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Endocannabinoids: Basic Physiology and Function Eliot L. Gardner New York Society of Addiction Medicine 7th Annual Conference NYC - February 2011 Eliot L. Gardner, Ph.D. Chief, Neuropsychopharmacology Section Intramural Research Program National Institute on Drug Abuse National Institutes of Health [email protected] 443.740.2516 Cannabis • • • • • Many species exist: Cannabis Sativa (European Many species exist: Cannabis Sativa (Europe), Cannabis Indica (India) plant), Cannabis indica (Indian plant) and Cannabis and Cannabis ruderalis (Siberia and central Asia) ruderalis (Siberia and central Asia plant) 460 known chemical constituents of cannabis 460 known constituents of cannabis 66 constituents havechemical a cannabinoid structure 9-THCaor Δ9-Tetrahydrocannabinol THC) most important constituent 66 constituents(Δhave cannabinoid structure Δ9-THC is the principal psychoactiveconstituent: component of cannabis Δ9-THC most important principal psychoactive component of cannabis Era of Cannabis Research: 200-1940 ● Circa 200 AD: Therapeutic properties of cannabis described in Chinese pharmacopoeia ● 1838-1840: Sir W.B. O’Shaughnessy methodically assesses medicinal properties of cannabis, and publishes findings ● 1899: Wood et al. isolate cannabinol from cannabis resin ● 1932: Cahn elucidates part of the structure of cannabinol ● 1940: Todd et al. and Adams et al. simultaneously elucidate the full structure of cannabinol and successfully synthesize it Era of Cannabinoid Research: 1960-1994 ● 1960: Mechoulam (Hebrew University) identifies THC as the principal psychoactive component of cannabis ● 1964: Gaoni and Mechoulam (Hebrew University) elucidate the chemical structure of THC ● 1970-1990: Cannabinoid pharmacology is thoroughly studied ● 1985: Gardner shows cannabinoid-opioid interaction in brain ● 1986: Gardner shows THC activates brain-reward systems ● 1988: Howlett’s group finds specific THC binding sites in brain ● 1990: Matsuda et al. clone the CB1 receptor ● 1992: Mechoulam’s group (Hebrew University) in collaboration with Pertwee’s group (Scotland) identify the first endocannabinoid – Mechoulam names it “anandamide” from the Sanskrit word “anand” meaning “bliss” ● 1993: Munro et al. clone the CB2 receptor Era of Endocannabinoid Research: 1994-2000 ● 1994: Scientists at Sanofi Recherche (France) develop the first CB1 receptor antagonist – SR141716A (Rimonabant) ● 1995: Mechoulam (Hebrew University) isolates and identifies the second endocannabinoid – 2-Arachidonoylglycerol (2-AG) ● 1996: Cravatt et al. (Scripps) clone the first endocannabinoid degrading enzyme – fatty acid amide hydrolase (FAAH) ● 1998: House of Lords report on medical cannabis ● 1998: Di Marzo et al. propose interactions between endocannabinoids and vanilloid receptors ● 1999: Zygmunt et al. and Smart et al. show that anandamide activates vanilloid receptors Current Endocannabinoid Research: 2000● 2003: Bisogno et al. clone the first endocannabinoid biosynthesizing enzymes ● 2005: Pertwee et al. (Scotland) discovers an allosteric site on CB1 receptors ● 2005: Sativex® approved for sale in Canada ● 2010: Gardner shows psychoactive (and potentially therapeutic) effects of cannabidiol ● ????: Discovery of new cannabinoid receptors ● ????: Discovery of new endocannabinoids ● ????: Discovery of new endocannabinoid enzymes ● ????: Cloning of new endocannabinoid transporters ● ????: Discovery of new cannabinoid-based therapies What is a cannabinoid? • Initially, compounds extracted by Cannabis spp producing characteristic psychoactivity • Later, compounds with a characteristic terpenoid structure • Currently, most any compound that produces cannabinoid psychoactivity, natural or synthetic • Occasionally, just compounds that will