Pharmacology of Palmitoylethanolamide and ... - DiVA-Portal

Pharmacology of

Palmitoylethanolamide and Related

Compounds

by Kent-Olov Jonsson

Umeå 2005

Department of Pharmacology and Clinical Neuroscience, Pharmacology, Umeå University

Printed by Solfjädern Offset AB, Umeå, Sweden Copyright © Kent-Olov Jonsson, 2005

ISBN 91-7305-821-1

ii

ABSTRACT

Anandamide (AEA) is an endogenous fatty acid which activates the same cannabinoid receptors as ∆9-tetrahydrocannabinol, the psychoactive substance in marijuana. In vivo, AEA exerts a number of actions including effects upon pain and inflammation. However, AEA has a short duration of action since it is rapidly metabolised, primarily by the intracellular enzyme fatty acid amide hydrolase (FAAH).

The general aim of this thesis has been to identify and characterize compounds capable of preventing the metabolism of AEA. The chemical approach was based on the endogenous anti-inflammatory compound palmitoylethanolamide (PEA), a compound related to AEA with the ability to inhibit AEA degradation by substrate competition, but without the ability to directly activate cannabinoid receptors.

A number of compounds were identified as inhibitors of rat brain FAAH in the initial in vitro studies, without having major affinity for the cannabinoid receptors. In particular, palmitoylisopropylamide (PIA) was found to reduce the metabolism of AEA in intact C6 glioma cells with potency similar to the prototypical AEA reuptake inhibitor AM404. This compound was in addition found to exert less effect upon C6 glioma cell proliferation than either AM404 or the closely related uptake inhibitor VDM11. To evaluate if PIA was effective in vivo, a model of mast cell dependent inflammation, oedema of the ear following local injection of compound 48/80, was set up using anaesthetised mice. Initially, a CB2 cannabinoid receptor-selective agonist was used to probe the model and demonstrated to produce an anti-oedema effect. In contrast, the compound was inactive in vitro in skin slice preparations. PIA showed a similar pattern, although there was a large variation in responses which affected the significance of the result obtained, as did the vehicle used to dissolve the compound.

Taken together, the present data would suggest that PIA can inhibit the degradation of AEA without having deleterious effects upon cell proliferation or affinity for the cannabinoid receptors. Further experimentation is necessary to elucidate the usef

Keywords: anandamide, cannabinoid, FAA mitoylethanolamide, palmitoylisopropylamide, inflam

ulness of this compound in vivo.

H, palmation, mast cells

iii

CONTENTS

CO

eral Background 9

18

34

37 β-Hexosaminidase release 38

38 40

NTENTS i

ORIGINAL PAPERS 6

EXPLANATIONS OF ABBREVIATIONS 7

INTRODUCTION 9 GenThe endocannabinoid system 9 Cannabinoid receptor ligands 13

In vivo effects of cannabinoid receptor activation 15 Anandamide (AEA) 17 N-acylethanolamines (NAEs) Synthesis and release of anandamide 19 Anandamide uptake 21 Anandamide degradation 23

FAAH inhibitors 24 Therapeutic potential of FAAH inhibitors 26

AIMS OF THE STUDY 28

METHODS AND METHODOLOGICAL CONSIDERATIONS 29 FAAH assay 29 Anandamide uptake in RBL-2H3 and C6 cells 31 Radioligand binding experiments 32 Cell cultures 33 Cell proliferation assay 34

Analysis of cell density Analysis of caspase activity 34

Animals 35 Plasma extravasation in mouse ear pinna 36 Mice paw skin release

Immunohistochemistry and PCR Data analyses and statistics

4

RESULTS AND DISCUSSION 41 Inhibition of FAAH catalysed anandamide hydrolysis 41 Interaction with CB1 and CB2 cannabinoid receptors 47 Inhibition of anandamide uptake 47 Inhibition of anandamide uptake and hydrolysis in intact C6 cells 49 Effects of PIA, AM404 and VDM11 upon cell proliferation 51 Effects of PIA and JWH133 upon inflammation in vivo and in vitro 53

GENERAL CONCLUSIONS AND FUTURE PERSPECTIVES 57

ACKNOWLEDGEMENTS 59

REFERENCES 61

APPENDIX 81

5

ORIG S

erals.

DM, Tiger G, Fowler CJ. Effects

oid anandamide. British Journal of Pharmacology

II. Vand M. Modifications

in N-palmitoylethanolamine: synthesis and evaluat the metabolism of anandamide. Journal

1448.

III. , Jacobsson SOP, Vandevoorde S, Lambert DFowler CJ. AM404 and VDM 11 non-specifically inhibit C6 glioma cprolif cellular accumulation of t

de. Archives of Toxicology (2003), 77: 201-207.

eptor-select33 reduces mast cell oedema in response to compound 48/

se from skin slices in vi

V. J. Characterisation of palmitoylisopropylamide

mast and in vivo. In manuscript.

pers were made with permission from the respective publishers.

INAL PAPER

The present thesis is based on the following papers which will be referred to in text by their roman num

I. Jonsson KO, Vandevoorde S, Lambert of

homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabin(2001), 133: 1263-1275.

evoorde S, Jonsson KO, Fowler CJ, Lambert D of the ethanolamine head ion of new agents interfering with of Medicinal Chemistry (2003), 46: 1440-

Jonsson KO, Andersson A M, ell

eration at concentrations used to block the he endocannabinoid anandami

IV. Jonsson KO, Persson E, Fowler CJ. The cannabinoid CB2 rec ive

agonist JWH1 80 in vivo but not the release of ß-hexosaminida tro. Submitted for publication.

Jonsson KO, Fowler C in cell dependent systems in vitro

Reprints of the published pa

6

EXPLANATIONS OF ABBREVIATIONS

trahydrocannabinol 2-AG AC ACEA yl-2-chloroethylamide AEA AM12AM25

agonist

CB1 cannabinoid receptor type 1 CB2 cannabinoid receptor type 2 Compound 48/80 mast cell degranulator CP55,940 non-selective cannbinoid receptor agonist FAAH fatty acid amide hydrolase (EC 3.5.1.4) FAAH-NS neurospecific FAAH knockout mice HU210 non-selective cannabinoid receptor agonist HU308 CB2 receptor-selective agonist IC50 concentration producing 50 % of the maximal

attainable inhibition JWH133 CB2 receptor-selective agonist MAFP methylarachidonylfluorophosphonate,

FAAH inhibitor NAEs N-acylethanolamines NAPE N-acylphosphatidylethanolamine NAPE-PLD N-acylphosphatidylethanolamine specific

phospholipase D NAT N-acyltransferase O-1812 CB1 receptor-selective agonist OEA oleoylethanolamide

∆9-THC ∆9-te 2-arachidonylglycerol adenylyl cyclase

arachidon anandamide (arachidonylethanolamide)

41 CB2 receptor-selective agonist 1 CB1 receptor-selective antagonist/inverse agonist

AM404 AEA uptake inhibitor AM630 CB receptor-selective antagonist/inverse 2

ATMK or ATFMK arachidonoyltrifluoromethylketone, FAAH inhibitor

BSA bovine serum albumine cAMP cyclic adenosine monophosophate

7

OL-135

palmitoylisopropylamide PMSF P RRBL-RT-PCSEA oylethanolamide SR141716A, rimonabant CB reS 4TASKTNF- TRPV tential, vanilloid receptor 1 URB532 UVDMWIN5

FAAH inhibitor PEA palmitoylethanolamide pI50 negative logarithmic value of IC50

PIA phenylmethylsulphonylfluoride, FAAH inhibitor

PA -α peroxisome proliferator-activated receptor alpha 2H3 rat basophilic leukaemia cells

R reverse transcriptase polymerase chain reaction stear

1 ceptor-selective antagonist/inverse agonist R14 528 CB2 receptor-selective antagonist/inverse agonist

-1 background membrane K+ channel α tumor necrosis factor alpha 1 transient receptor po

FAAH inhibitor RB597 FAAH inhibitor

11 AEA uptake inhibitor 5,212-2 non-selective cannabinoid receptor agonist

8

Pharmacology of Palmitoylethanolamide and Related Compounds

INTRODUCTION

General Background

t, Cacts t ut

ent of pain, inflammation, and m ar

bis, ∆9 etrahydrocannabinol n 1964 , a ∆9-TH d the UK for

treatment of chemotherapy-in d vomiting. Dronabinol, ∆9-THC e ouse of

ittee of K House of Lords conc f anecdotal evidence as e therapeutic efficacy o osis and chronic pain

”as a ma ation in proper clinical trials are now

dertaken for these in et al., n al., 2004). N utic agents of

HC i e same s as ∆ y their psychotropic

s. A major challenge fo es to harness the therapeutic pot abinoids without producing

The endocannabinoid sy

s and assumed that the anna unspecific

membranes du he mid activity was highly stereospecific

r and its endogenous ence erisation of the first made in ne et al., 1988).

The cannabis plan nnabis sativa, has been used for millennia as a recreational drug and affe he body in several ways. It has been used throughohistory for the treatm a number of diseases such asmuscle spasms, nausea any more (Reynolds 1890; Baker et al., 2003; Kumet al., 2001). The major active constituent of canna -t(∆9-THC) was isolated i (Gaoni & Mechoulam, 1964). Currently nabilonestructural analogue of C, is used in parts of the USA an

duced nausea anitself, is used as an appetit stimulator in AIDS patients in the USA (HLords, 1998). In their hearing in 1998, the Science and Technology Commthe U luded that the large amount oto th f cannabis in multiple sclerconditions warranted tter of urgency” investig(House of Lords, 1998). Clinical trials of standardised cannabis extractsbeing un dications (see e.g. Wade et al., 2003; Zajicek2004; Berma et onetheless, the usefulness as therapesuch extracts, of ∆9-T tself, or of synthetic compounds with thpharmacological action 9-THC, is greatly hampered beffect r many researchers is to find new approach

ential of these cannunwanted effects.

stem

Back in the 1970’ early 1980’s it was generally effects of cpsychotropic bis and synthetic cannabinoids was rather

on nerve cell e to their high lipophilicity. However, in t1980’s, several groups showed that cannabinoid(Razdan, 1986) which led to the search for a specific recepto

tmediators. As a consequ , the pharmacological characcannabinoid receptor was rat brain in the late 80’s (Deva

9

Kent-Olov Jonsson

10

ept brain by Matsuda and co-e a mbrane G-protein

led receptor superfamily e first endogenous ty mide, a lipid

ng to a group of fatty s), ted from pig brain rit liss, (Devan

located cytic ., 1993). In the same

epor A, as fatt ,

in 1994 the first da EA uptake was (Di Marzo et al., 199 molecular proof of this protein. In 1995, th the

, 2-arac ), belonging to a group of oacylgly tracted from rat brain and

t (Sugiura et al., 199 995). The knowledge of the system is co sed that

other endogenous compounds, including dihomo-γ-linolenoylethanolamide and docosatetraenoylethanolamide (Hanus et al., 1993), 2-arachidonyl-glycerylether (noladin ether, 2-AGE; Hanus et al., 2001), O-arachidonoyl-ethanolamine (virhodamine; Porter et al., 2002), N-arachidonoyl-dopamine (NADA; Bisogno et al., 2000; Huang et al., 2002) and oleamide (Legget et al., 2004), also function as cannabinoid receptor ligands, but its not clear as to whether they are true endocannabinoids.

NH

OOH

Shortly after, the CB1 rec or was cloned from ratworkers and found to b member of the seven transmecoup (Matsuda et al., 1990). In 1992, thcompound to exert activi at CB1 receptors, arachidonylethanolabelongi acid amines called the N-acylethanolamines (NAEwas extrac and named anandamide (AEA) after the Sanskword for b ananda e et al., 1992; structure, see Fig. 1). Around the same time, a peripherally receptor was cloned from human promyeloleukemia HL-60 cells and named the CB2 receptor (Munro et alyear, Deutsch and Chin r ted an enzyme responsible for hydrolysis of AEnowadays referred to y acid amide hydrolase (FAAH; Deutsch & Chin1993), and ta suggesting a transport system for Areported 4), although there is still no putative e second endogenous compound to activate cannabinoid receptors hidonoylglycerol (2-AGlipids called the mon cerols (MAGs), was excanine gu 5; Mechoulam et al., 1endocannabinoid ntinuously growing and it has been propo

NH

OOH

O

NH

OHO

NH

OH

Fig. 1. Structures of anandamide (AEA) and the related N-acylethanolamine palmitoylethanolamide (PEA).


In respect of the human C reports of two different splice variants, hCB1a and hCB1b, showing a slightly different pharmacological

l-length hCB1 (Shire et al., 1995; Ryberg et al., 2005). ns of different types of receptors, including a CB2-

like

B1 receptor, there have been

profile compared to the fulThere have also been indicatio

receptor, a mesenteric non-CB1 non-CB2 receptor and a non-CB1 non-CB2 brain receptor, although they have not been cloned so far (Howlett et al., 2002).

The identification of cannabinoid receptors and FAAH, the key enzyme in AEA degradation, as well as a putative transport protein has lead to the development and search for selective compounds revealing more information about the endogenous cannabinoid system as well as developing new therapeutic agents that act through the cannabinoid system.

So what is the physiological function of the endocannabinoid system (shown schematically in Fig. 2 below)? Even if this of course is a complex question, the dramatic expansion of studies concerning endocannabinoids and related compounds, as well as the well known effects of cannabis, have revealed a number of possible physiological roles. These include pain processing, memory functions, cognition, food intake, gastrointestinal and cardiovascular function, regulation of the immune system and control of motor function (reviews, see Piomelli, 2003; Gerdeman & Lovinger, 2003; De Petrocellis et al., 2004; Di Marzo et al., 2004; Fowler et al., 2005). This range of actions means that there is, at least in theory, considerable therapeutic potential for compounds affecting the endocannabinoid system. See figure 2 below for a schematic overview of the synthesis, release, action and inactivation of the endocannabinoid AEA.

