The negative health effects of THC(Tetrahydrocannabinol) on memory function of human brain

MINIREVIEW

Modulating the endocannabinoid system in human health and disease – successes and failures P�al Pacher and George Kunos

Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

Keywords

cannabinoids; clinical trials; disease;

endocannabinoid system; human;

pharmacology; therapeutic potential

Correspondence

P. Pacher, Laboratory of Physiological

Studies, National Institutes of Health/

NIAAA, 5625 Fishers Lane, MSC-9413,

Bethesda, MD 20892-9413, USA

Fax: +1 301 480 0257

Tel: +1 301 443 4830

E-mail: pacher@mail.nih.gov

(Received 14 January 2013, revised 11

March 2013, accepted 19 March 2013)

doi:10.1111/febs.12260

The discovery of the endocannabinoid system, comprising the G-protein

coupled cannabinoid 1 and 2 receptors (CB1/2), their endogenous lipid

ligands or endocannabinoids, and synthetic and metabolizing enzymes, has

triggered an avalanche of experimental studies implicating the endocanna-

binoid system in a growing number of physiological/pathological functions.

These studies have also suggested that modulating the activity of the en-

docannabinoid system holds therapeutic promise for a broad range of dis-

eases, including neurodegenerative, cardiovascular and inflammatory

disorders; obesity/metabolic syndrome; cachexia; chemotherapy-induced

nausea and vomiting; and tissue injury and pain, amongst others. However,

clinical trials with globally acting CB1 antagonists in obesity/metabolic syn-

drome, and other studies with peripherally-restricted CB1/2 agonists and

inhibitors of the endocannabinoid metabolizing enzyme in pain, have intro-

duced unexpected complexities, suggesting that a better understanding of

the pathophysiological role of the endocannabinoid system is required to

devise clinically successful treatment strategies.

Introduction

Although Cannabis sativa (the marijuana plant) is one

of the most ancient medicinal plants in the history of

medicine [1], the clinical use of synthetic cannabinoids

or medicinal plant extracts has been largely empirical

and limited to a few specific indications related to pain,

wasting disorders, and chemotherapy-induced nausea

and vomiting, as a result of their socially undesirable

psychoactive properties [2]. The discovery of endocann-

abinoids (ECs), which mimic some of the effects of syn-

thetic cannabinoids in vivo, their G-protein coupled

receptors, as well as their synthetic and metabolizing

enzymes, has prompted preclinical studies aiming to

explore the role of the endocannabinoid system (ECS)

in health and disease [2–4]. These studies have been greatly facilitated by the introduction of mice deficient

in cannabinoid receptors or EC degrading enzymes, as

well as selective cannabinoid receptor ligands and inhib-

itors of EC metabolism. The results of these studies

have implicated the ECS in a variety of physiopatholog-

ical processes, both in the peripheral and central ner-

vous systems and in various peripheral organs [2]. Such

studies have further suggested that modulating ECS

activity may have therapeutic potential in almost all dis-

eases affecting humans, including obesity/metabolic

syndrome [5]; diabetes and diabetic complications [6];

neurodegenerative [7,8], inflammatory [9], cardiovascu-

lar [10–12], liver [13,14], gastrointestinal [15] and skin [16] diseases; pain [17,18]; psychiatric disorders [19,20];

cachexia [2]; cancer [21,22]; and chemotherapy-induced

nausea and vomiting [23], amongst many others [2].

Abbreviations

2-AG, 2-arachidonoylglycerol; AEA, anandamide or arachidonoyl ethanolamide; CB1/2, cannabinoid receptor 1 or 2; CBD, cannabidiol; CNS,

central nervous system; EC, endocannabinoid; ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; MAGL, monoacylglycerol

lipase; MS, multiple sclerosis; THC, D9-tetrahydrocannabinol; TRPV1, transient receptor potential cation channel subfamily V member 1.

1918 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

These investigations have also uncovered the

remarkable complexity of the ECS, as exemplified by

differences in the therapeutic profile of activating/

inhibiting the same receptor in the central nervous

system (CNS) or in peripheral tissues, by the intrigu-

ing overlap between EC and eicosanoid signalling, or

by the often opposite effects mediated by cannabinoid

1 and 2 receptors (CB1/2) receptors in disease models

[2–4,6,24]. Similar complexities have emerged in clini- cal trials targeting the ECS. Although globally acting

(i.e. brain-penetrant) CB1 antagonists/inverse agonists

were shown to have therapeutic efficacy in obesity/

metabolic syndrome, they elicited anxiety/depression

in a small proportion of subjects, which has led to

their withdrawal from the market worldwide and

halted their further therapeutic development [5,25,26].

The first human trial with peripherally-restricted

mixed CB1/2 agonist(s) for pain failed as a result of

cardiovascular and metabolic side effects and hepato-

toxicity [27,28]. Amplifying the ECS tone by inhibit-

ing EC metabolism was ineffective in alleviating

osteoarthritic pain in human subjects [29,30]. Thus,

we need to better understand the pathophysiological

function of the ECS in humans, as well as refine the

indications and design of clinical trials, so that it is

possible to successfully translate recent progress in

cannabinoid biology into clinically effective treatment

strategies.

The present minireview discusses preclinical evidence

implicating the ECS in human disease, and reviews the

treatment strategies that target the ECS for therapeutic

gain in humans. Because of limitations of space, refer-

ence is also made to recent overviews on specific sub-

jects, rather than to original papers.

The ECS

D9-Tetrahydrocannabinol (THC), the putative psycho- active ingredient of marijuana, and its endogenous

counterparts, anandamide (arachidonoyl ethanola-

mide) (AEA) and 2-arachidonoylglycerol (2-AG), exert

their primary effects through CB1/2 receptors; 2-AG

favours CB2, whereas AEA binds with higher affinity

to CB1 [2], although, at higher concentrations, it may

also modulate transient receptor potential cation chan-

nel subfamily V member 1 (TRPV1) and other recep-

tors. Signalling by cannabinoid receptors is complex

because it may involve both G protein-dependent

pathways, such as inhibition of adenyl cyclase or the

modulation of ion channel function, and G protein-

independent mechanisms, including the activation of

various mitogen-activated protein kinases (p44/42

mitogen-activated protein kinases, p38, extracellular

signal-regulated kinase and c-Jun N-terminal kinase)

or ceramide signalling [2,31,32].

CB1 receptors, the most abundant G-protein cou-

pled receptor in the mammalian brain, mediate the

socially undesirable psychoactive effects of cannabis.

Although their expression was initially considered to

be restricted to the brain, more recent studies have

identified CB1 receptors in almost all peripheral tissues

and cell types, albeit at much lower densities than in

the brain, and documented their important regulatory

functions [2,3,5]. CB2 receptors are largely restricted to

immune and haematopoetic cells, although function-

ally relevant expression has been found in specific

regions of the brain and in the myocardium, gut,

endothelial, vascular smooth muscle and Kupffer cells,

exocrine and endocrine pancreas, bone, and reproduc-

tive organs/cells, as well as in various tumours [4].

Both cannabinoid receptors may undergo rapid inter-

nalization and intracellular trafficking upon agonist

exposure [33,34].

In the CNS, AEA and 2-AG are synthesized ‘on

demand’ and released to act as retrograde transmit-

ters on CB1 receptors [35–37]. They are not stored and are rapidly degraded after exerting a transient and local-

ized effect [38]. The synthesis of ECs largely depends on

the intracellular Ca2+-concentration. AEA is mainly

formed via a two step-pathway, involving a Ca2+-

dependent N-acyltranferase and N-acylphosphatidy-

lethanolamine-hydrolyzing phospholipase D, whereas

diacylglycerol lipase and phospholipase Cb are mainly responsible for the biosynthesis of 2-AG [3,37]. The

existence of additional, parallel biosynthetic pathways

for AEA has also been proposed [39,40].

AEA and 2-AG are removed from the extracellular

space by a process of cellular uptake and metabolism;

however, the putative transporter(s) involved have not

yet been cloned, and are the subject of much contro-

versy [41–43]. AEA is degraded primarily by fatty acid amide hydrolase (FAAH) and 2-AG is degraded by

monoacylglycerol lipase (MAGL) [3,44], although

additional enzymes have also been implicated in the

degradation of both AEA and 2-AG [45,46]. Endoc-

annabinoids may also be metabolized by cyclooxygen-

ases, lipooxygenases and cytochrome P450, leading to

the formation of bioactive metabolites that may acti-

vate CB receptor-independent mechanisms [24,47]. It is

also important to note that FAAH and MAGL are

also responsible for the degradation of numerous

potentially bioactive lipids. Thus, the biological conse-

quences of the inhibition of these enzymes are not nec-

essarily a result of enhanced EC levels. Some of the

enzymes involved in EC synthesis/degradation may

exist in several forms and their activity may vary in

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1919

P. Pacher and G. Kunos Targeting the endocannabinoid system

different tissues or even in different regions of the

same tissue [3,37,48–52]. In addition to AEA and 2-AG, several other EC-like

molecules have been discovered, although their activi-

ties have not been studied in sufficient detail [53,54].

Interestingly, recent studies have identified novel pep-

tide allosteric negative modulators of CB1 receptors

[55], the biological significance of which is yet to be

determined. Additionally, the anti-inflammatory lipid

lipoxin A4 may be an endogenous allosteric enhancer

of CB1 receptors [56]. A comprehensive overview of

the ECS is beyond the scope of the present minireview;

instead, several detailed reviews are available on this

subject [3,24,37,57].

The ECS in health and disease

Despite the ubiquitous expression of the various com-

ponents of the ECS, their genetic ablation or pharma-

cological blockade in normal, healthy animals has

minimal functional consequences, which suggests that

the ECS has minimal or no tonic activity under normal

physiological conditions [2,4]. On the other hand, an

increase or decrease in ECS tone is associated with var-

ious pathological states, as a result of the altered

expression of CB receptors, endocannabinoid metabo-

lizing enzymes and/or synthetic pathways, in a tissue-

specific and time-dependent manner. Examples of

selected pathologies in which dysregulation of the ECS

was reported (in most cases, up-regulation of CB1/2 and/or an increase in tissue levels of ECs) are shown in

Table 1, and have been summarized in more detail else-

where [2–4,58,59]. In some cases, altered ECS activity is transient and forms part of the body’s compensatory

response to a particular insult, thus reducing symptoms

and/or slowing progression of the disease (e.g. in neu-

ropathic pain); in other cases, activation of the ECS

may be pathogenic (e.g. in various forms of shock or

diabetic complications) or may reflect a deficiency (e.g.

in various tumours) of unknown significance [2].

From a therapeutic standpoint, the identification of

regional or tissue-specific changes in CB receptors is

important because their possible selective targeting

may mitigate unwanted side effects [59,60]. However,

these changes can serve as a basis for successful drug

development only as long as they are determined using

appropriate tools (e.g. specific antibodies), the specific-

ity of which needs to be carefully validated [4,61]. It is

also very important to understand the underlying

mechanisms of these alterations; for example, is the

increase in the tissue level of an EC the result of its

increased biosynthesis or a decrease in its enzymatic

degradation?

Cardiovascular consequences of targeting the ECS in health and disease

Because many promising drugs fail in clinical develop-

ment as a result of cardiovascular side effects, it is

important to briefly overview the cardiovascular conse-

quences of modulating the ECS. ECs exert complex

cardiovascular effects that are dominated by a decrease

in blood pressure and myocardial contractility, medi-

ated primarily by CB1 receptors located in the myocar-

dium, vasculature and neurones in the central and

autonomic nervous systems [2,62]. In cultured human

coronary artery endothelial cells [63] and cardiomyo-

cytes [64], CB1 activation promotes stress signalling

and cell death, and decreases contractility [10,12]. By

contrast, activation of cardiovascular CB2 receptors

does not have adverse haemodynamic consequences

[11]. CB1, CB2 or FAAH knockout mice have normal

blood pressure, myocardial contractility and/or barore-

flex sensitivity, indicating the minimal role of the ECS

in normal cardiovascular regulation [2]. However, in

several pathological conditions (e.g. shock, heart fail-

ure, cardiomyopathies, advanced liver cirrhosis), the

ECS may become activated to promote hypotension/

cardiodepression through cardiovascular CB1 receptors

[2,10]. CB1 receptor signalling may also promote dis-

ease progression in preclinical models of heart failure

[64–66] and atherosclerosis [67,68], and contributes to increased cardiovascular risk (e.g. plasma lipid altera-

tions, abdominal obesity, hepatic steatosis, insulin and

leptin resistance) in obesity/metabolic syndrome and

diabetes, both in rodents and humans [5,69–71]. By contrast, CB2 signalling in the heart and vasculature

may activate cardioprotective mechanisms and limit

inflammation [11].

Acute or chronic use of marijuana may decrease or

increase the heart rate and decrease blood pressure

depending on the duration of the use, dose and route

of administration [2,10]. An elevated resting heart rate

is a known independent risk factor for cardiovascular

disease in healthy men and women [72]. A recent con-

trolled study at the National Institute on Drug Abuse

evaluated the development of tolerance to the effects

of oral synthetic THC in 13 healthy male daily canna-

bis smokers who were residing on a secure research

unit over a period of 6 days [73]. Despite the develop-

ment of tolerance to the subjective intoxicating effect

of THC, no tolerance was observed to its hypotensive

and tachycardic effects [73]. Another recent study of

72 young male cannabis users and 72 matched controls

reported an increased heart rate variability in cannabis

users [74]. Surinabant, a selective CB1 antagonist, has

1920 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

T a b le

1 . E x a m p le s o f th e d y s re g u la ti o n o f th e E C S in

d is e a s e . C , c a n in e ; H , h u m a n ; P , p ig ; R , ro d e n t. N D , n o t d e te rm

in e d .

D is e a s e , s a m p le

E x p re s s io n /c h a n g e s in

C B 1 /2

C h a n g e s in

e n d o c a n n a b in o id

le v e ls

P ro p o s e d ro le

o f C B re c e p to rs

in d is e a s e

R e fe re n c e

M y o c a rd ia l in fa rc ti o n

(i s c h a e m ia -r e p e rf u s io n

in ju ry ) (R , P , H )

M y o c a rd iu m , in

h u m a n e p ic a rd ia l

a d ip o s e ti s s u e s o f is c h a e m ic

h e a rt s ,

u p -r e g u la ti o n o f C B 1 a n d p ro te in

k in a s e A , a c c o m p a n ie d b y C B 2 a n d

F A A H

d o w n -r e g u la ti o n , in c re a s e d

in d u c ib le

N O S /e n d o th e lia l N O S ra ti o n

a n d re d u c e d c e ll s u rv iv a l s ig n a lli n g

In c re a s e in

c ir c u la ti n g im

m u n e

c e lls

o r in

s e ru m

o f o b e s e

p a ti e n ts

w it h a d v e rs e

c a rd io v a s c u la r e v e n ts ; E le v a te d

e n d o c a n n a b in o id

p la s m a le v e ls

a re

s tr o n g ly

a s s o c ia te d w it h

c o ro n a ry

d y s fu n c ti o n in

o b e s e

h u m a n s u b je c ts

C B 2 : d e c re a s e in

le u k o c y te

in fi lt ra ti o n a n d

e n h a n c e m e n t o f p ro -s u rv iv a l p a th w a y s ;

C B 1 : c o n tr ib u ti o n to

c a rd io v a s c u la r d y s fu n c ti o n ,

c e ll d e a th /d y s fu n c ti o n in

h u m a n e n d o th e lia l c e lls

a n d c a rd io m y o c y te s ; c e n tr a l h y p o th e rm

ia (t h e

la tt e r is

o n ly

in ro d e n ts

a n d c a n b e p ro te c ti v e )

1 1 ,1 2 ,7 6 ,

8 5 –8

7 ,9 0 ,

1 8 4 –1

8 7

H e a rt

fa ilu re ,

c a rd io m y o p a th ie s

(R , H )

M y o c a rd iu m , c a rd io m y o c y te s ,

e n d o th e lia l c e lls

M y o c a rd iu m , c a rd io m y o c y te s ,

c ir c u la ti n g im

m u n e c e lls

a n d

p la te le ts

C B 2 : a tt e n u a ti o n o f in fl a m m a ti o n /i n ju ry ;

C B 1 : p ro m o ti o n o f c a rd ia c d y s fu n c ti o n a n d

c e ll d e a th

in c a rd io m y o c y te s a n d e n d o th e lia l c e lls

6 4 ,6 5 ,1 8 6 ,

1 8 8 –1

9 2

A th e ro s c le ro s is ,

re s te n o s is

(R , H )

In fi lt ra ti n g a n d o th e r im

m u n e c e lls ,

v a s c u la r s m o o th

m u s c le

a n d

e n d o th e liu m

S e ru m , a th e ro s c le ro ti c p la q u e s

C B 2 : c o n te x t- d e p e n d e n t a tt e n u a ti o n o r p ro m o ti o n

o f v a s c u la r in fl a m m a ti o n (m

o n o c y te

c h e m o ta x is ,

in fi lt ra ti o n a n d a c ti v a ti o n ) a n d fa c to rs

o f p la q u e

s ta b ili ty ; a tt e n u a ti o n o f v a s c u la r s m o o th

m u s c le

p ro lif e ra ti o n ;

C B 1 : in c re a s e o f v a s c u la r in fl a m m a ti o n a n d /o r p la q u e

v u ln e ra b ili ty

6 7 ,8 4 ,1 3 3 ,1 3 4 ,

1 9 3 –1

9 8

S tr o k e , s p in a l c o rd

in ju ry

(R , H )

