1

Endocannabinoids and N-acylethanolamines in

translational pain research: from monoacylglycerol lipase

to muscle pain

Nazdar Ghafouri

Department of pharmacology and Clinical Neuroscience

Umeå University

Umeå 2013

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Responsible publisher under swedish law: the Dean of the Medical Faculty

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

ISBN: 978-91-7459-567-3

ISSN: 0346-6612

http://umu.diva-portal.org/

Printed by:

Umeå, Sweden 2013

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“You've got to know your limitations. I don't know what your limitations are. I

found out what mine were when I was twelve. I found out that there weren't too

many limitations, if I did it my way.”

Johnny Cash

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Contents

Original papers 6

Abstract 7

Summary (Swedish) 8

Abbreviations 10

Introduction 11

The endocannabinoid system 11

Synthesis of NAEs 12

Synthesis of 2-AG 15

Cannabinoid receptors 15

Non-cannabinoid receptors 16

Catabolism of NAEs and 2-AG 18

The endocannabinoid system in pain processing 23

Exogenous cannabinoids in human pain trials 25

Exogenous PEA in animal pain models 26

Exogenous PEA in human pain trials 27

Blockade of endocannabinoid and NAE degradation as a 28

therapeutic approach for the treatment of pain

Chronic pain 30

Chronic muscle pain 31

Chronic neck/shoulder pain 31

Chronic widespread pain 32

Nociception and chronic muscle pain 33

Aims 34

Methods and methodological considerations 35

Assay of MAGL and FAAH (paper I) 35

Key methods used in paper II-IV 37

Subjects 37

Recruitment of subjects 38

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Selection of subjects 39

Training program (paper IV) 40

Microdialysis procedure 41

Pain intensity ratings 43

Standardized low-force repetitive exercise 44

Analysis of NAE levels 44

Summary of study protocols. 46

Results 48

Paper I 48

MAGL and FAAH assay characterization 48

Inhibition of FAAH and MAGL by 2-AG and related compounds 50

Paper II-IV 52

Pilot study 52

Anthropometric data 54

Pain intensity ratings 54

PEA and SEA microdialysate concentrations 57

Discussion 63

Future aspects 66

Acknowledgments 68

Reference list 70

Paper I

Paper II

Paper III

Paper IV

6

Original papers

The present thesis is based on following papers, which are referred to in the text by their

Roman numerals.

I Nazdar Ghafouri, Gunnar Tiger, Raj K. Razdan, Anu Mahadevan, Roger G.

Pertwee, Billy R. Martin, Christopher J. Fowler. Inhibition of monoacylglycerol

lipase and fatty acid amide hydrolase by analogues of 2-arachidonoylglycerol.

British Journal of Pharmacology. 2004 November; 143(6): 774–784

II Nazdar Ghafouri, Bijar Ghafouri, Britt Larsson, Maria V. Turkina, Linn

Karlsson, Christopher J. Fowler, Björn Gerdle. High levels of N-

Palmitoylethanolamid and N-Stearoylethanolamid in microdialysate samples

from myalgic trapezius muscle in women. PLoS ONE 6(11): e27257.

doi:10.1371/journal.pone.0027257

III Nazdar Ghafouri, Bijar Ghafouri, Britt Larsson, Christopher J. Fowler, Niclas

Stensson, Björn Gerdle. Palmitoylethanolamide and stearoylethanolamide levels

in the interstitium of the trapezius muscle of women with chronic widespread

pain and chronic neck-shoulder pain. Submitted.

IV Nazdar Ghafouri, Bijar Ghafouri, Christopher J. Fowler, Britt Larsson, Maria V.

Turkina, Linn Karlsson, Björn Gerdle. Effects of two different specific neck

training programs on palmitoylethanolamide and stearoylethanolamide

concentrations in the interstitium of the trapezius muscle in women with chronic

neck shoulder pain. Manuscript.

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Abstract

In the early nineties cannabinoid receptors, the main target for ∆9-tetrahydrocannabinol

(THC), the psychoactive component of marijuana were identified. Shortly after their

endogenous ligands, N-arachidonoylethanolamine (anandamide, AEA) and 2-diacylglycerol

(2-AG) were characterized. The enzymes primarily responsible for catalysing the degradation

of AEA and 2-AG are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase

(MGL) respectively. AEA is a member of the N-acylethanolamine (NAE) class of lipids,

which depending on the acyl chain length and number of double bonds can act as ligands for a

variety of biological targets. Exogenous cannabinoids have long been reported to have

analgesic effects, however the clinical usefulness of such substances is limited by their

psychoactive effects. Inhibition of endocannabinoid degradation would mean enhancing the

therapeutic effects without producing these unwanted side effects. In order to succeed in

developing such compounds the pharmacology of the enzymes responsible for the degradation

of endocannabinoids has to be thoroughly understood. When the preclinical part of this thesis

was planned, FAAH had been well characterized whereas little was known as to the

pharmacology of MGL. A series of compounds were tested in this first study aiming to find

MGL-selective compounds. Although no compounds showed selectivity for MGL over

FAAH, several interesting agents affecting both enzymes were identified.

In order to increase the knowledge concerning which patient group would benefit from such

treatment strategies it is important to investigate in which pain states the

endocannabinoids/NAEs are altered. Thus the general aim of the clinical part of this thesis

was to investigate the levels of endocannabinoids/NAEs in the interstitium of the trapezius

muscle in women suffering from chronic neck/shoulder pain (CNSP) and chronic wide spread

pain (CWP) and in healthy pain-free controls. Furthermore for the CNSP the effect of

training, which is a commonly recommended treatment for these patients, on the levels of

endocannabinoids/NAEs was also investigated. Microdialysis technique in the trapezius

muscle was used for sampling and masspectrometry was used for analysing. Two NAEs, N-

palmitoylethanolamine (PEA) and N-stearoylethanolamine (SEA), could be repeatedly

measured. The levels of these two lipids were significantly higher in CNSP compared to

CON. The result showed also that PEA and SEA mobilize differently in CWP compared to

both CNSP and CON. Taken together the results presented in thesis represent an early

characterization of the pharmacology of MGL and provides novel information on NAEs in

chronic muscle pain.

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Sammanfattning på svenska

Cannabis har länge använts som njutningsmedel men även för medicinska syften såsom

smärtbehandling. Under 90-talet upptäcktes att den primära psykoaktiva komponenten i

cannabis, ∆9-tetrahydrokannabinol (THC), kan bindas till och aktivera två G-proteinkopplade

receptorer (CB1 och CB2). Fortsatt forskning ledde sen till upptäckten av kroppsegna

cannabinoider (endocannabinoider). De två mest studerade endocannabinoiderna är

anandamid (AEA, arakidonoyletanolamid) och 2-AG (2-arakidonoylglycerol). AEA tillhör

kemiskt gruppen N-acyletanolaminer (NAE) där övriga substanser i gruppen inte aktiverar

cannabinoidreceptorer men har en rad andra biologiska effekter. De mest studerade

enzymerna som bryter ner endocannabinoider/NAE är fettsyraamidhydrolas (FAAH) och

monoacylglycerol lipas (MAGL). Idag har den smärtlindrande effekten av

cannabinoidreceptorstimulering demonstrerats i en rad olika djurmodeller för akut och

kronisk smärta. Möjligheterna att använda cannabinoider i klinisk praxis begränsas dock av de

psykogena effekterna. Blockering av nedbrytningen av de kroppsegna cannabinoiderna skulle

kunna leda till ökad terapeutisk effekt utan oönskade effekter. Utveckling av sådana

blockerande substanser förutsätter goda kunskaper om farmakologin för enzymerna som är

involverade i nedbrytningsprocessen. När den prekliniska delen av denna avhandlingen

planerades var kunskapen om MAGL begränsad medan FAAH var väl kartlagt. I första

studien testades därför en rad olika substansers selektivitet för de två enzymerna. Resultatet

av studien bidrog framför allt till ökade kunskaper om grundläggande farmakologiska

egenskaper hos MAGL.

För att veta vilka patienter som skulle ha nytta av en blockerad endocannabinoider/NAE

nedbrytning, är det viktigt att undersöka vid vilka smärttillstånd som nivåerna av dessa lipider

är förändrade. Kronisk muskel smärta är vanligt förekommande i befolkningen, särskilt bland

kvinnor och leder ofta till nedsatt arbetsförmåga och livskvalitet. Mekanismerna bakom

smärtorna är ofullständigt kända men studier pekar på att det råder en obalans mellan

smärthämmande och smärtfaciliterande processer. Studier pekar också på att signaler från

perifer vävnad såsom muskulatur vidmakthålla förändringar som sker på ryggmärgsnivå och

hjärnan vid kroniska smärttillstånd. För att underöka molekylära förändringar i muskulaturen

har flera forskargrupper använt trapeziusmuskeln som modell. Man har då använt sig av

mikrodialysteknik som innebär att ett mycket tunt membranförsett plaströr läggs in i

muskulaturen och genomsköljs med en vätska som liknar muskulaturens sammansättning, för

insamling av olika substanser från muskulaturen. De flesta mikrodialysstudierna hittills har

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fokuserat på att studera smärtande substanser och det saknas studier på potentiellt

smärthämmande substanser såsom endocannabinoider/NAE.

Övergripande syftet med den kliniska delen av avhandlingen var således att undersöka

nivåerna av endocannabinoider/NAE i muskulaturen hos kvinnor med kronisk nack/skulder

smärta, kronisk generaliserad (utbredd) smärta samt hos friska smärtfria kvinnor.

Mikrodialysteknik i trapezius muskeln användes för insamling av substanserna som sedan

analyserades i masspektrometer.

Resultaten visade att två stycken NAE, N-palmitoyletanolamin (PEA) och N-stearoyl-

etanolamin (SEA), kan detekteras och mätas med de använda teknikerna. Nivåerna av både

PEA och SEA var signifikant högre hos kvinnor med nack/skulder smärta jämfört med friska

smärtfria kvinnor. Resultaten visar också att substanserna möjligen mobiliseras på olika sätt

beroende på smärtutbredning och muskelaktivitet. Sammanfattningsvis uppvisar denna

avhandling en karakterisering av farmakologin för MAGL, som tidigare var ofullständigt

känd, samt ger ny kunskap om NAE vid kronisk muskelsmärta.

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Abbreviations

ACR American College of Rheumatology

AEA Arachidonoyl ethanolamide (Anandamide)

2-AG 2-Arachidonoyl glycerol

CB Cannabinoid

CNSP Chronic neck/shoulder pain

CON Controls

CWP Chronic wide spread pain

FAAH Fatty acid amide hydrolase

FMS Fibromyalgia syndrome

IASP International Association for the Study of Pain

LC Liquid chromatography

MAGL Monoacylglycerol lipase

MS Masspectrometry

NAAA N-Acylethanolamine-hydrolysing acid amidase

NAE N-Acylethanolamine

NAPE N-Acyl phosphatidylethanolamine

NAPE-PLD N-Acyl phosphatidylethanolamine phospholipase D

NAT N-Acyl transferas

NMCQ The Nordic Ministry Council Questionnaire

NRS Numeric rating scale

PAG Periaqueductal grey

PEA Palmitoylethanolamide

PPAR Peroxisome proliferator activated receptor

SEA Stearoylethanolamide

THC ∆9 -Tetrahydrocannabinol

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Introduction

The endocannabinoid system

Cannabis has long been utilised both for recreational and medicinal purposes. The main

psychoactive ingredient of cannabis, ∆9-tetrahydrocannabinol (THC) produces many of its

effects in the body as a result of its ability to activate a class of G-protein coupled receptors,

termed cannabinoid (CB) receptors, which were cloned in 1990 and 1993 (Matsuda et al.,

1990). Signalling mediated by cannabinoid receptors is involved in a broad range of different

physiological and pathological conditions such as appetite regulation, neurotoxicity, nausea,

regulation of the immune system as well as pain modulation (Fowler et al., 2005, Pacher et

al., 2006)

The existence of CB receptors led to a search for endogenous cannabinoid receptor ligands

("endocannabinoids") culminating in the discovery in 1992 of anandamide (N-arachidonoyl-

ethanolamide, AEA) (Devane et al., 1992). Three years later, 2-arachidonoylglycerol (2-AG)

was shown to act as an endocannabinoid (Mechoulam et al., 1995, Sugiura et al., 1995).

Although other endocannabinoids have been postulated (Hanus et al., 1993, Bisogno et al.,

2000, Hanus et al., 2001, Huang et al., 2002, Porter et al., 2002), AEA and 2-AG are the most

well-characterised endocannabinoids.

The endocannabinoid system comprises in brief the cannabinoid receptors type 1 (CB1) and

type 2 (CB2), their endogenous ligands, and the proteins responsible for their biosynthesis and

degradation. Because they are highly lipophilic compounds, endocannabinoids are not stored

in intracellular vesicles like other neurotransmitters. Instead they are synthesized and released

”on demand” by activity-dependent or receptor-stimulated cleavage of phospholipid

precursors in the cell membrane. Inactivation of endocannabinoid signalling is brought about

by cellular reuptake, followed by enzymatic degradation.

AEA is a member of the N-acylethanolamines (NAEs) class of lipids, which, depending on

the acyl chain length and number of double bonds can act as ligands for a variety of biological

targets. Other endogenous NAEs include N-palmitoylethanolamine (PEA), N-stearoyl-

ethanolamine (SEA) and N-oleoylethanolamine (OEA). The lipids can be defined on the basis

of the number of carbon atoms and double bonds in the acyl part of the molecule. PEA, for

example, is a (16:0) NAE, whereas SEA, OEA and AEA are (18:0), (18:1) and (20:4) NAEs,

respectively.

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PEA was isolated from mammalian tissues in 1965 (Bachur et al., 1965). Thus, long before

AEA was identified, extensive information regarding the biosynthesis of NAEs was available

(Schmid et al., 1990). Initial conclusions were that these lipids were produced by a

phospholipid-dependent pathway but more recently several important pathways have been

characterized. These are described below.

Synthesis of NAEs

The main route of NAE synthesis from lipids involves a two-step process, with N-acyl-

phosphatidylethanolamines (NAPEs) as the key intermediates. NAPEs are N-acylated

derivatives of phosphatidylethanolamines (PEs) and serve as precursors of different NAEs

depending on their different acyl species at the N position. These precursors are in turn

produced from enzymatic transfer of an acyl group from the sn-1 position of phospholipids

such as phosphatidylcholine (PC), 1-acyl-lyso PC, PE, andcardiolipin to the N-position of

different PEs. Thus, PE serves both as donor and receiver. This acyl transfer reaction is

catalyzed by a Ca2+

-dependent N-acyltransferase (Ca-NAT) and is the first step in the

transacylation–phosphodiesterase pathway that is considered the major route for NAE

production (Ueda et al., 2010). A NAPE-hydrolysing phospholipase D ( NAPE-PLD) then

hydrolyzes NAPE (produced by Ca-NAT) to NAE and phosphatidic acid. cDNA of NAPE-

PLD was cloned in 2004 (Okamoto et al., 2004) while the molecular characterization of Ca-

NAT is yet to be done. Ca2+

-independent NAT (iNAT) (Jin et al., 2009) and HRAS-like

suppressor family 2 (HRASLS2) (Uyama et al., 2009), are also reported to be capable of

forming NAPE by N-acylation of PE. The three enzymes are discussed in more detail below.