11

Kent-Olov Jonsson

AEA

mGluR+

Ca2+, Na+

+

AEA

Ethanolamine

Ca2+

FAAH

NAPE-PLD

NAT

CB1

-

NAPE

AEA AEA?

GABA

?

TRPV1

Arachidonicacid

AEA

mGluR++

Ca2+, Na+

++

AEA

Ethanolamine

Ca2+

FAAHFAAH

NAPE-PLD

NAT

CB1

--

NAPE

AEA AEA?

GABA

?

TRPV1

Arachidonicacid

Arachidonicacid

Fig.2. Schematic figure of the synthesis, action and metabolism of AEA in neurons (uptake and metabolism has also been shown in other cell types). AEA is synthesised in a two-step process via intracellularly membrane bound enzymes. In the first step, a lipid precursor is converted into NAPE by NAT. Secondly; NAPE is further transformed into AEA by NAPE-PLD. The synthesis is initialised “on demand” by a rise in intracellular calcium or by neurotransmitters such as glutamate. After synthesis, AEA is released directly out in the synapse by an unclear mechanism after which it can activate the G-protein-coupled CB

arachidonic acid and membrane of intracell

receptors aptically in the brain, le c neuron, such as GAB alised into the ce protein. Inside

e ion channel, which responds to h of capsaicin, the “hot” co hydrolysed into

ethanolamine by the enzyme FAAH, which is located in the ular organelles such as mitochondria. Abbreviations in the figure:

idylethanolamine phospholipase D; CB, cannabinoid receptor; TRPV1, transient receptor potential vanilloid 1; GABA, γ-aminobutyric acid; mGluR, metabotropic glutamate receptor; FAAH, fatty acid amide hydrolase.

. The activation of CB1 receptors, which are mainly located presynads to a decreased release of neurotransmitters located in the presynapti

A or glutamate. After activation of the CB receptors, AEA is internll by a process which may (or may not) involve a surface membrane

the cell, AEA can also activate the TRPV1 receptor, a non-selectiveat and low pH and is in addition responsible for the actions

mponent of red chili pepper. Inside the cell, AEA is also

AEA, anandamide; NAPE, N-acylphosphatidylethanolamine; NAT, N-acyltransferase; NAPE-PLD, N-acylphosphat

12


Ca

1

phase III clinical trials under the name rimonabant, and the CB2 antagonist/inverse agonist SR144528 (Rinaldi-Carmona et al., 1994; 1998). Another example in this group is the CB1 receptor antagonists/inverse agonist AM251. Among other chemical series, AM630, which is an aminoalkylindole, is also a commonly used CB2 antagonist/inverse agonist.

nnabinoid receptor ligands

Since the isolation of ∆9-THC (Gaoni & Mechaoulam, 1964), a number of different agonists and antagonists at the cannabinoid receptors have been developed. See figure 3 below for a selection of different CB ligands. The cannabinoid receptor agonists can be divided into several groups based on their chemical structure. They are divided into classical cannabinoids, non-classical cannabinoids, aminoalkylindoles and eicosanoids (Howlett et al., 2002). The classical group includes the plant derived agonist of C. sativa, including ∆9-THC, as well as synthetic analogues such as HU210. Both of these bind and activate CB1 and CB2 receptors without showing any particular selectivity between the receptors. Among the non-classical cannabinoids, CP55,940 is an important member, showing similar selectivity for both cannabinoid receptors, and the use of which led to the pharmacological characterization of the CB1 receptor (Devane et al., 1988). In the third group, the aminoalkylindoles, WIN55,212-2 is a compound showing similar affinity for both the CB1 and the CB2 receptor. The endocannabinoids, AEA and 2-AG, are important members of the eicosanoids.

Examples of potent and selective agonists at the CB1 receptor are the AEA analogues arachidonoyl-2-ethylchloride (ACEA) (Hillard et al., 1999) and O-1812 (Di Marzo et al., 2001b). Examples of CB2-selective agonists include HU-308, JWH133 and AM1241 (Hanus et al., 1999; Huffman et al., 1999; Malan et al., 2001b).

Concerning the antagonists/inverse agonists, the diarylpyrazoles is a major group, including the CB antagonist/inverse agonist SR141716A nowadays in

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Kent-Olov Jonsson

Agonists

ACEA

O-1812

HU-308

JWH133

AM1241

NH

OCl O

OH

O

O

OH

O

OOO

NO2O

N

I

N

NO2O

N

I

NN

Fig. 3. Structures of selected cannabinoid receptor ligands.

CP55940

Rimonabant(SR141716A)

SR144528

(-)-∆9-THC

HU-210

R-(+)-WIN55212

NH

OCl

NH

OOH

CN

NH

OOH

CN

OH

O

OH

O

OH

HO

O

OH

HO

O

O

NO

O

N

O

NO

O

N

OH

OH

OH

OH

OH

OH

Cl

Cl

Cl

N

N

O

NH

N

Cl

Cl

Cl

Cl

Cl

N

N

O

NH

N N

O

NH

NN

O

NH

N

CB1 selective Non-selective CB2 selective

Agonists

(-)-∆9-THC

HU-210

R-(+)-WIN55212

ACEA

O-1812

HU-308

JWH133

AM1241

NH

OCl O

OH

O

O

OH

O

OOO

NO2O

N

I

N

NO2O

N

I

NN

NH

OCl

NH

OOH

CN

NH

OOH

CN

OH

O

OH

O

OH

HO

O

OH

HO

O

O

NO

O

N

O

NO

O

N

OH

OH

OH

OH

OH

OH

Antagonists

CB1 selective CB2 selective

CP55940

Rimonabant(SR141716A)

SR144528

Cl

Cl

Cl

N

N

O

NH

N

Cl

Cl

Cl

Cl

Cl

N

N

O

NH

N N

O

NH

NN

O

NH

N

CB1 selective Non-selective CB2 selective

Antagonists

CB1 selective CB2 selective

14


In vivo effects of cannabinoid receptor activation

rt-term

or the CB1 receptor. Long term effects include impairment of the immune and

involvement of the CB1 receptor in this effect (Huestis et al., 2001).

As stated in the introduction, the current use of cannabinoid ligands as pharmaceuticals is for the treatment of nausea and for stimulation of appetite. The involvement in the regulation of food intake is shown by the use of rimonabant, which is now in phase III clinical trials for weight reduction (also in phase III trials for aid to smoking cessation; Le Fur, 2004). Another major therapeutical indication

The hitherto discovered cannabinoid receptors include the central CB1 receptor and the peripheral CB2 receptor. Agonist activation of these G-protein coupled receptors leads to several signal transduction pathways through Gi/o proteins. The most examined pathway is the inhibition of adenylyl cyclase (AC), leading to a decrease in cAMP production and inhibition of protein kinase A (PKA). In the case of CB1, inhibition of voltage-gated Ca2+ channels and activation of inwardly rectifying K+ channels, also seem to be important (Howlett et al., 2002; Pertwee, 1997). Cannabinoid receptor activation also modulates other signalling pathways via G-proteins, including the extracellular signal-regulated kinase pathway and stimulation of ceramide synthesis, a lipid that can activate apoptosis (Guzman et al., 2001).

The biological consequences of cannabinoid receptor activation are numerous.

The main adverse psychotropic effects in human seen with both cannabis and ∆9-THC include euphoria and relaxation, time distortion and impairment of sho

memory and motor skills. Acute effects also include anxiety, paranoia and panic reactions as well as tachycardia and lowering of blood pressure by vasodilation (Carlini, 2004; Kumar et al., 2001; Baker et al., 2003). The effects seen with ∆9-THC are also seen with both synthetic and endogenous ligands with affinity f

reproductive system (Kumar et al., 2001). In experimental animals, the use of cannabinoid ligands, as well as the use of genetically modified mice, has identified a tetrad of behavioural effects that point to activation of central CB1 receptors. These are hypothermia, hypoactivity, antinociception and catalepsy (see Martin et al., 1999) and correlate well with affinity for the CB1 receptor (Howlett et al., 2002). In respect of the “high” produced by smoked cannabis, clinical studies using rimonabant have confirmed the

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Kent-Olov Jonsson

is in the treatment of pain and several studies have been conducted in this respect. Cannabinoids modulate pain at supraspinal, spinal and peripheral levels (see Pertwee, 2001; Rice, 2001). The involvement of the cannabinoid system in this respect seems to be broad as shown by the fact that both CB1 and CB2 receptor agonists as well as palmitoylethanolamide (PEA), a fatty acid with no activity at the cannabinoid receptors, and other non-CB receptor cannabinoid related substances such as ajulemic acid can produce an antinociceptive response (Burstein et al., 2004). In the case of CB1 receptors, there is always the potential problem of separation between the wanted and unwanted (psychotropic) effects, unless the compounds are given locally (Dogrul et al., 2003) or designed so as not to cross the blood brain barrier (Fride et al., 2004). In the case of CB2 receptors it was initially believed that they did not participate in nociception, based on their restriction to immune cells. However, studies showing that PEA could be effective against carrageenan-induced hyperalgesia (Mazzari et al., 1996), suggested an involvement of the CB2 receptor. Since PEA was believed to be an endogenous ligand for the CB2 receptor (Facci et al., 1995) the involvement of CB2 receptors was examined. It has later been shown that PEA does not interact with the cannabinoid receptors in vitro, unless very high concentrations are used (Lambert et al., 1999; Sugiura et al., 2000; see also Paper I of this thesis).

There have also been reports showing effects of CB2-selective agonists. The first came in 1999, showing that HU-308 could reduce pain responses in the formalin test, which was reversed by SR144528 but not by rimonabant (Hanus et al., 1999). HU-308 was also effective in decreasing arachidonic acid-induced ear oedema in a SR144528, but not rimonabant, -sensitive manner. Similar results were seen using AM1241, which reversed carrageenan-induced hyperalgesia and this effect could be blocked by AM630 or SR144528 but not by AM251 or rimonabant (Quartilho et al., 2003; Nackley et al., 2003). AM1241 has also been shown to be effective against substance P-induced inflammation (Malan et al., 2001a). This compound has also been shown to suppress capsaicin-induced hyperalgesia in a SR144528 sensitive manner and to be effective in a model of neuropathic pain in an AM630 sensitive way (Hohmann et al., 2004; Ibrahim et al., 2003). A recent study has also shown the effect of the CB2 agonist JWH133 in models of inflammatory and neuropathic SR144528 but not pain, with the effects blocked by by rimonabant (Elmes et al., 2004).

16


Cannabinoids have also been shown to produce beneficial effects in animal mo

h a shorter duration (Fride & Mechoulam, 1993; Smith et al., 1994). It is a

dels of multiple sclerosis. For example, WIN55212 and JWH133 were shown to be effective against tremor and spasticity in CREAE mice (Baker et al., 2000).

Based on the findings that natural plant cannabinoids and synthetic cannabinoids exert a number of effects it is not surprising that the endogenous compounds produce similar effects. Indeed, this has been found in numerous studies and in the case of AEA some examples will be given below.

Anandamide (AEA)

AEA is the most studied of the endocannabinoids. It exerts the same effects as ∆9-THC and other cannabinoids in the tetrad test when exogenously administered, although wit

partial agonist at both CB1 and CB2 receptors in vitro (Mackie et al., 1993). AEA has been shown to produce a number of effects including antinociceptive and anti-inflammatory effects (Calignano et al., 1998; Jaggar et al., 1998; Farquhar-Smith & Rice, 2001; Richardson et al., 1998), stimulation of food intake (Williams & Kirkham, 1999; Hao et al., 2000; Di Marzo et al., 2001c), amelioration of spasticity in mice (Baker et al., 2001) and reduction of blood pressure (Varga et al., 1995). In addition to its interaction with the cannabinoid receptors, AEA has been reported to interact directly with other targets including L-type Ca2+ and background TASK-1 K+ channels as well as G-protein coupled non-CB1 and non-CB2 receptors (see Di Marzo et al., 2002b; Howlett et al., 2002; Maingret et al., 2001). Better investigated is the interaction between AEA and TRPV1 (vanilloid) receptors, a non-selective ion channel which also is activated by capsaicin (the pungent component of chili pepper), heat and protons (Zygmunt et al., 1999; Smart et al., 2000 see Di Marzo et al., 2002b; Ross et al., 2003). It has been proposed that AEA functions as an endovanilloid, although the concentration required for activation of the vanilloid receptor is much higher than for the cannabinoid receptors. Nevertheless, some of the effects of exogenously applied AEA upon hyperalgesia and vasodilation are mediated by the TRPV1 receptor (Di Marzo et al., 2001a, 2002a; Ross et al., 2003). This binding site for AEA, in contrast to the cannabinoid receptors, is intracellularly located (De Petrocellis et al., 2001; Jordt & Julius, 2002).

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Kent-Olov Jonsson

N-acylethanolamines (NAEs)

Long chain NAEs are fatty acid amides that are normally found in low levels in almost all mammalian cells and tissues as well as in fish, invertebrates, plants and microorganisms (Schmid et al., 2002). In addition to AEA, NAEs such as palmitoylethanolamide (PEA), stearoylethanolamide (SEA) and oleoylethanolamide (OEA) produce interesting pharmacological effects both in vitro and in vivo. In most tissues, AEA is found at low levels compared to other NAEs. For example, in pig brain AEA content, corresponding to 1 % of total NAE content, was 6±1 ng/g wet tissue. In comparison, the levels of PEA, OEA and SEA were 205±49, 96±17 and 227±43 ng/g wet tissue, respectively (Schmid et al., 1995). In contrast, 200-2000 times higher levels of AEA has been found in mouse uterus and in that tissue, the relative amount of AEA was 75-95 % of total NAE content (Schmid et al., 1997). NAE levels are elevated in response to different types of stimuli, particularly those related to cellular stress and damage. For example, cadmium chloride-induced inflammation in rat testis results in a 5-fold increase in AEA amount and a 39-fold PEA increase (Kondo et al., 1998). Neurodegeneration is also associated with a large increase in NAE content (see below). However, cell damage rather than the inflammatory stimulus may be a prerequisite for such increases, since in two models of inflammation (intraplantar formalin and inhalation of lipopolysaccharide), no obvious increase in NAE content was seen (Beaulieu et al., 2000; Holt et al., 2004).