interact with cannabinoid receptors Natural cannabinoids Representative cannabinoids Classical cannabinoids Non-classical cannabinoid Aminoalkylindole CB1 antagonists Endocannabinoids O O HO HO HN O HO Anandamide 2-Arachidonoylglycerol HO O HO O HO HO Noladin ether HN N-Arachidonoyldopamine O NH2 O Virodhamine Cannabinoid CB1 and CB2 Receptors Characteristics of CB1 and CB2 Receptors • • • • • • Both densely distributed throughout the body CB1 highly enriched in central nervous system Located on axon terminals Mediate retrograde signaling (Dendrite → Axon) G-protein coupled CB2 highly enriched in periphery – Especially in immune system • CB2 also in brain and CNS – Fewer than CB1; ~ Same density as μ opioid – Nonetheless, CB2s modulate neural signaling CB1 and CB2 Receptors not the only Receptors Activated by Cannabinoids • Cannabidiol (CBD) receptors • Transient Receptor Potential Cation V1 receptors (TRPV1; Capsaicin receptors) • G-coupled Protein Receptor 55 (GPR55) • G-coupled Protein Receptor 119 (GPR119) • Peroxisome Proliferator-Activated receptors (PPARs) • Others CB1-Mediated Signal Transduction AMPc ATP PKA AC MAPK NA+/H+ K+ exchanger CB1 Ca2+ Guindon, Beaulieu and Hohmann (2009) Pharmacology of the cannabinoid system, IASP Press Gene expression AA CB1 localization Mouse Monkey H.-C. Lu • Antibodies • Distinctive pattern of distribution • Cortex, hippocampus, basal ganglia, SN, cerebellum • Low in thalamus and most of brainstem Eggan S. and Lewis D. Cerebral Cortex 2007; 17:175 CB1 receptor localization (hippocampus) mRNA protein István Katona •In the forebrain, the majority of CB1 protein arises from a minority of interneuons (CCK+ GABAergic) CB1 receptor localization (hippocampus) protein Jim Wager-Miller •CB1 heavily expressed on some axons & terminals EM István Katona CB1 receptor localization (VTA) István Katona •CB1 expressed on two populations of terminals •Functionally, multiple VTA synapses are modulated by cannabinoids CB1 agonists modulate neurotransmission • The signaling pathways of CB1 suggest cannabinoids might decrease neurotransmission: •Inhibition of calcium channel, adenylyl cyclase •Activation of potassium channels, MAP kinase • Appropriate localization of the receptors • Multiple studies show inhibition of neurotransmitter release CB1 agonists modulate neurotransmission Typical experiment: Vc •Hippocampal slices •Patch clamp recording •Bath apply drugs stimulate record Measure GABAergic currents in CA1 Hájos CB1 receptor activation inhibits evoked GABA IPSC’s CB1 receptor summary • Abundantly expressed throughout the brain • Majority on axons and synaptic terminals • Primarily Gi/o coupled (not only!) • CB1 activation inhibits synaptic transmission Endogenous cannabinoids Receptors suggest endogenous ligands Two main families identified Both arachidonic acid derivatives Precursors in membranes “Made on demand” Amides (anandamide) Esters (2-AG) • Significant differences – Routes of synthesis – Mode of degradation (FAAH vs MAGL) – Efficacy CB1 agonist efficacy is variable Many studies have found 2-AG to be more efficacious than anandamide (or THC) at CB1 (GIRK activation in oocytes shown here) 2-AG MEA THC Luk, et al, 2004 eCB summary • Acyl ethanolamides (diverse; anandamide, AEA) • More promiscuous --- many targets • Acyl glycerol esters (2-AG) • Both are “Made on demand” • 2-AG ~100x more bulk levels, similar “signaling”(?) • Differing efficacies • Metabolic diversity, with “core” pathways What are the physiological effects of eCB’s on neuronal activity? • Exogenous cannabinoids inhibit neurotransmission • eCB’s are synthesized following increases in intracellular calcium and/or activation of Gq/11linked receptors • Might eCB’s synthesized in this fashion modulate neurotransmission? • Yes •Transient effects •Long lasting