Transcript 1 Biotechnology Research and Training Center

A Drug from the Cannabis Field Appears to Protect From Seizure Related Brain Damage
Long C1, Quizon PMP1, Romine H1, Kelly W1, Naidoo V1, Ruiz S1, Suggs S1, Elliott L1, Ellerbe J1, Makriyannis A2, and Bahr BA1,2
1 Biotechnology Research and Training Center, The University of North Carolina at Pembroke, NC, USA
Acknowledgements:
This work was supported by grant
#5R25GM077634-04 from the NIGMS
(National Institute of General Medical Sciences
supporting the UNCP RISE Program.
2 Center for Drug Discovery, Northeastern University, Boston, MA, USA
Excitotoxic events such as seizures have been shown to
cause brain damage, decreasing neurons and
astrocytes—a component of the Blood Brain Brrier
(BBB)—their ability to properly recover. Two endogenous
compounds
from
the
cannabinoid
system,
endocannabinoids
anandamide
(AEA)
and
2arachidonoylglycerol (2-AG), have been linked with ondemand response in protecting against excitotoxic
injuries. An excitotoxin, Kanic acid (KA), injected (i.p) is
used to mimic seizure related events, and has been
observed to raise AEA levels in the brain. In order to
modulate the endocannabinoid response, various classes
of the AM drug (e.g. AM6642, AM5206, AM6701) have
been shown to inhibit the AEA deactivating enzyme fatty
acid amide hydrolase (FAAH). KA caused calpainmediated spectrin breakdown, declines in synaptic
markers, and disruption of neuronal integrity in cultured
hippocampal slices. The pre- and postsynaptic proteins
were protected by the FAAH inhibitor to levels
comparable to those found in healthy control brains.
Using also, Glial Fibrillary Acidic Protein (GFAP) and
Brainspecific Angiogenesis Inhibitor 1 (BAI1) as
astrocyte markers, it was shown that KA also seemed to
reduce and inhibited astrocyte macrophages their ability
to play
their role
in
neuroprotection. FAAH
inhibition with AM5206 protected against the
neurodegenerative and astrocyte degredation cascade
assessed in the slice models various times postinsult. In
vivo, KA administration induced seizures and the same
neurodegenerative events exhibited in vitro. BAI1
activation and GFAP markers appeared to demonstrate
positive effects showing the protective effects of AM5206
on not only neuron, but astrocytes as well. This data
supports the idea that endocannabinoids are released
and converge on pro-survival pathways that prevent
excitotoxic progression for both neurons and astrocytes.
An alternative for medical marijuana is
needed due to cannabinoid side
effects and broad action in the brain
Mechanism of Action of 9-THC:
CB1 Localization
released
endoCB
2AG
MAGL
AA
-
Psychotropic
Effects
- synapsin II -
- actin -
sr
sp
insult +
con insult AM5206
- GluR1 -
sr
sp
Post
Repair
Pathway
synaptophysin
Actin
Load Control
sr
s
p
insult +
AM5206
GluR1
CB1
immediate
2 h delayed
5 h delayed
synapse
Compensatory Endocannabinoid Release
Pathogenic event
KA-induced seizures
Change in endocannabinoids
3-fold increase in anandamide
Reference
NMDA-induced excitotoxicity
13-fold increase in anandamide
Hansen 2001
Traumatic brain injury
2.5-fold increase in anandamide
Hansen 2001
Traumatic brain injury
10-fold increase in 2-AG
Panikashvili 2001
6-OHDA toxicity
3-fold increase in anandamide
Maccarrone 2003
Figure 2. AM5206 provides synaptic and cellular protection when
administered 2-5 h post-insult. Animals received either vehicle, or 8
mg/kg AM5206 immediately or 2-5 h following KA administration. At 48
h post-KA-injection, hippocampal tissue for immunoblotting and
histology was rapidly dissected from these animals and from no insult
control rats. The postsynaptic marker GluR1, presynaptic marker
synapsin II, and actin were assessed on single immunoblots
Immunoblots show that administration of the FAAH inhibitor protects
against KA-induced synaptic damage (GluR1, synapsin II). Note that
actin remained unchanged across treatment groups. Hippocampal tissue
fixed for histology was paraffin embedded, sectioned, and stained with
hematoxylin and eosin. Photomicrographs of the CA1 field show that the
excitotoxin-induced neuronal loss and pyknotic changes were
ameliorated by the FAAH inhibitor injected immediately or 2-5 h
following KA administration. sp, stratum pyramidale; sr, stratum
radiatum. Size bar: 45 mm
Marsicano 2003
Rat model of Parkinson’s disease 7-fold increase in 2-AG
Di Marzo 2000
Model of multiple sclerosis
2-fold increase in anandamide
Baker 2001
Model of multiple sclerosis
1.8-fold increase in 2-AG
Baker 2001
Stress (foot shock)
2-fold increase in anandamide
Hohmann 2005
Stress (foot shock)
2-fold increase in 2-AG
Hohmann 2005
Acute schizophrenia
8-fold increase in anandamide
Giuffrida 2004
3. Seizures were induced in a rat model
and AM6642 caused a dose dependent
reduction in seizure severity
Testing FAAH inhibitors in the intact animal
seizures
insult(s) AM Compounds
FAAH inhibitor
CB1 antagonist
Behavioral Testing
(A)
