DOCSLIB.ORG

Supporting Information

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Supporting Information Supporting Information Khaliullina et al. 10.1073/pnas.1416463112 SI Materials and Methods 10 min. The supernatant containing hemolymph was centri- Mammalian Cell Culture. HeLa cells were passaged in DMEM with fuged at 16,000 × g for 2 h to remove the debris and subjected to 10% FBS (Gibco), 50 U/mL penicillin, and 50 μg/mL strepto- lipid extraction. mycin (Gibco). ShhLIGHT2 cells, which are NIH 3T3 cells that express firefly Lipid Extraction. Lipids from purified VLDL, LDL, HDL, and Lpp luciferase under the control of a Gli-responsive promoter, and particles applied as crude extracts in signaling assays were Renilla luciferase controlled by the viral Thymidine kinase promoter extracted by a two-step Bligh and Dyer method (10) and applied (1) were maintained in DMEM + 10% (vol/vol) FBS, 150 μg/mL to ShhLIGHT2 cells and wing discs at concentrations found in zeocin (Invitrogen), and 400 μg/mL G418 (Invitrogen). hemolymph/human serum. Total lipid concentration was mea- HEK293S-TetR cells, which stably express an inducible SMO sured according to ref. 11. construct, were described (2). Cells were maintained in medium containing DMEM/F-12, 10% (vol/vol) FCS, 2 mg/mL Ge- Preparation of Saponification-Resistant Lipids. Dried VLDL lipid neticin, and 5 μg/mL Blasticidin, as described. extracts were incubated at 80 °C with 2 mL of 0.3 M methanolic NIH 3T3/Smo-mEos2 cells, which are NIH 3T3 cells that stably potassium hydroxide for 1 h. After cooling down to room tem- express Smo-mEos2 construct, were described (3). Cells were perature, the solution was extracted by two washes with diethyl maintained in DMEM + 10% (vol/vol) FCS. ether. The ether fractions, which contain saponification-resistant lipids, were combined and subjected to further analysis. Fly Stocks. Oregon R flies, hs-flippase, and adh-GAL4 obtained from Bloomington Stock Center, transgenic line UAS < HcRed > Preparation of Shh Applied in the ShhLIGHT2 Cell Assay. To prepare dsLpp is described in ref. 4. nonlipoprotein-associated Shh, we transfected HeLa cells with cDNA encoding human Shh (OriGene; SC300021) by using Lipid Extraction from Drosophila Larval Hemolymph. Third instar polyethylenimine (Polysciences) in OptiMEM (Invitrogen), then larvae were washed in 10% (vol/vol) NaCl and disrupted by using switched Shh-transfected HeLa cells to serum-free media + a loose dounce homogenizer, which releases the hemolymph but (DMEM 1% Insulin-Transferrin-Selenium mixture (ITS-X, is not designed to disrupt the cells, in 1% acetic acid in H O with Gibco) 4 h after transfection. After 48 h, conditioned media were 2 × 50 μM PF-3845. The extract was centrifuged at 1,000 × g for centrifuged at 1,000 g for 20 min and concentrated by using 10 min. The supernatant containing hemolymph was centrifuged Amicon Ultra -10K (Millipore) for use in the signaling assay. at 16,000 × g for 2 h to remove the debris and subjected to lipid Identically treated media from nontransfected HeLa cells extraction. were used as controls. To prepare Lipoprotein-Shh, transfected HeLa cells were cultured in DMEM + 10% (vol/vol) FBS. Con- Reagents. Lipids used in this study were as follows: cholesterol, ditioned media were centrifuged at 1,000 × g for 20 min. For desmosterol, 