Farmacannabis-UFRJ: The first laboratory in Brazil to analyze therapeutic products derived from Cannabis

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Pharmacy, Federal University of Rio de Janeiro, RJ, Brazil Farmacannabis-UFRJ Project Coordinator virginiamc@pharma.ufrj.

1 Br. J. Anal. Chem., 2017, 4 (16), pp 44-4 Feature Farmacannabis-UFRJ: The first laboratory in Brazil to analyze therapeutic products derived from Cannabis Virgínia Martins Carvalho Associate Professor Faculty of Pharmacy, Federal University of Rio de Janeiro, RJ, Brazil Farmacannabis-UFRJ Project Coordinator virginiamc@pharma.ufrj.br Therapeutic treatment using Cannabis extracts is sometimes the only alternative in the control of intractable epilepsy and other serious illnesses. Cannabis sativa is a banned plant in Brazil, but the import of Cannabis extracts has been authorized for medical use since 2015, and the first safeguards to cultivate Cannabis for medicinal purposes were obtained in Although sanitary regulations created the demand for the chemical analysis of Cannabis products, all laboratories in Brazil that analyze Cannabis and Cannabis products have forensic purposes, and laboratories focused on the monitoring of therapies are a medical necessity. To assess the safety of Cannabis derivative therapies, the Laboratory of Analytical Toxicology at the Faculty of Pharmacy, Federal University of Rio de Janeiro (LATOX- FAR-UFRJ) was structured, and is the first laboratory in B r a z i l t o m o n i t o r m e d i c a l t r e a t m e n t u s i n g phytocannabinoids. Through the project Farmacannabis, the LATOX-FAR-UFRJ analyzes Cannabis and its products to monitor the safety of therapies, and to minimize the risk of improper preparation of its extracts by people without pharmaceutical training. Most of the patients are children with intractable epilepsy and autism, but it also serves patients with cancer, chronic pain, Researchers at the LATOX-FAR-UFRJ Alzheimer's, Parkinson's and other diseases. The medicinal properties of the plant from the Cannabis genus have been described in the Chinese Pharmacopoeia Pen-Ts'ao Ching for 2000 years, considered the first pharmacopoeia known in the world, and described by the Assyrians for 3000 years [1]. The book Dicionário de Plantas Úteis do Brasil e das Exóticas Cultivadas, a compendium of references in the pharmaceutical area in Brazil, published by M. Pio Côrrea in 126 [2], describes the botanical characteristic and medicinal properties of Cannabis sativa L. being named generically by "true hemp". The Farmacopeia Brasileira published in 12 [3] describes the Cannabis sativa Linnaeus var. indica, with the generic names "maconha", "meconha", "diamba" and "cannabis", indicating the flowered summits as a raw material in preparing the Cannabis officinalis extract or fluid extract of India hemp, hemp powder and the Indian hemp dye. In forensic analysis guidelines, Cannabis is considered to be monospecific (Cannabis sativa L.) however, Cannabis is divided into several subspecies (C. sativa subsp. sativa, C. sativa subsp. indica, C. sativa subsp. ruderalis, C. sativa subsp. spontanea, C. sativa subsp. kafiristanca). The chemical and morphological 44