As mentioned above, AEA is active at CB receptors. The other “non-cannabinoid” fatty acid amines, although not being active at CB receptors, have been found to be endogenous signalling lipids and exert a number of effects. For example, OEA has been shown to reduce food consumption in rats and mice via activation of the nuclear peroxisome proliferator-activated receptor-α (PPAR-α) (Rodríguez de Fonseca et al., 2001; Fu et al., 2003) and exert weak antinociceptive effects (Calignano et al., 2001). SEA, the saturated homologue of OEA, has been found to produce the same effects as AEA on catalepsy, motility, analgesia, and body temperature of mice (Maccarone et al., 2002) and reduce food consumption in mice independent of an increased PPAR-α gene expression (Terrazzino et al., 2004 but see Fu et al., 2003). On the basis of a finding that a phospholipid fraction from egg yolk showed anti-allergic activity (Coburn et al., 1954), Kuehl and colleagues identified PEA as a constituent of egg yolk and reported further that it

18


affe

duce sub

synergistically with AEA (Jaggar et al., 1998; Calignano et al., 1998). Since PEA was believed at

ous ligand for the CB2 receptor (Facci et al., 1995), the involvement of CB receptors was examined. The anti-nociceptive actions of PEA was

tep enzymatic pathway (Schmid & Berdyshev, 2002). In the first step, a calcium-dependent membrane bound N-acyltransferase (NAT) catalyses the

cted joint anaphylaxis in the guinea pig (Kuehl et al., 1957). Following the discovery of the CB receptors, the effects of PEA saw new interest. In 1993, Aloe et al. reported that PEA, as well as other N-acylethanolamines, could re

stance P-induced mast cell degranulation in rat ear pinna (Aloe et al., 1993). Mazzari et al. (1996) showed a few years later a spectrum of actions of PEA in different models of inflammation. In 1998, PEA was also shown to be effective in two different models of inflammatory pain and in addition to act

that time to be an endogen2

found to be sensitive to SR144528, but not to rimonabant, which is consistent with a CB2 sensitive pathway (Calignano et al., 1998, 2001; Farquar-Smith et al., 2001).

Newer studies have investigated the possible involvement of CB2 receptors in the anti-inflammatory effect of PEA. In this respect, pre-treatment with PEA decreased carrageenan-induced oedema, in a SR144528 sensitive manner (Conti et al., 2002). The same effect was seen when PEA was administered after induction of inflammation, however, in this case, the effect could not be reversed by SR144528 (Costa et al., 2002). In a recent study, it has been shown that PEA binds and activates the nuclear PPAR-α. The authors attributed the anti-inflammatory effects of PEA solely to the activation of the PPAR-α, and indicated that this effect was independent of the CB2 receptor shown by the lack of effect of SR144528 (Lo Verme et al., 2005). In addition to the anti-inflammatory abilities, PEA has also shown other effects including alleviation of spasticity in a mouse model of multiple sclerosis (Baker et al., 2000, 2001) and suppression of convulsions in rats and mice (Lambert et al., 2001; Sheerin et al., 2004). The mechanisms responsible for these effects have not been elucidated, although the former is presumably related to the ability of PEA to reduce the rate of AEA metabolism by acting as an alternative FAAH substrate (see below).

Synthesis and release of anandamide

The synthesis of NAEs, including AEA and PEA, seems to be performed by the same two-s

19

Kent-Olov Jonsson

transfer of an acyl group from the sn-1 position of glycerophospholipid to the amino group of the phospolipid membrane precursor, phosphatidylethanolamine (PE), producing N-acylphosphatidylethanolamine (NAPE) (Schmid & Berdyshev, 2002). In the second step, a specific phospholipase D (NAPE-PLD), which recently has been cloned (Okamoto et al., 2004), hydrolyses the conversion into the corresponding NAE, although others have reported an alternative second pathway (Sun et al, 2004). With respect to AEA and PEA, synthesis has been shown in different tissues and cells, including cells of both neuronal and immune origin (Di Marzo et al., 1994; Di Marzo et al., 1996; Bisogno et al., 1996; Bisogno et al., 1997).

Although the mechanisms behind the release of NAEs are far from evident, it is established that they are synthesised and released “on demand” in neurons, at least in the case of the endocannabinoid AEA, under physiological and pathological conditions (reviews see Piomelli, 2003; Fowler, 2003). The calcium-dependency of the NAE synthesizing enzymes (see above) mean that conditions that result in an increased intracellular calcium level would be expected to result in an increased NAE synthesis. Consistent with this, depolarising stimuli, calcium ionophores and stimulation of receptors such as metabotropic glutamate receptors result in a stimulation of synthesis (see Di Marzo et al., 1994; Alger, 2002; Wilson & Nicoll, 2002; Piomelli, 2003). Calcium dysregulation is an important excitotoxic event (see Rang et al., 2004), and it has been shown that the levels of the endocannabinoids are elevated in certain brain areas in different animal models of diseases including stroke, multiple sclerosis and Parkinson’s disease (Di Marzo et al., 2004). For example, in rats with permanent middle cerebral artery occlusion, a model of stroke, different N-acylethanolamines was increased 15-44 times with an 18-fold increase of AEA levels in the striatum (Berger et al., 2004). In the central nervous system, the main function seems to be regulation of synaptic transmission and AEA has been shown to function as a retrograde messenger. In this role, endocannabinoids are released from the postsynaptic membrane and travel backwards into the synapse to activate presynaptic CB1 receptors and thereby decrease the presynaptic release of other neurotransmitters such as GABA and glutamate (Alger, 2002; Wilson & Nicoll, 2002). Indeed, the fact that endocannabinoids affect the activity of many neurotransmitters indicates a modulatory role for the endocannabinoid system in the brain (Baker et al., 2003). In the periphery, endocannabinoid systems are involved in reproduction, cardiovascular control and the gastrointestinal system (see Di Marzo et al., 2004).

20


For example, low AEA degrading activity has been shown to be related to miscarriage in humans (Maccarrone et al., 2000b). With respect to cardiovascular control, AEA levels were found to be elevated in rats during haemorrhagic shock and the blood from these rats induced vasodilation, which could be antagonised by rimonabant, in normal rats (Wagner et al., 1997).

Anandamide uptake

Since the degradation of AEA is brought about intracellularly (see below), released AEA needs to be internalized into the cell before this can occur. How this process works is not fully elucidated and is a matter of current debate. The accumulation of AEA has been found in a number of different cell lines, including cells of neuronal and immune origin from mouse, rat and man (see McFarland & Bar

bstrate specificity and selective inhibitability by structurally related compounds (see e.g. Di Marzo et al., 1994; Hillard et al., 1997;

xistence of a membrane protein is e inhibitors enhance extracellularly

med

ker, 2004; Fowler & Jacobsson, 2002 for review). Following early suggestions that the uptake of AEA was mediated by an energy

and sodium-independent process of facilitated diffusion (Di Marzo et al., 1994; Hillard et al., 1997), much effort has been made toward revealing this mechanism. There is today no clear consensus in this matter, although a number of different models has been proposed (Deutsch et al., 2001; Glaser et al., 2003; Hillard & Jarrahian, 2003; McFarland & Barker, 2004; Fowler et al., 2004; Ortega-Gutiérrez et al., 2004). These include: a) facilitated diffusion mediated by a membrane carrier protein, b) simple diffusion driven by intracellular FAAH, c) simple diffusion driven by intracellular sequestration of AEA or binding of AEA to other intracellular targets, d) endocytotic internalisation of AEA (review see McFarland & Barker, 2004).

In support of a surface membrane protein, it has been argued that the accumulation show all characteristics of this, including saturability, exhibiting comparable Km values with brain amines and amino acids transporters, temperature dependence, su

Beltramo et al., 1997; Fegley et al., 2004). The efurther supported by the fact that AEA uptak

iated receptor effects (CB1 receptors) and decrease intracellularly mediated receptor effects (TRPV1) (De Petrocellis et al., 2001; see also Paper III in this thesis). However, many different cells show AEA accumulation, which would

21

Kent-Olov Jonsson

suggest a rather unspecific process. The fact that AEA is highly lipophilic, makes diffusion through the lipid layer a distinct possibility.

The importance of FAAH in driving accumulation was suggested by Deutsch et al. (2001) who showed that FAAH transfected cells accumulated AEA to a larger extent than wild type cells with low FAAH activity, although it was still present in untransfected cells. Evidence against a separate membrane protein responsible for AEA accumulation was presented by Glaser et al. (2003). They showed that AEA transport inhibitors were not able to inhibit AEA uptake when using short incubation times (<1 min), but instead a saturable uptake was seen using longer incubation times. These authors indicated FAAH as the protein responsible for the saturable inhibition of uptake, and attributed the crossing over the plasma membrane as simple diffusion driven by the concentration gradient. In contrast to these results, Fegley et al. (2004) showed that there was no difference upon AEA uptake between neurons from FAAH (-/-) and FAAH (+/+) mice, indicating a lack of effect of FAAH. These results are in line with other studies identifying compounds that lack effects on FAAH but still can inhibit AEA uptake (Ortar et al., 2003; López-Rodríguez et al., 2003; Fegley et al., 2004). Furthermore, Fegley et al. (2004) reported that the prototypical AEA uptake inhibitor AM404 was still pharmacologically active in vivo when given to FAAH (-/-) mice.

Hillard & Jarrahian (2003) proposed that AEA is taken over the cell membrane by simple diffusion, and thereafter intracellularly sequestered or bound to proteins, in which the second process is the saturable part in AEA uptake. This model was proposed by the finding that radiolabelled AEA reaches higher intracellular concentrations than found extracellularly. An alternative to sequestration is the presence of intracellular shuttling protein(s), which take the AEA from the inside of the plasma membrane to the endoplasmic reticulum, where the FAAH is located (Fowler et al., 2004; Ortega-Gutiérrez et al., 2004). Since uptake inhibitors like AM404 and the closely related VDM11 can inhibit AEA binding to plastic wells (Karlsson et al., 2004; Fowler et al., 2004; Ortega-Gutiérrez et al., 2004; see also Paper I of this thesis), the structural requirements of such binding proteins may not need to be that stringent. In the fourth model, McFarland & Barker (2004) proposed that the sequestration of AEA was due to a caveolae-related endocytic process. Caveolae are flask shaped invaginations of the plasma membrane, and their model involves the binding of AEA to a protein located on

22


the extracellular part of the surface membrane in the caveolae, followed by an endocytic process where the membrane is pinched off and released into the cell.

Even if the exact process of AEA internalization is unclear, it is a fact that compounds that are identified as putative transport inhibitors enhances the effects of AEA in vitro and in vivo. For example, AM404 increased latency in the hot plate test when administered together with AEA (Beltramo et al., 1997). No effect was

ed alone. Another example is VDM11, which reduced spasticity in mice using a model of multiple sclerosis (Baker et al., 2001). Although, the

se AEA was reported for the first time in 1993 by Deutsch and Chi

seen when AM404 was us

specificity of these compounds have been questioned (e.g. Zygmunt et al., 2000; see Paper III of this thesis) there have recently been studies conducted using more selective uptake inhibitors, also showing similar in vivo effects (De Lago et al., 2004). For a recent compilation of the in vivo effects of putative AEA uptake blockers, see Fowler et al. (2005).

Anandamide degradation

The main enzyme responsible for the inactivation of AEA, in both peripheral tissue and the central nervous system, is fatty acid amide hydrolase, FAAH. This enzyme hydrolyses AEA into arachidonic acid and ethanolamine. Although it has been shown that other enzymes also are capable of inactivating AEA in vitro, such as cyclooxygenase-2 and lipoxygenase (see Kozak & Marnett, 2002; Maccarone et al., 2004), FAAH seems to be the key enzyme since FAAH (-/-) mice show a 15-fold increase in brain levels of AEA as well as a 50-100 fold decrease of catalytic activity in brain and liver compared to wild type (Cravatt et al., 2001). The ability of FAAH to metaboli

n (Deutsch & Chin, 1993). After the cloning of FAAH (Cravatt et al., 1996; Giang et al., 1997), it was confirmed that this enzyme was the same as the enzyme responsible of hydrolysing other NAEs including PEA and OEA (Bachur & Udenfriend, 1966; Schmid et al., 1983; Natarajan et al., 1984; Schmid et al., 1985).

FAAH is a membrane bound enzyme, and subcellular fractionation and

immunohistochemical studies have indicated that it is primarily localised to mitochondria and microsomal fractions (Hillard et al., 1995; Watanabe et al., 1998; Tiger et al., 2000; Glaser et al., 2003). FAAH shows an alkaline pH optimum ranging from pH 8.5 to 10, depending on source of enzyme (Deutsch et al., 2002).

23

Kent-Olov Jonsson

The enzyme was first cloned from rat liver and has since then been cloned from a number of species including human and mouse (Cravatt et al., 1996; Giang et al., 199

acylnitroanilides (Cr

7). The enzyme exists as a dimer and is believed to deliver AEA from the membrane to its active site (Bracey et al., 2002).

FAAH is widely distributed in mammalian tissue, and the distribution has been studied in detail. Many different types of tissues in rat and mouse, including liver, brain, small intestine, testis, eye and uterus have shown enzyme activity but activity was hardly detectable in heart and muscle (Deutsch & Chin, 1993; Desarnaud et al., 1995; Cravatt et al., 1996; Paria et al., 1996; Matsuda et al., 1997). This distribution correlates well with mRNA levels in rat (Cravatt et al., 1996; Katayama et al., 1997). The highest activities are found in liver and brain. In the brain, the localisation of FAAH is complementary to the CB1 receptor with FAAH located in neurons postsynaptic to CB1 receptors (Egertová et al., 1998, 2003). In contrast to the distribution in rat, FAAH mRNA in human organs is most abundant in pancreas, brain, kidney, and skeletal muscle with lower levels in liver and placenta (Giang et al., 1997).

