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Cannabinoids as

Therapeutics

Edited by R. Mechoulam

Birkhäuser Verlag

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Raphael Mechoulam

Medical Faculty

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Contents

List of contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX

Ethan Russo

Cannabis in India: ancient lore and modern medicine . . . . . . . . . . . . 1

Lumír O. Hanusˇ and Raphael Mechoulam

Cannabinoid chemistry: an overview . . . . . . . . . . . . . . . . . . . . . . . . . 23

Roger G. Pertwee

Cannabidiol as a potential medicine . . . . . . . . . . . . . . . . . . . . . . . . . 47

Mauro Maccarrone

Endocannabinoids and regulation of fertility . . . . . . . . . . . . . . . . . . . 67

Javier Fernández-Ruiz, Sara González, Julián Romero and

José Antonio Ramos

Cannabinoids in neurodegeneration and neuroprotection . . . . . . . . . . 79

Stephen A. Varvel and Aron H. Lichtman

Role of the endocannabinoid system in learning and memory . . . . . . 111

Richard E. Musty

Cannabinoids and anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Susan M. Huang and J. Michael Walker

Cannabinoid targets for pain therapeutics . . . . . . . . . . . . . . . . . . . . . 149

Luciano De Petrocellis, Maurizio Bifulco, Alessia Ligresti and

Vincenzo Di Marzo

Potential use of cannabimimetics in the treatment of cancer . . . . . . . 165

Linda A. Parker, Cheryl L. Limebeer and Magdalena Kwiatkowska

Cannabinoids: effects on vomiting and nausea in animal models . . . . 183

Itai A. Bab

The skeleton: stone bones and stoned heads? . . . . . . . . . . . . . . . . . . . 201

Daniela Parolaro and Tiziana Rubino

Cannabinoids and drugs of abuse . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

Francis Barth and Murielle Rinaldi-Carmona

Cannabinoids in appetite and obesity . . . . . . . . . . . . . . . . . . . . . . . . . 219

Geoffrey W. Guy and Colin G. Stott

The development of Sativex®– a natural cannabis-based medicine . . 231

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

VII

List of contributors

Itai A. Bab, Bone Laboratory, The Hebrew University of Jerusalem, P.O.B.

12272, Jerusalem 91120, Israel; babi@cc.huji.ac.il

Francis Barth, Sanofi-aventis, 371, rue du Professeur Joseph Blayac, 34184

Montpellier Cedex 04, France, e-mail: francis.barth@sanofi-aventis.com

Maurizio Bifulco, Istituto di Endocrinologia ed Oncologia Sperimentale,

Consiglio Nazionale delle Ricerche, and Dipartimento di Scienze

Farmaceutiche, Università degli Studi di Salerno, via Ponte Don Melillo,

84084 Fisciano (SA), Italy

Luciano de Petrocellis, Istituto di Cibernetica “Eduardo Caianiello”, Consiglio

Nazionale delle Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti,

Fabbricato 70, 80078 Pozzuoli (Napoli), Italy

Vincenzo Di Marzo, Istituto di Chimica Biomolecolare, Consiglio Nazionale

delle Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, Fabbricato

70, 80078 Pozzuoli (Napoli), Italy; e-mail: vdimarzo@icmib.na.cnr.it

Javier Fernández-Ruiz, Departamento de Bioquímica y Biología Molecular,

Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain;

e-mail: jjfr@med.ucm.es

Sara González, Departamento de Bioquímica y Biología Molecular, Facultad

de Medicina, Universidad Complutense, 28040 Madrid, Spain; e-mail:

sgrc@med.ucm.es

Geoffrey W. Guy, GW Pharmaceuticals plc, Porton Down Science Park,

Salisbury, Wiltshire, SP4 OjQ, UK

Lumír O. Hanusˇ, Department of Medicinal Chemistry and Natural Products,

Pharmacy School, Medical Faculty, Hebrew University, Ein Kerem

Campus, 91120 Jerusalem, Israel; e-mail: lumir@cc.huji.ac.il

Susan M. Huang, Department of Neuroscience Brown University, Providence,

RI 02912, USA

Magdalena Kwiatkowska, Department of Psychology, Wilfrid Laurier

University, Waterloo, Ontario N2L 3C5, Canada

Aron H. Lichtman, Department of Pharmacology and Toxicology, Virginia

Commonwealth University, PO Box 980613, Richmond, VA 23298, USA;

e-mail: alichtma@hsc.vcu.edu

Alessia Ligresti, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle

Ricerche, Via Campi Flegrei 34, Comprensorio Olivetti, Fabbricato 70,

80078 Pozzuoli (Napoli), Italy

Cheryl L. Limebeer, Department of Psychology, Wilfrid Laurier University,

Waterloo, Ontario N2L 3C5, Canada

Mauro Maccarrone, Department of Biomedical Sciences, University of Teramo,

Piazza A. Moro 45, 64100 Teramo, Italy; e-mail: mmaccarrone@unite.it

Raphael Mechoulam, Medical Faculty, Hebrew University, Ein Kerem

Campus, 91120 Jerusalem, Israel; e-mail: mechou@cc.huji.ac.il

Richard E. Musty, Department of Psychology, University of Vermont,

Burlington, VT 05405, USA; e-mail: musty@uvm.edu

Linda A. Parker, Department of Psychology, Wilfrid Laurier University,

Waterloo, Ontario N2L 3C5, Canada; e-mail: lparker@wlu.ca

Daniela Parolaro, Center of Neuroscience, University of Insubria, Via A. da

Giussano 10, 20152 Busto Arsizio (VA), Italy; e-mail: daniela.parolaro

@uninsubria.it

Roger G. Pertwee, School of Medical Sciences, Institute of Medical Sciences,

University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland;

e-mail: rgp@abdn.ac.uk

José Antonio Ramos, Departamento de Bioquímica y Biología Molecular,

Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain;

e-mail: jara@med.ucm.es

Julián Romero, Laboratorio de Apoyo a la Investigación, Fundación Hospital

Alcorcón, 28922 Alcorcón, Madrid, Spain; e-mail: jromero@fhalcorcon.es

Murielle Rinaldi-Carmona, Sanofi-aventis, 371, rue du Professeur Joseph

Blayac, 34184 Montpellier Cedex 04, France, e-mail: murielle.rinaldi-

carmona@sanofi-aventis.com

Tiziana Rubino, Center of Neuroscience, University of Insubria, Via A. da

Giussano 10, 20152 Busto Arsizio (VA), Italy

Ethan Russo, GW Pharmaceuticals, 2235 Wylie Avenue, Missoula, MT 59809,

USA; e-mail: erusso@montanadsl.net

Colin G. Stott, GW Pharmaceuticals plc, Porton Down Science Park,

Salisbury, Wiltshire, SP4 OjQ, UK; e-mail: cgs@gwpharm.com

Stephen A. Varvel, Department of Pharmacology and Toxicology, Virginia

Commonwealth University, PO Box 980613, Richmond, VA 23298, USA

J. Michael Walker, Department of Psychology, Indiana University, 1101 E 10th

Street, Bloomington IN 47405-7007, USA; e-mail: walkerjm@indiana.edu

VIII List of contributors

Preface

Twenty years ago the endocannabinoid system was unknown. We knew much

about the use over millennia of Cannabis plant preparations both as a medi-

cine and as “a drug that takes away the mind” (as so-well stated in ancient

Assyrian clay tablets). During the early part of the last century considerable

progress was made on the chemistry and pharmacology of Cannabis, but it

was only after the identification in 1964 of ∆9-tetrahydrocannabinol (∆9-THC)

as the active constituent of the plant that this field caught the interest of many

research groups and hundreds of papers on the chemistry, biochemistry,

metabolism and clinical effects of this compound were published. However, its

mechanism of action remained unknown for nearly two decades. In the mid-

1980s the presence of a cannabinoid receptor in the brain was identified and

shortly thereafter it was cloned. This was followed by the isolation of the

major endogenous cannabinoids, anandamide and 2-arachidonoyl glycerol,

and the clarification of their biosyntheses and degradations. These advances

led to an avalanche of publications in a wide variety of fields. We are now in

the midst of major advances in biochemistry/physiology associated with the

actions of the endocannabinoids.

This short volume tries to present an up-to-date picture in some of the major

fields of endocannabinoid research. The first chapter in this book, on the use

of Cannabis in India, can be viewed as an expression of thanks to the herbal

practitioners, who for centuries passed on the medical traditions associated

with the drug. The chemistry chapter is a short summary of active plant, syn-

thetic and endogenous cannabinoids being investigated today, many of which

are mentioned later in the book. Cannabidiol is an unusual cannabinoid – it

does not bind to the known receptors and yet exerts a variety of effects. Hence

a chapter is devoted to it. Most of the remaining chapters deal with the endo-

cannabinoid system and the endocannabinoids in a variety of conditions and

physiological systems. A chapter describes the research done on Sativex®,a

standardized plant extract, shortly to be introduced in Canada as a drug for

multiple sclerosis symptoms.

Numerous fields known to be affected by cannabinoids were not reviewed.

The vast expanse of emotions is one of them. Most marijuana users smoke the

drug in order to ‘get high’. But we know very little about the mechanisms

through which cannabis affects emotions. Under certain circumstances

∆9-THC causes aggression, although usually it leads to sedation. Anxiety is

another emotional aspect affected by cannabinoids. Although a short chapter is

devoted to the calming of anxiety by cannabinoids it does not attempt to pres-

ent a mechanistic picture. And we know next to nothing on the chemistry link-

X Preface

ing endocannabinoids with stress, fear, love, satisfaction or despair. Are the

endocannabinoids one of nature’s tools to shape emotions? This is probably

one of the fields which will be explored in the future. But books review the

past. Possibly the next edition of this book, in 5 or 10 years time, will report

on the progress made in associating endocannabinoids with emotions. Until

then we shall have to remain content with more mundane topics such as neu-

roprotection, reproduction, appetite and effects on cancer.

The multitude of endocannabinoid effects seems like a fertile field for

exploration by pharmaceutical firms. We soon expect to see the introduction of

a synthetic cannabinoid antagonist in the treatment of obesity and, possibly

later, drugs for neuroprotection, pain, multiple sclerosis, rheumatoid arthritis

and cancer. Will post-traumatic stress disorder, schizophrenia and Tourette’s

syndrome come next?

Raphael Mechoulam Jerusalem, January 2005

Cannabis in India: ancient lore and modern

medicine

Ethan Russo

GW Pharmaceuticals, 2235 Wylie Avenue, Missoula, MT 59809, USA

Introduction: Ayurvedic medicine

India is a land steeped in faith and mysticism. Ayurveda, combining the

Sanskrit words for life and knowledge, is a system of medicine intertwined

inextricably with these traits. That a core of belief combined with empirical

experimentation could produce a viable medical regimen still widely practiced

after well over 3000 years is astounding to Western physicians. Cannabis was

similarly bound to faith and mysticism in India in the past, in the Hindu and

Islamic traditions, as well as in numerous other minority religions [1]. Merlin

recently explained it well [2], “with the powerful tools of modern science and

human imagination, our understanding of our deep-rooted desire to experience

ecstasy in the original sense of the word (to break the mind free from the body

and communicate with the ‘gods’ or the ancestors) will become clear with

time”. This chapter will seek to examine the medical claims for cannabis of the

past, and place them in a contemporary light given current pharmacological

knowledge.

Ayurveda is based on a conceptual medical system that seeks to balance

three functional elements, called doshas, that the human body is composed of,

and are commonly represented as Vat a or Vayu (ether or air), Pitta (fire and

water) and Kapha (phlegm or water and earth). Nadkarni [3] has rejected these

simple relationships in favor of more abstract assignations [3]:

“…the word Vayu, does not imply ‘Wind’ in Ayurvedic literature, but

comprehends all the phenomena which come under the functions of the

Central and Sympathetic Nervous Systems; that the word Pitta does not

essentially mean ‘Bile’ but signifies the functions of Thermogenesis or

heat production and metabolism, comprehending in its scope the

process of digestion, coloration of blood and formation of various

secretions and excretions and that the word Kapha does not mean

‘Phlegm’ but is used primarily to imply the functions of Thermo-taxis

or heat regulation and secondarily formation of the various preservative

fluids, e.g., Mucus, Synovia, etc., …”

Cannabinoids as Therapeutics

Edited by R. Mechoulam

Good health in Ayurveda is dependent upon attaining an equilibrium state

of these factors. Disease is due to an imbalance or disharmony of the Tridosha

system as the results of some cause, internal or external. A disease of pro-

longed extent will overflow its site of origin and spread through the body.

Therapy is effected by a combination of religious, magical and prescriptive

regimens, herbal therapy being an important element of the latter.

According to Kapoor [4], the materia medica of India comprises in excess

of 2000 drugs, mostly of vegetal origin, with 700 medicinal plants known even

during Buddhist times, c.250 BCE. Cannabis remains important among them.

Cannabis: its history in the medicine of India

Cannabis sativa seems to have diffused from a geographic point in Central

Asia, according to classical plant explorers [5–8] and more modern authorities

[9–11]. Sharma [12] felt its origin was in the Himalayan foothills, but offered

little documentation. This botanical sleuthing has been supported by physical

evidence of cannabis flowers and seeds associated with haoma-soma religious

rites in ancient Bactrian sites in excavations by Sarianidi [13, 14] in Margiana

(present day Turkmenistan), dating to the second millennium BCE. Philological

support derives from the term bhanga, also seemingly originating among the

Central Asian Arya peoples [15].

The Zend-Avesta, the holy book of Zoroastrianism, which survives in frag-

ments, dating from around 600 BCE in Persia, alludes to the use of Banga in a

medical context, identified as hemp [16]. Of this use, Bouquet stated [10]: “It

is solely to its inebriating properties that hemp owes the signal honour of being

sung in the Vedas, and it was probably the peoples of Northern Iran who dis-

covered those properties, for they were already using the leaves (Cheng) and

the resin (Cers) as inebriants before the Hindus.” Mahdihassan [17] has

attempted to draw a philological link between the Ho-Ma of the Chinese, the

Hao-Ma of the Avesta and the So-Ma of Sanskrit, felt cognate to cannabis.

The earliest written reference to cannabis in India may occur in the

Atharvaveda, dating to about 1500 BCE [18]: “We tell of the five kingdoms of

herbs headed by Soma; may it, and kusa grass, and bhanga and barley, and the

herb saha, release us from anxiety.” Grierson [18] suggested this to be part of

an offering, and ingestion or burning would both be typical of ancient practices

for this purpose.

In the Sushruta Samhita (meaning the verses of Sushruta), perhaps dating

from the third to the eighth centuries BCE, cannabis was recommended for

phlegm, catarrh and diarrhea [18]. As noted, an anti-phlegmatic would be

interpreted in Ayurvedic medicine as possessing a wide variety of effects.

Similarly, Dwarakanath [19] has maintained that cannabis was employed in

Indian folk medicine in aphrodisiacs and treatments for pain in the same era

[19], while Sanyal observed [20] that “They also used the fumes of burning

Indian Hemp (Canabis Indica) [sic] as an anaesthetic from ancient times…”.

2E. Russo

Watt [21] felt that by this early date the sexual dimorphism of cannabis was

already evident to its cultivators, as well as the superiority of bhanga (mistak-

enly assigned as female) for cordage, and bhang (mistaken as male) for med-

ical and mystical application. It was also likely about this time that the prepa-

ration of ganja (labeled sinsemilla in contemporary North America) was devel-

oped by isolating female cannabis plants to prevent fertilization, and increase

resin production.

Aldrich [22] documented the development of tantric cannabis usage around

the seventh century as a mingling of Shaivite Hinduism and Tibetan

Buddhism. Apparently, the 11th century text, Mahanirvana Tantra, is current-

ly still consulted with regard to sexual practices, withholding of male ejacula-

tion and promotion of sexual pleasure in both genders.

An anonymous work, Anandakanda, added some 43 Sanskrit cannabis syn-

onyms (Tab. 1) [23], many attesting the remarkable rejuvenating effects of

cannabis. Dash [23] described the lengthy methods of cultivation, processing

and mixing of cannabis with eight other medicinal plants, that when combined

with personal isolation and celibacy for 3 years produce a result in which “it

is claimed that the man lives for 300 years free from any disease and signs of

old age”. He dated this work to the 10th century, while Rao [24] placed it in

the range of the 9th to the 12th centuries, and noted some 10 known manu-

scripts.

There is philological debate among Sanskrit scholars as to whether the iden-

tification of bhanga as cannabis can be authenticated before the year 1000 [25,

26]. Wujastyk [26] and Meulenbeld [25] dated the Anandakanda, or Root of

Bliss, to c.1200, also noting its full accounting of cannabis’ side effects. Their

candidate for the first uncontested source for cannabis is the

Cikitsasarasangraha of the Bengal author Vangasena, in the late 11th century,

who included bhanga as an appetizer and digestive, noting it as “a drug like

opium whose mode of action is to pervade the whole body before being

absorbed and digested” [26]. It was also suggested in two recipes for a long

and happy life. A contemporary work, the Dhanvantariyanighantu, observed a

narcotic effect [26].

In the 12th–13th centuries from Gujarat, Nagarjuna’s Yogaratnamala (The

Garland of Jewels of Yoga), suggested cannabis smoke as a method by which

to produce an impression of spirit possession in one’s enemies [26].

The Rajanighantu of the 13th century added additional synonyms (Tab. 1),

with attributed activities characterized as [18] (1) katutva (acridity), (2)

kashayatva (astringency), (3) ushnatva (heat), (4) tiktatva (pungency), (5)

vatakaphapahatva (removing wind and phlegm), (6) samgrdhitva (astrin-

gency), (7) vakpradatva (speech giving), (8) balyatva (strength-giving), (9)

medhakaritva (inspiring of mental power) and (10) sreshthadipanatva (the

property of a most excellent excitant).

According to interpretation of this source [27], “Its effects on man are

described as excitant, heating, astringent; it destroys phlegm, expels flatulence,

induces costiveness, sharpens the memory, excites appetite, etc.”

Cannabis in India: ancient lore and modern medicine 3

4E. Russo

Table 1. Indian names for cannabis in Sanskrit and Hindi

Indian name Meaning

ajaya the unconquered, invincible

ananda the joyful, joyous, laughter moving, bliss

bahuvadini causing excessive garrulousness

bhang,bhanga hemp, mature cannabis leaves

bhangini breaks three kinds of misery

bharita the green one

capala agile, capricious, mischievous, scatter-brained

capta light-hearted

chapala the light-hearted, causer of reeling gait, causer of vacillation

charas cannabis resin (hashish), either hand-rubbed or sifted

cidalhada gives happiness to mind

divyaka gives pleasure, lustre, intoxication, beauty

dnayana vardhani knowledge promoter

ganja unfertilized female cannabis flowers

ganjakini the noisy, vibrator

gatra-bhanga body disintegrator

harshani joy-giver

harshini the exciter of sexual desire, the rejoicer, delight-giver, causer of elation

hursini the exciter of sexual desire

Indrasana Indra’s food

jaya victorious, the conquering

kalaghni helps to overcome death

madhudrava helps excrete nectar

madini the intoxicator, sex intoxicator

manonmana accomplishes the objects of the mind

matulani wife of the datura

matkunari an enemy of bugs

mohini fascinating

pasupasavinaini liberates creatures from earthly bonds

ranjika causer of excitement

sakrasana the worthy food of Indra

samvida manjari flower causes garrulousness

sana cannabis

sarvarogaghni which cures all diseases

sawi green leaved

Shivbooty Shiva’s plant

siddha which has attained spiritual perfection

sidhamuli on whose root is siddha

siddhapatri vessel of highest attainment

siddhi success giver

siddhidi which endows siddhi on others

sidhdi emancipation, beatitude, fruit of worship

suknidhan fountain of pleasures

tandrakrit causer of drowsiness

trailokya vijaya victorious in the three worlds, conqueror of the three regions of the universe

trilok kamaya desired in the three worlds

ununda the laughter mover

urjaya promoter of success

vijaya victorious, promoter of success, all-conquering

vijpatta the strong leaved

virapattra leaf of heroes

vrijapata strong nerved

About the same time, in the Sharangadhara Samhita, fresh extracts of

bhang were employed medicinally [19], and it was linked to opium: “Drugs

which act very quickly in the body first by spreading all over and undergoing

change later are vyavayi; for example, bhanga, ahiphena” [28]. Additionally,

cannabis was cited as an intoxicant and employed as the primary ingredient in

a therapeutic mixture of herbs: “This recipe known as jatiphaladi churna if

taken in doses of one karpa, with honey, relieves quickly grahani (sprue

[chronic diarrhea]), kasa (cough), swasa (dyspnoea), aruchi (anorexia), kshaya

(consumption) and pratishyaya [nasal congestion] due to vata kapha (rhinitis)”

[28]. Inter-relationships of Tantra and Ayurveda in this work were explored by

Sharma [29].

The 15th-century Rajavallabha, written by Sutradhar Mandan for Rana

Kumhha of Mewar, attributed several additional qualities to cannabis [18]:

“Indra’s food (i.e., ganja) is acid, produces infatuation, and destroys

leprosy. It creates vital energy, the mental powers and internal heat, cor-

rects irregularities of the phlegmatic humour, and is an elixir vitae. It

was originally produced, like nectar from the ocean by the churning

with Mount Mandara, and inasmuch as it gives victory in the three

worlds, it, the delight of the king of the gods, is called vijaya, the vic-

torious. This desire-fulfilling drug was obtained by men on the earth,

through desire for the welfare of all people. To those who regularly use

it, it begets joy and destroys every anxiety.”

Dymock added [27], “The Rahbulubha alludes to the use of hemp in gonor-

rhoea.”

According to Chopra and Chopra [30], “In Dhurtasamagama (A.D. 1500),

ganja is described as a soporific which ‘corrects derangements of humours and

produces a healthy appetite, sharpens the wit and act as an aphrodisiac’.” In the

Ayurveda Saukhyam of Todarananda [31] it was said of cannabis that “It caus-

es unconsciousness, intoxication and talkativeness”.

During the Renaissance European awareness of the psychoactivity of

cannabis was kindled with the writings of Garcia da Orta, a Spanish Jew, who

in the service of Portugal visited India in 1563. In addition to his descriptions

of the plant as bangue, and a good illustration, he noted important medical

properties [32], “The profit from its use is for the man to be beside himself,

and to be raised above all cares and anxieties, and it makes some break into a

foolish laugh.” In another passage, stimulation of energy and appetite was

noted: “Those of my servants who took it, unknown to me, said that it made

them so as not to feel work, to be very happy, and to have a craving for food.”

Soon thereafter, it was observed [30], “In Bhavaprakash (A.D. 1600),

cannabis is mentioned as ‘anti-phlegmatic, pungent, astringent and digestive’.

On account of these marked narcotic properties it was probably also used as an

anaesthetic, sometimes combined with alcohol, by the ancient Indian and

Chinese surgeons.”

Cannabis in India: ancient lore and modern medicine 5

The 18th century Persian medical text Makhzan-al-Adwiya, written by M.

Husain Khan, was extremely influential in the Unani Tibbi, or Arabic-tradition

medicine on the subcontinent. In it, cannabis was described in its various prepa-

rations as an intoxicant, stimulant and sedative, but also the following [33]:

“The leaves make a good snuff for deterging the brain; the juice of the

leaves applied to the head as a wash, removes dandriff [sic] and vermin;

drops of the juice thrown into the ear allay pain and destroy worms or

insects. It checks diarrhea, is useful in gonorrhea, restrains seminal

secretions, and is diuretic. The bark has a similar effect.

The powder is recommended as an external application to fresh

wounds and sores, and for causing granulations; a poultice of the boiled

root and leaves for discussing inflammations, and cure of erysipelas,

and for allaying neuralgic pains.”

Ali Gorji (personal communication, 2004) has recently consulted this work

and added that it was helpful for stomach problems, nausea and uterine inflam-

mation. Campbell [1], translated additional Persian names from this source:

“Bhang is the Joy-giver, the Sky-flier, the Heavenly-guide, the Poor Man’s

Heaven, the Soother of Grief”. Dymock and co-authors added a few more syn-

onyms [34]: “the inebriating leaf”, “fakir’s grass”, “the green tent” and “the

throne giver”. Chopra and Chopra [30] rendered another passage from the

Makhzan as follows: “It is said that bhang is one of the best of God’s gifts, it

is a cordial, a bile absorber, and an appetizer, and its moderate use prolongs

life. It quickens the fancy, deepens thought and sharpens judgment.”

A nexus with Western medicine

The medical use of so-called Indian hemp was reintroduced to the West in the

19th century. In 1813,Ainslie [35] cited the use of ganjah and bangie as intox-

icants, but also to treat diarrhea, and in a local application for hemorrhoids. In

1839, the seminal work of Sir William B. O’Shaughnessy on cannabis was

written [36], then republished in England in 1843 [33]. His contribution was a

model of modern investigation, involving a review of classical Sanskrit and

Unani sources, a description of cannabis preparations including bhang (mature

cannabis leaves), ganja (unfertilized female flowers), and charas (processed

cannabis resin), an examination of contemporary Indian ethnobotanical uses

and experiments of cannabis extracts in dogs, finally culminating with a series

of human clinical trials with appropriate cautious dose titration. His treatise on

the subject demonstrated the apparent clinical utility of cannabis in a wide

range of disorders including cholera, rheumatic diseases, delirium tremens and

infantile convulsions. For the first time miraculous recoveries were evidenced

in a series of tetanus victims due to cannabis. Noting the anti-spasmodic and

muscle-relaxant effects, it was tried in rabies, where [33] “the influence of a

6E. Russo

narcotic, capable either of cheering or of inducing harmless insensibility,

would be fraught with blessing to the wretched patient”. Although no cure was

forthcoming, the patient was visibly relieved of distress, and able to take some

sustenance through his suffering. Its palliative benefit was not lost upon the

physician, “the awful malady was stripped of its horrors; if not less fatal than

before, it was reduced to less than the scale of suffering which precedes death

from most ordinary diseases”. Summing up his experience with cannabis,

O’Shaughnessy concluded that “in hemp the profession has gained an

anti-convulsive remedy of the greatest value”.

A series of other practitioners both in India and in Great Britain soon noted

success in extending the use of cannabis to treatment of migraine, and neuro-

pathic and other pain conditions [37, 38]. Few clinical syndromes seemed

unassailable: another Western physician in India observed the alleviation not

only of an alcohol hangover with accompanying headache, but the patient’s

cholera as well [39]. Churchill employed cannabis to treat excessive uterine

bleeding [40], and Christison applied it to childbirth [41] (reviewed in [42]).

In little more than a decade, a section on cannabis was deemed worthy of

inclusion in Johnston’s The Chemistry of Common Life, wherein the topic was

treated at length [43]: “In India it is spoken of as the increaser of pleasure, the

exciter of desire, the cementer of friendship, the laughter-mover, and the

causer of the reeling gait, – all epithets indicative of its peculiar effects.” About

the same time, medical usage became common in North America [44].

In 1870, Dutt provided information on certain bhang preparations [45],

“Numerous confections of bhang such a Kamesvara modaka,Madana moda-

ka,Balyasakrasana modaka…are considered aphrodisiacs and are used in

chronic bowel complaints, and nervous debility.” A recipe for Madana moda-

ka was then supplied, containing numerous herbs, but with “hemp leaves with

flowers and seeds fried in clarified butter, equal in weight to all the other

ingredients”, which was “used in cough, chronic bowels complaints and

impotence”.

In 1877, Kerr submitted an extremely detailed report from Bengal encom-

passing history, religious context, cultivation and employment of cannabis in

all its preparations [46]. This would form one source for the subsequent

Report of the Indian Hemp Drugs Commission [47]. Documentation of ganja

production, necessitating culling of male plants by the “ganja doctor” to pre-

vent fertilization and increase resin production, was emphasized. Despite

some apparent value judgments expressed, the author observed, “I am of

opinion, however, that no moral gain whatever will be effected by the total

suppression of ganja.”

Watt noted that cannabis was [21] “valuable as a remedy for sick headache,

and especially in preventing such attacks. It removes the nervous effects of a

malady.” Watt listed numerous contemporary European physicians on the sub-

continent and their successes in treating a large variety of disorders with

cannabis preparations. Dymock was one such [34]: “I have given the extract in

doses of from 1/2to 1 grain to a large number of European hospital patients suf-

Cannabis in India: ancient lore and modern medicine 7

fering from chronic rheumatism; it entirely relieved the pains and made them

excessively talkative and jolly, complaining that they could not get enough to

eat.” Dymock also appreciated popular Indian descriptions of the time [34]:

“When the ganja pipe begins to smoke all cares at once disappear” and

“Smoke ganja and increase your knowledge”.