7-dehydrocholesterol, vitamin D3 (Avanti Polar signaling ShhLIGHT2 assay, lipoprotein-Shh was isolated by Lipids), ergosterol, ergocalciferol D2, 1,25-dihydroxyvitamin D3 density centrifugation (12) and concentrated by using Amicon (Sigma-Aldrich), N-acylethanolamides 16:0, 16:1, 18:0, 18:1, Ultra -10K. 18:2, 20:4, N-acyldopamine 16:0, N-acyldopamine 20:4, 2-alkyl- ShhLIGHT2 Signaling and SAG Competition Assays. Twenty-four glycerol 18:2, 2-acylglycerol 20:4, 2-alkylglycerol 20:4 and 5 N-acylserine 20:4 (Cayman Chemical), N-acyl dopamine 18:0 hours before assay, ShhLIGHT2 cells were plated at 10 per well (Tocris Bioscience), N-acyl serine 16:0 (Avanti Polar Lipids), in 96-well plates and then switched to a serum-free medium + delta-9-tetrahydrocannabinol (Biomol), Cannabinol and Cannabidiol consisting of DMEM 1% ITS-X. In this medium, cells were (Sigma-Aldrich), ACEA, AM251, AM404, AM630, CB-13, O-1918, then treated with nontransfected serum-free HeLa cell super- CP-775146, GW6471, PF-3845, S 26948 and T 0070907 (Tocris natants and DMSO or DOPC liposomes (mock) or with non- Bioscience), SMO Agonist (SAG) (Santa Cruz Biotechnology), and lipoprotein-associated Shh supplemented with DMSO or DOPC BODIPY-cyclopamine (BioVision). Deuterated endocannabinoids liposomes (Shh) or with different lipids or synthetic compounds. used for LC-MS/MS quantification of endogenous species were Purified, commercially obtained lipids, or synthetic compounds purchased from Biomol and R&D Systems. were added in DMSO at indicated concentration. HPLC elution fractions were added in DOPC liposomes in Induction of RNAi. LppRNAi was induced as described (4). a ratio of 1:4. SAG was added to the ShhLIGHT2 cells at indicated concen- Immunohistochemistry and Image Analysis. Imaginal discs were trations alone or in the presence of different endocannabinoids. fixed and stained as described (5). Image analysis and statistics of Luciferase activity was assayed in cell lysates after 24 h, as image quantifications were performed as described (6). Anti- instructed by the manufacturer (Dual Glo Luciferase Assay; bodies were diluted as follows: anti-Ci 2A1 1:10 (7) and anti- Promega). The resulting Hh pathway activity was measured as the SMO 1:50 (8). firefly:Renilla ratio normalized to the ratio in cells that were not stimulated with Shh. Lipoprotein Isolation. VLDL, LDL, and HDL were isolated from human serum (Sigma) according to ref. 9. Drosophila Lipophorin BODIPY-Cyclopamine Binding Assay. HEK293S-TetR cells were (Lpp) was isolated as described (6). grown to confluence in medium containing Geneticin (2 mg/mL). The growth medium was then replaced with fresh medium Lipid Extraction from Drosophila Larval Hemolymph. Third instar containing tetracycline (2 μg/mL) and sodium butyrate (5 mM). larvae were washed in 10% (vol/vol) NaCl and disrupted by using After 2 d, fluorescence binding assays using BODIPY-cyclop- a loose dounce homogenizer, which releases the hemolymph but amine were conducted as described (13). BODIPY fluores- is not designed to disrupt the cells, in 1% acetic acid in H2O with cence was monitored by FACS and analyzed by using the 50 μM PF-3845. The extract was centrifuged at 1,000 × g for FlowJo software. Khaliullina et al. www.pnas.org/cgi/content/short/1416463112 1of9 Ciliary SMO Localization Assay. NIH 3T3/Smo-mEos2 