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CORE CANNABIS TRAINING
Advanced Endocannabinoid System Education
jahan@safeaccessnow.org
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The Endocannabinoid System
This section will cover the following topics:
• Patient Focused Certification
• The Expanded ECS
• Pharmacological Tools and Definitions
• Interactions between THC, CBD, and other components
• Review of Dosing from Clinical Trials (i.e., CINV and Appetite studies)
• Recent Breakthroughs and Promising Research (Diet)
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Jahan Marcu, Ph.D
• Chief Scientist, Americans for Safe
Access
• Chief Auditor for Patient Focused
Certification
• Serves on multiple expert
government, trade association
committees, and scientific
organizations including AHPA,
ACS, AOCS, AOAC, ASA, IACM,
IMCPC, ICCI, and others
• Author of the American Herbal
Pharmacopeia Cannabis Quality Control
and Therapeutic Monographs and other
international guidance documents
• Research has focused on the structure and
function of cannabinoid receptors, the anti-
cancer properties of cannabis compounds,
as well as method development & validation
for analyzing complex formulations
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American Herbal Products Association (AHPA)
Largest trade association representing the botanical and nutraceutical industry.
Played an integral role in the most successful grassroots movement in US History.
AHPA’s ongoing role:
• Galvanizing the botanical and nutraceutical industry
• Developing sensible national regulatory policy
• Fighting onerous FDA regulations
• Cannabis Committee Standards Adopted in 16 States
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American Herbal Pharmacopoeia (AHP)
Cannabis Monograph:
● Standards of Identity, Analysis, and Quality
Control
● Therapeutic Compendium
Standards That Ensure:
● Identity
● Purity
● Accuracyo Quality
o Potency
o Dosage
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AHPA & AHP GuidelinesStandards for Patient Focused Certification
Enhance and Promote Product Safety
• Ensures consistency of quality and effectiveness
• Ensures proper labeling
Standardized cannabis botanical medicine
• First standardized testing protocols developed for medical cannabis and medical
cannabis products
• Builds foundation for human clinical trials and case studies
• Increases patient and practitioner confidence
• Medical cannabis will no longer be a last resort
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PFC Seal of Approval
● Document and Facility Assessment
● Compliance with state and local regulatory
guidelines
● Compliance with AHPA and AHP standards
● Commitment to purity and identity of
products
● Implemented standardized methods:
○ Cultivation
○ Manufacturing, Packing, and Labeling
○ Laboratory
○ Distribution
○ Ancillary Operations
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What is PFC?
• Nonprofit Third Party Certification
• Peer Reviewed
• Seed to Consumption Quality Standards
• Staff Training
• Educational Materials
• Documentation & Facility Audits
• Complaint Hotline
• Government Relations
Phone: 202.857.4272 americansforsafeaccess.orgPhone: 202.857.4272
americansforsafeaccess.org
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The Endocannabinoid System (ECS)
• Discovered with the help of phytocannabinoids (Cannabis Sativa, Voacanga Africana,
Rhodenderon Anthpogonoides, Radula Marginata, and Helichrysum Umbraculigerum)
• Consists of endocannabinoids, cannabinoid receptors (i.e.,GPCRs), and enzymes for synthesis
and catabolism
• “Eat, sleep, relax, forget, and protect”
• Clinical Endocannabinoid Deficiency (CECD; Russo 2004)
Voacanga AfricanaRadula MarginataHelichrysum Umbraculigerum Rhodenderon Anthpogonoides Cannabis Sativa
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Inactive ReceptorAgonist
GI
GTP
GDP
Downstream
Cellular responses
GI
(-) Adenylyl cyclase
cAMP
GTP
Active Receptor
GTPase
GDPGI
The basics of GPCRs (CB1/CB2)
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The Endocannabinoid System
CB1
inhibit forskolin-stimulated AC
inhibit N,Q, L-type calcium channels
stimulate inwardly rectifying
potassium channels
activation of MAP kinase
CB2
inhibit forskolin-stimulated AC
activation of MAP kinase
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The Endocannabinoid System
• Neurotransmitters
- Endocannabinoids
• Proteins/Receptors
- Cannabinoid Receptors (CB1 and CB2)
• Synapse
-Endocannabinoids and CB1
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The Endocannabinoid system - Receptors
• Two main receptors have been identified to date, CB1 and CB2, members of the G-protein