2 distinctions of the subspecies are often not readily discernible and are not required for forensic purposes (UNODC, 200). The main active compounds present in the plant are the cannabinoid substances, of which the more abundant being Δ and Δ8-tetrahydrocannabinol (THC), cannabinol (CBN) and the cannabidiol (CBD) that allow the identification of the plant chemistry. The THC and CBD can still be identified in its acidic forms (CBDA and THCA) which are converted to the neutral forms by heating. After the elucidation of the CBD structure [4] and identification of Δ -THC, one of the substances responsible for their psychoactive and euphoric effects [5], several scientific papers have been developed showing the pharmacological and clinical properties of its compounds [6-]. The pharmacological effects are attributed to the interaction of cannabinoids with cannabinoid receptors mainly distributed in the central nervous system (CNS) known as CB1, and peripheral nervous system and immune system known as CB2 [10]. CBD offers paradoxical pharmacological effects in relation to the effects of Δ -THC, while the former acts as a depressant and anxiolytic, the second acts as a stimulant and hallucinogen [8]. The Δ -THC binds to the CB1 and CB2 receptors acting as a partial agonist, and seems to have a mixed neural activity, excitatory and inhibitory, in different areas of the brain, showing they do not only act in specific cannabinoid receptors [11]. Unlike Δ - THC, CBD has low affinity for CB1 and CB2 receptors, its action seems to result from an increase in anandamide, the endogenous cannabinoid neurotransmitter [12], shows anxyolytic [13] and anticonvulsant effects [14,15]. The pharmacological potential of cannabinoids in the treatment of Parkinson's disease [16], schizophrenia [17,18], Alzheimer's [1-21], neuropathic pain present in multiple sclerosis [], rheumatoid arthritis [22], and epilepsy [15,23,24] has been demonstrated. Probably, the first scientific work which has shown the effectiveness of the CBD in the control of seizures, was published in 180 by the group of Elizaldo Carlini, professor at the Federal University of São Paulo, together with Raphael Mechoulam, professor at the Hebrew University [14]. Currently extracts rich in CBD are being used at the clinic in the management of refractory epilepsy. After a long period in which the production of Cannabis based medicines was banned in Brazil, international experience stimulated the search for treatment of epilepsy with extracts from hemp, a flagship experience was the case of British girl, Charlotte Figi, who suffered from multiple episodes of convulsions since the first months of life, and diagnosed with Dravet syndrome when she was 2.5 years old. Her family, discouraged with the ineffectiveness of traditional pharmaceutical drugs, started administrating a Cannabis extract in Colorado, United States of America (USA). The Cannabis treatment controlled the epilepsy in the first week. In the USA, hemp extract has been approved by the Food and Drug Administration (FDA) as a food supplement, and the medical use of Cannabis in California was approved in 16. Charlotte's Cannabis treatment encouraged mothers, parents, patients and activists to put pressure on the regulation of medical Cannabis use in Brazil. The Conselho Federal de Medicina authorized the compassionate prescription of CBD in December 2014, and the Agência Nacional de Vigilância Sanitária, Brazilian regulatory agency (ANVISA) authorized its importation for personal medical use in In March 2016, by determination of Federal prosecutors to answer the plea of a patient, ANVISA has authorized the importation for personal medical use of the plant Cannabis sativa L, parts of the plant and THC, and partially corrected an inconsistency contained in regulating controlled substances (Portaria 344/8) from the year 2000, update dronabinol (Marinol ) on the list of stimulant drugs. Dronabinol is the synthetic THC structure identical to phytocannabinoid THC indicated primarily to the control of nausea and vomiting in patients undergoing through chemotherapy treatment. In addition, the first Cannabis extract named Mevatyl (Sativex in other countries) was registered as a pharmaceutical drug by ANVISA in 2017, while kept the plant and its active ingredients on the proscribed list (of prohibited drugs). Imported extracts are classified as hemp, that is plants of the genus Cannabis rich in CBD and poor in THC usually used as fiber. International law determined that the concentration of THC is less than 0.3% for the registration of products as dietary supplements and cosmetics. On the other hand, in Brazil emerges the use of handmade products and trade in clandestine products that are prepared with different varieties of Cannabis have not yet been characterized scientifically. Many people carry out experiments to produce Cannabis products for recreational and medical use, and have empirical knowledge in their own language of that which has been identified as "Cannabis culture", 45

they claim to differ in the macroscopic feature, organoleptic characteristics, develop agronomic methods and taxonomic classifications based on feeling and personal experience during use, and