Cannabis in its various forms remained the focus of intense scrutiny, and

continued to harbor critics. Because of concerns of its moral dangers, the

British and colonial authorities in India organized a commission to examine all

aspects of the issue [47]. Its findings exceeded 3000 pages after exhaustive

investigation and testimony, and may be summarized as follows [48]. (1)

Moderate use of cannabis drugs had no appreciable physical effects on the

body. As with all drugs, excessive use could weaken the body and render it

more susceptible to diseases. Such circumstances were not peculiar to

cannabis, however. (2) Moderate use of cannabis drugs had no adverse effect

on the brain, except possibly for individuals predisposed to act abnormally.

Excessive use, on the other hand, could lead to mental instability and ulti-

mately to insanity in individuals predisposed by heredity to mental disorders.

(3) Moderate use of cannabis drugs had no adverse influence on morality.

Excessive usage, however, could result in moral degradation. Although in cer-

tain rare cases cannabis intoxication could result in violence, such cases were

few and far between.

The commission advocated against governmental suppression of cannabis

drugs. Many positive statements accompanied descriptions of their religious

associations, and particularly their legion medical usage, both human and vet-

erinary [1]:

“It is interesting, however, to note that while the drugs appear now to be

frequently used for precisely the same purposes and in the same man-

ner as was recommended centuries ago, many uses of these drugs by

native doctors are in accord with their application in modern European

therapeutics. Cannabis indica must be looked upon as one of the most

important drugs of Indian Materia Medica.”

Particular attention remained focused on possible mental health sequelae of

cannabis despite the lack of such findings from the Commission. In the con-

clusions of Mills [49]:

“Indians used hemp narcotics for a variety of reasons and it is entirely

possible that its use at certain times disagreed with certain individuals

to the extent that they became muddled or even murderous. Yet the few

of those that did become muddled or murderous and that were snared in

the net of the colonial state came to be taken as representative of all

those in India that used cannabis preparations. From this, colonial gov-

ernment developed an image of all Indian users of hemp narcotics as

dangerous, lunatic and potentially violent.”

8E. Russo

Occasionally, colonial officials were enlightened enough to free themselves

from ethnocentric chauvinism. One Captain R. Huddleston, a Deputy

Commissioner in the Akola District, wrote in 1872 [50], “Therefore I should

not condemn these compounds [cannabis preparations] as being directly con-

nected with crime; that is to say, they are no more the cause of offence than is

the bazar liquor with which the Banjara is so often primed when he does high-

way robbery, or the beer and gin guzzled by the British rough before he beats

his wife and assaults a policeman.” Modern epidemiological investigation

refutes the etiological relationship of cannabis to violence and insanity [51],

but the debate continues.

In 1897, cannabis retained a key indication [52], “The treatment of Tetanus

by smoking gunjah…promises to supercede all other in India.” Waring [52]

went on to describe its effective application at the onset of spasms, and titra-

tion to patient requirements so long as was needed. In a previous source [53],

smoking every few hours was recommended for the duration of need, which in

four subjects ranged from 7 days to 11/2months. Lucas [54] introduced the

concept of smoking cannabis for tetanus to the British medical press in 1880.

Meanwhile, cannabis spread to other British colonies with the Indian dias-

pora. Emigrants brought the herb along with them as a work accessory or med-

icine. In South Africa they adopted the local name dagga [55], whereas in

Jamaica the Indian name, ganja, has been pre-eminent since the 19th century

[56, 57], and its tonic effects are part of national medical lore today [57].

Politics and cannabis collide

At the dawn of the 20th century, cannabis suffered further downturns. In

1914 it was dropped from the pharmacopoeia of Ceylon (now Sri Lanka),

over the vociferous objections of its adherents, such as Ratnam [58], whose

points of debate included passionate defenses of its medical benefits and

poignant political arguments comparing its benign nature to the relative dan-

gers of other popular recreational agents, alcohol in particular. The status of

cannabis was compounded by increasingly severe quality-control problems

with material exported from India to the UK [59]. These two factors, politi-

cal and pharmacological, were paramount in the decline of cannabis medi-

cines in the West.

Cannabis use remained common in 20th-century India, however. It was

noted [60]:

“Labourers who have to do hard physical work use hemp drugs in small

quantities to alleviate the sense of fatigue, depression and sometimes

hunger. … This produces a sense of well-being, relieves fatigue, stimu-

lates the appetite, and induces a feeling of mild stimulation which

enables the worker to bear the strain and perhaps the monotony of this

daily routine of life more cheerfully.”

Cannabis in India: ancient lore and modern medicine 9

Similarly, by 1954, cannabis remained integral in Indian faith, as one Brahmin

explained to a Western writer [61], “‘It gives good bhakti’, …the sort of devo-

tional act which consists in emptying the mind of all worldly distractions and

thinking only of God.”

As late as 1957, two authorities in India noted [30], “Cannabis undoubted-

ly has remarkable therapeutic properties. …the drug has no constipating

action, it does not depress the respiratory centre; and there is little or no lia-

bility to addiction formation.” They went on to describe the usage in veteri-

nary medicine for diarrhea in livestock, treating parasites, “footsore disease,

increasing milk-flow in cows, and pacifying them, but also it is often admin-

istered to bullocks as a tonic, to relieve fatigue and to impart additional stay-

ing power.” As a human household remedy, “A mild beverage made from

bhang leaves is believed to sharpen the appetite and help the digestion.”

Religious mendicants were said to employ it for gastrointestinal and rheu-

matic afflictions during their peregrinations. Continued attestations were

claimed for dysmenorrhea, gonorrhea, dysuria, asthma and spasmodic condi-

tions. A fresh leaf poultice was said to reduce eye pain and conjunctivitis,

swollen joints and local inflammations, while a piece of charas placed in den-

tal caries was said to alleviate toothache. They noted, “Much of the sanctity

attached to bhang is put down to its supposed properties ‘clearing the head

and stimulating the brain to think’.” Finally, contributions to sexual perform-

ance were still claimed, as cannabis preparations “are frequently used by both

young and middle-aged individuals for stimulating sexual desire and pro-

longing the sexual act”.

Usage in Unani medicine at this time included treatment of insomnia,

migraine, neuralgic pains, asthma, spasmodic conditions and previously noted

gynecological conditions [30]. A continued contribution to Islamic mysticism

was also noted as cannabis use “frees them from worldly bonds, and induces

communion with the divine spirit”.

In another book about medicinal plants of India [62], the author stated:

“Charas…is a valuable narcotic, especially in cases where opium can-

not be administered; it is of great value in malarial and periodical

headaches, migraine, acute mania, whooping cough, cough of phthisis,

asthma, anaemia of brain, nervous vomiting, tetanus, convulsion, insan-

ity, delirium, dysuria, and nervous exhaustion; it is also used as an

anaesthetic in dysmenorrhea, as an appetizer and aphrodisiac, as an

anodyne in itching of eczema, neuralgia, severe pains of various kinds

of corns, etc.”

Indian charas of good quality is said to have a resin content of about 35–45%

[63], which according to the calculations of Clarke [64], might yield a theo-

retical tetrahydrocannabinol (THC) content of up to 30%. Higher concentra-

tions have been achieved with modern techniques.

10 E. Russo

Nadkarni [3] observed of cannabis, “All parts of the plant are intoxicating

(narcotic), stomachic, antispasmodic, analgesic (anodyne), stimulating, aphro-

disiac and sedative.”

In 1977, Sharma observed [65] that “even today [cannabis] is used with

restraint and judgment by students of Indian medicine. There are reports

claiming the value of cannabis in the treatment of high blood pressure,

migraine headaches, and even cancer.”

In a modern review of Indian uses of cannabis, it was observed [66] that

“Cannabis was used medicinally for almost all the ills flesh is heir to”.

Cannabis remained a key ingredient in two aphrodisiacal preparations,

Madana modaka and Kamesvara modaka [67].

In a treatise entitled Indigenous Drugs of India [68] the authors noted the

requirement of dose titration due to increasingly inconsistent cannabis prepa-

rations. This drawback was addressed in a prior study [69] in which the authors

extracted local ganja to produce a 17% THC yield, which at intraperitoneal

doses of 75 mg/kg in rats resulted in a potentiation of sub-analgesic doses of

morphine.

In 1988 [70] cannabis was still mentioned as a remedy for malaria and

blood poisoning, among many other indications. In neighboring Nepal,

cannabis retains ethnobotanical applications among some 15 ethnic groups

[71], for diarrhea, dysentery, local wound treatment and in veterinary medi-

cine. In discussing the native use of cannabis and opium products by village

doctors in India, who provided 80% of the population with their medical care

in a report to the United Nations, the author felt that a legitimate role for them

persisted [19]:

“These drugs should be allowed to be used by Ayurvedic and Unani

physicians until such time as the benefits of modern medicine are

extended to rural areas. Banning their use by the large mass of

Ayurvedic and Unani physicians for therapeutic purposes may create a

vacuum which may not be easily filled for a long time to come.”

Cannabis in contemporary Ayurvedic medicine

According to Chopra and Chopra [30], the modern Ayurvedic properties of

cannabis are: paphahari, promoting loosening, separation and the elimination

of phlegm; grahini, promoting retention and binding the bowels; pachani, pro-

moting digestion; ushna, promoting heat; pitala, exciting the flow of bile;

mada-vardhani, promoting talkativeness or releasing the volitional restraint of

speech; moda-vardhani, promoting happiness; vag-vardhani, stimulating the

digestive fire; dipani, stimulating appetite; ruchya, promoting taste; nidrapra-

da, hypnotic. Kapoor [4] described its Ayurvedic attributes as follows [4]: its

rasa (taste) is tikta (bitter); its guna (physical properties) are laghu (light, easy

to digest), teeshan (acute, pungent) and rooksha (ununctuous); its veerya

Cannabis in India: ancient lore and modern medicine 11

(energy modality or potency) is ushana (heating, digestive); and its vipaka

(transformation reactions after digestion) are katu (constipative, semen

increasing). Among its properties and uses, it is conceived of as: madakari

(causing intoxication), nidrajanan (sleep-inducing), dipan (affecting appetite),

grahi (absorbable) and pachan (affecting digestion). Dwarakanath’s [19]

assignations were quite similar to these, but added Muslim descriptions such

as constipative, stomachic, appetizer, causing elation, aphrodisiac, retentive,

devitalizing, anodyne, hypnotic, anti-convulsant, causing delirium and intoxi-

cating. The same author listed the names of 48 modern Ayurvedic and eight

Unani Tibbi formulas containing cannabis for a wide range of indications.

A recent survey of bhang use in the holy city of Varanasi (formerly Benares)

found it quite prevalent across socioeconomic strata, especially the working

class, businessmen and among the more educated [72]. Most users in the third

or fourth decades of life employed it for anxiety or mood disorders for the

resulting pleasure, while older people cited benefits on gastrointestinal disorders

with improvement in appetite and bowel habits, or for alleviating insomnia.

Among the 100 subjects, 90% reported improvement in sleep without daytime

fatigue. Improvement in “marital adjustment” was also claimed. All employed

bhang orally, generally 1.5 g/day, for gastrointestinal indications, but 56%

employed 4–10 g/day, without evidence of associated toxic adverse events.

In 1996, native cannabis was again extracted to a yield of 17% THC, which

was then used to treat cancer pain in 42 human subjects [73]. In 11.9% there was

no analgesia with doses of 25 mg, but 64.3% had up to 50% pain reduction, and

9.5% had greater than 75% pain relief with no use of adjunctive medicine.

Dash [23] identified cannabis as one of the primary herbs of rejuvenation

and a synergist with other agents, promoting health, preventing disease and

offering “side benefits”. In order of therapeutic priority, its uses were listed as:

sprue syndrome, sterility, impotency, diarrhea, indigestion, epilepsy, insanity

and colic pain. In addition to the many indications above, the following were

also noted: gastritis, anorexia, anal fistula, throat obstruction, jaundice, bron-

chitis, tuberculosis, torticollis, splenic disorder, delirium, obstinate urinary dis-

orders, sinus problems, anemia, rhinitis, elephantiasis, edema, puerperal sep-

sis, gout and constipation.

The scientific basis of Indian cannabis claims

This chapter has enumerated the lore of Indian medicine with respect to ther-

apeutic benefits of clinical cannabis, but what is its scientific rationale? The

issues will be addressed systemically (Tab. 2).

The oldest cannabis claims are psychiatric from the Atharvaveda, citing its

usage for anxiety. Current research is supportive, particularly for cannabidiol

(CBD) as an anti-anxiety agent as well as an anti-psychotic (reviewed in

[74]). Similar benefit may accrue in calming dementia, as THC proved bene-

ficial in Alzheimer’s disease patients [75]. Recently, cannabichromene (CBC)

12 E. Russo

Cannabis in India: ancient lore and modern medicine 13

Table 2. Indications for cannabis in India

Cannabis indication Physiological basis Reference

Psychiatric

Anxiety CBD reduces anxiety in humans [74]

Extinction of aversive memories EC control in hippocampus [77]

Insomnia Increased sleep in pain/multiple [79, 80]

sclerosis patients

Addiction treatment Decreased usage of cocaine/alcohol [84, 86]

Neurological

Neuropathic pain EC modulation of CNS pathways [87, 88]

Clinical pain reduction [79, 80]

Muscle relaxation Spinal interneuron effects? [79, 89]

Neuroprotection THC/CBD antioxidant/NMDA [91]

antagonism

Migraine Effects on periaqueductal grey, 5-HT, [88, 92, 93]

inflammation, etc.

Seizures CBD anticonvulsant [95]

THC anticonvulsant, EC modulation [96, 97]

of seizure threshold

Dermatological

Anti-psoriatic? TNF-αantagonism [99]

Anti-pruritic Peripheral anti-nociception [100]

Rheumatic

Benefit in rheumatoid arthritis TNF-αantagonism [99]

Endocrinological

Appetite stimulation Hypothalamic effect? [101]

Oncological

Anti-nausea 5-HT3antagonism or other? [102, 103]

Tumor reduction Promotes apoptosis [104, 105]

Reduces angiogenesis [104]

Anti-prolactin effect [106]

Blocks pulmonary carcinogenesis [107]

Pulmonary

Asthma Bronchodilation [108, 110]

Gastroenterological

Intestinal spasm Smooth muscle relaxation [88, 112]

Secretory diarrhea EC modulation of secretion [112]

Gastritis Anti-inflammatory/gastric [114, 115]

cytoprotection

Jaundice ? immunomodulatory [116]

(Continued on next page)

has also demonstrated anti-depressant effects in an animal model [76].

Additional support for benefits of cannabis on mood is evident from work

demonstrating the regulation of extinction of aversive memories by the endo-

cannabinoid system [77].

Insomnia treatment is another ancient claim that finds documentation in

modern phase II–III clinical-trial results in multiple sclerosis patients and

those with chronic neuropathic pain [78–81]. The 19th-century observation of

benefit on addiction is echoed in modern studies of alcoholics [82] and cocaine

users [83], with experimental support for decreased use rates in clinical exper-

iments for each [84–86].

In the neurological realm, the ability of cannabis to treat pain, particularly

of neuropathic origin, is the subject of a great deal of current research. Results

to date are very encouraging, in terms of both basic science support (reviewed

in [87, 88]) and the benefits in clinical trials [78–80].

Although tetanus is rarely observed in the modern age of immunization, the

observed benefits on muscle relaxation underlie current application to treat-

ment of spasms and spasticity in multiple sclerosis and spinal cord trauma [79,

89], where cannabis extracts have proven as effective as any currently avail-

able agent [90]. Although rabies remains invariably fatal, the neuroprotective

effects of cannabis [91] may warrant new trials of cannabis extracts in its treat-

ment, and that of slow virus (prion) diseases. Indian medical literature on

migraine treatment is also supportive, as is a tremendous amount of patho-

physiological data [88, 92, 93]. As for clinical trials, however, the words of Dr

Mechoulam still ring true [94]: “no modern work exists”.

14 E. Russo

Table 2. (Continued)

Cannabis indication Physiological basis Reference

Gynecological

Dysmenorrhea Smooth muscle relaxation Reviewed in [42]

Uterine bleeding EC modulation in uterus Reviewed in [42]

Lower-urinary-tract symptoms Increased bladder capacity, [118]

decreased incontinence

Sexual

Impotence Pain reduction/spinal effects? [119]

Premature ejaculation EC modulation [120]

Infectious

Antibiotic Effects of cannabinoids/terpenoids [111, 121]

Anti-malarial Caryophyllene, α-terpineol [121, 123]

Insecticidal/pediculicidal Octopamine/GABA [126–128]

CBD, cannabidiol; CNS, central nervous system; EC, endocannabinoid; GABA, γ-aminobutyric acid;

5-HT, 5-hydroxytryptamine; 5-HT3, serotonin type-3 receptor; NMDA, N-methyl-D-aspartate; TNF-α,

tumor necrosis factor-α.

Another long-held claim pertains to cannabis in epilepsy. Previous experi-

mental work showed some support for CBD [95], but this has been greatly bol-

stered by current experiments by Wallace et al. [96, 97], demonstrating the

anti-convulsant properties of THC, and the modulation of seizure thresholds

by anandamide.

Examining additional ectodermal tissue, both eczema and itch were cited in

Indian literature as benefiting from cannabis treatment. Recent work demon-

strating the value of tumor necrosis factor-α(TNF-α) antagonists in psoriasis

[98] may justify the use of cannabis, particularly CBD-rich extracts, in the

treatment of related diseases, as CBD shares this mechanism of action [99].

Similarly, the benefits of THC on peripheral pain and itch are becoming

increasingly evident [88, 100].

Rheumatic diseases cited by O’Shaughnessy [36] and other authors remain

an issue, but experiments underline the benefits of CBD in experimental

rodent models of rheumatoid arthritis [99]. Phase II clinical trials are pend-

ing. Modern investigation demonstrates that cannabinoid treatments definite-

ly have a clinical role to play in issues of appetite, with benefit seen in

HIV/AIDS subjects [101], and in multiple sclerosis/neuropathic pain patients

[79].

The role of cannabis in oncology may now extend far beyond its demon-

strated ability to allay nausea in chemotherapy [102, 103], but include promo-

tion of apoptosis, and suppression of angiogenesis in a wide variety of tissue

types (reviewed in [104, 105]). Additionally, THC has anti-prolactin activity in

breast carcinomas [106], and introduces a metabolic block in pulmonary car-

cinogenesis [107].

The role of cannabis in asthma has been much debated, but it is clear that

THC is a bronchodilator [108], as is its terpenoid component, α-pinene [109],

and that smooth muscle contraction in the lungs is mediated by endocannabi-

noids [110]. Given these facts, plus the prominent anti-inflammatory benefits

of THC, CBD and terpenoids [111], it is apparent that additional investigation

with vaporizer or other non-smoked inhalant technology with cannabis

extracts is warranted.

The treatment of digestive issues with cannabis has figured prominently in

India to the current day. Whether it be through reduction of intestinal spasms,

constipation or inhibition of secretory diarrhea processes in cholera, cannabis

components offer neuromodulatory amelioration (reviewed in [88, 112]).

Given the combination of these factors mediated by THC, the TNF-αantago-

nism of CBD and the observed up-regulation of endogenous cannabinoids in

human inflammatory bowel disease [113], there is every reason to believe that

benefits will be forthcoming in clinical trials of cannabis extracts in Crohn’s

disease and ulcerative colitis. The gastritis claim finds support in studies doc-

umenting the benefit of cannabis in ulcer treatment [114], and the gastric cyto-

protective effect of the cannabis essential-oil component, caryophyllene [115].

Even claims for treatment of jaundice may find support in recent claimed ben-

efits seen in hepatitis C patients who use cannabis [116].

Cannabis in India: ancient lore and modern medicine 15

Hemorrhoids continue to plague mankind, and anecdotal evidence for the

benefits of cannabis from rural Kentucky echo the Indian claims [117]. Myriad

anti-inflammatory and anti-pruritic mechanisms may underlie the basis of such

treatment. The benefits of cannabis in dysmenorrhea and excessive uterine

bleeding are plausible given the expression of endocannabinoids in the uterus

(reviewed in [42]). The benefits of cannabis in symptoms of the lower urinary

tract have been strongly supported by increases in mean maximum cystomet-

ric capacity, decreased mean daytime frequency of urination, decreased fre-

quency of nocturia and mean daily episodes of incontinence in multiple scle-

rosis patients treated with cannabis-based medicine extracts [118].

The persistence of claims of cannabis increasing sexual pleasure and per-

formance is compelling, but not particularly amenable to simple experimental

verification. Does cannabis treat impotence? There are frequent claims of

such, including a successful pregnancy induced by one man who was previ-

ously impotent due to spinal damage, treated successfully with oromucosal

cannabis-based medicine [119]. Additionally, recent data demonstrate that a

cannabinoid agonist delayed ejaculatory responses in rats [120]. Thus, a con-

vincing case may be made for human clinical trials [88].

Claims of the benefits of cannabis in infectious diseases have received little

investigation since studies on bacteria in 1960 [121], wherein the authors

demonstrated that an isolated resin from cannabis inhibited growth of

Mycobacterium tuberculosis down to a dilution of 1:150000. Studies on

human herpes simplex virus in 1980 revealed the inhibition of viral growth by

THC even at low dosages [122]. A variety of cannabis components are

anti-infective (reviewed in [111]), supporting such applications, as well as the

use of cannabis in the treatment of malaria, where the essential oil components

caryophyllene and α-terpineol demonstrate anti-protozoal activity [123].

Cannabis may yet prove useful in the treatment of dandruff, as suggested in

Indian sources. Cannabichromene demonstrated anti-fungal activity [124], and

ρ-cymene showed anti-candidal effects [125]. Cannabis effects on the

causative yeast in dandruff, Malassezia ovalis, could be easily tested.

Clear benefits also seem likely in the treatment of lice, as this ancient indi-

cation has been supported by pediculicidal efficacy of cannabis terpenoid com-

ponents [126], the activity of terpenoids on insect octopaminergic receptors

[127], and their allosteric modulation of insect homo-oligomeric γ-aminobu-

tyric acid (GABA) receptors [128]. A whole range of new applications of

cannabis as an insecticide are possible [129]. Mechoulam decried the lack of

investigation of cannabis effects on intestinal parasites [94], and this remains

an area of deficiency in our cannabis knowledge.

Cannabis in India in context

As we have seen, the vast majority of claims for cannabis from India are fully

corroborated by modern scientific and clinical investigation. In closing, a pas-

16 E. Russo

sage from Campbell [1] written for the Report of the Indian Hemp Drugs

Commission more than a century ago offers a plaintive plea for this venerable

herb:

“By the help of bhang ascetics pass days without food or drink. The

supporting power of bhang has brought many a Hindu family safe

through the miseries of famine. To forbid or even seriously to restrict

the use of so holy and gracious a herb as the hemp would cause wide-

spread suffering and annoyance and to the large bands of worshipped

ascetics deep-seated anger. It would rob the people of a solace in dis-

comfort, of a cure in sickness, of a guardian whose gracious protection

saves them from the attacks of evil influences, and whose mighty power

makes the devotee of the Victorious, overcoming the demons of hunger

and thirst, of panic fear, of the glamour of Maya or matter, and of mad-

ness, able in rest to brood on the Eternal, till the Eternal, possessing him

body and soul, frees him from the having of self and receives him into

the ocean of Being. These beliefs the Musalman devotee shares to the

full. Like his Hindu brother the Musalman fakir reveres bhang as the

lengthener of life, the freer from the bonds of self. Bhang brings union

with the Divine Spirit. ‘We drank bhang and the mystery I am He grew

plain. So grand a result, so tiny a sin.’”

It is appropriate that modern-day cannabinoid researchers have acknowledged

the integral role that Indian culture has played in our understanding of the bio-

chemistry of cannabis. Thus, the first endocannabinoid, arachi-

donylethanolamide, was dubbed anandamide (ananda is Sanskrit for bliss;

Tab. 1) [130]. In like manner, the most recently identified endocannabinoid,

the cannabinoid antagonist O-arachidonylethanolamine, which is arachidonic

acid and ethanolamine joined by an ester linkage, has been nicknamed virod-

hamine (virodha is Sanskrit for opposition) [131].

It is fascinating to note that our own endogenous cannabinoid physiology

encompasses these positive and negative influences, in a manner analogous to

THC and CBD effects from cannabis, the Indian phytopharmaceutical that

leads us to this knowledge: nature and neurophysiology in symmetry and bal-

ance.

Acknowledgments

The author would like to thank Dominik Wujastyk, Jan Meulenbeld, Robert A. Nelson, Knut Movik,

Ali Gorji and the dedicated staff of Interlibrary Loan at Mansfield Library, University of Montana, for

their kind provision of resource materials for this chapter.

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Cannabinoid chemistry: an overview

Lumír O. Hanusˇ and Raphael Mechoulam

Department of Medicinal Chemistry and Natural Products, Medical Faculty, The Hebrew University

of Jerusalem, Ein Kerem Campus, 91120 Jerusalem, Israel

Introduction

Cannabis sativa probably originates from neolithic China [1]. However the

exact period of its domestication is unknown. The first known record of the use

of cannabis as a medicine was published in China 5000 years ago in the reign

of the Emperor Chen Nung. It was recommended for malaria, constipation,

rheumatic pains, absent-mindedness and female disorders. Later its use spread

into India and other Asian countries, the Middle East, Asia, South Africa and

South America. It was highly valued in medieval Europe. In Western Europe,

particularly in England, cannabis was extensively used as a medicine during the

19th century, while in France it was mostly known as a “recreational” drug [2].

Natural cannabinoids

The first successful attempt to identify a typical cannabis constituent was

achieved by Wood et al. [3], who isolated cannabinol from the exuded resin of

Indian hemp (charas), which was analysed as C21H26O2. Another big step was

made by Cahn, who advanced the elucidation of the structure of cannabinol

[4], leaving as uncertain only the positions of a hydroxyl and a pentyl group.

Several years later Todd’s group in the UK [5, 6] and independently Adam’s

group in the USA [7] synthesized several cannabinol isomers and compared

them with the natural one. One of the synthetic isomers was identical to the

natural product. The correct structure of the first natural cannabinoid, cannabi-

nol, was thus finally elucidated. These two groups assumed that the psy-

chotropically active constituents were tetrahydrocannabinols (THCs), which

however they could not isolate in pure form and therefore they could not elu-

cidate their structures.

A second cannabis constituent, the psychotropically inactive cannabidiol,

was also isolated, but its structure was only partially clarified [8]. Synthetic

THC derivatives, which showed cannabis-like activity in animal tests, were

prepared, but they obviously differed from the active natural product, on the

basis of their UV spectrum [9–12].

Cannabinoids as Therapeutics

Edited by R. Mechoulam

In a systematic study of the antibacterial substances in hemp Krejcˇ í and

ˇantavy´ found that an extract containing carboxylic acids was effective against

Staphylococcus aureus and other Gram-positive micro-organisms. They isolat-

ed cannabidiolic acid and reported a nearly correct structure [13, 14] (Fig. 1).

24 L.O. Hanusˇ and R. Mechoulam

Figure 1. A tentative biogenesis of the plant cannabinoids

Advances in isolation methods made possible a clarification of the chem-

istry of cannabis. In 1963 our group reisolated cannabidiol and reported its

correct structure and stereochemistry [15]. A year later we finally succeeded in

isolating pure THC (∆9-THC); we elucidated its structure, obtained a crys-

talline derivative and achieved a partial synthesis from cannabidiol [16]. The

absolute configuration of cannabidiol and of THC was established by correla-

tion with known terpenoids [17]. Several years later a minor psychotomimeti-

cally active constituent, ∆8-THC, was isolated from marijuana [18]. Whether

this THC isomer is a natural compound, or an artifact formed during the dry-

ing of the plant, remains an open problem.

Several additional, non-psychotropic cannabinoids were also identified at

that time. The best known are cannabigerol [19], cannabichromene [20, 21]

and cannabicyclol [22]. For a better understanding of the biogenesis of a

cannabinoids in the plant the isolation and identification of cannabinoid acids

turned out to be essential. Alongside cannabidiolic acid, the cannabinolic and

cannabigerolic acids were identified [23], followed by two ∆9-THC acids, A

and B [24, 25], as well as ∆8-THC acid [26, 27] and cannabielsoic acid [28].

The decarboxylated product of cannabielsoic acid, cannabielsoin, is found in

mammals as a metabolite of cannabidiol [29]. The syntheses of some of the

cannabinoid acids have been reported [30].

A tentative pathway for the biogenesis of cannabinoids in the plant has been

published [31–34]. However the only experimental support for ∆9-THC acid

formation from cannabigerolic acid (by direct oxidocyclization and not

through cannabidiolic acid as was assumed before) has been reported by

Shoyama’s group [35]. They showed that the presence of a carboxyl group in

the substrate is essential for enzymatic cyclization of the terpene moiety. This

finding may explain the presence of THC and THC acids in certain cannabis

strains (e.g. South African) that do not contain cannabidiol or its acid [36–38].

