The Cannabis Plant Botanical

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HANDBOOK OF CANNABIS AND RELATED PATHOLOGIES

ELSEVIERscience & technology books Companion Web Site: http://store.elsevier.com/product.jsp?&isbn=9780128007563 Handbook of Cannabis and Related Pathologies V.R. Preedy, Editor Available Resources: 20 additional chapters are available online - see contents list. A L LT O O L S FOR YOUR TEACHING N E E D S textbooks.elsevier.com ACADEMIC PRESS To adopt this book for course use, visit http://textbooks.elsevier.com.

HANDBOOK OF CANNABIS AND RELATED PATHOLOGIES BIOLOGY, PHARMACOLOGY, DIAGNOSIS, AND TREATMENT Edited by V.R. Preedy BSc, PhD, DSc, FRSB, FRSH, FRIPHH, FRSPH, FRCPath, FRSC Faculty of Life Sciences and Medicine, King’s College London, London, United Kingdom

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2017 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-800756-3 For information on all Academic Press publications visit our website at https://www.elsevier.com/ Publisher: Mara Conner Acquisitions Editor: April Farr Editorial Project Manager: Timothy Bennett Production Project Manager: Chris Wortley Designer: Mark Rogers Typeset by Thomson Digital

Dedicated to my friend Chris Bird

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Contents List of Contributors  xv 9.  Cannabis, Migration, and Psychosis Onset 79 Preface  xxv A. KOKONA, I. TARRICONE, M. DI FORTI, E. CARRA I 10.  The Global Epidemiology and Disease 89 Burden of Cannabis Use and Dependence 101 SETTING THE SCENE, BOTANICAL, 110 GENERAL AND INTERNATIONAL L. DEGENHARDT, A.J. FERRARI, W.D. HALL ASPECTS 11.  International Aspects of Cannabis Use and Misuse: the Australian Perspective 1.  The Cannabis Plant: Botanical Aspects 3 D.J. ALLSOP, W.D. HALL S. FARAG, O. KAYSER 12.  International Aspects of Cannabis Use 2.  The Biosynthesis of Cannabinoids 13 and Misuse: Egypt F. DEGENHARDT, F. STEHLE, O. KAYSER O.M.E. ABDEL-SALAM, A.F. GALAL, S.A. ELSHEBINEY, A.E.D.M. GAAFAR 3.  Increasing Plant Concentrations of THC and 24 13.  Cannabis Body Packing: A Caribbean Implications on Health Related Disorders Perspective 122 V. VINDENES, J. MØRLAND S.O. CAWICH, D. DAN, V. NARAYNSINGH 4.  Age as a Predictor of Cannabis Use 33 131 D. BERGEN-CICO, R.D. CICO II 5.  Lifetime Cannabis Use and Cognition PERSONAL, SOCIAL AND in Psychosis Spectrum Disorders COMMUNITY ASPECTS M.J. CUESTA, A.M. SÁNCHEZ-TORRES, R. LORENTE-OMEÑACA, 44 OF CANNABIS USE L. MORENO-IZCO 14.  Gender Differences in Cannabis 6.  A Profile of Synthetic Cannabinoid Users Use Disorders 53 R. SECADES-VILLA, S. FERNÁNDEZ-ARTAMENDI A.N. SANDERS, J.M. STOGNER 15.  The Role of Age in the Onset and Further 7.  Dual Disorders in Cannabis Misuse 61 Development of Cannabis Use Disorders 138 F. ARIAS-HORCAJADAS, N. SZERMAN, P. VEGA, S. BEHRENDT I. BASURTE, B. MESÍAS 16.  Effects of Cannabis Use on Neurocognition 8.  Cannabis Use and Cognitive Function 70 in Adolescents and Emerging Adults 151 C. EVREN N.E. WRIGHT, K.E. MAPLE, K.M. LISDAHL vii

viii CONTENTS 257 17.  Correlates and Consequences of Prenatal 267 Cannabis Exposure (PCE): Identifying and 27.  Cannabis Use and First-Episode Psychosis Characterizing Vulnerable Maternal Patients (FEP) Populations and Determining Outcomes for Exposed Offspring I. GONZÁLEZ-ORTEGA, M. MARTÍNEZ-CENGOTITABENGOA, A. GONZÁLEZ-PINTO L.K. BRENTS 160 18.  Cannabis and Clubbing: Relevance of 28.  Cannabis, Associative Memory, fMRI, Cannabis and Polydrug Use in the Clubbing and the Implicit Association Test Culture Today S.L. AMES, A.W. STACY D.A. HERZIG, S. BACHMANN 171 29.  Stress Response in Cannabis Users 278 19.  Cannabis and Sexual Behavior and Psychosis 288 G. SCIMECA, C. CHISARI, M.R.A. MUSCATELLO, M. BIOQUE, H.-H. TSENG, R. MIZRAHI C. CEDRO, G. PANDOLFO, R. ZOCCALI, A. BRUNO 180 20.  Friendships and Cannabis Use 30.  Motivation in Chronic Cannabis Use J.H. BOMAN IV, C. HECK R. HIRST, L. SODOS, S. GADE, L. RATHKE 21.  Students’ Knowledge of Cannabis 188 31.  Cannabis Use and Its Association to Mental Illness: A Focus on Mood and Anxiety Disorders 298 M. DROZD, J. SOBCZYN´SKI S. LEV-RAN, D. FEINGOLD 22.  Childhood Trauma and Cannabis: Risk Factors in Severe Mental Disorders? 198 308 32.  Cannabis Use and Well-Being M. AAS, I. MELLE J. ALLEN, M.D. HOLDER, Z. WALSH 208 33.  Craving and Cannabis: A Potential Paradox 317 M.J. LOFLIN, M. EARLEYWINE 23.  Parent’s Influence on Children’s 34.  Delta-9-Tetrahydrocannabinol and 326 Cannabis Use 215 Catalepsy-Like Immobilization S. MILLER, J.T. SIEGEL, W.D. CRANO N. EGASHIRA 24.  Cannabis Users and Premorbid 35.  The Interactive Nature of Cannabis 335 Intellectual Quotient 223 and Schizophrenia Risk Genes 345 L. FERRARO, L. SIDELI, D. LA BARBERA T. KARL, J.C. ARNOLD 25.  Cannabis and Traffic Accidents 234 36.  Neuroimaging Findings in Adolescent Cannabis Use and Early Phase Psychosis R.B. DE BONI, R.P. LIMBERGER, T.R.V. SOUSA C.E. CROCKER, J. COOKEY, P.G. TIBBO III 37.  Cannabis Smoking in Adult Schizophrenia: 357 A Cognitive and Functional Magnetic CANNABIS, BEHAVIOR, Resonance Imaging Perspective PSYCHOPATHOLOGY AND K. PAQUIN, T. LECOMTE, S. POTVIN NEUROPATHOLOGY 38.  The Long-Lasting Effects of Cannabis 372 26.  Drug-Related Pictures, Attentional Bias, Use on Movement and Brain Regions that and Cannabis Use 247 Control Movement D. ASMARO G. TODD, J.M. WHITE   

CONTENTS ix 481 39.  Assessment of Cannabis Acute Effects on 50.  Cardiovascular Effects of Cannabis Usage Driving Skills: Laboratory, Simulator, and 486 On-Road Studies S. MENAHEM 494 P. BONDALLAZ, H. CHTIOUI, B. FAVRAT, E. FORNARI, 379 C. GIROUD, P. MAEDER 51.  Cannabis and Stroke P.A. BARBER 40.  Chronic Cannabis Use and Axonal Fiber 52.  Cannabis Smoking and the Lung Connectivity 391 D.P. TASHKIN N. SOLOWIJ, A. ZALESKY, V. LORENZETTI, M. YÜCEL 41.  Microglial Activation and Cannabis 53.  Cannabis and Hepatic Injury 505 Exposure 401 S.A. NADA, O.M.E. ABDEL-SALAM, A.A. SLEEM L. CUTANDO, R. MALDONADO, A. OZAITA 42.  Cannabis and Psychosis: Correlation, 54.  Cannabis Allergy: More Than a Bad Trip 517 Causality, and Consequences A.L. VAN GASSE, V. SABATO, M.M. FABER, C.H. BRIDTS, D.G. EBO D. BASU, P. PARAKH 413 55.  Marijuana and Breastfeeding 527 M.G. HILL, K.L. REED 43.  Cannabis Use in Bipolar Disorder 422 T.V. LAGERBERG 56.  Hypocretins/Orexins and Addiction: Role in Cannabis Dependence 533 Á. FLORES, R. MALDONADO, F. BERRENDERO 44.  Cannabis Use in Epilepsy—Risks and Benefits 431 M. HOLTKAMP, M. HAMERLE 57.  Regulatory Role of Cannabinoids for Skin Barrier Functions and 45.  Cannabis, Cannabinoids, and Visceral Pain 439 Cutaneous Inflammation 543 R. ABALO, M. ISABEL MARTÍN-FONTELLES T. TÜTING, E. GAFFAL 46.  Cannabis and Postoperative Analgesia 450 V S.O CAWICH, U. DEONARINE, H.E. HARDING, D. DAN, PHARMACOLOGY AND CELLULAR V. NARAYNSINGH ACTIVITIES OF CANNABINOIDS AND ENDOCANNABINOIDS IV 58.  Cannabinoids and the Cannabinoid 553 CANNABIS, ORGANS, TISSUES Receptors: An Overview AND NON-CNS ASPECTS D. LU, D.E. POTTER 47.  Chronic Cannabis Abuse and Thyroid Function 461 59.  Signaling and Regulation of the 564 Cannabinoid CB1 Receptor U. BONNET M.R. HUNTER, D.B. FINLAY, M. GLASS 48.  Cannabis Hyperemesis Syndrome 466 U. BONNET 60.  Allosteric Modulation of the Cannabinoid CB1 Receptor 49.  Cannabis and Cannabinoids and the Effects 573 on Gastrointestinal Function: An Overview E.E. CAWSTON, M.R. HUNTER, M. GLASS 471 M. SAŁAGA, R. ABALO, J. FICHNA   

x CONTENTS 61.  Polymorphisms of the CB2 Cannabinoid VI Receptor 584 EFFECTS OF SPECIFIC NATURAL P. KUMAR, Z.-H. SONG AND SYNTHETIC CANNABINOIDS 62.  Chemistry of Cannabinoid Receptor Agonists 592 72.  The Role of 5-HT1A Receptor, and Nausea and Vomiting Relief by Cannabidiol (CBD), M. AGHAZADEH TABRIZI, P.G. BARALDI Cannabidiolic Acid (CBDA), and Cannabigerol (CBG) 63.  The Endocannabinoid System as a Target 606 703 for New Antiseizure Drugs E.M. ROCK, L.A. PARKER L.R. VILELA, A.C.P. DE OLIVEIRA, M.F. MORAES, F.A. MOREIRA, R.N. TAKAHASHI 73.  Genetic and Molecular Aspects of Addiction with Tetrahydrocannabinol 713 64.  Pharmacological Aspects of Anandamide and 2-Arachidonoyglycerol as Bioactive Lipids 616 T. JANUS, A. MACHOY-MOKRZYŃSKA, K. BOROWIAK M. ALHOUAYEK, G.G. MUCCIOLI 74.  Effects of ∆9-Tetrahydrocannabinol in Human Breast Cancer 65.  Pharmacological Aspects of NMDA 722 Receptors, mGluR5, and Endocannabinoids S. TAKEDA, E. IKEDA, H. OKAZAKI, K. WATANABE, H. ARAMAKI 630 Y. IZUMI, C.F. ZORUMSKI 75.  ∆9-THC and COX-2 Signaling 729 J. ZHANG, C. CHEN 66.  Peripheral CB1 Receptors and Ghrelin in Feeding Regulation: Pharmacological Implications 639 76.  Cannabinoids and the Addictive Effects of Nicotine L. ORIO, R. GÓMEZ DE HERAS, F. RODRÍGUEZ DE FONSECA L.V. PANLILIO, S.R. GOLDBERG 739 67.  Pharmacological Aspects of Novel 649 77.  Cannabinoid Regulation of Intraocular Antiobesity Agents Related to Cannabinoids Pressure: Human and Animal Studies, Cellular and Molecular Targets L. HERNANDEZ-FOLGADO A. ALOWAY, A. KUMAR, A.S. LAUN, Z.H. SONG 68.  Cannabinoid Reward and Dependence: 748 Focus on the Main Psychoactive Ingredients of Marijuana in Preclinical Studies 659 78.  Ocular Delivery of Tetrahydrocannabinol 760 G. PANAGIS G.R. ADELLI, P. BHAGAV, M.A. REPKA, W. GUL, M.A. ELSOHLY, S. MAJUMDAR 69.  Peroxisome Proliferator Activated Receptors and Cannabinoids 671 79.  The Role of γ-Aminobutyric Acid in the E. MUÑOZ, F. POLLASTRO, O. TAGLIALATELA-SCAFATI, G. APPENDINO Interoceptive Effects of Oral ∆9-Tetrahydrocannabinol in Humans 70.  The Protein–Protein Interactions of Cannabinoid 770 J.A. LILE, J.S. FOGEL, T.H. KELLY Receptor Interacting Protein 1a (CRIP1a) and Cannabinoid 1 Receptor: The Molecular Mechanism Study Through an Integrated 80.  The Role of ∆9-Tetrahydrocannabinol in Molecular Modeling Approach 680 Diabetes Mellitus 779 M.H. AHMED, Y. ZHANG Z.M. COSKUN, S. BOLKENT 71.  Synthetic Cannabinoids: a Summary 81.  Cannabidiol: An Overview of its 787 of Selected Phenomena with Respect to Antipsychotic Properties Behavioral Pharmacology and Abuse Liability 691 F.F. PERES, V. ALMEIDA, V.C. ABILIO B.T. BURROWS, L.R. WATTERSON, J. EGNATIOS, M.F. OLIVE   

CONTENTS xi 877 82.  Cannabidiol for the Treatment of 795 91.  Beneficial Effects of Cannabis and 883 Epilepsy: An Overview of Possible Related Compounds on Sleep 893 Mechanisms of Action and Preclinical and 905 Human Studies I.M.P. LINARES, J.A.S. CRIPPA, M.H.N. CHAGAS 917 931 R. GUIMARÃES DOS SANTOS, J.E.C. HALLAK, A.W. ZUARDI, 92.  Cannabinoid-Based Medicines for the 939 A.C. DE SOUZA CRIPPA, J.A. DE SOUZA CRIPPA Treatment of Gilles de la Tourette Syndrome 947 958 83.  Cannabidiol and Neuroprotection: Evidence 802 A.S. KANAAN, K.R. MÜLLER-VAHL from Preclinical Studies 971 93.  Cannabidiol and Multiple Sclerosis N. SCHRÖDER, V.K. DA SILVA, J.E.C. HALLAK, A.W. ZUARDI, J.A. DE SOUZA CRIPPA M. MECHA, A. FELIÚ, F.-J. CARRILLO-SALINAS, C. GUAZA 84.  Cannabinoids as Potent Inhibitors 813 94.  Cannabinoids and Their Effects on of Human CYP1 Enzymes Painful Neuropathy K. WATANABE, S. YAMAORI, K. MASUDA, T. KATSU, D. SELVARAJAH, R. GANDHI, S. TESFAYE S. NARIMATSU, I. YAMAMOTO 85.  The Synthetic Analog of 821 95.  Cannabis for Basal Ganglia Disorders ∆9-Tetrahydrocannabinol (THC): Nabilone. 828 (Parkinson Disease and Huntington Disease) Pharmacology and Clinical Application 839 O.M.E. ABDEL-SALAM R.E BALTER, M. HANEY 848 96.  Medical Cannabis for the Treatment 86.  Synthetic Cannabinoids in Dementia of Inflammatory Bowel Disease S. AMANULLAH, K. SHIVAKUMAR, S. HASSAN, A. LAHAT A. CANFIELD, J. COLE 97.  Cannabidiol for the Treatment 87.  Synthetic Cannabinoid Receptor of Drug Use Disorders Agonists (Spice) as New Recreational Psychoactive Substances R.G. DOS SANTOS, J.E.C. HALLAK, A.W. ZUARDI, J.A. DE SOUZA CRIPPA A. HELANDER 98.  Cannabinoids and Effects on the 88.  Accidents and Synthetic Cannabinoids Gastrointestinal Tract: A Focus on Motility in Blood of Drivers G. VERA, J. FICHNA, R. ABALO S.S. TUV, V. AUWÄRTER, V. VINDENES 99.  Potential Medical Uses of Cannabigerol: VII A Brief Overview MEDICINAL CANNABIS USE S. DEIANA 89.  Cannabis and Synthetic Cannabinoids VIII for Cancer Patients: Multiple Palliative Indications Together With Promising SCREENING, DIAGNOSIS, Laboratory Antineoplastic Effects 859 AND TREATMENT D. ZALMAN, G. BAR-SELA 100.  The Cannabis Abuse Screening Test (CAST) and Its Applications 90.  The Use of Medical Marijuana in the 869 Treatment of Psychiatric Disorders L. BASTIANI, R. POTENTE, M. SCALESE, V. SICILIANO, T. TELLIOG˘ LU, Z. TELLIOG˘ LU L. FORTUNATO, S. MOLINARO   

xii CONTENTS 101.  Screening of Synthetic Cannabinoids 981 109.  Cognitive Behavioral Therapy in 1056 Cannabis Use Disorder E.L. ØIESTAD, R. KARINEN, K. HAUGLAND, Å.M.L. ØIESTAD F.M. GUVEN, U.M. CAMSARI, O. SENORMANCI, G. OGUZ 102.  On-Site Drug Testing for Cannabis 998 110.  The Cannabis Withdrawal Syndrome— Symptoms and Time Course A.A. FERNÁNDEZ 1066 M. HESSE, B. THYLSTRUP 103.  Cannabinoids in Oral Fluid: Identification and Interpretation of Analytical Results 1007 111.  School-Based Cannabis Prevention C. MOORE Programs 1074 104.  Cannabinoids in Exhaled Breath 1018 C. ARIZA, F. SÁNCHEZ-MARTÍNEZ, A. PÉREZ O. BECK 112.  The CapOpus Trial for Cannabis Use Disorders 105.  Barriers to Treatment Seeking for 1086 Cannabis Dependence C.R. HJORTHØJ, M. NORDENTOFT 1025 P. GATES, J. COPELAND 106.  Pharmacotherapies for Cannabis 1030 113.  Treating Cannabis Use Disorders 1093 Use Disorders Through Technology-Assisted Interventions: The Telephone and Internet A.L. McRAE-CLARK P. GATES, J. COPELAND 107.  Self-Initiated Cannabis Use Cessation 114.  Reducing Cannabis Use With a Real-Time in Adolescents and Emerging Adults Intervention Using Mobile Technology 1101 1036 J. TSAI, M. LITTLE, S. SUSSMAN M. KELLS, L.A. SHRIER 108.  Treating Cannabis-Dependent Adolescents Cannabis Neuropathology Resources and 1111 with Family Therapy: The Case of Recommended Reading 1115 Multidimensional Family Therapy 1047 R. RAJENDRAM, V.B. PATEL, V.R. PREEDY H. RIGTER Index  Online Contents I II SETTING THE SCENE, BOTANICAL, PERSONAL, SOCIAL AND GENERAL AND INTERNATIONAL COMMUNITY ASPECTS OF CANNABIS USE ASPECTS e1.  Tetrahydrocannabinol Concentration e2.  Cannabis Use in Youth Subcultures e11 and Genetic Characterization of Cannabis e1 M. PAWSON, B.C. KELLY F. CASCINI, I. BOSCHI   