B ra in , m ic ro g lia , in fi lt ra ti n g im

m u n e

c e lls , e n d o th e liu m

S e ru m , b ra in

C B 2 : a tt e n u a ti o n o f in fl a m m a ti o n (e n d o th e lia l

a c ti v a ti o n , le u k o c y te

in fi lt ra ti o n ), a n d ti s s u e

in ju ry , a tt e n u a ti o n o f m o to r a n d a u to n o m ic

d e fi c it s in

a m o u s e m o d e l o f s p in a l c o rd

in ju ry ;

C B 1 : p ro m o te s h y p o th e rm

ia -d e p e n d e n t

p ro te c ti o n b u t, if h y p o th e rm

ia is

c o m p e n s a te d ,

in e ff e c ti v e o r e n h a n c e s in ju ry

9 0 ,1 9 9 –2

0 6

C ir rh o ti c c a rd io m y o p a th y

(R , H )

N D

M y o c a rd iu m , c ir c u la ti n g im

m u n e

c e lls

a n d p la te le ts

C B 2 : a tt e n u a ti o n o f h y p o te n s io n b y d e c re a s in g

liv e r in fl a m m a ti o n ;

C B 1 : c o n tr ib u ti o n to

c a rd io v a s c u la r d y s fu n c ti o n

1 8 9 –1

9 2

S e p ti c s h o c k b y liv e

b a c te ri a (R , H )

N D

S e ru m

C B 2 : d e c re a s e o r in c re a s e in

in fl a m m a ti o n a n d

ti s s u e in ju ry

m o s t lik e ly

b y a ff e c ti n g b a c te ri a l lo a d ;

C B 1 : c o n tr ib u ti o n to

c a rd io v a s c u la r c o lla p s e

1 0 ,2 0 7 –2

1 0

H e p a ti c is c h a e m ia -

re p e rf u s io n in ju ry

(R , P , H )

In fl a m m a to ry

im m u n e c e lls , a c ti v a te d

e n d o th e liu m

L iv e r, s e ru m , h e p a to c y te s , K u p ff e r

a n d e n d o th e lia l c e lls

C B 2 : a tt e n u a ti o n o f in fl a m m a ti o n (e n d o th e lia l

a c ti v a ti o n , le u k o c y te

c h e m o ta x is , in fi lt ra ti o n a n d

a c ti v a ti o n ), o x id a ti v e s tr e s s , a n d ti s s u e in ju ry ;

C B 1 : p ro m o ti o n o f liv e r in ju ry

1 3 5 ,1 3 8 ,2 1 1 –2

1 3

O b e s it y , n o n -a lc o h o lic

fa tt y

liv e r d is e a s e , d ia b e ti c

c o m p lic a ti o n s (R , H )

H e p a to c y te s , in fl a m m a to ry

c e lls ,

a d ip o c y te s , c e rt a in

n e u ro n e s , s it e s o f

d ia b e ti c c o m p lic a ti o n s (k id n e y s ,

re ti n a a n d m y o c a rd iu m )

L iv e r, a d ip o s e ti s s u e , b ra in , s k e le ta l

m u s c le , d ia b e ti c k id n e y s , h e a rt s ,

re ti n a s , s e ru m

C B 2 : e n h a n c e m e n t o f h ig h fa t d ie t- in d u c e d

s te a to s is

a n d in fl a m m a ti o n o r a tt e n u a ti o n o f

o b e s it y a s s o c ia te d o n e w it h a g e ;

C B 1 : in c re a s e in

fa t s to ra g e , d e c re a s e in

m e ta b o lis m ,

p ro m o ti o n o f in s u lin

a n d le p ti n re s is ta n c e a n d

in fl a m m a ti o n in

a d ip o s e ti s s u e a n d in

th e liv e r

5 ,6 ,7 0 ,1 0 1 ,1 0 8 ,

2 1 4 –2

2 1

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1921

P. Pacher and G. Kunos Targeting the endocannabinoid system

T a b le

1 . (C o n ti n u e d ).

D is e a s e , s a m p le

E x p re s s io n /c h a n g e s in

C B 1 /2

C h a n g e s in

e n d o c a n n a b in o id

le v e ls

P ro p o s e d ro le

o f C B

re c e p to rs

in d is e a s e

R e fe re n c e

L iv e r fi b ro s is , c ir rh o s is ,

a lc o h o l- in d u c e d liv e r in ju ry

(R , H )

A c ti v a te d s te lla te

c e lls , in fl a m m a to ry

c e lls , h e p a to c y te s , K u p ff e r c e lls

L iv e r, s e ru m , in fl a m m a to ry

c e lls

C B 2 : a tt e n u a ti o n o f fi b ro s is

a n d in ju ry /i n fl a m m a ti o n ;

C B 1 : in c re a s e in

fi b ro s is /i n ju ry

1 4 ,1 3 6 ,1 3 7 ,1 9 1 ,

2 2 2 ,2 2 3

P a n c re a ti ti s (R , H )

P a n c re a s

In fl a m e d p a n c re a s

C B 2 : a tt e n u a ti o n o f in fl a m m a ti o n ;

C B 1 : c o n te x t- d e p e n d e n t e ff e c t

1 4 5 ,1 4 6 ,1 4 8 ,2 2 4

In fl a m m a to ry

b o w e l

d is e a s e , c o lit is ,

d iv e rt ic u lit is

(R , H )

E p it h e lia l c e lls , in fi lt ra ti n g in fl a m m a to ry

c e lls , e n te ri c n e rv e s

In fl a m e d g u t

A tt e n u a ti o n o f in fl a m m a ti o n a n d v is c e ra l s e n s it iv it y

1 3 0 ,1 5 1 ,2 2 5 –2

2 9

N e p h ro p a th y (R , H )

K id n e y , h u m a n p ro x im

a l tu b u la r c e lls ,

p o d o c y te s

K id n e y

C B 2 : a tt e n u a ti o n o f in fl a m m a ti o n (c h e m o k in e

s ig n a lli n g a n d c h e m o ta x is , in fl a m m a to ry

c e ll

in fi lt ra ti o n a n d e n d o th e lia l a c ti v a ti o n ) a n d

o x id a ti v e s tr e s s ;

C B 1 : p ro m o ti o n o f in fl a m m a ti o n /i n ju ry

1 0 5 ,2 1 9 ,2 2 0 ,

2 3 0 –2

3 3

N e u ro d e g e n e ra ti v e /

n e u ro in fl a m m a to ry

d is o rd e rs

(m u lt ip le

s c le ro s is , A lz h e im

e r’ s ,

P a rk in s o n ’s

a n d

H u n ti n g to n ’s

d is e a s e ,

s p in a l c o rd

in ju ry ) (R , H )

M ic ro g lia , in fl a m m a to ry

c e lls , b ra in

le s io n s , n e u ro n e s ?

B ra in , s p in a l fl u id

C B 2 : a tt e n u a ti o n o f in fl a m m a ti o n (m

ic ro g lia

a c ti v a ti o n , s e c o n d a ry

im m u n e c e ll in fi lt ra ti o n ),

fa c ili ta ti o n o f n e u ro g e n e s is ;

C B 1 : a tt e n u a ti o n o f e x c it o to x ic it y , h y p o th e rm

ia ;

c o n te x t- d e p e n d e n t e ff e c t o n in ju ry /i n fl a m m a ti o n

2 ,7 ,9 1 ,9 2 ,1 5 2 ,

2 0 5 ,2 3 4 –2

5 0

P a in

(R )

In fl a m m a to ry

c e lls , c e rt a in

n e u ro n e s

S it e o f in d u c e d c h ro n ic

in fl a m m a to ry

p a in

C B 2 : a tt e n u a ti o n o f in fl a m m a to ry

p a in

v ia

u n k n o w n m e c h a n is m (s );

C B 1 : a tt e n u a ti o n o f v a ri o u s fo rm

s o f p a in

b y

in h ib it in g n e u ro tr a n s m is s io n

1 7 ,9 5 ,9 6 ,2 5 1 –2

6 6

P s y c h ia tr ic

d is o rd e rs

(a n x ie ty

a n d d e p re s s io n ,

s c h iz o p h re n ia ) (R , H )

G lia l, in fl a m m a to ry

c e lls , n e u ro n e s ?

B lo o d , c e re b ro s p in a l fl u id , b ra in

(i n c re a s e d in

s c h iz o p h re n ia , b u t

d e c re a s e d in

b ra in

in d e p re s s io n )

C B 2 : la rg e ly

u n e x p lo re d , in

ro d e n t m o d e ls

o f

d e p re s s io n /a n x ie ty , it m a y m o d u la te

C N S

in fl a m m a ti o n a n d e it h e r a tt e n u a te

o r p ro m o te

a n x ie ty

lik e b e h a v io u r;

C B 1 : c o n te x t- d e p e n d e n t e ff e c t o n a n x ie ty ,

im p ro v e d s le e p

1 9 ,2 6 7 –2

7 7

R h e u m a to id

a rt h ri ti s (H )

N D

S y n o v ia l fl u id , s y n o v ia

C B 2 : a tt e n u a ti o n o f th e a u to im

m u n e

in fl a m m a to ry

re s p o n s e ;

C B 1 : a tt e n u a ti o n o f p a in

2 7 8

C a n c e r (R , H )

In v a ri o u s tu m o u rs

o r c a n c e r c e lls

V a ri o u s tu m o u rs

C B 1 /2 : c o n te x t- d e p e n d e n t a tt e n u a ti o n o r

p ro m o ti o n o f tu m o u r g ro w th

(a p o p to s is ,

a n g io g e n e s is , p ro lif e ra ti o n , e tc .)

2 7 9 –2

8 2 , 2 ,2 2 ,1 4 9 ,

1 5 5 ,1 5 7

1922 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

recently been reported to inhibit THC-induced central

nervous system and heart rate effects in humans, pro-

viding proof of principle that those effects were indeed

mediated by CB1 receptor activation [75]. At the 20th

International Cannabinoid Research Society meeting

in Sweden, AstraZeneca presented data from the first

clinical studies investigating two novel, peripherally-

restricted, orally active mixed CB1/2 agonists

(AZD1940 and AZD1704). The study was terminated

as a result of adverse cardiovascular effects, weight

gain and mild hepatotoxicity [27,28].

An increasing number of case reports associates mar-

ijuana smoking with the precipitation of acute coronary

syndrome [76]. Alarmingly, this occurs mostly in young

healthy subjects without any previous cardiovascular

disease [77,78]. A retrospective study assessed the risk

of acute coronary syndrome after exposure to mari-

juana smoke. It was found that the risk of myocardial

infarction was highest during the first hour of exposure

[79]. The effect of marijuana use on mortality after

acute myocardial infarction was assessed in a prospec-

tive study involving 1913 adults who were hospitalized

with myocardial infarction at 45 US hospitals between

1989 and 1994, with a median follow-up of 3.8 years.

The results indicated that marijuana use may pose an

increased risk of infarction in susceptible individuals

with coronary heart disease [80]. A more recent study

evaluated the consequences of marijuana use and long-

term mortality among survivors of acute myocardial

infarction, and found that habitual marijuana use

among patients presenting with acute myocardial

infarction was associated with an apparent increase in

mortality rate (29% higher) over the subsequent

18 years, although this did not reach statistical signifi-

cance because of the limited sample size [81]. In the

absence of large-scale, long-term controlled studies with

repeated measures of marijuana use, a firm conclusion

on the long-term impact of cannabis use on cardiovas-

cular mortality cannot be drawn. Nevertheless, the

above findings are of concern. Because THC is a rela-

tively weak CB1 agonist compared to many synthetic

ligands, and also activates cardioprotective CB2 recep-

tors and is a potent antioxidant, it may be predicted

that the uncontrolled spread and use of mixtures of

potent synthetic CB1 agonists (spice, K2, etc.)

employed as recreational drugs would lead to signifi-

cantly greater cardiovascular morbidity. Indeed, in a

recent case series in healthy children, myocardial infarc-

tion was precipitated by synthetic cannabinoid use [82],

and another study reported tachycardia, loss of con-

sciousness and diffuse pain in two adolescents [83].

What is the situation regarding the ECS and cardio-

vascular pathology? As noted previously, EC/CB1

receptor signalling has been implicated as a patho-

genic factor in rodent models of cardiovascular dis-

eases, including atherosclerosis, shock and various

forms of cardiomyopathy. However, ECs were also

reported to exert protective effects, based mostly on

ex vivo and indirect studies, via CB2 and CB-receptor

independent mechanisms. Clearly, selective CB2 agon-

ists exert beneficial effects in rodent models of myo-

cardial infarction by limiting inflammatory cell

infiltration (in cardiomyocytes, the expression of CB2 is very low, if any) [11]. To analyze the role of the

ECS more directly, a recent study employed FAAH

knockout mice with a 2.5- to three-fold increase in

myocardial AEA content. When such mice were used

to induce various experimental models of cardiomyop-

athy, they displayed increased mortality, tissue injury

and neutrophil infiltration in the heart, which could

be partially rescued by CB1 antagonists [66]. Consis-

tent with this report, a recent study showed that

FAAH deficiency enhanced intraplaque neutrophil

recruitment in atherosclerotic mice and increased a

pro-inflammatory immune response [84]. These find-

ings indicate that the primary cardiovascular effects of

elevated EC tone are deletorious and are mediated by

CB1 receptors.

In obese human subjects, increased plasma levels of

AEA and 2-AG were strongly associated with coro-

nary circulatory dysfunction, suggesting that plasma

EC levels may be used as biomarkers of cardiovascular

risk in obesity [85]. In another study, increased plasma

AEA and 2-AG levels positively correlated with

impaired coronary endothelial function in obese sub-

jects [86]. In samples of epicardial fat from ischaemic

human hearts, the up-regulation of CB1 was accompa-

nied by down-regulation of CB2 and FAAH compared

to non-ischaemic hearts [87]. CB1 receptor density was

significantly higher in atherosclerotic coronary artery

sections from patients with unstable angina compared

to those with stable angina [67]. A G1359A polymor-

phism in the CB1 receptor gene was also associated

with coronary artery disease in the Chinese Han popu-

lation, although the effect of this polymorphism on

receptor function is unknown [88]. Both ECs were

reported to inhibit human cardiac Kv4.3 channels at

fairly low concentrations in ovary cells expressing

Kv4.3 or in human cardiomyocytes in a receptor-inde-

pendent manner [89], a harbinger of pro-arrhythmic

risk.

Thus, it is clear that the activation of CB1 receptors

by synthetic ligands or ECs is associated with adverse

cardiovascular consequences, which must be given very

careful consideration during the preclinical/clinical

development of new drugs targeting the ECS.

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1923

P. Pacher and G. Kunos Targeting the endocannabinoid system

Activation of CB1/2 receptors: THC, synthetic agonists and cannabinoid extracts

THC (dronabinol; Marinol; Solvay Pharmaceuticals,

Brussels, Belgium) and its synthetic analogue nabilone

(Cesamet; Valeant Pharmaceuticals, Irvine, CA, USA)

have been approved by the Food and Drug Adminis-

tration for treatment of chemotherapy-induced nausea

and vomiting and for stimulating appetite in wasting

disorders (e.g. AIDS, tumour cachexia, etc). Sativex

(GW Pharmaceuticals, Salisbury, Wiltshire, UK), an

oromucosal spray containing THC and the nonpsycho-

active plant cannabinoid, cannabidiol (CBD), has

recently been approved in Canada, the UK and several

other European countries for the symptomatic relief of

neuropathic pain and spasticity associated with multi-

ple sclerosis, and as an adjunctive analgesic treatment

for adults with advanced cancer. However, the thera-

peutic utility of THC and its synthetic analogues are

limited because of their unwanted psychotropic effects

mediated by central CB1 receptors. The present minire-

view summarizes only the clinically most relevant

indications.

Earlier preclinical studies suggested that ECs or

plant-derived cannabinoids exert neuroprotective

effects in the CNS by: (a) modulating excitability and

calcium homeostasis via effects on various ion chan-

nels (Ca2+, Na+, K+), intracellular Ca2+ stores and

gap junctions and N-methyl-D-aspartate receptors; (b)

attenuating excitatory glutamatergic transmissions

and modulating synaptic plasticity via presynaptic

CB1 receptors; (c) inducing CB1 receptor-mediated

hypothermia; (d) exerting antioxidant effects; and (e)

modulating immune responses and the release of pro-

inflammatory mediators by CB1, CB2 and non-CB1/

CB2 receptors on microglia, astrocytes, macrophages,

neutrophils, lymphocytes and neurones [2]. Numerous

recent studies have suggested that many of the previ-

ously described protective effects of synthetic CB1 ligands were attributable to centrally-mediated hypo-

thermia and/or receptor-independent antioxidant/

anti-inflammatory effects of the compounds, and that

ECs through the activation of CB1 receptors may also

promote tissue injury and neurodegeneration (e.g. in

stroke and other forms of I/R injury) [6,90–92]. Historical documents reveal that one of the earliest

uses of cannabis was to treat pain [93]. Studies in mod-

ern times initially focused on CB1 receptors and dem-

onstrated beneficial effects of cannabinoids in rodent

models of acute and chronic pain. The results sug-

gested that the observed antinociceptive effects have

complex mechanisms involving actions in the CNS,

spinal cord and peripheral sensory nerves [2,94].

Recent evidence also implicates CB2 receptors in the

antihyperalgesic activity of cannabinoids [95,96]; how-

ever, the exact mechanisms and cellular targets are elu-

sive because of a lack of reliable antibodies for CB2 [4].