effects Six Types of eCB-Mediated Synaptic Plasticity Have Been Clearly Identified • • • • • • Depolarization-induced suppression of inhibition Depolarization-induced suppression of excitation Metabotropic-induced suppression of inhibition Metabotropic-induced suppression of excitation Long-Term Depression (LTD) Slow self-inhibition (SSI) • Additional types are being constantly discovered Important Take-Home Messages • Endocannabinoids are neurotransmitters • Cannabinoids (e.g., THC) modulate neural activity • Endocannabinoids are involved in synaptic remodeling • Cannabinoids (e.g., THC) can modulate synaptic remodeling • Depending upon the specific CNS circuits involved, cannabinoids can have a host of actions on brain, cognition, and behavior (some beneficial, some not) Cannabinoids and pain ● central ● spinal ● periphery Peripheral and spinal localization of cannabinoid receptors Ständer et coll. J Dermatol Sci 2005 Hohmann & Herkenham Neuroscience 1999 Bridges et coll. Neuroscience 2003 Farquhar-Smith et coll. Mol Cell Neurosci 2000 AEA NAPEPLD ? Presynaptic neuron NAPE NAT 2-AG MGL Neurotransmitter vesicles ET CB1 Ca2+ ET DAGL AA COX 2-AG PG DAG PLC Phospholipid AEA NAPEPLD ? NAPE NAT ? Postsynaptic neuron Guindon et al., (2009) Pharmacology of the cannabinoid system, IASP Press Evaluation of nociceptive behavior in the formalin test Behaviours* Normal behaviour Pain behaviour (1) Pain behaviour (2) Observations Injected paw can support the weight of the animal. Injected paw has little or no weight on it. Injected paw is elevated, not in contact with any surface. Injection 50 µL Formaline 2.5 % NaCl 0,9% Pain behaviour (3) Pain score 1.2 1 Scoring system** Time spent in this category 0 0 1 Injected paw is licked, bitten or shaken. 2 0.8 0.6 0.4 * The same behaviours are observed with the hind paw. 0.2 0 0 5 10 15 20 25 30 35 40 Time (min) 45 50 55 60 ** Watson et al. (1997) Peripheral Antinociceptive Effects Composite Pain Score (CPS) NaCl 0.9 % Anandamide 0.1 µg Ibuprofen 2 µg Rofecoxib 2 µg 1, 2 1 0, 8 1.2 # 0, 6 0, 4 1 0, 2 † 0 0 5 10 15 20 25 30 35 40 45 50 55 60 0.8 contralateral 0.6 0.4 0.2 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min) † AUC (0-15) P < 0.05 and # AUC (15-60) P < 0.001 for analgesics vs NaCl 0.9 % ipsilateral Synergistic effect of anandamide + ibuprofen 25 Anandamide Ibuprofen Mix (1:10) 0.2 Anandamide Ibuprofen Mix 1:10 Add 1:10 Dose Ibuprofen (µg) Pain (Area Under the Curve) 20 15 10 0.15 0.1 0.05 5 0 0 0 -4 -3 -2 -1 Log dose (µg) Guindon et al. (2006) Pain 121: 85-93 0 1 0.005 0.01 0.015 0.02 Dose Anandamide (µg) Synergistic effect of anandamide + rofecoxib 25 Rofecoxib Mix (1:10) 20 0.2 Dose Rofecoxib (µg) Pain (Area Under the Curve) Anandamide 15 10 Anandamide Rofecoxib Mix 1:10 Add 1:10 0.15 0.1 0.05 5 0 0 0 -4 -3 -2 -1 0 1 Log dose (µg) Guindon et al. (2006) European Journal of Pharmacology 550: 58-77 0.005 0.01 0.015 Dose Anandamide (µg) Objectives of 2-AG, JZL184 and URB602 study Compare the peripheral antinociceptive effects of 2-AG, JZL184, URB602 and their combination in the formalin test Study the mechanisms by which JZL184 and URB602 produce their effects using specific CB1 and CB2 receptor antagonists Composite Pain Score (CPS) Peripheral Antinociceptive Effects NaCl 0.9% URB602 500 µg 1.2 1 0.8 1.2 # 0.6 0.4 1 0.2 0 † 0 5 10 15 20 25 30 35 40 45 50 55 60 0.8 contralateral 0.6 0.4 0.2 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min) † P < 0.001 and # P < 0.001 for URB602 (500 µg) vs NaCl 0.9 % ipsilateral Composite Pain Score (CPS) Peripheral Antinociceptive Effects Vehicle 1 1 JZL184 300 microg 0.8 # † 0.8 0.6 0.4 0.2 0 0.6 0 5 10 15 20 25 30 35 40 45 50 55 60 contralateral 0.4 0.2 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (min) † P < 0.001 and # P < 0.001 for JZL184 ((300 