View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Queen Mary Research Online Cannabinoids for the control of experimental multiple sclerosis Pryce, Gareth The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author For additional information about this publication click this link. https://qmro.qmul.ac.uk/jspui/handle/123456789/673 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] 1 Cannabinoids for the control of experimental multiple sclerosis GARETH PRYCE Neuroimmunology Unit, Neuroscience & Trauma Barts and the London School of Medicine and Dentistry Queen Mary University of London UK 2 This thesis is dedicated in memory of my Dad, Glyn Pryce (1927-2010), with love and thanks. 3 ACKNOWLEDGEMENTS I wouldlike to thank DavidBaker andGavin Giovannoni for their supervision and providingme with the opportunity to undertake this thesis. I wouldlike to thank J.Ludovic Croxford, Sam Jackson, Sarah Al-Izki from the lab past and present; Ana Cabranes (RIP) and the Javier Fernadez-Ruiz laboratory in Madrid, Spain; the Vincenzo Di Marzio Laboratory Naples, Italy; the Andy Irving Laboratory, Dundee; the laboratories of Elga De Vries andSandra Amor at the Free University Amsterdam, The Netherlands and the laboratories of Ruth Ross and Roger Pertwee in Aberdeen and Alison Hardcastle University College London for providing me with supportive data and particularly Cristina Visintin and David Selwoodof University College London andCanbex for providingdata from contract Research organizations. I thank Dr. D. Argentieri and Dr. D Ritchie for providing access to RWJ compounds andDr. M. Ferrari for providingCT3. David Selwoodand Cristina Visintin are thankedfor supply of VSN16. Jonathan Longand Ben Cravatt are thankedfor providingJZL184. Catherine Ledent, Sarah A. Thomas, Beat Lutz, Giovanni Marsicano and Benjamin Cravatt are thanked for supply of mice and materials. I would also like to thank The Multiple Sclerosis Society of Great Britain and Northern Ireland, the National Multiple Sclerosis Society, USA, Aims2Cure; the Brain Research Trust; the Bloomsbury Bioseed Fund; The Wellcome Trust, Canbex andthe Pharmaceutical Industry andBarts andthe London School of Medicine and Dentistry for support of various aspects of research. I acknowledge RW Johnson, Raritan, New Jersey, USA in part funded experiments involving RWJ compounds. Altantic Ventures part funded some of the early CT3 studies. I am also indebtedto the National Institute for Drug Abuse chemical supply programme for donating chemicals for this study. Lastly, but certainly not least I wouldlike to thank my Mum, Dad, my sister Jan and my partner Karen for their support andencouragement. I wouldparticularly like to thank DavidBaker for his unswervingfriendship, support, help, encouragement and cajoling when needed. It has been incredibly rewarding from an intellectual and personal level to work with him for the last thirteen years andhopefully for many more to come. My very great thanks to all! 4 ABSTRACT There have been numerous studies reporting that cannabinoids, both exogenous and endogenous, have a potential beneficial function during incidences of neurological damage. Usinggene knockout mice andcannabinoid-selective agents, this study demonstrates the diverse actions of cannabinoids with a particular focus on experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. The results presented here report on the action of stimulators of cannabinoid receptors in the nervous system (CNS) on; immune function, as a mechanism of suppressing autoimmune attack of the central nervous system, as agents to suppress neurodegenerative events leadingto disease progression andas agents that can control signs of disease that occur as the consequences of autoimmune neurodegeneration such as spasticity. Tetrahydrocannabinol the psychoactive component in cannabis andthe CB 1 cannabinoidreceptor appears to be central to many of the therapeutic actions of cannabis but also to the side-effect potential of cannabinoid drugs. This study reports on methods to avoid psychoactive side-effects of conventional brain-penetrant CB 1 receptor agonists whilst exploiting the therapeutic potential of the cannabinoid system in order to control spasticity. This was achievedby targetingmechanisms of endocannabinoid degradation, particularly using fatty acid amide hydrolase inhibitors. Furthermore, this study also reports the development of novel cannabinoidcompounds that are excluded from the brain and inhibit spasticity and also demonstrates the mechanism of exclusion of CNS-excluded cannabinoid CB 1 receptor agonists. This study