2011
University of Aberdeen Vincent Hoi Kit Li http://vli.tel
[NEW CANNABINOID THERAPEUTICS] A thesis presented as partial fulfilment for the degree of BSc (Hons) Physiology at the University of Aberdeen.
I
Declaration
I declare that all work in this thesis is my own. This thesis has never been
submitted as part of any previous degree application. The collection and analysis
of all results were performed by my lab partners (Tamema Choudhury & David
McNee) and me. All contributions from other sources have been appropriately
acknowledged and cited.
XVincent H. K. LiBSc (Hons) Physiology Candidate
II
Acknowledgements
This project was performed under supervision from Professor Ruth Ross and
Gemma Baillie with the help from Lesley Stevenson and others in the Cannabinoid
Group.
Firstly, I would like to express my gratitude to Professor Ross. She has provided us
invaluable advice on how we should go about with the project. As well as the
extensive feedback she provided me with.
Secondly, I would like to thank Gemma, who supervised us directly in the lab on a
daily basis. Above all, for her patience, the techniques she has taught us and
guidance throughout the project.
In addition, Lesley has supported us throughout 10 weeks in the laboratory,
ranging from brain collection to every aspect in the lab. Without her help, the
project would have been a struggle.
Furthermore, I would like to thank other members of the Cannabinoid Group,
Pietro Marini and Professor Roger Pertwee in particular, and everyone else in the
group who helped us in many ways.
Finally, a big thank you to my lab partners, Tamema Choudhury and David McNee.
It has been a pleasure working with both of you.
In memory of grandparent of Tamema and grandparent of Gemma.
III
A fully linked electronic edition of this thesis and additional experimental data can
be found on the following website: http://cnr1.eu
The British Journal of Pharmacology referencing style has been applied to this
thesis using RefWorks2.
The following guidelines were consulted for the writing up of this thesis:
• Information for Authors British Journal of Pharmacology http://www.brjpharmacol.org/view/0/authorInformation.html
• Style Guide University of Aberdeen http://www.abdn.ac.uk/documents/style-guide.pdf
• Lab Project (Hons) Guidelines School of Medical Sciences, University of Aberdeen (available via http://webct.abdn.ac.uk, login required)
IV
Table of Contents
Declaration ................................................................................................................... I
Acknowledgements .................................................................................................... II
Abbreviations ............................................................................................................. VI
1 Summary ............................................................................................................. 1
2 Introduction ........................................................................................................ 2
2.1 Cannabinoids .............................................................................................. 2
2.2 Cannabinoid receptors................................................................................ 3
2.3 Endocannabinoids ....................................................................................... 5
2.4 Fatty acid amino hydrolase (FAAH) ............................................................ 6
2.5 Allosteric Modulation ................................................................................. 8
2.6 Assays overview ........................................................................................ 10
2.6.1 GTPγS functional assay ......................................................................... 10
2.6.2 Equilibrium binding assay ..................................................................... 12
3 Aims .................................................................................................................. 13
4 Materials and Methods..................................................................................... 14
4.1 Materials ................................................................................................... 14
4.2 Mouse brain membrane preparation ....................................................... 15
4.3 [35S]GTPγS functional assay ...................................................................... 16
4.4 Equilibrium binding assay ......................................................................... 18
4.5 Mathematical analysis .............................................................................. 19
5 Results ............................................................................................................... 20
5.1 O-7756 ...................................................................................................... 20
V
5.2 O-7757 ...................................................................................................... 21
5.3 O-7758 ...................................................................................................... 22
5.4 O-7759 ...................................................................................................... 24
5.5 O-7760 ...................................................................................................... 25
5.6 O-7761 ...................................................................................................... 26
5.7 JK263-2 ...................................................................................................... 27
5.8 ORG27569 ................................................................................................. 30
5.9 URB597 ..................................................................................................... 32
5.10 F0870064 .................................................................................................. 34
5.11 Result summary ........................................................................................ 35
6 Discussion ......................................................................................................... 37
6.1 FAAH inhibitors ......................................................................................... 37
6.1.1 O-77 series ............................................................................................ 37
6.1.2 URB597 ................................................................................................. 38
6.2 Allosteric modulators ................................................................................ 39
6.2.1 ORG27569 ............................................................................................. 39
6.2.2 JK263-2 .................................................................................................. 40
6.2.3 F0870064 .............................................................................................. 41
6.3 Potential therapeutic uses ........................................................................ 42
6.3.1 Pain and Inflammation .......................................................................... 42
6.3.2 Obesity .................................................................................................. 42
7 Conclusion ......................................................................................................... 43
8 References ........................................................................................................ 44
VI
Abbreviations
2-AG: 2-arachidonyl glycerol
7TM: seven-transmembrane-spanning receptor
Δ9-THC: Delta-9-tetrahydrocannabinol
ADA: Adenosine deaminase
AEA: Arachidonoyl ethanolamide (anandamide)
BSA: Bovine serum albumin
cAMP: Cyclic adenosine monophosphate
CB1: Cannabinoid receptor 1
CB2: Cannabinoid receptor 2
CBD: Cannabindiol
CNS: Central nervous system
CL: Confidence limit
CP55,940: (-)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-[4-hydroxypropyl]cyclohexan-1-ol
DMSO: Dimethyl sulphoxide
DTT: Dithiotreitol
EC50: Concentration with half-maximal efficacy
EDTA: Ethylenediaminetetraacetic acid
Emax: Maximal agonist effect
FAAH: Fatty acid amino hydrolase
G protein: Guanine nucleotide binding protein
GDP: Guanosine diphosphate
GPCR: G-protein coupled receptor
GPR55: G-protein coupled receptor 55
GTP: Guanosine triphosphate
GTPase: Guanosine triphosphatase
GTPγS: Guanosine-5’-O-(3-thio)-triphosphate
[35S]GTPγS: Guanosine-5’-O-(3-[35S]thio)-triphosphate
MS: Multiple Sclerosis
ORG27569: 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-ylphenyl)ethyl]amide
pEC50: negative logarithm of the concentration with half-maximal efficacy value
SEM: Standard Error Mean
URB597: Cyclohexylcarbamic acid 3´-carbamoyl-biphenyl-3-yl ester
Veh: Vehicle
1
1 Summary
Background and purpose: Endocannabinoid system provides a mean to treating
various diseases such as cancer, pain and obesity, but the side effects associated
with the orthosteric ligands can be fatal. Studies have shown FAAH inhibitors
(URB597) as a way to enhance efficacy of endogenous cannabinoid (AEA).