cells were normal goat serum), incubated with the anti-acetylated Tubulin grown on glass coverslips to reach confluence. The cells were then antibody (Sigma, 1:2,000) in blocking solution for 1 h, washed with shifted to DMEM + 1% ITS-X and incubated for 24 h with or PBS, incubated with the secondary antibody in blocking solution, without SAG and different compounds. Cells were fixed for washed, and mounted in Vectashield. Slides were viewed under 10 min with 4% (vol/vol) formaldehyde in PBS, incubated for the confocal microscope Zeiss LSM Meta and analyzed by using 20 min in blocking solution (PBS + 0.1% Triton X-100 + 1% the Fiji software. 1. Taipale J, Cooper MK, Maiti T, Beachy PA (2002) Patched acts catalytically to suppress 8. Lum L, et al. (2003) Hedgehog signal transduction via Smoothened association with the activity of Smoothened. Nature 418(6900):892–897. a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2. Mol Cell 12(5): 2. Dwyer JR, et al. (2007) Oxysterols are novel activators of the hedgehog signaling 1261–1274. pathway in pluripotent mesenchymal cells. J Biol Chem 282(12):8959–8968. 9. Redgrave TG, Roberts DC, West CE (1975) Separation of plasma lipoproteins by 3. Kim J, et al. (2014) Simultaneous measurement of smoothened entry into and exit density-gradient ultracentrifugation. Anal Biochem 65(1-2):42–49. from the primary cilium. PLoS ONE 9(8):e104070. 10. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. 4. Panáková D, Sprong H, Marois E, Thiele C, Eaton S (2005) Lipoprotein particles are Can J Biochem Physiol 37(8):911–917. required for Hedgehog and Wingless signalling. Nature 435(7038):58–65. 11. Marsh JB, Weinstein DB (1966) Simple charring method for determination of lipids. J 5. Marois E, Mahmoud A, Eaton S (2006) The endocytic pathway and formation of the Lipid Res 7(4):574–576. Wingless morphogen gradient. Development 133(2):307–317. 12. Rudel LL, Lee JA, Morris MD, Felts JM (1974) Characterization of plasma lipoproteins 6. Khaliullina H, et al. (2009) Patched regulates Smoothened trafficking using lipopro- separated and purified by agarose-column chromatography. Biochem J 139(1):89–95. tein-derived lipids. Development 136(24):4111–4121. 13. Chen JK, Taipale J, Cooper MK, Beachy PA (2002) Inhibition of Hedgehog signaling by 7. Wang QT, Holmgren RA (1999) The subcellular localization and activity of Drosophila direct binding of cyclopamine to Smoothened. Genes Dev 16(21):2743–2748. cubitus interruptus are regulated at multiple levels. Development 126(22):5097–5106. Khaliullina et al. www.pnas.org/cgi/content/short/1416463112 2of9 Fig. S1. Lipids derived from mammalian lipoproteins reduce SMO levels in Drosophila wing imaginal discs and inhibit Shh signaling in ShhLIGHT2 cells. (A) Ratio of firefly:Renilla luciferase activity in ShhLIGHT2 cells treated as indicated. Treatment with DMSO served as a control for background activity (-). Error bars indicate SDs of three independent experiments. n > 3. (B′–B****) Wing discs from wild-type (B′) and LppRNAi (B*–B****) animals mock-treated (B′ and B*) or treated with HDL- (B**), LDL- (B***), or VLDL-derived (B****) lipids. (C) Quantification of SMO staining in the wing disk assay performed in (B′–B****) with lipids derived from HDL (magenta; P = 1.4), LDL (blue; P = 0.0048 for far anterior) or VLDL (green; P = 1.0 × 10−11).