Ca-NAT

Ca-NAT is an integral membrane protein and is catalytically active at neutral and alkaline pH.

Highest levels of activity of this enzyme are found in the brain and testis with lower levels in

skeletal muscle tissue (Cadas et al., 1997). Ca-NAT uses phospholipid, but not free fatty acid

or acyl-CoA, as acyl donor. Furtherermore this enzyme only transfers acyl chain from the sn-

1 position, but not from the sn-2 position of donor phospholipids such as PC, 1-acyl-lyso PC,

PE, and cardiolipin (Ueda et al., 2010). The calcium-dependency of Ca-NAT provides a

useful regulatory mechanism, and it is well established that levels of AEA and NAEs are

elevated by both physiologal and pathological processes affecting calcium concentrations

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(Natarajan et al., 1982). In addition to Ca2+

, Sr2+

, Mn2+

and Ba2+

are known to modulate Ca-

NAT activity (Ueda et al., 2010).

Ca-NAT does not discriminate based on the length or unsaturation of the transferred acyl

chain. This might explain why saturated fatty acyl chains (abundant in the sn-1 position of

glycerophospholipids) are much more common than polyunsaturated fatty acyl chains

(abundant in the sn-2 position) in the resulting NAPE molecules (Ueda et al., 2010).

iNAT

iNAT has functional similarity with lecithin retinol acyltransferase (LRAT) which catalyzes

transfer of a palmitoyl chain from the sn-1 position of PC to all-trans-retinol (Ueda et al.,

2010). iNAT was discovered and characterised in a study aimed to investigate the similarities

between LRAT and Ca-NAT (Jin et al., 2009). Several differencec in catalytic properties for

iNAT compared to Ca-NAT were found. The activity of recombinant iNAT was only slightly

increased by the addition of Ca2+

and was detected mainly in the cytosolic rather than

membrane fraction. Furthermore the enzyme reaction did not show positional specificity in

terms of the sn-1 and sn-2 positions of the donor substrate PC. Thus it is suggested that iNAT

constitutes an effective pathway for the formation of AEA and other unsaturated NAEs and

that the regulatory mechanisms are different from that of Ca-NAT (Jin et al., 2009, Muccioli,

2010). Whether or not iNAT contributes to the biosynthesis of NAPEs and NAEs in vivo is

unclear.

NAPE-PLD

NAPE-PLD is is a 45-46 kDa membrane-associated enzyme composed of 391–396 amino

acids.. This enzyme is a member of the metallo-β-lactamase family, a large superfamily

comprising a variety of hydrolases. Recombinant NAPE-PLD generates various long-chain

NAEs, including AEA, PEA and OEA from their corresponding NAPEs while being almost

inactive towards PC, PE, phosphatidylserine (PS), and phosphatidylinositol (PI) (Okamoto et

al., 2004, Ueda et al., 2010). Physiological regulation of NAPE-PLD activity is poorly

understood and specific NAPE-PLD inhibitors have not been developed.

Brain tissue from mice with a targeted disruption in the NAPE-PLD gene was analysed by

Leung et al in 2006. The authors showed that calcium-dependent conversion of NAPEs to

NAEs was reduced five-fold (Leung et al., 2006). However, only NAEs with saturated acyl

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side chains were affected, and these reductions were most dramatic for NAEs bearing very

long acyl chains (≥20 carbon atoms) (Leung et al., 2006). These results clearly suggested

alternative pathways for NAE synthesis. To date, at least two other pathways different from

the NAPE-PLD are involved in the synthesis of NAEs fron NAPEs in two-three step

reactions. One route involves glycerophospho-N-acylethanolaminelipids (GP-NAEs) as key

intermediates and the other, mainly characterized in macrophage-like RAW264.7 cells, uses

phospho-N-arachidonoylethanolamine (pAEA) as a key intermediate (Muccioli, 2010).

Enzymes in additional pathways of NAE synthesis

PLA2s

The phospholipase A2 (PLA2) family of enzymes are best known for their ability to produce

free fatty acids and lysophospholipids by hydrolysis of the sn-2 bond of phosphoglycerides.

PLA2s constitute a diverse family of enzymes and play an important role in a variety of

cellular processes, including the digestion and metabolism of phospholipids as well as the

production of precursors for inflammatory reactions (Hui, 2012). However, PLA2s are also

involved in the synthesis of NAEs by catalysing the production of lyso-NAPEs from NAPEs,

which are then further metabolised by a lysophospholipase D (lyso-PLD) to form NAEs

(Okamoto et al., 2007).

ABH4

ABH4 belongs to the α/β-hydrolase family that is a large protein family characterized by the

α/β-hydrolase fold. Recombinant ABH4 hydrolysis both NAPEs and lyso-NAPEs but does

not distinguish between saturated and polyunsaturated N-acyl species of lyso-NAPEs (Simon

and Cravatt, 2006). In mice, the highest lyso-NAPE-lipase activity is reported in brain, spinal

cord, and testis, followed by liver, kidney, and heart (Simon and Cravatt, 2006).

PLC/phosphatase pathway

Another pathway to convert NAPE to NAE is a two-step pathway composed of PLC-mediated

hydrolysis of NAPE to form NAE phosphate and subsequent dephosphorylation by

phosphatases to generate NAE. This pathway has primarily been described in macrophages

(Liu et al., 2006) and it is not known whether it is found in other cell types.

In summary, current evidence suggests that NAPE-PLD is at least partially responsible for the

in vivo formation of all NAEs and that in brain tissue PEA, OEA, and AEA are also generated

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from their corresponding lyso-pNAPEs by the brain lyso-PLD. This latter pathway appears

not to distinguish between N-acyl species and all the reported NAPE-PLD independent

pathways (sPLA2/lyso PLD and Abh4/GDE1 pathways) are also non-selective in this respect.

Further studies investigating factors such as the nature of the acyl chain, the phospholipid

membrane composition at the site of synthesis, or the tissue and condition, favouring one

pathway over the others for a given NAE are needed.

Synthesis of 2-AG

Although 2-AG synthesis similar to AEA can be regulated by mobilisation of calcium

(Bisogno et al., 1997), its synthetic pathway is different. 2-AG can be synthesized in two steps

via generation of 1-acyl-2-arachidonoylglycerol (diacylglycerol, or DAG) from

phosphatidylinositol by PLC activity and subsequent hydrolysis of DAG by a diacylglycerol

lipase (Kondo et al., 1998). DAG can also be produced from phosphatidic acid by a

phosphatidic acid hydrolase. An additional pathway leading to 2-AG synthesis is through a 2-

arachidonoyl-lysophosphatidylinositol intermediate. Phosphatidylinositol is selectively

degraded to the 2-acyl isomer of lysophosphatidylinositol in a Ca2+

-independent manner and

subsequently converted to a 2-monoacylglycerol by lysophosphatidylinositol-selective

phospholipase C (lyso-PLC) in rat brain (Ueda et al., 1993). 2-AG is synthesised and released

together with other monoacylglycerol homologues, and it has been suggested that they can

modulate the efficacy of 2-AG at CB receptors without having direct effect upon the receptors

on their on (Ben-Shabat et al., 1998).

Cannabinoid receptors

When the first cannabinoid receptor was identified (Matsuda et al., 1990), there were no

known endogenous agonists, so historically these receptors were defined as receptors that

mediate the effects of plant-derived or synthetic cannabinoids (Pertwee.RG et al.).

There are at least two types of CB receptors, termed CB1 and CB2, and they belong to the

seven transmembrane domain family of G-protein-coupled receptors. Upon stimulation both

receptors can inhibit adenylyl cyclase and activate mitogen-activated protein kinase by

signalling through Gi/o proteins. For CB1 receptors, the Gi/o proteins can also mediate increase

of potassium current, inhibition of calcium channel activity, and the receptor can also signal

through Gs proteins (Pertwee et al., 2010).

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CB1 receptor was first characterized in rat brain (Devane et al., 1988) and later cloned in both

rat (Matsuda et al., 1990) and human cells (Gerard et al., 1991). The receptor is predominately

expressed in the central nervous system (CNS) but is also found in peripheral tissues such as

cardiovascular and reproductive system (Svizenska et al., 2008) . It is one of the most

abundant G-protein-coupled receptor in the brain. Its distribution enables modulation of

cognition, memory, motor function and analgesia (Pertwee et al., 2010). CB1 is sparsely

distributed in the brainstem, which is consistent with the limited acute toxicity of smoking

high doses of marijuana.

CB2 receptors were identified in a human promyelocytic leukemic cell line (Munro et al.,

1993) a few years after the discovery of the CB1 receptors. CB2 receptors are mainly found in

cells of the immune system but also expressed by some neurons and can modulate immune

cell migration and cytokine release (Pertwee et al., 2010).

In recent years it has been evident that additional non-CB1/CB2 receptors are involved in

mediating the effects of exogenous and endogenous cannabinoids. The orphan G-protein

receptor GPR55, for example, has been proposed as a novel cannabinoid receptor with an

ability to interact with and be modulated by endogenous, plant and synthetic cannabinoid

ligands (Johns et al., 2007, Ryberg et al., 2007). There, are, however conflicting results

regarding the ligands with which this receptor interacts, and current nomenclature does not

class this receptor as a CB receptor (Pertwee et al., 2010).

Non-cannabinoid receptors

Endocannabinoids can also signal and exert different effects through receptors other than CB

receptors. Two of the more well-studied receptors are described below.

Transient receptor potential vanilloid 1

One of the receptors most studied in this context is the transient receptor potential vanilloid

receptor 1 (TRPV1). TRPV1 belongs to the transient receptor potential (TRP) superfamily of

cation channels which are involved in transduction of wide range of stimuli such as heat, cold,

touch, pH, and different exogenous irritative substances. TRPV1 (also known as vanilloid

receptor 1 or capsaicin receptor) was discovered as the receptor for the pungent ingredient in

hot chilli pepper, capsaicin (Caterina et al., 1997), and the human receptor was cloned in 2000

(Hayes et al., 2000). In the dorsal root ganglion (DRG), TRPV1 is found on C-and Aδ- fibers,

17

its activation leading to elevation of calcium levels and release of different neuropeptides (Ho

et al., 2012). It can be activated by noxious heat (>43 °C), protons and endogenous lipids and

has been has been investigated for its key role in different nociceptive processes (Caterina et

al., 1997, Palazzo et al., 2012). In 1999 it was demonstrated that AEA can activate the TRPV1

receptor (Zygmunt et al., 1999). Although the potency of AEA towards TRPV1 receptors is

lower than towards CB receptors, its efficacy is increased following phosphorylation of the

receptor (Lizanecz et al., 2006) or in inflammatory conditions (Singh Tahim et al., 2005),

raising the possibility that the net effect of AEA upon CB receptors vs. TRPV1 receptors may

change in pathological situations. Among NAEs, OEA acts as a TRPV1 agonist (Ahern,

2003) and PEA can potentiate the actions of AEA at these receptors (De Petrocellis et al.,

2001, Smart et al., 2002). N-arachidonoyl dopamine and noladin ether, but not 2-AG act as

full agonists at this receptor (Pertwee et al., 2010)

Peroxisome proliferator-activated receptor (PPAR)-alpha

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcriptional

factors that belongs to the family of nuclear receptors (Desvergne and Wahli, 1999). Different

fatty acids and their derivatives are agonists at PPARs and these receptors are believed to

functions as generalized lipid sensors (Pertwee et al., 2010). The PPAR family comprises of

three isoforms α, γ, and β/δ. Anandamide has been reported to activate PPAR-α and PPAR-γ

and 2-AG has been reported to activate PPAR-γ and PPAR- β/δ (Jhaveri et al., 2008, Pertwee

et al., 2010). PPAR-α is highly expressed in liver, brown fat, kidney, heart, skeletal muscle,

and large intestines in humans (Desvergne and Wahli, 1999) as well as being expressed in the

skin (Rivier et al., 1998). The two NAEs, OEA and PEA are endogenous ligands at PPAR-α

receptors and upon receptor activation exert anorexic and anti-inflammatory effects

respectively (Guzman et al., 2004, LoVerme et al., 2005).

Other receptors

Experiments with CB receptor ligands have led to results indicating that several other

receptors and ion-channels are responsive to these compounds. CB1 receptor

antagonist/inverse agonists have been shown to be able to bind to opioid, adrenergic,

dopamine, serotonin, adenosine, melatonin, tachykinin, and prostanoid receptors. CB1/CB2

agonists, anandamide and 2-AG can also activate certain types of opioid, muscarinic

acetylcholine, adrenergic, serotonin and adenosine receptors (Pertwee et al., 2010).

18

Catabolism of NAEs and 2-AG

Endocannabinoids and NAEs are cleared from the extracellular space by a process of

intracellular accumulation followed by enzymatic catabolism. The mechanism(s) involved in

the cellular uptake of these lipophilic molecules are controversial, particularly with respect to

whether or not a plasma membrane transporter molecule takes part in the translocation of

endocannabinoids into cells prior to carrier-mediated intracellular delivery to the catabolic

enzymes (Fowler, 2012a). The enzymes involved in the catabolism of NAEs and 2-AG are

described below.

NAE degradation

N-Acylethanolamines are inactivated by enzymatic hydrolysis to free fatty acids and

ethanolamines. Fatty acid amide hydrolase (FAAH) is the major degradative enzyme

responsible for this reaction in vivo, with AEA showing the highest reactivity (Ueda et al.,

1995, Cravatt et al., 1996, McKinney and Cravatt, 2005). FAAH is a 63kDa membrane-bound

serine hydrolase with optimal pH value at 8.5–10. This enzyme has a wide substrate

specificity and has been well characterized pharmacologically (Cravatt and Lichtman, 2002,

Fowler, 2004). In addition, an isoenzyme of FAAH with ~20% sequence identity at amino

acid level, referred to as FAAH-2, is expressed in humans but not rodents (Wei et al., 2006).

The two isoenzymes are suggested to have overlapping but yet distinct roles in vivo based on

their tissue distribution. FAAH-2 has been shown to hydrolyse AEA and PEA in homogenate

activity assays with activities ~6 and ~20% those of FAAH, respectively. Kaczocha et al.

(2010) demonstrated that FAAH-2 hydrolyzed AEA and PEA in intact cells transfected with

FAAH-2 with rates 30–40% those of cells transfected with FAAH, highlighting a potentially

greater contribution toward NAE catabolism in vivo (Kaczocha et al., 2010).

NAEs can also be metabolized by a second enzyme termed N-acylethanolamine-hydrolysing

acid amidase (NAAA) active only at acidic pH (optimal pH around 5) and found to be

catalytically distinct from FAAH (Ueda et al., 1999). The recombinant enzyme hydrolysis

different N-acylethanolamines with the highest reactivity with PEA (Tsuboi et al., 2005).

Little is known about the physiological importance of this enzyme, but recent data with novel

inhibitors have suggested that its blockade can produce anti-inflammatory and analgesic

effects in animal models in a PPAR- dependent manner (Solorzano et al., 2009, Sasso et al.,

2012a).