Alternatively, the discovery of allosteric site enables the “tuning” of the CB1
receptor with allosteric modulators. In this study I will investigate the
pharmacology of the potential drug candidates.
Experimental approach: For the 7 potential FAAH inhibitors and 3 potential
allosteric modulators, [35S]GTPγS assay and [3H]CP,55940 equilibrium binding assay
were performed on mouse brain membrane to determine its efficacy and affinity
at the mouse CB1 receptor.
Key results: FAAH inhibitor analogue O-7758 may enhance CP55,940 efficacy with
possibility of competing with [3H]CP55,950. Allosteric enhancer JK263-2 has
shown a marked increase in efficacy of both synthetic and endogenous CB1 agonist,
as well as enhancement of [3H]CP55,940 binding. Allosteric inhibitor ORG27569
also enhanced the affinity of the radiolabelled ligand, but completely abolished the
CP,55940 stimulation in [35S]GTPγS assay.
Conclusions and implications: Allosteric modulators and FAAH inhibitors may
provide a new way of treating various diseases using the endocannabinoid system
without the side effects of orthosteric ligand.
2
2 Introduction
2.1 Cannabinoids
Cannabis is one of the most common drugs being use in the UK. There are over 70
different compounds found inside the plant cannabis sativa (Elsohly and Slade,
2005). Delta-9-tetrahydrocannabinol (Δ9-THC) which is psychoactive and the non-
psychoactive compound, cannabindiol (CBD) are the two main constituents
responsible for its effects (Pertwee, 1999). Those cannabinoids found in plants are
known as phytocannabinoids.
Many synthetic cannabinoids such as CP55,940 are being made. Some of these
compounds show selectivity towards a group of receptor. In this case, CP55,940 is
an agonist which has higher affinity for CB1 receptor than CB2 receptor. As well as
being considerably more potent than its phytocannabinoid counterparts, Δ9-THC
(Pertwee, 1997).
Moreover, there is another type of cannabinoids, known as endocannabinoids,
which are made by the body itself (see Section 2.3).
3
2.2 Cannabinoid receptors
When cannabinoid enters the body, it exerts its effect by binding to cannabinoid
receptors. In mammals, there are at least two different types of cannabinoid
receptors present in the tissues, known as CB1 and CB2 (Pertwee and Ross, 2002).
Both receptors are G-protein coupled receptors (GPCR). Another GPCR known to
behave like cannabinoid receptor is GPR55 (Ross, 2009).
CB1 receptors are distributed heterogeneously in the brain. Areas that contain
high population of CB1 include the cerebral cortex, hippocampus, lateral caudate-
putamen, substantia nigra pars reticulate, globus pallidus, entopeduncular nucleus
and the molecular layer of the cerebellum. CB2 receptor is found in immune cells
and has a key role in cell differentiation and migration (Ross, 2007b). This project
will focus on CB1 receptors.
CB1 agonist and antagonist bind to the orthosteric site of the receptor. The
orthosteric site is defined as the primary binding site for the endogenous ligand on
a 7TM receptor (Ross, 2007a).
Not long ago, Sativex were licensed as a medicine in Canada, which contain nearly
1:1 of Δ9-THC and cannabindiol delivered via oromucosal spray for the treatment
of multiple sclerosis (Karschner et al., 2011).
However, there are also many side effects including depression, euphoria,
hallucination, memory loss and can lead to suicide.
4
Figure 2.1 Cannabinoids can either be made endogenously, synthetically or from plant. They can act on the orthosteric site of the CB1 receptor (Ross, 2007a).
5
Figure 2.3 Structure of 2-AG
2.3 Endocannabinoids
As the CB1 and CB2 receptors were
discovered, people start thinking
about why these receptors exist.
Were they made for the use of
cannabis?
Fortunately, these receptors are not made for the sole use of cannabis. The body
produce its own cannabinoids (also known as endocannabinoids). These
endocannabinoids were discovered by isolation.
The search for endogenous cannabinoids begun as early as in 1992 where a
lipophlic molecule was found to displace a potent cannabinoid ligand [3H]HU243
(Devane et al., 1992). This drug was identified as arachidonoyl ethanolamide and
named anandamide from “ananda”, the Sanskrit word for “bliss” (Pertwee, 2006).
Another endogenous ligand, 2-AG, were discovered soon after the discovery of
anandamide (Mechoulam et al., 1995)
Figure 2.2 Structure of Anandamide (AEA)
6
2.4 Fatty acid amino hydrolase (FAAH)
One of the disadvantages of using orthosteric modulators as a therapeutic tool is
that they often have many side effects as discussed previously. What if we could
make use of our own endocannabinoids by tweaking the endocannabinoid system?
FAAH may provide the answer. FAAH is one of the main enzymes responsible for
the breakdown of anandamide in the body (see Figure 2.4) (McKinney and Cravatt,
2005). The enzyme hydrolyses anandamide to arachidonic acid and ethanolamine
(Deutsch and Chin, 1993). Although not its main substrate, it also acts on 2-AG
(Goparaju et al., 1998).
By inhibiting the action of FAAH, the rate of breakdown of endogenous ligands is
slowed down and this increases the local concentration of the endogenous ligand
and as a result, increases in the ligand binding to the receptor. The enhancement
of endocannabinoids makes it a valuable tool as it does not have the mass
activation effect when a orthosteric ligand binds to the receptor.