Load more

Recommended publications
  • Marijuana: Drug of Abuse Or Therapeutic Option? Learning Objectives
    Handout for the Neuroscience Education Institute (NEI) online activity: Marijuana: Drug of Abuse or Therapeutic Option? Learning Objectives • Explain how cannabinoids affect the body and the brain • Educate patients about: – Evidence of efficacy for mental health and other conditions – Potential risks of cannabis use Timeline 1989? 90?: Discovery of ~2000 BC: binding site 2015: Elimination of Chinese 1943: Marijuana for THC— 1992: US Public Health emperors removed from CB1 Endogenous Service oversight cannabinoid recommend listing as a 1963: 1964: THC receptor for obtaining anandamide marijuana as medication in US Cannabidiol isolated marijuana for discovered medicine Pharmacopeia isolated research purposes 1995: 1851: Marijuana 1961: United 1970: Marijuana is Endogenous Aug 11 2016: listed as a Nations Single labeled Schedule I cannabinoid 2-AG DEA declines to medication in US Convention on by the US discovered reschedule Pharmacopeia Narcotic Drugs: Substance Abuse marijuana marijuana said to Act; this restricts be dangerous with both personal use no medical value and access for research purposes THE INTERSECTION OF THE HEALTHCARE AND CANNABIS INDUSTRIES What is Cannabis? 500 chemicals 100 cannabinoids Best understood: THC and CBD Scheduling of Controlled Substances No medicinal value, high Moderate to Lower High potential Low potential potential for low potential potential for for abuse for abuse abuse for abuse abuse Schedule I Schedule II Schedule III Schedule IV Schedule V Marijuana Cocaine Tylenol w/ Tramadol Robitussin AC Heroin
    [Show full text]
  • N-Acyl-Dopamines: Novel Synthetic CB1 Cannabinoid-Receptor Ligands
    Biochem. J. (2000) 351, 817–824 (Printed in Great Britain) 817 N-acyl-dopamines: novel synthetic CB1 cannabinoid-receptor ligands and inhibitors of anandamide inactivation with cannabimimetic activity in vitro and in vivo Tiziana BISOGNO*, Dominique MELCK*, Mikhail Yu. BOBROV†, Natalia M. GRETSKAYA†, Vladimir V. BEZUGLOV†, Luciano DE PETROCELLIS‡ and Vincenzo DI MARZO*1 *Istituto per la Chimica di Molecole di Interesse Biologico, C.N.R., Via Toiano 6, 80072 Arco Felice, Napoli, Italy, †Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, R. A. S., 16/10 Miklukho-Maklaya Str., 117871 Moscow GSP7, Russia, and ‡Istituto di Cibernetica, C.N.R., Via Toiano 6, 80072 Arco Felice, Napoli, Italy We reported previously that synthetic amides of polyunsaturated selectivity for the anandamide transporter over FAAH. AA-DA fatty acids with bioactive amines can result in substances that (0.1–10 µM) did not displace D1 and D2 dopamine-receptor interact with proteins of the endogenous cannabinoid system high-affinity ligands from rat brain membranes, thus suggesting (ECS). Here we synthesized a series of N-acyl-dopamines that this compound has little affinity for these receptors. AA-DA (NADAs) and studied their effects on the anandamide membrane was more potent and efficacious than anandamide as a CB" transporter, the anandamide amidohydrolase (fatty acid amide agonist, as assessed by measuring the stimulatory effect on intra- hydrolase, FAAH) and the two cannabinoid receptor subtypes, cellular Ca#+ mobilization in undifferentiated N18TG2 neuro- CB" and CB#. NADAs competitively inhibited FAAH from blastoma cells. This effect of AA-DA was counteracted by the l µ N18TG2 cells (IC&! 19–100 M), as well as the binding of the CB" antagonist SR141716A.
    [Show full text]
  • Cannabinoid Receptor and Inflammation
    Cannabinoid Receptor and Inflammation Newman Osafo1, Oduro Yeboah1, Aaron Antwi1, and George Ainooson1 1Kwame Nkrumah University of Science and Technology September 11, 2020 Abstract The eventual discovery of endogenous cannabinoid receptors CB1 and CB2 and their endogenous ligands has generated interest with regards to finally understanding the endocannabinoid system. Its role in the normal physiology of the body and its implication in pathological states such as cardiovascular diseases, neoplasm, depression and pain have been subjects of scientific interest. In this review the authors focus on the endogenous cannabinoid pathway, the critical role of cannabinoid receptors in signaling and mediation of neurodegeneration and other inflammatory responses as well as its potential as a drug target in the amelioration of some inflammatory conditions. Though the exact role of the endocannabinoid system is not fully understood, the evidence found leans heavily towards a great potential in exploiting both its central and peripheral pathways in disease management. Cannabinoid therapy has already shown great promise in several preclinical and clinical trials. 1.0 Introduction Ethnopharmacological studies have shown the use of Cannabis sativa in traditional medicine for over a thousand years, with its widespread use promoted by its psychotropic effects (McCoy, 2016; Turcotte et al., 2016). The discovery of a receptor within human body, that is selectively activated by cannabinoids suggested the presence of at least one endogenous ligand for this receptor. This is confirmed by the discovery of two endogenously synthesized lipid mediators, 2-arachidonoyl-glycerol and arachidonoylethanolamide, which function as high-affinity ligands for a subfamily of cannabinoid receptors ubiquitously distributed in the central nervous system, known as the CB1 receptors (Turcotte et al., 2016).