The enzyme boasts broad substrate specificity and is capable of metabolising a number of different NAEs (see above), the endocannabinoid 2-AG (Lang et al., 1999; Goparaju et al., 1999) as well as N-acylamides and N-

avatt et al., 1996; Lang et al., 1999; Boger et al., 2000a; Patricelli et al., 2001). In view of the fact that the biological activity of AEA is offset by its short

lifespan, a possible pharmacological target would be to inhibit the degradation of this endocannabinoid. In this respect, the development of FAAH inhibitors has been undertaken by a number of groups.

FAAH inhibitors

Since the first inhibitor of FAAH, phenylmethylsulfonylfluoride (PMSF), was described in 1993 (Deutsch & Chin, 1993), a number of different FAAH inhibitors have been synthesised and tested for the ability to inhibit AEA degradation. The main groups of FAAH inhibitors can be divided into organophosphates and organosulphates, substrate analogues and carbamates (Fowler, 2004b). The non-specific serine hydrolase inhibitor PMSF belongs to the first group and inhibits FAAH irreversibly with an IC50 value in the low micromolar range at physiological pH (Deutsch & Chin, 1993; Desarnaud et al., 1995; Deutsch et al., 1997a, 1997b). In

24


vivo, PMSF has been shown to potentiate the effects of exogenous AEA in the cannabinoid tetrad response, without being active per se (Compton et al., 1997; Wil

inhibitor, and later studies have reported IC50 values ranging between high nanomolar and low micromolar concentrations (see

les are the oleoyl- and palmitoyltrifluoromethyl-ketones, showing similar IC values (Patterson et al, 1996; see Paper I of this the

d to inhibit FAAH in the low nanomolar range with URB597 and URB532 as the mo

ey et al., 2000). Other examples in this group include methyl-arachidonylfluorophosphonate (MAFP), which is a more potent irreversible inhibitor with IC50 values in the low nanomolar range (Deutsch et al., 1997b). The lack of specificity of these compounds, as well as potential toxicological effects including acetylcholinesterase inhibition (Quistad et al., 2002), however, limits their usefulness.

There have been a number of publications where FAAH inhibitors have been based on AEA or other fatty acids as a chemical template. In 1994 came the first report of a number of different substrate analogues (Koutek et al., 1994). Among these compounds, arachidonoyltrifluoromethylketone (ATMK or ATMFK) was found to be the most potent FAAH

Fowler et al., 2001). Other examp50

sis). With respect to their selectivity, the trifluoromethyl ketones are also inhibitors of phospholipase A2 and ATMK shows affinity for the CB1 receptor (Lehr, 1997; Koutek et al., 1994).

Another approach with substrate analogues has led to α-ketoheterocycle

inhibitors of FAAH, and this has given rise to highly potent compounds which have shown to be active in vivo, producing a decrease in locomotor activity (Boger et al., 2000b; Fedorova et al., 2001). In respect of their selectivity towards other components of the endocannabinoid system, not much have been done so far. However, in a very recent study, reversible potent and selective FAAH inhibitors belonging to this group were reported and among these, OL-135 was found to produce CB1 receptor mediated analgesia (Lichtman et al., 2004a).

In the last group, the carbamates, a number of compounds have been reporte

st potent (Kathuria et al., 2003). They showed no affinity for the CB receptors. In vivo, these compounds potentiated AEA-induced hypothermia without showing the same signs of CB1 receptor activation as AEA itself did on catalepsy, hypothermia and increased food intake (Kathuria et al., 2003; Fegley et al., 2005). In

25

Kent-Olov Jonsson

addition to these results, URB597 showed a CB1 receptor sensitive anxiolytic effect in a mouse model (Kathuria et al., 2003).

In addition to the above, compounds that have actions on other systems, have

also

1 sensitive antinociceptive response et al., 2004a). The same is seen in FAAH (-/-)

rence in locomotor activity and body temperature compared to FA

been shown to inhibit FAAH at pharmacologically relevant concentrations. These include propofol (Patel et al., 2003) and some acidic non-steroidal anti-inflammatory agents (Fowler et al., 1997; 2003a), and there is some evidence to suggest that this action may contribute to some of the pharmacological properties of these compounds in vivo (Patel et al., 2003; Gühring et al., 2002; Ates et al., 2003; review see Fowler, 2004a).

Therapeutic potential of FAAH inhibitors

Since AEA and other endogenous NAEs, such as PEA, possess a number of interesting effects in vivo, one can argue that inhibition of the metabolism of these compounds would be of therapeutic potential. In this respect, inhibition of FAAH have been suggested to possess a possible therapeutic value in a number of disorders such as pain, anxiety, inflammation and neuroprotection (Cravatt & Lichtman, 2004; Gaetani & Piomelli, 2003; Fowler et al., 2005).

A drawback in this respect could be the unwanted psychotropic effects, which

is mainly mediated by the CB1 receptor. However, inhibition of FAAH and, hence, enhanced levels of the NAEs, is not accompanied by catalepsy, hypothermia or increased food intake but instead by a CB(Kathuria et al., 2003; Lichtman mice, with no diffe

AH (+/+) mice (Cravatt et al., 2001). In contrast to the lack of these effects, FAAH (-/-) mice have shown a decreased sensitivity in several models of acute and inflammatory pain as well as an anti-inflammatory phenotype (Cravatt et al., 2001; Lichtman et al., 2004b; Massa et al., 2004). The relative involvement of central and peripheral FAAH in these effects was recently shown using a transgenic mouse model (FAAH-NS) where FAAH expression was restricted to the central nervous system. The levels of AEA, PEA and OEA were normal in the CNS and spinal cord but elevated in the periphery, in contrast to the FAAH (-/-) mice

26


where a general increase was seen throughout the body (Cravatt et al., 2001; 2004). The antinociceptive effects of the NAEs were attributed to the CNS and in contras the anti-inflammatory effects involved the periphery (Cravatt et al., 2004).

As stated earlier, AEA possesses a number of benificial effects and is

synthesised and released “on demand”. If this means that AEA is produced only when and where it is needed, FAAH inhibitors would enhance the levels of AEA mainly in the

t

areas where the production of AEA already is increased. This could enhance the beneficial effects, without causing the general CB1 receptor-mediated effects. Even if it can not be excluded that inhibition of FAAH would produce the psychotropic effects seen with cannabis, the results so far with FAAH inhibitors support the possibility that AEA-mediated effects can be separated.

In summary, it is concluded that modulation of the endocannabinoid system could be of therapeutic value and that the development of selective and potent FAAH inhibitors could be one approach in this respect.

27

Kent-Olov Jonsson

AIMS OF THE STUDY

ased on the discussion above, the deB velopment of compounds which can inte

having effects upon cannabinoid receptors.

ability of compounds structurally affect the uptake and hydrolysis of the

lamine head group f palmitoylethanolamide with respect to its interaction with fatty acid amide

ract with the degradation of anandamide and other fatty acids would be of interest. At the beginning of this study, there were few selective FAAH inhibitors available and those described were either non-selective (i.e. PMSF, ATMK) or poorly characterised apart from their effect on FAAH (α-heterocycle inhibitors). In consequence the present thesis was undertaken with the ultimate goal of identifying and characterising selective FAAH inhibitors. The endogenous N-acylethanolamine palmitoylethanolamide was chosen as a template upon its ability to inhibit FAAH without

Paper I: To evaluate pharmacologically the based on palmitoylethanolamide to endogenous cannabinoid anandamide, as well as their ability to interfere with cannabinoid receptors. The general aim was to identify compounds able to inhibit anandamide metabolism without having activity at cannabinoid receptors. Paper II: To explore further the importance of the ethanoohydrolase. Paper III: To test some compounds identified in Paper I for effects not related to FAAH inhibition or anandamide uptake in biological systems, and in this respect to compare them with two established anandamide uptake inhibitors. To compare the ability of these compounds to inhibit intracellularly TRPV1-mediated effects of anandamide. Paper IV: To establish models of inflammation in vitro and in vivo for further investigation of compounds identified in Paper I-III and to investigate the effects of a CB2 receptor agonist in these models. Paper V: To investigate the effects of palmitoylisopropylamide in models of inflammation in vivo and in vitro.

28


METHODS AND METHODOLOGICAL

CONSIDERATIONS

The methods used in this thesis are presented briefly together with comments abo

Fig. 4. Hydrolysis of [3H]-AEA into arachidonic acid and [3H]-ethanolamine. The asterisk indicates the position of the tritium labelled for the FAAH assay.

[3H]-labelled AEA, with the ethanolamine part labelled, was used as substrate (Fig. 4). The addition of chloroform and methanol to the homogenates after the incubation phase allows the separation of the water-soluble [3H]-ethanolamine from the lipid soluble [3H]-AEA. The ability of PEA and related compounds to inhibit this hydrolysis was examined. The method was originally described by Omeir et al. (1995) and modified by Fowler et al. (1997).

Frozen brains (minus cerebellum) from adult rats were thawed and

homogenized at 4°C in HEPES buffer, pH 7.0. The homogenates were centrifuged twice at 36,000 g at 4 °C after which the tissue pellets were re-suspended in homogenisation buffer and incubated at 37 °C. After centrifugation at 36,000 g at 4°C, membranes were re-suspended in Tris-HCl buffer, pH 7.4. The protein content in the membrane preparations were determined according to the method of Harrington (1990), using bovine serum albumin as standard.

Test compounds or ethanol carrier and assay buffer were preincubated for 0 - 180 min at 37 °C. [3H]-AEA and untritated AEA were added and the samples were incubated at 37 °C. Reactions were stopped by placing the tubes in ice and adding

ut the methods. For further details of each method, see the original papers.

FAAH assay (Paper I and II)

AEA is converted to arachidonic acid and ethanolamine by the intracellular enzyme fatty acid amide hydrolase, FAAH. This method was used to measure the ability of PEA-related compounds to inhibit this conversion.

O

OH

OO

OH*NH2

OH*NH2

OHNH

OHO

*NH

OHO

NH

OH* +

FAAH

H2O

Arachidonic acidAnandamide Ethanolamine

29

Kent-Olov Jonsson

chloroform: methanol (1:1 ixing of the tubes the phases wer

sch & Chin, 1993) or high-performance liquid chromatography (Maccarone et al., 1999). Thechrdispusinproperf y. Another possibility is to separate the labelled ethanolamine part from the arachidonic part is by using open bed column chromatography (Dealloreproducibility. The two main disadvantages are that the product is not unequivocally identified, and that the assay uses chloroform. In this respect, the metproprodessho FAAH inhibitors, it is in theory possible that additional hydrolytic enzymes contribute to the observed activity. Cerbeehom brain samples from mice lacking FAAH do not hydrolyse AEA (Cravatt et al., 2001). A second common factinvo y acid-free BSA (see e.g. Omeir et al., 1995) or

v/v) in the tubes. After vortex me separated by centrifugation. Aliquots of the methanol/buffer phase

were removed and analysed for radioactivity by liquid scintillation spectroscopy with quench correction. Blanks contained distilled water instead of the homogenate preparations.

Comments on the FAAH assay

A number of FAAH assays have been described in the literature. Some assays, for example, use substrate radiolabelled in the arachidonoyl part of the molecule and the products are then separated by thin-layer chromatography (Deut

re are also methods without radiolabelled AEA, using different types of omatographic separation (Lang et al., 1996; Watanabe et al., 1998), a fluorescent lacement assay (Thumser et al., 1997) as well as a spectrophotometric assay, g fatty acid p-nitroanilides (Patricelli et al., 2001). The assay used separates the ducts by simple phase separation with chloroform and methanol, which is ormed easil

sarnaud et al., 1995). The main advantage of the present assay is its simplicity, wing a good throughput of samples, and its robustness, in terms of its

hod has been further developed by separating the substrate from the labelled duct using charcoal which binds to the substrate, but not the ethanolamine duct (Wilson et al., 2003; Boldrup et al., 2004). Common to all the assays cribed above is that they measure the hydrolysis of AEA and, although they w the appropriate sensitivities to standard

tainly, a hydrolytic activity distinct from FAAH that can metabolise PEA has n identified in the lung (Ueda et al., 1999). However, the use of brain ogenates should eliminate this possibility, since

or for all assays is the lipophilic nature of the substrate (and test compounds) lved. Most assays use either fatt

30


detergents (see ssay used here (but with a charcoal he inhibitory potency of indomethacin (Boldrup et ature of the compounds use

us, potencies of test compounds are best interpreted relative to standard compounds

e same method.

An

The hydrolysis of AEA by FAAH is performed inside the cell which makes it necessary for AEA to be transported across the cell membrane before this event can occur. The exact process of cellular uptake of AEA, however, is a matter of current debate, see introduction for further discussion.

In this model, cells are incubated with [3H]-labelled AEA. After the cells have ed and analysed in

regard t

the cells. The ability of PEA and related compounds to inhibit AEA uptake was exa

at 37 °C was allowed for 15 min. After the incubation, the plates were placed on ice after which the cells wer

ied version of the met

e.g. Boger et al., 2000b). Addition of detergent to the a extraction) does not affect tal., 2004), but the lipophilic n

d nevertheless means that inhibitory potencies of compounds can vary between laboratories (see e.g. Fowler et al., 2004 for an example with VDM11). Th

assayed in the same laboratory with th

andamide uptake in RBL-2H3 and C6 cells (Paper I)

been allowed to internalise AEA they are washed, solubilizo tritium content (Rakhshan et al., 2000). This method measures both

metabolised ([3H]-ethanolamine part) and non-metabolised [3H]-AEA taken up by

mined. RBL-2H3 or C6 cells were plated onto 24-well plates and incubated at 37 °C

for 18 hours. After the incubation period, the cells were washed once with warm assay buffer and pre-incubated with the test compounds or ethanol carrier at 37 °C for 10 min [3H]-AEA was added to the wells and uptake

e rinsed three times with ice-cold buffer containing 1 % BSA to terminate the transport. The cells were aspirated from the buffer and incubated at 75 °C in NaOH for 15 min. Aliquots of the solubilized cells were transferred to scintillation vials and assayed for tritium content by liquid scintillation spectroscopy with quench correction.