7
Figure 2.4 FAAH is an enzyme that hydrolyses anandamide and 2-AG to its inactive form (Ross, 2007a).
8
2.5 Allosteric Modulation
The previous method enhances the action of endocannabinoids by preventing the
breakdown of the active metabolites and hence increases its local concentration.
A recent discovery of the existence of an allosteric site on the CB1 receptor has
provided us with an alternative method (Price et al., 2005).
An allosteric binding site of the receptor is defined as a site of ligand binding on the
seven-transmembrane-spanning receptor where it is topographically distinct from
the orthosteric binding site (Ross, 2007a).
The binding of such allosteric modulators causes a conformational change in the
shape of the receptor, and ultimately, changes the affinity and/or efficacy of drug
binding to the orthosteric site. This enables the fine-tuning of the receptor (Ross,
2007a).
Again, this method does not require a direct orthosteric ligand and prevents the
mass activation of the receptors by enhance or inhibit the action of endogenous
ligands.
9
Figure 2.5 Allosteric site can act as a fine tuning or “volume switch” of the CB1
receptor (Ross, 2007a).
10
2.6 Assays overview
2.6.1 GTPγS functional assay
In order to gain an insight of the mode of actions of the drugs interested,
[35S]GTPγS functional assay is a good place to start.
In the normal GPCR model, when an agonist binds to the receptor, it causes the
dissociation of the G protein. Those subunits, alpha (α), beta (β) and (γ) move
away and associated with second messengers. GDP, which was attached to the
alpha subunit, now get dissociated and GTP is swapped into its position due to the
increase in the affinity to bind with GTP as it dissociate from the rest of the protein.
The GTPase then comes into play, which hydrolyses the GTP- α complex to GDP- α
complex. The subunits are then reassociate with each other and return to the
GPCR.
In the [35S]GTPγS assay, radiolabelled GTP molecule, [35S]GTPγS, is added to the
test. Instead of binding to GTP, the alpha subunit now binds to the [35S]GTPγS
irreversibly. By collecting the [35S]GTPγS -α complex and measure the radioactivity
given off by the radioactive isotope, the efficacy of the given durg can be measured.
(Harrison and Traynor, 2003).
11
Figure 2.6 Diagrammatic representation of how [35S]GTPγS works. 1) Agonist binds with GPCR. 2) G protein subunits disassociate from GPCR. 3) alpha subunit moves away from GPCR and increase the affinity for GTP. 4) [35S]GTPγS displace GDP and form irreversible complex with alpha subunit.
1
2 3
4
4
12
2.6.2 Equilibrium binding assay
The equilibrium binding assay is an assay for determining the affinity for a
particular drug. In this project, [3H]CP55,940, a radiolabelled synthetic CB1 agonist
is used. When the agonist is added to the membrane, it binds with the receptor.
However, as this is an reversible action, the agonist can also diffuse away from the
orthosteric site. After a certain time, the net number of agonist binding and the
net number of agonist diffusing away at a given time would be the same. This is
called the equilibrium state.
Figure 2.7 Structure of CP55,940
Some of the factors that can alter the equilibrium state of the radiolabelled ligand
binding include changes in concentration, competition with other ligands or
changes to the receptor.
13
3 Aims
1. Discuss the potential of CB1 receptor as a therapeutic target, the use of
orthosteric modulators, FAAH inhibitors and allosteric modulators.
2. To determine whether the potential drug candidates has any effect on the
G-protein activities (i.e. efficacy) mediated by synthetic CB1 agonist
CP55,940 via CB1 receptors by using the [35S]GTPγS assay.
3. If the G-protein level of activity activated via CP55,940 is altered by the
presence of the drug, further testing would be done (i.e. affinity or efficacy
with different drug) to determine its mode of action at the CB1 receptors.
14
4 Materials and Methods
4.1 Materials
CP55,940 was obtained from Tocris (Bristol, UK). Bovine serum albumin (BSA),
dithiothreitol, GDP, GTPγS, Tris buffer and other chemicals not listed were all
obtained from Sigma-Aldrich (St Louis, MO, USA). [3H]CP55940 (160 Ci/mmol),
[35S]GTPγS (1250 Ci/mmol) and Ultima Gold XR scintillation buffer were obtained
from PerkinElmer Life Sciences Inc. (Boston, MA, USA). ORG-27569 was obtained
from Organon Research (Newhouse, Lanarkshire, Scotland). GTPγS and adenosine
deaminase were from Roche Diagnostic (Indianapolis,IN, USA). The GF/B glass-
fibre filters were obtained from Brandel Inc. (Gaitherburg, MD, USA).
Centrifugation Buffer
Buffer A ([35S]GTPγS)
Buffer B ([35S]GTPγS)
Buffer A (Binding)
Buffer B (Binding)
Tris HCl pH7.4(mM)
2 50 50 50 50
Tris Base (mM)
2 50 50 50 50
EDTA (mM)
2 2 1 2 1
Anhydrous MgCl2(mM)
5 5 3 5 3
Sucrose (mM)
320 - - - -
NaCl pH7.7 (mM)
- - - 100 100
Table 4.1 Composition of Centrifugation Buffer, Buffer A and Buffer B used during the preparation of mouse brain membrane.
15
4.2 Mouse brain membrane preparation
Whole brains from adult male MF1 mice were dissected and suspended in ice cold
centrifugation buffer*. The tissues were homogenised with an Ultra-Turrex
homogeniser. The homogenates were then centrifuged at 1600g for 10 minutes
and the resulting supernatant was collected (stored in ice).
The pellets were then resuspended in centrifugation buffer and centrifuged at
1600g for 10 minutes for the second time. The supernatant from the second
centrifugation is then combined with the first, and the combined supernatant goes
under centrifugation at 28000g for 20 minutes. The supernatant from the third
centrifugation is discarded and the pellet is resuspended with Buffer A* and
incubated in the water bath at 37°C for 10 minutes.