19

Numerous potent and selective inhibitors of FAAH have been developed and they are

classified as reversible or irreversible according to their half-life inside the enzymes catalytic

site (Ahn et al., 2008). The first FAAH inhibitors mimicked the arachidonic part of AEA by

introducing, among other approaches, trifluoroketone groups in place of the ethanolamide

groups to inhibit the catalytic processes (Koutek et al., 1994, Boger et al., 1999). Further

investigations indicated that the hydroxyl group of Ser241

at the catalytic site has a crucial role

in amide bond hydrolysis and in the binding of reversible and irreversible inhibitors (Roques

et al., 2012). Later experiments in which the amide group was substituted with a carbamate

group resulted in development of URB597, the first selective FAAH inhibitor (Kathuria et al.,

2003). To date several other inhibitors have been developed in which the carbamate group is

replaced with a urea group. This results in an increasing potency due to the rigidity of the

enzyme-inhibitor complex which facilitates irreversible binding to Ser241

and prevents reverse

hydrolysis of the carbamylated enzyme (Roques et al., 2012). In vivo, FAAH inhibitors do not

produce the unwanted effects of CB1 receptor agonists such as catalepsy, hypothermia and

hyperphagia, which is important for the therapeutical usefulness of such compounds (Kathuria

et al., 2003, Lichtman et al., 2004).

2-AG degradation

Monoacylglycerol lipase

2-AG can also be metabolized by FAAH (Di Marzo, 1998, Goparaju et al., 1998) but the main

enzyme responsible for the hydrolysis of this endocannabinoid to arachidonic acid and

glycerol in the brain is monoacylglycerol lipase (MAGL) (Blankman et al., 2007). This

MAGL, a 33 kDa protein belonging to the AB hydrolase superfamily, was originally cloned

and purified in adipose tissue (Karlsson et al., 1997) and later in 2002 its important role in

terminating 2-AG signalling at cannabinoid receptors was highlighted (Dinh et al., 2002). The

enzyme has a wide substrate specificity among monoacylglycerols, and in this respect is

considered to play an important part of lipid metabolism affecting energy homeostasis,

signalling processes and cancer cell progression in humans in addition to its role in

terminating endocannabinoid signalling (Rengachari et al., 2012). In contrast to FAAH,

MAGL has been described to be both cytosolic- and membrane bound, with a pH optimum in

the area of pH 7-8 (Tornqvist and Belfrage, 1976, Sakurada and Noma, 1981, Somma-

Delpero et al., 1995, Blankman et al., 2007). MAGL is expressed in a wide range of tissues,

with high levels in adipose tissue, skeletal muscle, kidneys, and testis but it is also expressed

20

in the brain, adrenal gland, ovaries, heart and lungs in rat (Karlsson et al., 1997).The catalytic

site of MAGL has been identified as the triad Ser122

, His269

, and Asp239

(Karlsson et al., 1997,

Labar et al., 2010).

Until recently, MAGL inhibitors were few and far between. Early studies indicated that the

enzyme was inhibited by non-specific agents such as sulfhydryl group inhibitors, (p-

chloromercuribenzoic acid and N-ethylmaleimide) and organophosphates (Tornqvist and

Belfrage, 1976, Sakurada and Noma, 1981), and to have low sensitivity to potent FAAH

inhibitors (Bisogno et al., 1997, Di Marzo et al., 1999, Goparaju et al., 1999, Dinh et al.,

2002). Thus, more detailed pharmacological studies on the inhibitory potency of different

compounds upon FAAH and MAGL respectively were needed in order to develop potent

MAGL inhibitors.

Initial in vitro studies targeted the cysteine and serine residues and after the development of

carbamates to target FAAH these types of compounds were also investigated with regards to

MAGL. URB602, which inhibits both FAAH and MAGL, was initially reported as the first

selective inhibitor of 2-AG degradation (Hohmann et al., 2005), although its selectivity has

been questioned (Vandevoorde et al., 2007). The first highly potent selective MGL inhibitor,

the piperidine carbamate JZL184 was reported in 2009 by Long et al. This compound inhibits

MGL irreversibly in vitro. Upon intraperitoneal injection (16 mg/kg) to mice, the levels of 2-

AG in the brain were increased 7-fold within 0.5 hours and this increase lasted at least for 8

hours without any alterations in AEA levels (Long et al., 2009). JZL184 showed analgesic

properties (that could be blocked by a CB1 receptor antagonist) in different animal models

including tail-immersion test of acute thermal pain sensation, the acetic acid writhing test of

visceral pain, and the formalin test of noxious chemical pain. However in contrast to FAAH

inhibitors, treatment with JZL184 also resulted in hypothermia and hypomotility but not

catalepsy (Long et al., 2009). When both FAAH and MAGL were inhibited, however,

catalepsy was seen (Long et al., 2009). Furthermore, repeated administration of JZL84 to

mice resulted in a tolerance to its behavioural effects accompanied by a down-regulation of

central CB1 receptors (Schlosburg et al., 2010). Whether this effect is common to all MAGL

inhibitors or reflects an adaptive response to a long-term irreversible inhibition of the enzyme

awaits elucidation.

21

Other 2-AG hydrolytic enzymes

Apart from FAAH and MAGL two other serine hydrolases, alpha/beta hydrolases 6 and 12

(ABHD6, ABHD12), have been described to be involved in 2-AG inactivation (Savinainen et

al., 2012). Functional proteomic approaches have been used to investigate the different 2-AG

hydrolases in mouse brain membrane homogenates (Blankman et al., 2007). The study

confirmed that MAGL is the main enzyme involved in hydrolysing 2-AG in the brain

(approximately 85% at the substrate concentration used) and furthermore identified that

ABHD 6 and 12 mediate about 4 and 9 % respectively at pH 7.5 (Blankman et al., 2007). The

physiological and pathophysiological role of the newly discovered ABHD 6 and 12 is to date

not well known but mutations in the ABDH12 gene have been described to result in a

neurodegenerative disease called PHARC syndrome (demyelinating polyneuropathy, hearing

loss, ataxia, retinitis pigmentosa cataract) (Fiskerstrand et al., 2010). Apart from studies on

cell homogenates, the ability of ABHD6 to hydrolyse 2-AG in intact BV2 cells, which are cell

lines that have many of the features microglia cells express in vivo, has been demonstrated

(Marrs et al., 2010). ABHD6 is expressed by glutamatergic neurons, GABAergic interneurons

and astrocytes but not by primary microglia (in contrast to the situation in BV2 cells) and its

inactivation in neurons in primary culture results in accumulation of 2-AG (Marrs et al.,

2010). It has also been suggested that based on their distinct cellular and/or subcellular

localizations, MAGL, ABHD12 and ABHD6 might regulate the hydrolyzation of different 2-

AG pools (Blankman et al., 2007, Marrs et al., 2010).

Other enzymes involved in endocannabinoid catabolism

In addition to the hydrolytic pathways, AEA and 2-AG can also be metabolized by oxidative

enzymes including cyclooxygenase 2 (COX2), lipoxygenases (LOXs), and hepatic

cytochrome P450 enzymes (Kozak and Marnett, 2002). COX-2 metabolism of AEA and 2-

AG produces prostaglandin H2 ethanolamide (precursor to other prostaglandin ethanolamides

or prostamides) and prostaglandin glycerol esters, respectively (Yu et al., 1997, Kozak et al.,

2000). Major metabolites from AEA oxidation by 12- and 15-lipoxygenase (12-LOX and 15-

LOX) are 12- and 15-hydroxyeicosatetraenoic acid ethanolamide. 2-AG metabolism by 12-

LOX and 15-LOX produces 12- and 15-hydroperoxyeicosatetraenoic acid glycerol ester,

respectively (Yates and Barker, 2009). Cytochrome P450 catalyzed oxygenations of AEA was

first reported by Bornheim et al. (1993). In their study they demonstrated that hepatic P450

metabolized AEA to at least 20 different derivatives (Bornheim et al., 1993).

22

One of the many P450-derived metabolites of anandamide, 5, 6-EET-EA, has been shown to

bind to and functionally activate recombinant human CB2 receptor with more than 1000-fold

greater affinity than AEA (Snider et al., 2009).

From the above, it is clear that endocannabinoids have synthetic pathways that can be

regulated, and a variety of alternative catabolic pathways, although FAAH and MGL are

predominant. It is perhaps not surprising that following pathological changes,

endocannabinoid levels can change due both to regulation of calcium levels and the

expression of these synthetic and/or catabolic enzymes. An example of this is seen in an

experimental model of spinal cord injury (induced by contusion/compression) which

produced two different stages of endocannabinoid activation in rat. The first stage included

elevation of AEA and PEA levels, upregulation of NAPE-PLD (mRNA) and downregulation

of FAAH (mRNA) whereas in the delayed stage 2-AG was elevated followed by upregulation

of synthesizing enzyme, DGL-α ( mRNA), and to a lesser extent upregulation of MAGL

mRNA(Garcia-Ovejero et al., 2009).

23

The endocannabinoid system in pain processing

Animal studies so far have shown that the localization of endocannabinoids, together with

their receptors and metabolic enzymes, enables this endogenous system to control nociceptive

pathways at supraspinal, spinal and peripheral levels (Guindon and Hohmann, 2009). Early

studies in which different synthetic cannabinoids were injected into different brain regions in

rat presented that cannabinoids might act in the midbrain periventricular circuits such as

periaqueductal grey (PAG) to modulate pain sensitivity (Martin et al., 1995). Based on these

findings and that the anatomical subregions of PAG were different compared to the regions

involved in opioid analgesia, the role of endogenous AEA release in PAG was investigated by

microdialysis (Walker et al., 1999). Electrical stimulation of the dorsal and lateral PAG and

the dorsal adjacent area produced analgesia and this was blocked by a cannabinoid CB1

receptor antagonist. By using the same conditions it was also shown for the first time that this

stimulation results in AEA release and furthermore that intradermal injection of formalin

elevates AEA in the dorsal and lateral PAG (Walker et al., 1999). Animal models have also

demonstrated that endocannabinoid levels in specific brain regions are altered following nerve

injury (Petrosino et al., 2007) and that the release of these lipids in PAG might mediate non-

opioid stress induced analgesia (Hohmann et al., 2005).

Several studies have demonstrated that spinal cord endocannabinoid levels are affected in

experimental pain states. Induction of neuropathic pain in rats by chronic constriction of the

sciatic nerve results in both AEA and 2-AG increase in the spinal cord compared to sham-

operated rats (Petrosino et al., 2007). Also CB1 receptors in the spinal cord are upregulated

following peripheral nerve injury (Lim et al., 2003). In rats subjected to foot shock stress 2-

AG but not AEA levels in the spinal cord increase significantly compared to non-shocked rats

(Suplita et al., 2006). Spinal cord AEA and 2-AG levels are also increased following

cisplatin-induced peripheral neuropathy (Guindon et al., 2013). Intrathecal administration of

THC in mice results in potent antinociception but also hypothermia, catalepsy and

hypoactivity (Smith and Martin, 1992), i.e. the so-called tetrad of behaviours used to identify

central CB1 receptor-mediated effects (Adams and Martin, 1996).

Peripheral (intraplantar), but not systemic, administration of AEA inhibits oedema, capsaicin-

evoked plasma extravasion into hind paw, and furthermore both the induction of carrageenan-

induced thermal hyperalgesia and its maintenance once developed (Richardson et al., 1998).

The antinociceptive potency of anandamide following intraplantar, intravenous or

24

intraperitoneal administrations was investigated in the formalin-evoked pain model in mice by

Calignano et al. (1998). The results showed that AEA was potent in preventing pain

behaviour in this model when injected locally and was not accompanied by central signs of

cannabimimetic activity, indicating a peripheral site of action (Calignano et al., 1998).

Intraplantally administered AEA and ibuprofen (displaying synergistic effects) have

antinociceptive effects in rat paw formalin test (Guindon et al., 2006). Antinociceptive effects

were not seen when these compounds were given in the contralateral paw at doses higher than

the ED50 doses used in the ipsilateral paw, indicating local site of actions. Locally injected

anandamide, ibuprofen, rofecoxib and their combinations significantly decreased mechanical

allodynia and thermal hyperalgesia in rat model of neuropathic pain induced by partial sciatic

nerve ligation (Guindon and Beaulieu, 2006).

The peripheral contribution of endocannabinoid-mediated analgesia has also been investigated

by deleting CB1 receptor selectively in nociceptive neurons localized in the peripheral

nervous system in mice. These genetically modified mice showed a significantly reduced

response to noxious heat, reduced response thresholds to mechanical stimuli and greater

responses to intraplantar injections of capsaicin and formalin (Agarwal et al., 2007). Also low

doses of peripherally applied synthetic cannabinoids reduce inflammatory and neuropathic

pain, an effect that was almost lost in mice lacking CB1 receptors in nociceptive neurons

(Agarwal et al., 2007). Spinal nerve ligation in rat increases both AEA and 2-AG significantly

only in the ipsilateral DRG (Mitrirattanakul et al., 2006). Intraplantar injection of AEA

produces dose-dependent excitation of cutaneous C nociceptors and produces nocifensive

behaviour which is suggested to occur via TRPV1 activation (Potenzieri et al., 2009). Results

from different animal models of acute and chronic pain demonstrate that both CB1 and CB2

receptors on nociceptive nerve endings are involved in mediating cannabinoid-mediated

analgesia (Kress and Kuner, 2009). FAAH protein has been detected in rat DRG, spinal cord,

and peripheral nerve tissue (Lever et al., 2009), providing peripheral sites for targeting in

order to modulate the endocannabinoid system for treatment of pain. Further elucidation of

the peripheral mechanism of cannabinoids hold promising effects regarding the therapeutical

use of these lipids and fewer unwanted effects.

25

Exogenous cannabinoids in human pain trials

Many early studies investigating the effects of cannabinoids upon human pain were less than

ideal, and a critical review published in 2002 concluded that “Cannabinoids are no more

effective than codeine in controlling pain and have depressant effects on the central nervous

system that limit their use” (Campbell et al., 2001). However, in a more recent systematic

review by the same first author of that paper, 18 good quality randomized trials (published

between 2003 and 2010) were identified, and the data demonstrated that cannabinoids are a

modestly effective and safe treatment option for chronic non-cancer pain (Lynch and

Campbell, 2011). The majority of the studies included patients with neuropathic pain but also

fibromyalgia and rheumatoid arthritis were among conditions investigated. The trials

comprised of different forms of cannabinoids such as smoked cannabis, oromucosal extracts

with THC and cannabidiol (non-psychoactive constituent of cannabis plant) such as

Sativex®, synthetic cannabinoids (nabilone/Cesamet®, THC/Marinol®) and THC-11-oic acid

analogue (CT-3 or ajulemic acid). No serious adverse effects leading to withdrawal from the

studies were observed. Sedation, dizziness, dry mouth, nausea and disturbances in

concentration were most common side effects and also poor co-ordination, ataxia, headache,

paranoid thinking, agitation, dissociation, euphoria and dysphoria was seen.