In humans, the analgesic activity of THC and other

cannabinoids is less clear-cut because cannabinoids are

relatively weak analgesics compared to opiates, even

when they do show efficacy [2]. The clinical data on

THC, CBD and their combinations have been compre-

hensively reviewed elsewhere [97,98]. The primary

focus of these studies has been the safety/efficacy and

symptom relief (e.g. bladder incontinence, limb spastic-

ity, pain and sleep quality) in multiple sclerosis (MS)

or other pain-related conditions. Three studies have

demonstrated that cannabis extract in MS patients

improved urinary incontinence [98]. A number of con-

trolled and blinded trials evaluating the efficacy of oral

or sublingual cannabis/Sativex on spasticity in MS

found that, at doses lacking overt psychoactivity, these

drugs show no or minimal efficacy, as assessed by the

objective outcomes using the Ashworth scale. How-

ever, the treatment consistently improved subjective,

patient-assessed endpoints (spasms, pain, spasticity,

sleep quality). Follow-up studies using a patient

assessed numeric rating scale for spasticity showed sig-

nificant benefits of Sativex compared to placebo [98].

It could be argued that some of the benefits observed

were a result of mood improvement (patients feel sub-

jective improvement) but, because only some of the

symptoms were improved (spasticity, pain and sleep

quality), this may not be the case. In patients treated

with THC for 1 year, improvements using the Ash-

worth scale were reported [98]. Zhornitsky and Potvin

[97] performed a meta-analyses of the data from 33

studies with CBD alone or in various combinations

with THC, with the rationale for combining THC and

CBD being to attenuate the psychoactive effects of

THC by CBD, based on empirical evidence obtained

in some studies. Among these studies, 16 had been

conducted in healthy subjects and 17 in clinical popu-

lations, including four in MS, three in neuropathic and

cancer pain, four in schizophrenia and bipolar mania,

two in social anxiety disorder, and one each in cancer-

related anorexia, Huntington’s disease, insomnia and

epilepsy [97]. It was concluded that, depending on the

study and on the THC/CBD ratio, CBD may prolong/

intensify or inhibit THC-induced effects. In some of

these studies, THC or CBD+THC was more effective at reducing pain, although, in other studies, CBD

alone also exerted (or completely lacked) analgesic

properties. Notably, several of these studies used

1924 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

multiple pain assessment scores, and the treatments

were effective when evaluated by some but not by

other scales [97]. In one of the studies in which the

oral administration of CBD+THC in MS was not effective in improving symptoms, immunological anal-

ysis unexpectedly revealed a certain pro-inflammatory

effect of the drug [97]. The preliminary clinical evi-

dence was concluded to suggest that high-dose oral

CBD may have therapeutic benefits in social anxiety

disorder, insomnia and epilepsy, although it may also

cause mental sedation [97].

Taken together, the above studies in MS show con-

sistent improvements in subjective rather than quanti-

tative symptomatic outcome measures (including pain),

which supports the beneficial effects of cannabinoid-

based medicines in neuropathic pain associated with

MS. The relatively poor efficacy observed in some clini-

cal studies may be attributable to pharmacokinetic

problems such as first-pass effects via the liver and slow

absorption via the oral route of administration, which

may also limit the success of self-titration [98]. In most

of these studies, formulations containing THC fre-

quently caused generally mild to moderate side effects.

However, with individual dose-titration, which can be

better achieved by using the oromucosal Sativex spray,

side effects can be further attenuated. Initial dose-titra-

tion may also help in the early selection of responders

and exclusion of nonresponders. Future clinical studies

should explore how cannabinoid-based medicines affect

MS progression. In light of the preclinical data, the

combination of THC with CBD appears to be the most

promising, given the neuroprotective effects of CBD

observed in numerous preclinical studies [99].

There is considerable interest in developing THC-

based medicines for other forms of pain, such as pain

associated with cancer or diabetic neuropathy. How-

ever, under these conditions, we should also carefully

weigh the potential effect of the treatment on cancer

and/or diabetes progression. Regarding cancer,

although numerous studies suggest that THC may

slow down the growth/progression of certain types of

cancers in preclinical models, others suggest that THC

may in fact promote cancer growth, and cannabinoid

receptor deletion or inhibition is beneficial [2,4,22]. In

addition, the results of a clinical study evaluating the

association between ECS activity and survival and

pain in pancreatic cancer indicate that, although

patients with high CB1 receptor expression in enlarged

nerves in pancreatic ductal adenocarcinoma had a

lower combined pain score (intensity, frequency, dura-

tion), they had significantly shorter survival [100]. For

CBD, the evidence more clearly suggests potential ben-

efits in multiple preclinical tumour models [99]. In the

case of diabetes and diabetic complications, there is

strong evidence (both preclinical and clinical) indicat-

ing that CB1 activation promotes primary diabetes and

also contributes to all diabetic complications (includ-

ing neuropathy), and that CB1 antagonists can prevent

or reverse these changes, as well as insulin resistance

[6,69,101].

Interestingly, analysis of cross-sectional data from

the National Health and Nutrition Examination Sur-

vey (NHANES III, 1988–1994) indicated that mari- juana use was independently associated with a lower

prevalence of diabetes mellitus [102], and glucose toler-

ance and insulin sensitivity were found to be unchaged

in chronic marijuana smokers [103]. In view of the

demonstrated ability of acute marijuana smoking to

induce insulin resistance [104], these findings may

reflect desensitization of peripheral CB1 receptors in

chronic users. Further clinical studies are needed to

analyze the differential mechanisms involved in the

acute and chronic effects of marijuana use on glycae-

mic control.

Nevertheless, in light of the overwhelming preclinical

and clinical evidence suggesting that CB1 receptor acti-

vation contributes to diabetes development and its

complications (cardiovascular, neuropathy, retinopa-

thy, and nephropathy) [6], and a recent study by the

Centers for Disease Control and Prevention associat-

ing cases of acute kidney injury with synthetic cannab-

inoid use [105], the use of THC would be risky from a

clinical point of view in patients with established

diabetes. Diabetic patients also have impaired immune

functions and wound healing, which could be

adversely affected by immunosuppressive/immuno-

modulatory drugs such as THC. By contrast, CBD

demonstrated beneficial effects as a result of its

anti-inflammatory and antioxidant properties both in

preclinical models of primary diabetes and in models

of all major diabetic complications, which is encourag-

ing for its potential testing in diabetic patients [6].

As noted above, THC and its synthetic analogue

Nabilone are used to treat chemotherapy-induced nau-

sea and vomiting, as well as to stimulate appetite in

cachexia associated with AIDS or terminal tumours

[2]. In the case of AIDS, recent controlled studies in

nonhuman primates showed unexpectedly that chronic

THC administration before and during simian immu-

nodeficiency virus infection ameliorates disease

progression, and also attenuates viral load and tissue

inflammation, significantly reducing the morbidity and

mortality of virus-infected macaques [106], which is

very encouraging.

There is considerable preclinical and clinical

evidence showing that the combination of THC with

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1925

P. Pacher and G. Kunos Targeting the endocannabinoid system

opioids or nonsteroidal anti-inflammatory drugs may

enhance their efficacy in pain and also limit their side

effects [2,95,96]. It has become clear that cannabinoid

analgesia is predominantly mediated via peripheral

CB1 receptors in nociceptors [107], providing the ratio-

nale for selectively targeting peripheral CB1 receptors

by peripherally-restricted (brain impermeable) agonists,

thereby eliminating the undesirable CNS consequences

of CB1 stimulation [71]. Astra Zeneca (London, UK)

has developed two novel peripherally-restricted, orally

bioavailable CB1/2 agonists (AZD1940 and AZD1704).

Despite their mixed agonist activity at CB1 and CB2 receptors, the analgesic efficacy in rodent models was

mainly driven by CB1 receptors, as validated through

the use of CB1 selective antagonist and knockout mice

[27]. The clinical efficacy of AZD1940 as a pain relie-

ver was tested in two single-dose, phase II studies

(human capsaicin and third molar extraction models)

and in a multiple ascending doses study performed in

subjects with chronic low-back pain. The two single-

dose, phase II studies showed no efficacy at the pri-

mary endpoints (pain intensity and heat pain threshold

for capsaicin study) [28]. In the multiple ascending

dose study where AZD1940 was administered for

12 days, repeated dosing led to slow compound accu-

mulation, significant weight gain and elevation of

hepatic transaminases. AZD1704 also induced pro-

found hypotensive effects [28]. Thus, the analgesic effi-

cacy of peripherally-restricted CB1 agonists remains to

be established in humans. Although their cardiovascu-

lar and metabolic side effects confirm the role of CB1 receptors in these functions in humans, they further

limit their usefulness as therapeutic agents. The above

studies of Astra Zeneca with novel, peripherally-

restricted, orally bioavailable CB1/2 agonists did not

indicate CB2 involvement in preclinical models of

analgesia, whereas other studies suggest that CB2 acti-

vation may attenuate certain types of pain [95,96].

CB2-selective peripherally-restricted agonists (instead

of mixed CB1/2 agonists) may offer the better optimi-

zation of dosing in humans because metabolic and

cardiovascular side effects are less likely to occur.

Inhibition of the CB1 receptors: global and peripherally-restricted CB1 antagonists

Recent preclinical studies have provided compelling

evidence that ECs modulate food intake, energy bal-

ance, glucose and lipid metabolism through CB1 recep-

tors expressed in the brain and various peripheral

tissues, such as fat, liver and skeletal muscle

[5,70,108,109]. Treatment with brain-penetrant CB1

receptor antagonists/inverse agonists resulted in

improvements of multiple cardiovascular risk factors

both in preclinical studies and in clinical trials in

obese/overweight subjects [110–116]. Parallel preclini- cal studies clearly demonstrated that reduced food

intake was not the primary mechanism responsible for

the weight-reducing effect of CB1 antagonists, and sug-

gested that peripheral energy metabolism might be

directly under EC control [5]. These studies demon-

strated that ECs promote lipogenesis in adipose tissue

and liver but inhibit fatty acid oxidation and mito-

chondrial biogenesis, whereas CB1 antagonists exert

the opposite effects [5]. Meanwhile, clinical trials have

revealed that a small but statistically significant frac-

tion of subjects treated with the CB1 inverse agonist

rimonabant exhibited anxiety, depression and/or

suicidal ideations, eventually leading to the withdrawal

of rimonabant from the market in over 50 countries

and discontinuation of the therapeutic development of

this class of compounds [117].

By that time, there were several lines of evidence

strongly suggesting that selective inhibition of periph-

eral CB1 receptors may preserve much of the metabolic

benefit of global CB1 blockade at the same time as

minimizing side effects as a result of the blockade of

CB1 receptors in the CNS [5]. A proof of principle

study by Tam et al. [118] demonstrated that chronic

treatment of DIO mice with AM6545 (the first high-

affinity, selective, peripherally-restricted neutral CB1 antagonist) improved glucose tolerance, insulin sensi-

tivity and the plasma lipid profile, and also reversed

fatty liver, although it was less effective than its parent

compound rimonabant in reducing body weight

because it did not affect caloric intake. The same study

also provided evidence for the importance of CB1 receptors in hepatocytes in the development of diet-

induced insulin resistance. A subsequent study pro-

vided additional mechanistic insight by demonstrating

that CB1-mediated hepatic insulin resistance involves

ER stress-dependent impairment of insulin signalling,

as well as reduced insulin clearance [119]. In a follow-

up study, a highly potent, selective and brain imperme-

able CB1 receptor inverse agonist, JD5037, was even

more effective in improving metabolic parameters in

mouse models of obesity, and it not only improved

cardiometabolic risk, but also had antiobesity and

hypophagic effects by reversing leptin resistance [101].

This compound is currently undergoing toxicology

screening as a prelude to its clinical testing.

As discussed above, we have learned important les-

sons from the first clinical trials aiming to attenuate

pain with the peripherally-restricted mixed CB1/2 agon-

ists, which were terminated because of excessive weight

1926 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

gain, hepatotoxicity and cardiovascular adverse effects.

Interestingly, this side-effect profile strongly supports

the rationale for the development and therapeutic use

of peripherally-restricted CB1 antagonists in humans

[27,28].

Activation of CB2 receptors by selective agonists

Overwhelming evidence for the therapeutic potential of

EC/CB2 receptor signalling in some of the major

pathologies affecting humans has been reviewed

recently [4]. An important consideration for the thera-

peutic development of selective CB2 receptor agonists

is the absence of psychoactive effects, coupled with the

anti-inflammatory and tissue protective activity of these

ligands in numerous preclinical disease models [4].

CB2 receptors are predominantly expressed in

peripheral blood immune cells where the level of their

expression is strongly modulated by pro-inflammatory

and other stimuli, largely depending on the experimen-

tal conditions [120]. Initial studies focusing on the

immunomodulatory effects of THC and other cannabi-

noid ligands in vivo in rodents and in vitro in human

immune cell cultures demonstrated immunosuppressive

effects in T and B lymphocytes, natural killer cells and

macrophages, which most likely involved both CB1 and CB2 receptors, as well as CB receptor-independent

mechanisms [9,120,121]. ECs were also found to mod-

ulate T and B cell proliferation and apoptosis, immune

cell activation and inflammatory cytokine production,

chemotaxis and inflammatory cell migration, and mac-

rophage-mediated killing of sensitized cells [9,120,122].

These generally inhibitory effects were ligand- and cell

type-dependent and were also influenced by the experi-

mental conditions used [9,120,123,124]. A complicating

factor is the agonist-induced rapid internalization and

trafficking of CB2 receptors in vitro, which can con-

found any interpretation of the results [33,34]. The

effects of ECs or synthetic analogues on microglia acti-

vation/migration also appear to be largely experimen-

tal condition-dependent [123].

One important recent development has been the

identification of low levels of CB2 receptor expression

in tissues previously considered to be devoid of these

receptors. These include specific regions of the brain

[125–127], spinal cord and dorsal root ganglia [17,95,128], neurones in the myenteric and submucosal

plexus of the enteric nervous system [129–131], myo- cardium or cardiomyocytes [64,65,132], human vascu-

lar smooth muscle and endothelium [25,133–135], activated hepatic stellate cells [136,137], Kupffer

cells [138], reproductive organs/cells [139,140], colonic

epithelial cells [141], bone [142–144], mouse and human exocrine and endocrine pancreas [145–148], and various human tumours [149]. Further studies are

needed to fully explore the function of CB2 receptors

at these sites.

More importantly, disease-induced changes (usually

increases) in CB2 receptor expression have been

reported (Table 1), and synthetic CB2 receptor agon-

ists exerted protective effects in a variety of preclinical

disease models and pathological conditions [4], ranging

from cardiovascular disorders [11], various forms of is-

chaemic-reperfusion injury [90], gastrointestinal and

liver inflammation [13,150,151], autoimmune and neu-

rodegenerative disorders [7,152–154], kidney disorders [4], bone disorders [143,144], cancer [149,155–157], and pain [17,95].

As for the therapeutic potential of CB2 agonists, it

is important to note that, although, under conditions

of a sterile inflammatory response, CB2 agonists may

limit injury, in pathogen-induced inflammation, the

immunosuppressive effects of the CB2 receptor activa-

tion may enhance or even inflict tissue damage, and

may also lead to accelerated cancer growth in certain

types of tumours [4]. To successfully target CB2 in

selected human diseases, it is imperative to identify the

exact cellular location and disease-induced, time-

dependent changes in the expression of CB2 receptors.

This will necessitate the development of improved

research tools, such as more reliable and specific anti-

bodies. This is particularly important because, in many

injury models, CB2 agonists appear to be most effec-

tive when given before the initiation of the insult, and

may lose their efficacy or even promote inflammation

when given at later time [4]. Thus, a better understand-

ing of the underlying pathology and its effects on CB2 expression is required for the development of meaning-

ful therapeutic approaches. Before going to clinical

development for a particular indication, it is also

important to confirm previous preclinical findings with

novel and more selective CB2 agonists, because cur-

rently available ligands may not be entirely specific.

Better knowledge of the pharmacokinetics and metab-

olism of ligands is also essential, particularly given the

bell-shaped dose–response often seen with recently available CB2 agonists in various disease models [4].

The reason for the latter may be that, when used at

higher doses, currently used CB2 agonists may also

activate CB1 receptors, particularly when the relative

expression of CB1 over CB2 is high. Our understand-

ing of the complexities of CB2 receptor signalling is

still limited, and important interspecies differences in

CB2 receptor signalling and in the pharmacology of

CB2 ligands must also be considered [158].

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1927

P. Pacher and G. Kunos Targeting the endocannabinoid system

Problems with the use of peripherally-restricted

CB1/2 agonists for pain relief as a result of cardiovas-

cular and metabolic side effects have been discussed

above. A plausible alternative could be the testing of

peripherally-restricted selective CB2 agonists for anal-

gesia in humans because such compounds would be

expected to be devoid of cardiometabolic liabilities.

However, the preclinical data with AZD1940 and

AZD1704 indicate that the analgesic efficacy of this

class of compounds was mainly driven by the CB1 receptor [27] which, if confirmed in humans, would

limit the promise of this approach. Nevertheless, the

therapeutic development of selective CB2 receptor

ligands (agonists or inverse agonists/antagonists

depending on the pathology and its stage) is still a

promising strategy for a number of disease conditions,

provided that the issues discussed above are success-

fully resolved [4].

