Eliot Gardner, Ph.D. - Nysam

Report
Endocannabinoids:
Basic Physiology and Function
Eliot L. Gardner
New York Society of Addiction Medicine
7th Annual Conference
NYC - February 2011
Eliot L. Gardner, Ph.D.
Chief, Neuropsychopharmacology Section
Intramural Research Program
National Institute on Drug Abuse
National Institutes of Health
[email protected]
443.740.2516
Cannabis
•
•
•
•
•
 Many species exist: Cannabis Sativa (European
Many species exist: Cannabis Sativa (Europe), Cannabis Indica (India)
plant), Cannabis indica (Indian plant) and Cannabis
and Cannabis ruderalis (Siberia and central Asia)
ruderalis (Siberia and central Asia plant)
460 known chemical constituents of cannabis
460 known
constituents
of cannabis
66 
constituents
havechemical
a cannabinoid
structure
9-THCaor
Δ9-Tetrahydrocannabinol
THC) most important
constituent
 66 constituents(Δhave
cannabinoid
structure
Δ9-THC
is the principal
psychoactiveconstituent:
component of cannabis
Δ9-THC
most important
principal
psychoactive component of cannabis
Era of Cannabis Research: 200-1940
● Circa 200 AD: Therapeutic properties of cannabis described in
Chinese pharmacopoeia
● 1838-1840: Sir W.B. O’Shaughnessy methodically assesses
medicinal properties of cannabis, and publishes findings
● 1899: Wood et al. isolate cannabinol from cannabis resin
● 1932: Cahn elucidates part of the structure of cannabinol
● 1940: Todd et al. and Adams et al. simultaneously elucidate
the full structure of cannabinol and successfully synthesize it
Era of Cannabinoid Research: 1960-1994
● 1960: Mechoulam (Hebrew University) identifies THC as the
principal psychoactive component of cannabis
● 1964: Gaoni and Mechoulam (Hebrew University) elucidate
the chemical structure of THC
● 1970-1990: Cannabinoid pharmacology is thoroughly studied
● 1985: Gardner shows cannabinoid-opioid interaction in brain
● 1986: Gardner shows THC activates brain-reward systems
● 1988: Howlett’s group finds specific THC binding sites in brain
● 1990: Matsuda et al. clone the CB1 receptor
● 1992: Mechoulam’s group (Hebrew University) in collaboration
with Pertwee’s group (Scotland) identify the first endocannabinoid – Mechoulam names it “anandamide” from the Sanskrit
word “anand” meaning “bliss”
● 1993: Munro et al. clone the CB2 receptor
Era of Endocannabinoid Research: 1994-2000
● 1994: Scientists at Sanofi Recherche (France) develop the
first CB1 receptor antagonist – SR141716A (Rimonabant)
● 1995: Mechoulam (Hebrew University) isolates and identifies
the second endocannabinoid – 2-Arachidonoylglycerol (2-AG)
● 1996: Cravatt et al. (Scripps) clone the first endocannabinoid
degrading enzyme – fatty acid amide hydrolase (FAAH)
● 1998: House of Lords report on medical cannabis
● 1998: Di Marzo et al. propose interactions between endocannabinoids and vanilloid receptors
● 1999: Zygmunt et al. and Smart et al. show that anandamide
activates vanilloid receptors
Current Endocannabinoid Research: 2000● 2003: Bisogno et al. clone the first endocannabinoid biosynthesizing enzymes
● 2005: Pertwee et al. (Scotland) discovers an allosteric site on
CB1 receptors
● 2005: Sativex® approved for sale in Canada
● 2010: Gardner shows psychoactive (and potentially therapeutic) effects of cannabidiol
● ????: Discovery of new cannabinoid receptors
● ????: Discovery of new endocannabinoids
● ????: Discovery of new endocannabinoid enzymes
● ????: Cloning of new endocannabinoid transporters
● ????: Discovery of new cannabinoid-based therapies
What is a cannabinoid?