ONLINE CONTENTS xiii III e10.  Cannabis and the Use of Amphetamine-Like Substances e101 CANNABIS, BEHAVIOR, PSYCHOPATHOLOGY AND A. PORCU, M.P. CASTELLI NEUROPATHOLOGY e19 V e3.  Aggressive Behavior and Cannabis Use PHARMACOLOGY AND CELLULAR ACTIVITIES OF CANNABINOIDS W. LIU, H. PETRAS AND ENDOCANNABINOIDS e4.  COMT Genotypes, Cannabis Use, and e11.  Cannabinoid Signaling in Glioma Cells Psychosis: Gene-Environment Interaction e29 and Therapeutic implications e111 Evidence from Human Populations, and Its Methodological Concerns A. ELLERT-MIKLASZEWSKA, I. A. CIECHOMSKA, B. KAMINSKA M. FATJÓ-VILAS, C. PRATS, L. FAÑANÁS e5.  Neuroimaging and Genetics of the Acute e42 VI and Chronic Effects of Cannabis EFFECTS OF SPECIFIC NATURAL R. MARTÍN-SANTOS, J.A. DE SOUZA CRIPPA, S. BHATTACHARYYA AND SYNTHETIC CANNABINOIDS e6.  Gray Matter, Lateral Ventricle Volumes, e12.  Cannabidiol as an Antioxidant e122 and Executive Functioning in Cannabis Users with First-Episode Psychosis R.S. BORGES, A.B.F. DA SILVA P.J. CUNHA, P.G.P. ROSA, F.L.S. DURAN, L.C. SANTOS, e53 J.A.S. CRIPPA, G.F. BUSATTO, M.S. SCHAUFELBERGER e13.  The Anxiolytic Effects of Cannabidiol e7.  Cannabis Use and Attention-Deficit/ (CBD) e131 Hyperactivity Disorder: Potential Moderators A.W. ZUARDI, J.A. DE SOUZA CRIPPA, J.E.C. HALLAK, K.E. MAPLE, N.E. WRIGHT, K.M. LISDAHL A.C. CAMPOS, F.S. GUIMARÃES e64 e14.  New Ethological and Morphological Perspectives for the Investigation of Panicolytic-Like Effects of Cannabidiol e140 IV N.C. COIMBRA, J. MENDES-GOMES, J.A. DA SILVA, T. DOS ANJOS-GARCIA, F. ULLAH, R.C. ALMADA CANNABIS, ORGANS, TISSUES AND NON-CNS ASPECTS e15.  Spice Use Among United States e150 Military Personnel e8.  Cannabis and Oral Health: Deleterious Effects on Periodontitis and Dental Implants H.A. MORRIS, J.M. STOGNER G. NOGUEIRA-FILHO e72 e9.  Does Cannabis Use Increase the Risk VII of Developing Cancer in Humans? MEDICINAL CANNABIS USE R.C. CALLAGHAN, M. VERDICHEVSKI, T.M. FYFE, J.M. GATLEY e80 e16.  Cannabis Use in Fibromyalgia e158 M. FARRÉ, A. FARRÉ, J. FIZ, M. TORRENS   

xiv ONLINE CONTENTS e185 e193 VIII e18.  Self-report of Cannabis Use e202 SCREENING, DIAGNOSIS, T. VAN DER LINDEN AND TREATMENT e168 e19.  CANDIS Program: Modular Treatment e17.  Short Instruments to Screen for of Cannabis Use Disorders “Problematic” Cannabis Use in General Population Surveys E. HOCH, H. ROHRBACHER B. ANNAHEIM, S. LEGLEYE e20.  Engaging Cannabis Users in Treatment F. KAY-LAMBKIN, A. HEALEY, A. BAKER, W. SWIFT, L. THORNTON, A. TURNER   

List of Contributors M. Aas  NORMENT, KG Jebsen Centre for Psychosis G. Appendino  Department of Pharmaceutical Science, Research, Division of Mental Health and Addiction, Oslo University of Piemonte Orientale, Novara, Italy University Hospital & Institute of Clinical Medicine, University of Oslo, Oslo, Norway H. Aramaki  Department of Molecular Biology, Daiichi University of Pharmacy; Drug Innovation Research Center, R. Abalo  Area of Pharmacology and Nutrition, Faculty Daiichi University of Pharmacy, Fukuoka, Japan of Health Sciences, University Rey Juan Carlos, Alcorcón; Associated Unit I+D+i of the Institute of Medicinal Chemistry F. Arias-Horcajadas  Psychiatric Department, Doce de (IQM) and of the Institute of Research in Food Sciences (CIAL), Octubre Hospital, Madrid, Spain Spanish National Research Council (CSIC), Madrid, Spain C. Ariza  Evaluation and Intervention Methods Service, O.M.E. Abdel-Salam  Department of Toxicology and Narcotics, Public Health Agency, Barcelona, Spain National Research Centre, Dokki, Greater Cairo, Egypt J.C. Arnold  School of Medicine, Western Sydney University, V.C. Abilio  Department of Pharmacology, Federal Campbelltown; University of Sydney, Department of University of Sao Paulo; Integrated Laboratory of Clinical Pharmacology, Bosch Institute, Sydney; Brain and Mind Neurosciences (LiNC), Federal University of Sao Paulo, Research Institute, Camperdown, NSW, Australia Sao Paulo, Brazil D. Asmaro  Department of Psychology, Simon Fraser G.R. Adelli  Department of Pharmaceutics and Drug University, Burnaby, BC, Canada Delivery, School of Pharmacy, University of Mississippi, Oxford, MS, United States V. Auwärter  Department of Drug Abuse Research, Division of Forensic Sciences, Norwegian Institute of Public Health, M.H. Ahmed  Department of Medicinal Chemistry, School Oslo, Norway; Forensic Toxicology Department, Medical of Pharmacy and Institute for Structural Biology and Drug Center, University of Freiburg, Institute of Forensic Medicine, Discovery, Virginia Commonwealth University, Richmond, Freiburg, Germany VA, United States S. Bachmann  Clienia AG, Littenheid, Switzerland M. Alhouayek  Bioanalysis and Pharmacology of Bioactive Lipids Research Group, Louvain Drug Research Institute, A. Baker  Priority Research Centre for Translational Université catholique de Louvain, Brussels, Belgium Neuroscience and Mental Health, University of Newcastle, Callaghan, NSW, Australia J. Allen  Psychology Department, IKBSAS, University of British Columbia, Kelowna, BC, Canada R.E Balter  Division on Substance Abuse, New York State Psychiatric Institute and Department of Psychiatry, Columbia D.J. Allsop  Psychopharmacology Laboratory, School of University Medical Center, New York, NY, United States Psychology, University of Sydney, Sydney, NSW, Australia P.G. Baraldi  Department of Chemistry and Pharmaceutical R.C. Almada  Laboratory of Neuroanatomy & Science, University of Ferrara, Ferrara, Italy Neuropsychobiology, Department of Pharmacology, Ribeirão Preto Medical School of the University of São P.A. Barber  Department of Medicine, Centre for Brain Paulo (F­ MRP-USP), Ribeirão Preto, São Paulo, Brazil Research, University of Auckland, Auckland, New Zealand V. Almeida  Department of Pharmacology, Federal G. Bar-Sela  Division of Oncology, Rambam Health Care University of Sao Paulo; Integrated Laboratory of Clinical Campus and Faculty of Medicine, Technion—Israel Institute Neurosciences (LiNC), Federal University of Sao Paulo, of Technology, Haifa, Israel Sao Paulo, Brazil L. Bastiani  Institute of Clinical Physiology, The Italian A. Aloway  Department of Pharmacology and Toxicology, National Research Council (IFC-CNR), Pisa, Italy University of Louisville School of Medicine, Louisville, KY, United States D. Basu  Drug De-addiction and Treatment Centre, Department of Psychiatry, Postgraduate Institute of Medical S. Amanullah  Woodstock General Hospital, Woodstock, Education and Research, Chandigarh, India ON, Canada; School of Medicine, University of Western Ontario, London, ON, Canada; and Faculty of Medicine, I. Basurte  Gregorio Marañon Hospital, Madrid, Spain Dalhousie University, NS, Canada O. Beck  Department of Laboratory Medicine, Section of S.L. Ames  School of Community and Global Health, Clinical Pharmacology, Karolinska Institutet, Stockholm, Sweden Claremont Graduate University, Claremont, CA, United States S. Behrendt  Institute of Clinical Psychology and B. Annaheim  Institute for Biomedical Ethics (IBMB), Psychotherapy, Technische Universitaet Dresden, Dresden, University of Basel, Basel, Switzerland Germany D. Bergen-Cico  Department of Public Health, Addiction Studies, Syracuse University, Syracuse, NY, United States xv

xvi LIST OF CONTRIBUTORS F. Berrendero  Department of Experimental and Health U.M. Camsari  Department of Psychiatry and Psychology, Sciences, Laboratory of Neuropharmacology, School Mayo Clinic Health System, and Mayo Clinic College of of Health and Life Sciences, Pompeu Fabra University, Medicine, Rochester, MN, United States Barcelona, Spain A. Canfield  University of Toronto, Toronto, ON, Canada P. Bhagav  Department of Pharmaceutics and Drug Delivery, School of Pharmacy, University of Mississippi, Oxford, MS, E. Carra  Department of Psychosis Studies, Institute of United States Psychiatry, Psychology and Neuroscience, King’s College London, London, United Kingdom S. Bhattacharyya  Department of Psychosis Studies, King’s College London, Institute of Psychiatry, Psychology & F.-J. Carrillo-Salinas  Neurobiology and Functional Systems Neuroscience, London, United Kingdom Department, Cajal Institute, CSIC, Madrid, Spain M. Bioque  Barcelona Clínic Schizophrenia Unit, Hospital F. Cascini  Public Health Institute, Department of Forensic Clínic de Barcelona, Centro de Investigación Biomédica en Medicine, Università Cattolica del Sacro Cuore, Rome, Italy Red de Salud Mental (CIBERSAM), Barcelona, Spain M.P. Castelli  Department of Biomedical Sciences, Division S. Bolkent  Department of Medical Biology, Cerrahpasa of Neuroscience and Clinical Pharmacology, Cittadella Faculty of Medicine, Istanbul University, Istanbul, Turkey Universitaria, Monserrato, CA, Italy J.H. Boman IV  Department of Criminal Justice, University S.O. Cawich  Department of Clinical Surgical Sciences, of Wyoming, Laramie, WY, United States University of the West Indies, St. Augustine Campus, St Augustine, Trinidad and Tobago P. Bondallaz  Traffic Medicine and Psychology Unit, University Center of Legal Medicine, University Hospital of E.E. Cawston  Department of Pharmacology and Clinical Geneva, Geneva, Switzerland Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand U. Bonnet  Department of Psychiatry, Psychotherapy and Psychosomatics, Evangelisches Krankenhaus Castrop-Rauxel, C. Cedro  Department of Biomedical, Dental Sciences and Academic Teaching Hospital of the University of Duisburg- Morpho-functional Imaging, University of Messina, Messina, Essen, Castrop-Rauxel, Germany Italy R.S. Borges  Faculty of Pharmacy, Institute of Health M.H.N. Chagas  Department of Neurosciences and Behavior, Sciences, Federal University of Pará, Belém, Para, Brazil Faculty of Medicine, Ribeirão Preto, University of São Paulo, Ribeirão Preto; Barretos School of Health Sciences, Dr. Paulo K. Borowiak  Department of Clinical and Forensic Prata, Barretos, São Paulo, Brazil Toxicology, Pomeranian Medical University, Szczecin, Poland C. Chen  Neuroscience Center of Excellence, School of Medicine, Louisiana State University Health Sciences Center, I. Boschi  Public Health Institute, Department of Forensic New Orleans, LA, United States Medicine, Università Cattolica del Sacro Cuore, Rome, Italy C. Chisari  University of York, York, United Kingdom L.K. Brents  Brain Imaging Research Center, Psychiatric Research Institute, University of Arkansas for Medical H. Chtioui  Department of Clinical Pharmacology and Sciences, Little Rock, AR, United States Toxicology, University Hospital of Lausanne, Lausanne, Switzerland C.H. Bridts  Faculty of Medicine and Health Science, Department of Immunology-Allergology-Rheumatology, R.D. Cico  Columbia University, New York, NY, United States University of Antwerp, Antwerp, Belgium I.A. Ciechomska  Laboratory of Molecular Neurobiology, A. Bruno  Department of Biomedical, Dental Sciences and Neurobiology Center, Nencki Institute of Experimental Morpho-functional Imaging, University of Messina, Messina, Biology, Warsaw, Poland Italy N.C. Coimbra  Laboratory of Neuroanatomy & B.T. Burrows  Department of Psychology, Arizona State Neuropsychobiology, Department of Pharmacology, University, Tempe, AZ, United States Ribeirão Preto Medical School of the University of São Paulo (FMRP-USP); Neurobiology of Emotions Research G.F. Busatto  Department of Psychiatry, Faculty of Medicine, Centre (NAP-USP-NuPNE), Ribeirão Preto Medical School Laboratory of Psychiatric Neuroimaging (LIM-21), University of the University of São Paulo (FMRP-USP), Ribeirão Preto, of São Paulo; Center for Interdisciplinary Research on Applied São Paulo, Brazil Neurosciences (NAPNA), University of São Paulo, São Paulo, Brazil J. Cole  Queen Elizabeth Hospital, Charlottetown, PE, Canada R.C. Callaghan  Northern Medical Program, University of J. Cookey  Department of Psychiatry, Dalhousie University, Northern British Columbia, Prince George, BC; Dalla Lana Halifax, NS, Canada School of Public Health, University of Toronto, Toronto, ON; Human Brain Lab, Centre for Addiction and Mental Health, J. Copeland  National Cannabis Prevention and Information Toronto, ON, Canada Centre, University of New South Wales, Randwick, Sydney, NSW, Australia A.C. Campos  Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Z.M. Coskun  Department of Molecular Biology and Preto, São Paulo, Brazil Genetics, Faculty of Arts and Sciences, Istanbul Bilim University, Istanbul, Turkey   