In a series of elegant publications Shoyama’s group identified an enzyme

forming cannabichromenic acid and showed that this acid is formed directly

from cannabigerolic acid [39, 40].

It is possible that some of the natural neutral cannabinoids are artifacts

formed through decarboxylation, photochemical cyclization (cannabicyclol),

oxidation (cannabielsoic acid) or isomerization (∆8-THC and ∆8-THC acid) of

other constituents.

Endogenous cannabinoids

The discovery of a high-affinity, stereoselective and pharmacologically dis-

tinct cannabinoid receptor in a rat brain tissue [41] led to a search for natural

endogenous ligands in the brain, which bind to this cannabinoid receptor. We

assumed that the cannabinoid receptor in the brain is not present just to bind a

plant constituent, but to be activated by specific endogenous ligands. Our

approach involved first the synthesis of a potent labeled agonist (HU-243),

Cannabinoid chemistry: an overview 25

which made possible a sensitive bioassay. This compound is the most active

cannabinoid known so far [65]. In a standard bioassay we expected that

endogenous compounds with cannabinoid activity would displace tritiated

HU-243 bound to the central cannabinoid receptor (CB1).

Rat brains are too small and hence we started our isolations with porcine

brains. After nearly 2 years of tedious work, which involved numerous chro-

matographic separations, we isolated from brain an endogenous compound that

binds to the cannabinoid receptor with about the same potency as ∆9-THC. This

endogenous ligand was named anandamide [42], a name derived from the

Sanskrit word for bliss, ananda. When administered intraperitoneally to mice it

caused reduced activity in an immobility test and in open field tests, and pro-

duced hypothermia and analgesia, a tetrad of assays typical of the psychotropic

cannabinoids [43]. Later we isolated two additional, apparently minor, endo-

genous cannabinoids, homo-γ-linoleoylethanolamide and 7,10,13,16-docosa-

tetraenoylethanolamide [44].

The existence of a peripheral cannabinoid receptor (CB2) led to the search

for a ligand to this receptor. We isolated from canine gut another arachidonic

acid derivative, 2-arachidonoyl glycerol (2-AG) [45]. At around the same time

this compound was detected in brain [46] (see Fig. 2).

Hanusˇ et al. reported a third, ether-type endocannabinoid, 2-arachidonyl

glyceryl ether (noladin ether), isolated from porcine brain [47]. It binds to the

CB1cannabinoid receptor (Ki= 21.2 ± 0.5 nM) and causes sedation, hypother-

mia, intestinal immobility and mild antinociception in mice. It binds very

weakly to the CB2receptor. The presence of this endocannabinoid in brain has

been questioned [48]. However as this type of natural glycerol derivative (an

ether group on the 2-position) is unusual, we have repeated its isolation with

an identical result (unpublished observations).

In the course of the development of a bioanalytical method to assay anan-

damide in brain and peripheral tissues, a compound with the same molecular

weight as anandamide, but with a shorter retention time, was identified as

O-arachidonoyl ethanolamine (arachidonic acid and ethanolamine joined by

an ester linkage). This compound was named virodhamine [49].

On the basis of previous structure–activity relationship studies and on the

existence in body tissues of biosynthetic precursors, Huang et al. assumed that

N-arachidonoyl-dopamine (NADA) may exist as an endogenous

“capsaicin-like” cannabinoid in mammalian nervous tissues and may possibly

bind to the vanilloid receptor VR1 [50]. They found that NADA is indeed a

natural endocannabinoid in nervous tissues, with high concentrations found in

the striatum, hippocampus and cerebellum and lower concentrations in the

dorsal root ganglion. NADA binds to the cannabinoid receptors with a 40-fold

greater selectivity for the CB1(Ki= 250 ± 130 nM) than the CB2receptor

[50–52].

One of the typical endocannabinoid effects is pain suppression. Some

endogenous fatty acid derivatives (palmitoylethanolamide, oleamide), which

do not bind to CB1or CB2, either enhance this effect (the so-called entourage

26 L.O. Hanusˇ and R. Mechoulam

effect) or actually show activity by themselves, presumably by binding to

as-yet unidentified cannabinoid receptors [53].

Shortly after the isolation of anandamide, its biosynthesis, metabolism and

degradation in the body were studied [54, 55].

Synthetic cannabinoid receptors agonists/antagonists

In the late 1970s Pfizer initiated a cannabinoid project aimed at novel anal-

gesic compounds. Numerous active bicyclic compounds were synthesized.

The compound chosen for clinical evaluation was CP-55,940 [56, 57]. This

compound is more potent than morphine and is at least 200-fold more potent

than its enantiomer [55]. Structural and stereochemical evaluations led to high-

ly active analogs [58]. The cannabinoid-type side effects observed with this

group of “non-classical” cannabinoids led to the termination of the project

[58]. However, these compounds helped advance the cannabinoid field as they

Cannabinoid chemistry: an overview 27

Figure 2. The main endocannabinoids

were the first cannabinoids that were widely used as labeled ligands. Indeed,

in 1988 Allyn Howlett’s group used tritium-labeled CP-55,940 for the identi-

fication of the first cannabinoid receptor [59]. [3H]CP-55,940 is now an impor-

tant tool in the study of cannabinoid receptors [60].

The need for stereospecific cannabinoid ligands led to further syntheses of

enantiomers with essentially absolute stereochemical purity. This endevour

culminated by the preparation of very potent cannabimimetic compounds [61].

Replacement of the n-pentyl side chain with a 1,1-dimethyl heptyl side chain in

one of the major active primary metabolites of ∆8-THC, 11-hydroxy-∆8-THC,

led to the highly active ligand 11-hydroxy-∆8-THC-dimethylheptyl, or HU-210.

The psychotropically inactive enantiomer, HU-211, is however analgesic,

antiemetic and is at present being evaluated as an anti-trauma agent. Both com-

pounds were synthesized with very high enantiomeric purity (99.8%) [62]. The

high degree of enantioselectivity and potency of HU-210 was demonstrated in

mice, dogs and pigeons [63, 64].

The synthetic HU-210 was used to prepare a novel probe for the cannabi-

noid receptor. Hydrogenation of this compound yielded two epimers of

5'-(1,1-dimethylheptyl)-7-hydroxyhexahydrocannabinol [65]. The equatorial

epimer (designated HU-243) binds to the cannabinoid receptor with a KDvalue

of 45 pM, and is the most potent CB1agonist described so far. Tritiated

HU-243 was used as a novel probe for the cannabinoid receptor.

An effort to find new synthetic cannabinoids with increased therapeutic

activity and few adverse side effects led to the preparation of ajulemic acid

(HU-239), an analgetic and anti-inflammatory cannabinoid [66, 67]. This com-

pound has anti-tumor effects in mice [68], binds to the peroxisome prolifera-

tor-activated receptor γ(PPARγ), a pharmacologically important member of

the nuclear receptor superfamily [69], and induces apoptosis in human T lym-

phocytes [70]. However, it binds to CB1and has activity at the level of THC in

the tetrad assay in mice [71].

A group at the Sterling pharmaceutical company prepared analogs of the

anti-inflammatory drug pravadoline, an aminoalkylindole. To their surprise

28 L.O. Hanusˇ and R. Mechoulam

Structure 1

they discovered that these compounds acted not only as cyclooxygenase

inhibitors, but also as cannabinoid agonists [72]. In vitro structure–activity

relationship studies of these compounds led to numerous new compounds with

cannabinoid receptor agonist activity [73, 74]. The best-known compound in

this series is the conformationally restricted derivative WIN-55212-2 [75]. A

binding assay in rat cerebellum membranes has been developed. It makes use

of the stereospecific radioligand [3H](R)-(+)-WIN-55212-2.

The first potent and selective antagonist of the central cannabinoid receptor

(CB1), SR-141716A, was reported in 1994 by a group at Sanofi [76]. This

compound is not active on the peripheral cannabinoid receptor (CB2) and has

rapidly become a new tool in the study of cannabinoid receptor mechanisms

and in research on new therapeutic agents. Another novel CB1antagonist,

LY320135, which is not as selective as the previous one, was reported soon

Cannabinoid chemistry: an overview 29

Structure 2

Structure 3

thereafter. This substituted benzofuran reverses anandamide-mediated adeny-

late cyclase inhibition and also blocks WIN-55212-2-mediated inhibition of

N-type calcium channels [77].

The Sanofi group also described the first potent and selective antagonist of

the peripheral cannabinoid receptor (CB2), SR-144528 [78], and like the

above-mentioned CB1antagonist, it soon became a major tool in cannabinoid

research [79].

Our group reported the preparation of a CB2-selective ligand, HU-308 [80],

which is now being investigated as an anti-inflammatory drug by Pharmos, a

pharmaceutical firm. It shows no central nervous system effects due to its

essential lack of affinity for the CB1receptor. In HU-308 both phenolic groups

are blocked as methyl ethers. This is in contrast to cannabinoid CB1agonists

in which at least one of the phenolic groups has to be free.

Traumatic brain injury is a major cause of mortality and morbidity. There is

no effective drug to treat brain-injured patients. We found that on closed head

injury the amounts of 2-AG produced by the brain are increased 10-fold, and

that this endocannabinoid apparently has a neuroprotective role, as adminis-

tration of 2-AG to mice with head trauma reduces both the neurological dam-

age and the edema [81]. Numerous other groups have recorded work on vari-

30 L.O. Hanusˇ and R. Mechoulam

Structure 4

Structure 5

ous aspects of cannabinoids as neuroprotective agents (see Chapter by

Fernández-Ruiz et al. in this volume). On this basis a structurally novel, high-

ly potent CB1/CB2cannabinoid receptor agonist, BAY 38-7271, was prepared

and shown to have pronounced neuroprotective efficacy in a rat model of trau-

matic brain injury [82–85].

Pharmos have developed a cannabinoid, PRS 211,096, that binds to the

peripheral cannabinoid receptor and which is being assayed for treatment of

multiple sclerosis [86].

Cannabinoid chemistry: an overview 31

Structure 7

Structure 6

Structure 8

(R)-Methanandamide (AM-356) is a chiral analog of the endocannabinoid

ligand anandamide, It is more stable than anandamide to hydrolysis by fatty

acid amide hydrolase (FAAH), as the methyl group adjacent to the amide moi-

ety apparently interferes with the enzyme. It has a Kivalue of 20 ± 1.6 nM for

the CB1receptor [87]. The Kivalue for binding to the CB2receptor from

mouse spleen is 815 nM [88]. Thus (R)-methanandamide has a high selectivi-

ty for the CB1receptor.

6-Iodo-pravadofine (AM-630), an aminoalkylindole, attenuates the ability

of a number of cannabinoids to inhibit electrically evoked twitches of vas def-

erens isolated from mouse [89]. AM-630 behaves as a competitive antagonist

of cannabinoid receptor agonists in the guinea-pig brain [90]. AM-630 also

antagonizes the ability of the cannabinoid agonist WIN-55212-2 to stimulate

guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding in mouse

brain membrane preparations [91].

Gatley et al. [92] have developed a novel radioligand, [123I]AM-281, struc-

turally related to the CB1-selective antagonist SR-141716A, that is suitable for

in vivo studies of the central cannabinoid receptor and for imaging this recep-

tor in the living human brain [92].

32 L.O. Hanusˇ and R. Mechoulam

Structure 9

Scientists at the University of Connecticut have synthesized and studied a

series of aminoalkylindoles as selective CB2agonists. The compounds are stat-

ed to be useful for the treatment of pain, glaucoma, multiple sclerosis and other

diseases and disorders. Compound AM-1241 has a high affinity for the CB2

receptor in a mouse spleen preparation (Ki= 3.4 ± 0.5 nM), with good selectiv-

ity versus the CB1receptor in a rat brain preparation (Ki= 280 ± 41 nM). This

compound has recently been found to inhibit neuropathic pain in rodents [93].

AM-2233, a novel aminoalkylindole CB1agonist, was found to have a

greater potency than WIN-55212-2 in assays in vitro, but has a similar poten-

cy to it in a mouse locomotor assay. It was suggested that its behavioral effects

could have been mediated, in part, via an action on another receptor type in

addition to the CB1receptor. AM-2233 represents the first agonist CB1 recep-

tor ligand (Ki= 0.4 nM) with potential as an in vivo imaging agent for this

receptor [94, 95]. Stoit et al. [96] have reported the syntheses and biological

activities of potent pyrazole-based tricyclic CB1receptor antagonists. One can

find additional information on cannabinoid receptor agonists and antagonists

in Barth’s review [97].

Gallant et al. [98] have described two indole-derived compounds (see struc-

tures below), with binding potency for the human peripheral cannabinoid

receptor (CB2) in the nanomolar region, They are highly selective.

A new series of rigid 1-aryl-1,4-dihydroindeno[1, 2-c]pyrazole-3-carbox-

amides was recently designed [99]. Seven of the new compounds displayed

very high in vitro CB2-binding affinities. Four compounds showed very high

selectivity for the CB2receptor.

Cannabinoid structure–activity relationship data have indicated that the

cannabinoid side chain and the phenolic hydroxyl are key elements in CB1

receptor recognition. To test this hypothesis, the 1-deoxy analog, JWH-051, of

the very potent cannabinoid 11-hydroxy-∆8-THC-dimethylheptyl (HU-210)

was prepared and the affinity of this compound for the CB1receptor was deter-

mined [100]. Contrary to expectations, this 1-deoxy analog still had high affin-

ity for the CB1receptor (Ki= 1.2 ± 0.1 nM) and even greater affinity for the

Cannabinoid chemistry: an overview 33

Structure 10

CB2receptor (Ki= 0.032 ± 0.19 nM). On the basis of these data, it is apparent

that a phenolic hydroxyl group is not essential for cannabinoid activity.

To obtain selective ligands for the CB2and to explore the structure–activi-

ty relationship of the 1-deoxy-cannabinoids, the same research group

described the synthesis and pharmacology of 15 1-deoxy-∆8-THC analogues

[101]. Five of these analogues had high affinity (Ki≤20 nM) for the CB2

receptor. Four of them also had low affinity for the CB1receptor (Ki≥295 nM).

3-(1',1'-Dimethylbutyl)-1-deoxy-∆8-THC (JWH-133) had very high affinity

for the CB2receptor (Ki= 3.4 ± 1.0 nM) and low affinity for the CB1receptor

(Ki= 677 ± 132 nM).

In view of the importance of the CB2receptor, three series of CB2-selective

cannabinoid receptor ligands, 1-methoxy-, 1-deoxy-11-hydroxy- and

11-hydroxy-1-methoxy-∆8-THCs, were designed [102]. All of these com-

pounds have greater affinity for the CB2receptor than for the CB1receptor;

however, only 1-methoxy-3-(1',1'-dimethylhexyl)-∆8-THC (JWH-229) had

essentially no affinity for the CB1receptor (Ki= 3134 ± 110 nM) with high

affinity for CB2(Ki= 18 ± 2 nM).

34 L.O. Hanusˇ and R. Mechoulam

Structure 12

Structure 11

Recently the discovery of a further class of diarylpyrazolines with high

potency and selectivity for the CB1receptor was described [103]. These com-

pounds were found to be CB1antagonists. SLV319 was found to be a potent

CB1antagonist (Ki= 7.8 nM) close to that of the Sanofi compound

SR-141716A, with more than 1000-fold selectivity against CB2.

Additional synthetic compounds that bind to the CB1and/or CB2receptors

have been mentioned in patents. These were recently reviewed by Hertzog

[104].

Cannabinoid chemistry: an overview 35

Structure 13

Structure 14

Novartis AG has recently filed a patent application on a series of quinazo-

lines as cannabinoid agonists useful for the treatment of pain, osteoarthritis,

rheumatoid arthritis and glaucoma, among other indications [105]. Compound

1binds to both CB1(Ki= 34 nM) and CB2(Ki= 11 nM). The patent applica-

tion refers to the compound as having CB2agonist activity. Additionally, this

compound has been shown to be active in a rodent neuropathic pain model

when administered at an oral dose of 0.5 mg/kg.

The University of Connecticut has disclosed a series of indazole derivatives

that have been found to act as agonists of cannabinoid receptors [106]. The

compounds exhibit a range of selectivities for CB2over CB1. Compound 2,for

instance, exhibited Kivalues of 2.28 and 0.309 nM for the CB1and CB2recep-

tors, respectively. This compound produced dose-dependent anti-nociception

to thermal stimulus in rats. The compound reduced locomotor activity in rats

after intravenous administration, an effect attributed to activation of the CB1

receptor.

A series of aromatic CB2agonists has been disclosed by the Schering-Plough

Research Institute [107, 108]. The compounds are reported to have anti-inflam-

36 L.O. Hanusˇ and R. Mechoulam

Structure 15

Structure 16

matory and immunomodulatory activities, and to be active in cutaneous T cell

lymphoma, diabetes mellitus and other indications. Compound 3is stated to

bind to CB2with a Kivalue in the range 0.1–10 nM.

Researchers at AstraZeneca have disclosed a series of benzimidazoles and

azabenzimidazoles to be CB2agonists [109]. The compounds are described as

useful in the treatment of pain, cancer, multiple sclerosis, Parkinson’s disease,

Huntington’s chorea, transplant rejection and Alzheimer’s disease. Cannabinoid

receptor selectivity data are provided for some of the new compounds. For

instance, compound 4binds to CB2(Ki= 3.1 nM) with much greater affinity

than to CB1(Ki= 2.8 µM). No in vivo data are provided for the compounds.

The University of Connecticut has disclosed a series of biphenyls as

cannabinoid modulators [110]. These non-classical cannabinoids are described

as useful for the treatment of peripheral pain, neuropathy, neurodegenerative

diseases and other indications. Several of the compounds were found to bind

selectively to the CB2receptor. For instance, compound 5binds to CB2with a

Kivalue of 0.8 nM and to CB1with a Kivalue of 241 nM.

Cannabinoid chemistry: an overview 37

Structure 17

Structure 18

The Virginia Commonwealth University has filed a patent application on a

series of resorcinol derivatives as selective CB2agonists useful for the treatment

of pain, inflammation and autoimmune diseases [111]. Binding data for the

compounds to CB1and CB2are provided, and the compounds were assayed for

in vivo activity in mouse tail-flick, spontaneous activity and rectal temperature

assays. Compound 6had Kivalues of 40 and 0.8 nM, respectively, for the CB1

and CB2receptors. In addition, this compound was assessed by intravenous

administration and exhibited ED50 values of 2.7, 2.4 and 3.6 mg/kg in the spon-

taneous activity, tail-flick and rectal temperature assays, respectively.

The University of Connecticut has disclosed a series of dihydrotetrazines and

derivatives as CB2agonists [112]. Compound 7is reported to be a potent CB2

agonist (Ki= 19 nM) with 88-fold selectivity for the CB2over the CB1receptor.

Such compounds are reported to be useful in the treatment of pain, glaucoma,

multiple sclerosis, Parkinson’s disease,Alzheimer’s disease and other disorders.

Shionogi has also disclosed two series of thiazine-containing CB2agonists,

of which compounds 8and 9are examples [113, 114]. Selectivity data for sev-

eral of the compounds with regard to CB2/CB1affinities are described. For

38 L.O. Hanusˇ and R. Mechoulam

Structure 19

Structure 20

Structure 21

example, compound 8binds to CB2with a Kivalue of 0.3 nM and a Kivalue

of >5000 nM for CB1. Compound 9displayed a Kivalue of 1.2 nM at the CB2

receptor and 80 nM at the CB1receptor. When dosed orally at 100 mg/kg in a

mouse pruritis model, this compound reduced scratching by 98% relative to

control animals.

Shionogi has disclosed a series of amide-containing CB2modulators stated

to be useful in the treatment of inflammation, nephritis, pain, allergies,

rheumatoid arthritis, multiple sclerosis, brain tumors and glaucoma [115].

Compound 10 was found to bind to the CB2receptor with a Kivalue of 4 nM,

with very little affinity for CB1(Ki< 5 µM).

Recently 1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives (11) were

synthesized as new ligands of cannabinoid receptors [116]. Some of these com-

pounds possess a greater affinity for the CB2receptor than for the CB1recep-

tor. Compound 7-chloro-N-cyclohexyl-1-(2-morpholin-4-ylethyl)-1,8-naph-

thyridin-4(1H)-on-3-carboxamide (12) revealed a good CB2selectivity (CB1,

Ki= 1 µM; CB2, Ki= 25 ± 1.8 nM).

Indole derivatives were prepared and tested for their CB1and CB2receptor

affinities [117]. Three new highly selective CB2receptor agonists were identi-

fied, namely JWH-120 (CB1,Ki= 1054 ± 31 nM; CB2,Ki= 6.1 ± 0.7 nM),

JWH-151 (CB1,Ki>10000 nM; CB2,Ki= 30 ± 1.1 nM) and JWH-267 (CB1,

Ki= 381 ± 16 nM; CB2,Ki= 7.2 ± 0.14 nM).

Cannabinoid chemistry: an overview 39

Structure 22

Structure 23

Conclusions

C. sativa L. has been used throughout history not only for its fiber, but also as

a medicinal plant. It has been the object of scientific research over the past 150

years. After the isolation of the plant’s constituents, biochemical work led to

the identification of two receptors and of endogenous cannabinoids. Over the

last decade numerous synthetic agonists and antagonists have been prepared.

We may be approaching an important goal in cannabinoid research – the use

of cannabinoids in medicine – which has been the dream of several generations

of scientists.

40 L.O. Hanusˇ and R. Mechoulam

Structure 25

Structure 24

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46 M. Maccarrone

Cannabidiol as a potential medicine

Roger G. Pertwee

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill,

Aberdeen AB25 2ZD, Scotland, UK

Introduction

Cannabidiol (CBD) is one of more than 60 oxygen-containing hydrocarbon

constituents of cannabis that are collectively known as plant cannabinoids or

phytocannabinoids [1, 2]. It was first isolated in 1940, by Roger Adams from

Mexican marijuana and by Alexander Todd from Indian charas [3]. However,

the correct structure of CBD was not determined until 1963 and its absolute

stereochemistry until 1967 [4]. The CBD molecule is chiral and it is only the

3R,4R-(–)-enantiomer of this molecule that is found in cannabis. This enan-

tiomer is referred to throughout this review as CBD. The chemical nomen-

clature of CBD differs from that of 6aR,10aR-(–)-∆9-tetrahydrocannabinol

(∆9-THC), the main psychoactive constituent of cannabis. Thus, as shown in

Figure 1, whereas ∆9-THC has a pyran ring which determines its numbering,

CBD has no heterocyclic ring and its numbering is based on that of the ter-

pene ring. Much of the ∆9-THC and CBD that is extracted from harvested

cannabis derives from the C-2 and C-4 carboxylic acids of ∆9-THC or the

C-3'/C-5' carboxylic acid of CBD (Fig. 1), all of which undergo decarboxy-

lation when the plant material is stored or heated [1, 5]. The pharmacology

of ∆9-THC has been intensively investigated and it is now generally accept-

Cannabinoids as Therapeutics

Edited by R. Mechoulam

Figure 1. The structures of the phytocannabinoids (–)-∆9-tetrahydrocannabinol (∆9-THC) and

(–)-cannabidiol (CBD)

ed that, in contrast to CBD, it produces many of its effects by acting on

cannabinoid CB1receptors to modulate central and peripheral neurotrans-

mission and on cannabinoid CB2receptors to modulate cytokine release from

immune cells [6]. Additional pharmacological targets for ∆9-THC have also

been proposed [7]. Current knowledge about the pharmacological actions of

CBD is much more limited. There is already no doubt, however, that this

non-psychoactive phytocannabinoid is pharmacologically active and that its

pharmacological actions differ markedly from those of ∆9-THC [8, 9].

Moreover, as now discussed, it is likely that CBD will prove to have clinical

applications (i) for the management of epilepsy and certain other central

motor disorders, (ii) for the treatment of anxiety, psychotic illnesses and neu-

rotoxicity associated for example with stroke, (iii) for the treatment of

inflammation and (iv) for the attenuation of unwanted side effects produced

by ∆9-THC when this phytocannabinoid is used as a medicine. Other poten-

tial therapeutic targets for CBD include emesis, glaucoma, sleep and appetite

disorders, and cancer.

Epilepsy

To date there have been two investigations into the effects of CBD on epilep-

tic patients. One of these was performed with an epileptic patient who exhib-

ited symmetrical spike and wave electroencephalographic (EEG) activity

when in light sleep and was receiving medicine (unspecified) to prevent

tonic-clonic seizures [10]. When this patient fell into a light sleep after receiv-

ing chloral hydrate, intravenous infusion of CBD at 2.4 mg/min for 17 min was

associated with an increase in the occurrence of abnormal EEG. Whether CBD

altered the incidence of tonic-clonic seizures in this patient was not deter-

mined. The other investigation, a double-blind clinical trial, was carried out

with patients with secondary generalized epilepsy. These were patients who

were experiencing at least one generalized convulsive crisis per week even

though they were being given phenytoin, a barbiturate, primidone, clon-

azepam, carbamazepine, trimethadione and/or ethosuximide [11]. Of these

patients, seven were given 200 or 300 mg of CBD daily by mouth for up to 4.5

months. There was also one patient who crossed over from the placebo group

to CBD after 1 month. Seven other patients received placebo throughout the

investigation. Within the CBD group, four patients improved markedly, three

others showed some improvement and one patient did not improve. Only one

patient in the placebo group improved with time. The most serious side effect,

somnolence, was reported by four CBD patients and one placebo patient. As

to EEGs, improvement was observed in two of the placebo patients, but only

on one occasion, and in three of the CBD patients, with no change detected in

any of the other patients. Because all patients received their usual anti-epilep-

tic medicine(s) throughout this clinical trial, it is possible that instead of or as

well as having a direct anti-convulsant effect, CBD may have been enhancing

48 R.G. Pertwee

the anti-convulsant effects of some these other drugs as, indeed, it has been

found to do in some animal experiments (see below).

In line with its reported anti-epileptic effect in human subjects, CBD has

been found to show anti-convulsant activity in several in vivo animal models

of epilepsy. For example, as indicated in Table 1, it can prevent convulsions

induced in mice or rats by electroshock, by sound or by convulsant agents such

as pentylenetetrazol. In addition, it prevents clonic convulsions caused by

chronic placement of cobalt wire in the dura and kindled convulsions produced

by repetitive electrical stimulation of the subiculum or by repeated subcuta-

neous administration of pentylenetetrazol. There are also reports that CBD can

enhance the ability of phenytoin to prevent audiogenic seizures in rats and of

phenytoin and phenobarbitone to prevent convulsions induced by electroshock

in mice [15, 30, 36, 37] and that it exhibits anti-convulsant activity in certain

electrophysiological models of epilepsy (see [8]). Unlike ∆9-THC, which has

been found to produce a mixture of pro-convulsant and anti-convulsant effects

in animal experiments, there is evidence that CBD has only anti-convulsant

properties [32, 38, 39]. Indeed, there is one report that convulsions induced by

∆9-THC in rabbits can be prevented by CBD when these two cannabinoids are

co-administered, although not when CBD is injected before ∆9-THC [16].

Little is yet known about the mechanism underlying the anti-convulsant

effects of CBD. That this mechanism is specific in nature is suggested by the

existence of a relationship between the structures of CBD analogues and the

ability of these analogues to prevent convulsions in animals [8]. Such specifici-

ty is also supported by observations; firstly that CBD is not active in all animal

models of epilepsy and secondly that CBD is effective as an anti-convulsant in

Cannabidiol as a potential medicine 49

Table 1. Established rodent models of epilepsy in which CBD shows anti-convulsant activity

Measured response reduced or abolished by CBD Reference

Convulsions induced in rats by corneal electroshock [15, 19, 28]

Convulsions induced in mice by corneal electroshock [13, 18, 20–25, 27,

30, 34, 35]

Convulsions induced in mice by ear electroshock [29]

Convulsions induced in mice by pentylenetetrazol [12, 18, 29, 30, 34]

Convulsions induced in mice or rabbits by convulsant agents other [16, 18]

than pentylenetetrazol

Convulsions induced by chronic placement of cobalt wire in the dura of rats [14]

Kindled seizures induced in rats by repetitive subicular electroshock [31]

Kindled seizures induced in mice by repeated injections of pentylenetetrazol [26]

Audiogenic convulsions in rats [15, 17]

Amplitude of electrically evoked cerebrocortical potentials in [32, 33]

unanaesthetized rats

Kindled afterdischarges induced in rats by repetitive subicular electroshock [31]

All animals were unanaesthetized.

rats and mice at doses below those at which it produces a general impairment

of motor function, for example in rotarod, bar-walk or open-field performance

assays [8]. Also consistent with a specific mode of action is the finding that

CBD shows quite high anti-convulsant potency, both in frogs, in which it has

been shown to protect against electroshock-induced tonic convulsions with a

potency at least 100 times greater than that of phenytoin [40] [this finding could

not be replicated when the frog experiments were performed at a different time

of the year (SA Turkanis, personal communication)], and in rats, in which it has

been reported to exhibit signs of anti-epileptic activity at doses of 0.3 and

3 mg/kg administered intraperitoneally (i.p.) [19, 31]. In mice, however, CBD

appears to have somewhat less anti-convulsant potency, reported ED50 values

for the protection of this species from convulsions induced by electroshock

being 38 mg/kg (administered intravenously, i.v.) and 80–120 mg/kg i.p. [21,

30, 34, 35]. Karler et al. [25] found the peak concentration of CBD in mouse

brain to be 8 µg/g following its i.p. administration at a dose of 120 mg/kg. This

approximates to 8 µg/ml and hence 25 µM, a concentration at which CBD

would be expected to modulate central neurotransmission, for example by bind-

ing to cannabinoid CB1receptors and by inhibiting the transport of calcium,

anandamide or certain neurotransmitters across neuronal membranes (Tab. 2).