coupled receptors (GPCRs) family
• In general, activation of CB1 results in inhibition of neurotransmitter release, due to
hyperpolarization of membrane potential in the pre- or post-synaptic neuron
• CB2 receptors are primarily associated with cells derived from the bone marrow lineage
• GPR55, GPR18, and other orphan receptors may be a cannabinoid receptors
• Ion channels play an important role
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Localization of cannabinoid receptors - CB1
• Widespread , high level expression, pre- and post-synaptic in central nervous system
• Distribution parallels known pharmacology
• Nucleus of solitary tract
• Hypothalamus
• Motor systems
- Motor cortex, basal ganglia, cerebellum. spinal cord
- Motor neurons in spinal cord
• Eye
• Sympathetic ganglia, also enteric nervous system
• Immune system - bone marrow, thymus, spleen, tonsils
• Breast cancer cell lines
• Other peripheral sites - heart, lung, adrenal, kidney, liver, colon, prostrate pancreas, testes, ovaries,
placenta…one
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• Plasma membrane
• Endoplasmic reticulum
• Perinuclear association
• Other organelles?
http://people.eku.edu/ritchisong/cell1.gif
Subcellular localization of Cannabinoid
Receptors
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Localization of cannabinoid receptors -CB2
• Immune system - bone marrow, thymus, spleen, tonsils
- T and B lymphocytes, monocytes, NK cells, PMN, mast cells
- level of expression increased during activation/ differentiation
• Uterus
• Lung
• Bone (osteoclasts, osteoblasts, osteocytes)
- CB2 polymorphisms associated with osteoporosis
- CB2 KO mice have accelerated age-related trabecular loss (controversial)
• Microglia
• Brainstem neurons
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COOH
M
5
M
6
M
7
NH2
M
1
M
2
M
3
M
4
Extracellular
Intracellular
Cannabinoid Receptor Structure
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Assays
GTPγS Stimulation3H Binding
Ligand Classifications
Bicyclic (CP55,940)
Tricyclic (THC, HU-210)
Indoles or AAI (WIN55,212)
Fatty Acids (Anandamide, 2-AG)
Antagonist/Inverse Agonist (SR141716A)
CB Receptor Analysis Tools and Mutational
Analysis
Amino AcidsBinding
Activation
Conformation
Structure
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• Orthosteric (agonist binding site)
• Allosteric (Other site for AMs)
• Agonist
• Antagonist
• Allosteric Modulator (AM)
• Negative AMs (NAM)
• Positive AMs (PAM)
• Heterodimers/heteromers
Receptors, Ligand, and
their Interactions
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Mutation Description Disease Associations References
CNR1
Trinucleotide
repeat in 3’ UTR
AAT repeat
Schizophrenia, substance abuse
disorders, Parkinson’s disease,
inverse relation between number of
repeats and working memory
performance
Zhang et al., 2004; Comings et
al., 1997; Ujike et al., 2002;
Barrero et al., 2005, Ruiz-
Contreras et al., 2013
CNR1 SNPs or
Haplotypes
rs6454674; rs806380;
rs806377; rs1049353;
rs806379; rs1535255;
rs2023239;rs806368;
rs806369; rs1049353;
rs4707436; rs12720071;
rs3505747
Substance abuse disorders,
depression, anxiety and eating
disorders, obesity, schizophrenia,
attention deficit disorder
Hopfer et al., 2006; Zuo et al.,
2009; Zhang et al., 2004; Juhasz
et al., 2009; Lazary et al., 2009;
Ho et al., 2011, Okahisha et al.,
2011, Mutombo et al., 2012,
Marcos et al., 2012
CB2 SNPs rs2502992, rs2501432 Low bone mineral density or
osteoporosis associated in at least 3
distinct human populations
Huang et al., 2009; Karsak et al.,
2005; Karsak et al., 2009;
Yamada et al., 2007
ECS Genetics and Disease Associations
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The ‘Expanded’ Endocannabinoid System
• 5-HT1A receptors
• Adenosine A2A receptors
• GPR55
• CB1 and CB2 receptors
• PLA2
• Glutamate mGlu5 receptor
• GPR18
• 5-lipoxygenase (5-LOX)
• CYP1, CYP3A
• Transient receptor potential (TRP)
channels, Ion channels TRPA1,TRPM8
TRPV1, TRPV2, TRVP4
• DAGLα, diacylglycerol lipase α DAGLβ,
diacylglycerol lipase β
• FAAH, fatty acid amide hydrolase
• MAGL, monoacylglycerol lipase
• PLA2
• Adenosine transporter, (SLC29)
• Glutamate transporter
• PPARγ (NR1C3)
• Glycine α3 receptors
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Transient receptor potential of vanilloid-
type 1 (TRPV1) channel
• An important component of the ECS, treating chronic and inflammatory pain
• Ionotropic cannabinoid receptor
• The effects TRPV1 and anandamide (AEA) have been reported by 100s of studies,
activates and desensitizes
• Involved with physical stimuli
- Temperature (42C), mechanical and osmotic stimuli, electrical charge, light,
hypotonic cell swelling, and chemical stimuli (low pH).