Inhibition of EC metabolism, cellular uptake or biosyntheses

The hypothesis behind the therapeutic inhibition of

EC degradation was that increasing EC tissue levels

would be less likely to cause psychoactive effects than

would the use of synthetic CB1 ligands (endocannabi-

noids are biosynthesized and degraded in a site and

time-dependent manner), whereas the beneficial effects

of CB1/2 activation, such as analgesia, would be main-

tained [159]. In support of this, FAAH knockout mice

or mice treated with a FAAH inhibitor have elevated

AEA levels in the brain and other tissues, are super-

sensitive to exogenous AEA, and exhibit CB1 receptor-

mediated hypoalgesia [160,161] and reduced anxiety,

although they do not display catalepsy, an indicator of

psychoactivity in humans [162]. The antinociceptive

effect of FAAH inhibitors, likely mediated through

increases in AEA and PEA levels that activate CB1/2,

peroxisome proliferator-activated receptor a and/or TRPV1 [163], was investigated in acute and chronic

rodent models of pain [164]. Most of the initial results

were based on using URB597, which irreversibly inhib-

its FAAH both in the CNS and periphery [164].

Recent studies with a peripherally-restricted FAAH

inhibitor, URB937, showed efficacy in neuropathic

and inflammatory pain [165], confirming that the anal-

gesic effects of AEA are initiated at the peripheral sites

[107]. However, similar to direct-acting peripheral CB1/2 agonists, URB597 has both hypotensive [166] and

diabetogenic effects [167] mediated by CB1 receptors,

and FAAH knockout mice are also prone to diet-

induced obesity and diabetes [168]. The diabetogenic

effect of URB597 has been attributed to blocking

FAAH in the liver, and the novel FAAH inhibitor

AM3506, which does not block FAAH in the liver as a

result of its rapid uptake and metabolism by hepato-

cytes, was found to be devoid of glycaemic side effects

in rodents [167]. FAAH antagonism may also promote

fat accumulation and insulin resistance through

centrally-mediated hypothyroidism [169].

The analgesic effects of FAAH inhibition in preclini-

cal models prompted the development of PF-04457845,

an irreversible FAAH inhibitor with excellent analgesic

efficacy in animal models [29,170], which was selected

for clinical development. In a randomized, placebo-

controlled, phase II clinical trial PF-04457845 was

recently evaluated in patients with osteoarthritic pain

of the knee [30]. The results clearly demonstrated that

PF-04457845 inhibited FAAH activity in white blood

cells and raised the concentrations of various fatty acid

amides 3.5-10 fold, which persisted for up to 2 weeks

after discontinuation of the drug, and did not affect

cognitive function in test subjects. However, the study

failed to show any analgesic efficacy of PF-04457845,

whereas the nonsteroidal anti-inflammatory drug nap-

roxen, used as a positive control, was effective [30].

These results were also highlighted and discussed in a

recent editorial [171].

A promising alternative indication for the therapeu-

tic use of FAAH antagonists is post-traumatic stress

syndrome. The FAAH inhibitor AM3506 was recently

found to be effective in increasing fear extinction in a

CB1 receptor-dependent manner in a mouse model of

post-traumatic stress syndrome, and human carriers of

a low-expressing FAAH variant displayed quicker

habituation of amygdala reactivity to threat, as

detected by brain imaging [172].

The main rationale for the development of MAGL

inhibitors, which metabolize 2-AG, is similar to the

rationale for FAAH inhibitors. Numerous recent stud-

ies have demonstrated that MAGL inhibition or

genetic deletion exerts anti-emetic [173], antineoplastic

[174], and anxiolytic and antinociceptive effects in

rodents [175], and also protects against brain injury

[176,177], acute liver injury/inflammation [138] and

colitis either via enhancing CB1/2 signalling or by

attenuating eicosanoid synthesis in specific tissues,

such as the brain and liver [178], or by a combination

of both. In the case of cancer, MAGL inhibition mod-

ulates fatty acid release for the synthesis of protumo-

urigenic signalling lipids [174], as reviewed recently

[179,180].

Although the above preclinical findings are indeed

exciting, they also highlight important limitations. (a)

Raising the tissue levels of ECs may promote the

formation of cyclooxygenase-, lipoxygenase- and

1928 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

cytochrome P450-derived pro-inflammatory metabo-

lites [47,181]. (b) Some of the prostaglandins that were

attenuated by MAGL inhibitors have well documented

tissue protective functions. (c) Although the dual effect

of MAGL inhibition on attenuating eicosanoid and

enhancing EC signalling can be beneficial in certain

tissues (e.g. the brain and liver) where MAGL links

the EC and eicosanoid systems through the hydrolysis

of 2-AG, in other tissues, it can promote inflammation

and injury (e.g. in the myocardium) through the non-

CB mechanisms described above (the cardiotoxicity of

COX-2 inhibitors is well documented in humans). (d)

Chronic MAGL inhibition leads to functional antago-

nism of the ECS [175]. (e) As previously discussed,

very strong preclinical and clinical evidence suggests

that, in cardiovascular disease and diabetes/diabetic

complications, endocannabinoids (through CB1 and

most likely through the first two mechanisms described

above) promote cardiovascular injury. (f) There is

growing evidence that ECs exert pro-inflammatory

effects in various disease models through both CB1-

dependent and -independent mechanisms [6]. This is

supported by a recent study demonstrating that the

inhibition of EC synthesis is anti-inflammatory in mac-

rophages [182]. (g) Various isoforms of metabolizing

enzymes (e.g. FAAH) may have distinct functions [52],

and the functional properties of rodent and human

FAAH may also be different [183]. (h) Most of the

benefits observed with inhibitors of FAAH or MAGL

were reported in acute models; the safety of chronic

inhibition of these enzymes has not yet been deter-

mined, particularly in pathological situations. (i) The

use of irreversible inhibitors of FAAH and MAGL

could be a disadvantage for accurate dose titration

and would make it difficult to treat toxicity [164].

Conclusions and future directions

Recent clinical studies show that cannabinoid-based

medicines with controlled doses of plant-derived cann-

abinoids can provide symptomatic relief in a subset of

patients suffering from pain and spasticity associated

with MS and certain other types of pain, and there is

hope (based on preclinical studies) that these medica-

tions would also positively modulate disease progres-

sion. Synthetic cannabinoids are also useful in subset

of patients with wasting disorders and chemotherapy-

induced nausea and vomiting. There are numerous

promising new targets (plant-derived cannabinoids,

peripherally-restricted CB1 antagonists, selective CB2 agonists, inhibitors of endocannabinoid metabolism/

transport) ‘in waiting’, as discussed in the present

CB1 stimulation

CB1 inhibition Peripheral

CB2 stimulation

Undesirable effects

Inhibition of the EC metabolism/transport

Psychoactive, cardiovascular obesity, diabetes, inflammation

Fertility ? gastrointestinal motility

gastrointestinal motility

Immunosupression?,fertility?

Psychoactive, cardiovascular metabolic, inflammation ?

Modulation of the endocannabinoid (EC) system in human disease

Desirable effects

Pain, nausea/vomiting appetite (in cachexia)

Insulin resistance, inflammation lipogenesis, cardiometabolic risk

Inflammation, tissue injury

lipolysis, glucose tolerance

Pain, anxiety , inflammation?

Fig. 1. Cannabinoid therapeutics: finding the right balance.

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1929

P. Pacher and G. Kunos Targeting the endocannabinoid system

Table 2. Potential approaches/directions for future success.

Therapeutic approach

(target) Possible directions/approaches for success

Possibly therapeutic indications in

humans (realistic)

Potential/expected adverse

effects

THC based

medicines,

cannabinoid based

extracts (CB1, CB2

and unrelated

antioxidant

anti-inflammatory

mechanisms)

Optimization of route of administration, dosing

and indication

Better selection criteria for trials,

identification of potential positive responders

by initial titration

Placebo-controlled trials to establish short-

and long-term efficacy in given indications

Long-term controlled studies to determine

possible disease-modifying effects (e.g. in

multiple sclerosis) and adverse consequences

(e.g. immune and/or cardiovascular effects,

etc.)

Combination approaches in pain to achieve

better efficacy and fewer side effects (e.g.

with opioids, nonsteroid anti-inflammatory

drugs, etc.)

Optimization of the extract composition for

improved benefit/risk profile

Symptomatic relief in certain forms

of pain and spasticity (as in

neurodegenerative disorders such

as multiple sclerosis)

Stimulation of appetite in patients

with wasting disorders

Attenuation of chemotherapy-

induced nausea and vomiting

Topical administration in certain

skin disorders?

Nonpsychoactive constituents of

marijuana, such as CBD or their

analogues, may have therapeutic

utility in certain forms of acute

tissue injury, inflammatory

disorders, diabetes and diabetic

complications

In the case of

THC-containing

formulations, effects

related to CB1 stimulation

at higher doses

(e.g. psychoactive,

cardiovascular, metabolic

side effects) and potential

modulation of immune

responses

Peripherally

restricted CB1

agonists

(peripheral CB1)

Evaluation of the feasibility of the topical/local

use of peripherally restricted CB1 agonists in

certain forms of pain and skin conditions

(e.g. pruritus)

Topical/local use in certain forms of

pain and skin conditions/

diseases? (the systematic

administration/use is not likely

because of the established

adverse cardiovascular and

metabolic consequences of

this approach)

Cardiovascular

Metabolic

Kidney

Gastrointestinal

(decreased motility)

Pro-inflammatory?

Peripherally

restricted or global

CB2 agonists

(peripheral CB2)

Re-evaluation of human indications based on

previous failures of trials with mixed

peripherally restricted CB1/2 agonists

Search for new indications

More preclinical and clinical research to

understand the significance of tissue and

time specific changes in CB2 receptor

expression in pathological conditions

Development of novel, specific and orally

available ligands for proof of the principle

studies; evaluation of toxicology and

pharmacokinetics

Various forms of acute tissue

injuries associated with

inflammation (stroke, myocardial

infarction, traumatic injury, organ

transplantation, etc.)

Various forms of inflammatory

diseases if the anti-inflammatory

effects are confirmed in humans

Most likely related to

effects on immune and

haematopoietic system

Effects on fertility?

Peripherally

restricted CB1

antagonists, inverse

agonists

(peripheral CB1)

Development and testing of new ligands,

toxicology and safety studies in rodents, large

animals, and humans

Proof of the principle studies in large animals

and humans

Diabetes and diabetic

complications, Cardiometabolic

syndrome

Kidney disease?

Gastrointestinal (increased

motility)

Effects on fertility?

Inhibition of EC

metabolism, cellular

uptake or

biosynthesis (CB1/2,

TRPV1 and nuclear

receptors,

prostaglandin and

leukotriene

signalling)

Preclinical research to identify the putative

endocannabinoid transporter(s), and to better

understand the tissue, time, and disease-

specific metabolism of endocannabinoids to

various other bioactive mediators

(e.g. prostaglandins, leukotriens, etc.)

Re-evaluation of human indications based on

previous failures of trials with FAAH inhibitors

in pain

Search for new indications, better and more

selective ligands

Pain?

Certain disorders associated with

anxiety?

Certain forms of acute tissue

injury?

Similar, but acutely less

pronounced than with

CB1 agonists. However,

long-term use may be

associated with adverse

effects similar to

cyclooxygenase 2

inhibitors

(e.g. cardiovascular).

Pro-inflammatory effects

in certain cases?

1930 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

minireview. However, it is clear that, for the successful

translation of preclinical findings to clinical practice, a

better understanding of the pathological role of the

ECS in various diseases, of the potential side effects of

targeting this system, and of endocannabinoid phar-

macology is required, coupled with the development of

improved research tools to dissect these processes

(Fig. 1 and Table 2).

Future studies should focus on a rigorous evaluation

of the CB receptor dependent/independent and hypo-

thermia-independent effects of THC in preclinical mod-

els (e.g. in tissue injury, cancer, inflammation, etc.)

using global and tissue/cell specific knockout mice and

also aim to identify potential novel targets/mechanisms

of action of THC and other plant-derived cannabinoids,

coupled with the identification of nonpsychoactive con-

stituents in cannabis extracts with potential therapeutic

effects. Novel highly selective, orally available nontoxic

cannabinoid ligands should be developed and evaluated

in preclinical disease models. Large animal studies (e.g.

canine, pig, primate) should confirm the efficacy of can-

nabinoid ligands obtained in rodent disease models

before initiating human trials. The development of

specific novel antibodies for CB1/2 receptors and endoc-

annabinoid metabolic enzymes (FAAH, MAGL, diacyl-

glycerol lipase a/b) validated by using positive and negative controls is essential for accurately assessing the

time-dependent changes in CB1/2 receptors and meta-

bolic enzyme expression in diseased animal and human

tissues, with the aim of understanding the human rele-

vance of these changes. Our limited knowledge should

be expanded to enable an understanding of CB1/2 recep-

tor trafficking, signalling and their interspecies differ-

ences. The development of reliable radioligands suitable

for human imaging studies and research could contrib-

ute to a better understanding of the role of ECS in

human health and disease.

Acknowledgements

This study was supported by funds from the Intramu-

ral Research Program of NIAAA to P.P. and G.K.

The authors apologize to colleagues whose important

work could not be cited because of space limitations.

References

1 Hanus LO (2009) Pharmacological and therapeutic

secrets of plant and brain (endo)cannabinoids. Med

Res Rev 29, 213–271.

2 Pacher P, Batkai S & Kunos G (2006) The

endocannabinoid system as an emerging target of

pharmacotherapy. Pharmacol Rev 58, 389–462.

3 Di Marzo V (2008) Targeting the endocannabinoid

system: to enhance or reduce? Nat Rev Drug Discov 7,

438–455.

4 Pacher P & Mechoulam R (2011) Is lipid signaling

through cannabinoid 2 receptors part of a protective

system? Prog Lipid Res 50, 193–211.

5 Kunos G & Tam J (2011) The case for peripheral CB

(1) receptor blockade in the treatment of visceral

obesity and its cardiometabolic complications. Br J

Pharmacol 163, 1423–1431.

6 Horvath B, Mukhopadhyay P, Hasko G & Pacher P

(2012) The endocannabinoid system and plant-derived

cannabinoids in diabetes and diabetic complications.

Am J Pathol 180, 432–442.

7 Centonze D, Finazzi-Agro A, Bernardi G &

Maccarrone M (2007) The endocannabinoid system in

targeting inflammatory neurodegenerative diseases.

Trends Pharmacol Sci 28, 180–187.

8 Skaper SD & Di Marzo V (2012) Endocannabinoids

in nervous system health and disease: the big picture

in a nutshell. Philosophical transactions of the

Royal Society of London. Series B, Biol Sci 367,

3193–3200.

9 Klein TW (2005) Cannabinoid-based drugs as

anti-inflammatory therapeutics. Nat Rev Immunol 5,

400–411.

10 Pacher P, Mukhopadhyay P, Mohanraj R, Godlewski

G, Batkai S & Kunos G (2008) Modulation of the

endocannabinoid system in cardiovascular disease:

therapeutic potential and limitations. Hypertension 52,

601–607.

11 Steffens S & Pacher P (2012) Targeting cannabinoid

receptor CB(2) in cardiovascular disorders: promises

and controversies. Br J Pharmacol 167, 313–323.

12 Montecucco F & Di Marzo V (2012) At the heart of

the matter: the endocannabinoid system in

cardiovascular function and dysfunction. Trends

Pharmacol Sci 33, 331–340.

13 Lotersztajn S, Teixeira-Clerc F, Julien B, Deveaux V,

Ichigotani Y, Manin S, Tran-Van-Nhieu J, Karsak M,

Zimmer A & Mallat A (2008) CB2 receptors as new

therapeutic targets for liver diseases. Br J Pharmacol

153, 286–289.

14 Tam J, Liu J, Mukhopadhyay B, Cinar R, Godlewski

G & Kunos G (2011) Endocannabinoids in liver

disease. Hepatology 53, 346–355.

15 Izzo AA & Camilleri M (2008) Emerging role of

cannabinoids in gastrointestinal and liver diseases:

basic and clinical aspects. Gut 57, 1140–1155.

16 Biro T, Toth BI, Hasko G, Paus R & Pacher P (2009)

The endocannabinoid system of the skin in health and

disease: novel perspectives and therapeutic

opportunities. Trends Pharmacol Sci 30, 411–420.

17 Guindon J & Hohmann AG (2008) Cannabinoid CB2

receptors: a therapeutic target for the treatment of

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1931

P. Pacher and G. Kunos Targeting the endocannabinoid system

inflammatory and neuropathic pain. Br J Pharmacol

153, 319–334.

18 Guindon J & Hohmann AG (2009) The

endocannabinoid system and pain. CNS Neurol Disord

Drug Targets 8, 403–421.

19 Mechoulam R & Parker LA (2013) The

Endocannabinoid System and the Brain. Annu Rev

Psychol 64, 21–47.

20 Hillard CJ, Weinlander KM & Stuhr KL (2012)

Contributions of endocannabinoid signaling to

psychiatric disorders in humans: genetic and

biochemical evidence. Neuroscience 204, 207–229.

21 Guindon J & Hohmann AG (2011) The

endocannabinoid system and cancer: therapeutic

implication. Br J Pharmacol 163, 1447–1463.

22 Velasco G, Sanchez C & Guzman M (2012) Towards

the use of cannabinoids as antitumour agents. Nat

Rev Cancer 12, 436–444.

23 Parker LA, Rock EM & Limebeer CL (2011)

Regulation of nausea and vomiting by cannabinoids.

Br J Pharmacol 163, 1411–1422.