An additional systematic review focusing on the broader therapeutic application of

cannabinoid medications established indications for treatment in spasticity in multiple

sclerosis, nausea and vomiting following chemotherapy, loss of appetite in HIV/Aids, and

neuropathic pain (Grotenhermen and Muller-Vahl, 2012). The review also showed that

cannabinoids seem to have little or no effect in patients with acute pain although analgesic

effects in patients with postoperative pain have previously been reported (Holdcroft et al.,

2006). Sativex is now registered in several countries, including Sweden, as an adjuvant

treatment for spasticity associated with multiple sclerosis.

Patients with chronic pain often suffer from other related symptoms such as sleep

disturbances. In a randomized controlled trial, it was reported that nabilone is an effective

treatment for promoting sleep in patients with fibromyalgia who have chronic insomnia and

that this might be better treatment option then the commonly used amitriptyline (Ware et al.,

2010).

A systematic review of safety studies of cannabinoids, published between January 1980 and

week 42 of 2007, was conducted and comprised 31 studies after exclusion of studies focusing

26

on recreational cannabis (Wang et al., 2008). Most (96.6%) adverse effects were not serious

and dizziness was the most common reported event (15, 5%). Relapse of multiple sclerosis

(12.8%), vomiting 9.8% and urinary tract infection 9.1% was most common serious adverse

events.

Exogenous PEA in animal pain models

Since the discovery of PEA half way through last century, large amounts of data have

demonstrated the anti-inflammatory and antinociceptive effects of this fully saturated member

of NAEs (Lambert et al., 2002, Re et al., 2007). The antinociceptive effects have been

investigated in different animal models of acute and chronic pain. PEA decreases pain

behaviours in mice induced by injections of intraplantar formalin and intraperitoneal acetic

acid, kaolin, nerve growth factor and magnesium sulfate, but not capsaicin-evoked pain

behaviour or thermal nociception (Calignano et al., 1998, Jaggar et al., 1998, Calignano et al.,

2001, Farquhar-Smith and Rice, 2003). Intraperitoneal PEA decreases both the referred

thermal hyperalgesia following turpentine-induced bladder inflammation (Farquhar-Smith and

Rice, 2001) and mechanical hyperalgesia induced by partial sciatic nerve injury in mice

(Helyes et al., 2003). Experimental studies have also demonstrated that PEA has anti-

inflammatory effects (Re et al., 2007).

The underlying mechanism(s) behind the antinociceptive properties of PEA is yet to be fully

elucidated but it is suggested that this bioactive lipid has a protective role in maintaining

cellular homeostasis in response to different stressful events such as tissue trauma and

inflammation (Re et al., 2007, Skaper and Facci, 2012). This might be through different

mechanism such as inhibition of nociception by activating a fast-onset nongenomic

mechanism and a slow-onset genomic mechanism involved in tissue repair (LoVerme et al.,

2006, Sasso et al., 2012b). PEA has no direct effects upon CB receptors, but the

antinociceptive effects of PEA are reduced by the compound SR144528 (Calignano et al.,

1998, Farquhar-Smith and Rice, 2001, 2003). SR144528 was initially developed as a CB2

receptor antagonist/inverse agonist (Rinaldi-Carmona et al., 1998), but also interacts with

PPAR-α (Lo Verme et al., 2005), and this is the presumed target of PEA (LoVerme et al.,

2006). In both in vitro and animal model studies (carrageenan-induced oedema) PPAR-α

receptor activation has been shown to be required for the anti-inflammatory effects of PEA

(Lo Verme et al., 2005). In the same study it was also shown that topically applied PEA

reduced AEA levels in skin, which suggests that the effects in vivo are not produced by

27

preventing AEA hydrolysis. Subsequently, the same group also investigated the role of

PPAR-α receptor in antinociception in animal models of acute and more persistent

neuropathic pain. It was shown that the synthetic PPAR-α receptor agonists GW7647 and

Wy-14643 as well as PEA reduce both early and late phase nocifensive behaviour induced by

intraplantar injection of formalin in wild type mice whereas mice lacking the receptor did not

respond (LoVerme et al., 2006). Furthermore PEA decreased hyperalgesia induced by ligating

of the sciatic nerve in wild type mice but not in mice lacking PPAR-α receptor. Tissue levels

of PEA in the brain and spinal cord were not affected by the intraplantar administration but

were increased in the injected paw, suggesting a peripheral mechanism for the analgesic

effects (LoVerme et al., 2006). However, a separate study failed to find an increased PEA in

the rat paw skin after formalin injection (Beaulieu et al., 2000). Further studies investigating

the mechanism involved in PEA activation of PPAR-α receptor have confirmed the

antinociceptive effects and shown that other routes such as neurosteroid synthesis can be

involved in mediating the effects after activation of PPAR-α receptor (Sasso et al., 2012b).

Other potential mechanisms proposed to mediate the beneficial effects, are inhibition of mast-

cell degranulation via an “Autacoid Local Inflammation Antagonism” (ALIA) effect and

activation of other receptors such as the orphan GPR55 (Farquhar-Smith and Rice, 2003,

Petrosino et al., 2010).

Exogenous PEA in human pain trials

In the seventies, double-blind field trials with PEA (Impulsin®) were conducted with regards

to its efficacy in reducing the incidence and severity of respiratory tract infections (Masek et

al., 1974, Kahlich et al., 1979). Following the discovery of the endocannabinoid system and

with that the structural similarity between AEA and PEA, also the latter fatty acid gained

much interest for its potential antinociceptive effects (Lambert et al., 2002). To date, orally

administered PEA available as Normast (300 mg and 600 mg) has been tested in several

clinical chronic pain trials (Hesselink, 2012). Together with previous trials on respiratory

infections, these trials comprise of thousands of subjects, in which no adverse effects of this

naturally occurring bioactive lipid have been reported.

With regards to pain modulating effects of exogenous PEA in the clinical trials, it has been

shown to decreases pain intensity significantly in patients with different pain conditions such

as temporomandibular joint osteoarthritis, carpal tunnel syndrome, diabetes neuropathy,

herpes zoster infection, failed back surgery syndrome and osteoarthritis (Conigliaro et al.,

28

2011, Gatti et al., 2012, Marini et al., 2012). PEA is also a constituent of a topical cream,

MimyX that is used for the treatment of atopic dermatitis (Simpson, 2010).

Blockade of endocannabinoid and NAE degradation as a therapeutic approach for the

treatment of pain

There is now considerable data demonstrating that blockade or genetic deletion of FAAH give

rise to antinociceptive effects in a range of animal models of inflammatory, neuropathic,

visceral and cancer pain (Roques et al., 2012). The irreversible FAAH inhibitor, PF-

04457845, has been recently investigated in a randomized, placebo-controlled clinical trial of

patients with osteoarthritis of the knee. Although resulting in an increase of four NAEs

including AEA and PEA (which however also was seen in patients without alterations in

FAAH activity), this inhibitor did not have any analgesic effects (Huggins et al., 2012). A

number of reasons can be considered for this, including general aspects such as the lack of

predicitivity of the animal models (Rice et al., 2008) and the suitability of patient population

to the drug mechanism (Woolf, 2010). Additionally, the lack of effect may be a compound,

rather than a class effect. An alternative possibility, however, is that the AEA, following

FAAH inhibition, utilises other metabolic pathways which thereby reduce the effectiveness of

this therapeutic strategy. Little is known about this possibility, although the FAAH inhibitor

(URB597) has been shown to have synergistic effects with the NSAID diclofenac in a model

of visceral pain (Naidu et al., 2009), which would be consistent with this hypothesis. Other

FAAH inhibitors are reaching the clinical stage of their development, and it is to be hoped

that additional clinical trials will elucidate the usefulness of FAAH as a valuable target for the

treatment of pain.

The MAGL inhibitor, JZL184, has been demonstrated to reduce nociceptive responses in

several different animal models where pain is induced by noxious chemicals, inflammation

and neuropathy (Mulvihill and Nomura, 2012). Fulfilment of several criteria has been

proposed when translating the knowledge of MAGL inhibitors, gained from animal studies,

into clinical trials to find the right indications for patients (Fowler, 2012b). In summary the

criteria are that the therapeutical effects of cannabinoids should have been demonstrated for

the pain state(s) in question, that the endocannabinoid system should be altered in either

29

animal or human studies of the pathological state, and that repeated administration of the

compound does not result in tolerance. The latter may be an issue blocking the development

of MAGL inhibitors (see section above).

Among the few classes of NAAA inhibitors developed so far, one of the challenges has been

the stability of the compounds even though they show anti-inflammatory and antinociceptive

effects in animal models (Solorzano et al., 2009, Khasabova et al., 2012, Li et al., 2012, Sasso

et al., 2012a).

30

Chronic pain

Chronic pain is a common health care problem and is associated with disability, low quality

of life and substantial socioeconomic costs (Breivik et al., 2006). Pain is defined as “An

unpleasant sensory and emotional experience associated with actual or potential tissue

damage, or described in terms of such damage” according to The International Association for

the Study of Pain (IASP). Chronic pain is defined as pain that persists beyond the normal

tissue healing time (IASP, 1986), usually considered to be three months, although in research

a six month timescale is often used. In general, the recovery rates of chronic pain is low and

has been reported to be between 5.4-9.4% per year in different studies (Elliott et al., 2002,

Sjogren et al., 2010). Since a complex of different factors such as neurobiological,

psychological, coping styles, and contextual factors contributes to the development and

maintenance of chronic pain its now well established, a bio-psycho-social model is preferred

in clinical management of patients with chronic pain (Gatchel et al., 2007).

Progression of both central and peripheral sensitization are thought to be important processes

leading to the transition from acute to chronic pain, including the musculoskeletal disorders

described below (Graven-Nielsen and Arendt-Nielsen, 2010). According to IASP definition,

sensitization is increased responsiveness of nociceptive neurons to their normal input, and/or

recruitment of a response to normally subthreshold inputs (www.iasp-pain.org). Furthermore

in the additional note to the definition it is mentioned that sensitization is a

neurophysiological term that can only be applied when both input and output of the neural

system under study are known, and clinically may only be inferred indirectly from

phenomena such as hyperalgesia or allodynia. Central sensitization is then defined as

increased responsiveness of nociceptive neurons in the central nervous system to their normal

or subthreshold afferent input, which may include increased responsiveness due to

dysfunction of endogenous pain control systems, and peripheral neurons functioning normally

(www.iasp-pain.org). Peripheral sensitization is defined as increased responsiveness and

reduced threshold of nociceptive neurons in the periphery to the stimulation of their receptive

fields (www.iasp-pain.org).

In the literature however, there are often different and inconsistent definitions of central

sensitization. In a recent review, Woolf listed patient symptom features that would point

towards more central rather than peripheral changes in pain hypersensitivity (Woolf, 2011).

31

The features include pain mediated by low threshold Aβ fibers, a spread of pain sensitivity to

areas with no demonstrable pathology, aftersensations, enhanced temporal summation, and

the maintenance of pain by low frequency stimuli that normally do not elicit pain. The

processes leading to a more heightened pain state are occurring at multiple sites such as the

dorsal horn and different brain areas involving somatosensory, cognitive and pain modulating

sites resulting in dysfunctional descending pain modulating pathways and overactive

ascending facilitatory pathways (Nijs and Van Houdenhove, 2009).

To date there is evidence, but no consensus, supporting that nociceptive input from deep

tissues can drive the processes leading to central sensitization and maintenance of the chronic

pain state (Sandkuhler, 2009, Staud, 2010, 2011).

Chronic muscle pain

Common chronic pain conditions are chronic neck-shoulder pain (CNSP) including trapezius

myalgia, which has a prevalence of 10-20% in the community (Lidgren, 2008) and chronic

widespread pain (CWP) with a reported prevalence of 4.8–7.4% (Gerdle et al., 2008). While

localized musculoskeletal pain has minor functional consequences there is a strong

relationship between the number of pain sites and the functional ability of the patient

(Kamaleri et al., 2008). To date, the mechanism (s) behind chronic muscle pain is not fully

understood and standard pain treatments are of limited usefulness for patients suffering from

these conditions.

Chronic neck/shoulder pain

Neck/shoulder pain is more frequent in women than in men and may range from short

episodes with limited activity to severe and disabling episodes of chronic disability (Larsson

2007). Different definitions of neck pain are given based on anatomical location, aetiology,

severity, and duration of symptoms (Misailidou et al., 2010). Neck pain may be a feature of

any disorder that occurs above the shoulder blades and can also be a component of headaches,

temporomandibular joint syndrome, disturbances of vision, stroke, disorders affecting the

upper extremities, inflammatory diseases, fibromyalgia in addition to trauma (Guzman et al.,

2008). In majority of patients however, the source of their neck pain is unknown and is often

called non-specific neck pain. For this group of patients there are no signs of systemic disease

when conducting standard laboratory tests. In a systematic review published in 2009, it was

concluded that no evidence exists in the scientific literature supporting the use of diagnostic

imaging and that pathologic radiological findings are not associated with worse prognosis

32

(Tsakitzidis.G, 2009). However, strong evidence exists suggesting that female gender, older

age, high job demands, low social/work support, being an ex-smoker, as well as a history of

low back-and neck disorders, are risk factors for the development of nonspecific neck pain

(McLean et al., 2010). Repetitive work tasks, forceful exertions (with hand/wrists) and

awkward postures are also known risk factors (Larsson et al., 2007). Although several other

muscles such as levator scapulae and neck extensors are also affected, showing severe

tenderness (Andersen et al., 2011), chronic trapezius myalgia is the most frequent clinical

diagnosis in adults with self-reported chronic neck/shoulder pain (Juul-Kristensen et al.,

2006). Thus, the literature concerning analysis of risk factors suggests a multifactorial

aetiology with both non-modifiable and modifiable factors such as physical activity

(Haldeman et al., 2008).

Chronic widespread pain

According to the 1990 American College of Rheumatology (ACR) classification (Wolfe et al.,

1990) pain is considered widespread when it is present in the left and the right side of the

body as well as above and below the waist. In addition, axial skeletal pain must be present. In

the 1990 ACR criteria, which were intended for classification and not for diagnosis, patients

will be said to have fibromyalgia when fulfilling CWP criteria in addition to pain in at least 11

of 18 predefined tender points on manual palpation with a pressure of approximately 4 kg

(Wolfe et al., 1990). The ACR 2010 criteria comprise a widespread pain index and symptom

severity scale with assessment of cognitive symptoms, sleep disturbances, fatigue and somatic

symptoms (Wolfe et al., 2010, McBeth and Mulvey, 2012). Patients with CWP including

fibromyalgia syndrome (FMS) often report that their pain started as a local or regional pain

condition such as soft tissue neck injury and low back pain (Larsson. B, 2012). Risk factors

for the transitioning of regional pain states to CWP are largely unknown but some data

indicate that possible risk factors are female sex, higher age, family history of pain, depressed

mode and pain sites at baseline (Larsson. B, 2012). The spreading of pain to a generalized

state is associated with more negative consequences with respect to pain intensity, prevalence

of depressive symptoms, aspects of coping, life satisfaction/quality and general health

compared to more local or regional pain states (Peolsson et al., 2007). Central alterations (i.e.

central hyperexcitability and altered descending control of nociception, see section above) are

considered to play major role in CWP including FMS. However, increasing evidence

indicates that persistent noxious peripheral input drives the neuroplastic changes and the

maintenance of central sensitization in CWP (Ge et al., 2011).

