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CORE CANNABIS TRAINING

Advanced Endocannabinoid System Education

[email protected]

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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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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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• 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

http://people.eku.edu/ritchisong/cell1.gif

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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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patientfocusedcertification.orgConsole-Bram and Marcu 2012

• 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

http://doi.org/10.1111/bph.12944

http://doi.org/10.1007/s13311-015-0374-6

http://doi.org/10.1111/j.1476-5381.2011.01238.x

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Helpful References

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