Interestingly, when mice were injected i.p. with an anti-convulsant dose of

phenytoin (7 mg/kg), the brain concentration of this compound peaked at

6.6 µg/g [25], a concentration that approximates to 26 µM. Hence, it appears

that although the doses at which CBD and phenytoin exhibit anti-convulsant

activity in mice differ considerably, these disparate doses produce essentially

the same concentration of CBD and phenytoin within the brain, suggesting that

CBD may have much lower bioavailability in this species, at least when the

intraperitoneal route is used.

There are reports that (+)- and (–)-CBD are equipotent against convulsions

induced in rats by sound [52] or in mice by electroshock [27], making it unlike-

ly that CBD prevents convulsions by acting on pharmacological targets such as

CB1receptors that discriminate between these enantiomers [8, 42]. That CBD

does not act through CB1receptors to prevent convulsions is also supported by

a report that its ability to oppose electroshock-induced maximal convulsions in

mice is not attenuated by the selective CB1receptor antagonist, SR-141716A,

at a dose that does attenuate the anti-convulsant effect of ∆9-THC or

R-(+)-WIN-55212 [35].

Animal experiments have revealed several similarities between the

anti-convulsant properties of CBD and phenytoin [8]. Therefore, as has been

postulated for phenytoin, the anti-convulsant effect of CBD may depend at

least in part on an ability to block the spread of seizure activity in the brain,

possibly through suppression of post-tetanic potentiation. Indeed, there is

already a report that CBD can abolish post-tetanic potentiation in bullfrog iso-

lated ganglia, albeit at the rather high concentrations of 60–100 µM [53]. The

pharmacology of CBD has less in common with ethosuximide than with

phenytoin [8], suggesting that it may not share the ability of ethosuximide to

50 R.G. Pertwee

Cannabidiol as a potential medicine 51

Table 2. Some actions of CBD expected to affect neurotransmission

Action Tissue Effective concentration Reference

Antagonism of cannabinoid CB1receptor agonists Mouse vas deferens 120 nM (KBvalue) [47]

Inhibition of Ca2+ uptake Rat brainstem synaptosomes 100 nM [45]

Inhibition of Ca2+ uptake Mouse brain synaptosomes 1 µM [45]

Inhibition of 5-HT uptake Rat hypothalamic synaptosomes 1 µM [41]

Inhibition of dopamine and noradrenaline uptake Rat striatal or hypothalamic 1 µM [41, 49]

synaptosomes

Displacement of [3H]SR-141716A from CB1receptors CB1-containing membranes 1.26 µM (Kivalue) [51]

Displacement of 3H-CP-55,940 from CB1receptors CB1-containing membranes 2.28 µM (Kivalue) [50, 51]

4.35 µM (Kivalue)

Enhancement of evoked neuronal release of noradrenaline and ATP Mouse vas deferens 3.2 µM [47]

Inhibition of dopamine and noradrenaline uptake Mouse whole-brain synaptosomes 5 µM [46]

Inhibition of 5-HT and GABA uptake Mouse whole-brain synaptosomes 10 µM [46]

Enhancement of basal release of dopamine and noradrenaline Rat striatal and hypothalamic 10 µM [49]

synaptosomes

Antagonism of the cannabinoid CB1receptor agonist CP-55,940 Rat cerebellar membranes 10 µM [48]

Inhibition of choline uptake Rat hippocampal crude synaptosomal 16 µM (EC50) [28]

fraction

Inhibition of anandamide uptake RBL-2H3 cells 22 µM (EC50) [42]

Inhibition of anandamide metabolism N18TG2 cell membranes 27.5 µM (EC50) [42]

Attenuation of the affinity of dopamine D2receptor ligands for D2Mouse striatal membranes 30 µM [43, 44]

receptors

GABA, γ-aminobutyric acid; 5-HT, 5-hydroxytryptamine.

prevent petit mal epilepsy (absence seizures) in humans. However, because

CBD differs from phenytoin in not eliciting any excitatory responses in behav-

ioural and electrophysiological models of epilepsy [31, 39], it may also differ

from phenytoin in not exacerbating absence seizures.

Clearly, there is now sufficient evidence to warrant further clinical investi-

gations into the use of CBD for the management of epilepsy, particularly grand

mal. Important objectives will be to identify all the types of epilepsy against

which CBD is active and to determine whether this cannabinoid is more effec-

tive or has less serious, unwanted effects than established anti-epileptic drugs,

whether tolerance develops to anti-convulsant effects of CBD in humans as it

can in an animal model in which tolerance to phenytoin also develops [22, 25],

and whether the synergism between CBD and phenytoin or phenobarbitone

that has been observed in animal models of grand mal epilepsy also occurs in

humans. At the non-clinical level, there is an urgent need for new research

aimed at elucidating the mechanisms that underlie the anti-convulsant effects

of CBD.

Other central motor disorders

In experiments directed at investigating the ability of CBD to improve chorea

arising from Huntington’s disease, positive results were obtained in one inves-

tigation in which four patients with this disease received CBD orally at 300 or

600 mg/day [54] but not in a subsequent clinical trial in which 15

Huntingtonian patients were given CBD orally for 6 weeks at about

700 mg/day [55]. The ability of CBD to reduce dystonia has also been inves-

tigated [56]. When administered to five patients at a dose of 100– 600 mg/day

per os (p.o.) for 6 weeks together with the standard medication, CBD reduced

disease- or L-dopa-induced dystonia in all five patients. It also improved motor

function in two of the patients with disease-induced dystonia when given once

at 200 mg p.o. [57]. In two other patients, whereas CBD at 300–500 mg/day

improved dystonia, it exacerbated hypokinesia and resting tremor [56]. CBD

has also been reported to exhibit anti-dystonic activity in mutant hamsters

[58]. However, its effect was marginal and produced only by the rather high

dose of 150 mg/kg i.p. and not by 50 or 100 mg/kg i.p.

Anxiety

There is evidence that CBD has anxiolytic properties, at least in normal

human subjects. Zuardi et al. [59] have reported that at a dose of 300 mg p.o.

CBD relieves post-stress anxiety induced by a simulated public-speaking test

and there are other reports that CBD has a sedative or somnolent effect in nor-

mal subjects at 200–600 mg p.o. [60, 61]. There is also evidence that the anx-

iolytic effect produced by CBD in normal human subjects is mediated by lim-

52 R.G. Pertwee

bic and paralimbic brain areas [62]. Although the question of whether CBD

is effective against “pathological” anxiety states has still to be addressed,

there is already evidence that CBD can oppose anxiety induced in humans by

∆9-THC. Thus, Karniol et al. [63] found that groups of five human subjects

who took 30 mg of ∆9-THC p.o. together with 15, 30 or 60 mg of CBD expe-

rienced less ∆9-THC-induced anxiety and panic and greater feelings of pleas-

ure than when they took the same dose of ∆9-THC by itself. Similarly, Zuardi

et al. [64] found that whereas the incidence of feeling anxious, troubled, with-

drawn, feeble, incompetent and discontented was greater in eight human sub-

jects after ∆9-THC at 0.5 mg/kg p.o. than after placebo treatment, CBD at

1 mg/kg p.o. attenuated these effects of ∆9-THC when the two cannabinoids

were co-administered and by itself increased the incidence of feeling quick

witted and clear minded.

As discussed in greater detail elsewhere [8], CBD also shows signs of anx-

iolytic activity in animal models, experiments with rats or mice indicating that

it can suppress the conditioned emotional response, increase conflict response

rates and augment the proportion of time spent in the open arms of the elevat-

ed-plus maze. Interestingly, experiments with mice have also shown that the

anxiogenic effect produced by ∆9-THC in the elevated-plus maze can be

opposed by a dose of CBD (0.01 mg/kg i.p.) that by itself is sub-anxiolytic in

this bioassay [65]. CBD appears to have a bell-shaped dose-response curve for

its anxiolytic effect, at least in animal assays [8]. For example, in rat experi-

ments with the elevated-plus maze, it has been found to show greatest anxi-

olytic activity at 5 mg/kg i.p., less activity at 2.5 and 10 mg/kg and no activi-

ty at 20 mg/kg [66]. Why this should be remains to be established. There is

also nothing yet unknown about the mechanism(s) by which CBD reduces

anxiety other than that it appears to interact with its site(s) of action in a struc-

ture-dependent manner [8].

Psychotic illnesses

There is some very preliminary evidence that CBD may have anti-psychotic

activity. Thus in experiments with nine normal human subjects, Leweke et al.

[60] found co-administration of CBD (200 mg p.o.) to oppose the ability of

the cannabinoid receptor agonist, nabilone (1 mg p.o.), to produce binocular

depth inversion, a visual illusion that is thought to provide a model of psy-

chosis. CBD did not affect this measured response when administered by

itself, although it did decrease the vividness of mental imagery. Further evi-

dence comes from some in vivo experiments with rats. These indicate that

CBD shares the ability of established anti-psychotic drugs such as haloperi-

dol to oppose certain effects of apomorphine, for example stereotyped sniff-

ing and biting [67]. However, unlike at least some anti-psychotic drugs, CBD

has been found not to induce catalepsy in rats or to elevate plasma prolactin

in humans [61, 67].

Cannabidiol as a potential medicine 53

Neurotoxicity

As discussed in greater detail elsewhere [8, 68–72], there is convincing evi-

dence that CBD (and other cannabinoids that contain a phenol group) can pro-

tect neurons against oxidative stress and glutamate-induced excitotoxity by

acting through a mechanism that is independent of CB1or CB2receptors. CBD

has, for example, been found to protect against neurotoxicity induced by glu-

tamate in primary cultures of rat cerebrocortical neurons (EC50 = 2–4 µM) [73,

74]. This was irrespective of whether the neurotoxicity was induced through

N-methyl-D-aspartate (NMDA), 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)-

propionic acid (AMPA) or kainate receptors. CBD was not antagonized by

SR-141716A, an indication that its neuroprotective effect was not mediated by

CB1receptors. In addition, it has been found that CBD concentrations of 1 µM

or above oppose the release of calcium from intracellular stores stimulated by

metabotropic or ionotropic glutamate receptor activation [75] and protect

mouse hippocampal HT22 cells from oxidative death induced by hydrogen

peroxide [71]. CBD also shows neuroprotective activity in vivo. Thus in rats

with focal cerebral ischaemia induced by middle cerebral artery occlusion, it

reduced behavioural signs of neurological impairment and decreased cerebral

infarct volume when administered at ischaemia onset (5 mg/kg i.v.) and again

12 h after surgery (20 mg/kg i.p.) [74]. More recent experiments have shown

that CBD can also protect from signs of brain damage caused by cerebral

ischaemia in gerbils [76]. In these experiments the CBD dose-response curve

was bell-shaped, the optimal dose being 5 mg/kg i.p. There is also evidence

that in vivo treatment with CBD (2 mg/kg i.v.) can prevent retinal neurotoxic-

ity induced in adult rats by intravitreal injection of NMDA [72]. It will now be

important to establish whether CBD is neuroprotective in humans and, if it is,

to establish how best to exploit this effect in the clinic.

Strong evidence has emerged, for example from experiments in which reac-

tive oxygen species were generated in neuronal cultures [73], in mouse peri-

toneal granulocytes [77] or in a brain lipid oxidation assay [71], that, at con-

centrations in the low micromolar range, CBD possesses antioxidant (elec-

tron-donor) properties. Consequently, it is likely that the neuroprotective activ-

ity of CBD depends at least in part on an ability to act downstream of gluta-

mate receptors to protect cellular structures from damage induced by reactive

oxygen species generated in response to pathological events such as excessive

glutamate release. Interestingly, Hampson et al. [73] have reported that CBD

induces greater neuroprotection than α-tocopherol (vitamin E) or ascorbic

acid, both of which are endogenous neuroprotective antioxidants. Other

cannabinoids that possess neuroprotective properties include HU-211, which

is not a CB1receptor ligand, and ∆9-THC, which is. Whereas these phenolic

cannabinoids both possess antioxidant activity, it is noteworthy that they prob-

ably owe their neuroprotective activity, at least in part, to an ability to block

NMDA receptors (HU-211) or to inhibit glutamate release by activating presy-

naptic receptors (∆9-THC; see [6, 78]). Further support for the hypothesis that

54 R.G. Pertwee

CBD can prevent cell damage caused by reactive oxygen species comes first-

ly from evidence that in rats CBD (2 mg/kg i.v.) prevents NMDA-induced

apoptotic death of retinal cells, at least in part, by opposing the accumulation

of peroxynitrite [72] and secondly from the observation that at 100–700 nM,

although not at concentrations above 1 or 2 µM, CBD protects serum-deprived

human B lymphoblastoid cells or mouse NIH 3 T3 fibroblasts from oxidative

cell death [79]. Certain other classical cannabinoids, including the non-psy-

chotropic (+)-enantiomer of ∆9-THC, were also found to exhibit protective

activity in the latter investigation.

Inflammation

CBD has been reported to exhibit anti-inflammatory activity in several in vivo

bioassays (Tab. 3) [77, 80–83], with results from some of these experiments

indicating its dose-response curve to be bell-shaped. In addition, CBD has

been shown to produce anti-nociception in the mouse phenylbenzoquinone

abdominal stretch test [84], an effect that is consistent with its apparent

anti-inflammatory activity. However, there are also reports that CBD does not

exhibit anti-nociceptive activity in the mouse acetic acid abdominal stretch test

or attenuate signs of hyperalgesia induced in rats by the injection of yeast into

their hind paws [85]. In line with its inflammatory properties, there is evidence

that CBD can inhibit lipoxygenase [81, 86] and reduce release of the proin-

flammatory cytokines interleukin-1 [87, 88] and tumour necrosis factor-α[77,

87, 88]. In addition, there is evidence that it can inhibit cyclooxygenase, albeit

only at very high concentrations [86, 89–91]. However, CBD also possesses

actions that are likely to be proinflammatory: it can activate phospholipase A2

[90, 92–94] and inhibit release of the anti-inflammatory cytokine inter-

leukin-10 [95].

Results from recent experiments with the mouse microglial cell line BV-2

indicate that CBD may also reduce inflammation in the central nervous sys-

tem by affecting microglial cell migration [8, 96]. The data suggest that

microglial cells co-express CB2receptors and receptors for abnormal CBD

and that when these receptors are simultaneously activated they interact syn-

ergistically to trigger chemokinetic and chemotaxic migration of the

microglial cells [96]. The data also suggest that 2-arachidonoyl glycerol can

activate both these receptor types to stimulate migration of BV-2 cells and that

this effect of 2-arachidonoyl glycerol is opposed by CBD, acting on the pro-

posed abnormal CBD receptors. CBD was found to display the mixed ago-

nist/antagonist properties that are typical of a partial agonist. Thus, at 0.3 µM,

it opposed the stimulatory effect of 2-arachidonoyl glycerol on microglial cell

migration but when administered by itself it produced a slight enhancement of

basal migration (EC50 = 0.25 µM). There is evidence that microglial cells

migrate towards neuroinflammatory lesion sites to release proinflammatory

cytokines and cytotoxic agents and also that 2-arachidonoyl glycerol produc-

Cannabidiol as a potential medicine 55

56 R.G. Pertwee

Table 3. Anti-inflammatory effects of CBD in vivo

Bioassay Effect of CBD Dose Reference

Oedema induced in mice by sub-plantar injection of carrageenan Inhibition 100 mg administered 19 h before [80]

carrageenan by abdominal transdermal

patches using ethosomal carriers

Clinical signs of arthritis in mice (swelling, erythema, oedema, and/or Inhibition 5, 10 or 20 mg/kg/day i.p. [77]

rigidity of joints) and histologically assessed joint damage induced by or 25 or 50 mg/kg/day p.o.*

type II collagen in complete Freund’s adjuvant injected intradermally

at the base of the tail

Bovine type II collagen-induced IFN-γrelease from lymph node cells Inhibition 5 mg/kg/day i.p.†[77]

taken from arthritic mice

In vitro TNF release from synovial cells taken from knee joints of Inhibition 5 mg/kg i.p.†[77]

arthritic mice

LPS-induced elevation of mouse serum TNF Inhibition 10 mg/kg i.p. or s.c. [77]

Ca2+ ionophore-induced stimulation of LTB4production in mouse plasma Inhibition (ex vivo) 10 mg/kg p.o. [81]

Ca2+ ionophore-induced stimulation of TXB2production in mouse plasma Enhancement (ex vivo) 10 mg/kg p.o. [81]

Plasma levels of PGE2in rats with carrageenan-inflamed paw tissue Decrease 10–40 mg/kg p.o. [82]

Basal PGE production by mouse peritoneal macrophages Inhibition (ex vivo) 50 mg/kg p.o. [83]

IFN-γ, interferon-γ; LPS, lipopolysaccharide; LTB4, leukotriene B4; PGE, prostaglandin; s.c., subcutaneous; TNF, tumour necrosis factor; TXB2, thromboxane B2.

*Bell-shaped dose-response curve: CBD was more effective against clinical signs of arthritis (1) at 5 mg/kg/day i.p. than at 10 or 20 mg/kg/day i.p. and (2) at

25 mg/kg/day p.o. than at 50 mg/kg/day p.o. Optimal doses for reducing histologically assessed joint damage in arthritic mice were 5 mg/kg/day i.p. and 25 mg/kg/day

p.o.

†Dose of CBD administered to the arthritic mice before they were killed.

tion by microglial cells can be increased by a pathological stimulus [96].

Consequently, it is possible that when given alone or in combination with a

CB2receptor antagonist, CBD may have therapeutic potential for the man-

agement of neuroinflammation resulting from endocannabinoid-induced

enhancement of microglial cell migration.

Other potential therapeutic targets

Emesis

Experiments in which rats were conditioned to display rejection reactions

(gaping, chin rubbing and paw treading) in response to oral infusion of a

flavour previously paired with the emetic agent lithium chloride have shown

that the frequency of these rejection reactions can be reduced by both CBD and

4'-dimethylheptyl-CBD at 5 mg/kg i.p. [97]. Similar results have been

obtained with ∆9-THC, 11-hydroxy-∆8-THC-dimethylheptyl (HU-210) and

the 5-HT3receptor antagonist, ondansetron [98–100]. CBD has also been

found to modulate lithium-induced vomiting in the house musk shrew in a

manner that was insensitive to antagonism by the CB1-selective antagonist,

SR-141716A [101]. Interestingly, although vomiting was suppressed by CBD

at 5 and 10 mg/kg i.p., it was enhanced by higher doses (25 and 40 mg/kg i.p.).

In contrast, ∆9-THC exhibited only an anti-emetic effect which it seemed to

produce by acting through CB1receptors. More recently, CBD has been found

to share the ability of ondansetron and ∆9-THC to suppress cisplatin-induced

emesis in the house musk shrew [102]. Again, CBD differed from ∆9-THC

(and ondansetron) by producing a biphasic effect. It suppressed vomiting at

5 mg/kg i.p. and enhanced it at 40 mg/kg i.p. It is noteworthy that CBD (10 or

20 mg/kg i.p.) also differs from ∆9-THC in not reducing 2-arachidonoyl glyc-

erol-induced vomiting in shrews [103].

Glaucoma

CBD has been found to lower intraocular pressure when applied directly to the

eyes of cats, acutely at 250 µg or continuously at 20 µg/h [104]. ∆9-THC was

also shown to lower cat intraocular pressure. However, whereas ∆9-THC pro-

duced conjunctival hyperaemia, erythema and chemosis, CBD did not.

Sleep disorders

One notable side effect of CBD in epileptic patients is somnolence (see sec-

tion on epilepsy). Consistent with this observation, rats injected with CBD at

20 or 40 mg/kg i.p. have been found to show signs of behavioural quiescence

Cannabidiol as a potential medicine 57

followed by sleep, during which they exhibited cortical EEG patterns of the

kind observed in physiological sleep [105]. Slow-wave sleep latency was

decreased by the lower dose, whereas the higher dose increased the amount of

slow-wave sleep. Rapid eye movement (REM) sleep was not affected by either

dose. In a second investigation, CBD administered at doses of 25–100 mg/kg

i.p. was found to increase sleep duration in rats [104]. In this investigation,

however, CBD reduced the proportion of sleep time spent in REM sleep and

delayed REM-sleep onset, indications that CBD may not be particularly effec-

tive clinically for the treatment of sleep disorders.

Appetite disorders

Experiments with rats have shown that, at 50 mg/kg i.p., CBD decreases the

consumption of dry food, water and sucrose solutions [106] and that at

30 mg/kg i.p. it reduces consumption of sweetened milk candy [107]. The

effect of CBD on appetite and food consumption in humans has yet to be

investigated.

Cancer

As detailed elsewhere [8, 108–112], in vitro experiments have shown that at

concentrations of 1 µM or more CBD can affect the growth and proliferation

of cancer cells, the effect most usually observed being one of inhibition. There

is also evidence that CBD has the ability to induce apoptosis in cultures of

human HL-60 myeloblastic leukaemia cells and human U87 and U373 glioma

cells [111, 112]. The data suggest that it produces this effect at 3.2 µM in

γ-irradiated leukaemia cells, at 12.7 µM in non-irradiated leukaemia cells and

at 25 µM but not 10 µM in the glioma cells [111, 112]. These and higher con-

centrations of CBD did not induce detectable apoptosis in γ-irradiated or

non-irradiated monocytes obtained from normal individuals [111]. For the

human glioma cell lines at least, the anti-tumour effects of CBD appear to be

produced in a manner that is independent of CB1and vanilloid receptors,

although possibly not of CB2receptors [112]. It has also been found that the

growth of human glioma cells implanted subcutaneously into nude mice can

be inhibited by CBD when this is administered repeatedly in vivo at a subcu-

taneous dose of 0.5 mg/mouse [112]. Future research directed at establishing

whether CBD has potential as an anti-cancer drug should include the perform-

ance of additional CBD experiments with in vivo animal models of cancer and

attempt to identify those types of tumour that are particularly susceptible to

this compound. A recent finding by Kogan et al. [113] that a quinoid deriva-

tive of CBD, HU-331, shows marked anti-tumour activity in vitro and in vivo

(in mice) also merits further investigation.

58 R.G. Pertwee

Alzheimer’s disease

Iuvone et al. [114] have obtained evidence that one clinical application of CBD

may be for the prevention of neuronal cell death that occurs in Alzheimer’s dis-

ease. This evidence came from experiments performed with an in vitro model

of this disease in which rat cultured pheocromocytoma PC12 cells were

exposed to β-amyloid. It was found that CBD decreased β-amyloid-induced

neurotoxicity in these non-neuronal cancer cells at 0.1–100 µM in a manner

that appeared to depend, at least in part, on the ability of CBD to oppose

β-amyloid-induced intracellular accumulation of Ca2+, intracellular accumula-

tion of reactive oxygen species, lipid peroxidation and apoptosis, as measured

by caspase 3 accumulation and the occurrence of DNA fragmentation. This

CBD seemed to do in a CB1-receptor-independent manner. Whether CBD also

shows protective activity in a neuronal model of Alzheimer’s disease has yet

to be established.

Concluding discussion

In conclusion, results largely from animal experiments indicate that CBD has

a number of potential therapeutic applications. The evidence supporting its use

for the management of grand mal epilepsy, anxiety, neurotoxicity and inflam-

mation, both central and peripheral, is particularly convincing. However, it is

possible that CBD will also come to have other clinical uses, for example the

attenuation of unwanted effects of ∆9-THC, when this psychoactive cannabi-

noid is used as a medicine (see [8]), or the treatment of cancer, acute schizo-

phrenia, sleep or appetite disorders, disease- or drug-induced dystonia, glau-

coma or nausea. As to future research, this should be directed at (1) establish-

ing more conclusively whether CBD does indeed have therapeutic importance

by performing clinical trials that measure its efficacy, provide information

about the best dose regimens and delivery systems for particular applications

and identify any unwanted effects of significance, including the development

of tolerance to sought-after effects; (2) determining whether benefit-to-risk

ratios could be improved by co-administering CBD with other drugs, for

example with phenytoin for the management of grand mal epilepsy (see sec-

tion on epilepsy) or with a cannabinoid CB2receptor antagonist to treat cen-

tral neuroinflammation (see section on inflammation); (3) investigating the

mode(s) of action of CBD more precisely and completely; (4) matching par-

ticular actions of CBD to particular therapeutic applications or side effects; (5)

seeking out additional potential clinical uses for CBD for which there is cur-

rently little or no evidence.

There is also a need for CBD to be optimized as a medicine. In particular,

it is important that the therapeutic applications of this phytocannabinoid are

defined more precisely, for example by mounting clinical trials directed at

establishing in greater detail (1) the types of epilepsy, neurotoxicity, dystonia

Cannabidiol as a potential medicine 59

or cancer against which CBD is most effective or (2) the extent to which CBD

can attenuate unwanted effects of ∆9-THC or contribute additional beneficial

effects without also producing unacceptable reductions in the clinically

sought-after effects of the psychoactive cannabinoid. In addition, it will be

important to determine the degree to which the apparent bell shape of the rela-

tionship between the dose of CBD and at least some of its sought-after effects

(e.g. anxiolytic, neuroprotective and anti-inflammatory effects) limits the set-

ting up of an acceptable dose regimen in the clinic. It will also be of interest to

discover the cause(s) of these bell-shaped dose-response relationships which

could, for example, arise because some actions produced only by high doses

of CBD elicit responses (e.g. enhancement of tissue levels of anandamide

through inhibition of its neuronal uptake and enzymic deamidation) that

oppose effects produced by CBD at lower doses (e.g. antagonism of anan-

damide) (see [8]). Since CBD can modulate the activity of hepatic microsomal

cytochrome P450 enzymes through both inhibition and induction (see [8]),

there is also a need to be aware that CBD may undergo clinically significant

pharmacokinetic interactions with some established medicines. Finally, it will

be important to investigate the desirability/possibility of developing an ana-

logue of CBD that, for example, has improved efficacy or potency for

sought-after effects or that has a dose-response curve with a shape that is clas-

sically sigmoid rather than bell-shaped.

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Cannabidiol as a potential medicine 65

Endocannabinoids and regulation of fertility

Mauro Maccarrone

Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy

Introduction

The adverse effects of cannabinoids, and in particular of ∆9-tetrahydro-

cannabinol (∆9-THC), on reproductive functions have been known for a long

time [1, 2], and include retarded embryo development, fetal loss and pregnan-

cy failure (recently reviewed in [3, 4]). ∆9-THC has been reported to account

for the majority of the reproductive hazards of marijuana use, in particular in

males it leads to impotency by suppressing spermatogenesis, by reducing the

weight of reproductive organs and by decreasing the plasma concentration of

circulating hormones like testosterone. In females, ∆9-THC inhibits ovulation

by prolonging the estrous cycle and decreasing the proestrous surge of luteiniz-

ing hormone. In addition, exposure to natural cannabis extracts during preg-

nancy has been correlated with embryotoxicity and specific teratological mal-

formations in rats, hamsters and rabbits. Also the major endocannabinoid anan-

damide (N-arachidonoylethanolamine, AEA) has been shown to impair preg-

nancy and embryo development in mice [3], suggesting that endocannabinoids

might regulate fertility in mammals. Consistently, down-regulation of AEA

levels in mouse uterus has been associated with increased uterine receptivity,

which instead decreased when AEA was up-regulated [5]. The levels of uterine

AEA fluctuate with changes in the pregnancy status, which is important

because successful implantation is the result of an intimate cross-talk between

the active blastocyst and the receptive uterus [5]. AEA might be critical in reg-

ulating the so-called window of implantation through synchronization of tro-

phoblast differentiation and uterine preparation to the receptive state. This

hypothesis is consistent with the observation that low levels of cannabinoid

agonists exhibit accelerated trophoblast differentiation and outgrowth, while

higher doses inhibit trophoblast differentiation [6]. In the same context, the

higher level of AEA in the nonreceptive uterus correlates well with the embry-

otoxic effect of the nonreceptive uterine environment, as well as with the in

vitro observation that AEA inhibits embryo development and zona-hatching of

blastocysts [5]. In the mouse, mRNAs of AEA-binding CB1and CB2receptors

are expressed in the preimplantation embryos, and the levels of CB1receptors

are much higher than those found in brain [3]. Activation of blastocyst CB1

receptor is detrimental for mouse preimplantation and development [5, 6], but

Cannabinoids as Therapeutics

Edited by R. Mechoulam

it appears to accelerate trophoblast differentiation [3]. On the other hand, only

CB1mRNA is present in the mouse uterus [3]. In the context of CB receptor

modulation a recent study has shown a role for progesterone receptor in

∆9-THC modulation of female sexual receptivity [7], further demonstrating

that dysregulation of cannabinoid signalling disrupts uterine receptivity for

embryo implantation [8]. Also, sea urchin (Strongylocentrotus purpuratus)

sperm has been shown to have a CB receptor, and binding of AEA to this recep-

tor reduces sperm-fertilizing capacity, by inhibiting the egg jelly-stimulated

acrosome reaction [9, 10]. This observation has been recently extended to

humans [11], and the implications for male fertility will be discussed later in

this review. The biological activity of AEA via CB1and CB2receptors depends

on its concentration in the extracellular space, which is controlled by its syn-

thesis through a specific phospholipase D (PLD), by its cellular uptake through

a specific AEA membrane transporter (AMT) and by its intracellular degrada-

tion by the enzyme fatty acid amide hydrolase (FAAH) [12]. Among these pro-

teins, which together with AEA and congeners form the endocannabinoid sys-

tem, FAAH has emerged as a pivotal check-point in several human diseases

(for comprehensive reviews see [13–17]). Evidence in favour of its critical role

in mammalian fertility will be discussed in the following sections.