• Initially, compounds extracted by Cannabis spp
producing characteristic psychoactivity
• Later, compounds with a characteristic terpenoid
structure
• Currently, most any compound that produces
cannabinoid psychoactivity, natural or synthetic
• Occasionally, just compounds that will interact with
cannabinoid receptors
Natural cannabinoids
Representative cannabinoids
Classical cannabinoids
Non-classical cannabinoid
Aminoalkylindole
CB1 antagonists
Endocannabinoids
O
O
HO
HO
HN
O
HO
Anandamide
2-Arachidonoylglycerol
HO
O
HO
O
HO
HO
Noladin ether
HN
N-Arachidonoyldopamine
O
NH2
O
Virodhamine
Cannabinoid CB1 and CB2 Receptors
Characteristics of CB1 and CB2 Receptors
•
•
•
•
•
•
Both densely distributed throughout the body
CB1 highly enriched in central nervous system
Located on axon terminals
Mediate retrograde signaling (Dendrite → Axon)
G-protein coupled
CB2 highly enriched in periphery
– Especially in immune system
• CB2 also in brain and CNS
– Fewer than CB1; ~ Same density as μ opioid
– Nonetheless, CB2s modulate neural signaling
CB1 and CB2 Receptors not the only
Receptors Activated by Cannabinoids
• Cannabidiol (CBD) receptors
• Transient Receptor Potential Cation V1
receptors (TRPV1; Capsaicin receptors)
• G-coupled Protein Receptor 55 (GPR55)
• G-coupled Protein Receptor 119 (GPR119)
• Peroxisome Proliferator-Activated receptors
(PPARs)
• Others
CB1-Mediated Signal Transduction
AMPc
ATP
PKA
AC
MAPK
NA+/H+
K+
exchanger
CB1
Ca2+
Guindon, Beaulieu and Hohmann (2009)
Pharmacology of the cannabinoid system, IASP Press
Gene
expression
AA
CB1 localization
Mouse
Monkey
H.-C. Lu
• Antibodies
• Distinctive pattern of distribution
• Cortex, hippocampus, basal ganglia, SN,
cerebellum
• Low in thalamus and most of brainstem
Eggan S. and Lewis D. Cerebral
Cortex 2007; 17:175
CB1 receptor localization (hippocampus)
mRNA
protein
István Katona
•In the forebrain, the majority of CB1 protein arises
from a minority of interneuons (CCK+ GABAergic)
CB1 receptor localization (hippocampus)
protein
Jim Wager-Miller
•CB1 heavily expressed on some axons & terminals
EM
István Katona
CB1 receptor localization (VTA)
István Katona
•CB1 expressed on two populations of terminals
•Functionally, multiple VTA synapses are modulated by cannabinoids
CB1 agonists modulate
neurotransmission
• The signaling pathways of CB1 suggest
cannabinoids might decrease neurotransmission:
•Inhibition of calcium channel, adenylyl cyclase
•Activation of potassium channels, MAP kinase
• Appropriate localization of the receptors
• Multiple studies show inhibition of
neurotransmitter release
CB1 agonists modulate
neurotransmission
Typical experiment:
Vc
•Hippocampal slices
•Patch clamp recording
•Bath apply drugs
stimulate
record
Measure GABAergic currents in CA1
Hájos
CB1 receptor activation inhibits
evoked GABA IPSC’s
CB1 receptor summary
• Abundantly expressed throughout the brain
• Majority on axons and synaptic terminals
• Primarily Gi/o coupled (not only!)
• CB1 activation inhibits synaptic transmission
Endogenous cannabinoids
Receptors suggest endogenous ligands
Two main families identified
Both arachidonic acid derivatives
Precursors in membranes
“Made on demand”
Amides (anandamide)
Esters (2-AG)
• Significant differences
– Routes of synthesis
– Mode of degradation
(FAAH vs MAGL)
– Efficacy
CB1 agonist efficacy is variable
Many studies have
found 2-AG to be
more efficacious
than anandamide (or
THC) at CB1 (GIRK
activation in oocytes
shown here)
2-AG
MEA
THC
Luk, et al, 2004
eCB summary
• Acyl ethanolamides (diverse; anandamide, AEA)
• More promiscuous --- many targets
• Acyl glycerol esters (2-AG)
• Both are “Made on demand”
• 2-AG ~100x more bulk levels, similar “signaling”(?)