In Paper I some of the compounds were tested in a modifhod above with the major difference in analysing only the [3H]-ethanolamine

part. This was achieved by separating the radioactive substrate from the

31

Kent-Olov Jonsson

[3H]-ethanolamine product in a similar way as in the FAAH method, using chloroform:methanol. These results reflect the combined inhibition of both uptake and FAAH degradation of AEA.

Comments on the uptake method

These highly lipophilic compounds have the ability to adhere to plastic as well f the data somewhat troublesome. A possible way by using fatty acid-free bovine serum albumin,

whi

or WIN 55,212-2 (CHO-CB2). After incubation, the membrane suspension was

as glass, making interpretation oto decrease this interference is

ch binds to AEA and thereby prevent the binding of AEA to plastic (Karlsson et al., 2004). Indeed, some studies have utilized BSA when measuring the uptake (Glaser et al., 2003). Other authors, however, argue that BSA per se inhibits AEA uptake (Di Marzo et al, 1994). As a consequence, when the study concerning the compounds in Paper I was carried out, we chose following the prevailing view and to implement the experiments without BSA. Due to the problems with the method concerning plastic binding and use of BSA or not, we chose to not perform any uptake experiments with the compounds in Paper II awaiting a better method. Since then, later studies have shown 0.15 % fatty acid-free BSA may be a useful concentration to use in the study of AEA uptake (Sandberg & Fowler, 2005). Nonetheless, there is still considerable controversy as to the best way to assay AEA uptake (see Fowler et al., 2004), as indeed there is concerning the nature of the uptake process itself (Glaser et al., 2003; Hillard & Jarrahian, 2003; McFarland & Barker, 2004).

Radioligand binding experiments (Paper I and II)

The PEA-related compounds were all tested for their ability to interfere at human CB1 and CB2 receptors overexpressed in Chinese Hamster Ovary Cells. This method, which was undertaken by Dr Séverine Vandevoorde in Belgium in the studies reported here, measures the ability of the test compounds to displace a tritiated potent CB agonist from the cell membranes.

Membranes from CHO-CB1 and CHO-CB2 were incubated at 30 °C with [3H]-CP55,940 (CHO-CB1) or [3H]-WIN 55,212-2 (CHO-CB2) for 1 hour in Tris-HCl with MgCl2 and EDTA (pH 7.4) in the presence of PMSF and BSA. Non-specific binding was determined with high concentrations of HU-210 (CHO-CB1)

32


rapidly filtered through 0.5 % polyethyleneimine-pretreated GF/B glass fibre filters, and the radioactivity trapped on the filters was measured by liquid scintillation spectroscopy.

Comments on the radioligand method

Radioligand binding is nowadays considered a standard method for assessing affinity at receptors. In general, it is recommended that the non-specific binding is measured using a different compound than the radioligand. This was not the case for the CHO- CB2 cells. However, the high levels of receptor expression, and the

ic binding, mea

r CB2 (CHO- CB2) receptors (Paper I and II) were used. The different cells were cultured in: Eagle's minimum essential medium, 2 mM L-glut

Rakhshan et al., 2000; De Petrocellis et al., 2000; Jacobsson & Fowler, 2001; McFarland

correspondingly low level of non-specific binding relative to the specifn that the use of the same ligand to assess non-specific binding is unlikely to

introduce errors. Indeed, the same results were found when HU-210 was used at high concentrations instead of WIN55,212-2 (data not shown). By using transfected cells, overexpressing a specific receptor, compared to using tissue naturally expressing receptors, involvement of other receptors could by avoided.

Cell cultures (Paper I, II, III, IV and V)

Rat basophilic leukaemia (RBL-2H3) cells (Paper I, IV and V), rat C6 glioma cells (Paper I and III) and Chinese hamster ovary transfected with human CB1 (CHO- CB1) o

amine supplemented with 15 % FBS (RBL-2H3), Ham’s F10 medium supplemented with 10 % FBS (C6) or Ham’s F12 medium supplemented with 10 % FBS and 200 µg ml-1 G418 (CHO-CB1) and CHO-CB2). All cells were grown with 100 units ml-1 penicillin and 100 µg ml-1 streptomycin present.

Comments on the cells used

RBL-2H3 (rat basophilic leukemia) is a mastocyte tumour cell line which shows similar homology to rat mucosal mast cells (Seldin et al., 1985) and these cells have been thoroughly characterized in their ability to internalise [3H]-AEA (Bisogno et al., 1997;

et al, 2004). C6 rat glioma cells have also been characterized and shown to accumulate AEA (Deutsch & Chin, 1993; De

33

Kent-Olov Jonsson

Petrocellis et al., 2000; Bisogno et al., 2001; McFarland et al, 2004). They respond to AEA by enhanced production of apoptotic bodies (Maccarrone et al., 2000a) as well as decreased rate of proliferation (Jacobsson et al., 2001) and this effect seems to involve TRPV1 and CB1 receptors. The AEA binding site on TRPV1 receptors is intracellularly located (De Petrocellis et al., 2001; Jordt & Julius, 2002), and so it

en up into the cell before it can interact with this rece

ell proliferation assay kit was used, which quantifies the total nucleic acid content of the samples and is based on a dye which becomes

erature for 30 min bef

An

is necessary for the AEA to be takptor.

Cell proliferation assay (Paper III)

The method was used to examine the effects of two AEA uptake inhibitors and compare them with PIA from Paper I on cell function and to compare the ability of these compounds to affect AEA inhibition of cell proliferation. The method described by Jacobsson et al. (2001) was used. C6 glioma cells were plated on 96-well plates and incubated at 37 ºC. After 6 h, the cells were incubated with various concentrations of the compounds. Half of the media were replaced daily for four days with fresh substances dissolved in media. 24, 48, 72 and 96 h after the first addition of compounds, the plates were emptied of media and stored at -80 ºC.

Analysis of cell density

The assay was used to analyse the density of cells in the proliferation assay. The CyQuant® c

fluorescent when bound to nucleic acids. At the day of analysis, the plates were placed in room temp

ore addition of reagents and incubation for 5 min. Fluorescence was measured at excitation/emission of 485/520 nm. A series of calibration curves indicated that the relationship between cell density and fluorescence deviated very slightly from linear at high cell densities.

alysis of caspase activity

AEA has been shown to affect cell proliferation in C6 cells by induction of apoptosis (Maccarone et al., 2000) and the assay used, which can detect the

34


activities of several caspases, was employed to determine if the AEA uptake inhibitors effects upon cell proliferation was due to activation of caspases. Cells were cultured and treated with test compounds as described in the cell proliferation assay. After 48 or 96 h of incubation, the medium was removed and the plates were frozen. On the day of assay, the plates were thawed and aliquots of

-Glu-Val-Asp-Rhodamine 110) in lysate buffer was

h measures mitochondrial activity. We measured cell proliferation as an endpoint,

and a fluorescent marker. In addition, it was possible to evaluate the effect of the test compounds on the anti

totic bodies. , can not give a complete answer if the effects seen on ptosis, but will at least indicate if caspase activation is an

imp

were used and maintained under pathogen-free conditions at the animal facility at

medium were added. Substrate (Asp added and the samples were then incubated for 2 h at 37 °C. Free rhodamine

110 produced by the action of caspases upon this substrate was detected fluorometrically using excitation and emission wavelengths of 485 and 520 nm respectively. Lysate from apoptotic U937 cells treated with camptothecin was used as positive control.

Comments on cell proliferation assay

There are a large number of different assays that can be used to measure cell viability and proliferation. Examples of these include measurement of the release of LDH, a cytoplasmic enzyme present in all cells, or by using MTT assays, whic

which is easily performed using microtiter plates

proliferative effects of AEA, indicating an effect on AEA uptake. The method, although highly sensitive, does not give any direct answers on the mechanism behind the antiproliferative effects of the compounds.

To evaluate if apoptosis could be responsible for the antiproliferative effects seen, an assay that measures the activity of caspases, which is one of the initial biochemical changes in apoptosis, was assessed. This approach does not account for later events of apoptosis, and the most common characteristic, the fragmentation of DNA into 180 bp fragments, so called apopMeasuring caspase activitycell viability are due to apo

ortant step in inhibition of cell proliferation produced by these compounds.

Animals

For the in vivo experiments female BALB/cJBom mice (8-9 weeks, 18-23 g)

35

Kent-Olov Jonsson

Umeå University. The mice were fed with standard food and water ad libitum and kept at least a week at the animal facility before the experiments. All animal experiments carried out in this report were approved by the ethical committee at the Umeå University. The mice were anaesthetized with an intraperitoneal (i.p.) injection (0.1 ml/mouse) of “cocktail”, a mixture of 0.315 mg ml-1 fentanyl citrate and 10 mg ml-1 fluanisone (Hypnorm™, Janssen, Saunderton, UK), 5 mg ml-1 midazolam (Dormicum®, Roche, Basel, Switzerland) and sterile water at a ratio of 1:1:2. Anaesthesia was retained under the whole experiment with additional injections of anaesthetic mixture. In the experiments when only tissue was used,

xygen before cervical dislocation.

or vehicle. The mice were injected subcutaneously in one ear pinna with compound 48/80 and vehicle alone in the other ear pinna. Immediately after the

re given Evans blue intravenously and euthanized 2 h later. Eva

ks.

d formalin-induced (Mazzari et al., 1996) paw oedema, arachidonic acid-induced ear oedema (Hanus et al., 1999), LPS-induced pulmonary inflammation (Berdyshev et al., 1998) and turpentine-induced bladder inflammation (Jaggar et al., 1998). The different models assess effects of a variety of inflammatory mediators acting either alone or in concert, and of course the best

the animals were euthanized with CO2 and o

Plasma extravasation in mouse ear pinna

The method was used to examine the effects of JWH133, a CB2 agonist, as well as the PEA analogue PIA upon inflammation and a model of mast cell dependent inflammation was chosen. The method by Mazzari et al. (1996) was used with some modifications. Mice were injected i.p. with compounds or vehicle 30 min prior to induction of inflammation. In the experiments with SR144528, the compound (or vehicle) was administered i.p. additionally 45 min before PIA, JWH133

ear injections, mice wens blue was extracted by homogenising of the ear in formamide followed by a

2 h incubation at 50 °C. After centrifugation the supernatant was analysed by optical absorbance at 620 nm (Saria and Lundberg, 1983). Ears from mice not injected with Evans blue were used as blan

Comments on the plasma extravasation method

Models of inflammation that has been used to evaluate effects of PEA and CB2 receptor agonists includes: carrageenan-induced (Conti et al., 2002; Costa et al., 2002; Mazzari et al., 1996) an

36


information regarding the anti-inflammatory effect of a new compound would be to u

. All steps with drugs were mad

se a selection of models of inflammation. However, the limited time available precluded this approach, and we chose to use an apparently simple system involving mast cells, on the basis of previous findings that PEA and CB2 receptor agonists have been shown to be effective against mast cell dependent inflammation (Mazzari et al., 1996 and Malan et al., 2001a). Initially the method was assessed using substance P as a mast cell degranulator. The results using substance P did however not give any significant increase in plasma extravasation. This could be due to instability of this compound, or some other methodological errors. In consequence, compound 48/80, a chemically stable mast cell degranulator, was chosen as an inducer of inflammation. This compound produces oedema that is mast cell dependent, shown by lack of effect in mast cell deficient mice (Kim et al., 1999) and gave a significant increase in plasma extravasation in all experiments, although very variable between animals.

Mice paw skin release

The method was set up to examine the effects of JWH133, PIA and PEA upon effects of mast cells in vitro.

The skin on the upper side from both hind paws of the mice was separated from the connective tissue. The skin was gently loosened with a small forceps followed by additional incisions with a scissor and placed in an oxygenated super interstitial fluid (SIF) buffer. After weighing, the isolated skin was mounted on glass rods with the outside facing the rods and transferred into tubes containing SIF buffer continuously bubbled with 95 % O2 and 5 % CO2 at 37 ºC. The tissue was left to equilibrate for 90 min before drug treatment

e in bubbled SIF. After equilibration, the tissue was transferred to new tubes containing drugs or vehicle and incubated for 15 min before compound 48/80 or vehicle was added to the test tubes. Fluid was collected at 60 min after the addition of compound 48/80 and stored at -80 ºC until analysis. Following the collection of fluid, the tissue was weighed and placed on a paper and an outline of the area from each piece of tissue was made. The outline was weighed and an area was calculated from the known weight of 10 cm2 paper.

37

Kent-Olov Jonsson

Comments of the paw skin method

the release of ß-hexosaminidase. This enzyme is stored in granules and its release

(Schwartz & Austen, 1980). ompounds like CB2 receptor

ago

cular Devices).

The method was set up in Ruth Ross lab in Aberdeen and was based on the work by Kilo et al. (1997) and Ellington et al. (2002). By using whole skin tissue pieces a system with mast cells surrounded by their natural environment is utilized. This was chosen as a model system since mast cells seem to respond differently when separated from their environment (Robinson et al., 1989; Sweiter et al., 1993; Hamawy et al., 1994; Ra et al., 1994). Initially it was intended to study the release of TNF-α, an important inflammatory mediator that is synthesised and released in response to mast cell activation (Ackermann & Harvima, 1998; Gibbs et al., 2001). Initial experiments, conducted at the laboratory of Dr. Ruth Ross in Aberdeen, U.K., indicated however that the release of TNF-α in response to non-immunogenic stimulation of mast cells was under the limit of detection. In consequence, upon return to Umeå, a different end-point was used, namely

represents the immediate response to degranulationOf course, the possibility cannot be ruled out that c

nists and PIA differently affect the release of preformed and de-novo synthesised mediators.