· Molecular assessment
· Cellular assessment
· Drug Safety Evaluation
Actin
Load Control
GFAP, astrocyte marker
G
F
A
P
P<0.05
G
F
A
P
G
F
A
P
ANOVA P<0.006
NT
KA
Only
KA+
AM5206
A. Top Left: Integrated optical density units of β1 integrin band in rat hippocampus homogenates.
Between KA Only and KA+FAAH Inhibitor P<0.005. Top Right: Western blot of rat hippocampus with
no treatment, KA only and KA plus FAAH inhibitor stained for integrin β1. Actin to show load control
Middle left: Integrated optical density units of integrin β1 in rat neocortex homogenates. Middle Right:
Westen blot of integrin β1 in rat neocortex homogenates. Actin to show load control. Bottom Left: Using
imageJ software to determine the optical density of the GFAP marker P<0.05. Bottom Right: Western
blot of hippocampus with no treatment, KA only, and KA+AM5206 stained for BAI1, GFAP, and Actin
for load control.
3
2
***
1
***
· Balance
· Motor Skills / Coordination
· Learning
Harvest Brain / Organs
Common psychological effects
– euphoria
– distorted time perception
– enhancement of sensation
Many of these effects directly correlate with the
CB1 distribution in the brain
– cerebellum - movement and balance
– hippocampus - linear thinking and memory
– medulla - pain response
– neocortex - sleep/wake cycles
No reported deaths due to acute overdose of
marijuana (likely due to the lower density of
CB1 in the brainstem that control cardiovascular
and respiratory functions)
no insultt
control
Pre
AA
seizure
activity recovery period / treatment regimen
•
Common physical effects
– tachycardia
– hypertension
– thirst
– increased appetite
•
– constipation
– decreased intraocular pressure
– mydriasis
– conjuctival redness
– increased reaction time
– impaired coordination
•
– decreased libido
– mild analgesia
– mild anti-emetic effects
insult +
con insult AM5206
- synapsin II - actin -
VC
5 h delayed
insult
Kainic Acid
•
2 h delayed
immediate
insult +
con insult AM5206
- GluR1 -
FAAH
5. KA initiates a robust integrin response that
is protected against with FAAH inhibitors. It
also affects astrocyte markers that are also
protected with other class of FAAH inhibitors
AM5206 Administration:
AM6642
*Systemic Administration
*Epileptiform Type Brain Damage
Herkenham, M., et. al., PNAS. 1990. 87: 1932
4. AM5206 provides synaptic and cellular
protection when administered 2-5 h post-insult.
neuroprotection
seizure score
Summary
2. Breakdown of endogenous
cannabinoids can be blocked by selective
inhibitors (e.g. 6642) to enhance the
internal repair system that responds to
pathogenic events
0
KA K A+1mg/kg
1
veh KA+veh
KA+7mg/kg
AM6642
7-8 AM6642
+veh
AM6642, mg/kg
+KA
FIGURE 1. Dual FAAH and MAGL inhibitor AM6642 reduces severity of kainic
acid (KA) induced excitotoxicity in vivo. Animals were injected i.p. with 9
mg/kg KA immediately followed by either vehicle (n=15), 1 mg/kg AM6642
(n=7), or 7-8 mg/kg AM6642 (n=8). No-insult control rats received only one
vehicle injection (n=16). (A) Seizures were scored by blinded observers within a
4 h period. Mean 2nd h seizure scores post-injection are shown (± SEM).
ANOVA, P<0.0001; post hoc test compared to insult-only data: ***P<0.001.
Conclusions
1. Various classes of the AM drug have been
shown to inhibits FAAH and MAGL to protect
against cytoskeletal breakdown in vivo.
2. Dual
blockade
of
endocannabinoid
inactivation mechanisms may therefore be
an
ideal
target
for
neuroprotective
modulation of cannabinergic signaling to
offset neuropathological events including
excitotoxic brain damage.
3. AM5206 not only appears to have a
neuroprotective effect, but also appears to
have a astrocyte protective effect.
References
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