33

Nociception and chronic muscle pain

Muscle nociceptors are free nerve endings of group III and IV afferent fibers that detect

potential or actual tissue damage and express different classes of receptor molecules that are

targets for sensitizing and algesic substances (Mense, 2009). Intramuscular injection of

different substances such as capsaicin, glutamate, and hypertonic saline have been used in

order to investigate their sensitizing and pain eliciting properties in muscle (Graven-Nielsen

and Arendt-Nielsen, 2010). However, under pathological conditions there is a mix of different

sensitizing and algesic compounds that acts on muscle nociceptors (Mense, 2009). Due to its

clinical importance and easy access, the trapezius muscle has been used as a model when

investigating the peripheral molecular changes in chronic neck/shoulder pain by using

microdialysis technique. Most microdialysis studies so far have investigated metabolites and

algesic substances and in a recent systematic review serotonin, glutamate, pyruvate and

lactate were identified as potential biomarkers of chronic myalgia (Larsson, 2012).

Only few studies have investigated CWP by microdialysis technique. Significantly higher

interstitial concentrations of pyruvate and lactate in microdialysate samples from trapezius

muscle of CWP (including FMS) patients (Gerdle et al., 2010) have been reported, as have an

increase in dialysate lactate (from vastus lateralis muscle) in response to acetylcholine and a

higher nitric oxide synthase protein content (McIver et al., 2006).

In contrast to algesic substances, very little is known about pain inhibitory compounds such as

endocannabinoids and NAEs in chronic muscle pain states. A single study has investigated

the role of endocannabinoids in FMS by analysing AEA in plasma (Kaufmann et al., 2008).

AEA levels were significantly higher in women with FMS (n=22) compared to healthy

controls (n=22). To my knowledge, data from microdialysis studies of endocannabinoids and

NAEs in muscle tissue from chronic muscle pain patients are lacking.

34

Aims

Preclinical part; Paper I

When paper I was in planning phase, the pharmacological characteristics of MAGL and its

comparative effects with FAAH had not been well studied. Thus, for better understanding of

the pharmacology of MAGL, the aims of the study were:

-To determine optimal assay conditions for MAGL and FAAH.

-To test the abilities of different 2-AG analogues to affect MAGL and FAAH hydrolysis to

find MGL-selective inhibitors.

Clinical part; paper II-IV

As outlined in the introduction, prior to this thesis there were no studies investigating the

levels of endocannabinoids and NAEs locally in myalgic or healthy muscle. The general aims

of the clinical part of the present thesis were:

-To identify NAEs, including AEA, and 2-AG in human muscle dialysate.

-To compare the levels of NAEs, including AEA, and 2-AG between myalgic and healthy

muscle.

The specific aims, modified from the original general aims when it became apparent that PEA

and SEA, but not AEA and 2-AG, could be measured in the microdialysates (see Results

below) were:

-To investigate the levels of PEA and SEA in muscle dialysate in women with chronic neck/

shoulder pain (CNSP) and healthy controls (CON) during different stages of microdialysis

procedure.

-To investigate the levels of PEA and SEA in muscle dialysate before and after a brief low-

force exercise in CNSP, CON and women with chronic widespread pain.

-To investigate the effect of different training interventions conducted by CNSP on PEA and

SEA levels in muscle dialysate and pain intensity.

-To investigate the potential variation of PEA and SEA levels in muscle dialysate over time in

CON.

35

Methods and methodological considerations

Key methods used in this thesis are presented and discussed here. For detailed materials and

methods see the original papers.

Assay of MAGL and FAAH (paper I)

The assay is based on the measurement of the [3H]ethanolamine formed from hydrolysis of

[3H]AEA and the [

3H]glycerol formed from [

3H]2-OG hydrolysis (Omeir et al., 1995, Dinh et

al., 2002). These hydrophilic molecules (products) can be separated from nonhydrolysed

lipophilic AEA and 2-OG (substrates) by addition of chloroform/methanol or charcoal/HCL.

Homogenates of adult Sprague-Dawley rat cerebella were prepared in sodium phosphate

buffer (pH 8) containing sucrose and centrifuged for one hour, at 4 °C. Following the

centrifugation the supernatants (“cytosol fractions”) were saved and the pellets were

suspended in sodium phosphate buffer (pH 8) (“membrane fractions”). Protein concentration

in the membrane fraction was determined by using bovine serum albumin as standard and

samples were diluted in Tris-HCL buffer (pH 7.2) containing EDTA, unless otherwise stated.

For determination of hydrolysis rates of [3H]AEA and [

3H]2-OG aliquots of the membrane or

cytosol fractions were then added to glass tubes containing the test compound. Blanks

contained assay buffer instead of homogenate solution. Substrate was then added and the

samples were incubated for 10 min at 37 °C. Reactions were stopped by adding mixture of

chloroform/methanol, vortex mixing the tubes and placing them on ice. After separating the

phases by ten minutes centrifugation, aliquots of the methanol/buffer phase were taken and

measured for tritium content by liquid scintillation spectroscopy with quench correction.

During the study, an alternative method using active charcoal /HCl instead of chloroform /

methanol was developed for separating product and substrate (Boldrup et al., 2004). This

method was used in one of the experiments and gave the same results as the chloroform /

methanol assay (Fig. 3b of Paper I).

Comments

Prior to this study a number of different assays for FAAH had been described such as

chromatographic separation of substrate, coupled assays and spectrophotometric assays with

p-nitroanilide substrates and simple organic separation of radiolabelled product from

radiolabelled substrate (Boldrup et al., 2004). The assay used in this study was essentially

36

described by Omeir et al. (1995) and Dinh et al. (2002) and modified with regards to

differential separation of ethanolamine and AEA to charcoal in an acidic solution rather than

chloroform/methanol by Boldrup et al. (2004).

One of the great advantages of this assay is that it is simple and easy to perform. However

there are important factors that have to be taken into account in order to obtain reliable results

that can be reproduced and compared between different laboratories. For all enzyme assays,

tissue preparation, solubility and concentration of the compounds tested, incubation time, pH,

buffer type and temperature are important factors.

Hence, before testing different compounds for inhibition of FAAH and MAGL, initial

experiments were undertaken to determine the optimal assay conditions regarding protein

concentration, buffer, pH and different solvents. Furthermore, in this study the cytosolic

instead of the membrane fraction was used for MAGL activity in order to minimize the risk

for membrane-bound FAAH mediated activity. Given that there might be a risk of

contamination in the cytosolic fraction, the MAGL assay was validated by using FAAH

inhibitors.

As described in the introduction, hydrolysis by FAAH and MAGL are not the only routes of

AEA and 2-AG degradation and additional hydrolytic and oxidative enzymes might in theory

contribute to the activity results, although the products of non-hydrolytic pathways are not

necessarily separated from the substrates using the current assay procedure. In the case of

FAAH, this is not the case given the complete inhibition found with the selective inhibitor

FAAH (Fig. 2a of Paper I). Subsequent studies from this laboratory using rat cerebellar

cytosolic fractions and the charcoal assay to separate products from substrate have indicated

that the selective MAGL inhibitor JZL184 inhibits ~90% of the hydrolysis of 2-OG

(Bjorklund et al., 2010). Thus, a contribution to the observed product formation by other

enzymatic pathways than FAAH and MAGL is unlikely. Nevertheless, combining this assay

with alternative assays using recombinant enzymes would ensure that the inhibitory potency

of the compounds of interest is not assay-dependent (Bjorklund et al., 2010).

37

Key methods used in paper II-IV

Subjects

Subjects with chronic neck shoulder pain

Forty-one women with CNSP were recruited for microdialysis procedure as part of a

randomized controlled training trial undertaken with fifty-seven women with CNSP. The trial

was performed in the county of Östergötland, Sweden. Inclusion criteria were female sex, age

range 20-65 years, and pain in the neck-/shoulder area that had lasted more than 3 months.

Exclusion criteria were widespread pain according to the ACR classification, bursitis,

tendonitis, capsulitis, postoperative conditions in the neck/shoulder area, prior neck trauma,

disorder of the spine, neurological disease, rheumatoid arthritis or any other systemic

diseases, metabolic disease, malignancy, severe psychiatric illness, pregnancy, and difficulties

understanding the Swedish language.

Subjects with chronic widespread pain

Eighteen women with CWP were recruited. Inclusion criteria were female sex, age range 20-

65 years, and widespread pain according to the ACR classification. Exclusion criteria were

the same as mentioned above for CNSP with the exception of widespread pain. Clinical

examination was conducted according to the same protocol as the CNSP group with the

addition of a standardized examination of the lower extremities.

Healthy subjects

Twenty-four healthy women comprised a control group (CON). Inclusion criteria were female

sex, age range 20-65 years and pain-free. The exclusion criteria were the same as the CNSP

group, and in addition pain that had lasted more than seven days during the past 12 months.

Clinical examination was conducted according to the same protocol as for CWP.

Ethical approval

After receiving verbal and written information about the study, all subjects signed a consent

form that was in accordance with the Declaration of Helsinki. The study was granted ethical

clearances by the Linköping University Ethics Committee (Dnr: M10-08., M233-09, and Dnr:

2010/164-32).

38

Comments

CNSP and CWP were investigated since these are common pain conditions and comprise the

majority of patients referred to the Pain and rehabilitation Clinic in Linköping, Sweden. The

mechanism(s) behind these conditions are to date not fully understood. By using the trapezius

muscle as a model muscle, the research group in Linköping had previously studied these

conditions regarding algesic substances. However, nothing was known as to the levels of

NAEs and endocannabinoids and their potential role in muscle. Investigating the levels of

NAE and endocannabinoids in other types of chronic pain conditions such as neuropathic pain

conditions would be of great interest. Only women participated in the studies comprising the

clinical part of this thesis. Future studies should also investigate various aspects of pain in

men with CNSP.

Recruitment of subjects

The CNSP and CON group were recruited via advertisements in the local daily newspaper.

According to standard procedures, brief information regarding the study including gender,

age-span of interest and pain state was given on both advertisements. For the CNSP group the

additional information regarding training intervention was given.

Subjects in the CWP group were identified and recruited via fibromyalgia patient organization

and reviews of the medical reports of former out-patients at the multidisciplinary Pain and

Rehabilitation Centre, University Hospital, Linköping, Sweden.

Comments

The local newspaper used for advertisement was one of the biggest newspapers in the county.

This made the information available for the majority of people living there, and providing the

information in Swedish was in line with the inclusion/exclusion criteria of understanding the

Swedish language. The information regarding exercise intervention for the CNSP group might

have affected the number of subjects responding to the advertisement since it implied the

requirement of motivation for exercise in order to participate in the study.

Subjects in the CWP group were first identified by reviewing the medical reports of former

out-patients. It was important that participants were not in any treatment program at the time

of the study in order not to interfere with their outcome. Reviewing the charts did not,

however, result in a sufficient number of participants and remaining subjects were recruited

39

via patient organizations. Using different recruitment ways is not optimal, both regarding

comparing the CNSP to the CWP group but also regarding within group analyses.

Selection of subjects

The Nordic Ministry Council Questionnaire (NMCQ), a self-reported pain questionnaire

(Kuorinka et al., 1987) allowing assessment of pain in the last 12 months, together with a

structured telephone interview was used for primary screening.

Subjects were then invited for a standardized clinical examination in order to confirm

eligibility according to inclusion and exclusion criteria set for each group. Examination of the

upper extremities was conducted as described by Ohlsson et al. (1994) in all subjects and

included questions on pain, tiredness and stiffness on the day of examination, as well as

physical tests including; range of motion and tightness of muscles, pain threshold and

sensitivity, muscle strength and palpation of tender points.

The presence of trapezius myalgia (trapezius muscle was used for microdialysis in all

subjects, see microdialysis section) was assessed in all subjects. Diagnosis of trapezius

myalgia included tightness of the trapezius muscle, i.e., a feeling of stiffness in the

descending region of the trapezius muscle reported by the subject at examination of lateral

flexion of the head, and palpable tender parts in the trapezius muscle. The range of motion of

the cervical columna is normal or slightly decreased.

Comments

Various biases can affect self-reported questionnaires and many people can experience non-

specific neck/shoulder pain without any diagnosed disease. However, combining NMCQ, a

well-established questionnaire to survey pain symptoms, together with telephone interviews

and detailed standardized clinical examinations strengthens the capacity of identifying healthy

controls, subjects with neck/shoulder pain including trapezius myalgia, and subjects with

CWP.

40

Training programme (Paper IV)

The training programme was undertaken by Linn Karlsson (physiotherapist) as part of a

separate study (Karlsson et al, manuscript) outside the aims of the present thesis, but is

described here since some of the patients in that study were included in Paper IV. In summary

the training session in strength + stretch group started with specific strength training with

focus on the neck and shoulder muscles and included arm abduction, upright row, biceps

curls, reverse flyes, flyes and pullovers with dumbbells. Additionally, lifting the head up from

supine position was also a strength exercise. Three series of dynamic exercises for the trunk

and legs, e.g. sit-ups, back extensions and squats followed the strength training.

Stretching exercises for the neck, that is retraction of the neck, shoulder and upper limb

muscles i.e. stretching of m. trapezius upper and middle portion ended each training session

for strength + stretch group and constituted the complete training session for stretch alone

group. Stretching exercises for both groups also included m. sternocleidomastoideus, m.

rhomboids, m. pectoralis major and the flexors and extensors of the wrist.

Both groups were asked to undertake the training session three times a week and additionally

they were encouraged to perform optional aerobic exercise for 30 minutes with the same

frequency. The strength + stretch group was instructed to give priority to the specific strength

exercises if they did not manage to perform the complete session. Training support was

provided by skilled physiotherapists by phone or e-mail every 4 – 8 weeks. The support was

more frequent in the beginning of the training period.

The training modalities included in our definition of a completer were neck strength training

for strength + stretch group and stretching training for the stretch alone group. A completer

reported at least eight unbroken weeks of training with a frequency of at least 1.5 times

weekly preceding the outcome measurement time points (4 – 6 months). In total 29 women

performed both microdialysis sessions from which also samples were collected and analysed.

Of those 29, 24 subjects were completers.

41

Microdialysis procedure

The microdialysis technique enables the monitoring of different substances in the extracellular

environment and is widely used for sampling and quantitating neurotransmitters,

neuropeptides, and other substances in the brain and peripheral tissue such as skeletal muscle

(Ungerstedt, 1991, Plock and Kloft, 2005). During the process of microdialysis the catheter is

inserted in the tissue of interest and is perfused with a solution (perfusate) that resembles the

interstitial fluid. Substances diffuse into or out of the perfusion fluid in a concentration-

dependent manner and dialysate samples can be collected and analyzed.

All participants were asked not to take non-steroidal anti-inflammatory drugs for the last

seven days and/or paracetamol medication for the last 12 hours before the experiment. In

addition, they were asked not to consume coffee and/or tea and cigarettes and/or nicotine

agents for the last eight hours before the experiment. They were also instructed not to perform

any shoulder or neck-straining exercises for the last 48 hours before the study, except for

ordinary daily work and/or leisure activities.

