agonist or weak inverse agonist properties. This approach led to a family of compounds with in vitro neutral antagonist profiles, i.e. no or minimal effect on cAMP levels. The best known of these, AM4113 and AM6527,124–135when tested in rodents, reproduced all of the therapeutic effects associated with rimonabant and other inverse CB1 antagonists being developed by the pharmaceutical industry, including reduction in food consumption, weight loss, and ability to antagonize the effects of stimulant and nicotine addiction. When tested in animals, these compounds did not exhibit any of the undesirable side effects of inverse agonists in a number of animal models. The testing for side effect profiles included nausea, using emesis experiments in ferrets127 and rats,126, 128gastrointestinal motility in mice, as well as anxiety,129, 130 depression and anhedonia132 in rats. The above results are very encouraging, but require further validation to determine how the data can be translated into improved and novel CB1antagonists with favorable therapeutic index profiles.

Our second approach for obtaining CB1 antagonists with reduced undesirable side effect profiles was the development of peripherally-active compounds. This effort was reinforced by existing clinical data from the rimonabant trials, suggesting that some of the drug’s key therapeutic effects were peripherally mediated through a mechanism involving modulation in lipid metabolism and energy balance. The most prominent result of our effort was AM6545, a peripherally-acting neutral antagonist. When tested in rodents, this compound was found to produce weight loss, as well as improved lipid profile and insulin sensitivity. Currently, AM6545 is in advanced preclinical testing and being considered for development for the treatment of non-alcoholic fatty liver disease (NAFLD), as well as liver fibrosis.136–139Current SAR work on the 2-pyrrolidinone scaffold has led to AM10009 or molecule with distinct chiral features (J. Garcia et al, unpublished).

Cannabinoid Agonists with Controlled Deactivation

A program we recently initiated based on the “soft drug” approach aims at developing safer and more effective cannabinergic compounds with controllable deactivation, improved druggability and an overall safer pharmacological profile.

To this end, we incorporate a metabolically vulnerable ester group within the structure of a successful cannabinergic ligand.140, 141 Hydrolysis at the esteratic site leads to products devoid of pharmacological activity and low or no toxicity. In our controlled deactivation/detoxification design, the compound’s systemic half-life is determined by two factors. The first is the extent to which the ligand is sequestered within the body before it is released for systemic circulation (depot effect). This process is dependent on the compound’s physicochemical properties and can be modulated by adjusting log P and PSA. The second parameter is the rate of enzymatic hydrolysis by blood esterases. This can be calibrated by incorporating suitable stereochemical features in the vicinity of the hydrolyzable group (enzymatic effect). In recent publications involving analogs with an ester group at the side chain or as a lactone structure within the cannabinoid C-ring of the cannabinoid structure, we demonstrated our ability to modulate the rates of hydrolysis, as well as the drug’s depot effects. Key compounds synthesized in our laboratory show great promise as pharmacological probes and potential drug leads. Currently, we are pursuing this approach for the design of peripherally-acting analgesic agents, as well as potential medications in THC substitution therapy.

Endocannabinoid Deactivating Enzymes

Paracannabinoid Targets

Because of the pleiotropic nature of its endogenous ligands, cannabinoid biology is connected with other biochemical systems involving lipid modulators, such as the VR1 ion channel, a number of recently deorphanized GRPCs, including GPR55, as well as the arachidonic acid metabolizing enzyme cyclooxygenase-2 and the more recently identified NAAA. Because of their functional proximity with the endocannabinoid system, some of these targets are being explored in my laboratory. Recently, we have cloned, expressed, and purified GPR55, and are prepared to subject it to our LAPS approach in order to gain more detailed structural and functional information.

Also, we have cloned, expressed, and purified hNAAA, a lysosomal amidase which has shown promise as a therapeutic target for inflammation. Our mammalian expression system has allowed us to produce pure enzyme in milligram quantities.112 We used proteomic approaches to gain information on the catalytic mechanism of this unique cysteine amidase, where the apoenzyme is activated through an intramolecular scission, leading to two chain components, which form the active enzyme heterodimer.30, 112 Although NAAA hydrolyzes anandamide, its optimal native substrate is palmitylethanolamide, a lipid modulator generally associated with the nuclear receptor PPAR-alpha.

Recently, we have obtained good quality 1H NMR spectra of the purified enzyme, which allows us to follow the enzymatic catalysis in real NMR time, and to design novel inhibitors. The most successful of these, AM9053, is a chemically- and metabolically-stable compound with substantial in vitro potency (Ki = 30 nM). Very recently, its in vivo efficacy was demonstrated in a colon inflammation model.