β-Hexosaminidase release

β-Hexosaminidase content was analysed by using the method, with some modification, of Smith et al. (1997). Fluid samples from the paw skin experiments were allowed to reach room temperature and added to a 96-well microtiter plate. The reaction was started by adding p-nitrophenyl-N-acetyl-D-glucosaminide in a citrate-buffer as substrate. After 2 h of incubation at 37 °C, the reaction was stopped by addition of Tris-buffer, pH 9.0. Optical density was measured spectrophotometrically at 405 nm (subtraction at 570 nm), using a computer-operated ThermoMax microplate reader (Mole

Immunohistochemistry and PCR

Immunohistochemistry was assessed to determine the location of CB2 receptors in skin. Mouse skin tissue were cut into pieces, embedded in Tissue-Tek® and snap frozen on dry ice before storage at -80°C. Cryostat sections were made

38


and allowed to air dry for 1 h, followed by fixation in paraformaldehyde for 30 min. The sections were rinsed in phosphate buffered saline (PBS) between every step, except after blocking of unspecific binding with serum. Endogenous peroxidase activity was blocked by 3 % hydrogen peroxide in methanol for 5 min at room temperature (RT). Unspecific binding was blocked with normal goat serum for 5 min at RT. The primary antibody was directed against the first transmembrane domain of the human CB2 receptor. Sections were incubated with the primary antibody at 4°C O/N. Biotinylated goat anti-rabbit immunoglobulins were used as secondary antibody and incubated for 30 min. For detection, sections were incubated with horseradish peroxidase-labelled streptavidin and DAB. After detection, sections were washed in tap water, counterstained with haematoxylin and mounted in glycerol jelly. Control experiments were performed without the primary antibody as well as preabsorption with the blocking peptide used as antigen for production of the CB2 receptor antibody. In the preabsorption experiments the primary antibody and blocking peptide were incubated for one hour at RT.

To determine if CB2 receptor mRNA was present in skin tissue, pieces of skin

was prepared and analysed by using RT-PCR. Mouse spleen was used as a positive racted from skin and spleen with TRIzol LS Reagent

by following the manufacturer’s protocol. The RNA was quantified spe

nly RNA preparations showing intact species wer

control. Total RNA was ext

ctrophotometrically and the integrity of the RNA preparations was examined by agarose gel electrophoresis. O

e used for subsequent analysis. One microgram of total RNA was reverse transcribed into single-stranded cDNA. After incubation at, the AMV reverse transcriptase was denatured and the cDNA was stored at -20ºC until used for polymerase chain reaction (PCR).

The synthesized cDNA was amplified by PCR. The PCR analyses for mouse CB2 receptor and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were performed using PCR Core Kit standard protocol except for the use of HotStar Taq polymerase in the CB2 reactions. The expression was compared at the logarithmic phase of the PCR reaction. The PCR products were electrophoretically size fractionated in 1.5 % agarose gel and visualized using ethidium bromide. The estimated size of the PCR product was 479 bp for CB2 and 267 bp for GAPDH. The PCR products were purified using a QIAquick purification kit according to

39

Kent-Olov Jonsson

manufacturer’s protocol, and the sequence was confirmed using a DNA Sequencer.

Data analyses and statistics

test, or paired two-tailed t-test (Paper IV, Fig. 2A), using the

Km and Vmax values for inhibition of [3H]-AEA metabolism by PIA and OEA in Paper I were calculated from the mean data using the Direct Linear Plot analysis (Eisenthal & Cornish-Bowden, 1974) using the enzyme kinetics v 1.4 software package, Trinity Software, Campton, NH, USA. These values were used in secondary replots to determine Ki(slope) and Ki(intercept) values. For the analyses of pI50 values in Paper I and II non-linear regression analysis built into the GraphPad Prism computer programme (San Diego, CA, USA) were used (for details, see result and discussion below). The pI50 values for inhibition of cell proliferation in Paper III were calculated from the data expressed as % of control using the same computer programme with the same approach as in Paper I and II, and the top and bottom value was set to 100 % and 0 % respectively. The cell density data in Paper III was analysed either using one-way ANOVA followed by Bonferroni-Dunn post hoc tests the using Statview computer programme (SAS institute Inc. Cary, NC, USA) or two-tailed unpaired t-test using the GraphPad Prism programme. In Paper IV and V plasma extravasation and β-hexosaminidase release were analysed using one-way ANOVA followed by Dunnett’s multiple comparisonsGraphPad Prism software.

40


RESULTS AND DISCUSSION

gramme used (GraphPad Prism, GraphPad Software Inc., San Diego, CA, USA). When the bottom value gave a 95 % confidence interval value stra

ds. Although other possibilities cannot be ruled out

Inhibition of FAAH catalysed anandamide hydrolysis (Paper I

and II)

As the initial goal was to find new potent compounds which could affect the degradation of AEA, a number of compounds were tested for their ability to block FAAH-catalysed AEA hydrolysis by rat brain homogenates. The compounds were based on PEA as a chemical template, in view of the findings that this compound is inactive at CB receptors, but is able to inhibit AEA degradation by itself being a substrate for FAAH (Schmid et al., 1985; Tiger et al., 2000). Being a pharmacological study, the idea at the outset was to construct dose-response curves and thereby determine the pI50 values, i.e. the positive calculated logarithmic value of the concentration of the compound producing 50 % inhibition. However, as is apparent in Fig. 2 of Paper II, several of the compounds did not produce a maximal level of inhibition. Comparison of potencies between compounds with different maximal levels of effect is somewhat difficult. In Papers I and II, an approach was used whereby the data was fitted to sigmoidal curves of variable slope with an initial value of 100 % and the ”bottom” value determined by the computer pro

ddling zero (such as seen for compound 13 in Fig. 2 of Paper II), the bottom value was set to 0 and thereby the calculated maximum inhibition was 100 % for these compounds. In some cases not enough inhibition was seen to permit meaningful calculation of pI50 values and in these cases the inhibition seen at the highest concentration tested was given. In many cases, however, the 95 % confidence intervals of the compounds were both above zero. In these cases, the bottom value was used to calculate the maximum observable inhibition and the pI50 value represents the potency with respect to the inhibitable fraction. In the papers, it was suggested that the residual activity simply reflected a solubility issue for these highly lipophilic compoun

(for example, the presence of FAAH subtypes, presumably resulting from the posttranslational modification of a single gene product, can be suggested although there is as yet no evidence in support of such a suggestion), the compounds do

41

Kent-Olov Jonsson

have limited solubilities and this would seem to be the most rational explanation of the data.

While the use of pI50 and maximum observable inhibition allows for the

compact description of a large number of dose response curves, interpretation of the data is complicated. Is, for example, a compound with a pI50 value of 5.54 more useful than a compound with a pI50 value of 5.08 when the former only produces a maximum of 24 % inhibition whereas the latter produces a maximum

nd 37, respectively, of Paper II)? In terms of the ability to prevent AEA metabolism, it may be more useful simply to show to the

ty, and as pI50 values, reflecting potency. This value is the positive calculated logarithmic value of the concentration of the compound inhibiting 50 % of the measured response.

of 87 % inhibition (compounds 6 a

total inhibitory capability at a given concentration, such as 10 µM. In order to give the reader the opportunity to see such values, the original data for a series of compounds studied in Papers I and II have been redrawn to show the inhibition produced by 10 and 100 µM (Fig. 6). In Paper I, several homologues with fatty acid chain length varying from 3 (C3:0, propanoylethanolamide) to 18 carbons (C18:0, stearoylethanolamide, SEA) was tested against their ability to inhibit FAAH catalysed AEA hydrolysis. The mono-unsaturated homologue of SEA, where a double binding was introduced between carbon 9 and 10 in the fatty acid chain (C18:1, oleoylethanolamide, OEA), was also tested.

The chemical approach was to change the fatty acid chain (Fig. 5A), herein

termed homologues, or the ethanolamine head of PEA (Fig. 5B), herein termed analogues, and all chemical work was done at the Catholic University of Louvain in Brussels, Belgium, by Dr Lambert and co-workers. All these compounds show different degrees of solubility, which of course affect their ability and usefulness as potential pharmacological tools. In this respect, the data concerning the FAAH inhibition are presented and compared both at individual concentrations, somewhat reflecting solubili

42


For the homologues with chain lengths fhigher than 20 % of [3H]-AEA metabolism wlength instead contained 10 to 18 carbons a metabolism was seen at 100 µM of these hunsatured homologue OEA (C18:1) was thmetabolism by 70 % (Fig. 6A). This compoundinhibitor, which was somewhat unexpected (Schmid et al., 1985; Licthman et al., 2002) andas competitive inhibitors. However, in this resa concentration of 1 µM, inhibited rat brainmanner, whereas the inhibition produced by(Tiger et al., 2000). It was pointed out in thatmixed and competitive was rather small anparticularly since the blank values were relativactivities at high substrate concentrations”. ThAt 10 µM, PEA (C16) showed the highest degrhomologues with decreasing inhibition with adpattern was seen at 100 µM when instead theshowed the highest degree of inhibition tosaturated homologues containing 17 or 18 concentration, also showed lower inhibition cohighest degree of inhibition using PEA contahomologue with 12 carbons at 100 µM, is probthese compounds, limiting the observed

NH

O

OH

C3

C18C18:1

NH

O

OH

C3

C18C18:1

NH

O

RNH

O

R

A

B

43

Fig. 5. (A) Modificafatty acid chain

tions of the (broken lines)

creating homologues of PEA. C3, i.e 3 carbons in the acyl chain, is

(OEA) indicates the number of the double bounds in the molecule. (B)

the shortest and C18 the longest ofthe homologues. The :1 in C18:1

Modifications of the ethanolamine head (dotted line) creating analogues of PEA. In some cases, the hydrogen on the amine was replaced by an additional R-group.

rom 3 to 8 carbons, no inhibition as seen at 100 µM. When the chain 50 -100 % inhibition of [3H]-AEA omologues. At 10 µM, the mono-e most efficient, inhibiting AEA was also found to be mixed type of since it is a substrate for FAAH alternate substrates should interact pect it has been found that AEA, at PEA hydrolysis in a competitive 2 µM AEA was mixed in nature study that “the difference between d may reflect experimental error, ely high with respect to the specific e same conclusion may apply here. ee of inhibition among the saturated ditional carbons. A slightly different homologue containing 12 carbons gether with OEA (Fig. 6A). The carbon in the side chains, at this mpared to PEA. This shift, with the ining 16 carbons at 10 µM, to the ably a reflection of the solubility of inhibition at the highest tested

Kent-Olov Jonsson

concentration. When comparing the pI50 values of the homologues against the inhibitable part of AEA hydrolysis, compounds with side chains from 10-18 carb ns show similar values, except for PEA and OEA showing slightly higher valu

hanolamine head were tested. The most important findings from Paper I and Paper II and examples are indicated her

h the highest activity in the ethylamide analogue of PEA (Fig. 6B, [4]), showing a lower obtainable inhibition with both lower and higher chain length at 10 µM. The same pattern was found with 100 µM, although in this case the methylamide analogue (Fig. 6B, [3]) showed the same obtainable inhibition as the ethylamide analogue (Fig. 6B). This is consistent with studies by Schmid et al. (1985) showing that the length of the chain of the amino

oes and the C10 homologue showing slightly lower potency. The homologues

with 3-8 carbons in the side chain were inactive, as mentioned earlier. Thus, a decrease in number of carbons enhances solubility, but too few carbon atoms in the side chain produce an inactive compound. The introduction of a double bound between carbon 9 and 10 seems to enhance solubility, although no major difference in potency is seen shown by the similar pI50 values between the saturated and the mono-unsaturated homologue. This lack of importance of the double binding concerning potency is in line with studies using the same experimental conditions, showing the oleoyl-analogue of PTMK exhibiting similar potency as PTMK used in Paper I (Fowler et al., 2000; Holt et al., 2001).

Instead of changing the fatty acid chain, another approach was to modify the

ethanolamine head of PEA. In Paper I, four analogues of PEA with a butylamide, isopropylamide, cyclohexamide or a (2-methyl)ethanolamide chain instead of the ethanolamide part was tested. In Paper II, this approach was taken further and a number of analogues with modifications in the et

e. For further details of all of the analogues investigated, see Paper II. By replacing the –OH group in the ethyl chain with a halogen atom, no major

difference in potency between the compounds compared to PEA was seen except when a fluoride atom was introduced (data not shown), and in this case all inhibitory activity was abolished. At both 10 and 100 µM, here shown with a chloride atom as replacement of the –OH group, the obtained inhibition seemed to decrease slightly compared to PEA (Fig. 6B). The same pattern, with no major difference in potency, was seen when hydrogen was introduced instead of the –OH group. In this case, the length of the head group seemed to affect the highest obtainable inhibition of AEA hydrolysis, wit

44


alcohol moiety is of importance upon interaction with the amidohydrolase in rat liver. These results, taken together, indicate tha is not of major importance in the ability of the hydrolysis. The length of the carbon chain see inhibition. The lack of importance of the r arachidonoyl-based compounds, where the et replaced by cyclopropyl, methylcyclopropyl or FAAH catalysed metabolism of [14C]-AEA in ra

The increase of steric groups seemed t introduction of one additional methyl group at cy as well as maximum obtained inhibition (Fig. 6B, PIA), and two additional methyl gro

t the –OH group in the ethyl chainse analogues to interfere with AEAms to affect maximum obtainable

–OH group is also the case fohanolamine head group of AEAcyclobutyl was reported to inhibit t brain (Jarrahian et al., 2000). o be of some importance, sincethe alpha-carbon decreased poten

ups gave an inactive compound (Fig. 6B, [9]). The first of these compounds (PIA) was found to be a mixed type of inhibitor (Ki(slope) 15 µM, Ki(intercept) 87 µM; but see discussion with respect to oleoylethanolamide and AEA concerning the difference between mixed and competitive inhibition) and is probably not a substrate itself for FAAH, since the arachidonyl isopropylamide analogue of AEA have no substrate activity (Lang et al., 1999). This effect of steric hindrance is not only due to a general bulky steric effect, since some aryl analogues were active (see e.g. compound 24), although the maximum inhibition of AEA hydrolysis was no more than around 30 % for these compounds. The steric parameters for these group was indeed tightly regulated, shown by difference in activity between compounds 23 and 24 (Fig. 6B).