The most painful side (subjects with pain) or the dominant side (pain-free subjects) of the

trapezius muscle was used for microdialysis procedures. To guide the placement of catheters,

ultrasonographic measurements of the trapezius muscle area were conducted.

The skin and the subcutaneous tissues above the region where the catheter entered the

trapezius muscle were anaesthetized with a local injection (0.5 ml) of Xylocaine (20 mg/ml)

without adrenaline, and care was taken not to anaesthetize the underlying muscle. Two

commercially available microdialysis catheters (cut-off points of 20 and 100 kDa; CMA

Microdialysis AB, Solna) were inserted with a catheter into the pars descendens of the

trapezius muscle at half the distance between the processus spinosus of seventh cervical spine

and the lateral end of the acromion. Typically, a brief involuntary contraction and change of

resistance were perceived when the tip of the insertion needle of the catheter entered the

fascia and muscle. The catheters were placed in the trapezius muscle parallel to the muscle

fibers and perfused at 5 µl/min with a solution (perfusate) resembling the muscle interstitial

fluid.

The perfusion fluid consisted of Ringer acetate solution containing 3 mM glucose and 0.5 mM

lactate and 0.3 µl/ml [14

C]- Lactate (specific activity: 5.77 GBq/mmol) as well as 0.3 µl/ml

3H2O (specific activity: 37 MBq/gram).

42

After the insertion of catheters, participants rested comfortably in an armchair for a 120 min

period to allow the tissue to recover from possible changes in the interstitial environment

induced by the operative procedure. After this period, participants continued to rest for a 20

minutes of baseline period (140 min, i.e pre-exercise). This was followed by a 20 min period

of standardized repetitive low-force exercise performed on a pegboard (160 min). The

experiment ended with three recovery periods of 20 min during which participants rested

(180 min i.e post-exercise, 200 and 220 min). Participants were offered a standardized light

meal at the 100 min time point. No food or beverages except for water was allowed otherwise

during the experiment.

Immediately prior and after catheter insertion subjects were asked to rate their pain intensity

on a numeric rating scale. They continued to do so every 20 minutes throughout the

experiment during which microdialysate was collected in glass vials (CMA Microdialysis,

Sweden) at the same time intervals.

Each vial was weighed before and immediately after sampling order to confirm that sampling

was working according to the perfusion rate set. All samples were controlled for visible signs

of haemolysis that would result in the discarding of the sample. The samples were stored at

4 °C throughout the experiment and then stored as aliquots −70 °C until analysis.

Comments

The main advantage with the microdialysis technique is that it enables measurement of the

unbound fraction of substances at the site of action, compared to when using plasma or whole

tissue concentrations (Plock and Kloft, 2005). However, although it is well tolerated, the

tissue trauma caused by insertion and implementation of the microdialysis probe and poor

time resolution should be considered as drawbacks of this technique.

The exchange of substances through the membrane is dependent on several critical factors

including characteristics of the substances (solubility, molecular weight), the membrane

(surface, length, and pore-size), the perfusion flow rate set, temperature and the composition

of the perfusate. In an expert review from 2010, recommendations were made for optimal

sampling of extracellular NAEs and endocannabinoids (Buczynski and Parsons, 2010). The

microdialysis procedure in this thesis essentially follows the recommendations made by the

review. Briefly, major differences when sampling these bioactive lipids compared to sampling

43

hydrophilic substances seem to be the flow rate and the propensity of the lipids to adsorb to

the microdialysis tubes and membrane. In general the flow rate used is between 0.3 and 5

μL/min and a low flow rate results in larger recoveries (concentration of the substance in the

dialysate). However, sampling at a higher flow rate seems to increase the recovery of

lipophilic substances, possibly because of changes in amount that is adsorbed to surface

during the procedure (Kjellstrom et al., 1998, Zoerner et al., 2012). Furthermore, exact

interstitial concentrations of NAE and endocannabinoids cannot be obtained by the methods

used in this thesis. There are several different techniques to determine the in vivo relative

recovery and calculate the interstitial concentrations. The protocol in this thesis enables the

use of the internal reference technique (Scheller and Kolb, 1991) by radiolabeled lactate.

However, this cannot be used to determine interstitial levels of NAE and endocannabinoids

since the internal standard does not mimic the characteristics of these lipids.

Pain intensity ratings

All pain ratings during the microdialysis procedure concerned pain in the trapezius muscle of

the most painful side (subjects with pain) or the dominant side (pain-free subjects). The

subjects were asked to rate their pain intensity on a numeric rating scale (NRS) with numbers

(0 – 10; 0=no pain and 10= worst possible pain) provided along for guidance. Immediately

prior and after catheter insertion subjects were asked to rate their pain intensity. They

continued to do so every 20 minutes (same time points as dialysate sampling) throughout the

experiment that lasted 220 minutes.

Comments

Besides NRS, visual analogue scales (VAS) and verbal rating scales (VRS) are reliable and

valid scales that are frequently used for assessment of pain intensity. For assessing pain

intensity in clinical trials of chronic pain treatment, NRS recommended as a outcome measure

(Dworkin et al., 2005).

44

Standardized low-force repetitive exercise

The low-force exercise regime consisted of a repetitive arm movement task that was

performed unilaterally using the arm on the same side of the body as the inserted

microdialysis catheter. The subjects moved short wooden sticks (11.8 g) back and forth

between standardized positions 30 cm apart on a pegboard at a frequency of 1.3 Hz indicated

by an electronic metronome (Korg Inc., Tokyo, Japan). The participants performed the

exercise in a seated position with the pegboard placed 30 cm in front of them, measured from

the elbow with the upper arm hanging vertically and the elbow in a 90° flexion. The exercise

was supervised by qualified personnel (e.g. nurse or physiotherapist).

Comments

The advantage of using this low-force exercise is that compared to protocols employing

experimental induction of pain (such as injection of hypertonic saline, bradykinin, glutamate

into muscle) the exercise regime mimics situations in daily life, usually resulting in an

increase in pain intensity in subjects with chronic pain in neck/shoulder area. However, the

exercise is usually not painful for healthy subjects and this could make the comparison to

subjects with chronic pain difficult.

Analysis of NAE levels

Analysis of NAE and endocannabinoids has been performed by chromatographic separation

in combination with mass spectrometric (MS) (Buczynski and Parsons, 2010, Zoerner et al.,

2011). This combination allows detection and quantification of substances with low

concentrations in small sample volumes, such as microdialysate samples.

Chromatography is based on a mobile phase which moves through a stationary phase (a

column). When injected into the column, the substances in the microdialysate sample are

separated based on their different affinities to the two phases and the retention time, i.e. the

time it takes for a substance to travel through the stationary phase, is calculated. Fractions are

then processed in MS for further characterization. In MS analysis, molecules are ionized

(positive or negative parent ion), mass analyzed, fragmented (daughter ion), separated

according to their mass to charge ratio (m/z), and then detected.

On the day of analysis, 50 µl of dialysate samples were dried by SpeedVacc, redissolved in

methanol, vortexed and centrifuged. The supernatant was analysed by a nano-flow-Liquid

45

chromatography system (EASY-nLC™), coupled to a mass spectrometer with electrospray

ionization (Bruker Daltonics Ultra Performance High Capacity Ion Trap MS).

The compounds were eluted isocratically (acetonitrile with 0.1% formic acid) at a flow rate of

0.9 nL /min. The mass spectrometer was operated in the positive ion and multiple reaction

monitoring mode (MRM). The selected parent/daughter ion pairs used were 348.2/287.1,

379.2/269.1, 300.2/283.1 and 328.3/311.1 m/z for AEA, 2-AG, PEA and SEA respectively.

The linearity of the measuring range was assessed with standard curves ranging from 0.01–20

nM in human muscle dialysate. Standard curves generated using linear regression.

Comments

Both gas chromatography (GC) and liquid chromatography (LC) have been used for analysing

NAE and endocannabinoids, but the latter is the most applied chromatography technique for

separating different compounds in microdialysate samples (Buczynski and Parsons, 2010).

The main advantage with using LC, which was the method chosen here, is that the method is

simpler and it does not require prior chemical derivatization compared to GC (Buczynski and

Parsons, 2010, Zoerner et al., 2011).

For selecting specific parent ions, an alternative MRM method with an ion-trap MS was used

in this thesis. In contrast many studies have used the triple quadrupole MS, which produces

different fragments. In general, using ion trap results in increased sensitivity and specificity

for detection while triple quadrupole provides better limits of quantitation.

46

Summary of study protocols. For detailed procedures see original papers.

Paper I

The abilities of a series of analogues of 2-AG to inhibit cytosolic 2-OG and membrane-bound

anandamide hydrolysis by MAGL and FAAH, respectively was investigated. The studies

were undertaken at the Department of Pharmacology and Clinical Neuroscience, Umeå

University.

Paper II-IV

The self-reported pain questionnaire together with a structured telephone interview was used

for primary screening. Subjects were then invited for a standardized clinical examination as

well as weight, height (BMI), and blood pressure measurements in order to confirm

eligibility. All eligible subjects went through pressure point measurements in upper and lower

extremities and injection of hypertonic saline in tibialis anterior muscle. Microdialysis was

conducted in the trapezius muscle on the most painful side (CNSP, CWP) or the dominant

side (CON) defined by asking subjects whether they were right-or left handed.

The studies were undertaken at Rehabilitation Medicine, Department of Medicine and Health

Sciences, Linköping University, in collaboration with Pain and Rehabilitation Centre, County

Council of Östergötland, Linköping, Sweden

The number of CNSP, CWP and CON recruited for microdialysis and from which samples

were collected and analyzed in the studies are summarized in Table 1.

Pilot study: Initial pilot studies were undertaken in order to develop the method for analysing

NAEs and endocannabinoids, in microdialysate samples from the trapezius muscle, with

available equipment at the Faculty of Health, Linköping University, Sweden.

Paper II: Comprises women with CNSP and CON. PEA and SEA levels at 20 (trauma), 120

(end of trauma period), 140 (pre-exercise), 160 (low-force exercise), 180 (post exercise, i.e

first recovery period) and 220 min (third and last recovery period) together with pain intensity

ratings during the microdialysis procedure are presented.

Paper III: Comprises women with CNSP, women with CWP and CON. PEA and SEA levels

at 140 min (pre-exercise) and 180 min (post-exercise) together with pain intensity ratings

during microdialysis procedure are presented.

47

Paper IV: Comprises women with CNSP randomized to stretch alone or strength + stretch

training intervention. PEA and SEA levels at 140 min (pre-exercise) together with pain

intensity ratings during microdialysis procedure before and after 4-6 months training are

presented. The study also includes a CON group performing microdialysis twice, but with no

intervention during the 4-6 months in between.

Table 1: Total number of women with chronic neck/shoulder pain (CNSP), chronic wide spread pain

(CWP) and healthy pain free controls recruited for microdialysis are shown in brackets. For each paper

number of subjects from which microdialysates from the trapezius muscle were sampled and analyzed

are given. In paper IV the number of subjects represents those who performed two microdialysis

sessions (4-6 month in between sessions) from which samples were collected and analyzed. Samples

from 140 and 180 time points from 10 CNSP and 11 CON in paper II are included in Paper III.

Samples from 140 min (first microdialysis session) from 24 CNSP and 24 CON in paper III (including

5 CNSP and 11 CON participating in paper II) are included in paper IV

Subjects Paper II 20, 120, 140, 160, 180, 220 (min)

Paper III 140, 180 (min)

Paper IV 140 min before and after 4-6months

CNSP (n=41) 11 34 24

CWP (n=18) - 18 -

CON (n=24) 11 24 24

.

48

Results

Paper I

MAGL and FAAH assay characterization

Protein-dependency curves using different buffers

Initial experiments were undertaken to determine optimal conditions for assay of the

hydrolysis of the two substrates. These included time- and protein-dependency experiments

(to find conditions measuring initial rates of substrate hydrolysis) and the use of different

buffers, assay pH values, and solvent concentrations (to find the most appropriate vehicles for

the lipophilic test compounds). As expected from the known subcellular distributions of

FAAH and MAGL (Hillard et al., 1995, Goparaju et al., 1999) hydrolysis of [3H]AEA was

primarily seen in the membrane fraction whereas both cytosolic and membrane fractions

could hydrolyse 2-OG (see Fig. 1). For membrane AEA and 2-OG hydrolysis, no significant

difference was observed in the initial rates for the two buffer systems tested (tris or sodium

phosphate buffers, both pH 8.0), whereas for cytosolic 2-OG hydrolysis, the activity was

activity was significantly greater in the Tris buffer than in the sodium phosphate buffer

(2.04±0.045 vs. 1.23±0.090 nmol.mg protein-1

.min-1

). Tris buffer was thus used for the

experiments reported here. The pH optimum for 2-OG (and AEA) metabolism by the cytosol

fractions was lower than for AEA metabolism by the membrane fractions (Figure 1 of Paper

I), a finding consistent with the literature (Bry et al., 1979, Hillard et al., 1995). Acetonitrile

(0.05 – 5 % v.v-1

) and DMSO (0.05-2% v.v-1

) had no effect on the observed rates of

metabolism. Ethanol (0.05-5% v.v-1

) did not affect the rate of AEA metabolism but produced

a small increase in the rate of 2-OG metabolism by the cytosolic fractions. In all experiments,

solvent carrier concentrations were kept constant throughout and never exceeded 0.5% (above

the 0.25% ethanol present for the substrates alone). Under such conditions, [3H]2-OG was

metabolised by the cytosolic fractions with a median (n=3) Km value of 2.2 µM.

49

Fig. 1. Comparison of the initial rates of [3H]AEA and [

3H]2-OG hydrolysis by rat brain

membrane and cytosolic fractions. Data from Paper I.

Validity of MAGL assay

Given that 2-AG (and presumably 2-OG) can be metabolized by FAAH (Goparaju et al.,

1998), it is important to determine that the hydrolysis of 2-OG measured here is not due to

this enzyme. This risk has been minimized by the use of cytosolic preparations for 2-OG.

Furthermore, concentrations of 1 and 10 µM URB597 completely inhibited the hydrolysis of

AEA by the membrane fractions without affecting the hydrolysis of 2-OG by the cytosolic

fractions (Fig 2a, left columns of Paper I). Similarly, palmitoylethanolamide and

arachidonoylglycine inhibited the hydrolysis of AEA by the membrane fractions but did not

affect the hydrolysis of 2-OG by the cytosolic fractions. Finally, indomethacin, known to

inhibit FAAH in addition to its effects upon cyclooxygenases (Fowler et al., 2003), did not

affect 2-OG metabolism at concentrations ≤50 µM. Thus, there is no evidence to suggest that

FAAH is a contaminant in the 2-OG hydrolysis assays.

In the experiments using URB597, AEA was also investigated. A concentration of 10 µM was

without effect upon the cytosolic activity of 2-OG, whereas a concentration of 100 µM

reduced the activity, possibly by acting as a competing substrate, although with a potency

considerably lower than seen towards FAAH.