    [Show full text]
  • Efficacy of Cannabinoids in a Pre-Clinical Drug-Screening Platform for Alzheimer’S Disease
    Molecular Neurobiology https://doi.org/10.1007/s12035-019-1637-8 Efficacy of Cannabinoids in a Pre-Clinical Drug-Screening Platform for Alzheimer’s Disease David Schubert1 & Devin Kepchia1 & Zhibin Liang1 & Richard Dargusch1 & Joshua Goldberg & Pamela Maher1 Received: 30 January 2019 /Accepted: 6 May 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Finding a therapy for Alzheimer’s disease (AD) is perhaps the greatest challenge for modern medicine. The chemical scaffolds of many drugs in the clinic today are based upon natural products from plants, yet Cannabis has not been extensively examined as a source of potential AD drug candidates. Here, we determine if a number of non-psychoactive cannabinoids are neuroprotective in a novel pre-clinical AD and neurodegeneration drug-screening platform that is based upon toxicities associated with the aging brain. This drug discovery paradigm has yielded several compounds in or approaching clinical trials for AD. Eleven cannabinoids were assayed for neuroprotection in assays that recapitulate proteotoxicity, loss of trophic support, oxidative stress, energy loss, and inflammation. These compounds were also assayed for their ability to remove intraneuronal amyloid and subjected to a structure-activity relationship analysis. Pairwise combinations were assayed for their ability to synergize to produce neuropro- tective effects that were greater than additive. Nine of the 11 cannabinoids have the ability to protect cells in four distinct phenotypic neurodegeneration screening assays, including those using neurons that lack CB1 and CB2 receptors. They are able to remove intraneuronal Aβ, reduce oxidative damage, and protect from the loss of energy or trophic support.
    [Show full text]
  • Cannabinoid Interventions for PTSD: Where to Next?
    Accepted Manuscript Cannabinoid interventions for PTSD: Where to next? Luke Ney, Allison Matthews, Raimondo Bruno, Kim Felmingham PII: S0278-5846(19)30034-X DOI: https://doi.org/10.1016/j.pnpbp.2019.03.017 Reference: PNP 9622 To appear in: Progress in Neuropsychopharmacology & Biological Psychiatry Received date: 14 January 2019 Revised date: 20 March 2019 Accepted date: 29 March 2019 Please cite this article as: L. Ney, A. Matthews, R. Bruno, et al., Cannabinoid interventions for PTSD: Where to next?, Progress in Neuropsychopharmacology & Biological Psychiatry, https://doi.org/10.1016/j.pnpbp.2019.03.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 Cannabinoid interventions for PTSD: Where to next? Luke Ney1, Allison Matthews1, Raimondo Bruno1 and Kim Felmingham2 1School of Psychology, University of Tasmania, Australia 2School of Psychological Sciences, University of Melbourne, Australia ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2 Abstract Cannabinoids are a promising method for pharmacological treatment of post- traumatic stress disorder (PTSD). Despite considerable research devoted to the effect of cannabinoid modulation on PTSD symptomology, there is not a currently agreed way by which the cannabinoid system should be targeted in humans. In this review, we present an overview of recent research identifying neurological pathways by which different cannabinoid-based treatments may exert their effects on PTSD symptomology.