LIST OF CONTRIBUTORS xvii W.D. Crano  Division of Behavioral and Organizational S. Deiana  CNS Diseases Research Department, Boehringer Sciences, School of Social Science, Policy and Evaluation, Ingelheim Pharma GmbH & Co. KG, Birkendorfer straße, Claremont Graduate University, Claremont, CA, United Biberach an der Riss, Germany States U. Deonarine  Department of Clinical Surgical Sciences, J.A.S. Crippa  Department of Neuroscience and Behavior, University of the West Indies, St. Augustine Campus, Faculty of Medicine, Ribeirão Preto, University of São Paulo, St Augustine, Trinidad and Tobago Ribeirao Preto, Brazil M. Di Forti  Department of Psychosis Studies, Institute of C.E. Crocker  Department of Psychiatry, Dalhousie Psychiatry, Psychology and Neuroscience, King’s College University; Division of Neurology, Department of Medicine, London, London, United Kingdom Dalhousie University, Halifax, NS, Canada T. dos Anjos-Garcia  Laboratory of Neuroanatomy M.J. Cuesta  Department of Psychiatry, IdiSNA, Navarra & Neuropsychobiology, Department of Pharmacology, Institute for Health Research, Pamplona, Spain Ribeirão Preto Medical School of the University of São Paulo (FMRP-USP), Ribeirão Preto, São Paulo, Brazil P.J. Cunha  Department of Psychiatry, Faculty of Medicine, Laboratory of Psychiatric Neuroimaging (LIM-21), University R. Guimarães dos Santos  Department of Neuroscience and of São Paulo; Center for Interdisciplinary Research on Applied Behavior, Ribeirão Preto Medical School, University of São Neurosciences (NAPNA), University of São Paulo, São Paulo, Paulo, Ribeirão Preto, Sao Paulo, Brazil Brazil M. Drozd  Department of Pharmaceutics, Medical University L. Cutando  Department of Experimental and Health of Lublin, Lublin, Poland Sciences, Laboratory of Neuropharmacology, School of Health and Life Sciences, Pompeu Fabra University, F.L.S. Duran  Department of Psychiatry, Faculty of Medicine, Barcelona, Spain Laboratory of Psychiatric Neuroimaging (LIM-21), University of São Paulo; Center for Interdisciplinary Research on Applied A.B.F. da Silva  Institute of Chemistry of São Carlos, Neurosciences (NAPNA), University of São Paulo, São Paulo, University of São Paulo, São Carlos, Sao Paulo, Brazil Brazil J.A. da Silva  Laboratory of Neuroanatomy & M. Earleywine  Department of Psychology, School of Arts Neuropsychobiology, Department of Pharmacology, and Sciences, University at Albany, State University of New Ribeirão Preto Medical School of the University of São Paulo York, Albany, NY, United States (FMRP-USP), Ribeirão Preto, São Paulo, Brazil D.G. Ebo  Faculty of Medicine and Health Science, V.K. da Silva  Neurobiology and Developmental Biology Department of Immunology-Allergology-Rheumatology, Laboratory, Faculty of Biosciences, Pontifical Catholic University of Antwerp, Antwerp, Belgium University, Porto Alegre, Rio Grande do Sul, Brazil N. Egashira  Department of Pharmacy, Kyushu University D. Dan  Department of Clinical Surgical Sciences, University Hospital; Department of Neuropharmacology, Faculty of of the West Indies, St. Augustine Campus, St Augustine, Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan Trinidad and Tobago J. Egnatios  School of Medicine, University of California San R.B. De Boni  INI Evandro Chagas, FIOCRUZ, Rio de Diego, La Jolla, CA, United States Janeiro, Rio de Janeiro, Brazil A. Ellert-Miklaszewska  Laboratory of Molecular F. Rodríguez de Fonseca  Department of Psychobiology, Neurobiology, Neurobiology Center, Nencki Institute of Faculty of Psychology, Complutense University of Madrid, Experimental Biology, Warsaw, Poland Pozuelo de Alarcón, Madrid, Spain S.A. ElShebiney  Department of Toxicology and Narcotics, R. Gómez de Heras  Department of Psychobiology, Faculty National Research Centre, Cairo, Egypt of Psychology, Complutense University of Madrid, Pozuelo de Alarcón, Madrid, Spain M.A. ElSohly  ElSohly Laboratories Inc., Oxford, MS, United States A.C.P. de Oliveira  Department of Pharmacology, ICB, Federal University of Minas Gerais, Belo Horizonte, Minas C. Evren  Research, Treatment and Training Center for Gerais, Brazil Alcohol and Substance Dependence (AMATEM), Bakirkoy Training and Research Hospital for Psychiatry, Neurology and A.C. de Souza Crippa  Health Sciences Sector, Federal Neurosurgery, Istanbul, Turkey University of Paraná, Curitiba, Parana, Brazil L. Fañanás  Faculty of Biology, Anthropology Unit, J.A. de Souza Crippa  Department of Neuroscience and Department of Animal Biology, University of Barcelona, Behavior, Ribeirão Preto Medical School, University of São Biomedicine Institute of the University of Barcelona (IBUB), Paulo, Ribeirão Preto, Sao Paulo, Brazil Barcelona; CIBER of Mental Health (CIBERSAM), Madrid, Spain F. Degenhardt  Laboratory of Technical Biochemistry, Department of Biochemical and Chemical Engineering, M.M. Faber  Faculty of Medicine and Health Science, TU Dortmund University, Dortmund, Germany Department of Immunology-Allergology-Rheumatology, University of Antwerp, Antwerp, Belgium L. Degenhardt  National Drug and Alcohol Research Centre, University of New South Wales, Sydney, NSW, S. Farag  Technical University Dortmund, Technical Australia Biochemistry Dortmund, Dortmund, Germany   

xviii LIST OF CONTRIBUTORS A. Farré  Dual Disorder Unit, Addiction Program, Institute S. Gade  Palo Alto University, Palo Alto, CA, United States of Neuropsychiatry and Addiction—INAD, and Hospital del Mar Medical Research Institute—IMIM, Barcelona, Spain E. Gaffal  Laboratory of Experimental Dermatology, Department of Dermatology and Allergy, University M. Farré  Clinical Pharmacology Unit, Germans Trias i Pujol Hospital of the Friedrich-Wilhelm-University Bonn, Bonn, University Hospital—IGTP, and Human Pharmacology Unit, Germany Hospital del Mar Medical Research Institute—IMIM, and Autonomous University of Barcelona, Barcelona, Spain A.F. Galal  Department of Toxicology and Narcotics, National Research Centre, Cairo, Egypt M. Fatjó-Vilas  Faculty of Biology, Anthropology Unit, Department of Animal Biology, University of Barcelona, R. Gandhi  Academic Department of Diabetes and Biomedicine Institute of the University of Barcelona (IBUB), Endocrinology, Sheffield Teaching Hospitals NHS Barcelona; CIBER of Mental Health (CIBERSAM), Madrid, Foundation Trust, Sheffield, United Kingdom Spain P. Gates  National Cannabis Prevention and Information B. Favrat  Traffic Medicine and Psychology Unit, University Centre, University of New South Wales, Randwick, Sydney, Center of Legal Medicine, University Hospital of Geneva, NSW, Australia Geneva; Department of Ambulatory Care and Community Medicine, University Hospital of Lausanne, Lausanne, J.M. Gatley  Northern Medical Program, University of Switzerland Northern British Columbia, Prince George, BC; Dalla Lana School of Public Health, University of Toronto, Toronto, ON; D. Feingold  Department of Psychiatry, Sheba Medical Human Brain Lab, Centre for Addiction and Mental Health, Center, Tel Hashomer; Ariel University, Ariel, Israel Toronto, ON, Canada A. Feliú  Neurobiology and Functional Systems Department, C. Giroud  Forensic Toxicology and Chemistry Unit, Cajal Institute, CSIC, Madrid, Spain University Center of Legal Medicine, University Hospital of Lausanne, Lausanne, Switzerland A.A. Fernández  Forensic Laboratory Institute of Legal Medicine of Catalonia, Barcelona, Spain M. Glass  Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, S. Fernández-Artamendi  Addictive Behaviors Research University of Auckland, Auckland, New Zealand Group, Department of Psychology, University of Oviedo, Oviedo, Spain S.R. Goldberg  Preclinical Pharmacology Section, Behavioral Neuroscience Branch, Intramural Research Program, National A.J. Ferrari  School of Public Health, University of Institute on Drug Abuse, National Institutes of Health, Queensland, Herston; Institute for Health Metrics and Baltimore, MD, United States Evaluation, University of Washington, Seattle, WA, United States; Queensland Centre for Mental Health Research, Wacol, I. González-Ortega  Department of Psychiatry, University QLD, Australia Hospital of Alava-Santiago, CIBERSAM; University of the Basque Country; National Distance Education University L. Ferraro  Biomedical Department of Internal and Specialist (UNED)-Centro Asociado de Vitoria, Vitoria, Spain Medicine; Department of Experimental Biomedicine and Clinical Neuroscience, School of Medicine, University of A. González-Pinto  Department of Psychiatry, University Palermo, Palermo, Italy Hospital of Alava-Santiago, CIBERSAM; University of the Basque Country, Vitoria, Spain J. Fichna  Department of Biochemistry, Faculty of Medicine, Medical University of Lodz, Lodz, Poland C. Guaza  Neurobiology and Functional Systems Department, Cajal Institute, CSIC, Madrid, Spain D.B. Finlay  Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, V. Guillon  Service des enquêtes et des sondages, Paris, University of Auckland, Auckland, New Zealand France J. Fiz  EDIR, Center for Diagnosis and Rehabilitation, San F.S. Guimarães  Department of Pharmacology, Faculty of Martín Santa Fe, Argentina Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Á. Flores  Department of Experimental and Health Sciences, Laboratory of Neuropharmacology, School of Health and Life W. Gul  ElSohly Laboratories Inc., Oxford, MS, United Sciences, Pompeu Fabra University, Barcelona, Spain States J.S. Fogel  Department of Psychology, University of Kentucky F.M. Guven  Turkish Association for Cognitive and College of Arts and Sciences, Lexington, KY, United States Behavioural Therapies, Istanbul; Department of Psychiatry, Alcohol & Substance Use Disorders Treatment Center, Lara E. Fornari  CIBM, University Hospital of Lausanne, Anatolia Hospital, Antalya, Turkey Lausanne, Switzerland W.D. Hall  Centre for Youth Substance Abuse Research, L. Fortunato  Institute of Clinical Physiology, The Italian University of Queensland, Herston, QLD, Australia National Research Council (IFC-CNR), Pisa, Italy J.E.C. Hallak  Department of Neuroscience and Behavior, T. Fyfe  Northern Medical Program, University of Northern Ribeirão Preto Medical School, University of São Paulo, British Columbia, Prince George, BC, Canada Ribeirão Preto, Sao Paulo, Brazil A.E.D.M. Gaafar  Department of Photochemistry, Chemical M. Hamerle  Department of Psychiatry and Psychotherapy, Industries Division, National Research Centre, Cairo, Egypt Ludwig-Maximilian-University Hospital, Munich, Germany   

LIST OF CONTRIBUTORS xix M. Haney  Division on Substance Abuse, New York State T. Karl  Neuroscience Research Australia, Randwick; School Psychiatric Institute and Department of Psychiatry, College of of Medicine, Western Sydney University, Campbelltown, Physicians and Surgeons of Columbia University, New York, NSW, Australia NY, United States T. Katsu  Department of Pharmacy, Faculty of Pharmacy, H.E. Harding  Department of Surgery, University of the West Yasuda Women’s University, Hiroshima, Japan Indies, Mona Campus, Kingston, Jamaica F. Kay-Lambkin  NHMRC Centre for Research Excellence in S. Hassan  Dalhousie University, Dartmouth, NS, Canada Mental Health and Substance Use, National Drug and Alcohol Research Centre, University of New South Wales, Randwick, K. Haugland  National Criminal Investigation Service NSW, Australia (NCIS) Norway, Forensic Science Department, Oslo, Norway O. Kayser  Technical University Dortmund, Technical A. Healey  School of Psychology, The University of Newcastle, Biochemistry Dortmund, Dortmund, Germany University Drive, Callaghan, NSW, Australia M. Kells  Division of Adolescent/Young Adult Medicine, C. Heck  City and County of Denver, Crime Prevention and Boston Children’s Hospital, Boston, MA, United States Control Commission, Denver, CO, United States B.C. Kelly  Department of Sociology, Purdue University, A. Helander  Department of Laboratory Medicine, West Lafayette, IN, United States Karolinska Institutet, and Karolinska University Laboratory, Stockholm, Sweden T.H. Kelly  Departments of Behavioral Science and Psychiatry, University of Kentucky College of Medicine, and Department L. Hernandez-Folgado  Institute of Medical Chemistry, of Psychology, University of Kentucky College of Arts and CSIC, Madrid, Spain Sciences, Lexington, KY, United States D.A. Herzig  Clienia AG, Littenheid, Switzerland A. Kokona  Department of Medical and Surgical Sciences, Bologna University, Bologna, Italy M. Hesse  Center for Alcohol and Drug Research, Aarhus University, Copenhagen Department, Copenhagen, A. Kumar  Department of Pharmacology and Toxicology, Denmark University of Louisville School of Medicine, Louisville, KY, United States M.G. Hill  The University of Arizona, Tucson, AZ, United States P. Kumar  Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, R. Hirst  Palo Alto University, Palo Alto, CA, United States United States C.R. Hjorthøj  Mental Health Center Copenhagen, D. La Barbera  Department of Experimental Biomedicine Copenhagen University Hospital, Copenhagen, Denmark and Clinical Neuroscience, School of Medicine, University of Palermo, Palermo, Italy E. Hoch  Department of Psychiatry, Ludwig Maximilian University, Munich, Germany T.V. Lagerberg  NORMENT KG Jebsen Centre for Psychosis Research, Institute of Clinical Medicine, University of Oslo; M.D. Holder  Psychology Department, IKBSAS, University Division of Mental Health and Addiction, Oslo University of British Columbia, Kelowna, BC, Canada Hospital, Oslo, Norway M. Holtkamp  Epilepsy-Center Berlin-Brandenburg, A. Lahat  Department of Gastroenterology, Chaim Sheba Department of Neurology, Charité – Universitätsmedizin Medical Center, Tel-Hashomer, Israel Berlin, Berlin, Germany H.J. Larsen  Mental Health Center Copenhagen, Copenhagen M.R. Hunter  Department of Pharmacology and Clinical University Hospital, Copenhagen, Denmark Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand A.S. Laun  Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, E. Ikeda  Department of Molecular Biology, Daiichi United States University of Pharmacy, Fukuoka, Japan T. Lecomte  Research Centre of the University of Y. Izumi  Department of Psychiatry; The Taylor Family Montreal Institute for Mental Health; Department of Institute for Innovative Psychiatric Research, Washington Psychology, University of Montreal, Montreal, QC, University School of Medicine, St. Louis, MO, United States Canada T. Janus  Department of Clinical and Forensic Toxicology, S. Legleye  Institut national d’études démographiques Pomeranian Medical University, Szczecin, Poland (INED), Paris; University of Paris-Saclay, Univ. Paris-Sud, UVSQ, CESP, Inserm, Versailles, France B. Kaminska  Laboratory of Molecular Neurobiology, Neurobiology Center, Nencki Institute of Experimental S. Lev-Ran  Department of Psychiatry, Sheba Medical Biology, Warsaw, Poland Center, Tel Hashomer; Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel A.S. Kanaan  Clinic of Psychiatry, Social-Psychiatry and Psychotherapy, Hannover Medical School, Hannover; J.A. Lile  Departments of Behavioral Science and Psychiatry, Nuclear Magnetic Resonance Unit, Max Planck Institute University of Kentucky College of Medicine, and Department for Human Cognitive and Brain Sciences, Leipzig, of Psychology, University of Kentucky College of Arts and Germany Sciences, Lexington, KY, United States R. Karinen  Norwegian Institute of Public Health, Division of Forensic Sciences, Oslo, Norway   

xx LIST OF CONTRIBUTORS R.P. Limberger  LABTOXICO: Laboratory of Toxicology, R. Martín-Santos  Department of Psychiatry and Faculty of Pharmacy, Federal University of Rio Grande do Sul, Psychology, Hospital Clinic, IDIBAPS, University of Porto Alegre, Rio Grande do Sul, Brazil Barcelona, CIBERSAM, Barcelona, Spain I.M.P. Linares  Department of Neuroscience and Behavior, K. Masuda  Department of Physical Chemistry, Graduate Faculty of Medicine, Ribeirão Preto, University of São Paulo, School of Clinical Pharmacy, Shujitsu University, Okayama, Ribeirao Preto, Brazil Japan K.M. Lisdahl  Department of Psychology, University of A.L. McRae-Clark  Medical University of South Carolina, Wisconsin-Milwaukee, Milwaukee, WI, United States Charleston, SC, United States M. Little  Center for Population Sciences, Department of M. Mecha  Neurobiology and Functional Systems Department, Preventive Medicine, University of Tennessee Health Science Cajal Institute, CSIC, Madrid, Spain Center, Memphis, TN, United States I. Melle  NORMENT, KG Jebsen Centre for Psychosis W. Liu  NORC at the University of Chicago, Bethesda, MD, Research, Division of Mental Health and Addiction, Oslo United States University Hospital & Institute of Clinical Medicine, University of Oslo, Oslo, Norway M.J. Loflin  Department of Psychology, School of Arts and Sciences, University at Albany, State University of New York, S. Menahem  Paediatric Cardiology Unit, Monash Health, Albany, NY, United States Melbourne, Melbourne; Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia R. Lorente-Omeñaca  Department of Psychiatry, IdiSNA, Navarra Institute for Health Research, Pamplona, Spain J. Mendes-Gomes  Laboratory of Neuroanatomy & Neuropsychobiology, Department of Pharmacology, Ribeirão V. Lorenzetti  Melbourne Neuropsychiatry Centre, Preto Medical School of the University of São Paulo (FMRP- The University of Melbourne and Melbourne Health, USP), Ribeirão Preto, São Paulo, Brazil Melbourne; Brain & Mental Health Laboratory, Monash Institute of Cognitive and Clinical Neurosciences, School of B. Mesías  Instituto de Adicciones, Madrid, Spain Psychological Sciences, Monash University, Clayton, VIC, Australia S. Miller  Division of Behavioral and Organizational Sciences, School of Social Science, Policy and Evaluation, Claremont D. Lu  Department of Pharmaceutical Sciences, Rangel Graduate University, Claremont, CA, United States College of Pharmacy, Texas A&M University, Kingsville, TX, United States R. Mizrahi  Centre for Addiction and Mental Health (CAMH), Research Imaging Centre, Toronto, ON, Canada J. Mørland  Department of Drug Abuse Research, Division of Forensic Sciences, Norwegian Institute of Public Health, S. Molinaro  Institute of Clinical Physiology, The Italian Oslo, Norway National Research Council (IFC-CNR), Pisa, Italy K.R. Müller-Vahl  Clinic of Psychiatry, Social-Psychiatry C. Moore  Toxicology Research and Development, and Psychotherapy, Hannover Medical School, Hannover, Immunalysis Corporation, Pomona, CA, United States Germany M.F. Moraes  Department of Physiology and Biochemistry, A. Machoy-Mokrzyn´ ska  Department of Pharmacology, ICB, Federal University of Minas Gerais, Belo Horizonte, Pomeranian Medical University, Szczecin, Poland Minas Gerais, Brazil P. Maeder  Department of Radiology, University Hospital of F.A. Moreira  Department of Pharmacology, ICB, Federal Lausanne, Lausanne, Switzerland University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil S. Majumdar  Department of Pharmaceutics and Drug Delivery, School of Pharmacy, University of Mississippi, L. Moreno-Izco  Department of Psychiatry, IdiSNA, Navarra Oxford, MS, United States Institute for Health Research, Pamplona, Spain R. Maldonado  Department of Experimental and Health H.A. Morris  Department of Criminal Justice and Sciences, Laboratory of Neuropharmacology, School Criminology, University of North Carolina at Charlotte, of Health and Life Sciences, Pompeu Fabra University, Charlotte, NC, United States Barcelona, Spain E. Muñoz  Department of Cell Biology, Physiology and K.E. Maple  Department of Psychology, University of Immunology, University of Córdoba, Córdoba, Spain Wisconsin-Milwaukee, Milwaukee, WI, United States G.G. Muccioli  Bioanalysis and Pharmacology of Bioactive M. Martínez-Cengotitabengoa  Department of Psychiatry, Lipids Research Group, Louvain Drug Research Institute, University Hospital of Alava-Santiago, CIBERSAM; National Université catholique de Louvain, Brussels, Belgium Distance Education University (UNED)-Centro Asociado de Vitoria, Vitoria, Spain M.R.A. Muscatello  Department of Biomedical, Dental Sciences and Morpho-functional Imaging, University of M. Isabel Martín-Fontelles  Area of Pharmacology and Messina, Messina, Italy Nutrition, Faculty of Health Sciences, University Rey Juan Carlos, Alcorcón; Associated Unit I+D+i of the Institute of S.A. Nada  Department of Pharmacology, National Research Medicinal Chemistry (IQM) and of the Institute of Research Centre, Dokki, Greater Cairo, Egypt in Food Sciences (CIAL), Spanish National Research Council (CSIC), Madrid, Spain V. Naraynsingh  Department of Clinical Surgical Sciences, University of the West Indies, St. Augustine Campus, St Augustine, Trinidad and Tobago   