Endocannabinoid degradation during pregnancy

FAAH activity has been demonstrated in mouse uterus [18], and the level of

its mRNA has been shown to change during the peri-implantation period, in

both mouse uterus and embryos [19]. FAAH localizes in the endometrial

epithelium [20], where its activity and expression decrease during early preg-

nancy, due to a lower expression of the same gene rather than to FAAH

isozymes with different kinetic properties [20].

Despite the growing evidence that AEA adversely affects uterine receptivi-

ty and embryo implantation (reviewed in [3, 4]) and that AEA degradation by

FAAH may have physiological significance in these processes [18–20], the

regulation of FAAH during early pregnancy is still obscure. Recently, we

observed down-regulation of FAAH expression in pseudopregnant mice, and a

fall of FAAH activity and expression from day 0 to day 5.5 of gestation [20].

We also reported that this fall was smaller in ovariectomized animals and larg-

er in the same animals treated with estrogen, compared to controls [20]. These

findings suggest that sex hormones might regulate FAAH activity by modulat-

ing gene expression at the translational level. The results of the treatment of

virgin females with progesterone or estrogen, showing a similar down-regula-

tion of FAAH compared to controls, strengthened this hypothesis. Therefore,

it can be concluded that in mouse uterus sex hormones down-regulate FAAH

activity by reducing gene expression at the level of protein synthesis.

Interestingly, an AEA synthase activity was also measured in mouse uterus,

and was found to respond to sex hormones in the same way as FAAH [20].

68 M. Maccarrone

It can be proposed that down-regulation of FAAH during early pregnancy

might allow higher local levels of AEA, which indeed have been shown to

increase with pregnancy in the mouse uterus [5]. In turn, AEA might play a

role in the endometrial changes associated with pregnancy, for instance

through the inhibition of gap junctions and intercellular calcium signalling

[21, 22]. On the other hand, nanomolar concentrations of AEA inhibit embryo

development and blastocysts hatching in vitro [5, 20, 23]. This suggests that

the local concentration of AEA around the implanting embryos must be low,

implying that the blastocysts have the biochemical tools to dispose AEA and

to prevent its detrimental effects. Indeed, AMT and FAAH activity have been

demonstrated and characterized in these cells [20]. Moreover, AEA-induced

inhibition of embryo development and blastocyst hatching is prevented by a

CB1receptor antagonist, in line with the hypothesis that this activity of AEA

is mediated by CB1receptors [3].

Collectively, these findings lead to a dual function of AEA in regulating fer-

tility in mammals, a scenario that is schematically depicted in Figure 1. On one

hand, a decreased FAAH activity in mouse uterus during early pregnancy

might allow higher levels of AEA at the inter-implantation sites. Here,

enhanced AEA can be instrumental in modifying endometrium by inhibiting

gap junctions. On the other hand, a low level of AEA has to be granted at the

implantation sites, in order to reduce the toxic effects of this lipid to the blas-

tocysts. The reduction of AEA levels can be achieved by the active AMT and

FAAH expressed by blastocysts, as well as by the uterine epithelial cells. It

seems noteworthy that dual functions of AEA, depending upon its local con-

centration, have been already proposed to explain its anti-proliferative (high

AEA) or pro-proliferative (low AEA) effects on trophoblast growth at the

inter-implantation and implantation sites, respectively [18]. Consistently, AEA

has been shown to regulate blastocyst function and implantation within a very

narrow concentration range, by differentially modulating mitogen-activated

protein kinase signalling and calcium channel activity via CB1receptors [24].

The embryo–uterine interactions are further complicated by the recent find-

ing that mouse blastocysts rapidly (within 30 min of culture) release a soluble

compound that increases by approximately 2.5-fold the activity of FAAH

present in the mouse uterus without affecting gene expression at the transla-

tional level [25]. This “FAAH activator” is not present in uterine fluid, is

released by neither dead blastocysts nor mouse embryonic fibroblasts, and is

produced by trophoblast and inner cell-mass cells. Moreover, its activity is

fully neutralized by lipase and is further potentiated by trypsin, whereas other

proteases, phospholipases A2, C or D, DNase I or RNase A are ineffective.

Interestingly, the blastocyst-derived activator does not affect PLD, CB recep-

tors or AMT in mouse uterus, pointing to a selective action towards FAAH. As

yet the FAAH activator, the first ever reported to our knowledge, has not been

identified with any factor known to be released by blastocysts, like

platelet-activating factor, leukotriene B4or prostaglandins E2and F2α, and its

molecular identity remains elusive [25]. However, the fact that a specific

Endocannabinoids and regulation of fertility 69

FAAH activator is released by the implanting blastocysts further corroborates

the hypothesis that these cells need to protect themselves against the noxious

effects of uterine endocannabinoids. A lipid able to cross the cell membranes

and to rapidly and specifically activate FAAH in uterine epithelial cells is suit-

able to confer this protection, as schematically shown in Figure 2. A defective

70 M. Maccarrone

Figure 1. Local effects of anandamide on implantation and uterine changes. Binding of anandamide

(AEA) to type 1 cannabinoid receptors (CB1Rs) on the blastocyst leads to cell death, whereas its bind-

ing to CB1receptors on the uterine epithelium inhibits gap junctions and is instrumental in modify-

ing the uterus during gestation. The AEA membrane transporter (AMT) and the fatty acid amide

hydrolase (FAAH) present in the uterine epithelial cells cleave AEA into ethanolamine (EtNH2) and

arachidonic acid (AA), thus protecting the blastocyst against the noxious effects of AEA.

Progesterone (P) and estrogen (E2) down-regulate uterine FAAH. Also the blastocyst has active AMT

and FAAH (omitted for the sake of clarity), which dispose of AEA and facilitate implantation.

production of the activator by an unhealthy embryo might contribute to

implantation failure. Alternatively, a non-receptive, unhealthy uterus might be

unable to respond properly to a normal activator, leading to blastocyst death.

Though the molecular details of the embryo–uterine cross-talks remain to be

elucidated, present evidence strongly suggests that FAAH regulates these

cross-talks by controlling the endogenous tone of AEA.

Endocannabinoids and regulation of fertility 71

Figure 2. Interactions between blastocyst and uterine epithelium. At the site of implantation blasto-

cysts release a lipid compound able to activate fatty acid amide hydrolase (FAAH) in uterine epithe-

lial cells, termed the FAAH activator. This leads to the cleavage of anandamide (AEA), thus reducing

its uterine levels and noxious effects towards the implanting blastocyst (see also Fig. 1). AEA-syn-

thesizing phospholipase D (PLD), AEA membrane transporter (AMT) and AEA-binding type 1

cannabinoid receptors (CB1Rs) of uterine epithelial cells are not modulated by the FAAH activator.

FAAH and human reproduction

Human reproductive fluids, such as seminal plasma, mid-cycle oviductal fluid,

follicular fluid, amniotic fluid and milk have been reported to contain AEA,

N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA) in the low

nanomolar range, from 3 nM for AEA in the follicular fluid to 67 nM for OEA

in human milk [26]. This suggests that endocannabinoids might regulate mul-

tiple physiological and pathological reproductive functions in humans, imply-

ing that exogenous cannabinoids delivered by marijuana smoke could threaten

these processes. Consistently, Table 1 shows that blood AEA levels in women

experiencing miscarriage are approximately 4-fold higher than the levels

found in women with normal gestation [4]. Since the human reproductive tis-

sues are only sparsely accessible to experimenters, we took advantage of the

critical role of peripheral lymphocytes in embryo implantation and successful

pregnancy [27] to investigate in these cells how the endocannabinoid system

might affect human reproduction.

The pathophysiological effects of AEA, and possibly of several congeners,

are under control of FAAH. Indeed, FAAH-knockout mice have been shown

to have 15-fold higher levels of AEA than wild-type littermates, suggesting

that the hydrolase controls the in vivo levels of this endocannabinoid [28]. In

particular, lymphocyte FAAH might be involved in controlling pregnancy

failure by regulating the level of AEA at the feto–maternal interface, thus

interfering with the lymphocyte-dependent cytokine networks around the

mother and the fetus [27]. In this line, we have recently demonstrated that

decreased activity and expression of FAAH in maternal lymphocytes is an

early (<8 weeks of gestation) marker of spontaneous abortion in humans [29].

In a following study, we measured the activity of FAAH and AMT, and the

binding to CB receptors in lymphocytes isolated from healthy women at 7–8

weeks of gestation, and we found that FAAH activity was lower in women

72 M. Maccarrone

Table 1. The endocannabinoid system in human gestation. The endogenous levels of AEA were

assayed in the blood of healthy women and of women who had miscarried. The binding to CB1recep-

tors, and the activity of FAAH and AMT, were assayed in peripheral lymphocytes from the same sub-

jects

Parameter Women with normal gestation Women who miscarried

AEA content (pmol/ml) 0.9 ± 1.0 (100%) 4.0 ± 2.2 (444%)*

CB1binding (cpm/mg protein) 20380 ± 1930 (100%) 20400 ± 1795 (100%)

FAAH activity (pmol/min/mg protein) 133 ± 9 (100%) 48 ± 5 (36%)*

AMT activity (pmol/min/mg protein) 50 ± 4 (100%) 49 ± 4 (98%)

*P< 0.01 versus normal gestation (P>0.05 in all other cases).

who miscarried compared to those who did not [30]. None of the other pro-

teins of the endocannabinoid system were affected (Tab. 1). In addition, we

found that the levels of AEA and FAAH in peripheral lymphocytes undergo

specific variations during the various phases of the human ovulatory cycle. In

particular, the highest levels of FAAH activity, paralleled by the lowest AEA

concentrations, were found on day 21 of gestation, which is the period that

temporally coincides with the putative window of implantation in humans

[31]. Instead PLD, AMT and CB1receptors of lymphocytes did not change

during the menstrual cycle (Tab. 2). This evidence strengthens the concept

that high FAAH activity and low AEA levels may be among the factors that

contribute to the success of implantation. Furthermore, they point towards a

key role of FAAH, but not of the other proteins of the endocannabinoid sys-

tem, in lymphocyte-mediated control of the hormone-cytokine networks at

the feto–maternal interface. In this line, recent studies have shown that prog-

esterone up-regulates FAAH, but not PLD, AMT or CB1receptors, in human

lymphocytes [32]. Some molecular details of this activity have been unrav-

elled, showing that progesterone, through formation of a complex with its

intracellular receptor, enhances the level of the trancription factor Ikaros,

which in turn enhances FAAH gene expression by binding to a specific

sequence in the promoter region [32]. Also leptin, the product of the obese

gene which controls fertility [33] and immune function [34], has been recent-

ly shown to enhance FAAH gene transcription through a STAT3-mediated

activation of the FAAH promoter at a cAMP-response element (CRE)-like

site [32]. These findings suggest that the hydrolase can be regarded as a mole-

cular integrator of well-known fertility signals, and that it controls the activi-

ty of AEA in reproduction.

Endocannabinoids and regulation of fertility 73

Table 2. The endocannabinoid system in human ovulatory cycle. The endogenous levels of AEA, the

binding to CB1receptors, and the activity of PLD, FAAH and AMT, were assayed in peripheral lym-

phocytes of healthy women at different stages of the menstrual cycle

Parameter Day 7 Day 14 Day 21

AEA content 2.15 ± 0.20 (100%) 3.76 ± 0.35 (175%)*1.29 ± 0.14 (60%)*

(pmol/mg protein)

CB1binding 20000 ± 2030 (100%) 20000 ± 2050 (100%) 17400 ± 1795 (87%)

(cpm/mg protein)

PLD activity 130 ± 15 (100%) 117 ± 12 (90%) 130 ± 15 (100%)

(pmol/min/mg protein)

FAAH activity 115 ± 12 (100%) 46 ± 5 (40%) 253 ± 22 (220%)

(pmol/min/mg protein)

AMT activity 50 ± 5 (100%) 43 ± 4 (86%) 45 ± 5 (90%)

(pmol/min/mg protein)

*P< 0.05 versus day 7 (P>0.05 in all other cases).

P < 0.01 versus day 7 (P>0.05 in all other cases).

FAAH and the regulation of spermatogenesis

Despite the knowledge that chronic administration of ∆9-THC to animals

lowers testosterone secretion and reduces the production, motility and viabil-

ity of sperm [35], a role for the endocannabinoid system in controlling male

fertility remains to be elucidated. Evidence that AEA regulates human sperm

functions has been recently presented [11], and in vitro studies have demon-

strated that the AEA congener PEA may affect the time course of capacita-

tion of human spermatozoa, by modulating the properties of their membranes

[36]. In addition, rat testis is able to synthetize AEA [37], and this compound

has been detected in human seminal plasma at approximately 10 nM [26].

More recently, the presence of CB1receptors in Leydig cells and their

involvement in testosterone secretion have been demonstrated in mice [38].

Also the function of Sertoli cells has been shown to be altered by ∆9-THC,

though the molecular basis for this alteration has not been established [39].

As Sertoli cells of the mammalian seminiferous epithelium are involved in

the regulation of germ cell development by providing nutrients and hormon-

al signals needed for spermatogenesis, we have recently investigated whether

Sertoli cells are able to bind and degrade AEA, and whether this endo-

cannabinoid might control survival and death of these cells. This is also in

view of the well-documented pro-apoptotic activity of AEA [16]. In the same

context, the effect of follicle-stimulating hormone (FSH) has been checked,

because it dramatically impacts fetal and early neonatal Sertoli cell prolifer-

ation, and is critical in determining the spermatogenic capacity in the adult

mammals [40]. To date this study on Sertoli cells represents the only charac-

terization of the endocannabinoid system and its role in male reproductive

function [41]. Therefore, the main outcomes will be briefly summarized here

to put in a better perspective their physiological relevance and potential ther-

apeutic implications.

We found that Sertoli cells have the biochemical machinery to bind and

degrade AEA, and we have characterized this machinery in cells at a range of

ages (4–24 days), largely used as a model for immature mice in endocrino-

logical studies. Immature Sertoli cells express functional CB2receptors on

their surface, and the level of these receptors is constant in ageing cells [41].

Instead, FAAH activity declines age-dependently, due to a lower gene expres-

sion, and also the uptake of AEA through AMT declines in ageing Sertoli cells.

Incidentally, to the best of our knowledge this evidence represents the first

demonstration of the modulation of the endocannabinoid system by ageing. In

addition, we found that AEA uptake by Sertoli AMT, like that of other human

peripheral cells, is significantly increased by NO donors [41], which might be

relevant in vivo because NO plays several roles in regulating male fertility [42,

43]. In particular, NO regulates the contribution of Sertoli cells to fertility and

inflammation-mediated infertility [42–44], and a faster removal of AEA from

the extracellular space, which leads to termination of its biological activity,

might be the rationale for these effects of NO.

74 M. Maccarrone

An interesting observation is that AEA can force Sertoli cells to apoptosis,

and that this process is more evident upon ageing [41]. The pro-apoptotic

effect of AEA is not mediated by CB1,CB

2or so-called endothelial-type

cannabinoid receptors, nor by vanilloid receptors. Instead, CB2receptors

expressed by Sertoli cells have a protective role against the toxic effects of

AEA, and so does FSH. In fact, this hormone dose-dependently inhibits apop-

tosis by inducing a remarkable (approximately 5-fold) increase in FAAH activ-

ity [41]. Therefore, it can be suggested that altered levels of FSH can affect

testis development through the control of the pro-apoptotic potential of AEA.

This observation, together with the well-established relationship of Sertoli cell

number to the total spermatogenic output of the testis, can contribute to the

negative effects exerted on testicular development by altered FSH concentra-

tions, as well as by mutations of the FSH receptor gene [45]. Overall, the find-

ing that Sertoli cells partake in the peripheral endocannabinoid system may

open new perspectives to the understanding and treatment of male infertility.

In particular, the observation that FAAH modulates the biological effects of

AEA on Sertoli cells, and that this FAAH-mediated control is under hormon-

al regulation, extends to male reproduction the concept that AEA hydrolase is

an important check-point, as described above for female fertility.

Conclusions and perspectives

Available evidence clearly shows that in mammals endocannabinoid signalling

is intimately associated with embryo–uterine interactions during implantation.

The exact physiological significance of this signalling pathway is not yet clear,

and it is not known how widespread it might be among different species. At

any rate, in humans low FAAH in lymphocytes correlates with spontaneous

abortion, calling for attention on this enzyme as a key point in the control of

the endocannabinoid system during pregnancy [29–32]. Moreover, available

evidence seems to add endocannabinoids to the hormone-cytokine networks

responsible for embryo–uterine interactions, and this might represent a useful

framework for the interpretation of novel interactions between progesterone,

leptin, cytokines, FSH and (endo)cannabinoids [7, 32, 41].

An interesting outcome of the reported findings is that quantitation of

FAAH protein in maternal lymphocytes might become a diagnostic marker of

spontaneous abortion, easy to measure in routine analyses. Peripheral lym-

phocytes are easily isolated from blood samples and immunochemical tests for

FAAH could be run automatically, both important advantages for monitoring

gestation in a low-risk population at large.

Since defective FAAH correlates with pregnancy failure, of interest is also

the perspective that factors able to enhance FAAH activity might become use-

ful therapeutic tools for the management of spontaneous abortion. Enhancers

of promoter activity able to mimic the actions of Ikaros and STAT3, or lipid

activators of FAAH like that released by mouse blastocysts, might open the

Endocannabinoids and regulation of fertility 75

avenue to the development of new lead compounds of therapeutic value for the

treatment of male and female infertility in humans.

Acknowledgements

I thank Professor A. Finazzi-Agrò (Department of Experimental Medicine and Biochemical Sciences,

University of Rome “Tor Vergata”, Rome, Italy) for continuing support, all colleagues who gave their

valuable contribution to the fertility studies, and Mr G. Bonelli for the excellent production of the art-

work. This investigation was supported by Ministero dell’Istruzione, dell’Università e della Ricerca

(COFIN 2002 and 2003) and by Istituto Superiore di Sanità (IV AIDS Program), Rome.

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78 M. Maccarrone

Cannabinoids in neurodegeneration and

neuroprotection

Javier Fernández-Ruiz1, Sara González1, Julián Romero2and José Antonio

Ramos1

1Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad

Complutense, 28040-Madrid, Spain

2Laboratorio de Apoyo a la Investigación, Fundación Hospital Alcorcón, 28922-Alcorcón, Madrid,

Spain

Introduction

Among a variety of cellular and tissue functions, it has been suggested that the

endocannabinoid system might also exert an important function in the cellular

decision about death or survival (for review see [1–3]). This finding has

derived from several experimental observations indicating that cannabinoids

combine at the same time neuroprotective [4–6] and anti-proliferative [1, 3]

properties. Thus, over the last decade, a considerable volume of work has

accumulated evidence to assume that the endocannabinoid system plays a role

in the protection against acute or chronic brain damage [4–6]. This fact is par-

ticularly relevant considering the postmitotic characteristics of neuronal cells,

which makes repair processes after several types of brain injury extremely dif-

ficult. For instance, plant-derived, synthetic and/or endogenous cannabinoids

provide neuroprotection in in vitro and in vivo models that replicate cytotoxic

events, mainly energy failure and excitotoxicity, occurring during several types

of accidental brain injury (i.e. ischemia and head trauma), that acutely trigger

degeneration (see [4–6] for recent reviews). In addition, cannabinoids are also

neuroprotective in several chronic neurodegenerative pathologies that also

involve the occurrence of excitotoxicity, mitochondrial dysfunction, inflam-

mation and/or oxidative stress, such as Parkinson’s disease (PD), Huntington’s

disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD)

and multiple sclerosis (MS); see [4, 7] for review.

On the other hand, the activation of different elements of the endocannabi-

noid system, as part of an endogenous protectant response, has been docu-

mented in different experimental paradigms of neurodegeneration, although

with variable results, depending on age, species, type and severity of injury, and

mechanism(s) activated for cell death (reviewed in [5, 8, 9]). Thus, several stud-

ies have demonstrated that neuronal damage is accompanied by an increase in

the production of endocannabinoids (see [5, 8] for recent reviews), although

Cannabinoids as Therapeutics

Edited by R. Mechoulam

other authors did not find this response [10]. For instance, Hansen and cowork-

ers described an increase in the levels of anandamide (N-arachi-

donoylethanolamine, AEA) and its phospholipid precursor, but not of 2-arachi-

donoyl glycerol (2-AG), during acute degeneration in the neonatal rat brain [11,

12]. Similar results, increases in AEA with no changes in 2-AG, were found by

Marsicano et al. [13] in a mouse model of kainate-induced excitotoxicity, and

by Gubellini et al. [14] in a rat model of PD. However, Panikashvili et al. [15]

showed that 2-AG is massively produced in the mouse brain after closed head

injury. In addition, they found that this endocannabinoid has neuroprotective

effects, as indicated by a reduction in edema and infarct volume and by

improved clinical recovery after being administered to animals. This endoge-

nous response has been also found in humans since elevated levels of AEA and

other fatty acid amides have been also measured around the site of damage in a

microdialysis study perfomed on a single stroke patient [16].

That the increases reported in endocannabinoid production during neu-

rodegeneration [11–16] are part of an endogenous response may be also con-

cluded from the observation that blockade of the endocannabinoid uptake with

UCM707 increased protection against kainate-induced seizures in mice,

where AEA levels were reported to be elevated [13]. However, this point is

also controversial since, although van der Stelt et al. [10] found protection

after exogenous administration of AEA in a neonatal model of secondary exci-

totoxicity, they did not record any increases in AEA or 2-AG levels and, con-

comitantly, they did not find any effect of another uptake inhibitor, VDM11,

on lesion volume [10].

Cannabinoid receptor subtypes are also induced in nerve cells in response

to injury and/or inflammation [12, 17–19]. Thus, Jin et al. [17] reported that

CB1receptors are induced in neuronal cells after experimental stroke, whereas

we described an increase of these receptors in response to excitotoxic stimuli

in neonatal rats [12]. As regards CB2receptors, a receptor subtype that is most-

ly absent from the brain in healthy conditions (see below), recent reports have

shown induction of this receptor subtype in several pathologies [18, 19]. This

occurs in activated glial cells, mainly microglia surrounding senile plaques, in

human AD brain samples [18], which might indicate that CB2receptors play a

role in either reducing degenerative impact on neurons or, on the contrary, pro-

moting cytotoxic events. Induction of CB2receptors at lesioned sites has been

also documented in rat models of striatal degeneration replicating human HD

pathology [19].

In contrast with the protective properties of cannabinoids in non-trans-

formed nervous cells, these compounds are also able to elicit apoptosis in

transformed nerve cells (C6 glioma, human astrocytoma U373MG and mouse

neuroblastoma N18TG12 cells) in vitro [1, 20], and to promote the regression

of glioblastoma in vivo, through a mechanism that involves the activation of

mitogen-activated protein kinase and ceramide accumulation [21]. In addition,

cannabinoids have been recently reported to inhibit angiogenesis, which rep-

resents a key process in tumorigenesis [22]. These anti-proliferative effects of

80 J. Fernández-Ruiz et al.

cannabinoids represent a novel potential utility of cannabinoid-based com-

pounds in cancer treatment in the future [1, 3].

The present chapter will address the evidence concerning the first of these

dual effects; that is, the capability exhibited by cannabinoids to influence, by

either promoting or reducing, several biochemical mechanisms leading to the

reduction of neuronal cell death by apoptosis or necrosis. We will divide the

chapter into three parts. First, we will summarize the different cellular and

molecular mechanisms that have been reported to underly the neuroprotective

effects of plant-derived, synthetic or endogenous cannabinoids. Second, we

will overview the information concerning these neuroprotective effects in

acute neurodegeneration, in particular in the two major accidental causes of

this pathology, cerebral ischemia and traumatic brain injury. Third, we will

address the same properties in chronic neurodegeneration, with emphasis on

the five disorders, PD, HD, AD, MS and ALS, where relevant information has

been recently published.

Mechanisms involved in neuroprotection by cannabinoids

The molecular mechanisms underlying the neuroprotectant properties of

cannabinoids are quite diverse and, frequently, complementary. They include

some events not mediated by cannabinoid receptors [i.e. N-methyl-D-aspartate

(NMDA) receptor antagonism, antioxidant properties] and others that are

definitively mediated by either CB1or CB2receptors including their capabili-

ty (1) to reduce processes such as glutamate release, calcium influx and/or

inflammation, (2) to stimulate γ-aminobutyric acid (GABA) action and (3) to

improve blood supply to the injured brain (for review, see [4–6]). Other addi-

tional processes also influenced by cannabinoids, such as improvement of glu-

cose utilization, or alternatively the production of ketone bodies – which, pro-

duced by glial cells, may replace glucose as the major source of neuronal ener-

gy metabolism in ischemia (see [23, 24] for review) – or the lowering effect on

body temperature (see [25] for review), might be also considered. However,

they have been less explored in relation to their involvement in

cannabinoid-induced neuroprotection, and therefore they will not be addressed

here.

Anti-glutamatergic effects of cannabinoids

Excitotoxicity, reflected in excessive extracellular levels of glutamate and

hyperactivation of glutamate receptors, mainly the NMDA receptor subtype, is

a critical event in acute or chronic neurodegeneration (see [26] for review). It

is bidirectionally related to an uncontrolled shift in sodium, potassium and,

particularly, calcium concentrations that disrupt ionic homeostasis and leads to

severe cell swelling and death [26]. Cannabinoid agonists are certainly

Cannabinoids in neurodegeneration and neuroprotection 81

anti-glutamatergic substances since they are able to reduce excitotoxicity

[4–6, 9, 27]. This has been demonstrated both in vitro (i.e. using cultures of

hippocampal neurons [28] or spinal cord neurons [29]) and in vivo (i.e. rodent

models of ischemic damage [30]).

These anti-glutamatergic effects of cannabinoid agonists are mainly exerted

by inhibiting glutamate release, a fact that has been largely demonstrated using

cultured neurons from numerous brain regions (see [4, 9] for recent reviews)

and also in vivo, through the activation of CB1receptors located presynapti-

cally in glutamatergic terminals (see Fig. 1, and [31] for review). This inhibito-

ry effect of cannabinoid agonists on glutamate release is reversed by selective

CB1receptor antagonists, such as SR-141716 [4, 9]. In addition, the antagonist

by itself potentiated excitotoxicity in kainate-injected mice [13] and increased

lesion volume in a rat model of HD generated by local injections of the com-

plex II inhibitor malonate [32]. However, other authors, using neonatal models

of excitotoxicity, reported no effects of SR-141716 by itself [10] or a neuro-

protective effect that was counteracted by co-administration of cannabinoid

agonists [33].