After that, the suspension was then centrifuged at 23000g for 20 minutes. The
supernatant is discarded and resuspended with Buffer A*. The suspension is left at
room temperature for 40 minutes before the final centrifugation at 11000g for
15minutes. The supernatant was discarded and resuspended with Buffer B*. The
suspension were then homogenise using a hand held homogeniser.
Protein assay was then performed using the Bio-Rad DC kit (Hercules, CA, USA) to
determine its concentration. Depending on the assay, 1mg/ml and 0.15mg/ml
were made for [35S]GTPγS and equilibrium binding respectively. This is then stored
at -80°C until the day of experiment.
All centrifugation procedures were carried out at 4°C.
* Depending on the assay, different Buffer A and Buffer B were used; please refer
to Table 4.1 for chemical compositions.
16
4.3 [35S]GTPγS functional assay
The buffer required for this assay is Tris BSA (50mM Tris HCl, 50mM Tris Base and
0.1% BSA). The following chemicals were then added to the buffer (1mM EDTA,
5mM MgCl2, 100mM NaCl, 1mM DTT and 30µM GDP). The preparation of the
assay can be seen from Figure 4.1.
The mouse brain membrane (0.15mg/ml) were thawed and incubated with
adenosine deaminase (ADA, 0.5U/ml) at 30°C for 30 minutes. The membranes
were then incubated again, with agonist, , and vehicle or modulator for further 60
minutes at 30°C in assay buffer in the presence of 0.1nM [35S]GTPγS in a total assay
volume of 500µl.
Binding was initiated by the addition of [35S]GTPγS. Nonspecific binding was
measured in the presence of 30µM GTPγS. The reaction was stopped by rapid
vacuum filtration with Tris BSA using 24-well sampling manifold (Brandel cell
harvester) and GF/B filters that had been soaked in Tris BSA for at least 24 hours.
The reaction tubes were washed five times with ice-cold Tris BSA.
The filters were then place in the oven for at least 60 minutes and then soaked
with 5ml of scintillation fluid (Ultima Gold XR) for at least 60 minutes. The
radioactivity given off by the [35S]GTPγS-α complex were then measured by liquid
scintillation spectrometry.
17
Figu
re 4
.1 W
orkf
low
of [
35S]
GTPγ
S fu
nctio
nal a
ssay
pre
para
tion.
18
4.4 Equilibrium binding assay
Mouse brain membrane of 1mg/ml was thawed. The buffer used in the assay is
Tris BSA. The binding assay were performed with the CB1 agonist [3H]CP55,940
(0.7mM). The compound of interest was diluted in the same way as CP55,940 did
in Figure 4.1. The buffer, [3H] CP55,940, membrane and vehicle or drug of interest
is added in a total assay volume of 500µl. The binding was initiated by the addition
of membrane. This assay is then incubated in a 37°C water bath for 60 minutes.
The reaction was stopped by rapid vacuum filtration with Tris BSA using 24-well
sampling manifold (Brandel cell harvester) and GF/B filters that had been soaked in
Tris BSA for at least 24 hours. The reaction tubes were washed five times with ice-
cold Tris BSA.
The filters were then place in the oven for at least 60 minutes and then soaked
with 5ml of scintillation fluid (Ultima Gold XR) for at least 60 minutes. The
radioactivity given off by the [3H] CP55,940 were then measured by liquid
scintillation spectrometry.
19
4.5 Mathematical analysis
Analyses of data were conducted using GraphPad Prism 5 software (GraphPad
Software, San Diego, CA). The raw data (count) from the liquid scintillation were
converted to percentage stimulation and percentage displacement for[35S]GTPγS
and equilibrium binding assay respectively. Values were subtracted from the basal
value obtained. All the values above were calculated by nonlinear regression
analysis using the equation for a sigmoid concentration-response curve (GraphPad
Prism).
= −50 50logpEC EC
Equation 1 pEC50 is the negative logarithm of the agonist EC50 value.
Results are expressed as the mean ± S.E.M. in the case of Emax (the maximal agonist
effect) of n (n = sample size) experiments. The pEC50 values were expressed as
percentage with 95% confidence limits.
20
5 Results
5.1 O-7756
This is the first drug in the O-77 series. In the [35S]GTPγS function assay (Figure 5.1),
with the presence of DMSO vehicle, CP55,940 produced a pEC50 and Emax values of
7.49 ± 0.296 and 97.5 (95% confidence limits, 76.7-118.4) respectively. In the
presence of 1µM O-7756, the curve is largely the same as the curve with the
presence of vehicle. The pEC50 and Emax values were 6.87 ± 0.515 and 110.0 (95%
confidence limits, 52.3-167.7), showing no significant statistical difference between
them.
-10 -9 -8 -7 -6 -5 -4
-200
20406080
100120
DMSO1µM O-7756
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.1: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or O-7756. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 5).
21
5.2 O-7757
As the first drug in the series does not seem to have any effect, the second drug O-
7757 was tested. However, the results were similar to the first. In the [35S]GTPγS
function assay (Figure 5.2), with the presence of DMSO vehicle, CP55,940
produced a pEC50 and Emax values of 7.13 ± 0.198 and 96.0% (95% confidence limits,
77.9-114.1) respectively. In the presence of 1µM O-7757, the curve is largely the
same as the curve with the presence of vehicle. The pEC50 and Emax values were
7.07 ± 0.268 and 92.8% (95% confidence limits, 70.2-115.4), showing no significant
statistical difference between them.
-10 -9 -8 -7 -6 -5 -4
-200
20406080
100120 DMSO
1µM O-7757
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.2: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or O-7757. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 4).
22
5.3 O-7758
In the [35S]GTPγS function assay (Figure 5.3), with the presence of DMSO vehicle,
CP55,940 produced a pEC50 and Emax values of 7.62 ± 0.468 and 79.8 (95%
confidence limits, 60.0-99.7) respectively. In the presence of 1µM O-7758, the
curve is largely the same as the curve with the presence of vehicle. However, the
pEC50 and Emax values were 6.75 ± 0.219 and 100.8 (95% confidence limits, 80.1-
121.5), suggesting in the presence of 1µM O-7758, it enhances the percentage
stimulation by CP55,940.
