new cannabinoid therapeutics

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)

http://cnr1.eu/

http://www.refworks.com/refworks2

http://www.brjpharmacol.org/view/0/authorInformation.html

http://www.abdn.ac.uk/documents/style-guide.pdf

http://webct.abdn.ac.uk/

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


% s

timul

atio

n ab

ove

basa

l


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


% s

timul

atio

n ab

ove

basa

l


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


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


% s

timul

atio

n ab

ove

basa

l


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


% s

timul

atio

n ab

ove

basa

l


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


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


% s

timul

atio

n ab

ove

basa

l


27

5.7 JK263-2



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


% 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


% 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


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



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


% 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


% 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



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


% 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


% 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


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


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


% 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

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