• Cannabidiol (CBD) can activate and desensitize TRPV1, underlies part of CBD’s analgesic
and anti-inflammatory effects.
- and less potently THCV, cannabigerol (CBG), cannabigerovarin (CBGV), and
cannabidivarin (CBDV)
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CBD Inhibiting THC (Non-CB Receptor)
• Antagonism of CB1 can arise when one drug diminishes the effect of another drug by targeting
different receptors or enzymes. (Ex, Dry mouth and Cholinergic drugs) (McPartland 2015)
• CBD augments AEA levels, and both stimulateTRPV1 channels. Pre-synaptic TRPV1 channel
activation enhances glutamate release in the spinal cord and brain. This may counteract or
antagonize the inhibitory action of pre-synaptic CB1 receptors co-localizing on glutamatergic
neurons (Campos, 2009; Di Marzo, 2010).
• CBD inhibits adenosine uptake. Pre-synaptic A2A receptors form heteromers with CB1 receptors
on glutamatergic neurons, and A2A receptor agonism inhibits CB1 receptor-mediated effects
• CBD acts as a direct and indirect agonist at 5-HT1A receptors and facilitates 5-HT1A-mediated
anxiolytic effects (Campos and Guimaraes, 2008; Gomes etal., 2011; Campos et al., 2013);
inhibits tryptophan degradation.
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CBD Enhancing THC (Non-CB1 Receptor)
• CBD inhibition of FAAH
• CBD reduces peripheral hyperalgesia via TRPV1 channels (Costa et al., 2004), may hypothetically
potentiate THC central mechanisms via CB1
• CBD reduces inflammation and inflammatory cytokines (e.g. TNF-α, IL-6, IL-1β) through TRPV1-,
A2A and PPARγ mediated mechanisms. THC reduces inflammation through separate CB1- and
CB2 receptor-mediated mechanisms.