3 they claim to differ in the macroscopic feature, organoleptic characteristics, develop agronomic methods and taxonomic classifications based on feeling and personal experience during use, and describe compositions and levels of active ingredients. Although this empirical knowledge is being an instrument for the remedies of those who need these medicinal extracts, research and development is fully needed, along with scientific methodology, from the description of the varieties of Cannabis available in the national market, to the standard raw vegetal material. Cannabis features at least 50 phytocannabinoids and that, in addition to this variety of substances, the effect can be determined by the acidic or neutral form of the same cannabinoids such as THCA which is converted to THC by heating. The preparation technique of the extract (type of solvent extraction and temperature) is decisive in the final concentration of THC and THCA [25] since its concentrations can be modulated by heating. If forensic toxicology gas chromatography is sufficient to analyzing cannabinoid compounds, for medical purposes the decarboxylation of acid forms is a technical limitation easily solved by the use of liquid chromatography. To monitor Cannabis therapies and to give pharmaceutical support to patients, the Farmacannabis project was created at LATOX-FAR-UFRJ, where Cannabis products are analyzed and the production of homemade extracts is assisted, and medicinal extracts and plants used in its preparation are analyzed to determine the cannabinoids, toxic metal levels and microbiological quality. For structuring the project, many barriers were overcome with the persuasion of professional peers, that with professional ethics and a commitment to public health, agreed that it is necessary to brave prejudices in order to study and evaluate the safety of Cannabis treatments. Currently, Farmacannabis has ten PhD. Professors from pharmacy, neurosciences, botanic, natural products and juridical areas, several students and technical professionals, partnerships with public institutions like Instituto de Tecnologia em Fármacos, Farmanguinhos, Fiocruz (Far-Fiocruz), and Instituto Nacional de Controle de Qualidade em Saúde, Fiocruz (INCQS-Fiocruz), and partnerships with non-governmental Organizations (NGOs), such as A s s o c i a ç ã o B r a s i l e i r a p a r a C a n n a b i s Some researchers of the Farmacannabis project (ABRACANNABIS) and Associação de Apoio à Pesquisa e Pacientes de Cannabis Medicinal (APEPI). Financial support was obtained by collective funding (crowdfunding) and more than 800 people backed the project, with the collection of about US $ 30,000. Furthermore, Farmacannabis received financial support from the Manserv Company, a resident enterprise from Technological Park in UFRJ, and the technical support of the Brazilian company Nova Analítica. With strategically worked idea, the first laboratory in Brazil that officially analyzes medical Cannabis extracts was structured. Farmacannabis' preliminary results, obtained by the high-performance liquid chromatographic method development, showed that the commercial imported extracts have high CBD content with a chemical profile very different from homemade and illicit trade products (Tables I and II). In general, the homemade products showed low cannabinoid concentrations and are rich in THC, except for the Harle-Tsu variety with hemp profile introduced between patients by ABRACANNABIS. Hemp varieties are probably rare in Brazil because of the Cannabis cultivations which have been turned over to recreational use, and the agronomic techniques have been employed for the production of varieties rich in THC. The illegal Cannabis samples seized in the United States of America from 15 to 2014 increased THC content of 5% to 15% on average and the THC/CBD ratio increased from 14 times in 15 to about 80 times in 2014 [26]. The development of phytocannabinoid medicine in Brazil depends on the studies of available varieties of Cannabis that can become active pharmaceutical ingredients, and in this way, analytical chemistry laboratories and toxicological analyzes need to be prepared for the Cannabis pharmaceutical market that requires technical specificities different from the forensic application. 46