LIST OF CONTRIBUTORS xxi S. Narimatsu  Department of Health Chemistry, Graduate F. Pollastro  Department of Pharmaceutical Science, School of Medicine, Dentistry and Pharmaceutical Sciences, University of Piemonte Orientale, Novara, Italy Okayama University, Okayama, Japan A. Porcu  Department of Biomedical Sciences, Division G. Nogueira-Filho  School of Health Sciences, University of Neuroscience and Clinical Pharmacology, Cittadella Salvador, Laureate International Universities, Salvador, Bahia, Universitaria, Monserrato, CA, Italy Brazil R. Potente  Institute of Clinical Physiology, The Italian M. Nordentoft  Mental Health Center Copenhagen, National Research Council (IFC-CNR), Pisa, Italy Copenhagen University Hospital, Copenhagen, Denmark D.E. Potter  Department of Pharmaceutical Sciences, Rangel G. Oguz  Turkish Association for Cognitive and Behavioural College of Pharmacy, Texas A&M University, Kingsville, TX, Therapies, Istanbul; Department of Psychology, Canik Basari United States University, Samsun, Turkey S. Potvin  Research Centre of the University of Montreal Å.M.L. Øiestad  Norwegian Institute of Public Health, Institute for Mental Health; Department of Psychiatry, Division of Forensic Sciences, Oslo, Norway Faculty of Medicine, University of Montreal, Montreal, QC, Canada E.L. Øiestad  Norwegian Institute of Public Health, Division of Forensic Sciences, Oslo, Norway C. Prats  Faculty of Biology, Anthropology Unit, Department of Animal Biology, University of Barcelona, H. Okazaki  Drug Innovation Research Center, Daiichi Biomedicine Institute of the University of Barcelona (IBUB), University of Pharmacy, Fukuoka, Japan Barcelona; CIBER of Mental Health (CIBERSAM), Madrid, Spain M.F. Olive  Department of Psychology, Arizona State University, Tempe, AZ, United States V.R. Preedy  Faculty of Life Sciences and Medicine, King’s College London, London, United Kingdom L. Orio  Department of Psychobiology, Faculty of Psychology, Complutense University of Madrid, Pozuelo de R. Rajendram  Faculty of Life Sciences and Medicine, King’s Alarcón, Madrid, Spain College London, London, United Kingdom A. Ozaita  Department of Experimental and Health L. Rathke  Palo Alto University, Palo Alto, CA, United Sciences, Laboratory of Neuropharmacology, School States of Health and Life Sciences, Pompeu Fabra University, Barcelona, Spain K.L. Reed  The University of Arizona, Tucson, AZ, United States A. Pérez  Evaluation and Intervention Methods Service, Public Health Agency, Barcelona, Spain M.A. Repka  Department of Pharmaceutics and Drug Delivery, School of Pharmacy, University of Mississippi, G. Panagis  University of Crete, Department of Psychology, Oxford, MS, United States Laboratory of Behavioral Neuroscience, Rethymnon, Crete, Greece H. Rigter  Youth Interventions Foundation, Curium, Department of Child and Adolescent Psychiatry, Leiden G. Pandolfo  Department of Biomedical, Dental Sciences and University Medical Center, Leiden, The Netherlands Morpho-functional Imaging, University of Messina, Messina, Italy E.M. Rock  Department of Psychology and Collaborative Neuroscience Program, University of Guelph, Guelph, ON, L.V. Panlilio  Preclinical Pharmacology Section, Behavioral Canada Neuroscience Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, H. Rohrbacher  Practice for Cognitive Behaviour Therapy, Baltimore, MD, United States Munich, Germany K. Paquin  Research Centre of the University of Montreal P.G.P. Rosa  Department of Psychiatry, Faculty of Medicine, Institute for Mental Health; Department of Psychology, Laboratory of Psychiatric Neuroimaging (LIM-21), University University of Montreal, Montreal, QC, Canada of São Paulo; Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of São Paulo, São Paulo, P. Parakh  Department of Psychiatry, Ruby General Hospital, Brazil Kolkata, India F. Sánchez-Martínez  Evaluation and Intervention Methods L.A. Parker  Department of Psychology and Collaborative Service, Public Health Agency, Barcelona, Spain Neuroscience Program, University of Guelph, Guelph, ON, Canada A.M. Sánchez-Torres  Department of Psychiatry, IdiSNA, Navarra Institute for Health Research, Pamplona, Spain V.B. Patel  University of Westminster, School of Life Sciences, Department of Biomedical Science, London, United Kingdom M. Sałaga  Department of Biochemistry, Faculty of Medicine, Medical University of Lodz, Lodz, Poland M. Pawson  Department of Sociology, The Graduate Center, City University of New York, New York, NY, United States V. Sabato  Faculty of Medicine and Health Science, Department of Immunology-Allergology-Rheumatology, F.F. Peres  Department of Pharmacology, Federal University University of Antwerp, Antwerp, Belgium of Sao Paulo; Integrated Laboratory of Clinical Neurosciences (LiNC), Federal University of Sao Paulo, Sao Paulo, Brazil A.N. Sanders  Department of Criminal Justice and Criminology, University of North Carolina at Charlotte, H. Petras  American Institutes for Research, Washington, DC, Charlotte, NC, United States United States   

xxii LIST OF CONTRIBUTORS L.C. Santos  Department of Psychiatry, Faculty of Medicine, F. Stehle  Laboratory of Technical Biochemistry, Department Laboratory of Psychiatric Neuroimaging (LIM-21), University of Biochemical and Chemical Engineering, TU Dortmund of São Paulo; Center for Interdisciplinary Research on Applied University, Dortmund, Germany Neurosciences (NAPNA), University of São Paulo, São Paulo, Brazil J.M. Stogner  Department of Criminal Justice and Criminology, University of North Carolina at Charlotte, M. Scalese  Institute of Clinical Physiology, The Italian Charlotte, NC, United States National Research Council (IFC-CNR), Pisa, Italy S. Sussman  Departments of Preventive Medicine and M.S. Schaufelberger  Department of Psychiatry, Faculty of Psychology, and School of Social Work, Institute for Health Medicine, Laboratory of Psychiatric Neuroimaging (LIM-21), Promotion and Disease Prevention Research, University of University of São Paulo; Center for Interdisciplinary Research Southern California, Los Angeles, CA, United States on Applied Neurosciences (NAPNA), University of São Paulo, São Paulo; Department of Neuroscience and Behavior, Faculty W. Swift  NHMRC Centre for Research Excellence in Mental of Medicine, Ribeirão Preto, University of São Paulo, Ribeirao Health and Substance Use, National Drug and Alcohol Preto, Brazil Research Centre, University of New South Wales, Randwick, NSW, Australia N. Schröder  Neurobiology and Developmental Biology Laboratory, Faculty of Biosciences, Pontifical Catholic N. Szerman  Gregorio Marañon Hospital, Madrid, Spain University, Porto Alegre, Rio Grande do Sul, Brazil T. Tüting  Laboratory of Experimental Dermatology, G. Scimeca  Department of Biomedical, Dental Sciences and Department of Dermatology and Allergy, University Morpho-functional Imaging, University of Messina, Messina, Hospital of the Friedrich-Wilhelm-University Bonn, Bonn, Italy Germany R. Secades-Villa  Addictive Behaviors Research Group, M. Aghazadeh Tabrizi  Department of Chemistry and Department of Psychology, University of Oviedo, Oviedo, Pharmaceutical Science, University of Ferrara, Ferrara, Italy Spain O. Taglialatela-Scafati  Department of Pharmacy, University D. Selvarajah  Department of Human Metabolism, Medical of Napoli Federico II, Napoli, Italy School, University of Sheffield, Sheffield, United Kingdom R.N. Takahashi  Department of Pharmacology, CCB, Federal O. Senormanci  Department of Psychiatry, Bülent Ecevit University of Santa Catarina, Florianópolis, Santa Catarina, University School of Medicine, Zonguldak, Turkey Brazil K. Shivakumar  Health Sciences North, Department of S. Takeda  Laboratory of Xenobiotic Metabolism and Psychiatry, and Northern Ontario School of Medicine, Environmental Toxicology, Faculty of Pharmaceutical Sudbury, ON, Canada Sciences, Hiroshima International University (HIU), Kure, Hiroshima, Japan L.A. Shrier  Division of Adolescent/Young Adult Medicine, Boston Children’s Hospital, and Department of Pediatrics, I. Tarricone  Department of Medical and Surgical Sciences, Harvard Medical School, Boston, MA, United States Bologna University; Department of Mental Health, Bologna, Italy V. Siciliano  Institute of Clinical Physiology, The Italian National Research Council (IFC-CNR), Pisa, Italy D.P. Tashkin  Division of Pulmonary and Critical Care, Department of Medicine, David Geffen School of Medicine at L. Sideli  Department of Experimental Biomedicine and UCLA, Los Angeles, CA, United States Clinical Neuroscience, School of Medicine, University of Palermo, Palermo, Italy T. Telliog˘lu  Brown University, Alpert Medical School, Substance Abuse Division, Rhode Island Hospital, J.T. Siegel  Division of Behavioral and Organizational Providence, RI, United States Sciences, School of Social Science, Policy and Evaluation, Claremont Graduate University, Claremont, CA, Z. Telliog˘lu  Brown University, Alpert Medical School, United States Rhode Island Hospital, Providence, RI, United States A.A. Sleem  Department of Pharmacology, National S. Tesfaye  Academic Department of Diabetes and Research Centre, Dokki, Greater Cairo, Egypt Endocrinology, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom J. Sobczyn´ ski  Department of Pharmaceutics, Medical University of Lublin, Lublin, Poland L. Thornton  NHMRC Centre for Research Excellence in Mental Health and Substance Use, National Drug and Alcohol L. Sodos  Palo Alto University, Palo Alto, CA, United States Research Centre, University of New South Wales, Randwick, NSW, Australia N. Solowij  School of Psychology, University of Wollongong, Wollongong, NSW, Australia B. Thylstrup  Center for Alcohol and Drug Research, Aarhus University, Copenhagen Department, Copenhagen, Z.-H. Song  Department of Pharmacology and Toxicology, Denmark University of Louisville School of Medicine, Louisville, KY, United States P.G. Tibbo  Department of Psychiatry, Dalhousie University, Halifax, NS, Canada A.W. Stacy  School of Community and Global Health, Claremont Graduate University, Claremont, CA, G. Todd  School of Pharmacy and Medical Sciences, United States University of South Australia, Adelaide, SA, Australia   

LIST OF CONTRIBUTORS xxiii M. Torrens  Addiction Program, Institute of Neuropsychiatry Z. Walsh  Psychology Department, IKBSAS, University of and Addiction—INAD, and Hospital del Mar Medical British Columbia, Kelowna, BC, Canada Research Institute—IMIM, Barcelona, Spain K. Watanabe  Department of Hygienic Chemistry, Faculty J. Tsai  Department of Preventive Medicine, Keck School of of Pharmaceutical Sciences, Hokuriku University, Kanazawa; Medicine, University of Southern California, Los Angeles, CA, Pharmaceutical Education Support Center, Daiichi University United States of Pharmacy, Fukuoka, Japan H.-H. Tseng  Centre for Addiction and Mental Health L.R. Watterson  Department of Psychology, Arizona State (CAMH), Research Imaging Centre, Toronto, ON, Canada; University, Tempe, AZ, United States Department of Psychosis Studies, Institute of Psychiatry, King’s College London, London, United Kingdom J.M. White  School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia A. Turner  Priority Research Centre for Translational Neuroscience and Mental Health, University of Newcastle, N.E. Wright  Department of Psychology, University of Callaghan, NSW, Australia Wisconsin-Milwaukee, Milwaukee, WI, United States S.S. Tuv  Department of Drug Abuse Research, Division M. Yücel  Melbourne Neuropsychiatry Centre, The University of Forensic Sciences, Norwegian Institute of Public Health, of Melbourne and Melbourne Health, Melbourne; Brain & Oslo, Norway Mental Health Laboratory, Monash Institute of Cognitive and Clinical Neurosciences, School of Psychological Sciences, F. Ullah  Laboratory of Neuroanatomy & Neuropsychobiology, Monash University, Clayton, VIC, Australia Department of Pharmacology, Ribeirão Preto Medical School of the University of São Paulo (FMRP-USP), Ribeirão Preto, São I. Yamamoto  Hokuriku University, Kanazawa, Japan Paulo, Brazil S. Yamaori  Department of Pharmacy, Shinshu University T. Van der Linden  Department Drugs and Toxicology, Hospital, Matsumoto, Japan National Institute of Criminalistics, Brussels, Belgium A. Zalesky  Melbourne Neuropsychiatry Centre, The A.L. Van Gasse  Faculty of Medicine and Health Science, University of Melbourne and Melbourne Health, Melbourne, Department of Immunology-Allergology-Rheumatology, VIC, Australia University of Antwerp, Antwerp, Belgium D. Zalman  Division of Oncology, Rambam Health Care P. Vega  Instituto de Adicciones, Madrid, Spain Campus and Faculty of Medicine, Technion—Israel Institute of Technology, Haifa, Israel G. Vera  Area of Pharmacology and Nutrition, Faculty of Health Sciences, University Rey Juan Carlos, Alcorcón; J. Zhang  Neuroscience Center of Excellence, School of Associated Unit I+D+i of the Institute of Medicinal Chemistry Medicine, Louisiana State University Health Sciences Center, (IQM) and of the Institute of Research in Food Sciences New Orleans, LA, United States (CIAL), Spanish National Research Council (CSIC), Madrid, Spain Y. Zhang  Department of Medicinal Chemistry, School of Pharmacy and Institute for Structural Biology and Drug M. Verdichevski  Northern Medical Program, University Discovery, Virginia Commonwealth University, Richmond, of Northern British Columbia, Prince George, BC, Canada VA, United States T.R. Vieira Sousa  Center for Drug and Alcohol Research, R. Zoccali  Department of Biomedical, Dental Sciences and Hospital de Clinicas de Porto Alegre, Federal University of Morpho-functional Imaging, University of Messina, Messina, Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil Italy L.R. Vilela  Department of Pharmacology, ICB, Federal C.F. Zorumski  Department of Psychiatry; The Taylor University of Minas Gerais, Belo Horizonte, Minas Gerais, Family Institute for Innovative Psychiatric Research, Brazil Washington University School of Medicine, St. Louis, MO, United States V. Vindenes  Department of Drug Abuse Research, Division of Forensic Sciences, Norwegian Institute of Public Health, A.W. Zuardi  Department of Neuroscience and Behavior, Oslo, Norway Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Sao Paulo, Brazil   