• CBD acts as a positive allosteric modulator of glycine receptors, which contributes to cannabis-
induced analgesia (Xiong et al., 2011)
• CBD is an antioxidant and ROS scavenger, more potent than ascorbate or α-tocopherol
• CBD suppression of ROS, TNF-α, IL-6, IL-1β to dampen NF-κB. THC may dampen NF-κB through
a CB2-mediated mechanism
• CBD modulation of cytosolic Ca2+ levels via several mechanisms
• CBD inhibits cancer growth and induces apoptosis (Marcu et al. 2010) - Synergy
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CBD Influencing THC in Humans Part 1
• CBD decreased anxiety caused by THC (Karniol et al., 1974)
• CBD slightly increased time to onset, intensity, and duration of THC intoxication (Hollister and
Gillespie, 1975)
• CBD attenuated THC euphoria (Dalton et al., 1976)
• CBD reduced anxiety provoked by THC (Zuardi et al., 1982)
• CBD improved sleep and decreased epilepsy (Cunha et al., 1980; Carlini and Cunha, 1981)
• CBD decreased cortisol secretion and had sedative effects (Zuardi et al., 1993)
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CBD Influencing THC in Humans Part 2
• CBD provided antipsychotic benefits (Zuardi et al., 1995)
• CBD reduces the appetitive effects of THC (Morgan et al., 2010)
• CBD plus THC imparted synergistic inhibition of human glioblastoma cancer cell growth and
apoptosis (Marcu et al., 2010)
• Sativex compared to THC greater pain relief and improvement in sleep (Notcutt et al., 2004)
• Sativex compared to THC extract reduced cancer-related pain (Johnson et al., 2010)
• Sativex compared to THC reduced abnormalities in psychomotor performance associated with
schizophrenia (Roser et al., 2009)
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Studies combining THC and CBD
• Over 25 human and epidemiological studies
• Low ratio of CBD to THC (i.e.,1:4, 1:1)
• No studies of CBD to THC that were >1:1
• 2.7mg to 2.5mg (THC/CBD)
• Other concepts:
- CBD fluidizes membranes, allowing more THC penetration into muscles,
amplifying THC’s muscle relaxant properties
- CBD inhibits hepatic metabolism of drugs (pharmacokinetics)
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CBD (and component interaction) Summary
• CBD can directly stimulate receptors, enzymes, and other proteins.
• CBD can indirectly stimulate receptors by altering EC components and other
neurotransmitter levels.
• Synergy or additive effects occur due to interactions at the same target (i.e., CB1
receptors) or by triggering different pathways that converge on the same
physiological effects (i.e., inflammation).
- CBD inhibition of FAAH; THC & AEA synergy (Clapper et al., 2010)
- CBD reduces inflammation through TRPV1-, A2A, and PPARγ mediated
mechanisms. THC reduces inflammation through CB1 and CB2.
- CBD acts at glycine receptors (AM), contributes to pain relief (Xiong et al.,
2011)
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New Research: CBD as an Allosteric
Modulator of CB1
This article is protected by copyright. All rights reserved.
Figure 6. Cys-98 and Cys-107 coordinate the negative allosteric modulatory activity of
CBD at CB1. A,B) STHdhQ7/Q7
cells were transfected with arrestin2-Rluc- and CB1C98A
-
GFP2-, and CB1
C98S-GFP
2-, CB1
C107A-GFP
2-, and CB1
C107S-GFP
2-containing plasmids and
BRET2 was measured 30 min after treatment with THC (A) or 2-AG (B) ± CBD. CRCs were
fit using non-linear regression with variable slope (4 parameter) N = 4. C) Schematic of the
membrane-proximal region of CB1 summarizing data presented in this figure (adapted from
Fay and Farrens, 2013). Our observations and previous studies suggest that Cys-98 and Cys-
107 contribute to CB1 allosterism, while the orthosteric site is near the second extracellular
loop (orange box). In this diagram green represents extracellular surface of CB1. Black circles
represent residues unique to the N-terminus of CB1A. Grey circles represent residues unique
to the N-terminus of CB1B. Yellow circles represent Cys. Purple circles represent N-
glycosylated residues. Residues mutated in this study are marked in bold. Non-bold numbers
indciate amino acid number relative to N-terminus.
• Diminished signaling
from 2-AG and THC
• Orthosteric site
mutation, loss of
NAM activity
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Summary: The ECS and its Modulation
by Phytocannabinoids
• Example, acid
phytocannabinoids
(CBGA, THCA)
inhibit FAAH
Di Marzo et al. 2015
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Terpenes and Phytocannabinoids Part 1
Russo 2011
Terpenes can stimulate the
‘expanded ECS’ to support
phytocannabinoid activity
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Terpenes and Phytocannabinoids Part 2Russo 2011
Russo 2011
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Terpenes and Phytocannabinoids Part 3
• The essential oil of cannabis has analgesic, anti-inflammatory, and in some cases may even have antidepressant effects (,
(Segelman et al. 1974 Burstein et al. 1975,,Hall 2008; Russo et al. 2000).
• “Terpenoids improve THC pharmacokinetics by increasing vasodilatation of alveolar capillaries (which permits more
absorption of THC by the lungs), and by increasing blood–brain barrier permeability (Agrawal et al. 1989).” (Russo and
McPartland 2014)
• Examples from Russo (2011):
- β-myrcene is analgesic, anti-inflammatory, anti-convulsant, and a skeletal muscle relaxant.