4 Table I. Preliminary results from commercial imported products analyzed by HPLC-DAD Origin Pure CBD Form/vehicle Labeled content Concentration [mg/ml or mg/g] CBDA CBD THCA CBN THC USA Oil 200 mg/ml ND ND ND ND USA Oil 50 mg/ml ND ND 0.45 ND USA Oil 50 mg/ml ND ND ND ND USA Oil 50 mg/ml ND ND USA Oil 100 mg/ml ND ND ND ND USA Oil 200 mg/ml ND ND ND ND USA Oil 100 mg/ml ND ND ND ND USA Oil 100 mg/ml ND 0.70 ND ND ND CBD Enriched Extracts CBDA CBD THCA CBN THC USA Oil 50 mg/ml ND ND USA Oil 60 mg/ml ND 3.34 ND ND 1.44 USA Oil 61 mg/ml ND ND ND 1.74 Canada Oil 24.7 mg/ml USA Resin 170 mg/g ND USA Resin 160 mg/g THC < mg/g ND USA Resin 240 mg/g ND ND 1.60 USA Capsule ND ND ND ND 4.12 USA Capsule 50 mg/cap ND 5.88 ND ND 4.33 ND: Not detected (Limit of Detection = 0.01 mg/ml or 0.01 mg/g for resin); USA: United States of America. 47

5 Table II. Preliminary results from homemade or clandestine products analyzed by HPLC-DAD THC enriched extracts or THC-CBD (1:1) Concentration [mg/ml or mg/g] Origin Form/vehicle Cannabis strain CBDA CBD THCA CBN THC 48 RJ Oil Cronic ND ND 1.64 RJ Oil Pur Kush ND ND 1.3 RJ Oil Cinderela ND ND 0.80 ND 2.1 RJ Oil Cinderela ND ND 1.16 ND 2.64 RJ Oil Wildfire ND 0.7 ND ND 6.3 RJ Oil Harletsu oil + Cinderela resin RJ Oil Hybrid:CBD skunk haze + LSD 3.81 ND 2.3 ND 0.10 SC Oil UN SC Oil UN ND SC Oil UN RJ Resin Cinderela SC Resin UN ND SC Resin UN SC Resin UN ND CBD enriched extracts RJ Oil Harletsu ND ND RJ Oil Harletsu ND 0.10 RJ Oil Harletsu ND 0.25 RJ Oil Harletsu ND RJ Oil Harletsu * ND ND 0.33 RJ Oil Harletsu * ND Extracts with cannabinoids at trace level PB Glicerin UN ND ND 0.20 ND 0.14 PB Glicerin UN ND ND 0.20 ND 0.14 PB Glicerin UN ND 0.10 PB Glicerin UN ND 0.12 PB Glicerin UN ND 0.10 PB Glicerin UN ND 0.10 PB Glicerin UN ND ND 0.1 ND 0.10 PB Glicerin UN ND PB Glicerin UN ND 0.02 PB Water/spray UN 0.05 ND 0.20 ND 0.06 ND: Not detected (Limit of Detection = 0.01 mg/ml or 0.01 mg/g for resin); PB: Paraíba, BR; RJ: Rio de Janeiro, BR; SC: Santa Catarina, BR; UN: unknown; *Prepared by alcoholic extraction with patients in LATOX with pharmaceutical supervision.