50

Inhibition of FAAH and MAG lipase by 2-AG and related compounds

A series of acylglycerols, ethanolamines, trifluromethylketones, and two other compounds

(O-2203 and O-2204), synthesized by co-author Razdan as described previously (HAN and

RAZDAN, 1999, Ng et al., 1999) were tested. A summary of the data for the analogues and

homologues of 2-AG, AEA, O-2203 and O-2204 are shown in Table 2 and a representative

concentration-response curve is shown in Figure 2 (for detailed results regarding all

compounds tested see Table 1, 2 and Figure 4 paper I).

For acylglycerols, 2-AG showed a >4-fold selectivity for MAGL over FAAH. With respect

to rearrangement of the head group of 2-AG, the 1-AG analogue interacted with MAGL and

FAAH with the same potency as 2-AG. Introduction of an α-methyl group into the 1-AG did

not affect the potency towards MAGL, but increased the potency towards FAAH. AEA and

its shorter chain homologues palmitoylethanolamine showed expected FAAH selectivity,

inihibiting AEA metabolism presumably by acting as alternative substrates (Natarajan et al.,

1984, Schmid et al., 1985, Maurelli et al., 1995)

O-2203 and O-2204, inhibited FAAH and MAGL to similar extents (Fig. 2). For O-2203, the

pI50 values towards membrane [3H] AEA and cytosol [

3H] 2-OG were 4.08±0.15 (IC50 83

µM) and 4.04±0.05 (IC50 90 µM), respectively, giving a selectivity ratio of 0.9. O-2204

inhibited membrane [3H] AEA hydrolysis with a pI50 value of 4.46±0.19 (IC50 35 µM). With

respect to 2-OG hydrolysis, the compound did not produce complete inhibition, but for the

inhibitable component (75±6 %), the pI50 value was 4.85±0.10 (14 µM) giving a selectivity

ratio of 2.5. The compounds were also investigated by co-authors Pertwee and Martin to

determine if they had direct effects upon CB1 receptors and/or produced signs of central CB1

receptor activation. They were very weak ligands at the CB1 receptor expressed in CHO cells,

and did not produce cannabinoid-like behaviours in contrast to those seen with 2-AG (Table 3

of Paper I).

51

Table 2. Inhibition of membrane [3H]AEA cytosol [

3H]2-OG hydrolysis by 2-AG and related

compounds. Data from paper I are presented as means ± s.e.m. from analyses of data from three

experiments. C20:4 refers to the number of carbon atoms and unsaturated bonds, respectively, in the

acyl side chain (including the carbonyl group shown in the “Head group” column in the Table).

Substrate concentrations were 2 µM. ”Selectivity” is the ratio of pI50 values for the inhibitable

components (in brackets are values assuming 100% inhibition, when different). Compounds were

dissolved in aacetonitrile,

bethanol, or

cprovided dissolved in methyl acetate and diluted in acetonitrile.

pI50 (IC50 µM) [% max inhibition] for:

Compound Head group membrane [3H]AEA cytosol [

3H]2-OG Selectivity

2-AG

(C20:4)a

<4 (37 % inhibition

at 100 µM)

4.88±0.05 (13 µM)

[77±4%]

>7.6 (>4.2)

2-OG

(C18:1) a

5.61±0.26 (2.5 µM)

[53±9%]

4.84±0.03 (15 µM)

[100 %]

0.07 (2.5)

– Me-

1-AGa

4.49±0.11 (33 µM)

[100 %]

4.97±0.09 (11 µM)

[82±7%]

3.1 (1.9)

AEA

(C20:4)b

5.89±0.12 (1.3 µM)c

[66±5%]

4.22±0.04 (60 µM)

[100 %]

0.18 (0.12)

PEA

(C16:0)a

4.38±0.27 (42 µM)

[100 %]

<4 (no inhibition at 100

µM)

<0.4

O-2203

4.08±0.15 (83 µM) 4.04±0.05 (90µM) 0.9

O-2204

4.46±0.19 (35 µM)

[100 %]

4.85±0.10 (14 µM)

[75±6]

2.5

.

O

NH

OH

O

NH

OH

52

Fig. 2. Representative figure of inhibition of cytosolic [3H] 2-OG and membrane bound [

3H]

AEA hydrolysis by O-2203. Shown are means and s.e.m., n=3.

Paper II-IV

Pilot study

By using a nano-Lc system coupled to the ion trap mass spectrometer as described in methods

we were able to detect AEA, PEA, SEA and 2-AG in a single chromatographic run. AEA and

2-AG were, however, below the detection limit when analysing the microdialysate samples

and only PEA and SEA could consistently be measured. In a series (n≥4) of experiments

using with standard samples for these two detectable NAEs, it was found that the ion pairs

selected (300.2/283.1 and 328.3/311 for PEA and SEA, respectively) could be robustly

measured (Fig. 3).

53

PEA

SEA

Fig. 3. Example of typical MS and MS/MS spectra for PEA and SEA with the selected ion

pairs 300.2/283.1 and 328.3/311, respectively.

282.2

1+

296.3

328.3

1+

348.2

1+

379.3300.3

1+

133. +MS, 13.5-13.8min #1316-#1355, 100%=36951740

149.0 163.0 187.0263.0

1+

283.1

133. +MS2(300.1), 13.5-13.8min #1318-#1357, 100%=551630

1

2

3

4

7x10

Intens.

0

2

4

6

4x10

150 200 250 300 350 400 450 500 550 m/z

284.3

1+

300.2

1+

348.2

1+

361.2 379.3 565.6 595.4

328.3

1+136. +MS, 13.9-14.3min #1361-#1406, 100%=29811644

311.1

1+

136. +MS2(328.5), 13.9-14.3min #1362-#1407, 100%=64648080

1

2

3

7x10

Intens.

0

2

4

6

8

6x10

250 300 350 400 450 500 550 600 m/z

54

Anthropometric data (paper II-IV)

There were no significant differences in age or BMI between CNSP, CWP and CON (age

P=0.11; BMI: P=0.13, Kruskal-Wallis, Table 3)

Table 3. Age, BMI and pain duration in the three groups. Values shown are medians, with range in

parentheses. Kruskal-Wallis test was used for analyses of group differences in age and BMI and

Mann-Whitney U-test was used for analyses of group differences in pain duration.

CNSP CWP CON Statistics

Age (years)

45(29 - 60)

n=35

49 (29 - 62)

n=18

44 (27 - 56)

n=24

P=0.11

BMI (kg/m2)

24 (20 - 42)

n=34

27 (20 - 36)

n=18

23 (20 - 31)

n=22

P=0.13

Pain intensity ratings

Pain intensity ratings in CNSP, CWP and CON (paper II and III)

CON (n=24) had at group level no pain throughout the microdialysis experiment. Pain

intensity ratings for CNSP (n=35) and CWP (n=18) are shown in Figure 4.

No significant differences in pain intensities were found between CWP (n=18) and CNSP

(n=35) except for at the post-exercise measurement (180 min). At this time point CWP had

higher pain intensity than CNSP (P=0.029, Mann Whitney U test).

Pain intensity increased significantly in both the CNSP (P<0.001, Wilcoxons signed rank test)

and CWP (P=0.001, Wilcoxons signed rank test) during the low force exercise period (160

min) compared to pre-exercise (140 min).

Both groups also had significantly higher pain intensities at post-exercise (180 min) compared

to pre-exercise (CNSP: P=0.007 and CWP: P=0.004, Wilcoxons signed rank test).

55

Fig. 4. Pain intensity ratings (median with interquartile range) during the microdialysis

procedure for women with chronic neck shoulder pain (CNSP, n=35) and chronic widespread

pain (CWP, n=18). “Pre” represents pain intensity ratings prior to catheter insertion, “0” is

pain intensity immediately after catheter insertion, “140” represents baseline, “160”

represents the period during which subjects performed the 20 min low-force standardized

exercise and “180”, “200” and “220” represent the three recovery periods.

56

Pain intensity ratings in subgroup of CNSP randomized to stretch + strength training

(paper IV)

Comparisons were made for pain scores before and after the intervention. The median values

were lower after the intervention than before, but the differences only reached statistical

significance at the 160 min (low-force exercise) time-point (P<0.055, Wilcoxon matched-

pairs signed ranks test).

When the data for the strength + stretch intervention were analyzed in terms of a clinically

relevant decrease in pain (∆NRS ≥2), 4/13 patients reached this level at the 0 and 140 min;

8/13 at 160 min and 5/13 at 180, 200 and 220 min time-points.

Pain intensity increased significantly between 140 and 160 min time-points at the first but not

at the second microdialysis session (P<0.01 vs. P>0.06, Wilcoxon matched-pairs signed ranks

test).

Pain intensity ratings in subgroup of CNSP randomized to stretch alone training (paper

IV)

The effect of the stretch alone intervention upon the median values was less marked than seen

for the strength + stretch intervention and significance was not attained (P>0.2 at all time-

points, Wilcoxon matched-pairs signed ranks test).

The pain intensity in stretch alone was unchanged or increased (∆NRS ≥2) in 9/11, 10/11,

6/11, 9/11, 10/11, and 10/11 patients at 0, 140, 160, 180, 200 and 220 min time-points before

and after 4-6 months training. The corresponding values for the strength + stretch group were

9/13, 9/13, 5/13, and 8/13, 8/13, 8/13, respectively.

Pain intensity increased significantly between 140 and 160 min time-points during both

microdialysis sessions (P<0.005 for the first and P<0.05 for the second session, Wilcoxon

matched-pairs signed ranks test).

57

PEA and SEA microdialysate concentrations

Different time points of the microdialysis experiment in CNSP and CON (paper II)

PEA and SEA levels were determined during the different stages of the microdialysis

experiment (trauma, pre-exercise, low-force exercise, post-exercise) in 11 CNSP and 11 CON

(Table 4 and 5). There was no significant variation of the microdialysate concentrations of

PEA and SEA (P>0.2, Kruskal-Wallis) in either of the groups.

Group differences pre-and post-low force exercise between CNSP, CWP and CON

(paper III)

The levels of PEA and SEA were significantly higher in CNSP (n=34), both pre- and post-

exercise, compared to CON (n=24). PEA pre- and post-exercise; P <0.05, P<0.001 and SEA

pre- and post-exercise; P<0.001, P<0.01, Dunn’s Multiple comparison test (Tables 4 and 5).

When comparing CNSP to CWP (n=18), the levels of PEA and SEA were significantly higher

in CNSP post- but not pre-exercise (P <0.001 for substances post-exercise, Dunn’s Multiple

comparison test, Tables 4 and 5).

There were no significant differences in PEA and SEA levels between CWP and CON either

pre- or post-exercise (Tables 4 and 5).

58

Table 4. PEA levels in trapezius muscle microdialysates in subjects with chronic neck/shoulder pain

(CNSP), chronic wide spread pain (CWP) and pain-free controls (CON). Median values with min and

max in brackets are shown. For analysis of group differences two-tailed Mann-Whitney U-test was

used in paper II and Kruskal-Wallis test followed by Dunn’s Multiple comparison test was applied in

paper III ( P<0.05 was considered significant, NS denotes non-significant).

Time of sample (min after insertion of catheters)

20

(trauma

period)

120

140

(baseline)

160

(low force

exercise)

180

( recovery

period)

220

(recovery

period)

PEA (nM)

Paper II

CNSP

CON

P-value

Paper III

CNSP(n=34)

CWP (n=18)

CON (n=24)

Kruskall-

Wallis P value

Dunn’s

Multiple

comparison

tests

3.30

(0.42-11.90)

n=11

0.60

(0.10-2.50)

n=11

<0.001

1.90

(0.58-4.10)

n=9

0.73

(0.50-2.50)

n=8

0.011

1.95

(0.10-8.60)

n=10

0.51

(0.30-3.70)

n=10

0.15

1.75

(0.10-8.60)

1.26

(0.10-2.30)

0.65

(0.00-3.37)

0.019

CNSP vs.

CON <0.05

CNSP vs.

CWP NS

CWP vs.

CON NS

1.60

(0.60-6.60)

n=9

0.20

(0.10-1.80)

n=9

<0.005

2.40

(0.92-6.80)

n=10

0.50

(0.10-1.20)

n=11

< 0.001

2.25

(0.10-6.80)

0.65

(0.10-2.40)

0.50

(0.10-2.30)

<0.0001

CNSP vs.

CON <0.001

CNSP vs.

CWP <0.001

CWP vs.

CON NS

1.00

(0.40-4.50)

n=7

0.60

(0.10-0.90)

n=11

0.018

59

Table 5. SEA levels in trapezius muscle microdialysate in subjects with chronic neck/shoulder pain

(CNSP), chronic wide spread pain (CWP) and pain-free controls (CON). Median values with min and

max in brackets are shown. For analysis of group differences two-tailed Mann-Whitney U-test was

used in paper II and Kruskal-Wallis test followed by Dunn’s multiple comparison test was applied in

paper III.NS denotes not significant.

Time of sample (min after insertion of catheters)

20

(trauma

period)

120

140

(baseline)

160

(low force

exercise)

180

( recovery

period)

220

(recovery

period)

SEA (nM)

Paper II

CNSP

CON

P-value

Paper III

CNSP(n=34)

CWP (n=18)

CON (n=24)

Kruskall- Wallis P

Dunn’s Multiple

comparison test

3.75

(1.4-16.50)

n=11

0.90

(0.10-2.00)

n=11

< 0.001

4.80

(0.65-20.00)

n=9

0.30

(0.10-1.70)

n=8

0.002

2.93

(0.63-18.00)

n=10

0.45

(0.10-2.10)

n=10

0.003

2.00

(0.10-18.00)

1.00

( 0.10-3.80)

0.60

(0.00-2.70)

<0.0002

CNSP vs.

CON<0.001

CNSP vs.

CWP NS

CWP vs.

CON NS

3.80

(0.20-13.00)

n=8

0.40

(0.10-2.40)

n=9

0.018

5.40

(0.96-20.00)

n=10

0.40

(0.10-1.80)

n=11

<0.002

1.45

(0.10-20.00)

0.40

(0.10-2.90)

0.55

(0.10-1.80)

<0.0001

CNSP vs.

CON<0.01

CNSP vs.

CWP <0.001

CWP vs.

CON NS

1.92

(0.45-12.00)

n=7

0.20

(0.05-2.00)

n=11

<0.01

60

Within group analysis of pre-and post-low force exercise levels in CNSP, CWP and CON

(paper III)

PEA and SEA levels decreased significantly from pre- to post-exercise in CWP (P=0.037 for

PEA and P = 0.007 for SEA, Wilcoxons signed rank test). No significant changes in PEA and

SEA levels were seen in CON and CNSP. The individual values for the CWP group are

shown in Table 6.

Table 6. Individual values (nM) of PEA and SEA in CPW during pre–and post-low force exercise

PEA SEA

Pat. no. Pre-exercise Post-exercise Pre-exercise Post-exercise

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

0,1

0,3

1,8

1,6

0,9

1,5

0,7

1,7

1,3

1,3

1,2

0,1

1,0

0,8

1,5

2,3

1,1

1,8

0,4

2,4

1,4

0,8

0,1

1,4

0,1

1,1

0,8

0,5

1,2

1,2

0,1

0,1

0,1

1,1

0,2

0,1

0,1

0,9

2,1

1,2

1,1

1,6

0,8

0,8

0,3

1,1

1,0

1,0

3,8

2,3

2,4

1,0

0,3

0,3

0,5

2,9

0,6

0,7

0,2

1,3

0,1

0,5

0,3

0,5

0,6

0,3

0,1

0,1

0,1

0,5

0,1

0,1

61

Repeated measurements after 4-6 months in CON (paper IV)

PEA and SEA levels were measured in the microdialysate samples at the 140 min time point

during two separate microdialysis sessions (4-6 months between sessions) in CON (n=24).