µg) vs NaCl 0.9 % ipsilateral JZL184 with cannabinoid antagonists Inflammatory Phase Area Under the Curve 20 16 12 * 8 4 0 Vehicle JZL184 10µg AM251 80µg AM251 + JZL184 AM630 25µg AM630 + JZL184 * P < 0.001 for JZL184 (10 µg) vs Vehicle URB602 with cannabinoid antagonists Inflammatory Phase Area Under the Curve 20 16 * 12 8 4 0 NaCl 0.9 % URB602 70µg Guindon et al. (2006) Brithish Journal of Pharmacology 150: 693-701 AM251 AM251+ URB602 AM630 AM630+ URB602 * P < 0.001 for URB602 (70 µg) vs NaCl 0.9 % Conclusions JZL184, URB602, 2-AG and their combination reduce nociceptive behavior when given locally JZL184 is more potent than URB602 when given alone or combined with 2-AG Antinociceptive effects of JZL184 and URB602 are inhibited by AM251 and AM630 Cannabinoids and Addiction There is now an extensive published literature showing antiaddiction efficacy for cannabinoid ligands • • • • • • • • Gardner EL. Endocannabinoid signaling system and brain reward: emphasis on dopamine. Pharmacol Biochem Behav 81:263-284, 2005 De Vries TJ & Schoffelmeer AN. Cannabinoid CB1 receptors control conditioned drug seeking. Trends Pharmacol Sci 26:420-426, 2005 Cohen C et al. CB1 receptor antagonists for the treatment of nicotine addiction. Pharmacol Biochem Behav 81:387-395, 2005 Maldonado R et al. Involvement of the endocannabinoid system in drug addiction. Trends Neurosci 29:225-232, 2006 Basavarajappa BS. The endocannabinoid signaling system: a potential target for next-generation therapeutics for alcoholism. Mini-Revs Med Chem 7:769-779, 2007 Fattore L et al. Endocannabinoid regulation of relapse mechanisms. Pharmacol Res 56:418-427, 2007 Scherma M et al. The endocannabinoid system: a new molecular target for treatment of tobacco addiction. CNS & Neurol Disorders - Drug Targets 7:468-481, 2008 Paralaro D & Rubino T. The role of the endogenous cannabinoid system in drug addiction. Drug News Perspect 21:149-157, 2008 CB1 Antagonist-Induced Attenuation of Cocaine-Enhanced Brain Stimulation Reward CB1 Antagonist-Induced Attenuation of Cocaine-Enhanced Brain Stimulation Reward CB1 Antagonism Does Not Affect Motoric Ability PR Schedule Reward (# Infusion) 1 2 3 4 5 Pump Work Demand (# Lever Press) 1 2 4 6 9 Cocaine ? 14 15 16 …. 77 95 118 …. Cumulat 600 400 CB1 Antagonist-Induced Attenuation of IncentiveCocaine Motivation to 200 = 0.5 mg/kg/infusion Self-Administer i.v. Cocaine – 0Representative Animal 0 20 40 60 80 100 120 140 160 (Progressive-Ratio Model) Time (min) A After Vehicle 1600 1600 Cumulative Lever Presses Cumulative Lever Presses After AM 251 (1 mg/kg) B 1400 1200 1000 800 600 400 200 1400 1200 1000 800 600 400 200 Cocaine = 0.5 mg/kg/infusion Cocaine = 0.5 mg/kg/infusion 0 0 0 20 40 60 80 100 Time (min) B After AM 251 (1 mg/kg) 120 140 160 0 20 40 60 80 100 Time (min) 120 140 160 CB1 Antagonist-Induced Attenuation of Incentive Motivation to Self-Administer i.v. Cocaine (Progressive-Ratio Model) AM 251 120 60 AM251 (% Change in Break-Point) Break-Point 50 * 40 30 *** 20 (% Change over Baseline) 100 * 80 * *** 60 40 20 10 0 0 0 1 3 0 10 1 3 AM 251 (mg/kg, i.p.) AM 251 (mg/kg, i.p.) SR141716A 120 100 100 ) n) Break-Point (Lever Presses for Last Infusion) 70 AM251 (Original Break-Point) 10 CB1 Receptor Antagonism Dose-Dependently Attenuates Relapse to Cocaine-Seeking Behavior (Reinstatement Model) CB1 Receptor Antagonism Does Not Attenuate Relapse to Non-Drug Reward-Seeking Behavior (Reinstatement Model) CB1 Receptor Antagonist Micro-Injected Into Nucleus Accumbens Attenuates Cocaine-Seeking Behavior (Reinstatement Model) CB1 Receptor Antagonism By Itself Does Not Produce Drug-Seeking Behavior (Reinstatement Model) CB1 Receptor Antagonism Markedly Attenuates CocaineEnhanced Nucleus Accumbens Glutamate (Brain Microdialysis) CB1 Receptor Antagonism Markedly