-10 -9 -8 -7 -6 -5 -4
-200
20406080
100120 DMSO
1µM O-7758
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.3: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or O-7758. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 6).
23
In the equilibrium binding assay (Figure 5.4), the reference CP,55940 has a pEC50
and Emax values of 7.62 ± 0.468 and 79.8 (95% confidence limits, 60.0-99.7)
respectively. The O-7758 has a pEC50 and Emax values of 6.01 ± 0.376 and 87.4 (95%
confidence limits, 57.9-116.9). The drug O-7758 is displacing [3H]CP55,940,
demonstrating a similar curve as unlabelled CP55,940.
-11 -10 -9 -8 -7 -6 -5 -4
-200
20406080
100120 O-7758
CP55940
log concentration (M)
% d
ispl
acem
ent o
f [3 H
]CP5
5940
Figure 5.4: Equilibrium binding of [3H]CP55,940 (0.7 nM) in mouse brain membranes in the presence of unlabelled ligand the O-7758. Each symbol represents the mean percentage of displacement of [3H]CP55,940 ± S.E.M. (n = 6).
24
5.4 O-7759
This is the fourth drug in the O77 series. In the [35S]GTPγS function assay (Figure
5.5), with the presence of DMSO vehicle, CP55,940 produced a pEC50 and Emax
values of 7.73 ± 0.663 and 61.8 (95% confidence limits, 41.4-82.2) respectively. In
the presence of 1µM O-7759, the curve is largely the same as the curve with the
presence of vehicle. The pEC50 and Emax values were 6.64 ± 0.534 and 89.9 (95%
confidence limits, 22.2-154.3), showing no significant statistical difference between
them.
-10 -9 -8 -7 -6 -5 -4
-200
20406080
100120 DMSO
1µM O-7759
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.5: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or O-7759. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 7).
25
5.5 O-7760
This is the fifth, In the [35S]GTPγS function assay (Figure 5.6), with the presence of
DMSO vehicle, CP55,940 produced a pEC50 and Emax values of 7.39 ± 0.160 and 70.3
(95% confidence limits, 62.0-78.5) respectively. In the presence of 1µM O-7760,
the curve is largely the same as the curve with the presence of vehicle. The pEC50
and Emax values were 6.99 ± 0.408 and 89.28 (95% confidence limits, 36.7-141.9),
showing no significant statistical difference between them.
-10 -9 -8 -7 -6 -5 -4
-200
20406080
100120 DMSO
1µM O-7760
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.6: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or O-7760. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 6).
26
5.6 O-7761
This is the final drug in the series. In the [35S]GTPγS function assay (Figure 5.7),
with the presence of DMSO vehicle, CP55,940 produced a pEC50 and Emax values of
7.54 ± 0.398 and 67.8 (95% confidence limits, 43.6-91.9) respectively. In the
presence of 1µM O-7761, the curve is largely the same as the curve with the
presence of vehicle. The pEC50 and Emax values were 6.59 ± 0.395 and 73.7 (95%
confidence limits, 31.9-115.5). Again, it shows no significant statistical difference
between them.
-10 -9 -8 -7 -6 -5 -4
-200
20406080
100120 DMSO
1µM O-7761
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.7: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or O-7761. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 8).
27
5.7 JK263-2
In the [35S]GTPγS function assay (Figure 5.8), with the presence of DMSO vehicle,
CP55,940 produced a pEC50 and Emax values of 7.79 ± 0.226 and 41.4 (95%
confidence limits, 35.0-47.8) respectively. In the presence of 1µM JK263-2, the
curve is largely the same as the curve with the presence of vehicle. The pEC50 and
Emax values were 7.40 ± 0.465 and 55.2 (95% confidence limits, 42.25-68.23).
The curve with the presence of JK263-2 has shifted upward relative to the DMSO
curve. This suggests that there is an enhancement of the percentage stimulation
by CP55,940. An equilibrium binding assay has been carried out to determine its
effect on the CP55,940 binding.
-10 -9 -8 -7 -6 -5 -4
-20
0
20
40
60
80
100 DMSO1µM JK263-2
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.8: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or JK263-2. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 6).
28
The enhancement observed in the [35S]GTPγS assay was exciting. The result for
the equilibrium binding assay was also interesting (Figure 5.9). The reference
CP55,940 curve has a pEC50 and Emax values were 8.85 ± 0.164 and 102.6 (95%
confidence limits, 95.1-110.1) respectively. In the presence of JK263-2, the pEC50
and Emax values were 6.68 ± 0.406 and 109.2 (95% confidence limits, 63.8-154.6)*.
The asterisk here indicates that the values are negative. This means instead of
displacing the [3H]CP55,940, it enhances the binding of the radiolabelled ligand.
-11 -10 -9 -8 -7 -6 -5 -4
-100-80-60-40-20
020406080
100120
JK-263-2CP55940
log concentration (M)
% d
ispl
acem
ent o
f [3 H
]CP5
5940
Figure 5.9: Equilibrium binding of [3H]CP55,940 (0.7 nM) in mouse brain membranes in the presence of unlabelled ligand the JK-263-2. Each symbol represents the mean percentage of displacement of [3H]CP55,940 ± S.E.M. (n = 12).
29
Further testing has been done with JK263-2, instead of running [35S]GTPγS assay
with synthetic agonist CP55,940, endogenous ligand anandamide is used in the
assay. In the [35S]GTPγS function assay (Figure 5.10), with the presence of DMSO
vehicle, anandamide produced pEC50 and Emax values of 5.96 ± 0.207 and 61.5 (95%
confidence limits, 47.3-75.6) respectively. In the presence of 100nM JK263-2, the
curve is largely the same as the curve with the presence of vehicle. The pEC50 and
Emax values were 5.91 ± 0.214 and 110.3 (95% confidence limits, 81.6-139.0),
-10 -9 -8 -7 -6 -5 -4
-200
20406080
100120
DMSO100nM JK263-2
AEA log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.10: Stimulation of binding of [35S]GTPγS to mouse brain membranes by anandamide (AEA) in the presence of vehicle (DMSO) or JK263-2. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 4).