provides further evidence for the efficacy of cannabinoid compounds during an ongoing CNS disease and also their efficacy for treating the consequences of CNS autoimmune disease, which hopefully, will give additional impetus for further clinical investigations of cannabinoidagents in not only multiple sclerosis but also other neurodegenerative diseases of the CNS. 5 TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………………………………………………....3 ABSTRACT…………………………………………………………………………………………………………………….4 TABLE OF CONTENTS……………………………………………………………………………………………………5 LIST OF FIGURES……………………………………………………………………………………………………….10 LIST OF TABLES……………………………………………………………………………………………............12 ABBREVIATIONS………………………………………………………………………………………................13 CHAPTER ONE INTRODUCTION ……………………………………………………………………………………………………15 1.1 The Cannabinoidsystem……………………………………………………………………………………..15 1.1.1 Cannabinoidreceptors CB 1……………………………………………………………………………...16 1.1.2 CB 2 receptor………………………………………………………………………………………………………19 1.1.3 Non CB 1/2 receptor mediatedcannabinoidsignaling………………………………………20 1.1.3.1 GPR55……………………………………………………………………………………………………………20 1.1.3.2 Vanilloidreceptor TRPV-1…………………………………………………………………………….21 1.1.3.3. Peroxisome proliferator-activated receptors (PPARs)……………………………… 22 1.1.3.4. GPR18…………………………………………………………………………………………………………..23 1.2. CB 1/2 receptor agonists/antagonists…………………………………………………………………..24 1.2.1. Inverse agonism……………………………………………………………………………………………..25 1.3. Endocannabinoids……………………………………………………………………………………………….25 1.4. Multiple Sclerosis andexperimental models……………………………………………………..28 1.4.1. Multiple Sclerosis…………………………………………………………………………………………….28 1.4.2. Experimental allergic encephalomyelitis (EAE)………………………………………………34 1.5 Cannabinoids andsymptom management in MS……………………………………………….37 1.6 Cannabinoids in Autoimmunity……………………………………………………………………………40 1.7 Cannabinoids in Neuroprotection and disease progression in MS and animal models………………………………………………………………………………………………………………………..43 1.8 Aims of this study………………………………………………………………………………………………..51 CHAPTER TWO MATERIALS AND METHODS ……………………………………………………………………………..52 2.1. Animals................................................................................................52 2.1.1. Laboratory Mice..................................................................................52 2.1.2. Transgenic Mice……………………………………………………………………………………………….52 2.1.2.1. CB 1 CannabinoidReceptor Knockout Mice………………………………………………….52 6 2.1.2.2. CB 1 CannabinoidReceptor Conditional Knockout Mice………………………………53 2.1.2.3. CB 2 CannabinoidReceptor Knockout Mice………………………………………………….54 2.1.2.4. G-protein CoupledReceptor 55 Knockout Mice………………………………………….55 2.1.2.5. Transient Receptor Potential VanilloidReceptor 1 Knockout Mice…………….55 2.1.2.6. Fatty AcidAmide Hydrolase Knockout Mice……………………………………………….55 2.1.2.7. P-Glycoprotein Knockout Mice…………………………………………………………………….56 2.2. Genotypingof Animals……………………………………………………………………………………….56 2.2.1. Production of Crude DNA………………………………………………………………………………..56 2.2.2 Polymerase Chain reaction………………………………………………………………………………57 2.2.3. P-glycoprotein andCNS exclusion pump activity………………………………………….59 2.2.4. Ribonucleic Acid(RNA) extraction andMicroarray………………………………………..59 2.3. Chemicals…………………………………………………………………………………………………………..60 2.3.1. Vehicles…………………………………………………………………………………………………………..60 2.3.1. CB 1-targetedCannabinoidReceptor Reagents……………………………………………..60 2.3.2. CB 2-selective CannabinoidReceptor Reagents……………………………………………..61 2.3.2. CNS ExcludedCB 1-Receptor Agonists…………………………………………………………….61 2.3.3. EndocannabinoidDegradation Inhibitors……………………………………………………….61 2.3.4. Inhibitors of CNS efflux Pumps………………………………………………………………………62 2.3.5. Non-CannabinoidReceptor Reagents…………………………………………………………….62 2.4. Receptor BindingAssays........................................................................62 2.5. Pharmacokinetics..................................................................................63 2.6. Induction of Experimental Autoimmune Encephalomyelitis……………………………..63 2.6.1. Preparation of Spinal CordHomogenate………………………………………………………..63 2.6.2. Preparation of Inoculum for Spinal Cord-InducedDisease ABH Mice…………..64 2.6.3. Preparation of Inoculum for MOG-InducedDisease in C57BL/6 Mice…………..65 2.6.4. Injection of animals………………………………………………………………………………………..65 2.6.5. Clinical Disease Scoring………………………………………………………………………………….66 2.7. Behavioral Testing………………………………………………………………………………………………67 2.7.1. Open fieldactivity monitoring…………………………………………………………………………67 2.7.2. Temperature measurement…………………………………………………………………………….67 2.7.3 RotoRodActivity