24 Piscitelli F & Di Marzo V (2012) ‘Redundancy’ of

endocannabinoid inactivation: new challenges and

opportunities for pain control. ACS Chem Neurosci 3,

356–363.

25 Pacher P & Steffens S (2009) The emerging role of the

endocannabinoid system in cardiovascular disease.

Semin Immunopathol 31, 63–77.

26 Di Marzo V (2008) Play an adagio with a Stradivarius:

the right patient for CB1 receptor antagonists? Nat

Clin Pract Cardiovasc Med 5, 610–612.

27 Groblewski T, Hong X, Lessard E, St-Onge S, Yang

H, Panetta R, Cao CQ, Swedberg MD, Cebers G,

Nyberg S et al. (2010) Pre-clinical pharmacological

properties of novel peripherally-acting CB1-CB2

agonists. Proccedings of 20th Annual Symposium of

the International Cannabinoid Research Society,

Lund, Sweden, 2010.

28 Groblewski T, Karlsten R, Segerdhal M, Kalliom€aki J,

Jonzon B, Bielenstein M, Cebers G, Swedberg M,

Annas A, Christoph G et al. (2010) Peripherally-acting

CB1-CB2 agonists for pain: do they still hold promise?

Proceedings of the 20th Annual Symposium of the

International Cannabinoid Research Society, Lund,

Sweden, 2010.

29 Li GL, Winter H, Arends R, Jay GW, Le V, Young T

& Huggins JP (2012) Assessment of the pharmacology

and tolerability of PF-04457845, an irreversible

inhibitor of fatty acid amide hydrolase-1, in healthy

subjects. Br J Clin Pharmacol 73, 706–716.

30 Huggins JP, Smart TS, Langman S, Taylor L &

Young T (2012) An efficient randomised, placebo-

controlled clinical trial with the irreversible fatty acid

amide hydrolase-1 inhibitor PF-04457845, which

modulates endocannabinoids but fails to induce

effective analgesia in patients with pain due to

osteoarthritis of the knee. Pain 153, 1837–1846.

31 Howlett AC, Barth F, Bonner TI, Cabral G, Casellas

P, Devane WA, Felder CC, Herkenham M, Mackie K,

Martin BR et al. (2002) International Union of

Pharmacology. XXVII. Classification of cannabinoid

receptors. Pharmacol Rev 54, 161–202.

32 Pertwee RG, Howlett AC, Abood ME, Alexander SP,

Di Marzo V, Elphick MR, Greasley PJ, Hansen HS,

Kunos G, Mackie K et al. (2010) International Union

of Basic and Clinical Pharmacology. LXXIX.

Cannabinoid receptors and their ligands: beyond CB1

and CB2. Pharmacol Rev 62, 588–631.

33 Atwood BK, Wager-Miller J, Haskins C, Straiker A &

Mackie K (2012) Functional selectivity in CB2

cannabinoid receptor signaling and regulation:

implications for the therapeutic potential of CB2

ligands. Mol Pharmacol 81, 250–263.

34 Kleyer J, Nicolussi S, Taylor P, Simonelli D, Furger

E, Anderle P & Gertsch J (2012) Cannabinoid receptor

trafficking in peripheral cells is dynamically regulated

by a binary biochemical switch. Biochem Pharmacol

83, 1393–1412.

35 Devane WA, Hanus L, Breuer A, Pertwee RG,

Stevenson LA, Griffin G, Gibson D, Mandelbaum A,

Etinger A & Mechoulam R (1992) Isolation and

structure of a brain constituent that binds to the

cannabinoid receptor. Science 258, 1946–1949.

36 Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M,

Kaminski NE, Schatz AR, Gopher A, Almog S,

Martin BR, Compton DR et al. (1995) Identification

of an endogenous 2-monoglyceride, present in canine

gut, that binds to cannabinoid receptors. Biochem

Pharmacol 50, 83–90.

37 Wang J & Ueda N (2009) Biology of endocannabinoid

synthesis system. Prostaglandins Other Lipid Mediat

89, 112–119.

38 Di Marzo V, Bifulco M & De Petrocellis L (2004) The

endocannabinoid system and its therapeutic

exploitation. Nat Rev Drug Discov 3, 771–784.

39 Liu J, Wang L, Harvey-White J, Huang BX, Kim HY,

Luquet S, Palmiter RD, Krystal G, Rai R,

Mahadevan A et al. (2008) Multiple pathways

involved in the biosynthesis of anandamide.

Neuropharmacology 54, 1–7.

40 Simon GM & Cravatt BF (2008) Anandamide

biosynthesis catalyzed by the phosphodiesterase GDE1

and detection of glycerophospho-N-acyl ethanolamine

precursors in mouse brain. J Biol Chem 283,

9341–9349.

41 Fowler CJ (2012) Anandamide uptake explained?

Trends Pharmacol Sci 33, 181–185.

42 Fowler CJ (2013) Transport of endocannabinoids

across the plasma membrane and within the cell.

FEBS J 280, 1895–1904.

1932 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

43 Kaczocha M, Vivieca S, Sun J, Glaser ST & Deutsch

DG (2012) Fatty acid-binding proteins transport

N-acylethanolamines to nuclear receptors and are

targets of endocannabinoid transport inhibitors. J Biol

Chem 287, 3415–3424.

44 Cravatt BF & Lichtman AH (2003) Fatty acid amide

hydrolase: an emerging therapeutic target in the

endocannabinoid system. Curr Opin Chem Biol 7,

469–475.

45 Ueda N, Tsuboi K & Uyama T (2010) N-

acylethanolamine metabolism with special reference to

N-acylethanolamine-hydrolyzing acid amidase

(NAAA). Prog Lipid Res 49, 299–315.

46 Ueda N, Tsuboi K, Uyama T & Ohnishi T (2011)

Biosynthesis and degradation of the endocannabinoid

2-arachidonoylglycerol. BioFactors 37, 1–7.

47 Rouzer CA & Marnett LJ (2011) Endocannabinoid

oxygenation by cyclooxygenases, lipoxygenases, and

cytochromes P450: cross-talk between the eicosanoid

and endocannabinoid signaling pathways. Chem Rev

111, 5899–5921.

48 Cravatt BF & Lichtman AH (2002) The enzymatic

inactivation of the fatty acid amide class of signaling

lipids. Chem Phys Lipids 121, 135–148.

49 McKinney MK & Cravatt BF (2005) Structure and

function of fatty acid amide hydrolase. Annu Rev

Biochem 74, 411–432.

50 Fezza F, De Simone C, Amadio D & Maccarrone M

(2008) Fatty acid amide hydrolase: a gate-keeper of

the endocannabinoid system. Subcell Biochem 49,

101–132.

51 Palkovits M, Harvey-White J, Liu J, Kovacs ZS,

Bobest M, Lovas G, Bago AG & Kunos G (2008)

Regional distribution and effects of postmortal delay

on endocannabinoid content of the human brain.

Neuroscience 152, 1032–1039.

52 Fu J, Bottegoni G, Sasso O, Bertorelli R, Rocchia W,

Masetti M, Guijarro A, Lodola A, Armirotti A,

Garau G et al. (2012) A catalytically silent FAAH-1

variant drives anandamide transport in neurons. Nat

Neurosci 15, 64–69.

53 Di Marzo V & De Petrocellis L (2010)

Endocannabinoids as regulators of transient receptor

potential (TRP) channels: A further opportunity to

develop new endocannabinoid-based therapeutic drugs.

Curr Med Chem 17, 1430–1449.

54 Hanus LO & Mechoulam R (2010) Novel natural and

synthetic ligands of the endocannabinoid system. Curr

Med Chem 17, 1341–1359.

55 Bauer M, Chicca A, Tamborrini M, Eisen D, Lerner

R, Lutz B, Poetz O, Pluschke G & Gertsch J (2012)

Identification and quantification of a new family of

peptide endocannabinoids (Pepcans) showing negative

allosteric modulation at CB1 receptors. J Biol Chem

287, 36944–36967.

56 Pamplona FA, Ferreira J, Menezes de Lima O Jr,

Duarte FS, Bento AF, Forner S, Villarinho JG,

Bellochio L, Wotjak CT, Lerner R et al. (2012) Anti-

inflammatory lipoxin A4 is an endogenous allosteric

enhancer of CB1 cannabinoid receptor. Proc Natl

Acad Sci USA 109, 21134–21139.

57 Ueda N, Tsuboi K & Uyama T (2013) Metabolism of

endocannabinoids and related N-acylethanolamines:

Canonical and alternative pathways. FEBS J 280,

1874–1894.

58 Di Marzo V (2008) Endocannabinoids: synthesis and

degradation. Rev Physiol Biochem Pharmacol 160, 1–24.

59 Miller LK & Devi LA (2011) The highs and lows of

cannabinoid receptor expression in disease:

mechanisms and their therapeutic implications.

Pharmacol Rev 63, 461–470.

60 Pertwee RG (2012) Targeting the endocannabinoid

system with cannabinoid receptor agonists:

pharmacological strategies and therapeutic possibilities.

Philosophical Transactions of the Royal Society of

London. Series B, Biol Sci 367, 3353–3363.

61 Atwood BK & Mackie K (2010) CB2: a cannabinoid

receptor with an identity crisis. Br J Pharmacol 160,

467–479.

62 Pacher P, Batkai S & Kunos G (2005) Cardiovascular

pharmacology of cannabinoids. Handb Exp Pharmacol

168, 599–625.

63 Rajesh M, Mukhopadhyay P, Hasko G, Liaudet L,

Mackie K & Pacher P (2010) Cannabinoid-1

receptor activation induces reactive oxygen species-

dependent and -independent mitogen-activated

protein kinase activation and cell death in human

coronary artery endothelial cells. Br J Pharmacol

160, 688–700.

64 Mukhopadhyay P, Rajesh M, Batkai S, Patel V,

Kashiwaya Y, Liaudet L, Evgenov OV, Mackie K,

Hasko G & Pacher P (2010) CB1 cannabinoid

receptors promote oxidative stress and cell death in

murine models of doxorubicin-induced

cardiomyopathy and in human cardiomyocytes.

Cardiovasc Res 85, 773–784.

65 Mukhopadhyay P, Batkai S, Rajesh M, Czifra N,

Harvey-White J, Hasko G, Zsengeller Z, Gerard NP,

Liaudet L, Kunos G et al. (2007) Pharmacological

inhibition of CB1 cannabinoid receptor protects

against doxorubicin-induced cardiotoxicity. J Am Coll

Cardiol 50, 528–536.

66 Mukhopadhyay P, Horvath B, Rajesh M, Matsumoto

S, Saito K, Batkai S, Patel V, Tanchian G, Gao RY,

Cravatt BF et al. (2011) Fatty acid amide hydrolase is

a key regulator of endocannabinoid-induced

myocardial tissue injury. Free Radic Biol Med 50,

179–195.

67 Sugamura K, Sugiyama S, Nozaki T, Matsuzawa Y,

Izumiya Y, Miyata K, Nakayama M, Kaikita K,

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1933

P. Pacher and G. Kunos Targeting the endocannabinoid system

Obata T, Takeya M et al. (2009) Activated

endocannabinoid system in coronary artery disease

and antiinflammatory effects of cannabinoid 1 receptor

blockade on macrophages. Circulation 119, 28–36.

68 Dol-Gleizes F, Paumelle R, Visentin V, Mares AM,

Desitter P, Hennuyer N, Gilde A, Staels B, Schaeffer P

& Bono F (2009) Rimonabant, a selective cannabinoid

CB1 receptor antagonist, inhibits atherosclerosis in

LDL receptor-deficient mice. Arterioscler Thromb

Vasc Biol 29, 12–18.

69 Kunos G, Osei-Hyiaman D, Liu J, Godlewski G &

Batkai S (2008) Endocannabinoids and the control of

energy homeostasis. J Biol Chem 283, 33021–33025.

70 Di Marzo V (2008) The endocannabinoid system in

obesity and type 2 diabetes. Diabetologia 51,

1356–1367.

71 Kunos G, Osei-Hyiaman D, Batkai S, Sharkey KA &

Makriyannis A (2009) Should peripheral CB(1)

cannabinoid receptors be selectively targeted for

therapeutic gain? Trends Pharmacol Sci 30, 1–7.

72 Cooney MT, Vartiainen E, Laatikainen T, Juolevi A,

Dudina A & Graham IM (2010) Elevated resting heart

rate is an independent risk factor for cardiovascular

disease in healthy men and women. Am Heart J 159,

612–619e3.

73 Gorelick DA, Goodwin RS, Schwilke E, Schwope

DM, Darwin WD, Kelly DL, McMahon RP, Liu F,

Ortemann-Renon C, Bonnet D et al. (2013) Tolerance

to effects of high-dose oral {Delta}9- tetrahydrocannabinol and plasma cannabinoid

concentrations in male daily cannabis smokers. J Anal

Toxicol 37, 11–16.

74 Schmid K, Schonlebe J, Drexler H & Mueck-

Weymann M (2010) The effects of cannabis on heart

rate variability and well-being in young men.

Pharmacopsychiatry 43, 147–150.

75 Klumpers LE, Roy C, Ferron G, Turpault S, Poitiers

F, Pinquier JL, van Hasselt JG, Zuurman L, Erwich

FA & van Gerven JM (2012) Surinabant, a selective

CB(1) antagonist, inhibits THC-induced central

nervous system and heart rate effects in humans. Br J

Clin Pharmacol doi:10.1111/bcp.12071.

76 Singla S, Sachdeva R & Mehta JL (2012)

Cannabinoids and atherosclerotic coronary heart

disease. Clin Cardiol 35, 329–335.

77 Pratap B & Korniyenko A (2012) Toxic effects of

marijuana on the cardiovascular system. Cardiovasc

Toxicol 12, 143–148.

78 Leblanc A, Tirel-Badets A, Paleiron N, Castellant P,

Cornily JC, Andre M, Grassin F, Feuvrier Y,

Blanchard C, Zagnoli F et al. (2011) Cannabis and

myocardial infarction without angiographic stenosis in

young patient: guilty or not guilty? A case report.

Annales de Cardiologie et d’Angeiologie 60, 154–158.

79 Mittleman MA, Lewis RA, Maclure M, Sherwood JB

& Muller JE (2001) Triggering myocardial infarction

by marijuana. Circulation 103, 2805–2809.

80 Mukamal KJ, Maclure M, Muller JE & Mittleman

MA (2008) An exploratory prospective study of

marijuana use and mortality following acute

myocardial infarction. Am Heart J 155, 465–470.

81 Frost L, Mostofsky E, Rosenbloom JI, Mukamal KJ

& Mittleman MA (2013) Marijuana use and long-term

mortality among survivors of acute myocardial

infarction. Am Heart J 165, 170–175.

82 Mir A, Obafemi A, Young A & Kane C (2011)

Myocardial infarction associated with use of the

synthetic cannabinoid K2. Pediatrics 128, e1622–e1627.

83 Heath TS, Burroughs Z, Thompson AJ & Tecklenburg

FW (2012) Acute intoxication caused by a synthetic

cannabinoid in two adolescents. J Pediatr Pharmacol

Ther 17, 177–181.

84 Lenglet S, Thomas A, Soehnlein O, Montecucco F,

Burger F, Pelli G, Galan K, Cravatt B, Staub C &

Steffens S (2013) Fatty acid amide hydrolase deficiency

enhances intraplaque neutrophil recruitment in

atherosclerotic mice. Arterioscler Thromb Vasc Biol 33,

215–223.

85 Quercioli A, Pataky Z, Vincenti G, Makoundou V, Di

Marzo V, Montecucco F, Carballo S, Thomas A,

Staub C, Steffens S et al. (2011) Elevated

endocannabinoid plasma levels are associated with

coronary circulatory dysfunction in obesity. Eur Heart

J 32, 1369–1378.

86 Quercioli A, Pataky Z, Montecucco F, Carballo S,

Thomas A, Staub C, Di Marzo V, Vincenti G,

Ambrosio G, Ratib O et al. (2012) Coronary

vasomotor control in obesity and morbid obesity:

contrasting flow responses with endocannabinoids,

leptin, and inflammation. JACC Cardiovasc Imaging

5, 805–815.

87 Cappellano G, Uberti F, Caimmi PP, Pietronave S,

Mary DA, Dianzani C, Micalizzi E, Melensi M,

Boldorini R, Nicosia G et al. (2013) Different

expression and function of the endocannabinoid

system in human epicardial adipose tissue in relation

to heart disease. Can J Cardiol 29, 499–509.

88 Liu R & Zhang Y (2011) G1359A polymorphism in

the cannabinoid receptor-1 gene is associated with

coronary artery disease in the Chinese Han

population. Clin Lab 57, 689–693.

89 Amoros I, Barana A, Caballero R, Gomez R, Osuna

L, Lillo MP, Tamargo J & Delpon E (2010)

Endocannabinoids and cannabinoid analogues block

human cardiac Kv4.3 channels in a receptor-

independent manner. J Mol Cell Cardiol 48, 201–210.

90 Pacher P & Hasko G (2008) Endocannabinoids and

cannabinoid receptors in ischaemia-reperfusion

1934 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

injury and preconditioning. Br J Pharmacol 153,

252–262.

91 Bisogno T & Di Marzo V (2010) Cannabinoid

receptors and endocannabinoids: role in

neuroinflammatory and neurodegenerative disorders.

CNS Neurol Disord Drug Targets 9, 564–573.

92 Fowler CJ, Rojo ML & Rodriguez-Gaztelumendi A

(2010) Modulation of the endocannabinoid system:

neuroprotection or neurotoxicity? Exp Neurol 224,

37–47.