45

Kent-Olov Jonsson

C3:

0

C4:

0

C6:

0

C8:

0

C10

:0

C12

:0

C14

:0

C16

:0

C17

:0

C18

:0

C18

:1

0

20

40

60

80

100

120

10 µM100 µM

A

4.26

4.79

4.77

5.30

4.71

4.95

5.54

no p

I 50

no p

I 50

no p

I 50

no p

I 50

% In

hibi

tion

of [3 H

]AEA

met

abol

ism

Fig. 6. Effects of homologues (A) and analogues (B) of PEA (C16:0) upon rat brain FAAH catalysed hydrolysis of 2 µM [3H]-AEA. In the homologues, the numbers of carbons in the fatty acid chain are indicated. In the analogues both the structure of the changed ethanolamine head group and the abbreviation or compound number (taken from Paper II) are indicated. The numbers above the columns in the figure are the pI50 values of the compounds, taken from Paper II.

[PEA

]

[34] [2

]

[3]

[4]

[5]

[6]

[19]

[PIA

]

[9]

[PC

A]

[24]

[23]

80

10 µM100 µM

B

5.01

4.5

5.4

5.56

5.54

5.58

4.89

no p

I 50

5.08

5.

no p

I 50no

pI 5

0

~3.6

7 530

% In

hibi

tion

of [3 H

]AE

met

abol

ism

A

0

20

40

60

NO

HN

OH

NN N NN NN NN NN NC

lN

Cl

NN NN NN NN NN

46


Interaction with CB1 and CB2 cannabinoid receptors (Paper I

and II)

All compounds in Paper I and Paper II were tested at 10 µM, as well as at 100 µM in some cases, upon their ability to interact with human CB1 and CB2 receptors. The analogues and homologues were assessed for their ability to inhibit specific binding of the synthetic cannabinoid receptor ligands [3H]-CP55,940 (CB1) or [3H]-WIN55,212-2 (CB2) on CHO cells transfected with the human CB1 or CB2 rece

seen. The analogue emanating as one of the most interesting from these studies, PIA, showed low affinity and inhibited specific binding at CB1 receptors with 25 % and 15 % and at CB2 receptors with 12 % and 21 % at 10 and 100 µM, respectively.

Inhibition of anandamide uptake (Paper I)

If inhibition of AEA hydrolysis and thereby prolongation of the lifespan of this compound is beneficiary, another approach is to reduce accumulation (and hence FAAH accessibility) of AEA. By identifying compounds inhibiting the process which accumulate AEA this is a way of avoiding, or at least delaying, AEA degradation. Indeed, putative AEA uptake inhibitors, such as AM404, have been shown to be active in vivo in a variety of models (see Fowler et al., 2005). All compounds in Paper I were screened for their ability to inhibit AEA uptake in

ptors. Concerning the homologues in Paper I, all except one produced <15 % at 10 µM or <25 % at 100 µM inhibition of specific binding with [3H]-WIN55,212-2 on CHO cells expressing CB2 receptors. In the case of the CB1 receptors, the data was less clear with inhibition of [3H]-CP55,940 between 19-32 % at 10 µM and between 8-41 % at 100 µM of the homologues. The only exception was the mono-saturated homologue oleoylethanolamide which gave 13-14 % inhibition at 10 µM and around 64 % at 100 µM at both CB1 and CB2 receptors. The analogues did not produce any inhibition of CB2 specific binding higher than 16 % at 10 µM or higher than 22 % at 100 µM for the compounds tested at that concentration in Paper I. Concerning the interaction with CB1 receptors, the analogues showed, to some extent, more affinity compared with the interaction at the CB2 receptor. None of the compounds were highly active with the most of these exhibiting an inhibition of specific binding less than 30 %. Even with the most active analogues, an inhibition less than 45 % was

47

Kent-Olov Jonsson

RBL-2H3 cells and were initially tested at 1, 10 and 100 µM. In this first screening, none of the compounds were active at 1 µM, although it was found that at concentrations of 10 or 100 µM the homologues containing more than 10 carbons in the fatty acid chain, exhibited some ability to inhibit [3H]-AEA uptake. The shorter homologues were inactive at all concentrations tested. As with the longer homologues, 10 or 100 µM of the analogues tested in this paper, showed ability to inhibit [3H]-AEA uptake.

As a result of the observation that AM404 can affect binding of [3H]-AEA to cell culture wells, which we became aware of after the initial screening, all compounds was tested retroactively for their ability to block [3H]-AEA binding to the wells. Neither the saturated homologues nor the analogues affected this binding to a major extent. In contrast, AM404 as well as the mono-saturated homologue OEA did indeed inhibit the binding of [3H]-AEA to the wells.

As a consequence of the effect of binding to plastic, the most promising compounds from the initial uptake data as well as AM404 and PEA were tested using RBL-2H3 and/or C6 cells with parallel assays using wells. In Fig. 7 the data are presented as cell specific, i.e. the [3H]-AEA uptake of each individual compound in wells with no cells is subtracted from the uptake for this compound when cells are present. The data indicated that 30 µM of PIA, as well as 100 µM AM404, inhibited cell specific [3H]-AEA uptake in both RBL-2H3 (Fig. 7A) and C6 (Fig. 7B) cells. In contrast to these compounds, 30 µM of PEA did not affect and 30 µM of OEA showed only a small effect on cell specific [3H]-AEA uptake in C6 cells (Fig. 7C). As mentioned above, both AM404 and OEA per se inhibited the [3H]-AEA uptake (see Paper I for figures) in wells without cells, making it troublesome to interpret the uptake data of these compounds. Among the compounds analysed, PIA was the substance that showed most potential in affecting AEA uptake, and in contrast to AM404 and OEA, this compound had little effect on the binding of [3H]-AEA to plastic.

48


RBL-2H3 C6 C6

Fig. 7. Effects of PEA and related compounds (µM) upon the uptake of 10 µM [3H]-AEA into RBL-2H3 basophilic leukaemia cells (A) and rat C6 glioma cells (B, C). Shown are means±s.e.mean of cell specific, i.e. the [3H]-AEA uptake of each individual compound in wells not containing cells is subtracted from the uptake for this compound when cells are present. Data are recalculated from figure 6 of Paper I.

Inhibition of anandamide uptake and hydrolysis in intact C6 cells

(Paper I)

Since the in vivo effects of AEA metabolism is dependent on both uptake and hydrolysis, a compound producing inhibition of both processes might be favourable compared to compounds only affecting each separate process. To investigate the combined effects upon these processes, the compounds in figure 7 were tested for their ability to inhibit [3H]-ethanolamine production in intact C6 cells. By separating this product from the added [3H]-AEA substrate, a measurement of the compounds effect on both AEA uptake and degradation can be accomplished. PIA and OEA together with AM404 was found to be the most potent in this respect, showing a concentration dependent inhibition of [3H]-

m 3-30 µM (Fig. 8A), with ion tested. PEA and PCA

exh

ethanolamine production, at concentrations ranging froPIA being the most effective at the highest concentrat

ibited the same concentration dependent pattern as the compounds above, although the latter did not inhibit [3H]-ethanolamine production to the same extent (Fig. 8B). The relative contribution of uptake or FAAH inhibition in this model is somewhat difficult to speculate on, since the FAAH inhibition also is dependent on the ability of the compounds to be taken up by the cells. In this respect, less is known about the cellular uptake of the examined compounds, although the lipophilicity indicates at least a possible passive diffusion over the cell membrane.

Non

e

PCA

10

PCA

30

PIA

10

PIA

30

AM

404

100

0

5

10

15A

ake

in.-1

)

B C

[3H

](p

mo

AEA

upt

l.wel

l-1.m

Non

e

PCA

10

PCA

30

PIA

10

PIA

30

AM

404

100

0.0

2.5

5.0

7.5

10.0

[3H

]AEA

u(p

mol

.wel

l-1pt

ake

.min

.-1)

Non

e

OEA

10

OEA

30

PEA

30

0

4

8

12

[3H

]AEA

u(p

mol

.wel

l-1pt

ake

.min

.-1)

49

Kent-Olov Jonsson

In the case of PEA, some studies have examined the cellular uptake in RBL-2H3 and Neuro-2 neuroblastoma cells and found to it be mediated in a different way compared to AEA (Bisogno et al., 1997; Jacobsson et al., 2001).

Fig. 8. Effects of OEA, PIA, AM404 (A), PCA and PEA (B) upon the hydrolysis of [3H]-AEA in intact C6 rat glioma cells. Data are means±s.e.mean and are recalculated from table 3 of Paper I.

Main conclusions from Papers I and II.

From the data presented above, it can be concluded that the PEA homologues and analogues tested have shed some light upon the structure-activity relationship in the respect of these compounds as FAAH inhibitors and to some extent the effect of chemical structure and solubility. None of the analogues or homologues was found to be dramatically more potent compared to PEA itself. However, potency is not the only important attribute of a compound – selectivity is just as important. PIA, the lead compound chosen in these studies, not the least as a result of its ability to inhibit AEA metabolism in intact cells, has subsequently been shown to inhibit (albeit weakly, but to a similar extent to PEA itself), the metabolism of radioactive PEA by lung N-palmitoylethanolamine-selective acid amidase (Vandevoode et al., 2003). However, in contrast to PEA, PIA has no effect at TRPV1 receptors (Smart et al., 2002) and has been used to demonstrate an endogenous tone of an FAAH-sensitive compound (presumably AEA) at bladder TRPV1 receptors (Dinis et al., 2004). Similarly, PIA does not share the ability of PEA to decrease forskolin-stimulated cAMP accumulation in a microglial cell line (Franklin et al., 2003). Thus, data so far accumulated would suggest that PIA is a more selective inhibitor of AEA metabolism than PEA.

Non

e

OEA

3

OEA

10

OEA

30

PIA

3

PIA

10

PIA

30

AM

404

3

AM

404

10

AM

404

30

0.0

2.5

5.0

7.5

10.0A

hano

lam

ine

uctio

nll-1

.min

.-1)

(µM)

[3H

] p(p

mol

.we

et rod

Non

e

PCA

3

PCA

10

PCA

30

PEA

3

PEA

10

PEA

30

0.0

2.5

5.0

7.5

10.0B

min

en in

.-1)

(µM)[3

H]e

than

prod

uc(p

mol

.wel

lola tio -1.m

50


Effects of PIA, AM404 and VDM11 upon cell proliferation

(Paper III)

After the identification of compounds from Paper I and II, we further wanted to test if the most promising analogues from the initial studies affected cell proliferation. As a comparison, other presumptive AEA uptake inhibitors such as AM404 and VDM11 were also tested in this approach. The specificity of AM404 has been questioned since it has shown to cause an influx of calcium into a variety

at a concentration of 1 µM; this effect was not blocked by either the CB1 receptor

g a 96 h incubation, it was found that both AM404 and its structurally relat

of cells (Chen et al., 2001) at concentrations used for AEA uptake studies, which may be due to the fact that the compound is an agonist at TRPV1 receptors (Zygmunt et al., 1999; 2000; Jordt & Julius, 2002). More recently Kelley & Thayer (2004) reported that AM404 affected calcium spiking in rat hippocampal neurons

antagonist rimonabant, the TRPV1 antagonist capsazepine, or by treatment of pertussis toxin.

Followined analogue VDM11 inhibited, in a concentration dependent manner, the

proliferation of C6 glioma cells. This was also seen with PCA. In contrast, PIA did not inhibit cell proliferation at all at daily added concentrations of 1, 3 and 10 µM, and produced only a small effect at 30 µM. These data are summarised in Fig. 9A together with data performed at the same time with OEA. The dramatic inhibition seen at 30 µM of this compound may be related to the ability of this compound to inhibit ceramidase (Sugita et al., 1975).

AM404, VDM11 and PIA were also tested for their ability to inhibit cell

proliferation at different time points. The dose and time response data showed that both AM404 and VDM11 exerted a major effect upon cell proliferation at concentrations of 10 and 30 µM at all time points investigated (24, 48, 72 and 96 h after administration). At 3 µM, the same pattern was seen for VDM11 whereas AM404 had a very small (albeit significant) effect. At 1 µM, a small, but significant inhibition of cell proliferation could be seen after the 72 h treatment with both of these compounds. PIA did not affect cell proliferation at all when tested at 1, 3 and 10 µM at any time point. At the highest concentration tested, 30 µM, PIA showed a significant reduction after 24 h, although this effect was not as pronounced as with the other AEA uptake inhibitors. These results indicate that AM404 and

51

Kent-Olov Jonsson

VDM11 indeed affect cell proliferation at the same concentrations used for AEA uptake studies (De Petrocellis et al., 2000), and to a larger extent than is seen for PIA.

To evaluate further the mechanism behind the cell proliferation inhibiting effects found, AM404, VDM11, PCA and PIA were incubated at concentrations that affected cell proliferation with a mixture of cannabinoid (AM251+AM630) and TRPV1 (capsazepine) receptor antagonists or α-tocopherol, an antioxidant. The antiproliferative effects could not be antagonised by these treatments, except in the case of 30 µM PCA, where a partial block was seen with α-tocopherol. Since apoptotic cell death can be induced by uncontrolled calcium influx (see Kim et al., 2002 for example) and AM404 induces a rise in calcium influx (Chen et al., 2001), we also wanted to test if the antiproliferative effects could be a due to induction of apoptosis. In this respect, we tested AM404, VDM11 and PIA upon their ability to

positive control supplied with the kit (camptothecin treated U937 en the treated and untreated cells in

caspase ac

ition per se were included.

activate caspases, which is an initial event in apoptosis. Very low levels of caspase activity were seen after either 48 or 96 h treatment with the compounds (<2.5 %), compared to the cells). Although there was a difference betwe

tivity, this difference essentially reflected the cell content of the wells, which was measured in parallel plates using the same treatment.