On the other hand, some specific cannabinoids, such as dexanabinol

(HU-211) and AEA, are also able to directly act on NMDA glutamatergic

receptors (see [4–6] for review). The case of HU-211 represents, together with

cannabidiol (CBD) whose neuroprotective effects will be discussed below, an

82 J. Fernández-Ruiz et al.

Figure 1. Cellular and molecular mechanisms involved in neuroprotective actions of cannabinoids.

interesting option because HU-211 has a cannabinoid structure, but does not

bind cannabinoid receptors [4, 34]. Its neuroprotective activity originates from

its capability to directly act on the glutamate system, by blocking the NMDA

receptor at a site close to, but distinct from, that of non-competitive antago-

nists, such as MK-801 and phencyclidine, and from the recognition site for

glutamate or glycine [4, 35]. Based on this antagonistic capability, HU-211

directly reduces NMDA receptor-mediated Ca2+ influx into neurons ([4, 36];

see more details below). However, it also provides neuroprotection because it

is antioxidant [4, 37] and reduces the levels of tumor necrosis factor-α

(TNF-α) [4, 34]. The result of these neuroprotective mechanisms activated by

HU-211 is an improvement of motor and memory functions in association with

reduced edema and blood–brain-barrier breakdown in rats subjected to closed

head injury (see [4, 34] for review). AEA was also shown to directly interact

with NMDA receptors in cortical, cerebellar and hippocampal slices, thereby

producing a potentiation of NMDA-induced calcium responses [38]. However,

this occurs only in the presence of SR-141716 [38]. This effect would be inde-

pendent of its neuroprotective effects mediated by the activation of cannabi-

noid receptors (i.e. cannabinoid receptor-mediated reduction in Ca2+ influx,

and anti-inflammatory and vascular effects; see details below).

Finally, it is interesting to also consider the recent evidence suggesting that

one of the mechanisms of neuroprotection elicited by NMDA receptor block-

ade would imply the enhancement of GABA transmission [39]. Cannabinoids

are able to increase inhibitory transmission mediated by GABA in some

regions such as the basal ganglia [40, 41]. This would speak in favour of the

critical importance of the imbalance between inhibitory and stimulatory inner-

vations during processes of transneuronal delayed death. Cannabinoid ago-

nists, by inhibiting glutamate release [4, 31] and/or increasing GABA presence

at synapses – presumably by blocking GABA reuptake [40, 41] – might recti-

fy this imbalance, thus delaying/arresting transneuronal death occurring in

specific regions such as the substantia nigra pars reticulata [42].

Reduction of calcium influx by cannabinoids

As mentioned above, excitotoxicity causes hyperactivation of glutamate recep-

tors that results in intracellular accumulation of cytotoxic levels of Ca2+, which

activate numerous destructive pathways involving calpains, caspases and other

proteases, protein kinases, lypases, endonucleases, NO synthase, reactive oxy-

gen species, etc. (for review, see [26]). In addition, voltage-sensitive ion chan-

nels are activated in response to the depolarization associated with

NMDA-induced Ca2+ influx, and elevate intracellular levels of this and other

ions. Cannabinoid agonists are able to close these voltage-sensitive ion chan-

nels, then reducing the overall Ca2+ current and the overactivation of destruc-

tive pathways which decrease the degree of neuronal death providing neuro-

protection (see Fig. 1, and [4–6, 9, 25] for review). These effects would be

Cannabinoids in neurodegeneration and neuroprotection 83

exerted preferentially through the activation of CB1receptors that, in this case,

would be postsynaptically located (on neurons containing NMDA glutamate

receptors) in contrast with those involved in the inhibition of glutamate release

which would be presynaptically located (see Fig. 1). Several types of calcium

channel (mainly N-, L- and P/Q-types) have been reported to be coupled to

CB1receptors and are inhibited by the activation of these receptors [43–46].

In addition, AEA has been also reported to directly interact with T-type Ca2+

channels [47]. Cannabinoid agonists also affect K+currents by opening

inwardly rectifying K+channels [44, 48, 49], an effect that might be also part

of the neuroprotective action of cannabinoids.

This direct Ca2+-lowering effect of cannabinoid agonists would add to the

reduction of this ion produced indirectly as a consequence of the anti-gluta-

matergic effects of cannabinoids, which, through reducing glutamate release or

blocking NMDA receptors (see above), would result in a reduction of gluta-

matergic receptor-mediated Ca2+ entry into the cells. As a consequence of this

direct and/or indirect inhibition of Ca2+ influx produced by cannabinoid ago-

nists, they would reduce the activation of Ca2+-dependent cytotoxic cascades

thus preventing neuronal damage [4–6]. In support of this hypothesis, several

studies have demonstrated that the increase of Ca2+ influx produced by differ-

ent neurotoxic stimuli, including NMDA and other excitotoxins, was reduced

by different plant-derived, synthetic or endogenous cannabinoids [13, 28, 38,

50, 51], and that most of these effects were counteracted by SR-141716, thus

suggesting involvement of CB1receptor activation [28, 38].

Antioxidant properties of cannabinoids

Brain injury in acute and chronic neurodegeneration triggers the accumulation

of harmful products, such as reactive oxygen intermediates (see [52] for

review), which, if not eliminated, damage DNA, proteins or membrane lipids,

leading oxidative cell death. These oxidative species are produced, in response

to excitotoxicity and/or mitochondrial dysfunction, from several sources,

including arachidonic acid metabolism, mitochondrial defects, and the action

of NO synthase and other enzymes (see [52, 53] for review). This process,

so-called oxidative stress, appears when the normal balance between oxidative

events and endogenous antioxidant mechanisms (i.e. antioxidative enzymes

such as superoxide dismutase, catalase and peroxidase, glutation and

small-molecule antioxidants such as vitamins A, C and E and ubiquinol) is dis-

rupted, being responsible for secondary damage in conditions of acute neu-

rodegeneration [54]. Certain classic cannabinoids, such as CBD, ∆9-tetrahy-

drocannabinol (∆9-THC), cannabinol, nabilone, levonantradol, dexanabinol

and others, that contain phenolic groups in their chemical structure, are able to

reduce oxidative stress [55]. These cannabinoids are potent antioxidant com-

pounds against reactive oxygen species formed during the ischemic metabo-

lism or in several chronic brain injuries where oxidative stress represents a crit-

84 J. Fernández-Ruiz et al.

ical event in the pathogenesis, such as PD [4–6]. However, it must be noted

that these antioxidant properties of specific cannabinoids would be certainly

CB1receptor-independent [37, 55–57]. This antioxidant capability has been

proposed to explain the neuroprotective effects that ∆9-THC and other relat-

ed-cannabinoids showed in animal models of cerebral ischemia ([58, 59]; see

[4–6] for review). Hampson et al. [56], using cultures of rat cortical neurons

exposed to toxic levels of glutamate, also found that both ∆9-THC and CBD

provided neuroprotection, via a CB1receptor-independent mechanism, pre-

sumably based on the antioxidant properties of both compounds, which are re-

latively equivalent.

Cannabidiol is a plant-derived cannabinoid that presents an interesting phar-

macological profile, comparable to that previously mentioned for HU-211 (see

above). CBD is non-psychoactive, because does not bind significantly to CB1

receptors. However, it exhibits an antioxidant potency comparable, and, even

superior, to that of classic dietary antioxidants such as ascorbate and α-toco-

pherol [56]. CBD was equivalent to ∆9-THC as an antioxidant compound, but

it would be more advantageous than ∆9-THC for a potential clinical use

because it can be used at higher doses and for longer times than those possible

with ∆9-THC, due to its lack of psychoactivity. An additional advantage for

CBD is that its use in prolonged treatment does not induce tolerance [60], a

phenomenon often observed with ∆9-THC [61]. On the other hand, it should

be mentioned that recent evidence suggests that CBD might also act by block-

ing endocannabinoid uptake, thus increasing endocannabinoid levels [62], or

by binding to hypothetical CBD receptors [63] still waiting to be isolated

and/or cloned. All these properties, but particularly its antioxidant potential,

enable CBD to be used as a neuroprotective compound, with minimal psy-

chotropic side effects, against the brain damage produced by reactive oxygen

species in brain ischemia [56] and also in several chronic neurodegenerative

diseases (see [64] and details below). In this sense, we have recently found that

in rat models of PD, either ∆9-THC or CBD are able to delay/arrest the pro-

gression of neuronal death [64]. It is possible that they might also be effective

in HD, as suggested by preliminary evidence [65], since it has been demon-

strated that production of free radicals, originated as a consequence of a mito-

chondrial dysfunction, is one of the major cytotoxic events that takes place

during the pathogenesis of this motor disorder (see [66] for review).

Anti-inflammatory properties of cannabinoids

Another mechanism potentially linked to various chronic and acute brain degen-

erative pathologies is the activation of inflammatory processes. Inflammation

may induce or aggravate brain damage through increasing the release of neuro-

toxic mediators, such as TNF-α, interleukin (IL)-1β, IL-6, eicosanoids, NO and

reactive oxygen species. Alternatively, it could enhance the neuronal vulnera-

bility to these cytotoxic stimuli. These factors are predominantly produced by

Cannabinoids in neurodegeneration and neuroprotection 85

glial cells (mainly reactive microglia) and impact on neurons to induce neu-

rodegeneration (see [67] for review). For instance, IL-6 and TNF-αhave been

shown to promote demyelination, thrombosis, leukocyte infiltration and

blood–brain-barrier disruption (see [68] for review). By contrast, glial cells

(mainly astrocytes) are able to produce prosurvival factors which play a role in

neuronal protection [69]. Both phenomena occur in ischemia [70], trauma [71],

PD [72], HD [73], AD [74, 75] and other diseases [67]. In addition, inflamma-

tion can also elicit neurodegeneration through the activation of autoimmune

responses against brain antigens, as happens in the case of MS and other

demyelinating diseases (see [4, 76] for review). Cannabinoid agonists are able

to reduce the inflammation that occurs in these diseases. This effect is possibly

caused by local effects on glial cells, exerted by either reducing the release of

cytotoxic factors or increasing the production of prosurvival molecules (see

Fig. 1, and [4–6, 24, 25, 68] for review). This is consistent with the idea that the

endocannabinoid signaling system would play crucial roles in glial cells both in

healthy and pathological conditions (for review, see [24, 68]).

The anti-inflammatory potential of cannabinoid agonists in neurodegenera-

tive diseases has been recently addressed in several studies that have revealed

potent anti-inflammatory effects of selective agonists for CB1(arachidonoyl-2-

chloroethylamide, ACEA) or CB2(JWH-133, JWH-015) receptors and also of

non-selective cannabinoid agonists (see [4–6, 24, 68] for review). In part, this

is the consequence of an effect of cannabinoids by protecting astrocytes and

oligodendrocytes from death, which is also beneficial for neurons [77, 78]. On

the other hand, cannabinoid agonists, possibly by activating CB1receptors

[79], modulate proinflammatory cytokine production by glial cells, mainly

IL-1, TNF-α, IL-6 and IL-12 which play a major role in the development of

damage in neurodegenerative/neuroinflammatory conditions, such as those

occurring in cerebral ischemia (see [4, 68] for review). Of particular interest is

the inhibitory effect of cannabinoid agonists on the production of TNF-αsince

this is a major contributor to the pathophysiology of brain injury [80]. These

inhibitory effects might be exerted by inhibiting the activation of the nuclear

factor-κB (NF-κB), which is involved in the induction of cytokine gene expres-

sion. HU-211, which does not bind to cannabinoid receptors, was able to inhib-

it this transcription factor [34]. In addition, several cannabinoid agonists were

able to reduce mRNA levels for certain cytokines in lipopolysaccharide-treat-

ed rat microglial cells but these effects were not cannabinoid receptor-mediat-

ed [81]. Another important inflammation-related mediator is NO, which is pro-

duced in response to immune-mediated cellular toxicity playing a role in neu-

rodegeneration (for review, see [1, 68]). Strategies that reduce the expression

of the inducible or neuronal forms of NO synthase may be neuroprotective (see

[25] for review). In this sense, cannabinoid agonists have been reported to

inhibit the release of NO in microglia [82], astrocytes [83], neurons [84] and

macrophages [85].

Glial cells may also secrete various trophic factors, such as the transform-

ing growth factor-β, the anti-inflammatory cytokine IL-10, and neurotrophins,

86 J. Fernández-Ruiz et al.

that could potentially rescue damaged neurons [79, 86], and whose production

might be enhanced by cannabinoids. For instance, Molina-Holgado et al. [87]

have recently reported that IL-1 receptor antagonist, an important anti-inflam-

matory cytokine that protects against experimentally induced ischemic, exci-

totoxic and traumatic brain insults, is produced in response to cannabinoid

receptor activation in primary cultured glial cells. Interestingly, cannabinoid

receptor activation failed to do this in knockout mice for this anti-inflammato-

ry cytokine [87].

The anti-inflammatory properties of cannabinoid agonists also involve the

activation of the CB2receptors which suggest an additional role of this recep-

tor subtype in the inflammatory processes. This is obviously of great interest

since this receptor subtype is not involved in psychotrophic effects of cannabi-

noids. This importance has been renewed in the light of recent evidence indi-

cating that CB2receptors are also expressed in the brain, even in healthy con-

ditions, located on cerebellar neurons [88], astrocytes [89], oligodendrocytes

[78], reactive microglia [18, 19] and perivascular microglial cells [90]. Of spe-

cial interest is the case of CB2receptors located in microglia since these cells

are known to perform critical roles, as they are considered the resident

macrophages in the central nervous system (CNS) [91]. Initially, it was sug-

gested that microglial cells were involved in a protective role, eliminating died

cells and allowing regeneration of viable axons after brief episodes of neuronal

injury (i.e. physical trauma or ischemia/hypoxia). However, recent evidence

suggests that a sustained activation of microglia contributes to the pathogene-

sis in chronic neurodegenerative diseases, as mentioned above (see [67, 68] for

review). Recent reports [92, 93] suggest that CB2receptors play an important

role in some key processes (i.e. microglial cell proliferation and migration at

neuroinflammatory lesion sites), involved in the initial steps of microglial acti-

vation in response to infection, inflammation or tissue injury [94].

Therefore, it appears well-demonstrated that microglia, astrocytes and

oligodendrocytes respond to cannabinoid agonists, so that the beneficial

effects of these compounds in neuroinflammation/neurodegeneration might be

related to some of the following events: (1) inhibition of proinflammatory

mediator production, (2) enhancement of anti-inflammatory factor production,

(3) inhibition of microglial recruitment and (4) enhancement of astrocyte or

oligodendrocyte survival.

Vascular effects of cannabinoids

Brain damage, such as that caused by stroke or traumatic injuries, is also asso-

ciated with the release of several endothelium-derived mediators, such as

endothelin-1 (ET-1), NO and others, which affect the local vascular tone (for

review, see [95]). The major of these mediators is ET-1, which, formed at

endothelial cells, is able to produce vasoconstriction, thus limiting the blood

supply to the injured area and aggravating brain damage [96]. Cannabinoids,

Cannabinoids in neurodegeneration and neuroprotection 87

in particular 2-AG, are potent modulators of the vascular tone (see [97, 98] for

review), which is suggestive that they might provide neuroprotection in part

because of this property. In this sense, cannabinoids counteract the

ET-1-induced vasoconstriction, thus helping to restore blood supply to the

injured brain (see Fig. 1, and [99] for review). This effect was exerted by the

activation of CB1receptors since it was prevented by SR-141716 [100], which

indicates that this receptor subtype is located in brain microvasculature (see

[101] for review). In addition, as mentioned above, cannabinoid agonists are

able to reduce NO production, thus reducing the vascular effects of this addi-

tional endothelium-derived mediator [99]. Both effects might be part of the

neuroprotective response provided by cannabinoid agonists, in particular in

cases of acute neurodegeneration such as stroke and head trauma.

Cannabinoids in acute neurodegeneration

Traumatic brain injury is the leading cause of death in young people and rep-

resents, together with cerebral ischemia, two more frequent reasons for acute

neurodegeneration resulting in permanent disability [102, 103]. Cell death dur-

ing these acute insults is mainly necrotic and is characterized by a loss of plas-

ma membrane integrity leading to subsequent inflammatory events. Apoptosis,

characterized by activation of an endogenous mechanism of destructive

enzymes called caspases, may also occur during acute degeneration but always

as a secondary event. Unfortunately, neurodegeneration caused by either

ischemia or trauma is currently without a satisfactory clinical treatment,

despite several trials using compounds exhibiting anti-glutamatergic activity,

calcium-blocking actions, antioxidant properties or anti-inflammatory effects

[26, 104–108]. As cannabinoids combine all these properties, recent preclini-

cal studies have tried to demonstrate that they may provide neuroprotection in

acute degeneration produced by several types of accidental injuries, such as

those producing glutamatergic excitotoxicity [13, 28, 56, 88], ischemic stroke

[30, 109, 110], hypoxia [111], head trauma [15], oxidative stress [55, 56],

ouabain-induced secondary excitotoxicity [10, 50] and others (see Tab. 1 for

an overview, and [4–6] for recent reviews).

In vivo, treatment with cannabinoids reduced infarct size and associated

edema, and produced a functional improvement (reduction of neurological

deficits) in animal models reproducing acute degeneration [4–6], i.e. rodents

with global (transient) or focal (permanent or transient) cerebral ischemia

induced by occlusion of carotid and vertebral arteries or intracraneal vessels,

respectively (see [9] for review). Neuroprotection by cannabinoids was also

seen in vitro using cultured neurons subjected to hypoxia and/or glucose dep-

rivation, or exposed to excitotoxic stimuli, where cannabinoids increased sur-

vival of neurons (see [6] for review). For instance, cannabinoid agonists pro-

tected cultured rat hippocampal neurons [28] and mouse spinal cord neurons

[29] from excitotoxicity. Nagayama and coworkers [30] reported that

88 J. Fernández-Ruiz et al.

WIN-55,212-2 was protective in vitro and also in an in vivo model of ischemic

damage. Anandamide and 2-AG have been also found to protect rat cortical

neurons from in vitro ischemia [111]. In another studies using in vivo models,

Cannabinoids in neurodegeneration and neuroprotection 89

Table 1. Neuroprotective effects of cannabinoid-related compounds in acute or chronic neurodegen-

erative disorders

Disease Therapeutic applications References

Acute neurodegeneration

Reduction of infart size (and associated edema) and [4– 6, 15, 30, 109]

neurological deficits by cannabinoid agonists in

rodents with global or focal cerebral ischemia, or

subjected to closed head injury.

Increase of cell survival by several cannabinoid [6, 28–30, 55,

agonists in cultured neurons (from different regions) 56, 88, 111]

subjected to hypoxia and/or glucose deprivation, or

exposed to excitotoxins.

Neuroprotection provided by direct or indirect [9, 10, 13, 50, 113]

cannabinoid agonists in rodents subjected to

excitotoxic stimuli. Greater brain injury in CB1

receptor-deficient mice subjected to ischemia.

Chronic neurodegeneration

Huntington’s Reduction of striatal injury by ∆9-THC in a non- [65]

disease (HD) apoptotic rat model (lesions with 3-nitropropionic

acid).

CB2receptor-mediated neuroprotection by canna- [19, 32]

binoids in a rat model of striatal injury that pro-

gresses through apoptotic death (local applications of

malonate); CBD exerted poor neuroprotective action

in this model, whereas SR-141716 increased striatal

damage.

Parkinson’s Reduction of dopaminergic injury in the 6-hydroxy- [64]

disease (PD) dopamine rat model by ∆9-THC and CBD.

Increased cell survival in vitro exerted by HU-210 [64]

through enhancing glial influences to neurons.

Alzheimer’s Prevention of β-amyloid toxicity in vitro by AEA, [176, 177]

disease (AD) noladin-ether or CBD.

Multiple Reduction of motor deterioration in EAE rats by [186 –188]

sclerosis (MS) plant-derived cannabinoids or uptake inhibitors.

Improvement of motor function, reduction of activated [189, 190]

microglia, and promotion of remyelination by several

cannabinoid agonists in a Theiler’s murine encephalo-

myelitis model.

CB1receptor-deficient mice were more vulnerable to [184]

inflammatory and excitotoxic insults following immune

attack in EAE.

Amyotrophic Delayed motor impairment and increased survival [192]

lateral sclerosis after ∆9-THC administration in a genetic mouse model

(ALS) of ALS.

2-AG was administered to mice subjected to closed head injury and significant

reduction of brain edema and infart volume, better clinical recovery and

reduced hippocampal cell death were documented [15]. Interestingly, the

effects of 2-AG as a neuroprotective agent were enhanced by several 2-acyl-

glycerols, which are present in the brain but that do not bind cannabinoid

receptors. It was assumed that this effect, called the entourage effect, might be

produced by partially blocking the mechanisms involved in 2-AG inactivation

(uptake and hydrolysis) [25, 112].

Except in a few cases [30, 111], most of the neuroprotectant effects of sev-

eral cannabinoid agonists were attenuated by SR-141716, thus supporting a

mediation of CB1receptors, which can be also concluded from the studies of

Parmentier-Batteur et al. [113]. These authors reported a greater brain injury

(increased infart size and neurological deficits) in CB1receptor-deficient mice

subjected to transient focal cerebral ischemia [113]. Similar results were

recently reported by Marsicano et al. [13] in the same knockout mouse model

but subjected to kainate injections. Conversely, in an in vivo neonatal model of

NMDA-induced excitotoxicity, CB1receptor blockade reduced infart size and

number of degenerating cortical neurons [33]. By contrast, other authors used

a different in vivo neonatal model that, by blocking the Na+/K+-ATPase with

ouabain, replicates the changes produced in ionic homeostasis during energy

deprivation and/or mitochondrial dysfunction characteristic of acute (and also

chronic) neurodegenerative diseases. They found a reduction of neuronal

injury in neonatal rats by ∆9-THC [50] or AEA [10], an effect that was pre-

vented by SR-141716, mainly in the case of ∆9-THC [50], thus indicating CB1

receptor mediation. Lastly, it is important to note that, in all these examples,

the neuroprotective capability of cannabinoid agonists is likely the conse-

quence of their capability to reduce excitotoxicity, oxidative stress and/or

inflammation, which are key events involved, to different extents, in the neu-

rodegeneration occurring in these acute pathologies.

Despite the neuroprotectant properties that cannabinoids display in acute

degeneration, the clinical development with cannabinoid-based compounds is

still poor and only dexanabinol (HU-211) is presently being tested in a phase

III clinical trial to reduce brain damage caused by head trauma or cerebrovas-

cular injuries [34, 114] (see also www.pharmoscorp.com/product/dexanabi-

nol.htm). This clinical trial has already demonstrated that HU-211 significant-

ly improves the neurological outcome of head injured patients.

Cannabinoids in chronic neurodegeneration

Cannabinoids, based again on their anti-glutamatergic, antioxidant and/or

anti-inflammatory properties, might be useful to delay/arrest the progression of

neuronal degeneration also in chronic diseases, where processes such as exci-

totoxicity, mitocondrial dysfunction, energy failure, oxidative stress and

inflammation are cooperative events in the pathogenesis (see [115–123] for

90 J. Fernández-Ruiz et al.

review). This would include diseases such as AD, ALS, HD, PD, MS and other

pathologies (see Tab. 1 for an overview, and [4–7, 24, 25, 68, 76] for review).

This adds to other benefits reported for cannabinoid-based compounds by alle-

viating specific clinical signs, such as the anti-hyperkinetic effect in HD [124,

125], the orexigenic action in AD [126] or the antispastic effects in MS

[127–130] produced by direct or indirect agonists of cannabinoid receptors. By

contrast, CB1receptor blockade has been reported to be effective to improve

motor inhibition in PD [131, 132] and memory deficits in AD [133]. However,

these effects on symptom relief will be addressed here only marginally.

HD is an inherited neurodegenerative disorder characterized by motor abnor-

malities, cognitive dysfunction and psychiatric symptoms, which presents in

mid life and is ultimately fatal (for review, see [115, 116]). The most striking

neuropathological change in HD patients is the preferential and progressive

degeneration of the striatum due to the selective death of striatal projection

neurons (these neurons contain CB1receptors [134]), which is accompanied

by a biphasic pattern of motor deterioration that evolves from an early hyper-

kinetic phase (choreic movements) to a late akinetic and more disabling phase

[115, 116]). Although it has been demonstrated that HD is a disease of genet-

ic origin (it is caused by an expansion of a polyglutamine tract in the N-termi-

nal portion of the huntingtin protein [116]), mechanisms underlying striatal

degeneration are still unknown. In addition, the therapeutic outcome for HD

patients has been scarce and includes mainly (1) anti-dopaminergic drugs to

reduce the excessive movement characteristic of first phases of the disease

[135] and (2) anti-glutamatergic agents to reduce excitotoxicity [136].

However, both treatments have resulted to be poor in terms of improving qual-

ity of life for HD patients. In this context, cannabinoid agonists might provide

therapeutic benefits in both aspects since they produce hypokinesia [7, 137]

and also provide neuroprotection [4–6, 27, 137].

As mentioned above, recent studies have addressed the anti-hyperkinetic

effects of direct or indirect cannabinoid agonists in animal models of HD [124,

125], based on the demonstration, in humans and laboratory animals, that the

endocannabinoid transmission becomes hypofunctional in the basal ganglia in

HD [138–145]. More recently, the neuroprotective potential of cannabinoids

has been also tested in this disease [19, 32, 65], and, although the matter is still

far from being clarified, some results have provided promising expectatives.

The rationale for these studies is based on the idea that the losses and/or dys-

function of CB1receptors in the basal ganglia is a very early event that takes

place before the appearance of major neuropathological signs and when cell

death has not occurred or is minimal. This has been found in both humans and

different models of transgenic mice that express mutated forms of huntingtin

like the human pathology [140–142]. In addition, we have recently found that

Cannabinoids in neurodegeneration and neuroprotection 91

rats with striatal atrophy caused by injection of mitochondrial toxins exhibited

profound changes in G-protein activation by CB1receptor agonists, several

days before overt striatal degeneration and appearance of severe motor symp-

toms, and in the absence of significant modifications of binding sites and

mRNA levels for this receptor [65]. All these observations, collectively, support

the notion that early functional changes in CB1receptors might be involved in

the pathogenesis of HD but, more importantly, they might play an instrumen-

tal role in striatal neurodegeneration [65]. In other words, (1) these defects in

CB1receptor signaling [65] could render neurons more vulnerable to the

degenerative process associated with HD and (2) the stimulation of these recep-

tors might reduce/delay the progression of striatal degeneration. This hypothe-

sis has been also considered by van der Stelt et al. [9], who, considering the

data obtained in HD and also in other pathologies, proposed that the malfunc-

tioning of the endocannabinoid system (i.e. AEA or 2-AG synthesis is inhibit-

ed, CB1receptors are inactive or their expression is lost) might be a signal to

trigger an unbalance in glutamate homeostasis and initiate excitotoxicity.

The neuroprotective potential of cannabinoids in HD would be based on one

or more of the above-described mechanisms by which cannabinoids may

reduce neuronal injury (i.e. acting as chemical antioxidants, inhibiting gluta-

mate release, reducing Ca2+ influx and/or producing anti-inflammatory effects;

for review, see [4, 5]). This is possible in HD because it is a neurodegenerative

disorder where mitochondrial dysfunction, excitotoxicity, inflammation and

oxidative stress have been proposed as cooperative events in the pathogenesis

[116]. In a recent study [65] we have found a promising action of the

non-selective plant-derived cannabinoid, ∆9-THC, by protecting striatal neu-

rons against the in vivo toxicity of 3-nitropropionic acid, a mitochondrial toxin

that replicates the complex II deficiency characteristic of HD patients [146].

Striatal injury in this animal model progresses by mechanisms that mainly

involve non-apoptotic death, since it is caspase 3-independent and produced

via the Ca2+-regulated protein calpain and activation of non-NMDA receptors

[147, 148]. However, it remains to be demonstrated whether the neuroprotec-

tive effect of ∆9-THC in this animal model of HD is caused by the activation

of CB1,CB

2or the combined action of both receptors, as well as through other

mechanisms available to ∆9-THC. The involvement of CB2receptors, but not

CB1receptors, has been demonstrated in other rat models of striatal injury gen-

erated by unilateral injections of malonate, another complex II inhibitor.

Malonate produces cell death that progresses mainly through the activation of

apoptotic machinery (it activates NMDA receptors and caspase 3 [149]). Thus,

we found that activation of CB2(using HU-308) but not CB1(using ACEA)

receptors provided neuroprotection, and that this effect was reversed by

SR-144528, a selective CB2receptor antagonist [19]. This indicates a crucial

role for this receptor subtype in neuroprotective effects of cannabinoids in this

model. An important aspect of these observations is that CB2receptors are

induced in response to malonate application in glial cells, possibly in reactive

microglia [19]. As this receptor subtype is usually absent in the brain in

92 J. Fernández-Ruiz et al.

healthy conditions, its induction in this model of striatal injury might be inter-

preted as part of an endogenous response against the degeneration caused by

the inhibition of the mitochondrial complex II. In addition, CBD was not effec-

tive in this HD model, thus indicating that neuroprotection exerted by cannabi-

noids is not due to their antioxidant properties [19].