• Differing efficacies
• Metabolic diversity, with “core” pathways
What are the physiological effects of eCB’s on
neuronal activity?
• Exogenous cannabinoids inhibit neurotransmission
• eCB’s are synthesized following increases in
intracellular calcium and/or activation of Gq/11linked receptors
• Might eCB’s synthesized in this fashion modulate
neurotransmission?
• Yes
•Transient effects
•Long lasting effects
Six Types of eCB-Mediated Synaptic
Plasticity Have Been Clearly Identified
•
•
•
•
•
•
Depolarization-induced suppression of inhibition
Depolarization-induced suppression of excitation
Metabotropic-induced suppression of inhibition
Metabotropic-induced suppression of excitation
Long-Term Depression (LTD)
Slow self-inhibition (SSI)
• Additional types are being constantly discovered
Important Take-Home Messages
• Endocannabinoids are neurotransmitters
• Cannabinoids (e.g., THC) modulate neural activity
• Endocannabinoids are involved in synaptic remodeling
• Cannabinoids (e.g., THC) can modulate synaptic remodeling
• Depending upon the specific CNS circuits involved,
cannabinoids can have a host of actions on brain,
cognition, and behavior (some beneficial, some not)
Cannabinoids and pain
● central
● spinal
● periphery
Peripheral and spinal localization of
cannabinoid receptors
Ständer et coll. J Dermatol Sci 2005 Hohmann & Herkenham
Neuroscience 1999
Bridges et coll.
Neuroscience 2003
Farquhar-Smith et coll.
Mol Cell Neurosci 2000
AEA
NAPEPLD ?
Presynaptic neuron
NAPE
NAT
2-AG
MGL
Neurotransmitter
vesicles
ET
CB1
Ca2+
ET
DAGL
AA
COX
2-AG
PG
DAG
PLC
Phospholipid
AEA
NAPEPLD ?
NAPE
NAT ?
Postsynaptic neuron
Guindon et al., (2009) Pharmacology of
the cannabinoid system, IASP Press
Evaluation of nociceptive behavior in the
formalin test
Behaviours*
Normal
behaviour
Pain
behaviour (1)
Pain
behaviour (2)
Observations
Injected paw can
support the
weight of the
animal.
Injected paw has
little or no weight
on it.
Injected paw is
elevated, not in
contact with any
surface.
Injection 50 µL Formaline 2.5 %
NaCl 0,9%
Pain
behaviour (3)
Pain score
1.2
1
Scoring system**
Time spent in this
category
0
0
1
Injected paw is
licked, bitten or
shaken.
2
0.8
0.6
0.4
* The same behaviours are observed with the hind paw.
0.2
0
0
5
10
15
20
25
30
35
40
Time (min)
45
50
55
60
** Watson et al. (1997)
Peripheral Antinociceptive Effects
Composite Pain Score (CPS)
NaCl 0.9 %
Anandamide 0.1 µg
Ibuprofen 2 µg
Rofecoxib 2 µg
1, 2
1
0, 8
1.2
#
0, 6
0, 4
1
0, 2
†
0
0
5
10
15
20
25
30
35
40
45
50
55
60
0.8
contralateral
0.6
0.4
0.2
0
0
5
10
15
20
25
30
35
40
45
50
55
60
Time (min)
† AUC (0-15) P < 0.05 and # AUC (15-60) P < 0.001 for analgesics vs NaCl 0.9 %
ipsilateral
Synergistic effect of anandamide +
ibuprofen
25
Anandamide
Ibuprofen
Mix (1:10)
0.2
Anandamide