30
5.8 ORG27569
In the [35S]GTPγS function assay (Figure 5.11), with the presence of DMSO vehicle,
CP55,940 produced a pEC50 and Emax values of 6.96 ± 0.379 and 41.7 (95%
confidence limits, 25.4-58.1) respectively. In the presence of 1µM ORG27569, the
curve has flatten and shifted down to the bottom. The pEC50 and Emax values were
6.28 ± 0.983 and -9.89 (95% confidence limits, -16.4-(-3.39)). This is significantly
different relative to vehicle.
-10 -9 -8 -7 -6 -5 -4
-20
0
20
40
60DMSO1µM ORG27569
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.11: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or ORG27569. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 6).
31
With ORG27569, the functional assay shows a significant effect, this is the same for
the equilibrium binding assay. The result for the equilibrium binding assay was
also interesting (Figure 5.12). The reference CP55,940 curve has a pEC50 and Emax
values were 8.85 ± 0.164 and 102.6 (95% confidence limits, 95.1-110.1)
respectively. In the presence of ORG27569, the pEC50 and Emax values were 5.77 ±
0.208 and 95.9 (95% confidence limits, 70.3-121.5)*. The asterisk here indicates
that the values are negative. This means instead of displacing the [3H]CP55,940, it
enhances the binding of the radiolabelled ligand.
-10 -9 -8 -7 -6 -5 -4
-100-80-60-40-20
020406080
100120
ORG27569CP55940
log concentration (M)
% d
ispl
acem
ent o
f [3 H
]CP5
5940
Figure 5.12: Equilibrium binding of [3H]CP55,940 (0.7 nM) in mouse brain membranes in the presence of unlabelled ligand the ORG-27569. Each symbol represents the mean percentage of displacement of [3H]CP55,940 ± S.E.M. (n = 6).
32
5.9 URB597
In the [35S]GTPγS function assay (Figure 5.13), with the presence of DMSO vehicle,
CP55,940 produced a pEC50 and Emax values of 7.05 ± 0.218 and 52.57 (95%
confidence limits, 43.5-61.6) respectively. In the presence of 1µM URB597, the
curve has shifted downwards relative to the curve with the presence of vehicle.
The pEC50 and Emax values were 7.55 ± 0.351 and 25.5 (95% confidence limits, 16.1-
35.0). The effect was significant and hence an equilibrium binding assay would be
a good way to determines the affinity.
-10 -9 -8 -7 -6 -5 -4
-20
0
20
40
60
80
100 DMSO1µM URB597
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.13: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or URB597. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 5).
33
The result for the equilibrium binding assay was also interesting (Figure 5.14). The
reference CP55,940 curve has a pEC50 and Emax values were 8.85 ± 0.164 and 102.6
(95% confidence limits, 95.1-110.1) respectively. In the presence of URB597, the
pEC50 and Emax values were 5.95 ± 0.437 and 22.8 (95% confidence limits, 7.10-
38.5). The curve is shown as a flat line at the bottom, with little displacement of
[3H]CP55,940.
-11 -10 -9 -8 -7 -6 -5 -4
-200
20406080
100120 URB597
CP55940
log concentration (M)
% d
ispl
acem
ent o
f [3 H
]CP5
5940
Figure 5.14: Equilibrium binding of [3H]CP55,940 (0.7 nM) in mouse brain membranes in the presence of unlabelled ligand the URB597. Each symbol represents the mean percentage of displacement of [3H]CP55,940 ± S.E.M. (n = 6).
34
5.10 F0870064
In the [35S]GTPγS function assay (Figure 5.2), with the presence of DMSO vehicle,
CP55,940 produced a pEC50 and Emax values of 7.65 ± 0.278and 73.5 (95%
confidence limits, 60.3-86.7) respectively. In the presence of 1µM F0870064, the
curve is largely the same as the curve with the presence of vehicle. The pEC50 and
Emax values were 7.61 ± 0.309 and 65.2 (95% confidence limits, 55.4-75.0), showing
no significant statistical difference between them.
-10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100 DMSO1µM F0870064
CP55940 log concentration (M)
% s
timul
atio
n ab
ove
basa
l
Figure 5.15: Stimulation of binding of [35S]GTPγS to mouse brain membranes by CP55,940 in the presence of vehicle (DMSO) or F0870064. Each symbol represents the mean percentage of stimulation above basal ± S.E.M. (n = 6).
35
5.11 Result summary
pEC50 Emax (95% CL) (%)
DMSO 7.49 ± 0.296 97.5 (76.7-118.4) O7756 6.87 ± 0.515 110.0 (52.3-167.7) DMSO 7.13 ± 0.198 96.0 (77.9-114.1) O7757 7.07 ± 0.268 92.8 (70.2-115.4) DMSO 7.62 ± 0.468 79.8 (60.0-99.7) O7758 6.75 ± 0.219 100.8 (80.1-121.5) DMSO 7.73 ± 0.663 61.8 (41.4-82.2) O7759 6.64 ± 0.534 89.9 (22.2-154.3) DMSO 7.39 ± 0.160 70.3 (62.0-78.5) O7760 6.99 ± 0.408 89.28 (36.7-141.9) DMSO 7.54 ± 0.398 67.8 (43.6-91.9) O7761 6.59 ± 0.395 73.7 (31.9-115.5) DMSO 7.79 ± 0.226 41.4 (35.0-47.8) JK263-2 7.40 ± 0.465 55.2 (42.25-68.23) DMSO 6.96 ± 0.379 41.7 (25.4-58.1) ORG27569 6.28 ± 0.983 -9.89 (-16.4-(-3.39)) DMSO 7.05 ± 0.218 52.57 (43.5-61.6) URB597 7.55 ± 0.351 25.5 (16.1-35.0) DMSO 7.65 ± 0.278 73.5 (60.3-86.7) F0870064 7.61 ± 0.309 65.2 (55.4-75.0)
Table 5.1 pEC50 and Emax values for vehicle (DMSO) and the drugs in the [35S]GTPγS assay with CP55,940. The value for the corresponding (paired) vehicle is above the drug of interest.