    [Show full text]
  • Effects of Cannabinoids on the Development of Chick Embryos In
    www.nature.com/scientificreports OPEN Efects of cannabinoids on the development of chick embryos in ovo Received: 16 April 2019 Sofa B. Gustafsson & Stig O. P. Jacobsson Accepted: 3 September 2019 We have examined the efects of the synthetic cannabinoids HU 210 and HU 211, the plant-derived Published online: 17 September 2019 cannabidiol and the endogenous cannabinoid anandamide on the viability and development of chick embryos. Fertilized White Leghorn chicken eggs were injected with the test compounds or carrier vehicle, via a drilled small hole in the egg, directly into the egg yolk. After nine days of exposure, the embryonal viability, length and wet weight of embryos, and wet weight of brains were measured, and the development stages were assessed according to the Hamburger and Hamilton (HH) scale. The potent synthetic cannabinoid receptor agonist HU 210 and the non-psychotropic cannabidiol were embryotoxic at the highest concentrations examined (10 µM and 50 µM, respectively), with no viable embryos after the HU 210 injection, and 20% viability after the cannabidiol injections. The efects of HU 210 on the chick embryo were attenuated by α-tocopherol and the cannabinoid receptor antagonist AM251, whereas only α-tocopherol gave a statistically signifcant protection against the embryotoxic efects of cannabidiol. This study shows that exposure to plant-derived or synthetic cannabinoids during early embryonal development decreases embryonal viability. Extrapolation of data across species is of course difcult, but the data would argue against the use of cannabinoids, be it recreationally or therapeutically, during pregnancy. Cannabis has a long history of medical and therapeutic use, and the discovery of the cannabinoid (CB) signalling system, comprising CB receptors, endogenous ligands and enzymes for ligand biosynthesis and inactivation, has led to an enormous expansion of the CB research feld and in turn to the identifcation of new targets for therapeutic drugs1–3.
    [Show full text]
  • Endocannabinoid System Dysregulation from Acetaminophen Use May Lead to Autism Spectrum Disorder: Could Cannabinoid Treatment Be Efficacious?
    molecules Review Endocannabinoid System Dysregulation from Acetaminophen Use May Lead to Autism Spectrum Disorder: Could Cannabinoid Treatment Be Efficacious? Stephen Schultz 1, Georgianna G. Gould 1, Nicola Antonucci 2, Anna Lisa Brigida 3 and Dario Siniscalco 4,* 1 Department of Cellular and Integrative Physiology, Center for Biomedical Neuroscience, University of Texas (UT) Health Science Center San Antonio, San Antonio, TX 78229, USA; [email protected] (S.S.); [email protected] (G.G.G.) 2 Biomedical Centre for Autism Research and Therapy, 70126 Bari, Italy; [email protected] 3 Department of Precision Medicine, University of Campania, 80138 Naples, Italy; [email protected] 4 Department of Experimental Medicine, University of Campania, 80138 Naples, Italy * Correspondence: [email protected] Abstract: Persistent deficits in social communication and interaction, and restricted, repetitive pat- terns of behavior, interests or activities, are the core items characterizing autism spectrum disorder (ASD). Strong inflammation states have been reported to be associated with ASD. The endocannabi- noid system (ECS) may be involved in ASD pathophysiology. This complex network of lipid signal- ing pathways comprises arachidonic acid and 2-arachidonoyl glycerol-derived compounds, their G-protein-coupled receptors (cannabinoid receptors CB1 and CB2) and the associated enzymes. Alter- Citation: Schultz, S.; Gould, G.G.; ations of the ECS have been reported in both the brain and the immune system of ASD subjects. ASD Antonucci, N.; Brigida, A.L.; Siniscalco, D. Endocannabinoid children show low EC tone as indicated by low blood levels of endocannabinoids. Acetaminophen System Dysregulation from use has been reported to be associated with an increased risk of ASD.