Ibuprofen
Mix 1:10
Add 1:10
Dose Ibuprofen (µg)
Pain (Area Under the Curve)
20
15
10
0.15
0.1
0.05
5
0
0
0
-4
-3
-2
-1
Log dose (µg)
Guindon et al. (2006) Pain 121: 85-93
0
1
0.005 0.01 0.015 0.02
Dose Anandamide (µg)
Synergistic effect of anandamide +
rofecoxib
25
Rofecoxib
Mix (1:10)
20
0.2
Dose Rofecoxib (µg)
Pain (Area Under the Curve)
Anandamide
15
10
Anandamide
Rofecoxib
Mix 1:10
Add 1:10
0.15
0.1
0.05
5
0
0
0
-4
-3
-2
-1
0
1
Log dose (µg)
Guindon et al. (2006) European Journal of Pharmacology 550: 58-77
0.005
0.01
0.015
Dose Anandamide (µg)
Objectives of 2-AG, JZL184
and URB602 study
 Compare the peripheral
antinociceptive effects of 2-AG,
JZL184, URB602 and their
combination in the formalin test
 Study the mechanisms by
which JZL184 and URB602
produce their effects using
specific CB1 and CB2 receptor
antagonists
Composite Pain Score (CPS)
Peripheral Antinociceptive Effects
NaCl 0.9%
URB602 500 µg
1.2
1
0.8
1.2
#
0.6
0.4
1
0.2
0
†
0
5
10
15
20
25
30
35
40
45
50
55
60
0.8
contralateral
0.6
0.4
0.2
0
0
5
10 15 20 25 30 35 40 45 50 55 60
Time (min)
† P < 0.001 and # P < 0.001 for URB602 (500 µg) vs NaCl 0.9 %
ipsilateral
Composite Pain Score (CPS)
Peripheral Antinociceptive Effects
Vehicle
1
1
JZL184 300 microg
0.8
#
†
0.8
0.6
0.4
0.2
0
0.6
0 5 10 15 20 25 30 35 40 45 50 55 60
contralateral
0.4
0.2
0
0
5
10
15
20
25
30
35
40
45
50
55
60
Time (min)
† P < 0.001 and # P < 0.001 for JZL184 ((300 µg) vs NaCl 0.9 %
ipsilateral
JZL184 with cannabinoid
antagonists
Inflammatory Phase
Area Under the Curve
20
16
12
*
8
4
0
Vehicle
JZL184 10µg
AM251 80µg
AM251 + JZL184
AM630 25µg
AM630 + JZL184
* P < 0.001 for JZL184 (10 µg) vs Vehicle
URB602 with cannabinoid
antagonists
Inflammatory Phase
Area Under the Curve
20
16
*
12
8
4
0
NaCl 0.9 % URB602 70µg
Guindon et al. (2006) Brithish Journal
of Pharmacology 150: 693-701
AM251
AM251+
URB602
AM630
AM630+
URB602
* P < 0.001 for URB602 (70 µg) vs NaCl 0.9 %
Conclusions
 JZL184, URB602, 2-AG and their
combination reduce nociceptive behavior
when given locally
 JZL184 is more potent than URB602
when given alone or combined with 2-AG
 Antinociceptive effects of JZL184 and
URB602 are inhibited by AM251 and
AM630
Cannabinoids and Addiction
There is now an extensive published literature showing antiaddiction efficacy for cannabinoid ligands
•
•
•
•
•
•
•
•
Gardner EL. Endocannabinoid signaling system and brain reward: emphasis on
dopamine. Pharmacol Biochem Behav 81:263-284, 2005
De Vries TJ & Schoffelmeer AN. Cannabinoid CB1 receptors control conditioned
drug seeking. Trends Pharmacol Sci 26:420-426, 2005
Cohen C et al. CB1 receptor antagonists for the treatment of nicotine addiction.
Pharmacol Biochem Behav 81:387-395, 2005
Maldonado R et al. Involvement of the endocannabinoid system in drug addiction.