93 Mechoulam R & Hanus L (2000) A historical overview

of chemical research on cannabinoids. Chem Phys

Lipids 108, 1–13.

94 Hohmann AG & Suplita RL II (2006)

Endocannabinoid mechanisms of pain modulation.

AAPS J 8, E693–E708.

95 Anand P, Whiteside G, Fowler CJ & Hohmann AG

(2009) Targeting CB2 receptors and the

endocannabinoid system for the treatment of pain.

Brain Res Rev 60, 255–266.

96 Rahn EJ & Hohmann AG (2009) Cannabinoids as

pharmacotherapies for neuropathic pain: from the

bench to the bedside. Neurotherapeutics 6, 713–737.

97 Zhornitsky S & Potvin S (2012) Cannabidiol in

humans-the quest for therapeutic targets.

Pharmaceuticals 5, 529–552.

98 Baker AL, Thornton LK, Hides L & Dunlop A (2012)

Treatment of cannabis use among people with

psychotic disorders: a critical review of randomised

controlled trials. Curr Pharm Des 18, 4923–4937.

99 Izzo AA, Borrelli F, Capasso R, Di Marzo V &

Mechoulam R (2009) Non-psychotropic plant

cannabinoids: new therapeutic opportunities from an

ancient herb. Trends Pharmacol Sci 30, 515–527.

100 Michalski CW, Oti FE, Erkan M, Sauliunaite D,

Bergmann F, Pacher P, Batkai S, Muller MW, Giese

NA, Friess H et al. (2008) Cannabinoids in pancreatic

cancer: correlation with survival and pain. Int J

Cancer 122, 742–750.

101 Tam J, Cinar R, Liu J, Godlewski G, Wesley D,

Jourdan T, Szanda G, Mukhopadhyay B, Chedester

L, Liow JS et al. (2012) Peripheral cannabinoid-1

receptor inverse agonism reduces obesity by reversing

leptin resistance. Cell Metab 16, 167–179.

102 Rajavashisth TB, Shaheen M, Norris KC, Pan D,

Sinha SK, Ortega J & Friedman TC (2012) Decreased

prevalence of diabetes in marijuana users: cross-

sectional data from the National Health and Nutrition

Examination Survey (NHANES) III. BMJ Open 2,

e000494.

103 Muniyappa R, Sable S, Ouwerkerk R, Mari A,

Gharib AM, Walter M, Courville A, Hall G, Chen

KY, Volkow ND et al. (2013) Metabolic effects of

chronic cannabis smoking. Diabetes Care 36, 1–8.

104 Hollister LE & Reaven GM (1974) Delta-9-

tetrahydrocannabinol and glucose tolerance. Clin

Pharmacol Ther 16, 297–302.

105 Murphy TD, Weidenbach KN, Houten CV, Gerona

RR, Moran JH, Kirschner RI, Maraffa JM, Stork

CM, Birkhead GS, Newman A et al. (2013) Acute

kidney injury associated with synthetic cannabinoid

use – multiple States, 2012. MMWR Morb Mortal

Wkly Rep 62, 93–98.

106 Molina PE, Winsauer P, Zhang P, Walker E, Birke L,

Amedee A, Stouwe CV, Troxclair D, McGoey R,

Varner K et al. (2011) Cannabinoid administration

attenuates the progression of simian immunodeficiency

virus. AIDS Res Hum Retroviruses 27, 585–592.

107 Agarwal N, Pacher P, Tegeder I, Amaya F,

Constantin CE, Brenner GJ, Rubino T, Michalski

CW, Marsicano G, Monory K et al. (2007)

Cannabinoids mediate analgesia largely via peripheral

type 1 cannabinoid receptors in nociceptors. Nat

Neurosci 10, 870–879.

108 Osei-Hyiaman D, DePetrillo M, Pacher P, Liu J,

Radaeva S, Batkai S, Harvey-White J, Mackie K,

Offertaler L, Wang L et al. (2005) Endocannabinoid

activation at hepatic CB1 receptors stimulates fatty

acid synthesis and contributes to diet-induced obesity.

J Clin Invest 115, 1298–1305.

109 Pagotto U, Marsicano G, Cota D, Lutz B & Pasquali

R (2006) The emerging role of the endocannabinoid

system in endocrine regulation and energy balance.

Endocr Rev 27, 73–100.

110 Despres JP, Golay A & Sjostrom L (2005) Effects of

rimonabant on metabolic risk factors in overweight

patients with dyslipidemia. N Engl J Med 353,

2121–2134.

111 Van Gaal LF, Rissanen AM, Scheen AJ, Ziegler O &

Rossner S (2005) Effects of the cannabinoid-1 receptor

blocker rimonabant on weight reduction and

cardiovascular risk factors in overweight patients:

1-year experience from the RIO-Europe study. Lancet

365, 1389–1397.

112 Scheen AJ, Van Gaal LG, Despres JP, Pi-Sunyer X,

Golay A & Hanotin C (2006) Rimonabant improves

cardiometabolic risk profile in obese or overweight

subjects: overview of RIO studies. Rev Med Suisse 2,

1916–1923.

113 Despres JP, Ross R, Boka G, Almeras N & Lemieux I

(2009) Effect of rimonabant on the high-triglyceride/

low-HDL-cholesterol dyslipidemia, intraabdominal

adiposity, and liver fat: the ADAGIO-Lipids trial.

Arterioscler Thromb Vasc Biol 29, 416–423.

114 Nissen SE, Nicholls SJ, Wolski K, Rodes-Cabau J,

Cannon CP, Deanfield JE, Despres JP, Kastelein JJ,

Steinhubl SR, Kapadia S et al. (2008) Effect of

rimonabant on progression of atherosclerosis in

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1935

P. Pacher and G. Kunos Targeting the endocannabinoid system

patients with abdominal obesity and coronary artery

disease: the STRADIVARIUS randomized controlled

trial. JAMA 299, 1547–1560.

115 Rosenstock J, Hollander P, Chevalier S & Iranmanesh

A (2008) SERENADE: the Study Evaluating

Rimonabant Efficacy in Drug-naive Diabetic Patients:

effects of monotherapy with rimonabant, the first

selective CB1 receptor antagonist, on glycemic control,

body weight, and lipid profile in drug-naive type 2

diabetes. Diabetes Care 31, 2169–2176.

116 Hollander PA, Amod A, Litwak LE & Chaudhari U

(2010) Effect of rimonabant on glycemic control in

insulin-treated type 2 diabetes: the ARPEGGIO trial.

Diabetes Care 33, 605–607.

117 Di Marzo V & Despres JP (2009) CB1 antagonists for

obesity–what lessons have we learned from

rimonabant? Nat Rev Endocrinol 5, 633–638.

118 Tam J, Vemuri VK, Liu J, Batkai S, Mukhopadhyay

B, Godlewski G, Osei-Hyiaman D, Ohnuma S,

Ambudkar SV, Pickel J et al. (2010) Peripheral CB1

cannabinoid receptor blockade improves

cardiometabolic risk in mouse models of obesity. J

Clin Investig 120, 2953–2966.

119 Liu J, Zhou L, Xiong K, Godlewski G,

Mukhopadhyay B, Tam J, Yin S, Gao P, Shan X,

Pickel J et al. (2012) Hepatic cannabinoid receptor-1

mediates diet-induced insulin resistance via inhibition

of insulin signaling and clearance in mice.

Gastroenterology 142, 1218–1228.

120 Klein TW, Newton C, Larsen K, Lu L, Perkins I,

Nong L & Friedman H (2003) The cannabinoid

system and immune modulation. J Leukoc Biol 74,

486–496.

121 Cabral GA & Staab A (2005) Effects on the immune

system. Handb Exp Pharmacol 168, 385–423.

122 Cencioni MT, Chiurchiu V, Catanzaro G, Borsellino

G, Bernardi G, Battistini L & Maccarrone M (2010)

Anandamide suppresses proliferation and cytokine

release from primary human T-lymphocytes mainly via

CB2 receptors. PLoS ONE 5, e8688.

123 Miller AM & Stella N (2008) CB2 receptor-mediated

migration of immune cells: it can go either way. Br J

Pharmacol 153, 299–308.

124 Buckley NE (2008) The peripheral cannabinoid

receptor knockout mice: an update. Br J Pharmacol

153, 309–318.

125 Van Sickle MD, Duncan M, Kingsley PJ, Mouihate

A, Urbani P, Mackie K, Stella N, Makriyannis A,

Piomelli D, Davison JS et al. (2005) Identification and

functional characterization of brainstem cannabinoid

CB2 receptors. Science 310, 329–332.

126 Onaivi ES (2006) Neuropsychobiological evidence for

the functional presence and expression of cannabinoid

CB2 receptors in the brain. Neuropsychobiology 54,

231–246.

127 Viscomi MT, Oddi S, Latini L, Pasquariello N,

Florenzano F, Bernardi G, Molinari M & Maccarrone

M (2009) Selective CB2 receptor agonism protects

central neurons from remote axotomy-induced

apoptosis through the PI3K/Akt pathway. J Neurosci

29, 4564–4570.

128 Beltramo M (2009) Cannabinoid type 2 receptor as a

target for chronic – pain. Mini Rev Med Chem 9,

11–25.

129 Wright KL, Duncan M & Sharkey KA (2008)

Cannabinoid CB2 receptors in the gastrointestinal

tract: a regulatory system in states of inflammation.

Br J Pharmacol 153, 263–270.

130 Marquez L, Suarez J, Iglesias M, Bermudez-Silva FJ,

Rodriguez de Fonseca F & Andreu M (2009)

Ulcerative colitis induces changes on the expression of

the endocannabinoid system in the human colonic

tissue. PLoS ONE 4, e6893.

131 Duncan M, Mouihate A, Mackie K, Keenan CM,

Buckley NE, Davison JS, Patel KD, Pittman QJ &

Sharkey KA (2008) Cannabinoid CB2 receptors in the

enteric nervous system modulate gastrointestinal

contractility in lipopolysaccharide-treated rats. Am J

Physiol Gastrointest Liver Physiol 295, G78–G87.

132 Bouchard JF, Lepicier P & Lamontagne D (2003)

Contribution of endocannabinoids in the endothelial

protection afforded by ischemic preconditioning in the

isolated rat heart. Life Sci 72, 1859–1870.

133 Rajesh M, Mukhopadhyay P, Hasko G, Huffman JW,

Mackie K & Pacher P (2008) CB2 cannabinoid

receptor agonists attenuate TNF-alpha-induced human

vascular smooth muscle cell proliferation and

migration. Br J Pharmacol 153, 347–357.

134 Rajesh M, Mukhopadhyay P, Batkai S, Hasko G,

Liaudet L, Huffman JW, Csiszar A, Ungvari Z,

Mackie K, Chatterjee S et al. (2007) CB2-receptor

stimulation attenuates TNF-alpha-induced

human endothelial cell activation, transendothelial

migration of monocytes, and monocyte-endothelial

adhesion. Am J Physiol Heart Circ Physiol 293,

H2210–H2218.

135 Rajesh M, Pan H, Mukhopadhyay P, Batkai S,

Osei-Hyiaman D, Hasko G, Liaudet L, Gao B &

Pacher P (2007) Cannabinoid-2 receptor agonist

HU-308 protects against hepatic ischemia/reperfusion

injury by attenuating oxidative stress, inflammatory

response, and apoptosis. J Leukoc Biol 82, 1382–1389.

136 Julien B, Grenard P, Teixeira-Clerc F, Van Nhieu JT,

Li L, Karsak M, Zimmer A, Mallat A & Lotersztajn S

(2005) Antifibrogenic role of the cannabinoid receptor

CB2 in the liver. Gastroenterology 128, 742–755.

137 Mallat A & Lotersztajn S (2008) Endocannabinoids

and liver disease. I. Endocannabinoids and their

receptors in the liver. Am J Physiol Gastrointest Liver

Physiol 294, G9–G12.

1936 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

138 Cao Z, Mulvihill MM, Mukhopadhyay P, Xu H,

Erd�elyi K, Hao E, Holovac E, Hasko G, Cravatt BF,

Nomura DK et al. (2013) Monoacylglycerol lipase

controls endocannabinoid and eicosanoid signaling

and hepatic injury in mice. Gastroenterology 144,

808–817.

139 Wang H, Dey SK & Maccarrone M (2006) Jekyll and

hyde: two faces of cannabinoid signaling in male and

female fertility. Endocr Rev 27, 427–448.

140 Maccarrone M (2009) Endocannabinoids: friends and

foes of reproduction. Prog Lipid Res 48, 344–354.

141 Rousseaux C, Thuru X, Gelot A, Barnich N, Neut C,

Dubuquoy L, Dubuquoy C, Merour E, Geboes K,

Chamaillard M et al. (2007) Lactobacillus acidophilus

modulates intestinal pain and induces opioid and

cannabinoid receptors. Nat Med 13, 35–37.

142 Bab I, Ofek O, Tam J, Rehnelt J & Zimmer A (2008)

Endocannabinoids and the regulation of bone

metabolism. J Neuroendocrinol 20 (Suppl 1), 69–74.

143 Bab I & Zimmer A (2008) Cannabinoid receptors and

the regulation of bone mass. Br J Pharmacol 153,

182–188.

144 Bab I, Zimmer A & Melamed E (2009) Cannabinoids

and the skeleton: from marijuana to reversal of bone

loss. Ann Med 41, 560–567.

145 Michalski CW, Laukert T, Sauliunaite D, Pacher P,

Bergmann F, Agarwal N, Su Y, Giese T, Giese NA,

Batkai S et al. (2007) Cannabinoids ameliorate pain

and reduce disease pathology in cerulein-induced acute

pancreatitis. Gastroenterology 132, 1968–1978.

146 Michalski CW, Maier M, Erkan M, Sauliunaite D,

Bergmann F, Pacher P, Batkai S, Giese NA, Giese T,

Friess H et al. (2008) Cannabinoids reduce markers of

inflammation and fibrosis in pancreatic stellate cells.

PLoS ONE 3, e1701.

147 Bermudez-Silva FJ, Suarez J, Baixeras E, Cobo N,

Bautista D, Cuesta-Munoz AL, Fuentes E, Juan-Pico

P, Castro MJ, Milman G et al. (2008) Presence of

functional cannabinoid receptors in human endocrine

pancreas. Diabetologia 51, 476–487.

148 Petrella C, Agostini S, Alema GS, Casolini P, Carpino

F, Giuli C, Improta G, Linari G, Petrozza V &

Broccardo M (2010) Cannabinoid agonist WIN55,212

in vitro inhibits interleukin-6 (IL-6) and monocyte

chemo-attractant protein-1 (MCP-1) release by rat

pancreatic acini and in vivo induces dual effects on the

course of acute pancreatitis. Neurogastroenterol Motil

22, 1248–1256.

149 Guzman M (2003) Cannabinoids: potential anticancer

agents. Nat Rev Cancer 3, 745–755.

150 Izzo AA & Camilleri M (2009) Cannabinoids in intestinal

inflammation and cancer. Pharmacol Res 60, 117–125.

151 Izzo AA & Sharkey KA (2010) Cannabinoids and the

gut: new developments and emerging concepts.

Pharmacol Ther 126, 21–38.

152 Fernandez-Ruiz J, Romero J, Velasco G, Tolon RM,

Ramos JA & Guzman M (2007) Cannabinoid CB2

receptor: a new target for controlling neural cell

survival? Trends Pharmacol Sci 28, 39–45.

153 Cabral GA, Raborn ES, Griffin L, Dennis J &

Marciano-Cabral F (2008) CB2 receptors in the brain:

role in central immune function. Br J Pharmacol 153,

240–251.

154 Fernandez-Ruiz J (2009) The endocannabinoid system

as a target for the treatment of motor dysfunction. Br

J Pharmacol 156, 1029–1040.

155 Pisanti S & Bifulco M (2009) Endocannabinoid system

modulation in cancer biology and therapy. Pharmacol

Res 60, 107–116.

156 Stella N (2010) Cannabinoid and cannabinoid-like

receptors in microglia, astrocytes, and astrocytomas.

Glia 58, 1017–1030.

157 Fowler CJ, Gustafsson SB, Chung SC, Persson E,

Jacobsson SO & Bergh A (2010) Targeting the

endocannabinoid system for the treatment of cancer – a practical view. Curr Top Med Chem 10, 814–827.

158 Ndong C, O’Donnell D, Ahmad S & Groblewski T

(2011) Cloning and pharmacological characterization

of the dog cannabinoid CB(2)receptor. Eur J

Pharmacol 669, 24–31.

159 Makriyannis A, Mechoulam R & Piomelli D (2005)

Therapeutic opportunities through modulation of the

endocannabinoid system. Neuropharmacology 48,

1068–1071.

160 Cravatt BF, Demarest K, Patricelli MP, Bracey MH,

Giang DK, Martin BR & Lichtman AH (2001)

Supersensitivity to anandamide and enhanced

endogenous cannabinoid signaling in mice lacking

fatty acid amide hydrolase. Proc Natl Acad Sci USA

98, 9371–9376.

161 Lichtman AH, Shelton CC, Advani T & Cravatt BF

(2004) Mice lacking fatty acid amide hydrolase exhibit

a cannabinoid receptor-mediated phenotypic

hypoalgesia. Pain 109, 319–327.

162 Kathuria S, Gaetani S, Fegley D, Valino F, Duranti

A, Tontini A, Mor M, Tarzia G, La Rana G,

Calignano A et al. (2003) Modulation of anxiety

through blockade of anandamide hydrolysis. Nat Med

9, 76–81.