Another aspect that remains to be elucidated concerns the mechanism(s)

(i.e. reduction of excitotoxicity, antioxidant effects or anti-inflammatory

action) that underlies the neuroprotective properties of cannabinoids in HD. In

this sense, using an in vitro design to test 3-nitropropionic acid toxicity, we

recently found a dose-dependent increase in the survival of cultured cerebellar

granule cells when these cells were incubated in the presence of another

non-selective cannabinoid agonist, HU-210 (I Lastres-Becker, F

Molina-Holgado, JA Ramos and JJ Fernández-Ruiz, unpublished observa-

tions). Interestingly, this neuroprotective effect is slightly enhanced if the

exposure to HU-210 is indirect, by incubating glial cells with the cannabinoid

and the resulting conditioned medium being exposed to neurons (I

Lastres-Becker, F Molina-Holgado, JA Ramos and JJ Fernández-Ruiz, unpub-

lished observations). This would indicate that the neuroprotective effect of

cannabinoids might be produced in part through modulating glial influence to

neurons (i.e. by increasing prosurvival factors such as glial anti-inflammatory

molecules, and/or by reducing cytotoxic ones such as NO, TNF-αor proin-

flammatory cytokines; see [24, 68] for review). Two specific observations sup-

port this hypothesis. First, it has been largely demonstrated that activation of

glial cells (astrocytes, oligodendroglia or microglia) occurs in HD [73, 150] as

in other neurodegenerative pathologies. Second, neuronal survival in these in

vitro experiments was extremely enhanced if the conditioned media were gen-

erated after exposure to HU-210 of glial cells obtained from IL-1β-deficient

mice (I Lastres-Becker, F Molina-Holgado, JA Ramos and JJ Fernández-Ruiz,

unpublished observations).

The major clinical neuropathology in PD includes bradykinesia (slowness of

movement), rigidity and tremor caused by the progressive degeneration of

dopaminergic neurons of the substantia nigra pars compacta that leads to a

severe dopaminergic denervation of the striatum (see a recent review in [117]).

Although the etiology of PD is presently unknown, major pathogenic process-

es that trigger the progressive loss of nigral dopaminergic neurons are oxida-

tive stress, mitochondrial dysfunction and inflammatory stimuli [72, 151,

152]. Dopaminergic-replacement therapy with L-dopa represents a useful rem-

edy to release rigidity and bradikynesia in PD patients [153], at least in the

early and middle phases of this disease. Later on, the chronic use of this ther-

apy loses efficiency and elicits the appearance of an irreversible dyskinetic

state characterized by involuntary movements. On the other hand, PD is in the

Cannabinoids in neurodegeneration and neuroprotection 93

same situation that HD in terms of pharmacotherapy to delay/arrest the

progression of neurodegeneration, substances that reduce excitotoxicity,

calcium influx, oxidative stress and/or inflammation being the only options

assayed to date (for review, see [154, 155]), although the results obtained so

far are not as promising as expected [155]. Since cannabinoid agonists share

many of the above properties (for review, see [4–6]), they have been proposed

as potentially useful neuroprotective substances also in PD, although the issue

has been explored only very recently. However, their hypokinetic profile is a

disadvantage in this case, because, despite their neuroprotectant activity in

long-term treatments, they acutely enhance rather than reduce motor symp-

toms in this disease, as revealed by a few clinical studies [7, 156, 157]. This is

compatible with the observation of overactivity in the endocannabinoid trans-

mission in the basal ganglia in PD in both patients [158] and animal models

[14, 131, 158–160]. This explains the hypokinetic profile of this disease and

the recent proposal of CB1receptor antagonists to alleviate bradykinesia and

rigidity, in particular at later phases of PD when L-dopa therapy is less effec-

tive (for review, see [7, 161]), although the first experiences in laboratory ani-

mals have been controversial [131, 162]. There would be only one exception

for cannabinoid agonists to be used for symptom relief in PD; that is, patients

for whom tremor is the major symptom. CB1receptor agonists might be use-

ful to alleviate this symptom, due to the well-know inhibitory effect of

cannabinoid agonists on glutamate release from subthalamonigral neurons

[163], whose hyperactivity is responsible for tremor [117]. However, the only

small clinical study carried out to test the effects of cannabinoids on parkin-

sonian tremor led to negative results [164].

Coming back to the question of neuroprotection by cannabinoids in PD,

recent preclinical studies carried out with ∆9-THC have revealed that this com-

pound could be also neuroprotective in PD [64]. The administration of ∆9-THC

reversed the impairment of dopaminergic transmission in the basal ganglia of

rats with hemiparkinsonism caused by the unilateral application of 6-hydrox-

ydopamine [64]. These effects did not occur in the contralateral structures,

thus indicating that the effects of ∆9-THC were produced by reducing

dopaminergic cell death in the lesioned side rather than producing up-regula-

tory effects in surviving neurons [64]. The quantification of tyrosine hydroxy-

lase mRNA levels in the substantia nigra of these animals corroborated this

finding [64]. However, the fact that the same neuroprotective effects were

elicited by CBD, another plant-derived cannabinoid with negligible affinity for

the CB1receptor, suggests that these neuroprotective effects could be CB1

receptor-independent, based on the antioxidant properties of both

plant-derived cannabinoids [64]. This observation is similar to the results

reported by Hampson et al. [56], who compared the neuroprotective effects of

∆9-THC and CBD in rat cortical neuron cultures exposed to toxic levels of glu-

tamate. This observation is particularly important in PD, a disease in which

oxidative stress, together with excitotoxicity and mitochondrial failure, is a

major hallmark in the pathogenesis of the disease [117].

94 J. Fernández-Ruiz et al.

However, we have also found evidence that glial-mediated effects are also

involved in the neuroprotection provided by cannabinoids in PD. In this sense,

although the cause of dopaminergic cell death in PD is still unknown, it has

been postulated that alterations in glial cell function (i.e. microglial activation)

may also play an important role in the initiation and/or early progression of the

neurodegenerative process [165], especially in a region like the substantia

nigra which is particularly enriched in microglia and other glial cells [166]. In

fact, several glial-derived cytotoxic factors, such as TNF-α, IL-1β, NO and

others, have been reported to be elevated in the substantia nigra and the cau-

date putamen of PD patients [167]. Based on this previous evidence, we

recently performed an in vitro study to evaluate the effects of cannabinoid ago-

nists on the neuronal toxicity of 6-hydroxydopamine. We found a marked

increase in neuronal survival when cells were incubated with conditioned

media generated by exposing glial cells to the non-selective cannabinoid

HU-210, compared with the poor increase in neuronal survival produced by

direct exposure of neuronal cells to HU-210 [64]. This supports the hypothe-

sis that neuroprotection by cannabinoids in PD might be significantly depend-

ent, not only on the antioxidant potential of certain cannabinoids, but also on

the anti-inflammatory and glial cell-mediated effects reported for most of

cannabinoids [24, 68]. Because of the role suggested for CB2receptors in

glial-mediated effects of cannabinoids [68], it is possible that this receptor sub-

type may be involved in part of the effects observed in PD, as found in HD

[19], although this question must be explored in further studies.

AD is the leading cause of dementia in the elderly, affecting to more than 4

million people in the United States alone. The pathological hallmarks of AD

are currently well known and include neuritic plaques (enriched in β-amyloid

peptide, Aβ) and fibrillary tangles (enriched in hyperphosphorylated tau pro-

tein), neuronal loss, synaptic dysfunction and gliosis (see [94, 118, 168] for

review). The cellular and molecular events involved in the pathogenesis of AD

have been partially unveiled. Briefly, it is currently thought that aberrant pro-

cessing of the β-amyloid precursor protein leads to the formation of Aβ

deposits which, in conjuction with other factors, stresses nearby neurons,

resulting in tau hyperphosphorylation and inducing the formation of neurofib-

rillary tangles [168]. Additionally, this process initiates an inflammatory

response in which astrocytes and microglia play a critical role [118], as

described for other neurodegenerative diseases (see above). The current thera-

pies for AD are (1) acetylcholinesterase inhibitors that serve to improve mem-

ory deficits caused by depleted levels of acetylcholine resulting from neuronal

loss [169] and (2) NMDA receptor blockers, such as the uncompetitive antag-

onist memantine that has provided efficacy against β-amyloid-induced neu-

rodegeneration in rats and shown great promise in clinical trials [170].

Cannabinoids in neurodegeneration and neuroprotection 95

Cannabinoids have been recently proposed as candidates for both symptom

relief and slowing of degeneration (see [171] for review).

The evidence relating cannabinoids to AD is relatively recent and has been

obtained from either biochemical or pharmacological studies. Thus, Westlake

et al. [172] reported a decrease of CB1receptor gene expression in AD post-

mortem tissues, in particular in the basal ganglia, but which could not be attrib-

utable to the pathologic process. Thus, while CB1receptor protein levels

remained unchanged, CB1mRNA exhibited a reduction that, as the authors

argued in their study, was probably parallel to the neuronal loss that accompa-

nies the progression of the disease. Studies conducted in aged rats have pro-

vided similar findings [173]. More recently, Benito et al. [18] reported that

CB1receptor levels were unaltered in brain regions affected by Aβdeposits. In

this immunohistochemical study, a slight decrease in the staining intensity of

the samples was observed, but CB1receptor protein distribution was basically

the same as in control cases.

In contrast with the lack of changes in CB1receptors, the analysis in post-

mortem tissues from AD patients revealed that CB2receptors are selectively

overexpressed in the microglial cells that are associated with Aβ-enriched neu-

ritic plaques [18]. This selectivity is especially striking, as parenquimal (silent)

microglia seem not to express CB2receptors. Recent data [90] indicate that

CB2receptors may be also expressed by a limited population of microglial cells

in the healthy brain, i.e. perivascular microglial cells, which play a pivotal role

in infectious processes affecting the CNS [174]. It may be hypothesized that

the induction of CB2receptors in microglial cells surrounding neuritic plaques

in AD may be part of an anti-inflammatory response of the CNS in order to

protect neurons from degeneration. In addition, FAAH expression and enzy-

matic activity are increased in neuritic plaques from AD tissue samples; in par-

ticular, FAAH seems to be abundantly expressed by plaque-associated astroglia

[18]. These results suggest that FAAH may participate in the important role

that astrocytes play in the gliotic response to Aβdeposition [118].

Despite the observation of changes in specific elements of the endo-

cannabinoid system, in particular CB2receptors, during the pathogenesis in

AD, only few preclinical or clinical data exist regarding the potential thera-

peutic usefulness of cannabinoids in this disease. A part of these studies deals

with the treatment of specific symptoms, as revealed by the clinical study of

Volicer et al. [126], who demonstrated a beneficial effect of dronabinol

(∆9-THC in oil solution for oral administration) by stimulating appetite and

improving disturbed behavior in AD patients. In addition, the abundance of

CB1receptors in the hippocampus and the parahippocampal and enthorinal

cortices, as well as their involvement in the control of cholinergic activity, sug-

gests an additional usefulness of cannabinoid-based compounds in memory

deficits typical of these patients. In this sense, cannabinoid receptor agonists

impair memory processing and cognition [175], whereas CB1receptor block-

ade with SR-141716 improves memory deficits in mice administered with Aβ,

presumably by an increase in hippocampal acetylcholine levels [133].

96 J. Fernández-Ruiz et al.

Recent data on the putative neuroprotective and anti-inflammatory proper-

ties of cannabinoids have opened new perspectives that could be of interest in

AD. For instance, the contribution of CB1receptors in an in vitro model of AD

has been also studied by Milton [176]. This study shows that AEA and

noladin-ether are able to prevent Aβ-induced neurotoxicity through a CB1

receptor-mediated mechanism. Thus, after exposure to different fibrilogenic

peptides, the two endocannabinoids, at nanomolar concentrations, were shown

to prevent their toxic effect on a neuronal cell line [176]. Furthermore, this pro-

tective effect was reversed by the CB1receptor specific antagonist AM251, and

seemed to be mediated by the mitogen activated protein kinase pathway, since

a selective inhibitor for this signaling pathway also prevented the protective

effects of the two endocannabinoids [176]. Similar results have been recently

published by Iuvone et al. [177] using cultured PC12 cells. These authors

found a marked reduction in cell survival following exposure of cells to Aβ,

that was associated with increased reactive oxygen species production and

lipid peroxidation, as well as caspase 3 activation, DNA fragmentation and

increased intracellular calcium [177]. Interestingly, the treatment of the cells

with CBD prior to Aβexposure significantly elevated cell survival while it

decreased oxidative stress, lipid peroxidation, caspase 3 levels, DNA frag-

mentation and intracellular calcium [177]. The authors concluded that CBD

exerts a combination of neuroprotective, antioxidative and anti-apoptotic

effects against Aβtoxicity, and that inhibition of caspase 3 appearance from its

inactive precursor, pro-caspase 3, by CBD might be involved in the signalling

pathway for this neuroprotection [177].

Therefore, taken together, the data obtained in the above studies suggest that

cannabinoids could have an important role in the prevention of Aβ-induced

neurotoxicity and counteract some of its devastating effects. Without exclud-

ing a role for CB1receptors or for other mechanisms available to certain

cannabinoids, these data suggest that part of these beneficial effects of

cannabinoids might be mediated by CB2receptors located on glial cells acti-

vated by the inflammatory process elicited by maturation of senile plaques.

These effects would be similar to those described above concerning the

anti-inflammatory role of cannabinoids exerted through the modulation of sev-

eral cytotoxic mediators such as NO, TNF-α, cytokines and others (see [24,

68] for recent reviews).

MS is the neurological disease that represents the most frequent cause of

non-traumatic, chronic disability in young adults (for review, see [178]). It is

an autoimmune disease that causes demyelination and axonal loss, in particu-

lar in the spinal cord, resulting in a variety of neurological signs, among which

pain and motor impairment are the most characteristic [119]. It was initially

thought that neurological signs in MS were exclusively caused by inflamma-

Cannabinoids in neurodegeneration and neuroprotection 97

tory processes due to activated immune cells entering the CNS [119, 120].

This explains why current therapies addressed to delay disease progression

include basically immunomodulatory agents (i.e. substances targeted against

immune elements, such as interferon, glatiramer or mitoxantrone [179]).

However, recent evidence also supports the ultimate occurrence of excitotoxi-

city and neurodegeneration (oligodendrocyte death and axonal loss) in this dis-

ease [121, 180–182]. It is for this reason that cannabinoids, in addition to their

well-described relieving effects on specific symptoms in MS [127–130, 183],

might be also used as neuroprotectant molecules to delay/arrest disease pro-

gression by protecting oligodendrocytes from death and by reducing axonal

degeneration [76, 183, 184].

As regards the symptom-relieving effects of cannabinoids in MS, most of

the studies have focused in the management of pain and motor-related symp-

toms such as spasticity, tremor and dystonia (for review see [76, 183]). They

have tried to provide solid experimental support to previous anecdotal, uncon-

trolled or preclinical data that suggested a beneficial effect for marijuana when

smoked by MS patients to alleviate specific symptoms such as spasticity, dys-

tonia, tremor, ataxia, pain and others (for review see [76, 183]). This has been

the basis for a clinical trial recently completed in the UK, which has proved

that cannabinoids did not have a beneficial effect on spasticity in MS patients,

but increased the patient’s perception of improvement for different signs of

this disease [185]. In animal studies, a series of studies by Baker and cowork-

ers revealed a potent anti-spasticity effect of plant-derived, synthetic and

endogenous cannabinoid agonists in a mouse model of MS, chronic relapsing

experimental autoimmune encephalomyelitis (CREAE) [127–130]. They also

demonstrated that these effects were mediated by cannabinoid CB1and, to a

lesser extent, CB2receptors [127]. Using this mouse model, they have also

described anti-spastic effects of compounds that are able to inhibit the process

of termination of the biological action of endocannabinoids [128–130]. These

data were concordant with the increase in endocannabinoid levels recorded in

the brain and, in particular, in the spinal cord of these animals [128]. The

chronic administration of plant-derived cannabinoids [186, 187] or specific

endocannabinoid-uptake inhibitors [188] may also reduce or delay the inci-

dence and severity of clinical signs in rats with experimental autoimmune

encephalomyelitis (EAE), a monophasic model of MS where only inflamma-

tion takes place. However, the amelioration of experimental MS in this rat

model is presumably due to the fact that cannabinoid agonists acted either as

immunosuppressive agents, by preventing the accumulation of inflammatory

cells in the CNS [186, 187], or by exerting a direct anti-inflammatory effect

[188]. Beneficial effects of cannabinoids have been also reported by other

authors [189, 190] in a MS mouse model generated by infection with the

Theiler’s murine encephalomyelitis virus. In these animals, cannabinoid ago-

nists produced an improvement of motor function, and reduced activated

microglia and promoted remyelination in the spinal cord [189], so cannabi-

noids might provide beneficial effects in MS that would go beyond symptom

98 J. Fernández-Ruiz et al.

relief. In this sense, a recent study by Pryce and coworkers [184] has demon-

strated that CB1receptor-deficient mice tolerated inflammatory and excitotox-

ic insults poorly and developed substantial degeneration following immune

attack in EAE.

Despite the progress in the pharmacological evaluation of cannabinoid-based

medicines in MS in patients and animal models, there are no data on the possi-

ble changes in CB1and CB2receptors in the postmortem brain of patients with

MS, while only a few studies have examined the status of the endocannabinoid

transmission in animal models of this disease [128, 188, 191]. Thus, Baker and

coworkers reported an increase of endocannabinoid levels in the brain and, in

particular, in the spinal cord in the mouse model of MS [128], that was inter-

preted by these authors as indicative of an endocannabinoid influence on the

control of some symptoms of MS in an environment of existing neurological

damage (see [76] for review). In our laboratory, using EAE rats, we recently

reported a decrease of CB1receptor binding and mRNA levels [191], although

the decreases in CB1receptors were mainly circumscribed to the basal ganglia

(lateral and medial caudate-putamen), and to a lesser extent to cortical regions.

We have also recorded a reduction of endocannabinoid levels in these and in

other brain structures [188]. However, as the pathology in MS models mainly

occurs in the spinal cord, the relevance of the observations in the basal ganglia

remains to be elucidated, although it is possible that they are a secondary adap-

tative event originated by primary changes at the spinal level. Thus they might

be related to the motor deterioration which is one of the most prominent neu-

rological signs in these rats [188, 191] and also in the human disease [76, 183].

Based on this fact, we hypothesized that the changes in CB1receptors and their

ligands in the basal ganglia might be associated with disturbances in several

neurotransmitters acting at this circuitry. If this were the case, the well-known

effects of cannabinoid agonists on these neurotransmitters might underly the

improving effects of these compounds in motor symptoms of MS (see [7, 27]

for review). However, our hypothesis was wrong because we did not record any

changes in dopamine, serotonin (5-hydroxytryptamine), GABA or glutamate in

the basal ganglia of MS rats [188].

ALS

ALS is one of the most common neurodegenerative disorders, occurring both

sporadically and as a familial disorder with demonstrated inherited cases in

about a 10% of patients. Although it shows multiple clinical variants, it is char-

acterized by degeneration of spinal motor neurons primarily and cortical neu-

rons secondarily (see [122, 123] for recent reviews). Neuronal loss occurs

from a combination of metal-elicited oxidative injury, excitotoxicity, aggrega-

tion and/or dysfunction of critical proteins, and genetic factors [122, 123].

Classic therapy in this disease includes riluzole, an inhibitor of glutamate

release and sodium channel blocker, but it is unsatisfactory and does not arrest

Cannabinoids in neurodegeneration and neuroprotection 99

the progression of this lethal disease whose duration averages approximately

2–3 years after diagnosis [122, 123]. Recent evidence has provided support to

the possibility that cannabinoids may also function in ALS as neuroprotectant

agents. This evidence has been obtained by Raman and coworkers [192] in a

mouse genetic model of ALS (HSODG93A transgenic mice) that overexpresses

a mutated form of the enzyme copper/zinc superoxide dismutase 1 (SOD-1),

which is linked to approximately 20% of familial cases of ALS [193, 194].

This enzyme plays a critical role as the endogenous scavenger of the superox-

ide anion, thus reducing the occurrence of oxidative stress. The mutation of

SOD-1 increases the formation of superoxide anions and the oxidative tissue

damage, and this is the key process that elicits all symptomatology character-

istic of this ALS genetic mouse model. Raman and coworkers found that

∆9-THC was effective in delaying motor impairment and prolonging survival

if administered before or after the onset of signs in the ALS mouse model

[192]. In addition, ∆9-THC was also effective at reducing oxidative damage

and excitotoxicity in spinal cord cultures [192]. No data exist on possible

changes in specific elements of the endocannabinoid system in humans affect-

ed by this disease, but very recently Witting et al. [195] have published the first

paper demonstrating endocannabinoid accumulation in the spinal cord of

HSODG93A transgenic mice, which was interpreted by these authors as part of

an endogenous defense mechanism against the oxidative damage characteris-

tic of this disease.

Concluding remarks and future perspectives

Among a variety of pharmacological effects, cannabinoids have been demon-

strated as potentially useful and clinically promising neuroprotective mole-

cules. In this chapter we have reviewed the cellular and molecular mechanisms

that might be involved in these neuroprotective effects, paying emphasis in

their potential (1) to reduce excitotoxicity exerted by either inhibiting gluta-

mate release or, in some specific cases, blocking glutamatergic receptors, (2)

to block NMDA receptor-induced calcium influx exerted directly, as a conse-

quence of the antagonism of these receptors, or indirectly, through the inhibi-

tion of selective channels for this ion, (3) to decrease oxidative injury by act-

ing as scavengers of reactive oxygen species, a property independent of

cannabinoid receptor and restricted to specific classic cannabinoids, (4) to

reduce inflammation by acting predominantly through the activation of CB2

receptors on the glial processes that regulate neuronal survival and (5) finally,

to restore blood supply to injured areas by reducing the vasocontriction pro-

duced by several endothelium-derived factors such as ET-1 or NO. Through

one or more of these processes cannabinoids may provide neuroprotection in

conditions of acute or accidental neurodegeneration, such as that occurring in

traumatic injury or ischemic episodes. In fact, dexanabinol is already in a

phase III clinical trial for therapeutic intervention in these pathologies.

100 J. Fernández-Ruiz et al.

Cannabinoids might be also used, in addition to several symptomatic utilities

also described here, to delay/arrest the progression of neurodegeneration in

chronic diseases affecting cognitive processes, such as AD, motor control or

performance, such as PD, HD and ALS, or those initially produced by inflam-

matory processes, such as MS. Most of these diseases have scarcely been stud-

ied for applications of cannabinoids, or for changes in specific elements of the

endocannabinoid system, but a rise in the number of studies is expected as

soon as the promising results generated by these molecules progress from the

present preclinical evidence to clinical applications.

Acknowledgements

The experimental work carried out by our group and that has been mentioned in this chapter has been

supported during recent years by the Plan Regional de Investigación – CAM (grant nos

08.5/0029/1998 and 08.5/0063/2001) and MCYT (grant no. SAF2003–08269). The authors are

indebted to all colleagues who contributed in this experimental work.

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Cannabinoids in neurodegeneration and neuroprotection 109

Role of the endocannabinoid system in learning and

memory

Stephen A. Varvel and Aron H. Lichtman

Department of Pharmacology and Toxicology, Virginia Commonwealth University, PO Box 980613,

Richmond, VA 23298, USA

Introduction

Following the discovery of an endocannabinoid system in the central nervous

system, which consists of the endogenous ligands arachidonoylethanolamide

(anandamide) and the monoacylglycerol 2-arachidonoyl glycerol (2-AG) that

bind to the CB1receptor [1, 2], a great deal of effort has been focused on

understanding the physiological function of this system. The identification of

these and other putative endogenous cannabinoids, including noladin ether [3]

and virodhamin [4], has sparked further in understanding the physiological

functions of the endogenous cannabinoid system. A growing body of evidence

suggests that this system serves several physiological functions including the

modulation of pain [5–7], feeding [8], drug dependence [9–11], excitotoxici-

ty [12], and cognition [13, 14]. In this review, we discuss recent in vivo and in

vitro research investigating the role that the endocannabinoid system plays in

learning and memory. Recent behavioral evidence indicates that the endo-

cannabinoid system modulates key components of learning and memory,

which include memory consolidation and extinction. On the molecular level,

endocannabinoids have been demonstrated to modulate electrophysiological

correlates of learning, suggesting that they play an important role in synaptic

plasticity. Investigations into the role of the endocannabinoid system in learn-

ing processes should make important advances in the following three areas: (1)

development of new cannabinoid-based pharmacotherapies with minimal

undesirable side effects, (2) understanding the long-term consequences of mar-

ijuana use (which remains the most commonly used illicit drug [15]), and (3)

shedding light on basic issues in neuroscience such as how are memories

formed, stored, and forgotten?

Behavioral effects of cannabinoid agonists in learning paradigms

It has long been recognized that marijuana and its chief psychoactive compo-

nent, ∆9-tetrahydrocannabinol (∆9-THC), produce disturbances in various

Cannabinoids as Therapeutics

Edited by R. Mechoulam

111

aspects of learning and memory of humans (see [16, 17] for review) and in

animal models of learning and memory (see Tab. 1 for an overview of rat,

mouse, and nonhuman primate studies). Such deficits resulting from admin-

istration of ∆9-THC and other cannabinoids have come under increasing

scrutiny in recent years as new tools have become available, and as it has

become clear that these deficits are the result of interactions with an endo-

cannabinoid system that may play a crucial role in the physiological basis of

learning and memory.

One strategy for investigating the role that the endocannabinoid system may

play in learning and memory processes is through the use of animal learning

models. While it is certain that exogenous administration of an agonist cannot

closely mimic the actions of an endogenous system tightly integrated within

neural circuits sensitive to specific spatio-temporal contexts, useful informa-

tion about the endogenous system can be taken from these studies. For exam-

ple, those particular memory tasks that are particularly sensitive to disruption

by exogenous agonists may provide insight into processes that are modulated

by endocannabinoids. Conversely, endocannabinoids are probably not crucial

to aspects of memory that are insensitive to disruption by exogenous agonists.

So which aspects of learning and memory appear most sensitive to agonists?

The most consistent delineation made regarding the effects of CB1agonists is

that they tend to disrupt aspects of short-term (i.e. working) memory, while

leaving retrieval of well-learned information (i.e. long-term or reference mem-

ory) largely intact. Working memory is an evolving concept reflecting those

processes necessary to learn and react to new information that changes over

time – a mnemonic whiteboard of sorts. Clearly, the term working memory

encompasses many distinct processes, including attentional mechanisms, as

well as associational, consolidation, encoding and retrieval processes.

Determining the impact of CB1agonists on these components is the focus of

ongoing investigation. The section below reviews many of the key studies that

address this issue by using a variety of animal models of cognition, including

instrumental operant tasks, spatial maze paradigms, and conditioned avoid-

ance tasks.

A major consideration in animal models of cognition is that learning and

memory is not directly measured, but is inferred based on changes in per-

formance. In particular, alterations in attentional, sensorimotor, and motiva-

tional processes can affect performance, independently of cognition. These

potential confounds are of considerable concern in investigating the role of the

endogenous cannabinoid system, as cannabinoid agonists and antagonists as

well as CB1–/– mice are known to affect many non-mnemonic functions that

could impact specific animal models of learning, including locomotor activity,

motivation, feeding behavior, and anxiety. In addition, there are many types of

short-term and long-term memory that are reflected in a diversity of animal

models, including recognition tasks, spatial tasks, food-motivated operant

behavior, and fear-conditioning procedures. Moreover, within each particular

form of memory, multiple processes are involved including acquisition, con-

112 S.A. Varvel and A.H. Lichtman

Role of the endocannabinoid system in learning and memory 113

Table 1. Effects of cannabinoid agonists in behavioral learning and memory paradigms

(a) Rats

Model Drug Observations Reference

Operant: ∆9-THC Delay-dependent disruptions of performance [18]

delayed match (correlated with decreased hippocampal

and delayed activity)

non-match to Tolerance developed to ∆9-THC-induced [21]

sample non- disruptions with repeated testing

match to place

∆9-THC, anand- ∆9-THC and R-methanandamide disrupted [24]

amide, R-me- performance, blocked by SR-141716

thanandamide

WIN-55,212-2 Delay-dependent disruptions [19]

Tolerance developed to daily dosing of [170]

3.75 mg/kg

∆9-THC, Decrease in performance associated with [20]

WIN-55,212-2 impaired hippocampal firing, blocked by

SR-141716

∆9-THC, Both produced working-memory deficits [22]

anandamide Both produced working-memory deficits, [23]

(+PMSF) reversed by SR-141716

Operant: repeated ∆9-THC Tolerance to performance deficits [25]

acquisition Deficits sensitive to estrogen [26]

Radial arm maze ∆9-THC Disruptions after acute and chronic dosing [32]

Increased errors [37]

∆9-THC-induced disruption blocked by [61]

SR-141716

Microinjections in hippocampus, not [33, 90]

other regions, produce deficits

Impaired retention, decreased activity [171]

CP-55,940 Impaired performance, blocked by [36]

SR-141716 and epstastigmine

∆9-THC, ∆9-THC, WIN-55,212, and CP-55940 [33]

WIN-55,212, impaired performance

CP-55940,

anandamide

Water maze HU-210 Impaired acquisition [38]

∆9-THC Deficits in working memory [172]

Nabilone, ∆8-THC ∆8-THC, not nabilone, disrupted place learning [173]

∆9-THC, WIN-55,212 disrupts learning, ∆9-THC [174]

WIN-55,212 produces place-aversion

(Continued on next page)

solidation, encoding, and retrieval processes. Thus, the endocannabinoid sys-

tem may play selective roles in different components of memory, as well as in

different types of memory.