Now and the future

Almost three decades after its discovery, the endocannabinoid system offers rich opportunities for the chemist. It has been identified as a major biochemical system, playing major roles in brain function, autonomic physiology, the immune system, and as a key player in maintaining homeostasis. Much of this biology needs to be further elaborated and new, related pathways remain to be discovered. The medicinal chemist should have a major role in developing or participating in the development of molecular tools in this exciting endeavor. These include selective pharmacological probes, in vivo imaging agents for PET and SPECT, as well as in vitro fluorescent and radiolabelling reagents. There continues to be a need for new ligands for structural biology work involving NMR, x-ray, and mass spectrometry that will assist in understanding the function of some key endocannabinoid proteins, including the two GPCRs and the enzymes and proteins involved in endocannabinoid biosynthesis, metabolism, and transport. The pleiotropic nature of the endocannabinoids encourages the exploration of noncannabinoid functional proteins associated with the endocannabinoid system, and identified in this perspective as paracannabinoid targets, as potential therapeutic targets. These may include other GPCRs and channels that are modulated by lipid ligands, as well as enzymes involved in their biotransformations.

As a therapeutic target, the endocannabinoid system has found to date only modest success, and the failure of Sanofi’s CB1 antagonist Acomplia was a major setback in this endeavor. However, a number of new findings, coupled with current efforts for cannabinoid-based drug development, point to a more successful future. CB1 antagonist approaches, including the peripherally-acting compounds, show promise as medications for metabolic disorders, liver damage and fat metabolism. In this regard, early candidates, such as AM6545, with combined neutral antagonist/peripheral profiles, may reduce the peripheral side effect profile in this class of compounds. As for the brain penetrant neutral CB1 antagonists, these may find usefulness in addiction disorders, including those from nicotine, opioids, and alcohol.

Notwithstanding the initial unsatisfactory results from the limited in vivo trials, CB2 agonists continue to show promise as potential medications for inflammatory and neuropathic pain, as well as in neurodegenerative conditions, including ALS and multiple sclerosis. Arguably, the development of CB1 agonists with reduced side effects, either through peripheralization or the controlled deactivation approaches I have outlined earlier, still holds great promise as analgesic medications for pain management, lacking some of the side effects associated with opioid anaglesia.

Therapeutic approaches involving indirectly-acting cannabinoid agonists, such as FAAH inhibition, are attractive because of their more favorable side effect profiles, notably the absence of THC-like effects in the CNS. However, initial in vivo trials with a FAAH inhibitor for osteoarthritis have not proven successful. Notwithstanding this failure, such compounds still offer promise as anti-inflammatory and neuroprotective medications, if properly targeted. MGL inhibitors, acting, at least in part, through a non-cannabinoid mechanism, are being developed as potential anticancer medications. Also, the observed synergy accompanying the simultaneous inhibition of both deactivating enzymes FAAH and MGL invites the development of optimized dual inhibitors. Indirectly-acting cannabinoid agonists also hold promise in drug abuse as potential substitution therapies. Early results identify NAAA inhibitors as potential anti-inflammatory agents in conditions such as irritable bowel syndrome. Recent data with our selective NAAA inhibitor AM9053 are very exciting. Such compounds may also prove useful as peripherally-acting analgesics.

Finally, CB1 positive or negative allosteric modulators offer interesting prospects for developing medications lacking CB1-related undesirable side effects. Although substantial research has identified several scaffolds that exhibit in vitro allosteric profiles, in vivoexperiments have shown only modest efficacy results. This is an area which would benefit from novel or improved scaffolds.

The ubiquitous presence of the endocannabinoid system presents difficulties in targeting it for therapeutic gain. For this reason, it is important that indications for cannabinergic drug development be explored very thoughtfully. In this regard, a better understanding of cannabinoid receptor-related functional selectivity should assist in the development of drugs with safer pharmacological profiles, and also identify additional therapeutic opportunities. Thus, compounds belonging to the same pharmacological class could be optimized for different indications, i.e. developing drugs from the same family with many flavors to address specific needs. Notwithstanding some of the earlier failures in developing cannabinoid-related medications, there is ample accumulated knowledge to predict that the endocannabinoid system offers great promise as a potential source of future therapies.

Acknowledgments

Nonstandard Abbreviations Used

References