36
pEC50 Emax (95% CL) (%)
CP55940 8.85 ± 0.164 102.6 (95.1-110.1) O-7758 6.01 ± 0.376 87.4 (57.9-116.9) JK263-2 6.68 ± 0.406 109.2 (63.8-154.6)* ORG27569 5.77 ± 0.208 95.9 (70.3-121.5)* URB597 5.95 ± 0.437 22.8 (7.10-38.5)
Table 5.2 pEC50 and Emax values the drugs tested against [3H]CP55,940 in the equilibrium binding assay. Asterisk indicates the values go in the opposite direction, which is an enhancing the binding of [3H]CP55,940 instead of displacement.
pEC50 Emax (95% CL) (%)
DMSO 5.96 ± 0.207 61.5 (47.3-75.6) JK263-2 5.91 ± 0.214 110.3 (81.6-139.0)
Table 5.3 pEC50 and Emax values for vehicle (DMSO) and the drug (JK263-2) in the [35S]GTPγS assay with anandamide.
37
6 Discussion
6.1 FAAH inhibitors
6.1.1 O-77 series
This is a completely new series of drugs developed based on a FAAH inhibitor. The
initial intention was to see if these FAAH inhibitor analogues would demonstrate
similar behaviour as the original FAAH inhibitor molecule.
There were six drugs in this series, from O-7756 up to O-7761. The first two drugs
in the series, O-7756 and O-7757, shows no change in the CP55,940 induced G
protein activity in the [35S]GTPγS functional assay. O-7759, O-7760 and O-7761
showed similar behaviour, with no signs of significant effect to the efficacy of
CP55,940.
Despite the aforementioned five drugs not giving much hope, O-7758 shows an
increased in maximal effect of the [35S]GTPγS stimulation by CP55,940, although
not significant. It would make sense to have a closer look with an equilibrium
binding assay. The results shows a displacement of [3H]CP55,940 by O-7758.
O-7758 has shown an interesting behaviour, as a potential FAAH inhibitor, it
competes for the orthosteric site. The result needs to be verified by more
repetition.
38
6.1.2 URB597
This is a well-known FAAH inhibitor (also known as KDS-4103) (Piomelli et al., 2006).
It is one of the most promising FAAH inhibitor as an innovative antidepressant
(Maccarrone et al., 2010).
The results obtain from the function assay was not consistent with literatures, and
as a result, a conclusion cannot be drawn on this drug. However, the equilibrium
binding assay did work correctly, and as previously literature have stated that it
has no affinity for orthosteric site (Piomelli et al., 2006).
The only information obtain from this drug is that it does not compete for the
orthosteric site. There can be a variety of reason for the error appear in the
functional assay, including the preparation of membrane and buffer, as well as the
shelf-life of the drug, how well the experiments were carried out. Unfortunately
there is no single answer but repetition should eliminate the error.
Figure 6.1 Structure of URB597
39
6.2 Allosteric modulators
6.2.1 ORG27569
ORG27569 was one of the first drugs discovered for its ability to bind to the
allosteric site of the CB1 receptor (Price et al., 2005). In the [35S]GTPγS assay, with
the presence of 1µM ORG27569, the percentage stimulation above basal by
CP55,940 has completely abolished. A similar compound, ORG29647 has shown
similar results (Price et al., 2005).
The equilibrium binding assay was fascinating. In the presence of ORG27569, the
displacement of [3H]CP55,940 was negative, in contrast with the presence of non-
radiolabelled CP55,940. Instead of displacing the radiolabelled agonist, the binding
of the ligand was enhanced. This is consistent with the results found in the
literature, which has the most marked effect out of the ORG compounds (Price et
al., 2005).
Figure 6.2 Structure of ORG27569
40
6.2.2 JK263-2
JK263-2 is a newly discovered allosteric enhancer, there are no published data
available at the time of this report is being written. The results has shown that in
the presence of the drug, both CP55,940 and anandamide efficacies were
enhanced.
The equilibrium binding assay suggests that, rather than competing for the
orthosteric site, JK263-2 enhances the affinity of [3H]CP55,940 binding. This
behaviour was similar to the ORG27569, a known allosteric inhibitor (Price et al.,
2005).
With JK263-2, there was an opportunity for testing with anandamide, an
endogenous CB1 agonist. At 100nM anandamide was able to increase the efficacy
of CP55,940.
JK263-2 as an allosteric enhancer would have similar outcome as if it was a FAAH
inhibitor. At this stage the specificity of the drug is not known, but if proven to be
CB1 specific, the drug would be better than FAAH inhibitor which also hydrolyses 2-
AG.
41
6.2.3 F0870064
This is a relatively new drug with very limited published data available. The only
literature source available suggests it is a putative allosteric enhancer of the CB1
receptor (Baillie et al., 2009).
From the results, F0870064 does not appears to have a significant effect on the
ability of CP55,940 to stimulate [35S]GTPγS turnover. This is consistent with the
literature source.
However, the previous study have shown that in the presence of F0870064 with
either anandamide (endogenous), WIN55212-2 (synthetic) or Δ9-THC
(phytocannabinoid), the efficacy of the agonist were significantly increased (Baillie
et al., 2009).
This would have been an interesting drug to undergo further testing such as
equilibrium binding assay to determine its affinity, or functional assay with
anandamide to confirm how it affecting the efficacy of endogenous CB1 agonist.