Trends Neurosci 29:225-232, 2006
Basavarajappa BS. The endocannabinoid signaling system: a potential target for
next-generation therapeutics for alcoholism. Mini-Revs Med Chem 7:769-779, 2007
Fattore L et al. Endocannabinoid regulation of relapse mechanisms. Pharmacol Res
56:418-427, 2007
Scherma M et al. The endocannabinoid system: a new molecular target for treatment
of tobacco addiction. CNS & Neurol Disorders - Drug Targets 7:468-481, 2008
Paralaro D & Rubino T. The role of the endogenous cannabinoid system in drug
addiction. Drug News Perspect 21:149-157, 2008
CB1 Antagonist-Induced Attenuation of Cocaine-Enhanced
Brain Stimulation Reward
CB1 Antagonist-Induced Attenuation of Cocaine-Enhanced
Brain Stimulation Reward
CB1 Antagonism Does Not Affect Motoric Ability
PR Schedule
Reward
(# Infusion)
1
2
3
4
5
Pump
Work Demand
(# Lever Press)
1
2
4
6
9
Cocaine
?
14
15
16
….
77
95
118
….
C u m u la t
600
400
CB1 Antagonist-Induced Attenuation of Incentive Motivation to
Self-Administer i.v. Cocaine – Representative Animal
(Progressive-Ratio Model) T im e (m in )
200
C o ca in e = 0 .5 m g /kg /in fu sio n
0
0
A
Afte r V e h ic le
40
60
80
100
120
140
160
A fte r A M 2 5 1 (1 m g /k g )
B
1600
1600
C u m u la tive L e ve r P re s s e s
C u m u la tive L e ve r P re s s e s
20
1400
1200
1000
800
600
400
200
1400
1200
1000
800
600
400
200
C o c a in e = 0 .5 m g /k g /in fu s io n
C o ca in e = 0 .5 m g /kg /in fu sio n
0
0
0
20
40
60
80
100
T im e (m in )
B
Afte r AM 2 5 1 (1 m g /k g )
120
140
160
0
20
40
60
80
100
T im e (m in )
120
140
160
CB1 Antagonist-Induced Attenuation of Incentive Motivation to
Self-Administer i.v. Cocaine (Progressive-Ratio Model)
AM 251
AM251 (Original Break-Point)
AM251 (% Change in Break-Point)
120
60
B re a k -P o in t
50
*
40
30
***
20
(% C h an g e o ver B aselin e)
100
*
80
*
***
60
40
20
10
0
0
0
1
3
0
10
SR141716A
1
3
AM 2 5 1 (m g /k g , i.p .)
AM 2 5 1 (m g /k g , i.p .)
120
100
n)
B re a k -P o in t
(L e ve r P re s s e s fo r L a s t In fu s io n )
70
100
10
CB1 Receptor Antagonism Dose-Dependently Attenuates
Relapse to Cocaine-Seeking Behavior (Reinstatement Model)
CB1 Receptor Antagonism Does Not Attenuate Relapse to
Non-Drug Reward-Seeking Behavior (Reinstatement Model)
CB1 Receptor Antagonist Micro-Injected Into Nucleus Accumbens
Attenuates Cocaine-Seeking Behavior (Reinstatement Model)
CB1 Receptor Antagonism By Itself Does Not Produce
Drug-Seeking Behavior (Reinstatement Model)
CB1 Receptor Antagonism Markedly Attenuates CocaineEnhanced Nucleus Accumbens Glutamate (Brain Microdialysis)
CB1 Receptor Antagonism Markedly Attenuates Cocaine
Sensitization
CB1 Receptor Gene-Deletion (CB1 Gene Knock-Out) Abolishes
Cocaine’s Psychostimulant Effects
CB1 Receptor Gene-Deletion (CB1 Gene Knock-Out) Abolishes
Cocaine-Enhanced Nucleus Accumbens Dopamine (Dialysis)
CB1 Receptor Gene-Deletion (CB1 Gene KO) Attenuates
Evoked Nucleus Accumbens Dopamine Release (Voltammetry)
CB1 Receptor Gene-Deletion (CB1 Gene KO) Attenuates
Evoked Nucleus Accumbens Dopamine Release (Voltammetry)
CB1 Antagonist SR141716 (Rimonabant) By Itself Markedly
Inhibits Nucleus Accumbens Dopamine (Brain Microdialysis)
Other CB1 Receptor Antagonists (Either Neutral Antagonists or
Inverse Agonists) Do Not Do This !!