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Preface Cannabis is probably one of the most commonly however, addressed in The Handbook of Cannabis and used drugs of misuse. It has a wide range of adverse Related Pathologies: Biology, Pharmacology, Diagnosis, effects including impairing learning and memory. At and Treatment which embraces all aspects of canna- the same time, the medical use of cannabis has been bis in a one-stop-shop approach. Where appropriate, advocated due to its ability to relieve pain as an ex- positive aspects of cannabis and related metabolites ample. Understanding the nature of the pleasure- are described. seeking, disinhibition, and other effects have also paved the way for the specialized field of cannabis The book is divided into eight major parts as follows: pharmacology. This includes elucidating the nature 1. Setting the Scene, Botanical, General, and of cannabinoid receptors, which has led to the devel- opment of synthetic cannabinoid agonists. However, International Aspects the aforementioned is a rather simplistic synopsis. 2. Personal, Social, and Community Aspects of The long-term use of cannabis may also increase the risk of schizophrenia, paranoia, and other psychoses. Cannabis Use Its use can affect cells, organs, individuals, families, 3. Cannabis, Behavior, Psychopathology, and subcultures, groups, and communities. This is be- cause the interrelationships between cannabis and in- Neuropathology dividual components, diagnosis, screening, social and 4. Cannabis, Organs, Tissues, and non-CNS Aspects community effects, psychopathology, neuropathol- 5. Pharmacology and Cellular Activities of ogy, non-CNS effects, polydrug use, medicinal appli- cations, treatments, and pharmacology are complex. Cannabinoids and Endocannabinoids Furthermore, although the active agents in cannabis 6. Effects of Specific Natural and Synthetic are known, the individual steps between exposure by ingestion or inhalation and effects on cells and the Cannabinoids body are multifactorial, and cut across many scientific 7. Medicinal Cannabis Use disciplines. It is thus important to learn from these 8. Screening, Diagnosis, and Treatments interrelationships to embrace a multidisciplinary ap- proach to understand all the threads and ramifications The Editor recognizes the difficulties in ascribing of cannabis use, misuse, and applications. For exam- chapters to particular sections, and even their location ple, some cellular mechanisms elucidated by studying within separate sections. This is because some chapters one anatomical CNS component may also be relevant can be categorized in many ways. However, this issue to other areas of the CNS, or other fields of cannabis is resolved with the excellent indexing carried out by toxicity and pharmacology. Another example relates Elsevier. to the impact of cannabis on social dysfunction, which may also be relevant to other psychosocial scenarios, The Handbook of Cannabis and Related Pathologies: Biol- or useful in devising new treatment strategies. An ad- ogy, Pharmacology, Diagnosis, and Treatment transcends ditional example relates to preclinical studies which both multiple disciplinary and intellectual divides, as may be relevant to understanding clinical patholo- each chapter has: gies, psychomorbidities, or therapeutic drugs. Un- • Key Facts raveling these complex relationships is difficult, as • Mini-Dictionary there is a wide myriad of material related to canna- • Summary Points bis. In simple terms, the material on cannabis use and misuse has hitherto been either scattered, diffused, Finally, there is a chapter on Resources and Recom- or crosses different disciplines. These limitations are, mended Reading, suggested by some of the book’s con- tributors. The Handbook of Cannabis and Related Pathologies: Biology, Pharmacology, Diagnosis, and Treatment has been designed for those working in the field of cannabis and cannabinoids, drug abuse workers, neurologists, specialists in addictive behaviors, health scientists, public health and community workers, doctors, xxv

xxvi PREFACE pharmacologists, research scientists, and other special- for undergraduates, postgraduates, lecturers, and aca- ists. The book is valuable as a personal reference book, demic professors. and also for academic libraries that cover the domains of health sciences or addictions. Contributions are from Professor V.R. Preedy, BSc, PhD, DSc, leading national and international experts, including FRSB, FRSH, FRIPHH, FRSPH, FRCPath, FRSC those from world renowned institutions. It is suitable King’s College London, London, United Kingdom   

PART I SETTING THE SCENE, BOTANICAL, GENERAL AND INTERNATIONAL ASPECTS  1 The cannabis plant: Botanical aspects 3  2 The biosynthesis of cannabinoids 13  3 Increasing plant concentrations of THC and 24 implications on health related disorders 33  4 Age as a predictor of cannabis use  5 Lifetime cannabis use and cognition in psychosis 44 53 spectrum disorders 61  6 A profile of synthetic cannabinoid users 70  7 Dual disorders in cannabis misuse 79  8 Cannabis use and cognitive function  9 Cannabis, migration, and psychosis onset 89 10 The global epidemiology and disease burden 101 of cannabis use and dependence 11 International aspects of cannabis use and 110 122 misuse: the australian perspective 12 International aspects of cannabis use and misuse: Egypt 13 Cannabis body packing: A caribbean perspective

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CHAPTER 1 The Cannabis Plant: Botanical Aspects S. Farag, O. Kayser Technical University Dortmund, Technical Biochemistry Dortmund, Dortmund, Germany SUMMARY POINTS LIST OF ABBREVIATIONS • This chapter focuses on the botanical aspects of CBD Cannabidiol Cannabis. CBDA Cannabidiolic acid • Cannabis trichomes can come in glandular and CBN Cannabinol GPP Geranylpyrophosphate nonglandular shapes, including oil resin. GRIN Germplasm Resources Information Network • Resin glands are the main producer of ISSR Inter simple sequence repeat NPGS National Plant Germplasm System cannabinoids. RAPD Random amplified polymorphic • Recently, hybrid cannabis strains have been RFLP Restriction fragment length polymorphism RH Relative humidity developed. RFLP Restriction fragment polymorphisms • Modern hydroponic techniques, coupled with THCA Tetrahydrocannabinolic acid THC ∆9-Tetrahydrocannabinol selective artificial lighting, are used in order to USDA United States Department of Agriculture solve the issue of low-potency cannabis. • However, we argue that it is necessary to apply INTRODUCTION transgenic Cannabis plants to facilitate the metabolic pathway for cannabinoid production Cannabis sativa L. (marijuana; Cannabaceae) is an or agronomic traits. annual dioeciously flowering plant. The first appear- ance of Cannabis was believed to be central Asia about KEY FACTS 5000 BC. For millennia, the plant has also been used for fiber, oil production, and traditional uses. It contains a • Most popular varieties of Cannabis are a combination number of medicinally important compounds, such as, of two or three of C. sativa, C. indica or C. ruderalis. cannabinoids (Appendino, Chianese, & Taglialatela- Scafati,  2011), terpenoids (Ross & ElSohly,  1996), flavo- • Cannabis cultivated for fiber or oil, or narcotics noids (Vanhoenacker, Van Rompaey, De Keukeleire, & production. Sandra,  2002), alkaloids (Turner & Elsohly,  1976), and others (Brenneisen, 2007). Cannabinoids are a unique • Cannabinoids are the main active ingredient. class of terpenophenolic compounds to Cannabis plants, • Cultivation and breeding of narcotic strains is not accumulated mainly in the cavity of trichomes (Kim & Mahlberg,  1997). More than 80 cannabinoids have permitted in most countries. been isolated from C. sativa (Elsohly & Slade, 2005). The • Only female plants are economically important for main psychoactive compound is ∆9-tetrahydrocannabi- nol (THC), with well-known medicinal effects (Elbatsh, producing resin in narcotic strains. • Indoor horticultural lighting is a new system to mimic sunlight. • Indoor hydroponic technology is used for maximizing cannabinoids. Handbook of Cannabis and Related Pathologies. http://dx.doi.org/10.1016/B978-0-12-800756-3.00001-6 Copyright © 2017 Elsevier Inc. All rights reserved. 3

4 1.  The Cannabis Plant: Botanical Aspects Moklas, Marsden, & Kendall, 2012). At present, cultiva- erect stems. The stems are usually angular, furrowed, tion and breeding of Cannabis is prohibited in most coun- branched, with woody interior, sometimes hollow in the tries, except by permission for purposes of research and internodes, and vary from 1 to 6 m in height. The branch- pharmaceutical uses (ElSohly, 2002). Cannabis plants are ing is either opposite or alternate. The roots are advanta- usually propagated through the seed (sexual reproduc- geous, with branched taproot, generally 30–60 cm deep, tion; outdoor cultivation) or by vegetative propagation, up to 2.5 m in loose soils, very near to the surface, and using stem cuttings (asexual reproduction; indoor cul- more branched in wet soils. Leaves are green and pal- tivation) (Potter, 2004). However, both techniques have mate (seven lobes). However, the size and shape of the advantages and disadvantages. This chapter is dedicat- leaflets differs markedly, according to genetic origin. The ed to botanical aspects, including morphology, taxono- leaf arrangement is either opposite, or alternate or spiral. my, genetics, conservation, geographical distribution, The leaflets are 6–11 cm (length) and 2–15 mm (width). and cultivation forms. Leaf margins are coarsely serrated. The adaxial and abax- ial surfaces are green, with scattered, resinous trichomes. BOTANY OF CANNABIS Inflorescences consist of numerous flower heads that Macroscopical Features can be found on long, leafy stems from each leaf axil. The staminate (male flower) consists of five pale-green, Information was published elsewhere, giving detailed hairy sepals about 2.5–4  mm long, and five pendulous technical descriptions of Cannabis morphology (Clarke, stamens, with slender filaments and stamen. The pistil- 1981; UNODC, 2009) Fig. 1.1. However, this information late (female flowers) are almost sessile, and are in pairs. has been simplified in the present text. C. sativa is an an- The fruit (seed), is an achene, contains a single seed with nual, dioeciously (ie, male and female flowers are found a hard shell tightly covered by the thin wall of the ovary, on separate plants), pollinated plant with strong taproot, and it is ellipsoid, slightly compressed, smooth, about 2–5 mm long, generally brownish and mottled. FIGURE 1.1  (A) female C. sativa; (B) portion of the female flowers; (C) pistillate female flower (stigmas, style, perigonal bract, and stipule); (D) portion of the female flowers show anther; (E) mature seed. I.  Setting the scene, botanical, general and international aspects

5 Botany of Cannabis FIGURE 1.2  Microscopic photographs of C. sativa trichomes. (A) Trichomes on the flower; (B) capitate-stalked trichome; (C) capitate-sessile trichome; (D) bulbous trichome; (E) trichomes on the bract; (F) trichomes on the stem; (G) trichomes on the adaxial surface of a floral leaf. A big capitate-sessile trichome is indicated with an arrow; (H) trichomes on the abaxial surface of a leaf. Present abundant small capitate-sessile and bulbous trichomes. Source: Adapted from Happyana et al. (2013). Microscopical Features centuries, as the result of breeding and selection. How- ever, the Cannabis processed by these methods creates In general, Cannabis trichomes comprise a diverse set many debates about further botanical classification. So of structures and different types of trichomes (eg, glan- far, there is no general agreement about the taxonomic dular and nonglandular) on a single leaf, when viewed rank of various groups within the genus Cannabis, and through a hand lens (Fig.  1.2). Cannabis trichome re- consequently its monospecific or polyspecific character, searchers have commonly described two types of the since the time of Linnaeus (late 18th century) (Hazekamp, nonglandular trichome that have not been associated Justin, Lubbe, & Ruhaak, 2010). UNODC (1956) divided with terpenoid development (Table  1.1). Three types domesticated Cannabis into three different groups: of glandular trichome have been described on female • fiber hemp, long, unbranched plants, with poor seed plants, namely bulbous, sessile, and capitate stalked (Happyana et al., 2013). Male plants have been found to production exhibit a fourth type—the antherial glandular trichome, • oil seed hemp, short, early maturing plants, with rich which has only been found on anthers (Fairbairn, 1972). Glandular trichomes are made from a series of differenti- seed production ated cells with different functional properties, namely the • drug hemp, short, strongly branched plants, with secretory cells, and stalk cells (Kim & Mahlberg, 1991). small dark green leaves. Classification of Cannabis Schultes, Klein, Plowman, & Lockwood (1974) distin- guished three species within the genus: C. sativa L., C. The first official publication which recorded the use indica Lam., and C. ruderalis. Other authors referred to of Latin binomials is Linnaeus’s Species Plantarum, and the same taxa only at subspecific level within one single it can be dated back to the year 1753. Afterward, the in- species, C. sativa (Hoffmann, 1961). Small and Cronquist ternational community acknowledged it as the starting (1976) divided the single species C. sativa into the sub- point for modern botanical nomenclature. The species species sativa and indica, each consisting of a domesti- name Cannabis means “cane-like,” while the genus name cated (Table 1.2) and wild varieties. Within the subspe- “sativa” has the meaning “planted or sown,” and signifies cies sativa, the domesticated and the wild varieties are that the plant is propagated from seed, and not from pe- C. sativa subsp. sativa var. sativa (domesticated), C. sativa rennial roots (Raman,  1998). According to the modern subsp. sativa var. spontama (wild), C. sativa subsp. indica system of classification, Cannabis belongs to the family var. indica (domesticated), and C. sativa subsp. indica of Cannabaceae, along with the Humulus genus (hops) var. kafiristanica (wild). However, it is commonly ac- (Turner, Elsohly, & Boeren, 1980a,b). Different varieties of cepted that Cannabis is monotypic, and consists only of Cannabis have been developed over the course of many a single species: C. sativa (Brenneisen,  1983; Beutler & Dermarderosian, 1978). I.  Setting the scene, botanical, general and international aspects

6 1.  The Cannabis Plant: Botanical Aspects TABLE 1.1 A Summary of Cannabis Trichomes Classification, Structure, Distribution, Timing of Development, and Lifespan Trichomes Classification Structure Distribution Timing of Lifespan References development/ density Nonglandular (1) Noncystolithic trichomes: Lower side of Decreases The viability and (Fairbairn, 1972; trichomesa long, unicellular, smooth, vegetative leaves with age functioning Hammond & and pistillate secretion is Mahlberg, 1977; Turner curved, covering bracts correlated with et al., 1977, 1980b, 1981; trichomes senescence of Croteau, 1988; epidermal cells Werker, 2000; Guy & (2) Cystolithic trichomes: Stott, 2005; Happyana more squat, unicellular, claw et al., 2013) shape, cystolith covering trichomes, containing calcium carbonate Glandular (1) Bulbous: Vegetative leaves trichomesb with smallest gland and pistillate bracts (2) Capitate-sessile (unstalked): the structure is commonly simple, and the trichomes head connected directly to the mesophyll cells. (3) Capitate-stalked: Bracts and floral Increases with the structure more complex, leaves age they developed resin head (also known as the glandular head) that resembles a golf ball sitting on a tee (the trichome’s stalk). Antherial sessile Large size, with a diameter Underside of the trichomesc of approximately anther lobes 70–80 µm a Nonglandular trichomes lack cannabinoids. b Glandular trichomes are the principal or sole site of storage of most cannabinoids, the content of ∆9-THC in pistillate flowers ranged between 10 and 12%, and in leaves ranged between 1 and 2%. c Male plants are of no consequence in medicine production because they develop few glandular trichomes and, consequently, produce few cannabinoids or terpenes. TABLE 1.2 Synopsis of C. sativa Sectional Species, Subspecies, and Varieties Recognized Based on Chemical, Genetic, and Morphological Variation Section sativa Section spontanea C. sativa (L.)a C. ruderalis (L)a C. chinensis (Delile) C. sativa subsp. spontanea (Serebr.) C. gigantea (Delile) var. spontanea C. americana (Houghton) var. ruderalis C. sativa subsp. Intersita (So.) Section indica subsp. culta (Serebr) C. indica (Lam.)a subsp. Sativa (L.) C. macrosperma (Stokes) var. sativa C. sativa subsp. indica (Lam.) var. praecox var. indica var. monoica var. kif var. gigantea var. afghanica var. Chinensis var. kafiristanica var. pedemontana a Includes the endemic and domesticated populations (Raman, 1998; Sytsma et al., 2002; Hillig, 2005). I.  Setting the scene, botanical, general and international aspects

7 Botany of Cannabis The current scientific classification of Cannabis (Sytsma developing new varieties. Newly hybrid varieties have et al., 2002) been developed as a result of the crossbreds, such as, “super-sativa” (Clarke & Watson, 2002; de Meijer, 2004).   Class   Hamamelidae Recently, varieties of Cannabis have been licensed to    Subclass    Rosales GW Pharmaceuticals Ltd, as part of indoor breeding     Order     Cannabaceae programs (de Meijer & Hammond, 2005). In the United      Family      Cannabis States, the majority of Cannabis cultivars were selected       Genus       sativa from single landrace sources, or from multihybrid prog-        Species enies made from different landraces (de Meijer,  2004). The marijuana potency monitoring project at the Uni- Other Recent Taxonomic Studies versity of Mississippi (USA) is breeding Sinsemilla, CHEMOTAXONOMIC CLASSIFICATION Skunk 1, Four Way, Four Way-F, Thai/Skunk, Terbag W1, K2, and MX Cannabis of hybrid varieties (ElSohly, Recently, chemotaxonomic classification splits the Holley, & Turner, 1985; Elsohly, Holley, Lewis, Russell, & phenotypes based on the quantitative differences in the Turner, 1984). In the Netherlands, there are three differ- cannabinoid ratio of tetrahydrocannabinolic acid (THC), ent Cannabis varieties from sativa: Bedrocan, Bedrobinol, cannabinol (CBN), and cannabidiol (CBD), in the ratio of and Bediol, and one variety from C. indica is Bedica – [THC] + [CBN]/[CBD]. If the ratio exceeded 1, plants are all studied and registered by Bedrocan BV (Fischedick, classified as “chemo-type,” otherwise as “fiber-type,” and Hazekamp, Erkelens, Choi, & Verpoorte,  2010). Nowa- this was the first study to differentiate between the drug- days, many Cannabis hybrid cultivars (Table  1.3) and and fiber-type, by Fetterman et al. (1971). Therefore, this some selected pure strains have been commercialized in ratio was subsequently used to discriminate chemotype, many private companies, and there are up to 20 more intermediate type, and fiber-type (Turner, Cheng, Lewis, or less well defined strains for either indoor or outdoor Russell, & Sharma,  1979). Hillig and Mahlberg (2004) cultivation, in The Netherlands, but a sufficient data set split Cannabis into putative species and subspecies, us- is not available, due to illegal cultivation. Today, the cul- ing multivariate data analysis. Moreover, it was reported tivation and production of hemp is restricted and con- that, depending on age, the Cannabis plant can be classi- trolled because of its association with narcotic use. Most fied into different morphotypes, at different time points of the hemp breeders cultivate fiber hemp with the ul- of its development. Although this classification was not timate goal to reduce THC content below 0.2%, or even comprehensive enough to elucidate infrageneric taxo- to get noncannabinoid plants by breeding and crossing nomic structure, and does not define the contents of can- experiments (de M­ eijer, 1995). nabinoids for each chemotype, it provides a usable tool for classification (Hazekamp et al., 2010). Genetics of Cannabis MOLECULAR CLASSIFICATION Genome of Cannabis sativa Several molecular techniques have been evaluated to The genome of Cannabis (2n = 18 + XX for female, and establish the genetic relationship among different variet- 2n = 18 + XY for male) has a karyotype composed of nine ies of Cannabis plants. Some recent studies have classi- autosomes and a pair of sex chromosomes (X and Y). Sex fied and identified C. sativa samples that cannot be dif- chromosomes changes during the developmental stages ferentiated by HPLC analysis alone, by using genomic are claimed to occur in many dioecious plants, as a strat- DNA, random amplified polymorphic DNA (RAPD), egy for survival (Flemming et  al., 2007). The genome and restriction fragment polymorphisms (RFLP) analy- was measured in both female (XX) and male plants (XY) sis, but little work appears to have been conducted with (Vyskot & Hobza, 2015). The estimated haploid genome marker types that would be usable for breeding (Gillan, sizes are 818 Mb for female plants, and 843 Mb for males Cole, Linacre, Thorpe, & Watson, 1995; Faeti, Mandolino, (Sakamoto et al., 1998). The genomic resources available & Ranalli, 1996). Recently, Kojoma, Iida, Makino, Sekita, for Cannabis are mainly confined to transcriptome infor- and Satake (2002) reported that different Cannabis were mation: the NCBI database contains 12,907 ESTs and 23 identified by means of inter simple sequence repeat unassembled RNA-Seq datasets of Illumina reads (Marks (ISSR). ISSR is a technique offering the reproducibility et al., 2009). The genetic basis of cannabinoid variation in and simplicity of RAPDs with high reliability (Galvan, C. sativa showed that the amount of THC versus CBD is Bornet, Balatti, & Branchard, 2003). likely governed by one locus with two codominant al- leles, B(d) and B(t) (de Meijer et al., 2003). One possible Current Cannabis Varieties explanation for these results is that the two alleles en- code either THCA or CBDA synthase so that homozy- Recently, Cannabis growers have become more gous plants would contain either tetrahydrocannabinolic aware to create variations between different strains for I.  Setting the scene, botanical, general and international aspects