    [Show full text]
  • The Complex Pharmacology of Cannabis Ben J
    The complex pharmacology of cannabis Ben J. Whalley [email protected] 28 June 2013 © University of Reading 2009 www.reading.ac.uk/cinn Outline • Constituents of cannabis – Cannabinoids and non-cannabinoids – Historical changes in composition • The endocannabinoid system – The physiological system affected by THC – Dispelling myths aka ‘Just because it has ‘cannab’ in the name…’ – Cannabinoids that do act via the endocannabinoid system • Beyond the endocannabinoid system – Cannabinoids that don’t act via the endocannabinoid system • Considerations for human medical development and use – Modulating the ubiquitous – Natural ≠ safe – Winning strategies: ‘Smaller & Quicker’ or ‘Slower & Larger’? [email protected] 2 Constituents of cannabis • Cannabis: for the most part, we are talking about the plant Cannabis sativa. • You’ve probably gathered that it one of the most widely used recreational and medicinal drugs worldwide. – ~150 million people smoking cannabis daily (WHO) • May be the first non-food plant cultivated by humans (~8000 BC)[2]. • Best known for its psychoactive constituent, Δ9- tetrahydrocannabinol (‘THC’). [email protected] Much, much more than just D9-THC • In addition to D9-THC, cannabis also contains: • >100 other ‘cannabinoids’ – Cannabinoids are chemical entities that are structurally similar to THC[3]. – phenol ring, 5-carbon alkyl chain, central pyran ring and mono- unsaturated cyclohexyl ring. – Not found in any other plant. • >400 other non-cannabinoids[3]. – Cannot rule out specific effects of
    [Show full text]
  • The Use of Cannabinoids in Animals and Therapeutic Implications for Veterinary Medicine: a Review
    Veterinarni Medicina, 61, 2016 (3): 111–122 Review Article doi: 10.17221/8762-VETMED The use of cannabinoids in animals and therapeutic implications for veterinary medicine: a review L. Landa1, A. Sulcova2, P. Gbelec3 1Faculty of Medicine, Masaryk University, Brno, Czech Republic 2Central European Institute of Technology, Masaryk University, Brno, Czech Republic 3Veterinary Hospital and Ambulance AA Vet, Prague, Czech Republic ABSTRACT: Cannabinoids/medical marijuana and their possible therapeutic use have received increased atten- tion in human medicine during the last years. This increased attention is also an issue for veterinarians because particularly companion animal owners now show an increased interest in the use of these compounds in veteri- nary medicine. This review sets out to comprehensively summarise well known facts concerning properties of cannabinoids, their mechanisms of action, role of cannabinoid receptors and their classification. It outlines the main pharmacological effects of cannabinoids in laboratory rodents and it also discusses examples of possible beneficial use in other animal species (ferrets, cats, dogs, monkeys) that have been reported in the scientific lit- erature. Finally, the article deals with the prospective use of cannabinoids in veterinary medicine. We have not intended to review the topic of cannabinoids in an exhaustive manner; rather, our aim was to provide both the scientific community and clinical veterinarians with a brief, concise and understandable overview of the use of cannabinoids in veterinary
    [Show full text]
  • Pharmacodynamics of Cannabinoids
    Open Access Archives of Pharmacy and Pharmaceutical Sciences Review Article Pharmacodynamics of cannabinoids Alexandra Sulcova* ISSN ICCI - International Cannabis and Cannabinoids Institute, Jachymova 26/2, 110 00 Praha, 2639-992X Czech Republic “Pharmacodynamics of cannabinoids “(i.e. a set of biological effects elicited in the *Address for Correspondence: Alexandra Sulcova, M.D, Ph.D, Professor of Pharmacology, living organism by interaction with its biochemical and biophysical functions up to the FCMA, FECNP, FCINP, ICCI - International cellular level) is studied for a long time during both, physiological and pathological Cannabis and Cannabinoids Institute, Jachymova conditions. Cannabinoids received their names according to their natural occurrence 26/2, 110 00 Praha, Czech Republic, Tel: 420 732167678; Email: [email protected] as constituents of Cannabis sativa L. (marijuana). The species was classiied in the “Linnaeus’s Species Plantarum (1753)”, the word “sativa” means things that are Submitted: 12 April 2019 Approved: 07 May 2019 cultivated [1]. For ages, people have used cannabis-based preparations for healing and Published: 08 May 2019 pain suppression until the discovery (in 1897) of aspirin (acetylsalicylic acid) which contemporary medicine uses until today. Chemical investigation of marijuana conirmed Copyright: © 2019 Sulcova A. This is an open access article distributed under the Creative various cannabinoid-type components called cannabinoids (presently estimated at Commons Attribution License, which permits about 150). Regarding their possible pharmacodynamic effects, tetrahydrocannabinol unrestricted use, distribution, and reproduction (THC) and cannabidiol (CBD) are the most explored. The determination of THC structure in any medium, provided the original work is properly cited by means of nuclear magnetic resonance imaging increased sharply the number of professional scientiic reports dealing with the studies of THC pharmacodynamic mechanisms of action [2].