There was a significant decrease of PEA levels at the second microdialysis session compared

to the first; median [min-max] 0.65 [0-3.7] nM during the first session vs. 0.15 [0.10-2.50]

nM during the second session, P<0.05, Wilcoxons signed rank test.

There was no significant difference in SEA levels between the two microdialysis sessions; 0.6

[0-2.7] nM vs. 0.65 (0.10-4.2) nM.

PEA levels in subgroups of CNSP randomized to stretch alone and stretch + strength

training (paper IV)

At the fist microdialysis session, the median PEA values for the patients randomized to stretch

alone (n=11) and to stretch + strength (n=13) groups were 0.4 and 2.0 nM, respectively. Only

the stretch + strength group was significantly different from control CON (P<0.05, Dunn's

multiple comparisons test following significant Kruskal-Wallis).

At the second microdialysis session, the corresponding median values were 3.3 and 2.0 nM,

respectively. In this case, the median values for both subgroups were significantly different

from CON (P<0.001 for stretch and P<0.011 for stretch + strength groups respectively), but

the two treatment training groups did not differ significantly from each other. However, the

difference in PEA levels during the two microdialysis sessions in CON (above section) makes

interpretation of the effects of intervention on PEA levels difficult.

SEA levels in subgroups of CNSP randomized to stretch alone and stretch + strength

training (paper IV)

At the first microdialysis session, the median SEA values for the patients randomized to

stretch alone and to stretch + strength groups were 1.1 and 2.5 nM, respectively. Only the

stretch + strength group was significantly different from control (P<0.001, Dunn's multiple

comparisons test following significant Kruskal-Wallis).

At the second microdialysis session, the corresponding median values were 5.0 and 2.4 nM,

respectively. In this case, the median values for the stretch alone group was significantly

62

different from CON (P<0.001, Dunn's multiple comparisons test following significant

Kruskal-Wallis tests), whereas the median values for the stretch + strength group were not

significantly different from CON. For the stretch alone group, the median SEA level after the

training regime was significantly increased (P<0.005, Wilcoxon matched-pairs signed rank

test). In contrast the median SEA levels for the stretch + strength group were not significantly

changed by the training regimen (P>0.9, Wilcoxon matched-pairs signed rank test).

Relationship between the SEA levels and the pain intensity rating scores following the

training programs (paperIV)

The correlation between the change in the NRS before and after the training programmes

(∆NRS) at 160 min and the corresponding change in SEA concentrations (∆SEA) at 140 min

was calculated for all 24 completers of CNSP. For all completers, regardless of training

regimen, there was no significant correlation between the two parameters (Spearman's rho

0.019, P>0.93). However, a significant negative correlation was found for the stretch +

strength group alone (Spearman's rho -0.59, P<0.05) while the correlation in stretch alone

group was not significant (Spearman's rho 0.46, P>0.15).

63

Discussion

Paper I

Prior to paper I, the pharmacological characteristics of MAGL and its comparative effects

with FAAH had not been well studied. The present study has provided much needed basic

pharmacological data for the interaction of MAGL with 2-AG (and AEA) analogues.

Although the study did not identify an MAGL-selective compound, three compounds that

inhibited FAAH and MAGL to the same extent were identified. It was not until several years

later in 2009 when the first highly potent selective MGL inhibitor, JZL184, which inhibits

MGL irreversibly, was reported (Long et al., 2009). This compound has subsequently been

used to delineate the role of MAGL in a number of pathological conditions ranging from pain

to cancer (review, see Fowler, 2012b), although the potential therapeutic usefulness of the

compound is limited by tolerance to its behavioural effects accompanied by a down-

regulation of central CB1 receptors (Schlosburg et al., 2010). Whether similar tolerance

phenomenon are seen with reversible MAGL inhibitors awaits elucidation.

Paper II-IV

Using a protocol with both a baseline (pre-exercise), followed by a brief pain provocation test

known to increase pain intensity in patients with chronic muscle pain (20 min exercise) and a

post exercise registration the major results, that will be discussed below, were:

PEA and SEA could be robustly measured in human muscle microdialysate samples

whereas AEA and 2-AG were not detectable.

In CNSP and CON there were no changes in PEA and SEA levels during the different

stages of microdialysis procedure

In CNSP the levels of PEA and SEA were significantly higher both pre- and post-

exercise compared to CON.

The CNSP had significantly higher levels of both PEA and SEA post-exercise, but not

pre-exercise, than CWP

In CWP the levels of PEA and SEA decreased significantly from pre- to post-exercise.

In CON there was a variation of baseline PEA but not SEA levels when measured twice

with a 4-6 month interval between measurements.

64

The CNSP and CWP group differed in several characteristics. The brief low force exercise

resulted in an increase in pain intensity in both CNSP and CWP, but post-exercise the pain

intensity was higher in CWP. In contrast the brief exercise was not perceived as painful in CON.

In CNSP the levels of PEA and SEA were significantly higher both pre- and post-exercise

compared to CON. CWP did not show significant differences compared to CON but looking at

within-group differences the levels of PEA and SEA decreased significantly from pre- to post-

exercise. This decrease was not seen in CNSP and CON. An increase in pain intensity by activity

is often reported by CWP patients making them more vulnerable to nociceptive input, which

might explain the low compliance in training interventions (Nijs et al., 2012). A shift from pain

inhibition towards pain facilitation and a dysfunctional pain inhibitory pathway is associated

with development of widespread pain (Ge et al., 2012). The molecular changes that occur in

transition from localized to widespread pain are not fully understood. The present results might

indicate that in CNSP the levels of these bioactive lipids are at highest attainable as a protective

response to the on-going nociception, whereas this is not the case for the CWP group in which

the levels decrease. This might reflect the peripheral changes in a defect pain inhibitory pathway.

The operative word here, however, is “might” and further studies are needed.

In our group we have recently applied proteomic approach to investigate dialysate samples from

CNSP (n=37), CWP (n = 18) and CON (n = 22) from the catheter with 100 kDa cut-off, which

were pooled for each group (Olausson et al., 2012). Approximately 300 proteins were found in

the interstitium of human muscles of which 97 were identified and those proteins were involved

in inflammatory processes and metabolic, structural, regulatory, contractile and transporter

proteins. In CNSP and CWP 50% and 31%, respectively, of the identified proteins were at least

two-fold higher or lower compared to CON. The two groups of patients showed at least two-fold

alterations in concentrations of the same proteins (18%) when compared to CON and

approximately two-thirds of these alterations were in the same direction. Thus, these results

show marked alterations of the trapezius muscle proteome of CNSP and CWP when compared to

CON.

Exercise therapy forms to date a natural part of rehabilitation of patients with CNSP. The

mechanism (s) leading to decrease in pain are still not fully elucidated. Data on the impact of

exercise treatment on concentrations of pain-relieving substances (opioids, serotonin, and

norepinephrine) in plasma or cerebrospinal fluid in patients with musculoskeletal pain is not

65

conclusive (Fuentes et al., 2011). Regarding NAEs, increases in circulating PEA, AEA and OEA

as response to acute exercise in humans has been reported (Sparling et al., 2003, Heyman et al.,

2012). This class of lipids have not previously been investigated in this context in muscle. Thus,

the aim of Paper IV was to investigate the effect of different training interventions conducted by

CNSP on PEA and SEA levels in muscle dialysate and pain intensity. Due to dropouts, subjects

not meeting completer criteria, and samples being excluded due to technical failure, the two

training groups were small, which is a major study limitation.

Baseline PEA levels decreased significantly in CON between first and second microdialysis

session. This might be due to seasonal variations but that is somewhat speculative since it has not

been investigated in humans previously, although there is some data suggestive of temporal

variations in circulating NAE levels in animals (Vaughn et al., 2010). Also dietary fat intake

might affect tissue levels of NAEs, as has been seen in the brains of rodents (Hansen and Diep,

2009). At the second microdialysis session, the median values for both patient groups were

significantly different from controls but the two treatment groups did not differ significantly

from each other. The most conservative conclusion to be made from these data is that neither

treatment paradigms normalizes PEA levels to those seen in healthy controls.

For SEA, the median values for the healthy controls between the two microdialysis sessions were

not significantly different. For the stretch alone group, the median SEA level after the exercise

regime was significantly increased. In contrast, the median SEA levels for the stretch + strength

group were not significantly changed by the exercise regimen and there was a negative

correlation between the change in pain scores following the brief low-force exercise during the

microdialysis procedure before and after the training program and the change in SEA levels.

Prior to this thesis there were no studies investigating the levels of endocannabinoids and NAEs

in human muscle. The main strength of applying the microdialysis technique as undertaken in

this thesis is that it provides novel information concerning the local production of NAEs in

human pain states. Samples from the 20k Da catheter were used in all subjects for analysing

NAEs. The use of this small catheter increases the likelihood that potential NAE degradative

enzymes are not sampled together with the compounds of interest. In contrast, approximately

300 proteins can be detected in samples from 100 kDa catheter (Olausson et al., 2012). The low

force exercise protocol used is painful for the CNSP and CWP and provides a good experimental

model for investigating chronic pain states, since it mimics movements in everyday life which

66

causes pain and discomfort for these groups of patients. However the exercise was not painful for

CON. For further investigation of mobilization of PEA and SEA, a different protocol for eliciting

pain, i.e injection of hypertonic saline into muscle, could be tested in CON.

In initial pilot studies AEA and 2-AG could not be detected in the microdialysate samples. In

most tissues AEA is present at two to three orders of magnitude lower concentrations than PEA.

Optimizing the perfusion fluid by adding albumin or cyclodextrines, which was not done in this

study, has been reported to enhance relative recovery of lipophilic molecules (Zoerner et al.,

2012). Whether or not such procedures would allow for measurement of endocannabinoids in

muscle microdialysate fluid awaits elucidation.

Future aspects

The nature of the thesis project means that it has raised more questions than it has answered.

Given the development of MAGL inhibitors, for example, it would be of interest to

investigate the levels of endocannabinoids and NAE in muscle by microdialysis technique in

animals given MAGL inhibitors alone or in different combination with FAAH-. NAAA- and

COX inhibitors. Other potential projects for future study include:

Optimization of microdialysis protocol and MS/MS for sampling and analysing AEA

and 2-AG in human muscle tissue.

Investigation as to whether different protocols for induction of muscle pain, other than

low-force exercise, mobilize endocannabinoid and NAE levels in muscle.

Investigation of the levels of PEA and SEA in CWP before and after conducting a

training intervention.

Investigation of mRNA levels of endocannabinoid and NAE synthesizing enzymes in

human muscle tissue in CNSP, CWP and CON.

67

Investigation into the expression of CB1, CB2 and PPAR-α receptors in human muscle

biopsies from CNSP, CWP and CON (see Fig. 5 below for data from a pilot study

undertaken by us investigating the expression of CB1 receptors in trapezius muscle).

Hopefully, studies of this type will further delineate the role of NAEs and the

endocannabinoid system in the pathogenesis of CNSP and CWP.

Fig. 5. Results from pilot study showing immunohistochemical staining for CB1 receptor expression in human

myalgic trapezius muscle biopsy (N. Ghafouri, unpublished data).

68

Acknowledgements

Background: The mechanisms behind what makes you to begin with research and complete a

thesis are to date not fully understood. Writing a thesis is at times associated with some pain,

repetitive computer work, and poor social activity. Both internal and external factors are

believed to be important in order to get the work done. The aim of this study was to elucidate

the factors involved in completing this thesis

Method: A retrospective study was conducted by me (n=1), looking at factors/persons who

have helped me during my work with thesis.

Results and discussion: Many persons who all have helped me through the years were

identified and characterized (but not fragmented by MS/MS).

First of all, my supervisor Christopher Fowler. Thank you for all your support and

understanding through the years. Without your great knowledge in the endocannabinoid field

and your ability to always answer any questions almost immediately, I really could not have

done this.

My co-supervisor Britt Larsson. Thank you for introducing me to the incredible field of

microdialysis. For our discussions about a scientific approach and your guidance. Your

support and advice gave me the self-confidence to take on any challenges that came up during

the different projects.

My co-supervisor Bijar Ghafouri. There are no words to describe how thankful I am for all

your endless support inside and outside PAINOMICS Lab. Truly your knowledge in

developing methods and analysing as well as the ability of always being there as a supervisor

is amazing. You give me so much inspiration. Thank you for all your wonderful “qozarqoots”

and “var så qots“.

Thank you Professor Björn Gerdle, the head of the division of Pain and Rehabilitation centre

in Linköping, for introducing me to the field of clinical research. For being sympathic, giving

me trust and freedom to explore the field of muscle pain. I now understand what being a

“laserstråle” means.

Thank you Britt Jacobsson for your warm welcome when I first started at the pharmacology

department and for your skilful laboratory work. Thank you Eva-Britt Lind for your great help

69

and patience during the microdialysis studies. Surely the sound of the monometer will echo in

our head for a long time.

Thank you Linn Karlsson, Anna Frumerie, Frida Forsberg and Natalie Baldew for your help

in recruitment and clinical examination of subjects. Thank you Patrik Olausson, Niclas

Stensson, the rest of the “gang” at the PAINOMICS lab and the neighbouring lab, especially

Helen Karlsson. I really enjoyed our “calibrating” evenings and the on-going fights between

basic researchers and clinicians.

Also thank you to the rest of the staff at the pharmacology department in Umeå and Pain and

Rehabilitation centre for your support and encouragements. Many thanks to Marie Lindgren at

Rehabilitation medicine in Linköping and the rest of the “lunch gang” for letting me take time

off for research and for accepting me as a teadrinker.

To my father who I know would have been proud and with his incredible sense of humour

would have said “it does not matter how many dr titles you aim at. You will always be my

little girl”. You recognised the power within knowledge and education. This is my way of

continuing what you started so many years ago, with our beloved Koya in my heart. To my

mother who taught me to always believe in myself and strive to be better but that my value as

a human being is not measured by my achievements.

Thank you Ya Nemam for making me interested in research many years ago. You have

always been there for me with all your heart. You show me that everything is possible.

Thank you to all my siblings who have been there for me. Together we have lived in different

places in the world and in different circumstances. But no matter if it was fleeing from real

bombs or having fights with boshqapparanda and watermelons, you have been my home. You

have taught me the importance of finding the funny side of things during rougher times.

Thank you to my helperke girls Sara, Soma and Kurde. I don’t know what I would do without

all the laughter you bring in to my life. Eat you with bread, tara piwaz and yoghurt drink

forever.

Last but certainly not least, thank you yarakam.

Conclusion: Clearly the results show that I could not have done this work without the

encouragement and support I got from all of you. No further studies are needed to show that.

“For all that has been - Thanks. For all that shall be - Yes” (Dag Hammarskjöld).

70

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