8 1.  The Cannabis Plant: Botanical Aspects TABLE 1.3 Origin of Hemp Varieties Were Reported in acid (THCA) or cannabidiolic acid (CBDA) as the major Literaturea cannabinoid, and heterozygotes would have an approxi- mately equal mixture of the two (Fig. 1.3). Another ex- Variety Country planation is that THCA and CBDA synthases are closely linked genes, perhaps produced as a result of a gene Finola Finland duplication event. A recent study analyzed the THCA synthase sequences from drug (high-THC) and fiber Glukhov 33, Kuban, Uso 11, Zenica, USO 13, USO Ukraine (low-THC) varieties, and found that the amino acid se- 15, USO 31, YUSO 14, YUSO 16 quence of THCA synthase from high-THC varieties dif- fered by 37 major substitutions, compared to low-THC Asso, Carmagnola, CS (Carmagnola Selezionata), Italy varieties (Kojoma, Seki, Yoshida, & Muranaka, 2006). Carmono, Carma, Codimono, Eletta Campana, Ferrara, Ermes, Fibrimor Geographical Distribution Fibranova Small and Cronquist (1976) state that genus Canna- bis geographically grows to the north of latitude 30°N Fasamo, Ferimon Germany and south of latitude 60°N (Hillig,  2005). The genus is believed to have originated in the Northwest Himalayas, Santhica 27, Epsilon 68, Fedora 17, Fedora 19, France and occurs widely in Africa. Fedrina 74, Felina 32, Felina 34, Fibrimon 21, Fibrimon 24, Fibrimon 56, Futura, Futura 77, Agricultural Status Futura 75, Santhica 23, Dioica 88 Nowadays, fiber hemp is cultivated in a number Kompolti Sargaszaru, Kinai unisexualis, Kompolti, Hungary of countries around the world, and China represents Kompolti Hybrid TC, Kompolti Hyper, Elite, the largest producer of hemp with focus on fiber-type Fibriko (Mediavilla, Bassetti, & Leupin, 1999). Nevertheless, cul- tivation of medicinal Cannabis is prohibited in most of Fibramulta 151, Irene, Lovrin 110, Moldovan, Romania countries, except by permission for purposes of research Secuieni 1 and pharmaceutical uses. Beniko, Bialobrzeskie, LKCSD, Dolnoslaskie Poland Conservation Initiatives Chamaeleon, Dutch “Yellow” line Netherlands Cannabis populations are facing the threat of genetic drift—which has a direct effect on the changes to the Ermakovskaya Mestnaya Russia phenotype and chemical profile, due to allogamous (de Meijer & Vansoest, 1992). The conservation of Cannabis Delta 405, Delta-llosa Spain germplasm is divided into two main strategies: in situ and ex situ. Kenvir Turkey Ex Situ Conservation in Gene Banks Swissmix Swiss The Cannabis gene bank at Vavilov Research Insti- tute of Plant Industry (St. Petersburg, Russia) main- Ratslaviska Czech tained about 200 accessions, for more than 50 years (de Meijer,  1998). In addition, the Hungarian gene bank at Silistrensi, Mecnaja copt Bulgaria the Research Center for Agrobiodiversity (Tápiószele, Hungary) maintained about 70 accessions. Collections of Pesnica Slovenia up to 20 accessions are preserved in other depositories in Germany, Turkey, and Japan. In comparison with other Flajsmanova, Novosadksa, Novosadska plus, Former crops, the available number of well-documented Cannabis Novosadska konoplja ­Yugoslavia accessions is limited (de Meijer & Vansoest, 1992). Now- adays, several accessions are maintained by the United Kinai Egylaki, Kinai Ketlaki China States Department of Agriculture (USDA)/National Plant Germplasm System (NPGS), and associated data Kozuhura Zairai Japan can be accessed from the Germplasm Resources Infor- a Low THC cultivars, less than 0.2% dry weight. mation Network (GRIN) database. FIGURE 1.3  Inheritance of chemical phenotype in C. sativa “co- dominant monogenic control,” homozygous THC producing BtBt genotypes are typically selected for recreational use. Source: From de Meijer et al. (2003). I.  Setting the scene, botanical, general and international aspects

9 Botany of Cannabis In Situ Conservation as In Vitro Gene Banks Indoor Cultivation In vitro conservation of encapsulated microcuttings This method of breeding is used for increasing resin of Cannabis shootlets was attempted under slow growth potency, and avoiding unwanted male plants (Chandra, conditions between 5 and 15°C (Lata, Chandra, Khan, Lata, Khan, & ElSohly, 2010). The complete growth cy- & ElSohly,  2008; Lata, Chandra, Mehmedic, Khan, & cle, quality, and quantity of biomass can be regulated ElSohly,  2012), but adaptation to in vitro conditions under controlled environmental conditions (6–8 weeks). could induce mutants of the offspring plants, causing The successful indoor system requires an effective hy- genetic and chemical drift (Larkin & Scowcroft, 1981). droponic system to deliver nutrients and oxygen, and support the plants’ growth (Fig. 1.4). However, there is a Cultivation Techniques of Cannabis number of different techniques that have been proposed for the indoor horticulture of Cannabis, for example, the Outdoor Cultivation standing aerated technique, nutrient film technique, and Cannabis plant can be propagated from seeds, and aeroponics technique. In hydroponic growing, the nutri- ent solution is best at a pH within a certain range (5.5– the life cycle is completed within 4–6 months, depend- 6.5) for maximum uptake and good plant growth (Argo ing on the time of the plantation and the variety. It can & Fischer, 2002). Indoor Cannabis crop cultivation needs reach up to 5 m (16 ft.) in height, in a 4–6 months grow- artificial light and compressed CO2 gas for photosyn- ing season (Raman, 1998; Clarke & Watson, 2002). Her- thesis, and for controlling flowering and plant biomass maphroditic varieties of this plant have been bred for (Jones, 1997). Here, selective vegetative female plants are industrial hemp production, as this allows more uni- used for making clones. Later, all clones are kept under form crops (Leggett, 2006). The process of germination is standard environmental conditions (light, temperature, usually completed in 3–7 days (Clarke & Watson, 2002). RH, and CO2 concentration) in a growing room for vegeta- The seedling stage is completed within 2–3 months. Lat- tion (18 h/day photoperiod) and for flowering (12 h/day er, the plant is characterized by increased biomass and photoperiod). total growth under long day time lengths (vegetative growth). It is easy to recognize the male and female sex In vitro Micropropagation at this stage. Later in summer, the reproductive phase The micropropagation system offers a number of of Cannabis begins when the plant is exposed to short day time lengths (less light per day than darkness) of 12– clear advantages, including (1) human-controlled 14 h or less, depending on its latitude and genetic origin method with fast propagation in a comparably short (Brenneisen,  1983). Once the male flowers ripened and time, due to high potential multiplication rates, (2) it pollinated, the female flowers died directly. The pro- is independent of seasonal factors like climate and ge- duced seeds after flowering have combinations of traits ography, and (3) the produced plants are usually free from two parents, as a result of cross fertilization (Clarke from ­microorganism-borne diseases (Zafar, Aeri, & & Watson, 2002). This method is mostly used for the cul- D­ atta, 1992). On the other hand, in vitro propagation of tivation of Cannabis for hemp fiber, or Cannabis seed with C. sativa through the seed is possible in most of cultivars, less than 0.2% THC. although the greatest problem with such a method is the FIGURE 1.4  Indoor cultivation of C. sativa. Source: Photo provided from Bedrocan BV, the Netherlands. I.  Setting the scene, botanical, general and international aspects

10 1.  The Cannabis Plant: Botanical Aspects FIGURE 1.5  In vitro micropropagation of leaf-derived calli from C. sativa L. (A) Callus culture, (B) meristemoid formation, (C) shootlets multiplication on Gamborg’s B5 medium supplemented with various combinations of auxins and cytokinins. Source: Photos provided from Sayed Farag PhD project, Technische Universität Dortmund. high level of heterozygosity that could lead to a rapid Micropropagation  In vitro technique for multiplying plant tissues and dramatic profile shift of secondary metabolites through in vitro culture, either indirectly (with intervening callus from one generation to the next (Chandra et  al.,  2010). stage) or directly (without an intervening proliferative stage). This In fact, in vitro propagation using explants or somatic is achieved by altering the concentration of growth regulators, embryogenesis has been reported (Lata et al., 2002). Be- mainly auxins and cytokinins. sides the progress in the field of plant biotechnology, Random amplified polymorphic DNA (RAPD)  A molecular very little progress has been made to date toward devel- technique for the rapid assignation of DNA-based character states oping an in vitro regeneration from C. sativa. Previous for phylogenetic analysis. The technique uses the polymerase reports on de novo organogensis of C. sativa emerged chain reaction (PCR) to amplify any genomic region containing in early 1980s (Fisse, Braut, Cosson, & Paris, 1981), and single primer of nucleotide arbitrary sequence. subsequently from callus of different genotypes and ex- Restriction fragment length polymorphism (RFLP)  A molecular plant sources, including cotyledons and stem (Wielgus, technique for genome mapping, and variation analysis (genotyping, Luwanska, Lassocinski, & Kaczmarek,  2008), young forensics, paternity tests, hereditary disease diagnostics, etc.). The leaves (Lata, Chandra, Khan, & ElSohly,  2010), inter- technique uses restriction of endonucleases to cut DNA at specific nodes, and axillary buds and petioles (Slusarkiewicz- (generally 4–6 bp) recognition sites. Jarzina, Ponitka, & Kaczmarek, 2005), and roots (Ranalli Trichome  Defined as hair-like structures that extend from the & Mandolino,  1999). Alternatively, the use of meriste- epidermis of aerial tissues; are present on the surface of most matic callus for micropropagation was studied recently terrestrial plants. (Farag & Kayser, unpublished results, Fig. 1.5). References Recommendations for Future Action Appendino, G., Chianese, G., & Taglialatela-Scafati, O. (2011). Can- Given the high therapeutic and commercial value of nabinoids: occurrence and medicinal chemistry. Current Medicinal Cannabis, legal indoor breeding started in some pharma- Chemistry, 18(7), 1085–1099. ceutical companies. The biotechnological research for ge- netic improvement has been minimal to date. Researches Argo, W. R., Fischer, P. R. (2002). Understanding pH management for on transgenic Cannabis is still needed to facilitate the met- container-grown crops. Meister, Willoughby, Ohio. abolic engineering of cannabinoids and agronomic traits. Beutler, J. A., & Dermarderosian, A. H. (1978). Chemotaxonomy of MINI-DICTIONARY Cannabis .1. Cross breeding between Cannabis sativa and Cannabis ruderalis, with analysis of Cannabinoid content. Economic Botany, Encapsulation  In vitro technique for the production of synthetic 32(4), 387–394. seeds (Ca-alginate beads) for long-term storage of germplasm. Genome  The complete set of chromosomal and extra- Brenneisen, R. (1983). Psychotropic drugs. I. Cannabis sativa L. (Canna- chromosomal DNA/RNA of an organism, a cell, an organelle, binaceae). Pharmaceutica Acta Helvetiae, 58(11), 314–320. or a virus. Inter simple sequence repeats (ISSR)  A molecular technique Brenneisen, R. (2007). Chemistry and analysis of phytocannabinoids for evolutionary biology. Its simple sequence repeats (SSR), also and other Cannabis constituents. In M. A. Elsohly (Ed.), Marijuana known as microsatellites, are tandem repeats of a few base pairs and the cannabinoids (pp. 17–49). Totowa: Humana Press. distributed throughout the genome. Chandra, S. H., Lata, I., Khan, A., & ElSohly, M. A. (2010). Propagation of elite Cannabis sativa for the production of ∆9-Tetrahydrocannabinol (THC) using biotechnological tools. In A. Rajesh (Ed.), Medicinal Plant Biotechnology (pp. 98–114). UK: CABI. Clarke, R. C. (1981). Marijuana botany. An advanced study: the propagation and breeding of distinct cannabis. Berkeley, CA: And/Or Press. Clarke, R. C., & Watson, D. P. (2002). Botany of natural Cannabis medi- cines. In F. Grotenhermen, & E. Russo (Eds.), Cannabis and canna- binoids: pharmacology, toxicology, and therapeutic potential (pp. 3–13). New York: The Haworth Press. Croteau, R. (1988). Catabolism of monoterpenes in essential oil plants. Developments in Food Science, 18, 65–84. I.  Setting the scene, botanical, general and international aspects

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12 1.  The Cannabis Plant: Botanical Aspects Raman, A. (Ed.), (1998). The Cannabis plant: botany, cultivation, and Turner, C. E., Elsohly, M. A., & Boeren, E. G. (1980a). Constituents of processing for use. In Cannabis: The Genus Cannabis (pp. 29–54). Cannabis sativa L .17. a review of the natural constituents. Journal of Amsterdam: Harwood Academic Publishers. Natural Products, 43(2), 169–234. Ranalli, P., & Mandolino, G. (1999). Advances in hemp research. In P. Turner, J. C., Hemphill, J. K., & Mahlberg, P. G. (1980b). Trichomes and Ranalli (Ed.), Advances in biotechnological approaches for hemp breeding cannabinoid content of developing leaves and bracts of Cannabis sa- and industry (pp. 185–208). New York: The Haworth Press. tiva L (Cannabaceae). American Journal of Botany, 67(10), 1397–1406. Ross, S. A., & ElSohly, M. A. (1996). The volatile oil composition of Turner, J. C., Hemphill, J. K., & Mahlberg, P. G. (1981). Interrelation- fresh and air-dried buds of Cannabis sativa. Journal of Natural Prod- ships of glandular trichomes and cannabinoid content. 2. Develop- ucts, 59(1), 49–51. ing vegetative leaves of Cannabis sativa L (Cannabaceae). Bulletin on Narcotics, 33(3), 63–71. Sakamoto, K., Akiyama, Y., Fukui, K., Kamada, H., & Satoh, S. (1998). Characterization: genome sizes and morphology of sex chromo- UNODC, United Nations, (2009). Recommended methods for identifi- some in hemp (Cannabis sativa L.). Cytologia, 63, 459–464. cation and analysis of cannabis and cannabis products, New York. http://www.unodc.org/documents/scientific/ST-NAR-40-Ebook. Schultes, R. E., Klein, W. M., Plowman, T., & Lockwood, T. E. (1974). pdf Cannabis: an example of taxonomic neglect. Harvard University Bo- tanical Museum Leaflets, 23, 337–367. UNODC, United Nations (1956). Problems of modern hemp breeding, with particular reference to the breeding of varieties of hemp contain- Slusarkiewicz-Jarzina, A., Ponitka, A., & Kaczmarek, Z. (2005). In- ing little or no hashish, New York. http://www.unodc.org/unodc/ fluence of cultivar, explant source and plant growth regulator on en/data-and-analysis/bulletin/bulletin_1956-01-01_3_page007.html c­ allus induction and plant regeneration of Cannabis sativa L. Acta Biologica Cracoviensia Series Botanica, 47(2), 145–151. Vanhoenacker, G., Van Rompaey, P., De Keukeleire, D., & Sandra, P. (2002). Chemotaxonomic features associated with flavonoids of can- Small, E., & Cronquist, A. (1976). A practical and natural taxonomy for nabinoid-free cannabis (Cannabis sativa subsp sativa L.) in relation to Cannabis. Taxon, 25, 405–435. hops (Humulus lupulus L.). Natural Product Letters, 16(1), 57–63. Sytsma, K., Morawetz, J., Pires, J., Nepokroeff, M., Conti, E., Zjhra, M., Vyskot, B., & Hobza, R. (2015). The genomics of plant sex chromo- Hall, J., & Chase, M. (2002). Urticalean rosids: circumscription, ro- somes. Plant Science, 236, 126–135. sid ancestry, and phylogenetics based on rbcL, trnL-F, and ndhF sequences. American Journal of Botany, 89(9), 1531–1546. Werker, E. (2000). Trichome diversity and development. Advances in Botanical Research, 31, 1–35. Turner, C. E., & Elsohly, M. A. (1976). Anhydrocannabisativine, a new alkaloid isolated from Cannabis sativa. Lloydia, 39(6), 474–1474. Wielgus, K., Luwanska, A., Lassocinski, W., & Kaczmarek, Z. (2008). Estimation of Cannabis sativa L. tissue culture conditions essential Turner, J. C., Hemphill, J. K., & Mahlberg, P. G. (1977). Gland distri- for callus induction and plant regeneration. Journal of Natural Fibers, bution and cannabinoid content in clones of Cannabis sativa L. 5(3), 199–207. ­American Journal of Botany, 64(6), 687–693. Zafar, R., Aeri, V., & Datta, A. (1992). Application of plant tissue and Turner, C. E., Cheng, P. C., Lewis, G. S., Russell, M. H., & Sharma, G. K. cell culture for production of secondary metabolites. Fitoterapia, 63, (1979). Constituents of Cannabis sativa .15. Botanical and chemical 33–43. profile of Indian variants. Planta Medica, 37(3), 217–225. I.  Setting the scene, botanical, general and international aspects