163 Starowicz K, Makuch W, Osikowicz M, Piscitelli F,

Petrosino S, Di Marzo V & Przewlocka B (2012)

Spinal anandamide produces analgesia in neuropathic

rats: possible CB(1)- and TRPV1-mediated

mechanisms. Neuropharmacology 62, 1746–1755.

164 Roques BP, Fournie-Zaluski MC & Wurm M (2012)

Inhibiting the breakdown of endogenous opioids and

cannabinoids to alleviate pain. Nat Rev Drug

Discovery 11, 292–310.

165 Clapper JR, Moreno-Sanz G, Russo R, Guijarro A,

Vacondio F, Duranti A, Tontini A, Sanchini S,

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1937

P. Pacher and G. Kunos Targeting the endocannabinoid system

Sciolino NR, Spradley JM et al. (2010) Anandamide

suppresses pain initiation through a peripheral

endocannabinoid mechanism. Nat Neurosci 13,

1265–1270.

166 Batkai S, Pacher P, Osei-Hyiaman D, Radaeva S, Liu

J, Harvey-White J, Offertaler L, Mackie K, Rudd

MA, Bukoski RD et al. (2004) Endocannabinoids

acting at cannabinoid-1 receptors regulate

cardiovascular function in hypertension.

Circulation 110, 1996–2002.

167 Godlewski G, Alapafuja SO, Batkai S, Nikas SP,

Cinar R, Offertaler L, Osei-Hyiaman D, Liu J,

Mukhopadhyay B, Harvey-White J et al. (2010)

Inhibitor of fatty acid amide hydrolase normalizes

cardiovascular function in hypertension without

adverse metabolic effects. Chem Biol 17, 1256–1266.

168 Tourino C, Oveisi F, Lockney J, Piomelli D &

Maldonado R (2010) FAAH deficiency promotes

energy storage and enhances the motivation for food.

Int J Obes (Lond) 34, 557–568.

169 Brown WH, Gillum MP, Lee HY, Camporez JP,

Zhang XM, Jeong JK, Alves TC, Erion DM,

Guigni BA, Kahn M et al. (2012) Fatty acid amide

hydrolase ablation promotes ectopic lipid storage

and insulin resistance due to centrally mediated

hypothyroidism. Proc Natl Acad Sci USA 109,

14966–14971.

170 Ahn K, Smith SE, Liimatta MB, Beidler D,

Sadagopan N, Dudley DT, Young T, Wren P,

Zhang Y, Swaney S et al. (2011) Mechanistic and

pharmacological characterization of PF-04457845:

a highly potent and selective fatty acid amide

hydrolase inhibitor that reduces inflammatory and

noninflammatory pain. J Pharmacol Exp Ther

338, 114–124.

171 Di Marzo V (2012) Inhibitors of endocannabinoid

breakdown for pain: not so FA(AH)cile, after all. Pain

153, 1785–1786.

172 Gunduz-Cinar O, Macpherson KP, Cinar R, Gamble-

George J, Sugden K, Williams B, Godlewski G,

Ramikie TS, Gorka AX, Alapafuja SO et al. (2012)

Convergent translational evidence of a role for

anandamide in amygdala-mediated fear extinction,

threat processing and stress-reactivity. Mol Psychiatry

doi:10.1038/mp.2012.72.

173 Sticht MA, Long JZ, Rock EM, Limebeer CL,

Mechoulam R, Cravatt BF & Parker LA (2012)

Inhibition of monoacylglycerol lipase attenuates

vomiting in Suncus murinus and 2-arachidonoyl

glycerol attenuates nausea in rats. Br J Pharmacol 165,

2425–2435.

174 Nomura DK, Long JZ, Niessen S, Hoover HS, Ng

SW & Cravatt BF (2010) Monoacylglycerol lipase

regulates a fatty acid network that promotes cancer

pathogenesis. Cell 140, 49–61.

175 Schlosburg JE, Blankman JL, Long JZ, Nomura DK,

Pan B, Kinsey SG, Nguyen PT, Ramesh D, Booker L,

Burston JJ et al. (2010) Chronic monoacylglycerol

lipase blockade causes functional antagonism of the

endocannabinoid system. Nat Neurosci 13, 1113–1119. 176 Piro JR, Benjamin DI, Duerr JM, Pi Y, Gonzales C,

Wood KM, Schwartz JW, Nomura DK & Samad TA

(2012) A dysregulated endocannabinoid-eicosanoid

network supports pathogenesis in a mouse model of

Alzheimer’s disease. Cell Reports 1, 617–623.

177 Carloni S, Alonso-Alconada D, Girelli S, Duranti A,

Tontini A, Piomelli D, Hilario E, Alvarez A &

Balduini W (2012) Pretreatment with the

monoacylglycerol lipase inhibitor URB602 protects

from the long-term consequences of neonatal hypoxic-

ischemic brain injury in rats. Pediatr Res 72, 400–406.

178 Nomura DK, Morrison BE, Blankman JL, Long JZ,

Kinsey SG, Marcondes MC, Ward AM, Hahn YK,

Lichtman AH, Conti B et al. (2011) Endocannabinoid

hydrolysis generates brain prostaglandins that promote

neuroinflammation. Science 334, 809–813.

179 Mulvihill MM & Nomura DK (2013) Therapeutic

potential of monoacylglycerol lipase inhibitors. Life

Sci 92, 492–497.

180 Fowler CJ (2012) Monoacylglycerol lipase – a target

for drug development? Br J Pharmacol 166,

1568–1585.

181 Gatta L, Piscitelli F, Giordano C, Boccella S,

Lichtman A, Maione S & Di Marzo V (2012)

Discovery of prostamide F2alpha and its role in

inflammatory pain and dorsal horn nociceptive neuron

hyperexcitability. PLoS ONE 7, e31111.

182 Hsu KL, Tsuboi K, Adibekian A, Pugh H, Masuda K

& Cravatt BF (2012) DAGLbeta inhibition perturbs a

lipid network involved in macrophage inflammatory

responses. Nat Chem Biol 8, 999–1007.

183 Di Venere A, Dainese E, Fezza F, Angelucci BC,

Rosato N, Cravatt BF, Finazzi-Agro A, Mei G &

Maccarrone M (2012) Rat and human fatty acid

amide hydrolases: overt similarities and hidden

differences. Biochim Biophys Acta 1821, 1425–1433.

184 Di Filippo C, Rossi F, Rossi S & D’Amico M (2004)

Cannabinoid CB2 receptor activation reduces mouse

myocardial ischemia-reperfusion injury: involvement of

cytokine/chemokines and PMN. J Leukoc Biol 75,

453–459.

185 Montecucco F, Lenglet S, Braunersreuther V, Burger

F, Pelli G, Bertolotto M, Mach F & Steffens S (2009)

CB(2) cannabinoid receptor activation is

cardioprotective in a mouse model of ischemia/

reperfusion. J Mol Cell Cardiol 46, 612–620.

186 Defer N, Wan J, Souktani R, Escoubet B, Perier M,

Caramelle P, Manin S, Deveaux V, Bourin MC,

Zimmer A et al. (2009) The cannabinoid receptor type

2 promotes cardiac myocyte and fibroblast survival

1938 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

and protects against ischemia/reperfusion-induced

cardiomyopathy. FASEB J 23, 2120–2130.

187 Lamontagne D, Lepicier P, Lagneux C & Bouchard

JF (2006) The endogenous cardiac cannabinoid

system: a new protective mechanism against

myocardial ischemia. Arch Mal Coeur Vaiss 99,

242–246.

188 Weis F, Beiras-Fernandez A, Sodian R, Kaczmarek I,

Reichart B, Beiras A, Schelling G & Kreth S (2010)

Substantially altered expression pattern of cannabinoid

receptor 2 and activated endocannabinoid system in

patients with severe heart failure. J Mol Cell Cardiol

48, 1187–1193.

189 Batkai S, Jarai Z, Wagner JA, Goparaju SK,

Varga K, Liu J, Wang L, Mirshahi F, Khanolkar AD,

Makriyannis A et al. (2001) Endocannabinoids acting

at vascular CB1 receptors mediate the vasodilated

state in advanced liver cirrhosis. Nat Med 7, 827–832.

190 Moezi L, Gaskari SA & Lee SS (2008)

Endocannabinoids and liver disease. V.

endocannabinoids as mediators of vascular and

cardiac abnormalities in cirrhosis. Am J Physiol

Gastrointest Liver Physiol 295, G649–G653.

191 Batkai S, Mukhopadhyay P, Harvey-White J, Kechrid

R, Pacher P & Kunos G (2007) Endocannabinoids

acting at CB1 receptors mediate the cardiac contractile

dysfunction in vivo in cirrhotic rats. Am J Physiol

Heart Circ Physiol 293, H1689–H1695.

192 Batkai S & Pacher P (2009) Endocannabinoids and

cardiac contractile function: pathophysiological

implications. Pharmacol Res 60, 99–106.

193 Montecucco F, Matias I, Lenglet S, Petrosino S,

Burger F, Pelli G, Braunersreuther V, Mach F,

Steffens S & Di Marzo V (2009) Regulation and

possible role of endocannabinoids and related

mediators in hypercholesterolemic mice with

atherosclerosis. Atherosclerosis 205, 433–441.

194 Mach F & Steffens S (2008) The role of the

endocannabinoid system in atherosclerosis.

J Neuroendocrinol 20 (Suppl 1), 53–57.

195 Steffens S, Veillard NR, Arnaud C, Pelli G, Burger F,

Staub C, Karsak M, Zimmer A, Frossard JL & Mach

F (2005) Low dose oral cannabinoid therapy reduces

progression of atherosclerosis in mice. Nature 434,

782–786.

196 Montecucco F, Burger F, Mach F & Steffens S (2008)

CB2 cannabinoid receptor agonist JWH-015

modulates human monocyte migration through

defined intracellular signaling pathways. Am J Physiol

Heart Circ Physiol 294, H1145–H1155.

197 Pacher P & Ungvari Z (2008) Pleiotropic effects of the

CB2 cannabinoid receptor activation on human

monocyte migration: implications for atherosclerosis

and inflammatory diseases. Am J Physiol Heart Circ

Physiol 294, H1133–H1134.

198 Montecucco F, Di Marzo V, da Silva RF, Vuilleumier

N, Capettini L, Lenglet S, Pagano S, Piscitelli F,

Quintao S, Bertolotto M et al. (2012) The activation

of the cannabinoid receptor type 2 reduces

neutrophilic protease-mediated vulnerability in

atherosclerotic plaques. Eur Heart J 33, 846–856.

199 Naccarato M, Pizzuti D, Petrosino S, Simonetto M,

Ferigo L, Grandi FC, Pizzolato G & Di Marzo V

(2010) Possible anandamide and

palmitoylethanolamide involvement in human stroke.

Lipids Health Dis 9, 47.

200 Hillard CJ (2008) Role of cannabinoids and

endocannabinoids in cerebral ischemia. Curr Pharm

Des 14, 2347–2361.

201 Muthian S, Rademacher DJ, Roelke CT, Gross GJ &

Hillard CJ (2004) Anandamide content is increased

and CB1 cannabinoid receptor blockade is protective

during transient, focal cerebral ischemia. Neuroscience

129, 743–750.

202 Zhang M, Adler MW, Abood ME, Ganea D, Jallo J

& Tuma RF (2009) CB2 receptor activation attenuates

microcirculatory dysfunction during cerebral ischemic/

reperfusion injury. Microvasc Res 78, 86–94.

203 Zhang M, Martin BR, Adler MW, Razdan RK, Jallo

JI & Tuma RF (2007) Cannabinoid CB(2) receptor

activation decreases cerebral infarction in a mouse

focal ischemia/reperfusion model. J Cereb Blood Flow

Metab 27, 1387–1396.

204 Zhang M, Martin BR, Adler MW, Razdan RK,

Ganea D & Tuma RF (2008) Modulation of the

balance between cannabinoid CB(1) and CB(2)

receptor activation during cerebral ischemic/

reperfusion injury. Neuroscience 152, 753–760.

205 Baty DE, Zhang M, Li H, Erb CJ, Adler MW,

Ganea D, Loftus CM, Jallo JI & Tuma RF (2008)

Cannabinoid CB2 receptor activation attenuates motor

and autonomic function deficits in a mouse model of

spinal cord injury. Clin Neurosurg 55, 172–177.

206 Murikinati S, Juttler E, Keinert T, Ridder DA,

Muhammad S, Waibler Z, Ledent C, Zimmer A,

Kalinke U & Schwaninger M (2010) Activation of

cannabinoid 2 receptors protects against cerebral

ischemia by inhibiting neutrophil recruitment. FASEB

J 24, 788–798.

207 Kohro S, Imaizumi H, Yamakage M, Masuda Y,

Namiki A & Asai Y (2004) Reductions in levels of

bacterial superantigens/cannabinoids by plasma

exchange in a patient with severe toxic shock

syndrome. Anaesth Intensive Care 32, 588–591.

208 Kohro S, Imaizumi H, Yamakage M, Masuda Y,

Namiki A, Asai Y & Maruyama I (2006) Anandamide

absorption by direct hemoperfusion with polymixin

B-immobilized fiber improves the prognosis and

organ failure assessment score in patients with sepsis.

J Anesth 20, 11–16.

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1939

P. Pacher and G. Kunos Targeting the endocannabinoid system

209 Csoka B, Nemeth ZH, Mukhopadhyay P, Spolarics Z,

Rajesh M, Federici S, Deitch EA, Batkai S, Pacher P

& Hasko G (2009) CB2 cannabinoid receptors

contribute to bacterial invasion and mortality in

polymicrobial sepsis. PLoS ONE 4, e6409.

210 Tschop J, Kasten KR, Nogueiras R, Goetzman HS,

Cave CM, England LG, Dattilo J, Lentsch AB,

Tschop MH & Caldwell CC (2009) The cannabinoid

receptor 2 is critical for the host response to sepsis.

J Immunol 183, 499–505.

211 Kurabayashi M, Takeyoshi I, Yoshinari D,

Matsumoto K, Maruyama I & Morishita Y (2005) 2-

Arachidonoylglycerol increases in ischemia-reperfusion

injury of the rat liver. J Invest Surg 18, 25–31.

212 Batkai S, Osei-Hyiaman D, Pan H, El-Assal O, Rajesh

M, Mukhopadhyay P, Hong F, Harvey-White J, Jafri

A, Hasko G et al. (2007) Cannabinoid-2 receptor

mediates protection against hepatic ischemia/

reperfusion injury. FASEB J 21, 1788–1800.

213 Ishii Y, Sakamoto T, Ito R & Yanaga K (2010)

F2-isoprostanes and 2-arachidonylglycerol as

biomarkers of lipid peroxidation in pigs with hepatic

ischemia/reperfusion injury. J Surg Res 161, 139–145.

214 Mendez-Sanchez N, Zamora-Valdes D, Pichardo-

Bahena R, Barredo-Prieto B, Ponciano-Rodriguez G,

Bermejo-Martinez L, Chavez-Tapia NC, Baptista-

Gonzalez HA & Uribe M (2007) Endocannabinoid

receptor CB2 in nonalcoholic fatty liver disease. Liver

Int 27, 215–219.

215 Deveaux V, Cadoudal T, Ichigotani Y, Teixeira-Clerc

F, Louvet A, Manin S, Nhieu JT, Belot MP, Zimmer

A, Even P et al. (2009) Cannabinoid CB2 receptor

potentiates obesity-associated inflammation, insulin

resistance and hepatic steatosis. PLoS ONE 4, e5844.

216 Agudo J, Martin M, Roca C, Molas M, Bura AS,

Zimmer A, Bosch F & Maldonado R (2010)

Deficiency of CB2 cannabinoid receptor in mice

improves insulin sensitivity but increases food intake

and obesity with age. Diabetologia 53, 2629–2640.

217 Rajesh M, Batkai S, Kechrid M, Mukhopadhyay P,

Lee WS, Horvath B, Holovac E, Cinar R, Liaudet L,

Mackie K et al. (2012) Cannabinoid 1 receptor

promotes cardiac dysfunction, oxidative stress,

inflammation, and fibrosis in diabetic cardiomyopathy.

Diabetes 61, 716–727.

218 Cote M, Matias I, Lemieux I, Petrosino S, Almeras N,

Despres JP & Di Marzo V (2007) Circulating

endocannabinoid levels, abdominal adiposity and

related cardiometabolic risk factors in obese men. Int

J Obes (Lond) 31, 692–699.

219 Barutta F, Piscitelli F, Pinach S, Bruno G, Gambino

R, Rastaldi MP, Salvidio G, Di Marzo V, Cavallo

Perin P & Gruden G (2011) Protective role of

cannabinoid receptor type 2 in a mouse model of

diabetic nephropathy. Diabetes 60, 2386–2396.

220 Barutta F, Corbelli A, Mastrocola R, Gambino R,

Di Marzo V, Pinach S, Rastaldi MP, Perin PC &

Gruden G (2010) Cannabinoid receptor 1 blockade

ameliorates albuminuria in experimental diabetic

nephropathy. Diabetes 59, 1046–1054.

221 Annuzzi G, Piscitelli F, Di Marino L, Patti L, Giacco

R, Costabile G, Bozzetto L, Riccardi G, Verde R,

Petrosino S et al. (2010) Differential alterations of the

concentrations of endocannabinoids and related lipids

in the subcutaneous adipose tissue of obese diabetic

patients. Lipids Health Dis 9, 43.