In addition to the effects per se of the compounds, they were tested upon their

ability to reduce the effect of exogenously administered AEA in this system. AEA has previously been shown to produce antiproliferative effects in these cells in a TRPV1 mediated way (Jacobsson et al., 2001). Given by the fact that the AEA binding site for TRPV1 receptors is intracellularly located (De Petrocellis et al, 2001; Jordt & Julius, 2002), a hindering of AEA uptake would decrease the antiproliferative effects of AEA. Indeed, such an effect was seen when AM404, VDM11 or PIA was administered together with different concentrations of AEA and incubated for 96 h. The uptake inhibitors were tested at concentrations where they showed no or only a small effect on proliferation per se, and examples of the effects upon 2 µM AEA are given in Fig. 9B. The compounds showed a similar pattern in their way of inhibiting the antiproliferative effects of AEA. It should be noted that a large variability was seen upon the effects of AEA, and since the aim was to investigate whether AM404, VDM11 and PIA could decrease these effects, only those experiments where AEA produced an inhib

52


This variability has been subsequently investigated in detail and been shown nbe related to analogue show

ot to the lipophilicity of AEA, since its water soluble phosphate ester s the same variability (Fowler et al., 2003b).

inding that CB2 receptor agonists, together with PEA, can affect mast cell dependent inflammation in different models which seem

Fig. 9. (A) Effects of PEA and AEA related compounds upon the proliferation of C6 rat glioma cells. The cell density was measured after 96 h. Data are expressed as % of cells treated with vehicle. Data are means±s.e.mean and are taken from Paper III. (B) Effects of AM404, VDM11 and PIA upon the antiproliferative effects of 2 µM AEA. Data are means±s.e.mean and are taken from figure 4 of Paper III.

Effects of PIA and JWH133 upon inflammation in vivo and in

vitro (Papers IV and V)

After the initial identification and characterisation of potential analogues from Paper I-III, we wanted to test if PIA could be effective in vivo. To establish an in vivo system for this purpose, we set up a model of mast cell dependent inflammation. This was based on the f

s, at least to some extent, to involve CB2 receptors (Malan et al., 2001a; Aloe et al., 1993; Mazzari et al., 1996; Hanus et al., 1999). As a reference the CB2 receptor agonist, JWH133 (Huffman et al., 1999) was used.

The first set of experiments showed that 20 or 200 µg but not 600 µg

(corresponding to 1, 10 and 30 mg/kg) JWH133 was significantly effective in reducing compound 48/80-induced plasma extravasation when the CB2 agonist was i.p. administered in an ethanol vehicle (Fig. 10A). To investigate if CB2 receptors were involved in this response, the CB2 receptor antagonist/inverse agonist SR144528 was administered i.p. prior to JWH133. In this set of experiment, however, the effect of JWH133 did not reach statistical significance

1 3 10 30 1 3 10 30 1 3 10 30 1 3 10 30 1 3 10 30

0

25

50

75

100

AAM 404 VDM 11 PCA PIA OEA

(µM)

Cel

l den

sity

(% o

f con

trol

)

0

25

50

75

100

AEA (2µM)

AEA + PIA (3µM)

AEA + VDM11 (1µM)AEA + AM404 (1µM)

B

Cel

l den

sity

(% o

f con

trol

)

53

Kent-Olov Jonsson

when injected alone at a dose of 200 µg, although the pattern was the same as in the initial experiment (Fig. 10B). Concerning the effects of SR144528, the results are hard to interpret in terms of antagonising the effects of JWH133, since the SR1

e of carrier. As a con quence, PIA and PEA were dissolved in a preparation of eth

44528 itself seemed to decrease the response of compound 48/80 (Fig. 10B). Although this is at first sight somewhat an unexpected finding, it is in line with data by Iwamura and co-workers, who found that SR144528 decreased carrageenan-induced paw oedema in mice (Iwamura et al., 2001). Similar results were also found in a recent study, were topical application of SR144528 reduced TPA (tetradecanoylphorbol acetate)-induced ear swelling as well as leukotriene B4 production and neutrophil infiltration in the mouse ear (Sugiura et al., 2004). Smith et al (2001) also reported that SR144528 reduced neutrophil migration in a mouse peritonitis model, and cited preliminary data that this was also seen in a CB1/ CB2 double knockout, indicating a CB1 and CB2 receptor independent effect.

In the case of PIA, as well as PEA (data not shown), initially no effects were

seen on plasma extravasation when the compounds were administered in an ethanol vehicle (Fig. 10C). Given the fact that these compounds are highly lipophilic, and that PEA has proven effective in other mast cell models, we wanted to test if the lack of effect in this model was due to the choic

seanol:Cremophor®:saline (1:1:18) instead. In this case, both treatments seemed

to decrease the compound 48/80-induced plasma extravasation, although this was not found to be statistically significant due to one animal in each PIA treated group which did not respond to the treatment (Fig. 10D). One explanation could be that the injection of PIA in these animals did not result in a systemic administration of the drug, although there is no proof for this concept. If this is a plausible explanation, the present data are in line with the finding by Mazzari et al. (1996) that PEA is effective in reducing mast cell dependent inflammation in vivo, and that PIA and JWH133 produces similar effects.

As a complement to the effects of the compounds in vivo, we wanted to evaluate if any effects could be seen using an in vitro system. Instead of using mast cell lines or isolated mast cells, in which the cells ability to respond is altered (Robinson et al., 1989; Sweiter et al., 1993; Hamawy et al., 1994; Ra et al., 1994), we chose to establish a skin tissue model where the cells are surrounded by a natural environment. When the mouse paw skin tissue was stimulated by compound

54


48/80 a significant release of the granular enzyme β-hexosaminidase was seen, although this was not followed by a detectable production of the cytokine TNF-α. Pre-treatment of the skin tissue with JWH133, PIA or PEA did not alter the basal release of β-hexosaminidase.

To investigate if CB2 receptors were present in skin tissue, PCR and

immunohistochemical experiments also were undertaken. The PCR experiments indicated that the skin samples expressed mRNA for the CB2 receptor. Immunoreactivity towards the CB2 receptor antibody used was detected in the epidermal rather than the dermal layers.

al values, taken from Paper IV and V, are shown. Statistically significant differences in one-way ANOVA followed by Dun

Fig. 10. Effects of JWH133, PIA, PEA and SR144528 upon compound 48/80-induced plasma extravasation in mouse ear pinna. The individu

nett’s multiple comparisons post-hoc test are indicated as **P<0.01 and *P<0.05 when compared with vehicle treatment.

vehicle 20 200 6000.0

0.5

1.0

1.5

2.0

2.5

* **

A

xtra

vasa

tion

s bl

ue, µ

g/m

l)

[JWH133] (µg)

E(E

van

vehicle JWH133 (200) vehicle JWH133 (200)0

1

2

3

4 Control SR144528Bxt

rava

satio

nva

ns b

lue,

µg/

ml)

Treatm ent (µg)

E(E

Vehicle 20 200 6000

1

2

3

[PIA] (µg)

Extr

avas

atio

n(E

vans

blu

e, µ

g/m

l)

C

Vehicle PIA (20) PIA (200) PEA (200)0

1

2

3

4

Pretreatm ent (µg)

Extr

avas

atio

n(E

vans

blu

e, µ

g/m

l)

D

55

Kent-Olov Jonsson

Main conclusions from Papers IV and V

Perhaps the most important conclusion to be made is that the ”apparently simple” (see above) model for inflammation chosen turned out to be far from simple, with both a large variation in response and the choice of vehicle producing problems of their own. The most robust findings of the studies are that JWH133 reduces the oedema response to compound 48/80 in vivo but does not affect the release of the granular enzyme ß-hexosaminidase in response to this agent in the skin slices, and that mRNA for the CB2 receptor is present in the skin samples. Our interpretation of the present data is that, at least for JWH133, the action of this compound is not directly upon mast cells, but rather an indirect one, presumably mediated by CB2 receptors. Our data would suggest that PIA is also able to modify mast cell function in vivo, although the reason for the non-responsiveness of two of the animals is not clear.

56


GENERAL CONCLUSIONS AND FUTURE

PERSPECTIVES

The present thesis has been undertaken with the main goal of identifying and cha terising selective FAAH inhibitors. The main conclusions that can be drawn are:

electivity for FAAH versus CB receptors, making them suitable as pharmacological research tools.

• The lead compound, PIA, shows ability to hinder cellular accumulation of AEA, and experimental data also suggest that the total effect upon AEA degradation equals other known AEA uptake inhibitors, with the advantage of showing less effects not related to FAAH inhibition or AEA uptake.

• JWH133, a CB2-selective agonist, can reduce plasma extravasation in a mouse model of mast cell dependent inflammation and PIA may be active in a similar way.

A number of additional observations can be made. For example, as stated in the introduction, it is not clear how AEA is accumulated intracellularly, which makes is somewhat difficult to interpret uptake data. Whatever mechanism is

Lago et al., 2002; 2004). This would suggest that compounds that can inhibit both AEA metabolism and accumulation may be more effective than compounds acting on each process per se. Some of the compounds studied in this thesis appear to be able to inhibit both processes, but further studies will have to be conducted before definitive conclusions can be made. The in vivo data in the thesis suggests that PIA could be effective against mast cell dependent

rac

• The compounds, which where chemically based on the endogenous anti-inflammatory fatty acid PEA, may not be the most potent FAAH inhibitors developed, but show s

responsible for AEA uptake into cells, there are data suggesting that compounds like URB707 and OMDM-2 that inhibit AEA uptake, but do not inhibit FAAH (López-Rodríguez et al., 2003; Ortar et al., 2003), are still effective in potentiating the effects of AEA in vivo (De

57

Kent-Olov Jonsson

inflammation, although, as noted earlieHowever, PEA, which is a well known

r, the results did not reach significance. anti-inflammatory compound, did not

pro

ed to CB1 agonists that they lack the psychotropic effects mediated by the CB1 receptor. Our data showed the presence of CB2 mRNA in skin and suggested that the location of the receptors was in the epidermis, although this conclusion is dependent upon the true specificity of the antibody used. Thus, more studies are needed to confirm the location of functional CB2 receptors present in skin tissue. Another approach to examine if the anti-inflammatory effects seen with JWH133 are due to CB2 receptor activation is to test the compound in CB2 (-/-) mice which, to my knowledge, has not been performed with any CB2 agonist so far.

What could be future approaches? A major drawback with the compounds

identified in this thesis is the limited solubility, hampering their usefulness both in vitro and in vivo. This is a general phenomenon, when dealing with lipophilic compounds such as the cannabinoids. A future approach would be to develop less lipophilic substances, to decrease these issues. If FAAH inhibition, AEA uptake inhibition and CB2 activation is beneficiary, a possible chemical approach would be to develop compounds with combined activity at these targets.

Although there are many unsolved questions, and whatever the future holds in

its hands, the present work has hopefully contributed to an increased knowledge in the continuously growing scientific field of cannabinoids.

duce a significant response either, indicating that the in vivo model itself was troublesome, due to large variability. More investigations are therefore needed to establish the usefulness of PIA in vivo. If, for the sake of argument, an effect was seen with PIA, what is the mechanism responsible for its actions in vivo? Even if the first data clearly showed a FAAH and AEA uptake inhibiting role of PIA in vitro it is possible that other mechanisms could be responsible for its actions in vivo. For example, a recent study attributed the anti-inflammatory effects of PEA to activation of PPAR-α, although this is the only study published so far in this respect (Lo Verme et al., 2005). The same mechanism could operate here as well, and this hypothesis should be tested.

A more clear-cut result of this study (and of others) is the effectiveness of CB2 agonists in vivo, which have the advantage compar

58


ACKNOWLEDGEMENTS

ainly performed at the DThe work in this thesis was m epartment of Pharmacology and Clini

reach this point. I wish to express my sincere gratitude to all those who have contributed during this journey. In particular, I wish to thank the following people: First I supervisendocan ent and ideas, and for keeping me off the streets! Dr Gupharmac Dr Stig son, for critical discussions concering science, teaching and technical issues, which have been very valuable during this work. Dr SéveBelgium peration, which have been a foundation for this thesis. I al

cal Neuroscience at Umeå University, Umeå, Sweden. During my years as a graduate student I have met several people who have helped me to

would like to express my gratitude to Professor Chris Fowler, my or, who has wakened my interest for the scientific field of nabinoids. Thank you for your never-ending encouragem

nnar Tiger, my co-supervisor, for fruitful discussions concering ology in general and for making the department a funnier place on earth.

Jacobs

rine Vandevoorde and Dr Didier Lambert, our co-workers in Brussels, , for fruitful co-o

so would like to thank Dr Ruth Ross in Aberdeen, for giving me the chance to explore Scottish science in her lab. Douglas McHugh, my friend from Aberdeen, who I had the opportunity to meet during my stay in Scotland. Thank you for helping me out in the lab as well as showing me around outside the University! Also thanks to all other people at the lab in Aberdeen for making my stay enjoyable. Professor Torbjörn Egelrud and Dr Annelii Ny for help with the immunohistochemistry experiments.

59

Kent-Olov Jonsson

Olov Nilsson and Sandra Holt, PhD students at the department, for scientific and teaching discussions. I also want to thank the rest of the members of the department, especially Britt Jacobsson and Ingrid Persson, who have helped me with some scientific work, and also Emma Söderström, Alf Olsson and Björn Carlsson for help with all non-scientific issues at the department. I also would like to thank all my friends who have, although not always in a direct way, contributed to this thesis. Especially Tobias Holmberg, a garrulous, singing friend, Dr Jörgen Kajberg, another “shy and quiet” friend and Dr Bengt Wikman, a friend who built an excellent mobile sauna. I am also very grateful to my family, for giving me support through life and always being helpful. Finally, Emma, thank you for being part of my life and helping me with everything, especially during the last year. I am indeed also grateful for your scientific contribution to this thesis.

60


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