CHAPTER 2 The Biosynthesis of Cannabinoids F. Degenhardt, F. Stehle, O. Kayser Laboratory of Technical Biochemistry, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany SUMMARY POINTS • Two cannabigerol-like compounds were detected in • This chapter focuses on the pathway which leads the aerial parts of Helichrysum umbraculigerum Less., a plant common in the eastern parts of South Africa. to the biosynthesis of phytocannabinoids in C. sativa L. • N-alkyl amides (cannabinomimetics), found in the • CBGA is the central precursor of medicinal plants Echinaceae angustifolia and Echinaceae phytocannabinoid biosynthesis in Cannabis. purpurea (purple cornflower), are known to interact • CBGAS, only three enzymes—THCAS, CBDAS, with the CB2 receptor. and CBCAS—are involved in the biosynthesis of phytocannabinoids in Cannabis plants. LIST OF ABBREVIATIONS • Sequences of CBDAS and THCAS are known. • The carboxyl group in CBGA seems to be AAE Acyl-activating enzyme essential for the enzymatic reactions catalyzed by BBE Berberine bridge enzyme CBDAS, CBCAS, and THCAS. CBC Cannabichromene • The diversity of more than 60 cannabinoids is the CBCA Cannabichromenic acid result of nonenzymatic modifications. CBCAS Cannabichromenic acid synthase • Propyl cannabinoids occur by the prenylation of CBCVA Cannabichrovarinic acid divarinic acid (DA) with geranyl diphosphate CBD Cannabidiol (GPP). CBDA Cannabidiolic acid CBDAS Cannabidiolic acid synthase KEY FACTS OF CBDV Cannabidivarin PHYTOCANNABINOIDS—BESIDES CBDVA Cannabidivarinic acid C .   S AT I VA CBG Cannabigerol CBGA Cannabigerolic acid (3-geranyl olivetolate) • Phytocannabinoids are plant-derived natural compounds CBGAS Cannabigerolic acid synthase that act as ligands to cannabinoid receptors (CB1 and CB2) CBGVA Cannabigerovarinic acid or share chemical similarity with cannabinoids. CBN Cannabinol CBNRA Cannabinerolic acid (cis-CBGA) • C. sativa L. is intensively investigated for the presence CHS Chalcone synthase of phytocannabinoids. To date, only a few plants are CsAAE1 C. sativa hexanoyl-CoA synthetase 1 discovered that contain phytocannabinoids other than CsAAE3 C. sativa hexanoyl-CoA synthetase 2 the ones known from Cannabis. CsHCS1 C. sativa hexanoyl-CoA synthetase 1 CsHCS2 C. sativa hexanoyl-CoA synthetase 2 • The New Zealand liverwort Radula marginata and DA Divarinic acid Japanese liverwort Radula perrottetii contain perrotteti- DABB Dimeric α + β barrel nene, a naturally occurring bibenzyl cannabinoid. DMAPP Dimethylallyl diphosphate Handbook of Cannabis and Related Pathologies. http://dx.doi.org/10.1016/B978-0-12-800756-3.00002-8 Copyright © 2017 Elsevier Inc. All rights reserved. 13

14 2.  The Biosynthesis of Cannabinoids DOXP 1-Deoxy-d-xylulose-5-phosphate for the biosynthesis of cannabinoids, the terpenopheno- GOT Geranylpyrophosphate:olivetolate lic constituents that show psychoactive effects. But since GPP geranyltransferase other plants also have secondary metabolites that inter- HTAL Geranyl diphosphate act with the human cannabinoid receptors, a new defini- IPP Hexanoyltriacetic acid lactone tion had to be made. Hence, phytocannabinoids are now MEP Isopentenyl diphosphate defined as any plant-derived natural compound that MVA 2C-methyl-d-erythritol-4-phosphate can act as a ligand to human cannabinoid receptors (CB1 NPP Mevalonate and CB2) or share chemical similarity with cannabinoids OA Neryl diphosphate (Gertsch, Pertwee, & Di Marzo,  2010). Interestingly, all OAC Olivetolic acid parts of the Cannabis plant, with the exception of seeds, OLS Olivetolic acid cyclase can contain cannabinoids, but they mainly accumulate PKS Olivetol synthase in the glandular trichomes of female flowers (Gagne SNP Polyketide synthase et al., 2012; van Bakel et al., 2011). STS Single nucleotide polymorphism THC Stilbene synthase The following chapter focuses on the pathway that THCA Tetrahydrocannabinol leads to the enzymatic biosynthesis of cannabinoids. For THCAS Tetrahydrocannabinolic acid a long time, it was postulated that the key intermedi- THCV Tetrahydrocannabinolic acid synthase ate is cannabidiol (CBD) or cannabidiolic acid (CBDA), THCVA Tetrahydrocannabivarin both resulting from a condensation of a monoterpene, Tetrahydrocannabivarinic acid and olivetol or olivetolic acid (OA), respectively. In 1964, Gaoni and Mechoulam postulated cannabigerol (CBG) INTRODUCTION as the key intermediate, the condensation product of ge- ranyl diphosphate (GPP), and olivetol or OA. Based on Cannabis sativa L. (hemp) is one of the oldest do- this, they concluded that the cannabinoids CBD, tetrahy- mestic plants in the history of mankind, and has been drocannabinol (THC) and cannabinol (CBN) are all de- cultivated for at least 10,000  years (Schultes, Klein, rived from CBG, and just differ in the way of cyclization Plowman, & Lockwood,  1974). Together with Humulus (Gaoni & Mechoulam, 1964). Finally, incorporation stud- lupulus (hop), C. sativa belongs to the small family of ies with 13C-labeled glucose have shown that GPP and Cannabaceae. Cannabis is an annual, usually dioecious, OA are indeed the precursors for formation of cannabig- wind-pollinated herb, with both male and female flow- erolic acid (CBGA). Thus, the general structure of canna- ers growing on separate plants. The plant is well known binoids is assembled by two parts: (1) a diphenol (resor- cin) carrying an alkyl chain (OA); and (2) a monoterpene moiety (GPP) (Fig.  2.1). Subsequently, Fellermeier and FIGURE 2.1  General structure of cannabinoids and their precursors, olivetolic acid, and geranyl diphosphate. Cannabinoids are composed of two parts: a cyclic monoterpene part (red), and a diphenol (resorcin) part, carrying an alkyl chain (blue). The dibenzopyran-numbering system is used. I.  Setting the scene, botanical, general and international aspects

15 Cannabinoid precursor biosynthesis coworkers postulated CBGA as the central cannabinoid via the lipoxygenase pathway (Marks et al., 2009; Stout, precursor (Fellermeier, Eisenreich, Bacher, & Zenk, 2001; Boubakir, Ambrose, Purves, & Page, 2012). Nevertheless, Fellermeier & Zenk, 1998). Interestingly, free OA has nev- further studies are necessary to clarify the origin of the er been detected in Cannabis plant material until now. hexanol moiety. It is worthy to note that, although more than 60 can- Hexanoyl-CoA is a medium-chain fatty acyl-CoA that nabinoids are known, only three enzymes, besides can- can be detected in high amounts in Cannabis flowers nabigerolic acid synthase (CBGAS), namely tetrahydro- (Stout et al., 2012). It is synthesized by an acyl-activating cannabinolic acid synthase (THCAS), cannabidiolic acid enzyme (AAE) called hexanoyl-CoA synthetase (Marks synthase (CBDAS), and cannabichromenic acid synthase et  al.,  2009; Page & Stout,  2013). AAEs can use short, (CBCAS), are involved in cannabinoid biosynthesis. The medium, long as well as very long-chain fatty acids as resulting acidic cannabinoids are the most abundant ones carboxylic acid substrates. Two novel enzymes were accumulating in Cannabis. The neutral and psychoactive identified, C. sativa hexanoyl-CoA synthetase 1 (CsHCS1 forms are the results of nonenzymatic decarboxylation or CsAAE1) and C. sativa hexanoyl-CoA synthetase 2 during storage, heat or sunlight; explaining the heating (CsHCS2 or CsAAE3) that are capable of producing of plant material (ie, smoking or baking), during Canna- hexanoyl-CoA using hexanoate and CoA as substrates. bis consumption (Fischedick, Hazekamp, Erkelens, Choi, Based on transcript levels, CsHCS1 seems to be tri- & Verpoorte, 2010; Taura et al., 2007a). Thus, the broad chome-specific. Although CsHCS2 exhibits lower tran- diversity of the different cannabinoids is mainly due to script levels, in comparison to CsHCS1, it is abundant in nonenzymatic transformation or degradation of both all tissues. The gene of CsHCS1 consists of a 2163-nucle- acidic and neutral cannabinoids by the effects of light otide open reading frame, and encodes a 720-amino acid (UV irradiation) and auto-oxidation (Crombie, Ponsford, polypeptide chain. The gene of CsHCS2 is composed of Shani, Yagnitinsky, & Mechoulam,  1968; Razdan, a 1632-nucleotide open reading frame, and encodes a Puttick, Zitko, & Handrick, 1972). It is still unclear if all 543-amino acid polypeptide chain. Both CsHCSs gener- these forms are present in living plants as natural or ar- ally require divalent cations for activity. This was shown tefacts, due to storage and sample preparation (ElSohly by adding Mg2+, Mn2+, and Co2+ to the enzyme assays. & Slade, 2005). Thus, CsHCS1 preferentially accepts Mg2+, and CsHCS2 Co2+. The highest enzyme activity was detected at 40°C CANNABINOID PRECURSOR and pH 9 for both enzymes. Furthermore, both enzymes BIOSYNTHESIS can be inhibited by high concentrations of CoA (Page & Stout, 2013; Stout et al., 2012). Polyketide Pathway Toward Olivetolic Acid Taken together, the published data suggest that The origin of hexanoate in trichomes has not been CsHCS1 is the enzyme involved in the biosynthesis of elucidated so far. Suzuki, Kurano, Esumi, Yamaguchi, and cannabinoids: (1) it is the most abundant AAE in tri- Doi (2003) showed that the side-chain moiety of alkyl- chomes; (2) it is highly specific for short-chain fatty acyl- resorcinols is formed by fatty acid units, but it remains CoA, particularly hexanoate (KM value in the nM range); unclear if the moiety is the result of biosynthesis or deg- and (3) it is localized in the cytosol, as suggested for the radation of fatty acids. Studies regarding the incorpora- olivetol synthase (see later). In contrast, CsHCS2 is lo- tion of 13C-labels into cannabinoids indicate that hexano- calized in the peroxisomes and accepts a broad range of ate is synthesized from acetyl-CoA as a starter unit, and substrates, while showing a KM value for hexanoate in five molecules of malonyl-CoA. These building blocks the mM range (Page & Stout, 2013; Stout et al., 2012). are precursors of the fatty acid biosynthesis (Fellermeier et al., 2001). The alkylresorcinol moiety of cannabinoids is de- rived from OA, the product of polyketide synthases Based on this, two pathways are feasibly possible, (PKSs) that catalyze the aldol condensation of hexanoyl- after analysis of a cDNA/EST library generated from CoA with three molecules of malonyl-CoA (Fellermeier female flowers (glands) of C. sativa. First, the hexanoyl et  al.,  2001; Raharjo, Chang, Choi, Peltenburg-Looman, residue could be obtained by an early termination of the & Verpoorte, 2004) (Fig. 2.2). The second precursor mal- fatty acid biosynthesis. Subsequently, the hexanoyl moi- onyl-CoA is predominantly derived from acetyl-CoA ety of the resulting hexanoyl-ACP would be cleaved by a by carboxylation. The ATP-dependent reaction is cata- thioesterase or transferred to CoA by an ACP-CoA trans- lyzed by an acetyl-CoA carboxylase (EC 6.4.1.2). The en- acylase. Finally, acyl-CoA synthetase would catalyze the zyme utilizes the first step in the fatty acid biosynthesis conversion of the obtained n-hexanol to hexanoyl-CoA (Chen, Kim, Weng, & Browse, 2011; Konishi, Shinohara, (Marks et  al.,  2009). Second, n-hexanol could be pro- Yamada, & Sasaki, 1996). Taura et al. (2009) discovered duced by the breakdown of C18 unsaturated fatty acids a plant type III PKS in flowers and rapidly expanding leaves of C. sativa. The gene of olivetol synthase (OLS) encodes a 385-amino acid polypeptide chain that does I.  Setting the scene, botanical, general and international aspects

16 2.  The Biosynthesis of Cannabinoids TABLE 2.1 Enzymes Involved In Cannabinoid Biosynthesis in C. sativa L Enzyme Accession no.a EC no. References Olivetol synthase OLS AB164375 2.3.1.206 Taura et al. (2009) Olivetolic acid cyclase OAC AFN42527.1 4.4.1.26 Gagne et al. (2012) Cannabigerolic acid synthase CBGAS US2012/0144523 2.5.1.102 Fellermeier and Zenk (1998); A1b Page and Boubakir (2012) Cannabichromenic acid synthase CBCAS 1.3.3.- Morimoto et al. (1998) Cannabidiolic acid CBDAS AB292682 1.21.3.8 Taura et al. (2007a) synthase Tetrahydrocannabinolic acid synthase THCAS AB057805 1.21.3.7 Sirikantaramas et al. (2004) The table lists the enzymes and the corresponding GenBank accession numbers involved in biosynthesis of C. sativa phytocannabinoids. aGenBank. bPatent number. not contain a signal peptide (Table 2.1). The OLS protein Finally, using both OLS and OAC with hexanoyl-CoA has a theoretical molecular mass of 43 kDa, as confirmed and malonyl-CoA in one assay, the formation of OA, by SDS-PAGE analysis. However, size-exclusion chro- pentyldiacetic acid (triketide pyrone), and hexanoyltri- matography experiments revealed a molecular mass of acetic acid lactone (HTAL; tetraketide pyrone) could be about 89 kDa, indicating a homodimeric enzyme (Gagne demonstrated (Page & Gagne,  2013) (Fig.  2.2). It is as- et al., 2012; Taura et al., 2009). OLS (PKS-1) was prelimi- sumed that OLS catalyzes the formation of an interme- narily crystallized by Taguchi et al. (2008) and the struc- diate that is subsequently converted into OA by OAC ture was finally published by Yang et al. (2016). It is of (Gagne et al., 2012; Taguchi et al., 2008). interest that the enzyme does not produce OA, but olive- tol, triketide pyrone, and tetraketide pyrone. Analysis of Biosynthesis of Geranyl Diphosphate the amino acid sequence displayed a high similarity with those of Medicago sativa chalcone synthase (CHS), and The monoterpene moiety of cannabinoids (Fig. 2.2) is other plant PKSs (60–70%). Additionally, the catalytic tri- derived from GPP. Its precursors, isopentenyl diphos- ade residues of CHS (Cys164-His303-Asn336) are conserved phate (IPP), and dimethylallyl diphosphate (DMAPP), (Taura et al., 2009). Since CHSs catalyze intramolecular are predominantly (>98%) biosynthesized via the C6 → C1 Claisen condensations, Raharjo, and coworkers 2C-methyl-d-erythritol-4-phosphate (MEP) pathway were the first to suggest in 2004 (Raharjo et al., 2004) that [also termed as nonmevalonate pathway or 1-deoxy-d- OLS could be a stilbene synthase (STS). These enzymes xylulose-5-phosphate (DOXP) pathway] (Fellermeier catalyze C2 → C7 aldol condensations, followed by a et al., 2001). These results are supported by Marks et al. decarboxylation step. Additionally, studies by Austin, (2009). They isolated RNA from the glands of a tetra- Bowman, Ferrer, Schröder, & Noel (2004) showed that hydrocannabinolic acid (THCA)-producing Cannabis the cyclization reaction can be changed from a Claisen- strain and generated a cDNA library. After sequencing, type (CHS) to an aldol-type (STS) by substitution of a they were able to identify all but one enzyme involved few amino acids in CHS (= aldol switch). in the MEP pathway. Additionally, Stout et  al. (2012) found high expression of MEP pathway genes in Can- Nevertheless, since OLS alone is not capable to form nabis flowers. Furthermore, in higher plants the MEP OA, another enzyme/PKS might be involved in the pathway, mainly involved in secondary metabolism, biosynthesis. The missing enzyme should catalyze a C2 is localized in plastids (described in detail elsewhere, → C7 intramolecular aldol condensation upon which for example, Eisenreich, Bacher, Arigoni, & Rohdich, the carboxylate moiety is preserved. This is important (2004), or Hunter (2007), whereas the mevalonate (MVA) since CBGAS does not accept olivetol as a prenyl do- pathway, predominantly contributing to primary me- nor (Fellermeier & Zenk, 1998). Gagne et al. (2012) iso- tabolism, is localized in the cytosol. The compartmental lated a gene encoding a 101-amino acid polypeptide separation between these two pathways is not absolute. chain. This small protein (12 kDa) shows similarities to The metabolites of both pathways can be transported bi- a polyketide cyclase that belongs to the dimeric α + β directionally across the plastid membranes (Eisenreich barrel (DABB)-type protein family. Furthermore, the et al., 2004). identified gene exhibits high expression levels in glan- dular trichomes. Together, this made the polyketide Subsequently, the head-to-tail condensation of IPP cyclase a promising candidate for the missing olivetolic and DMAPP to form GPP is catalyzed by geranyl di- acid cyclase (OAC). phosphate synthase (Fig. 2.2) (Burke et al., 1999). I.  Setting the scene, botanical, general and international aspects