Caveats Regarding Development of
Cannabinoid Agonists as Potential
Pharmacotherapeutic Agents
• CB1 and CB2 receptors are ubiquitous throughout
the body – Potential for numerous side effects
• Some cannabinoid ligands have poor bioavailability
• CB1 receptor agonists have addictive potential
Potential Cannabinoid Therapies - Tools
•
•
•
•
•
•
•
•
•
•
•
•
Endocannab Uptake Inhibitors – AM404, UCM707, AM1172
FAAH Inhibitors – URB597, OL135, BMS1, SA47, PF750
MAGL Inhibitors – URB602, OMDM169, JZL184
Dual CB1/CB2 Agonists – WIN55512, CP55940, HU210
Anandamide Analogues – Methanandamide, Metfluoroanand.
Selective CB1 Agonists – ACEA, ACCP
Selective CB2 Agonists – HU308, JWH015, JWH133, AM1241
2-AG Synthesis Inhibitors – O3640, O3891, OMDM188, O5596
CB1 Antagonists/Inverse Agonists – SR141716A, AM251
CB1 Neutral Antagonists – AM4113, PIMSR1
CB2 Antagonists/Inverse Agonists – SR144528, AM630
CB1 Receptor Allosteric Modulators – ORG27596, ORG29647
Potential Cannabinoid Therapies – Clinical Indications
• Diseases of Energy Metab.
–
–
–
–
–
–
–
Appetite Dysregulation
Obesity
Dyslipidemia
Periph Energy Metab Dysreg
Cachexia
Anorexia
Type 2 Diabetes
• Pain
– Somatosensory Pain
– Neuropathic Pain
• Inflammation
• CNS Disorders
– Closed Head Brain Trauma
– Neurotoxicity
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Stroke
Spinal Cord Injury
Multiple Sclerosis
Parkinson’s Disease
Huntington’s Disease
Tourette’s Syndrome
Tardive Dyskinesia
Dystonia
Amyotrophic Lateral Sclerosis
Alzheimer’s Disease
Epilepsy
Anxiety
Depression
Insomnia
Post-Traumatic Stress Disorder
Schizophrenia
Potential Cannabinoid Therapies – Clinical Indications
• CNS Disorders – con’t
– Nausea & Emesis
– Drug & Alcohol Addiction
• Cardiovascular & Respiratory
–
–
–
–
–
–
–
Hypertension
Hypotension
Circulatory Shock
Myocardial Reperfusion Injury
Atherosclerosis
Cardiopathies
Asthma
• Eye Disorders
– Glaucoma
– Retinopathy
– Intraocular Pressure
• Cancer
– Cancer Cell Proliferation
– Colorectal Cancer
• GI and Liver Disorders
–
–
–
–
–
–
Inflammatory Bowel Disease
Ulcerative Colitis
Hepatitis
Cirrhosis – Encephalopathy
Cirrhosis – Liver Fibrosis
Cirrhosis – Vasodilatation
• Musculoskeletal Disorders
– Arthritis
– Osteoporosis
– Post-Fracture Bone Healing
• Reproductive Disorders
Acknowledgments
• Ken Mackie, MD – Dept of Psychological and Brain Sciences,
Indiana University Bloomington
• Josée Guindon, PhD – Dept of Psychology, Univ of Georgia
• Andrea G. Hohmann, PhD – Neuroscience and Behavior
Program, Univ of Georgia
• Raphael Mechoulam, PhD – Dept of Medicinal Chemistry,
Hebrew University of Jerusalem
• Roger Pertwee, PhD – School of Medical Sciences, Univ of
Aberdeen, Scotland
• Steven Goldberg, PhD – Behavioral Neuroscience Research
Branch, NIDA, NIH
• Javier Fernández-Ruiz, PhD – Facultad de Medicina, Universidad Complutense, Madrid
• Vincenzo Di Marzo, PhD – Endocannabinoid Research Group,
Consiglio Nazionale delle Ricerche, Naples, Italy
Neuropsychopharmacology Section, Intramural Research Program
National Institute on Drug Abuse, National Institutes of Health
Acknowledgment
Raphael Mechoulam, PhD
Dept of Medicinal Chemistry, Hebrew Univ of Jerusalem

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