222 Siegmund SV & Schwabe RF (2008)

Endocannabinoids and liver disease. II.

Endocannabinoids in the pathogenesis and treatment

of liver fibrosis. Am J Physiol Gastrointest Liver

Physiol 294, G357–G362.

223 Munoz-Luque J, Ros J, Fernandez-Varo G, Tugues S,

Morales-Ruiz M, Alvarez CE, Friedman SL, Arroyo

V & Jimenez W (2008) Regression of fibrosis after

chronic stimulation of cannabinoid CB2 receptor in

cirrhotic rats. J Pharmacol Exp Ther 324, 475–483.

224 Zyromski NJ, Mathur A, Pitt HA, Wade TE, Wang S,

Swartz-Basile DA, Prather AD & Lillemoe KD (2009)

Cannabinoid receptor-1 blockade attenuates acute

pancreatitis in obesity by an adiponectin mediated

mechanism. J Gastrointest Surg 13, 831–838.

225 Borrelli F & Izzo AA (2009) Role of

acylethanolamides in the gastrointestinal tract with

special reference to food intake and energy balance.

Best Pract Res Clin Endocrinol Metab 23, 33–49.

226 Wright K, Rooney N, Feeney M, Tate J, Robertson

D, Welham M & Ward S (2005) Differential

expression of cannabinoid receptors in the human

colon: cannabinoids promote epithelial wound healing.

Gastroenterology 129, 437–453.

227 Kimball ES, Schneider CR, Wallace NH & Hornby PJ

(2006) Agonists of cannabinoid receptor 1 and 2

inhibit experimental colitis induced by oil of mustard

and by dextran sulfate sodium. Am J Physiol

Gastrointest Liver Physiol 291, G364–G371.

228 Storr MA, Keenan CM, Zhang H, Patel KD,

Makriyannis A & Sharkey KA (2009) Activation of

the cannabinoid 2 receptor (CB2) protects against

experimental colitis. Inflamm Bowel Dis 15,

1678–1685.

229 Storr MA, Keenan CM, Emmerdinger D, Zhang H,

Yuce B, Sibaev A, Massa F, Buckley NE, Lutz B,

Goke B et al. (2008) Targeting endocannabinoid

degradation protects against experimental colitis in

mice: involvement of CB1 and CB2 receptors. J Mol

Med 86, 925–936.

230 Mukhopadhyay P, Rajesh M, Pan H, Patel V,

Mukhopadhyay B, Batkai S, Gao B, Hasko G &

Pacher P (2010) Cannabinoid-2 receptor limits

inflammation, oxidative/nitrosative stress, and cell

1940 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

death in nephropathy. Free Radic Biol Med 48,

457–467.

231 Mukhopadhyay P, Pan H, Rajesh M, Batkai S, Patel

V, Harvey-White J, Mukhopadhyay B, Hasko G, Gao

B, Mackie K et al. (2010) CB1 cannabinoid receptors

promote oxidative/nitrosative stress, inflammation and

cell death in a murine nephropathy model. Br J

Pharmacol 160, 657–668.

232 Horvath B, Mukhopadhyay P, Kechrid M, Patel V,

Tanchian G, Wink DA, Gertsch J & Pacher P (2012)

beta-Caryophyllene ameliorates cisplatin-induced

nephrotoxicity in a cannabinoid 2 receptor-dependent

manner. Free Radical Biol Med 52, 1325–1333.

233 Lim JC, Lim SK, Han HJ & Park SH (2010)

Cannabinoid receptor 1 mediates palmitic acid-induced

apoptosis via endoplasmic reticulum stress in human

renal proximal tubular cells. J Cell Physiol 225,

654–663.

234 Benito C, Nunez E, Tolon RM, Carrier EJ, Rabano

A, Hillard CJ & Romero J (2003) Cannabinoid CB2

receptors and fatty acid amide hydrolase are

selectively overexpressed in neuritic plaque-associated

glia in Alzheimer’s disease brains. J Neurosci 23,

11136–11141.

235 Benito C, Romero JP, Tolon RM, Clemente D,

Docagne F, Hillard CJ, Guaza C & Romero J (2007)

Cannabinoid CB1 and CB2 receptors and fatty acid

amide hydrolase are specific markers of plaque cell

subtypes in human multiple sclerosis. J Neurosci 27,

2396–2402.

236 Ramirez BG, Blazquez C, Gomez del Pulgar T,

Guzman M & de Ceballos ML (2005) Prevention of

Alzheimer’s disease pathology by cannabinoids:

neuroprotection mediated by blockade of microglial

activation. J Neurosci 25, 1904–1913.

237 Maresz K, Carrier EJ, Ponomarev ED, Hillard CJ &

Dittel BN (2005) Modulation of the cannabinoid CB2

receptor in microglial cells in response to

inflammatory stimuli. J Neurochem 95, 437–445.

238 Maresz K, Pryce G, Ponomarev ED, Marsicano G,

Croxford JL, Shriver LP, Ledent C, Cheng X, Carrier

EJ, Mann MK et al. (2007) Direct suppression of

CNS autoimmune inflammation via the cannabinoid

receptor CB1 on neurons and CB2 on autoreactive

T cells. Nat Med 13, 492–497.

239 Kim K, Moore DH, Makriyannis A & Abood ME

(2006) AM1241, a cannabinoid CB2 receptor selective

compound, delays disease progression in a mouse

model of amyotrophic lateral sclerosis. Eur J

Pharmacol 542, 100–105.

240 Shoemaker JL, Seely KA, Reed RL, Crow JP &

Prather PL (2007) The CB2 cannabinoid agonist AM-

1241 prolongs survival in a transgenic mouse model of

amyotrophic lateral sclerosis when initiated at

symptom onset. J Neurochem 101, 87–98.

241 Price DA, Martinez AA, Seillier A, Koek W, Acosta

Y, Fernandez E, Strong R, Lutz B, Marsicano G,

Roberts JL et al. (2009) WIN55,212–2, a cannabinoid

receptor agonist, protects against nigrostriatal cell loss

in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

mouse model of Parkinson’s disease. Eur J Neurosci

29, 2177–2186.

242 Benito C, Tolon RM, Pazos MR, Nunez E, Castillo

AI & Romero J (2008) Cannabinoid CB2 receptors in

human brain inflammation. Br J Pharmacol 153,

277–285.

243 Palazuelos J, Aguado T, Pazos MR, Julien B,

Carrasco C, Resel E, Sagredo O, Benito C, Romero J,

Azcoitia I et al. (2009) Microglial CB2 cannabinoid

receptors are neuroprotective in Huntington’s disease

excitotoxicity. Brain 132, 3152–3164.

244 Palazuelos J, Davoust N, Julien B, Hatterer E,

Aguado T, Mechoulam R, Benito C, Romero J,

Silva A, Guzman M et al. (2008) The CB(2)

cannabinoid receptor controls myeloid progenitor

trafficking: involvement in the pathogenesis of an

animal model of multiple sclerosis. J Biol Chem 283,

13320–13329.

245 Sagredo O, Gonzalez S, Aroyo I, Pazos MR,

Benito C, Lastres-Becker I, Romero JP, Tolon RM,

Mechoulam R, Brouillet E et al. (2009) Cannabinoid

CB2 receptor agonists protect the striatum against

malonate toxicity: relevance for Huntington’s disease.

Glia 57, 1154–1167.

246 Tolon RM, Nunez E, Pazos MR, Benito C, Castillo

AI, Martinez-Orgado JA & Romero J (2009) The

activation of cannabinoid CB2 receptors stimulates in

situ and in vitro beta-amyloid removal by human

macrophages. Brain Res 1283, 148–154.

247 De March Z, Zuccato C, Giampa C, Patassini S,

Bari M, Gasperi V, De Ceballos ML, Bernardi G,

Maccarrone M, Cattaneo E et al. (2008) Cortical

expression of brain derived neurotrophic factor and

type-1 cannabinoid receptor after striatal excitotoxic

lesions. Neuroscience 152, 734–740.

248 Mestre L, Docagne F, Correa F, Loria F,

Hernangomez M, Borrell J & Guaza C (2009) A

cannabinoid agonist interferes with the progression of

a chronic model of multiple sclerosis by

downregulating adhesion molecules. Mol Cell Neurosci

40, 258–266.

249 Loria F, Petrosino S, Hernangomez M, Mestre L,

Spagnolo A, Correa F, Di Marzo V, Docagne F &

Guaza C (2010) An endocannabinoid tone limits

excitotoxicity in vitro and in a model of multiple

sclerosis. Neurobiol Dis 37, 166–176.

250 Pertwee RG (2005) The therapeutic potential of drugs

that target cannabinoid receptors or modulate the

tissue levels or actions of endocannabinoids. AAPS J

7, E625–E654.

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1941

P. Pacher and G. Kunos Targeting the endocannabinoid system

251 Calignano A, La Rana G, Giuffrida A & Piomelli D

(1998) Control of pain initiation by endogenous

cannabinoids. Nature 394, 277–281.

252 Hanus L, Breuer A, Tchilibon S, Shiloah S,

Goldenberg D, Horowitz M, Pertwee RG, Ross RA,

Mechoulam R & Fride E (1999) HU-308: a specific

agonist for CB(2), a peripheral cannabinoid receptor.

Proc Natl Acad Sci USA 96, 14228–14233.

253 Malan TP Jr, Ibrahim MM, Deng H, Liu Q, Mata

HP, Vanderah T, Porreca F & Makriyannis A (2001)

CB2 cannabinoid receptor-mediated peripheral

antinociception. Pain 93, 239–245.

254 Clayton N, Marshall FH, Bountra C &

O’Shaughnessy CT (2002) CB1 and CB2 cannabinoid

receptors are implicated in inflammatory pain. Pain

96, 253–260.

255 Ibrahim MM, Deng H, Zvonok A, Cockayne DA,

Kwan J, Mata HP, Vanderah TW, Lai J, Porreca F,

Makriyannis A et al. (2003) Activation of CB2

cannabinoid receptors by AM1241 inhibits

experimental neuropathic pain: pain inhibition by

receptors not present in the CNS. Proc Natl Acad Sci

USA 100, 10529–10533.

256 Ibrahim MM, Porreca F, Lai J, Albrecht PJ, Rice FL,

Khodorova A, Davar G, Makriyannis A, Vanderah

TW, Mata HP et al. (2005) CB2 cannabinoid receptor

activation produces antinociception by stimulating

peripheral release of endogenous opioids. Proc Natl

Acad Sci USA 102, 3093–3098.

257 Ibrahim MM, Rude ML, Stagg NJ, Mata HP, Lai J,

Vanderah TW, Porreca F, Buckley NE, Makriyannis

A & Malan TP Jr (2006) CB2 cannabinoid receptor

mediation of antinociception. Pain 122, 36–42.

258 Nackley AG, Makriyannis A & Hohmann AG (2003)

Selective activation of cannabinoid CB(2) receptors

suppresses spinal fos protein expression and pain

behavior in a rat model of inflammation. Neuroscience

119, 747–757.

259 Nackley AG, Suplita RL II & Hohmann AG (2003)

A peripheral cannabinoid mechanism suppresses spinal

fos protein expression and pain behavior in a rat

model of inflammation. Neuroscience 117, 659–670.

260 Nackley AG, Zvonok AM, Makriyannis A &

Hohmann AG (2004) Activation of cannabinoid CB2

receptors suppresses C-fiber responses and windup in

spinal wide dynamic range neurons in the absence and

presence of inflammation. J Neurophysiol 92,

3562–3574.

261 Quartilho A, Mata HP, Ibrahim MM, Vanderah TW,

Porreca F, Makriyannis A & Malan TP Jr (2003)

Inhibition of inflammatory hyperalgesia by activation

of peripheral CB2 cannabinoid receptors.

Anesthesiology 99, 955–960.

262 Elmes SJ, Jhaveri MD, Smart D, Kendall DA &

Chapman V (2004) Cannabinoid CB2 receptor

activation inhibits mechanically evoked responses of

wide dynamic range dorsal horn neurons in naive rats

and in rat models of inflammatory and neuropathic

pain. Eur J Neurosci 20, 2311–2320.

263 Hohmann AG, Farthing JN, Zvonok AM &

Makriyannis A (2004) Selective activation of

cannabinoid CB2 receptors suppresses hyperalgesia

evoked by intradermal capsaicin. J Pharmacol Exp

Ther 308, 446–453.

264 Scott DA, Wright CE & Angus JA (2004) Evidence

that CB-1 and CB-2 cannabinoid receptors mediate

antinociception in neuropathic pain in the rat. Pain

109, 124–131.

265 Whiteside GT, Lee GP & Valenzano KJ (2007) The

role of the cannabinoid CB2 receptor in pain

transmission and therapeutic potential of small

molecule CB2 receptor agonists. Curr Med Chem 14,

917–936.

266 Rahn EJ, Zvonok AM, Thakur GA, Khanolkar AD,

Makriyannis A & Hohmann AG (2008) Selective

activation of cannabinoid CB2 receptors suppresses

neuropathic nociception induced by treatment with the

chemotherapeutic agent paclitaxel in rats. J Pharmacol

Exp Ther 327, 584–591.

267 Muller-Vahl KR & Emrich HM (2008) Cannabis and

schizophrenia: towards a cannabinoid hypothesis of

schizophrenia. Expert Rev Neurother 8, 1037–1048.

268 Andreasson S, Allebeck P, Engstrom A & Rydberg U

(1987) Cannabis and schizophrenia. A longitudinal

study of Swedish conscripts. Lancet 2, 1483–1486.

269 De Marchi N, De Petrocellis L, Orlando P, Daniele F,

Fezza F & Di Marzo V (2003) Endocannabinoid

signalling in the blood of patients with schizophrenia.

Lipids Health Dis 2, 5.

270 Ishiguro H, Horiuchi Y, Ishikawa M, Koga M,

Imai K, Suzuki Y, Morikawa M, Inada T,

Watanabe Y, Takahashi M et al. (2010) Brain

cannabinoid CB2 receptor in schizophrenia. Biol

Psychiatry 67, 974–982.

271 Khan A, Kendall DA & Fone KCF (2010) The effects

of the cannabinoid CB2 receptor antagonist, AM630,

on isolation rearing-induced behavioural deficits in

rats. Schizophr Res 117, 391–392.

272 Grinspoon L & Bakalar JB (1995) Marihuana as

medicine. A plea for reconsideration. JAMA 273,

1875–1876.

273 Grinspoon L, Bakalar JB, Zimmer L & Morgan JP

(1997) Marijuana addiction. Science 277, 749; author

reply 750-2.

274 Onaivi ES, Ishiguro H, Gong JP, Patel S, Meozzi PA,

Myers L, Perchuk A, Mora Z, Tagliaferro PA,

Gardner E et al. (2008) Functional expression of brain

neuronal CB2 cannabinoid receptors are involved in

the effects of drugs of abuse and in depression. Ann

NY Acad Sci 1139, 434–449.

1942 FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works

Targeting the endocannabinoid system P. Pacher and G. Kunos

275 Garcia-Gutierrez MS, Perez-Ortiz JM, Gutierrez-Adan

A & Manzanares J (2010) Depression-resistant

endophenotype in mice overexpressing cannabinoid

CB(2) receptors. Br J Pharmacol 160, 1773–1784.

276 Hu B, Doods H, Treede RD & Ceci A (2009)

Depression-like behaviour in rats with

mononeuropathy is reduced by the CB2-selective

agonist GW405833. Pain 143, 206–212.

277 Garci AGMA & Manzanares J (2011) Overexpression

of CB2 cannabinoid receptors decreased vulnerability

to anxiety and impaired anxiolytic action of

alprazolam in mice. J Psychopharmacol 25, 11–20.

278 Richardson D et al. (2008) Characterisation of the

cannabinoid receptor system in synovial tissue and

fluid in patients with osteoarthritis and rheumatoid

arthritis. Arthritis Res Ther 10, R43.

279 Blazquez C, Carracedo A, Barrado L, Real PJ,

Fernandez-Luna JL, Velasco G, Malumbres M &

Guzman M (2006) Cannabinoid receptors as novel

targets for the treatment of melanoma. FASEB J 20,

2633–2635.

280 Zheng D, Bode AM, Zhao Q, Cho YY, Zhu F, Ma

WY & Dong Z (2008) The cannabinoid receptors are

required for ultraviolet-induced inflammation and skin

cancer development. Cancer Res 68, 3992–3998.

281 McKallip RJ, Lombard C, Fisher M, Martin BR,

Ryu S, Grant S, Nagarkatti PS & Nagarkatti M

(2002) Targeting CB2 cannabinoid receptors as a

novel therapy to treat malignant lymphoblastic

disease. Blood 100, 627–634.

282 Guida M, Ligresti A, De Filippis D, D’Amico A,

Petrosino S, Cipriano M, Bifulco G, Simonetti S,

Orlando P, Insabato L et al. (2010) The levels of

the endocannabinoid receptor CB2 and its ligand

2-arachidonoylglycerol are elevated in endometrial

carcinoma. Endocrinology 151, 921–928.

FEBS Journal 280 (2013) 1918–1943 ª Journal compilation ª 2013 FEBS. No claim to original US Government works 1943

P. Pacher and G. Kunos Targeting the endocannabinoid system

Copyright of FEBS Journal is the property of Wiley-Blackwell and its content may not be copied or emailed to

multiple sites or posted to a listserv without the copyright holder's express written permission. However, users

may print, download, or email articles for individual use.