17 Cannabinoid precursor biosynthesis FIGURE 2.2  Biosynthesis of cannabigerolic acid (CBGA). The biosynthesis of the central intermediate CBGA is colored in dark green. The minor products CBNRA and CBGVA are shaded in light green. The precursor pathways are highlighted in light blue (GPP) and blue (OA). MEP, 2C-methyl-d-erythritol-4-phosphate; DOXP, 1-deoxy-d-xylulose-5-phosphate; MVA, mevalonate (Burke, Wildung, & Croteau,  1999; de Meijer et al., 2009; Fellermeier & Zenk, 1998; Page & Gagne, 2013; Taura et al., 2009). Cannabigerolic Acid Biosynthesis by mass spectrometry (MS) measurements as CBGA and its cis-isomer cannabinerolic acid (CBNRA; Fellermeier Cannabigerolic acid synthase (CBGAS) or geranylpy and Zenk, 1998, used CBNA instead of CBNRA). The en- rophosphate:olivetolate geranyltransferase (GOT) pre- zyme activity was found to be Mg2+-dependent. CBGAS dominantly catalyzes the C-prenylation of OA by GPP seems to be specific for OA as a prenyl acceptor, but also to form CBGA (Fig.  2.2). CBGA is presumed to be the accepts different prenyl donors like GPP and, to a lesser central precursor for cannabinoid biosynthesis, since dif- degree, neryl diphosphate (NPP) (Fig. 2.2). The produc- ferent cyclization of the prenyl moiety leads to THCA tion ratio of CBGA/CBNRA changes from 2:1 to 1:1 or its isomers cannabichromenic acid (CBCA) and CBDA when NPP is used as a prenyl donor instead of GPP. (Page & Boubakir,  2012; Sirikantaramas, Morimoto, & Shoyama, 2007). However, the aromatic prenyltransferase CBGAS seems to be a soluble enzyme, but Fellermeier and Zenk Fellermeier and Zenk (1998) detected the enzyme in (1998) could not completely exclude a membrane-bound crude homogenates of rapidly expanding young leaves activity. Besides, two soluble hop prenyltransferases, of C. sativa. This part of the plant contains the later en- involved in the biosynthesis of hop bitter acids, are de- zymes of the THCA biosynthetic pathway (Morimoto, scribed by Zuurbier, Fung, Scheffer, & Verpoorte (1998). Komatsu, Taura, & Shoyama, 1997; Taura et al., 1995a). Nevertheless, these are the only descriptions of soluble There are indications that CBGAS, like other prenyl- plant C-prenylating enzymes; until now, it was not pos- transferases, is a membrane-bound prenyltransferase sible to get the sequence information or to isolate the cor- (Yamamoto, Kimata, Senda, & Inoue,  1997). However, responding genes or enzymes. Fellermeier and Zenk (1998) could not detect any enzyme activity in particulate fractions, but in the soluble fraction Contradictorily, all known sequences of plant aro- of the crude extract. Two major products were identified matic prenyltransferases belong to membrane-bound I.  Setting the scene, botanical, general and international aspects

18 2.  The Biosynthesis of Cannabinoids enzymes (Yamamoto et  al.,  1997; Yamamoto, Senda, & cations, whereas the highest enzyme activity was ob- Inoue,  2000; Zhao, Inoue, Kouno, & Yamamoto,  2003). tained by using Mg2+ (Page & Boubakir, 2012). This is in accordance with the second report dealing with the CBGAS (Page & Boubakir, 2012). They published a CANNABINOID PATHWAY sequence of CBGAS that was mainly expressed in glan- dular trichomes of female flowers and young leaves CBGA, the central precursor of cannabinoid biosyn- of Cannabis plants. The gene encodes a 395-amino acid thesis, is converted by three enzymes (Fig. 2.3): CBDAS, polypeptide chain showing a membrane-bound type of CBCAS, and THCAS. They predominantly use CBGA prenyltransferases. They were able to express the recom- as substrate, and catalyze the stereoselective, oxida- binant CBGAS in Sf9 insect as well as in Saccharomyces tive cyclization of the monoterpene moiety of CBGA to cerevisiae cells, and verified the CBGAS activity in the CBDA, CBCA, or THCA, respectively. The THCAS and microsomal fractions. Using MS measurements, CBGA CBDAS reactions are oxygen-dependent, producing hy- (3-geranyl olivetolate; comparison with CBGA stan- drogen peroxide proportional to either CBDA or THCA dard) was identified as the major product, and 5-geranyl (Sirikantaramas et  al.,  2004; Taura et  al., 2007b). Re- olivetolate (identification only by LC-MS analysis) as the markably, the CBCAS reaction is oxygen independent, minor product. Furthermore, Page and Boubakir (2012) and can be inhibited by hydrogen peroxide. Thus, the showed that CBGAS is specific only to GPP as a prenyl enzyme seems not to be an oxygenase or a peroxidase donor, and approves OA, olivetol, phlorisovalerophe- (Morimoto, Komatsu, Taura, & Shoyama,  1998). Fur- none, naringenin, and resveratrol as prenyl acceptor. Ad- thermore, all three enzymes also convert CBNRA, the ditionally, the enzyme reaction is dependent on divalent FIGURE 2.3  Biosynthesis of cannabinoids. The enzymatically catalyzed reactions are highlighted in dark green. All nonenzyme-dependent modifications reactions are colored in light green. Biosynthesis of C3-cannabinoids starting from cannabigerovarinic acid (CBGVA) is carried out by the same enzymes and for better clarity not shown (Crombie et al., 1968; de Meijer, 2011; Morimoto et al., 1998; Shoyama, Fujita, Yamauchi, & Nishioka, 1968; Shoyama, Oku, Yamauchi, & Nishioka, 1972; Taura et al., 1995a; 1996). I.  Setting the scene, botanical, general and international aspects

Cannabinoid pathway 19 cis-isomer of CBGA, with a lower specificity (Morimoto acid substitutions. These substitutions seem to be the rea- et  al.,  1998; Shoyama et  al.,  2012; Sirikantaramas, son for decreased THCAS activity in “fiber-type” strains Morimoto, & Shoyama, 2007; Taura et al., 1995b; Taura, (Kojoma, Seki, Yoshida, & Muranaka,  2006). Minise- Morimoto, & Shoyama, 1996). Since no enzymatic activ- quencing of samples from both types of Cannabis plants ity was detectable using the neutral cannabinoid CBG, showed three different single nucleotide polymorphism the carboxyl group in CBGA seems to be essential for the (SNP) genotypes. “Fiber-type” plants are homozygous enzymatic reactions catalyzed by THCAS, CBDAS, and for the inactive THCAS form. “Drug-type” plants are CBCAS (Morimoto et al., 1997; Taura et al., 1995a; Taura either homozygous or heterozygous for the active form et al., 1996). of THCAS. It seems that only a single copy of the gene encoding the active THCAS form is necessary for the Besides, it was postulated that THCA is biosyn- biosynthesis of THCA (Rotherham & Harbison, 2011). thesized and stored in the storage cavity of the glan- dular ­trichomes of Cannabis plants (Sirikantaramas, Cannabidiolic Acid Synthase ­Morimoto, & Shoyama, 2007). The gene of the wildtype CBDAS encodes a 544-amino Tetrahydrocannabinolic Acid Synthase acid polypeptide (Table  2.1). According to Taura et  al. (2007b), processed CBDAS consists of 517 amino ac- The THCAS gene encodes a 545-amino acid polypep- ids following cleavage of the 28 amino acid long signal tide chain (Table 2.1). According to Sirikantaramas et al. peptide. The mature CBDAS has a theoretical molecu- (2004), a 28 amino acid long signal peptide is cleaved lar mass of 59 kDa. An actual mass of about 74 kDa was in the processed THCAS, leading to a protein of 517 detected by SDS-PAGE that is possibly caused by post- amino acids. The mature THCAS has a theoretical mo- translational glycosylation of seven Asn residues (Taura lecular mass of 59 kDa. An actual mass of about 75 kDa et al., 1996; Taura et al., 2007b). CBDAS is a monomeric was detected using SDS-PAGE (Taura et  al., 1995b). enzyme with a pH optimum of 5.0 (Taura et  al.,  1996). This could be explained by posttranslational modifica- A comparison between THCAS and CBDAS revealed a tions, since eight possible Asn glycosylation sites were sequence similarity of 84% (Taura et al., 2007b) (Figs. 2.4 confirmed (Sirikantaramas et  al.,  2004). Furthermore, and 2.5). Like THCAS, CBDAS is a flavinated enzyme in deglycosylated THCAS indeed showed a molecular which His114 and Cys176 are most likely the FAD-binding mass of 59 kDa, and remained fully active (Taura et al., sites. Since CBDAS exhibits structural and biochemical 2007b). THCAS is a monomeric enzyme with the high- properties related to those of THCAS, it is probable that est activity between pH 5.5 and pH 6.0 (Taura et  al., the reaction mechanism of CBDAS is similar to that of 1995b). Sequence comparison identified similarities THCAS (Taura et al., 2007b). to the berberine bridge enzyme (BBE) of Eschscholzia californica (Shoyama et  al.,  2012). BBE belongs to the Cannabichromenic Acid Synthase (CBCAS) family of oxidoreductases and has a covalently bound FAD (Kutchan & Dittrich, 1995). The THCAS amino acid The sequence of CBCAS is still unknown. Morimoto sequence revealed a flavinylation consensus sequence et al. (1998) purified the enzyme to apparent homogene- (Arg110-Ser-Gly-Gly-His114) in which His114 is probably ity, but its sequence is not yet available in public databas- the FAD-binding site (Sirikantaramas et al., 2004). This es. According to van Bakel et al. (2011), possible CBCAS could be confirmed by X-ray crystallography (PDB ID: candidates are currently analyzed biochemically. 3VTE) at a resolution of 2.75Å. The results show that the enzyme is composed of two domains and one FAD However, CBCAS was isolated and partially purified binding pocket present in between. Besides His114, a from young leaves of C. sativa (Morimoto et  al.,  1997; second residue, Cys176, could be identified to be cova- 1998). In contrast to CBDAS and THCAS, CBCAS seems lently bound to the FAD (Shoyama et al., 2012). Based to be homodimer with a determined native molecular on X-ray structure data and mutational analysis of mass of 136  kDa and a maximum activity at pH 6.5. A THCAS, a possible catalytic reaction mechanism of molecular mass of 71 kDa was estimated for the mono- THCAS was proposed by Shoyama et al. (2012), assign- mers using SDS-PAGE. According to kinetic data, CBCAS ing a central role to Tyr484 in the catalytic mechanism has a higher affinity for CBGA than THCAS and CBCAS (Fig. 2.5). Nevertheless, since the crystal structure was (Morimoto et al., 1998). CBCA and its neutral form CBC published without substrate analoga, further studies are both racemic. Studies of Morimoto et al. (1997) sug- are necessary to verify the suggested mechanism. gested that both enantiomers of CBCA are formed by a CBCAS catalyzed reaction with a molar ratio of 5:1. Cannabis plants can be divided into two groups: But it is still unknown which of the two isomers is the “fiber-type” and “drug-type” plants (see dictionary). major product (Gaoni & Mechoulam,  1971; Morimoto Alignment of THCAS coding sequences from “fiber- et al., 1997; Taura et al., 2007a). type” and “drug-type” plants showed 37 major amino I.  Setting the scene, botanical, general and international aspects

20 2.  The Biosynthesis of Cannabinoids FIGURE 2.4  Alignment of amino acid sequences of THCA synthase and CBDA synthase. The alignment was performed with CLUSTAL W using the BLOSUM 62 matrix (Henikoff & Henikoff, 1992; Larkin et al., 2007). The signal peptide cleavage sites are indicated by a triangle. Second- ary elements (α-alpha-helices; β-beta-sheets; TT-turns; η-310 helix) are shown for the THCAS. Fully conserved residues are shaded in black. The sequences show an overall identity of 84%. The figure was made with ESPript 3.0 (Robert & Gouet, 2014). THCAS, tetrahydrocannabinolic acid synthase; CBDAS, cannabidiolic acid synthase. I.  Setting the scene, botanical, general and international aspects

Mini-dictionary 21 FIGURE 2.5  X-ray structure of the active center of THCAS. The backbone is shown as cartoon diagram (PDB ID: 3VTE). The FAD molecule (orange) is covalently attached to His114 and Cys176 (yellow). The active site residues are highlighted in green (Shoyama et al., 2012). The close-up of active center was prepared with PyMOL. THCAS, tetrahydrocannabinolic acid synthase. Cannabinoids with Propyl Side Chains (THCV) and/or cannabidivarin (CBDV) are usually only detectable in Cannabis indica (de Zeeuw, 1972). In contrast to the classic C5-phytocannabinoids, which contain an n-pentyl side chain, cannabinoids with MINI-DICTIONARY an n-propyl side chain are called C3-phytocannabinoids or propyl cannabinoids. The C-prenylation of divarinic Cannabinoids  Cannabinoids are a group of terpenophenolic acid (DA) instead of OA by GPP yields in cannabiger- compounds. They show affinities to cannabinoid receptors (CB1, ovarinic acid (CBGVA) (Fig. 2.2) (de Meijer, Hammond, CB2) or are structurally related to tetrahydrocannabinol (THC). & Micheler, 2009). The formation of propyl cannabinoids Cannabinoids can be differentiated into phytocannabinoids, does not occur by shortening the side chain of pentyl can- synthetic cannabinoids, and endocannabinoids. nabinoids (Kajima & Piraux, 1982). CBGVA is the central CBGA  Cannabigerolic acid (CBGA) is the central precursor of branch-point intermediate in the biosynthesis of C3-can- phytocannabinoid biosynthesis. It is nonpsychoactive. nabinoid acids, like CBGA for pentyl cannabinoid acids. “Drug-type” plants  These are THCA-rich plants. The THCA The enzymes CBDAS, CBCAS, and THCAS are not se- is converted into psychoactive ∆9-THC by a nonenzymatic lective for the length of the alkyl side chain, and can use decarboxylation that enables the plants to be labelled as THC-rich. both as a substrate. The resulting cannabinoids are called “Fiber-type” plants  “Fiber-type” plants are also known as cannabidivarinic acid (CBDVA), cannabichrovarinic acid “nondrug” plants. These plants have a low (<0.2%) or no THCA (CBCVA), and tetrahydrocannabivarinic acid (THCVA). content, but they contain a high amount of cannabidiolic acid The diverse amount of 2-carboxylic acids in different (CBDA). Cannabis strains is caused by dissimilar enzyme specifici- Phytocannabinoids  Phytocannabinoids are a unique group ties at the level of CBGA or CBGA-analogs formation (de of secondary metabolites (cannabinoids) occurring naturally Meijer et al., 2009; Shoyama, Hirano, & Nishioka, 1984). in plants. Other names include natural cannabinoids or herbal Relatively high amounts of tetrahydrocannabivarin cannabinoids (see Key facts). ∆8-THC  In Cannabis plants, ∆8-tetrahydrocannabinol (∆8-THC) is detectable in low amounts (<1% of the present ∆9-THC). Like I.  Setting the scene, botanical, general and international aspects

22 2.  The Biosynthesis of Cannabinoids ∆9-THC, it is also psychoactive. Maybe ∆8-THC is an artefact of the National Academy of Sciences of the United States of America, 109, extraction and/or analysis process. The term THC includes a 12811–12816. combination of ∆8-THC and ∆9-THC. Gaoni, Y., & Mechoulam, R. (1964). Isolation, structure, and partial ∆9-THC  ∆9-Tetrahydrocannabinol (∆9-THC) and ∆1-THC synthesis of an active constituent of hashish. Journal of the American describe the same compound, differing in the numbering system Chemical Society, 86, 1646–1647. used (dibenzopyran-numbering and monoterpene-numbering Gaoni, Y., & Mechoulam, R. (1971). The isolation and structure of system, respectively). ∆9-THC is responsible for the psychoactive ∆1-tetrahydrocannabinol and other neutral cannabinoids from effects of Cannabis products. It binds to the human cannabinoid hashish. 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