    [Show full text]
  • Cannabinoid Treatment of Opiate Addiction
    Neuroanatomy and Behaviour, 2021, 3(1), e14. Neuroanatomy ISSN: 2652-1768 and H doi: 10.35430/nab.2021.e14 ε Behaviour REVIEW Cannabinoid treatment of opiate addiction Erin McLemon1 and Rose Chesworth1,* 1School of Medicine, Western Sydney University, Campbelltown NSW 2560, Australia *[email protected] Abstract Opioid abuse is a growing global problem. Current therapies for opioid abuse target withdrawal symptoms and have several adverse side effects. There are no treatments to address opioid-induced neural adaptations associated with abuse and addiction. Preclinical research demonstrates interactions between the endogenous opioid and cannabinoid systems, suggesting that cannabinoids may be used to treat opioid addiction and dependence. The aim of this review is to assess how cannabinoids affect behavioural and molecular measures of opioid dependence and addiction-like behaviour in animal models. It appears that cannabidiol and cannabinoid receptor 1 (CB1R) antagonists have potential for treating drug-craving and drug-seeking behaviour, based on evidence from preclinical animal models. Ligands which inhibit the action of cannabinoid degradation enzymes also show promise in reducing opioid withdrawal symptoms and opioid self-administration in rodents. Agonists of CB1R could be useful for treating symptoms of opioid withdrawal; however, the clinical utility of these drugs is limited by side effects, the potential for cannabinoid addiction and an increase in opiate tolerance induced by cannabinoid consumption. The mechanisms by which cannabinoids reduce opioid addiction-relevant behaviours include modulation of cannabinoid, serotonin, and dopamine receptors, as well as signalling cascades involving ERK-CREB-BDNF and peroxisome proliferator-activated receptor-a. Identifying the receptors involved and their mechanism of action remains a critical area of future research.
    [Show full text]
  • Appendix-2Final.Pdf 663.7 KB
    North West ‘Through the Gate Substance Misuse Services’ Drug Testing Project Appendix 2 – Analytical methodologies Overview Urine samples were analysed using three methodologies. The first methodology (General Screen) was designed to cover a wide range of analytes (drugs) and was used for all analytes other than the synthetic cannabinoid receptor agonists (SCRAs). The analyte coverage included a broad range of commonly prescribed drugs including over the counter medications, commonly misused drugs and metabolites of many of the compounds too. This approach provided a very powerful drug screening tool to investigate drug use/misuse before and whilst in prison. The second methodology (SCRA Screen) was specifically designed for SCRAs and targets only those compounds. This was a very sensitive methodology with a method capability of sub 100pg/ml for over 600 SCRAs and their metabolites. Both methodologies utilised full scan high resolution accurate mass LCMS technologies that allowed a non-targeted approach to data acquisition and the ability to retrospectively review data. The non-targeted approach to data acquisition effectively means that the analyte coverage of the data acquisition was unlimited. The only limiting factors were related to the chemical nature of the analyte being looked for. The analyte must extract in the sample preparation process; it must chromatograph and it must ionise under the conditions used by the mass spectrometer interface. The final limiting factor was presence in the data processing database. The subsequent study of negative MDT samples across the North West and London and the South East used a GCMS methodology for anabolic steroids in addition to the General and SCRA screens.
    [Show full text]