- β-caryophyllene is analgesic and anti-inflammatory, eases gut muscle spasms,
- D-limonene is an antioxidant, antidepressant and anticonvulsant, and blocks carcinogenesis induced by
benz[a]anthracene, one of the “tars” generated by the combustion of herbal cannabis.
- D-linalool is sedative, anxiolytic, analgesic and anti-inflammatory, and induces apoptosis in cancer cells.
-α-pinene is anti-inflammatory, aids memory as an acetylcholinesterase inhibitor, and causes bronchodilation.
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The variables in synergy, and
the entourage effect
• Dosing/Administration
• Inhalation topography
• Genetic
• Disease/conditions (i.e., elevated receptor levels)
• Lifestyle
• Diet
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Ex) MW 300
1M = 300g in 1 L
1mM = 300mg in 1 L
1uM= 300ug in 1 L
1nM = 300ng in 1 L
Metabolism (Dosing Vs. Concentration)
MW of THC: 314.5 g/mol
MW of CBD: 314.5 g/mol
i.e., From cancer research
1.7/0.4 uM THC/CBD
534.7 ng/mL THC
125.8 ng/mL CBD
34mg THC = ~140 ng/ml
(i.e., 3.8% THC 1 gram NIDA)
• That’s blood…What about at
the target site?
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Metabolism
• THC bioavailability is 25% from smoking/inhalation
• Peak plasma 6-10min
• High intra- and intergroup variability due to many
factors (i.e., smoking topography)
• THCTHC-COOH (inactive, 30-45min [equal] to
THC) & 11-OH-THC
• CBD & CBN not detected after 1hour of smoking
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Clinical Trials for Appetite and CINV
• Since 1975, more than 40 clinical studies have examined the effectiveness of
synthetic THC, Nabilone (THC derivative) or smoked Cannabis CINV and appetite
stimulant
• Inhaled Cannabis
- Typical Inhaled Dose in clinical research 1gram (2-5% THC)
- Appetite 1-4 doses per day (2.5-10mg orally) (4-21 days)
- CINV before or after treatment
- Oral THC recommended dosage for nausea and vomiting induced by cancer
chemotherapy are 5–15 mg/m2/dose, without exceeding 4–6 doses per day (CPA
2005)
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Emerging Research: Diet and the ECS
• Humans cannot make AEA de novo
• Fish oils alter endocannabinoid levels (Balvers
et al. 2012)
• High/low fat diet influences endocannabinoid
genes expression (Engali et al. 2014)
• High-fructose diet (Lowette et al. 2015),
increased CB1mRNA and decreases learning
and cognition
• Omega-3 deficiency in mammals can abolish
ECS functions in rats, GPCR uncoupling
(Lafourcade et al. 2010)
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Emerging Research: Diet and the ECS
• Humans cannot make AEA de novo
• Fish oils alter endocannabinoid levels (Balvers et al.
2012)
• High/low fat diet influences endocannabinoid genes
expression (Engali et al. 2014)
• High-fructose diet (Lowette et al. 2015), increased
CB1mRNA and decreases learning and cognition
• Omega-3 deficiency in mammals can abolish ECS
functions in rats, GPCR uncoupling (Lafourcade et al.
2010)
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McPartland, J. M., Duncan, M., Di Marzo, V., & Pertwee, R. G. (2015). Are cannabidiol and Δ(9) -
tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review. British
Journal of Pharmacology, 172(3), 737–753. http://doi.org/10.1111/bph.12944
Di Marzo, V., & Piscitelli, F. (2015). The Endocannabinoid System and its Modulation by
Phytocannabinoids. Neurotherapeutics : the Journal of the American Society for Experimental
NeuroTherapeutics, 12(4), 692–698. http://doi.org/10.1007/s13311-015-0374-6
Russo, E. B. (2011). Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage
effects. British Journal of Pharmacology, 163(7), 1344–1364. http://doi.org/10.1111/j.1476-
5381.2011.01238.x
Pertwee, R. (2014). Handbook of Cannabis. Oxford University Press.
Helpful References
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