42
6.3 Potential therapeutic uses
6.3.1 Pain and Inflammation
URB597 has been through much in vitro and in vivo experimentation and has
shown positive signs as a drug treatment for pain (Schlosburg et al., 2009).
JK263-2 as an allosteric enhancer would be able to amplify the signal modulated by
the endogenous ligand activating the CB1 receptors. This would prevent the mass
activation if a direct orthosteric agonist is administered and should present little or
no side effects..
6.3.2 Obesity
Obesity has been one of the major costs to the NHS in the UK and is also a global
epidemic (Ogden et al., 2007). A low cost treatment is needed to reduce the cost.
In many cases, surgery is needed and this may provide the answer to it.
ORG27569 as an allosteric inhibitor would be able to lower the CB1 activated by
the endogenous ligand and therefore it should lower the signal for appetite, which
would reduce food intake by the person and ultimately provide a cure to obesity.
43
7 Conclusion
In this project, 10 drugs were tested. 5 out of 6 from the O77 series did not show
any signs of FAAH inhibitor actions. O-7758 has enhanced CP55,940 but also
competes with it for the orthosteric site. URB597 results obtained were incorrect.
The allosteric drugs were the only ones that have demonstrate positive results.
Both ORG27569 and JK263-2 have shown marked increase in affinity and change in
efficacy. F0870064 did not do much with synthetic ligand but results would have
been fascinating if it was done with anandamide.
There were many drugs that additional testing could have been done to investigate
their pharmacology further. Assays such as pERK, cAMP and β-arrestin would have
provided some more insightful results. Some of the results would have benefited
from extra readings. However, due to the time limitation of this project it was
impossible to carry out those extra analyses.
The future directions will obviously include further in vitro testing of the current
and new potential FAAH inhibitors and allosteric modulators with anandamide. 2-
AG is another endogenous cannabinoid which has been proved difficult to test in
vitro. One of the original intentions of the project was to investigate in vitro 2-AG
analysis. Unfortunately, due to unforeseen circumstances this was unable to
proceed.
44
8 References
Baillie, GL, Whyte, J, Pertwee, RG, Ross, RA (2009). Allosteric enhancement of the
cannabinoid CB1 receptors. Proceedings of the British Pharmacological Society,
London, 7, 039P
Deutsch, DG, Chin, SA (1993). Enzymatic synthesis and degradation of anandamide,
a cannabinoid receptor agonist. Biochem Pharmacol 46: 791-6.
Devane, WA, Hanus, L, Breuer, A, Pertwee, RG, Stevenson, LA, Griffin, G, et al.
(1992). Isolation and structure of a brain constituent that binds to the cannabinoid
receptor. Science 258: 1946.
Elsohly, MA, Slade, D (2005). Chemical constituents of marijuana: the complex
mixture of natural cannabinoids. Life Sci 78: 539-48.
Goparaju, SK, Ueda, N, Yamaguchi, H & Yamamoto, S (1998). Anandamide
amidohydrolase reacting with 2-arachidonoylglycerol, another cannabinoid
receptor ligand. FEBS Lett 422: 69-73.
Harrison, C, Traynor, JR (2003). The [35S]GTPγS binding assay: approaches and
applications in pharmacology. Life Sci 74: 489-508.
Karschner, E, Darwin, W, McMahon, R, Liu, F, Wright, S, Goodwin, R, et al. (2011).
Subjective and Physiological Effects After Controlled Sativex and Oral THC
Administration. Clinical Pharmacology & Therapeutics 89: 400-7.
Maccarrone, M, Gasperi, V, Catani, MV, Diep, TA, Dainese, E, Hansen, HS, et al.
(2010). The endocannabinoid system and its relevance for nutrition. Annu Rev Nutr
30: 423-40.
45
McKinney, MK, Cravatt, BF (2005). Structure and function of fatty acid amide
hydrolase. Annu Rev Biochem 74: 411-32.
Mechoulam, R, Ben-Shabat, S, Hanus, L, Ligumsky, M, Kaminski, NE, Schatz, AR, et
al. (1995). Identification of an endogenous 2-monoglyceride, present in canine gut,
that binds to cannabinoid receptors. Biochem Pharmacol 50: 83-90.
Ogden, CL, Yanovski, SZ, Carroll, MD & Flegal, KM (2007). The epidemiology of
obesity. Gastroenterology 132: 2087-102.
Pertwee, RG (1999). Pharmacology of cannabinoid receptor ligands. Curr Med
Chem 6: 635-64.
Pertwee, RG (1997). Pharmacology of cannabinoid CB1 and CB2 receptors.
Pharmacol Ther 74: 129-80.
Pertwee, R, Ross, R (2002). Cannabinoid receptors and their ligands* 1.
Prostaglandins, leukotrienes and essential fatty acids 66: 101-21.
Pertwee, RG (2006). Cannabinoid pharmacology: the first 66 years. Br J Pharmacol
147 Suppl 1: S163-71.
Piomelli, D, Tarzia, G, Duranti, A, Tontini, A, Mor, M, Compton, TR, et al. (2006).
Pharmacological profile of the selective FAAH inhibitor KDS-4103 (URB597). CNS
Drug Rev 12: 21-38.
46
Price, MR, Baillie, GL, Thomas, A, Stevenson, LA, Easson, M, Goodwin, R, et al.
(2005). Allosteric modulation of the cannabinoid CB1 receptor. Mol Pharmacol 68:
1484-95.
Ross, RA (2009). The enigmatic pharmacology of GPR55. Trends Pharmacol Sci 30:
156-63.
Ross, RA (2007a). Allosterism and cannabinoid CB(1) receptors: the shape of things
to come. Trends Pharmacol Sci 28: 567-72.
Ross, RA (2007b). Tuning the endocannabinoid system: allosteric modulators of the
CB1 receptor. Br J Pharmacol 152: 565-6.
Schlosburg, JE, Kinsey, SG & Lichtman, AH (2009). Targeting fatty acid amide
hydrolase (FAAH) to treat pain and inflammation. AAPS J 11: 39-44.