6 ACKNOWLEDGMENTS The Farmacannabis' coordinator is grateful to Dr. Ernesto Diaz Rocha, MS. Fábio Luiz Costa de Souza and Andrey Fabiano Lourenço de Aguiar for the laboratorial and analytical help. REFERENCES 1. Honório, K. M.; Arroio, A.; Da Silva, A. B. F. Quim. Nova, 2006, 2 (2), pp Corrêa, M. P. Dicionário das Plantas Úteis do Brasil e das Exóticas Cultivadas. Imprensa Nacional, Rio de Janeiro, 126, pp Silva R. A. D. Pharmacopeia dos Estados Unidos do Brasil. Companhia Editora Nacional, Rio de Janeiro, 12, pp Mechoulam, R.; Shvo, Y. Tetrahedron, 163, 1 (12), pp Gaoni, Y.; Mechoulam, R. J. Am. Chem. Soc., 164, 86 (8), pp Mechoulam, R.; Shani, A.; Edery, H.; Grunfeld, Y. Science, 170, 16 (45), pp Ilan, A. B.; Gevins, A.; Coleman, M.; ElSohly, M. A.; De W it, H. Behav. Pharmacol., 2005,16, pp Mechoulam, R.; Peters, M.; Murillo-Rodriguez, E.; Hanus, L. O. Chem. Biodivers., 2007, 4, pp Whiting, P. F.; W olff, R. F.; Deshpande S.; Di Nisio, M.; Duffy, S.; Hernandez, A. V.; Keurenties, J. C.; Lang, S.; MIsso, K.; Ryders, S.; Schmidlkofer, S.; W eswood, M.; Kleijnen, J. J. Am. Med. Assoc., 2015, 313 (24), pp Leweke, F. M.; Koethe, D. Addict. Biol., 2008, 13, pp Pertwee, R. G. Br. J. Pharmacol., 2008, 153, pp Mechoulam, R.; Panikashvili, D.; Shohami, E. Trends Mol. Med., 2002, 8, pp Fogaça, M. V.; Reis, F. M.; Campos, A. C.; Guimarães, F. S. Eur. Neuropsychopharmacol., 2014, 24 (3), pp Cunha J. M.; Carlini, E. A.; Pereira, A. E.; Ramos, O. L.; Pimentel, C.; Gagliardi R.; Sanvito W. L.; Lander, N.; Mechoulam, R. Pharmacology, 180, 21, pp Tzadok, M.; Ulieal-Siboni, S.; Linder, I.; Kramer, U.; Epstein, O.; Menascu, S.; Nissenkorn, A.; Yosef, O. B.; Hyman, E.; Granot, D.; Dro, M.; Lerman-Sague, T.; Ben-Zeev, B. Seizure, 2016, 35, pp Zuardi, A. W.; Crippa, J. A.; Hallak, J. E.; Pinto, J. O.; Chagas, M. H.; Rodrigues, G. G.; Dursun, S. M.; Tumas, V. J. Psychopharmacol., 200, 23 (8), pp Zuardi, A. W.; Hallak, J. E.; Dursun, S. M.; Morais, S. L.; Sanches, R. F.; Musty, R. E.; Crippa, J. A. J Psychopharmacol., 2006, 20 (5), pp Leweke, F. M.; Piomelli, D.; Pahlisch, F.; Muhl, D.; Gerth, C. W., Hoyer, C.; Klosterkötter, J.; Hellmich, M; Koethe, D. Transl. Psychiatry, 2012, pp Iuvone, T.; Esposito, G.; Esposito, R.; Santamaria, R.; Di Rosa, K.; Izzo, A. A. J Neurochem., 2004, 8 (1), pp Esposito, G.; De Filippis, D.; Maiuri, M. C.; De Stefano, D.; Carnuccio, R.; Iuvone, T. Neurosci. Lett., 2006, 3 (1-2), pp Esposito, G.; Scuderi, C.; Savani, C.; Steardo, L. Jr.; De Filippis, D.; Cottone, P.; Iuvone, T.; Cuomo, V.; Steardo, L. Br. J. Pharmacol., 2007, 151 (8), pp Blake, D. R.; Robson, P.; Ho, M.; Jubb, M. W.; McCabe, C. S. Rheumatology, 2006, 45, pp Hill, A. J.; W illiams, C. M.; W halley, B. J.; Stephens, G. J. Pharmacol. Ther., 2012, 133, pp Hussain, S. A.; Zhou, R.; Jacobson, C.; W eng, J.; Cheng, E.; Lav, J.; Hung, P.; Lerner, J. T.; Sankar, R. Epilepsy Behav., 2015, 47, pp Romano, L. L.; Hazekamp, A. Cannabinoids, 2013, 1 (1), pp ElSohly, M. A.; Mehmedic, A.; Foster, S.; Gon, C.; Chandra, S.; Church, J. Biol. Psychiatry, 2016, 7, pp

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