Attenuates Cocaine Sensitization CB1 Receptor Gene-Deletion (CB1 Gene Knock-Out) Abolishes Cocaine’s Psychostimulant Effects CB1 Receptor Gene-Deletion (CB1 Gene Knock-Out) Abolishes Cocaine-Enhanced Nucleus Accumbens Dopamine (Dialysis) CB1 Receptor Gene-Deletion (CB1 Gene KO) Attenuates Evoked Nucleus Accumbens Dopamine Release (Voltammetry) CB1 Receptor Gene-Deletion (CB1 Gene KO) Attenuates Evoked Nucleus Accumbens Dopamine Release (Voltammetry) CB1 Antagonist SR141716 (Rimonabant) By Itself Markedly Inhibits Nucleus Accumbens Dopamine (Brain Microdialysis) Other CB1 Receptor Antagonists (Either Neutral Antagonists or Inverse Agonists) Do Not Do This !! Caveats Regarding Development of Cannabinoid Agonists as Potential Pharmacotherapeutic Agents • CB1 and CB2 receptors are ubiquitous throughout the body – Potential for numerous side effects • Some cannabinoid ligands have poor bioavailability • CB1 receptor agonists have addictive potential Potential Cannabinoid Therapies - Tools • • • • • • • • • • • • Endocannab Uptake Inhibitors – AM404, UCM707, AM1172 FAAH Inhibitors – URB597, OL135, BMS1, SA47, PF750 MAGL Inhibitors – URB602, OMDM169, JZL184 Dual CB1/CB2 Agonists – WIN55512, CP55940, HU210 Anandamide Analogues – Methanandamide, Metfluoroanand. Selective CB1 Agonists – ACEA, ACCP Selective CB2 Agonists – HU308, JWH015, JWH133, AM1241 2-AG Synthesis Inhibitors – O3640, O3891, OMDM188, O5596 CB1 Antagonists/Inverse Agonists – SR141716A, AM251 CB1 Neutral Antagonists – AM4113, PIMSR1 CB2 Antagonists/Inverse Agonists – SR144528, AM630 CB1 Receptor Allosteric Modulators – ORG27596, ORG29647 Potential Cannabinoid Therapies – Clinical Indications • Diseases of Energy Metab. – – – – – – – Appetite Dysregulation Obesity Dyslipidemia Periph Energy Metab Dysreg Cachexia Anorexia Type 2 Diabetes • Pain – Somatosensory Pain – Neuropathic Pain • Inflammation • CNS Disorders – Closed Head Brain Trauma – Neurotoxicity – – – – – – – – – – – – – – – – Stroke Spinal Cord Injury Multiple Sclerosis Parkinson’s Disease Huntington’s Disease Tourette’s Syndrome Tardive Dyskinesia Dystonia Amyotrophic Lateral Sclerosis Alzheimer’s Disease Epilepsy Anxiety Depression Insomnia Post-Traumatic Stress Disorder Schizophrenia Potential Cannabinoid Therapies – Clinical Indications • CNS Disorders – con’t – Nausea & Emesis – Drug & Alcohol Addiction • Cardiovascular & Respiratory – – – – – – – Hypertension Hypotension Circulatory Shock Myocardial Reperfusion Injury Atherosclerosis Cardiopathies Asthma • Eye Disorders – Glaucoma – Retinopathy – Intraocular Pressure • Cancer – Cancer Cell Proliferation – Colorectal Cancer • GI and Liver Disorders – – – – – – Inflammatory Bowel Disease Ulcerative Colitis Hepatitis Cirrhosis – Encephalopathy Cirrhosis – Liver Fibrosis Cirrhosis – Vasodilatation • Musculoskeletal Disorders – Arthritis – Osteoporosis – Post-Fracture Bone Healing • Reproductive Disorders Acknowledgments • Ken Mackie, MD – Dept of Psychological and Brain Sciences, Indiana University Bloomington • Josée Guindon, PhD – Dept of Psychology, Univ of Georgia • Andrea G. Hohmann, PhD – Neuroscience and Behavior Program, Univ of Georgia • Raphael Mechoulam, PhD – Dept of Medicinal Chemistry, Hebrew University of Jerusalem • Roger Pertwee, PhD – School of Medical Sciences, Univ of Aberdeen, Scotland • Steven Goldberg, PhD – Behavioral Neuroscience Research Branch, NIDA, NIH • Javier Fernández-Ruiz, PhD – Facultad de Medicina, Universidad Complutense, Madrid • Vincenzo Di Marzo, PhD – Endocannabinoid Research Group, Consiglio Nazionale delle Ricerche, Naples, Italy Neuropsychopharmacology Section, Intramural Research Program National Institute on Drug Abuse, National Institutes of Health Acknowledgment Raphael Mechoulam, PhD Dept of Medicinal Chemistry, Hebrew Univ of Jerusalem