114 S.A. Varvel and A.H. Lichtman

Table 1. (Continued)

(a) Rats

Model Drug Observations Reference

T-maze ∆9-THC Working-memory deficits [41]

Working-memory deficits, inhibition of [175]

acetylcholine release. Blocked by SR-141716,

sulpiride

No tolerance after twice-daily injections for [43]

14 days

Object recognition ∆9-THC Disrupted at 10 mg/kg, lower doses [176]

potentiated by ethanol

CP-55,940, Decreased learning/performance [177]

methanandamide

(b) Mice

Model Drug Observations Reference

Water maze ∆9-THC Working-memory deficits, reversed by [39]

SR-141716

Working-memory deficits reversed by [42]

bicuculline

Deficits in acquisition and working memory, [178]

reversed by SR-141716

∆9-THC, Working-memory deficits not present in [40]

WIN-55,212, CB1-knockout mice

methanandamide

Passive avoidance Anandamide Post-training anandamide (short, not long, [44]

interval) impaired retention, blocked by D1

and D2agonists

Intra-hippocampal anandamide, post training, [45]

disrupted retention

Post-training anandamide impaired retention [48]

in DBA/2 mice, improved it in C57BL/6 mice;

both effects blocked by naltrexone

Anandamide disrupted consolidation, effect [46]

modulated by stress, blocked by naltrexone

Interaction between subthreshold doses of [47]

of anandamide + morphine on consolidation

(Continued on next page)

Operant tasks

For decades behavioral researchers have made use of operant (instrumental)

tasks to study the effects of drugs on mnemonic function. One implementation

of this paradigm that relies heavily on working memory processes is the

delayed-match (or non-match) to sample (DMTS or DNMS) instrumental task.

These experiments generally consist of a subject being presented with a sam-

ple stimulus, an interval of time during which the stimulus is removed, and a

subsequent test phase when the sample stimulus is presented simultaneously

with a novel stimulus. The subject must indicate, usually by pressing a lever,

which was the sample (match) or which was the novel (non-match) stimulus.

∆9-THC and WIN-55,212-2, a potent synthetic cannabinoid analog, have both

been shown to disrupt accuracy of such performance in a delay-dependent

manner, consistent with a selective disruption of working memory, and are

blocked by the CB1antagonist SR-141716 [18–20]. Importantly, these behav-

ioral deficits were associated with a selective reduction in hippocampal cell

ensemble firing during the sample phase, but not during the non-match phase

of these experiments [18, 20]. Further work has also shown that tolerance

develops to the disruptive effects of ∆9-THC in this task [21], though it should

be noted that the occurrence of rate suppression in this study obfuscated the

assessment of choice accuracy.

Another series of experiments demonstrated that ∆9-THC and anandamide

[in the presence of PMSF (phenylmethylsulphonyl fluoride), a nonspecific

amidase inhibitor], produced selective deficits in working memory perform-

ance in a two-component operant task [22]. One component (conditional dis-

Role of the endocannabinoid system in learning and memory 115

Table 1. (Continued)

Model/species Drug Observations Reference

DNMS, concurrent discri- ∆9-THC ∆9-THC only disrupted [31]

mination/Rhesus monkeys DNMS task

Operant task battery: TRD Sensitivity of tasks (high–low):

DMTS, CPR, IRA, PR TRD>DMTS = IRA = CPR>PR [28]

Repeated acquisition/Squirrel ∆9-THC disrupted task, blocked [30]

monkeys by SR-141716

Operant task battery (see Marijuana smoke Sensitivity of tasks (high–low): [27]

above)/Rhesus monkeys TRD = DMTS>IRA = CPR>PR

Repeated acquisition, ∆9-THC, ∆9-THC and WIN-55,212 produced [29]

conditional discrimination/ WIN-55,212-2 deficits in the repeated acquisition

Rhesus monkeys task, blocked by SR-141716

CPR, conditioned position responding; IRA, incremental repeated acquisition; PR, progressive ratio;

TRD, temporal response diffentiation; DNMS, delay non-match to sample

crimination) required rats to press one of two levers in the presence of an

auditory or visual stimulus in a test of reference memory, while the other

component (delayed non-match to position) required rats to press the lever

opposite the one that was appropriate for the first component – a test of work-

ing memory. Consistent with a CB1receptor mechanism of action, the selec-

tive deficits produced both by ∆9-THC and anandamide in this task were

reversed by SR-141716 [23]. Others have examined slightly different aspects

of learning with other variations of operant tasks. For example, ∆9-THC and

methanandamide (an anandamide analog resistant to degradation) disrupted

the repeated acquisition of a series of three responses in an

SR-141716-reversible manner, though only at doses which also reduced

response rates [24]. A refinement of this procedure added a performance com-

ponent in which rats executed a previously learned series of responses that

remained the same across sessions. ∆9-THC reduced accuracy and rate of

responding in both components, and as with the DNMS studies mentioned

above, tolerance developed to these effects after daily administration [25].

Unfortunately, the sensitivity of the performance requirements of these tasks

to ∆9-THC-induced disruptions (as measured by response rates) precluded a

straightforward interpretation regarding the selectivity of ∆9-THC’s effects.

Interestingly, ∆9-THC-induced deficits in this task were shown to be sensitive

to estrogen, suggesting that sex differences may play an important modulato-

ry role on cannabinoids effects on learning [26].

A series of experiments in rhesus monkeys tested the effects of ∆9-THC or

marijuana smoke on performance in a battery of instrumental tasks designed

to assess different aspects of learning and memory [27, 28]. Both ∆9-THC and

marijuana smoke produced the most profound deficits in a temporal response

differentiation task and a DMTS task. They also produced moderate disrup-

tions of conditioned position responding and incremental repeated acquisition

tasks, and were least disruptive of a progressive ratio task. Further studies with

rhesus monkeys as well as squirrel monkeys have also demonstrated that

∆9-THC as well as WIN-55,212-2 disrupted a repeated acquisition perform-

ance that was reversed by SR-141716 [29, 30]. Finally, another study showed

that performance of a similar DNMS task was disrupted at doses of ∆9-THC

that did not interfere with a concurrent discrimination task [31], also consis-

tent with a specific deficit in working memory processes.

Spatial tasks

Tests of spatial learning and navigation take advantage of strategies that animals

use for foraging and avoidance of predators in their natural environment and

represent an important approach to investigations of learning and memory. One

such spatial memory task that has been used to study the effects of ∆9-THC on

spatial learning is the eight-arm radial maze, which requires rats to learn which

arms contain food rewards, and to remember which arms have already been vis-

116 S.A. Varvel and A.H. Lichtman

ited after an interposed delay. Nakamura et al. found that low dose of

1.25 mg/kg ∆9-THC was shown to produce small deficits in retention (i.e. more

errors) after a short delay that became more pronounced after daily administra-

tion [32]. These experiments suggested a cumulative detrimental effect of

chronic ∆9-THC on spatial memory, though the deficits disappeared after a peri-

od of drug washout. Lichtman et al. [33] showed that ∆9-THC, WIN-55,212-2,

and a potent bicyclic cannabinoid analog CP-55,940 all disrupted choice accu-

racy, while ∆9-THC and CP-55,940 did so at doses lower than were required to

increase task completion time (WIN-55,212-2 produced both effects at similar

doses). While ∆9-THC-induced memory impairment was blocked by

SR-141716, the cholinesterase inhibitor physostigmine failed to diminish this

effect [34]. However, the dose of physostigmine employed (0.06–0.24 mg/kg)

produced excessive cholinergic activity (e.g. excessive salivation) and thus con-

founded the interpretation of this study. However, a subsequent study reported

that a decreased doses range of physostigmine (0.01–0.05 mg/kg), as well as

another cholinesterase inhibitor tetrahydroaminoacridine, improved

∆9-THC-induced memory impairment in the radial-arm maze task [35].

Similarly, epstastigmine, a more selective cholinesterase inhibitor, reversed

∆9-THC-induced deficits in the radial-arm maze [36]. Studies of serotonergic

function found no relation between ∆9-THC-induced deficits in the radial-arm

maze and 5-hydroxytryptamine (5-HT) turnover [37].

Another spatial learning and memory task that has become increasingly pop-

ular is the Morris water maze. Unlike the radial-arm maze, this task does not

entail food deprivation, but requires the subjects to navigate in a pool of water

to locate a hidden platform by learning its relationship relative to salient visual

cues. The highly potent cannabinoid agonist, (–)-11-hydoxy-∆8-THC-dimethyl-

heptyl (HU-210), has been found to impair the ability of rats to acquire the hid-

den platform task, without disrupting their performance in a version of the

maze in which the location of the platform was directly visible [38]. These

deficits were accompanied by signs that are thought to reflect heightened anx-

iety, such as increased time spent around the outer edges of the pool (thigmo-

taxia) and increased vocalizations. Thus it was hypothesized that cannabinoids

may produce an anxiety-like state that could contribute to their effects on

learning [38].

∆9-THC also disrupted the performance of mice in a working-memory ver-

sion of the water maze in which the location of the platform was changed

before each session [39]. These effects were relatively selective as they

occurred at significantly lower doses than those required to disrupt a reference

memory version of the task (in which the platform remained in a constant posi-

tion), or produce other effects characteristic of cannabinoid activity such as

antinociception, hypothermia, catalepsy, and hypomotility. Subsequent

water-maze experiments showed that the working-memory deficits produced

by ∆9-THC, as well as WIN-55,212 and methanandamide, at doses that did not

impair a cued version of the task were fully reversed by SR-141716, and were

not observed in CB1–/– mice [40].

Role of the endocannabinoid system in learning and memory 117

Another task that includes some spatial processing requirements is the

delayed-alternation T-maze task. ∆9-THC has been demonstrated to disrupt

performance of rats in a delayed-alternation T-maze task, in which rats were

required to remember which arm had been baited during a preceding sample

trial [41]. These effects occurred at doses that did not interfere with a previ-

ously learned black/white discrimination, a reference-memory task. Similar

effects of ∆9-THC have been demonstrated in mice at a dose that did not affect

choice latency [42]. Intriguingly, others replicated this effect and found that

unlike in the operant tasks described above, tolerance did not develop to

∆9-THC, even after 14 days of twice-daily injections [43].

Conditioned avoidance

Evidence in support of a further delineation of the specificity of cannabinoid

effects on memory comes from several studies in mice that examined the

effects of anandamide in an inhibitory avoidance procedure. In this paradigm,

a chamber or runway is paired with an aversive consequence, most typically

an electric shock. Following the conditioning procedure, the subject is returned

to the apparatus and its memory is assessed by noting its latency to re-enter the

area in which it had previously received the shock. Anandamide administered

immediately after the training trial, but not 2 h after, impaired memory

(decreased latencies) of DBA mice assessed 24 h later [44], suggesting a dis-

ruption of consolidation or encoding processes, since the mice were only sus-

ceptible for a short time following training. This effect appears to be

delay-dependent, as intra-hippocampal administration of anandamide pro-

duced similar deficits in mice tested 24 h after training, while no deficits were

observed when they were tested just 2 h after training [45]. Further work with

this model demonstrated that the disruptive effects of anandamide on memory

consolidation can be amplified by stress, potentiated by a low dose of mor-

phine, and completely reversed by naloxone [46, 47]. Nonetheless, this effect

appears to be dependent on the strain of mouse, as anandamide inexplicably

appeared to improve memory performance in C57BL/6 mice [48].

In summary, cannabinoid agonists from several different chemical classes, as

well as the endocannabinoid anandamide, tend to selectively disrupt tasks heav-

ily dependent on working memory, as assessed in a variety of behavioral para-

digms, at doses that do not affect reference memory tasks or produce many

other commonly assessed cannabinoid effects. These effects have been shown

to be mediated via the CB1receptor, as they are blocked by CB1antagonists and

do not occur in CB1–/– mice. Further, the working-memory deficits observed in

the operant DNMS and spatial radial-arm maze tasks have been specifically

linked to effects in the hippocampus, suggesting that endocannabinoids may

play a particularly critical role in this brain area (see below). Many issues relat-

ed to the chronic administration of cannabinoids on learning remain unresolved.

118 S.A. Varvel and A.H. Lichtman

Most notably, tolerance has been shown to develop to the effects of ∆9-THC in

several operant tasks, but not in the delayed-alternation T-maze.

Endocannabinoid modulation of cognitive processes

Although it is well established that stimulation of CB1receptors by exoge-

nously administered cannabinoids reliably produces potent and fairly specific

memory deficits, the role that the endogenous cannabinoid system plays in

learning and memory is less clear. Even prior to the discovery of anandamide,

tonic activation of the endocannabinoid had been proposed to play a role in an

active forgetting process, in which extraneous information is deleted from

memory storage [78]. Using laboratory animal models, we hope to gain a bet-

ter understanding of the role that the endocannabinoid system plays in learn-

ing and memory as well as in neurodegenerative disease states. Below, we

describe evidence suggesting that blocking CB1receptors prolongs memory

duration in a variety of animal models of cognition. In addition, there is some

suggestion that this receptor may represent a potential therapeutic target to

treat memory deficits associated with Alzheimer’s disease. However, other

research indicates that disruption of the endocannabinoid system can interfere

with an important component of learning, the process of extinction.

Endocannabinoid modulation of memory duration

The disruption of CB1receptor signaling through the use of CB1–/– mice and

mice treated with CB1receptor antagonists are common approaches to inves-

tigate whether the endocannabinoid system is tonically active in whole ani-

mals. The role that the endocannabinoid system plays in learning and memo-

ry is typically inferred by the manner in which either approach alters perform-

ance in a memory test. The endocannabinoid system appears to play an

inhibitory role on memory duration, as CB1–/– mice and SR-141716-treated

animals have exhibited improved performance in several memory tasks (see

Tab. 2). Using a social recognition test, Terranova et al. [13] were the first to

provide in vivo evidence that the endocannabinoid system tonically modulates

memory. In this task a mature rodent spends more time investigating unfamil-

iar juvenile conspecifics than familiar ones. Subjects are presented with a juve-

nile conspecific on two separate 5-min trials separated by a delay of varying

durations. A decrease in investigative time during the second trial compared

with the first trial indicates that the subject ‘remembered’ the conspecific,

while no difference in investigative time between the two presentations indi-

cates that it no longer remembers the conspecific. SR-141716 given 5 min after

the first trial dose-dependently enhanced memory 120 min later during the sec-

ond trial [13]. The fact that SR-141716 was ineffective when administered at

15 or 90 min after trial 1 suggests the involvement of consolidation processes.

Role of the endocannabinoid system in learning and memory 119

Additionally, SR-141716 prevented the retroactive inhibition of memory elicit-

ed by a procedure in which the test subject was exposed to a different juvenile

conspecific between the two trials. In a related task, CB1–/– mice exhibited

enhanced object-recognition memory compared to wild-type control mice

[49]. Similarly, CB1–/– mice have been shown to display enhanced long-term

potentiation (LTP), an electrophysiological model of synaptic plasticity

thought to underlie learning [50].

SR-141716 was also found to enhance memory in a modified eight-arm

radial maze task [51]. When a long delay (6 h) was interposed between the first

and second halves of the task, SR-141716-treated rats committed significantly

fewer re-entry errors than committed by vehicle-treated rats. SR-141716 was

120 S.A. Varvel and A.H. Lichtman

Table 2. Studies examining the impact of disrupting endocannabinoid signaling in various animal

models of learning and memory

Model CB1–/– mice CB1receptor Reference

antagonists

Social recognition Not tested Enhanced [13]

Retroactive inhibition Not tested Enhanced

Aged rats (24 months) and mice (10–12 months) Not tested Enhanced

Object recognition Enhanced Not tested [49]

Rat radial-arm maze, delayed non-match Not tested Enhanced [51, 52]

Mouse conditioned freezing

Acquisition (tone) No effect No effect

Extinction (tone) Impaired Impaired [14]

Extinction (context) Impaired Impaired [63]

Mouse Morris water maze

Acquisition No effect No effect [40, 65]

Reversal learning Impaired Not tested [40]

Extinction (space trials) Impaired Impaired [65]

Operant tasks

Pigeon fixed consecutive number responding Not tested No effect [53]

Rat repeated-acquisition procedure Not tested No effect [24]

Rat non-match to position Not tested No effect [23]

Monkey repeated-acquisition procedure Not tested No effect [29]

Rat delayed non-match to sample Not tested No effect [20]

Mouse lever-pressing acquisition of lever pressing Impaired No tested [54]

Scopolamine-induced memory impairment

Rat social recognition Not tested No effect [13]

Rat radial-arm maze Not tested No effect [61]

Monkey repeated acquisition Not tested No effect [30]

Rat passive avoidance retention

i.c.v. β-amyloid peptide-(25–35) or -(1–42) Not tested Enhanced [60]

i.c.v., intracerebrovascular.

only effective when given immediately before phase 1, and failed to enhance

memory when administered either immediately after phase 1 or 30 min before

phase 2. In another study, the memory-enhancing effects of SR-141716 in a

delay radial-arm maze task were found to be dose-related and occurred when

administered immediately after the first session, suggesting that the drug

enhanced consolidation processes [52]. However, studies using operant para-

digms have shown no benefits of SR-141716 treatment on performance [20,

23, 24, 53]. In fact, CB1–/– mice have been reported to display impaired acqui-

sition in acquiring lever-pressing response using a simple fixed-ratio schedule

[54]. However, other phenotypes of these mice, including, hypomotility [55],

decreased feeding [8], and altered emotionality [56], may have indirectly dis-

rupted acquisition through motivational or sensorimotor alterations. One

salient difference between studies in which SR-141716 enhances memory and

those that fail to find any memory improvement is the temporal components of

the task. Memory paradigms that reveal enhancement require memory

processes that are in the order of minutes or hours, while the studies in which

SR-141716 was ineffective require the retention of information that is in the

order of seconds.

Endocannabinoid involvement in neurodegenerative diseases

Alterations of the endocannabinoid system have been reported in at least two

neurodegenerative disease states, suggesting the involvement of this system.

Specifically, reductions of CB1receptors were reported in brains of patients

diagnosed with either Alzheimer’s disease [57] or Huntington’s chorea [58]. A

more recent report found that brains from Alzheimer’s disease patients con-

tained upregulated levels of CB2receptors, as well as increased expression of

fatty acid amide hydrolase (FAAH), in microglia associated with β-amyloid

plaques [59]. Preclinical studies investigating the role of the endocannabinoid

system on memory generally utilize young, healthy animals, which arguably

may not be optimal to investigate potential nootropic agents. Conversely, there

is a great multitude of animal models of cognitive dementia and/or memory

impairment that includes surgical procedures, traumatic brain injury, intrac-

erebral administration of agents, transgenic mice, and drugs. Few studies have

examined the functional role of the endocannabinoid system or cannabi-

noid-based therapies in these pathological states or animal models of demen-

tia. Nonetheless, the fact that SR-141716 attenuated the deficits displayed by

aged mice and rats in the social recognition task suggests that this agent may

have some utility in treating memory deficits that are associated with aging

[13]. Similarly, SR-141716 has been shown to prevent memory deficits in a

passive avoidance task using a rat Alzheimer’s model [60]. In this model, an

intracerebroventricular injection of β-amyloid fragments disrupted memory

when the rats were given the retention test 1 week, but not 1 day, following

acquisition training. SR-141716 given 30 min prior to a 1-week retention test

Role of the endocannabinoid system in learning and memory 121

prevented this memory deficit; however, it failed to enhance performance

when given 30 min prior to acquisition. This pattern of findings suggests that

acute administration of CB1receptor antagonists may be effective in prevent-

ing retrieval deficits associated with neurodegenerative states [60]. In contrast

SR-141716 does not appear to be effective in the scopolamine model of mem-

ory impairment in which this nonselective muscarinic antagonist is known to

induce a variety of memory deficits. SR-141716 failed to enhance scopo-

lamine-induced deficits in a variety of animal models including the rat social

recognition task [13] a rat radial-arm maze task [61], and a monkey repeat-

ed-acquisition task [30].

Endocannabinoid modulation of extinction

The studies outlined in Table 2, which found that blockade of CB1receptor

signaling enhanced in memory tasks, suggest that SR-141716 may potentially

serve as a memory-enhancing agent. However, disruption of CB1receptor sig-

naling has also been shown to impair another important component of cogni-

tion, extinction. Extinction is defined as a process by which learned behaviors

that are no longer reinforced become actively suppressed. SR-141716-treated

mice and CB1–/– mice exhibited impaired extinction of conditioned freezing to

a tone that had been paired with foot shock [14]. Following the conditioning

procedure, presentation of the tone during extinction (i.e. in the absence of

shock) was found to increase endogenous levels of anandamide and 2-AG in

the amygdala, a brain area associated with fear. Moreover, nonreinforced pres-

entation of the tone following fear conditioning also led to differences in acti-

vation of extracellular signal-regulated kinases (ERKs) between CB1+/+ and

CB1–/– mice [62]. Compared with wild-type mice, the CB1–/– mice expressed

different levels of phosphorylated ERKs, its downstream effector Akt, and the

phosphatase calcineurin in different aspects of the amygdala and hippocam-

pus. These findings are consistent with the notion that release of endocannabi-

noids plays a role in extinction learning. A subsequent study found that

SR-141716 also impaired conditioned freezing to the test chamber in which

the mice had received the shock [63]. Of consequence, conditioned freezing to

a context is believed to involve hippocampal processes, while the hippocam-

pus is not believed to play a role in conditioned freezing to a tone [64].

Interestingly, SR-141716 failed to disrupt the within-session (short-term)

extinction of conditioned freezing, but did disrupt extinction when the mice

were tested 24 h later, suggesting it was consolidation of the extinction learn-

ing that was impaired.

Similarly, the endocannabinoid system also appears to play a role in

long-term extinction in the Morris water maze task [65]. In this model,

SR-141716-treated mice, CB1–/– mice, and appropriate C57Bl/6 control mice

were trained in a fixed-platform procedure, the platform was removed after

the mice learned the platform location, and then the mice were subjected to

122 S.A. Varvel and A.H. Lichtman

either a spaced extinction procedure (i.e. a single 60-s extinction trial given

every 2–4 weeks) or a massed extinction procedure (i.e. four daily 120-s tri-

als given on 5 consecutive days). In the spaced extinction task, the control

mice exhibited extinction across the probe trials, while both SR-141716-treat-

ed mice and CB1–/– mice continued to return to where the platform had been

located, indicating extinction deficits. Importantly, an additional group of

wild-type mice that was given only a single probe trial 9 weeks following

acquisition exhibited near-perfect performance, indicating that this task

assessed extinction and not merely time-dependent forgetting. In contrast,

disruption of CB1receptor signaling did not alter the rate of extinction when

the massed extinction procedure was used. Collectively, these results suggest

that the endocannabinoid system may play a specific role in long-term or

spaced extinction procedures.

An implication of endocannabinoid modulation of extinction is that disrup-

tion of CB1receptor signaling may interfere with learning tasks that require

the suppression of previously learned responses. In support of this notion,

CB1–/– mice as well as SR-141716-treated mice learn the location of the

fixed-platform Morris water maze task at identical rates to wild-type mice [40,

65]. Following acquisition, however, CB1–/– mice exhibited a significant

impairment in a reversal task in which the location of the hidden platform was

moved to the opposite side of the tank [40]. While the wild-type mice readily

learned the new platform location, the CB1–/– mice continued to swim to the

original platform location, despite being repeatedly shown the new platform

location. Endocannabinoid modulation of extinction also has implications

related to drug abuse. Specifically, it has recently been demonstrated that

∆9-THC and cannabidiol, a structurally related component of marijuana that

does not bind to the CB1receptor, enhances extinction of cocaine-induced and

amphetamine-induced conditioned place preference learning in rats [66].

Thus, augmenting the endocannabinoid system may be useful in treating a

wide range of perseverant, maladaptive behaviors related to aversive situa-

tions, including post-traumatic stress disorders [14] and persistent drug-seek-

ing behavior [66].

Potential confounds in manipulations altering CB1receptor signaling

Although findings in which SR-141716-treated animals or CB1–/– mice exhib-

it altered performance in mnemonic tests are generally interpreted as evidence

supporting endocannabinoid tone, other explanations can also account for

these types of finding. In the former case, SR-141716 does not appear to act

solely as a CB1receptor antagonist. For example, SR-141716 has been found

to decrease [35S]guanosine-5'-(γ-O-thio)triphosphate (GTPγS) binding in

membranes isolated from human cannabinoid CB1receptor-transfected

Chinese hamster ovary cells [67, 68], an effect opposite to that of cannabinoid

agonists [69–71], suggesting inverse agonist activity. Since cannabinoid ago-

Role of the endocannabinoid system in learning and memory 123

nists impair memory, a cannabinoid inverse agonist would be expected to

enhance memory. On the other hand, SR-141716 was found to be 7000-fold

more selective as a CB1receptor antagonist than as an inverse agonist [72],

raising questions regarding the relevance of its inverse agonism in the whole

animal. Another plausible explanation is that SR-141716 may act at non-CB1

sites of action, though it does not bind to CB2, histamine, dopamine, opioid,

5-HT, adenosine, and several other receptors and ion channels [73, 74].

SR-141716 has been reported to antagonize WIN-55,212-mediated inhibition

of hippocampal excitatory transmission in CB1–/– mice, suggesting activity at

either an uncharacterized cannabinoid receptor or noncannabinoid site of

action [75]. Curiously, AM-251, a CB1receptor antagonist that is structurally

very similar to SR-141716, failed to block WIN-55,212-2-induced inhibition

of excitatory transmission [76].

In the case of CB1–/– mice, alternative interpretations related to the use of

knockout models must be considered. For example, potential confounding fac-

tors include hitchhiking genes that are derived from the original cell line, epis-

tasis in which the effect of gene disruption is modified by the genetic back-

ground in which it is placed, and pleiotropic effects in which other conse-

quences of gene disruption indirectly affect the behavior of interest [77].

Nonetheless, a similar phenotype between the CB1–/– mice and

SR-141716-treated wild-type mice in any given behavioral test would support

the notion that these processes are under tonic endocannabinoid tone.

Neuroanatomical locus of effects

The endocannabinoid system is heterogeneously distributed throughout the

central nervous system, reflecting the diversity of physiological processes in

which endocannabinoids have been implicated as playing a role. Several lines

of evidence suggest that the hippocampus, an area long implicated with learn-

ing processes, plays a major role in the mediating both the effects of exoge-

nous cannabinoids on memory and endocannabinoid modulation of memory.

First, analysis of the distribution of CB1receptors shows that the hippocampus

contains a high density of CB1receptors, as has been demonstrated with

[3H]CP-55,940 [78] and [3H]SR-141716 [79] autoradiography, detection of

CB1mRNA expression [80], as well as with CB1receptor antibodies [81, 82].

The hippocampus has also been shown to contain relatively large amounts of

the endocannabinoids anandamide [83, 84] and 2-AG [84].

Immunocytochemical studies have revealed that FAAH, the enzyme respon-

sible for anandamide catabolism [85, 86] and monoglyceride lipase, an

enzyme that is believed to play a role in the hydrolysis of 2-AG [87, 88], are

significantly present within the hippocampus. Collectively, the high abundance

of CB1receptors, endogenous ligands, and enzymes associated with endo-

cannabinoids within the hippocampus strongly suggest that this system plays

a tonic role in physiological mechanisms of this brain area. Further support for

124 S.A. Varvel and A.H. Lichtman

this hypothesis comes from the observation that anandamide levels were

markedly decreased in the hippocampus of CB1–/– mice, but not in other brain

regions, suggesting that levels of anandamide in the hippocampus may be reg-

ulated in part by tonic activation of CB1receptors [89].

Additional evidence suggesting the hippocampus as an important locus for

cannabinoid effects on learning and memory comes from experiments investi-

gating the memory-impairing effects of CB1agonists. Not only do the effects

of cannabinoid agonists resemble those of hippocampal lesions, but also the

∆9-THC-induced deficits in the DMTS paradigm described above have been

associated with specific decreases in firing of individual hippocampal neurons

during the sample, but not the match, part of the experiment [18, 20] Site

microinjection studies also confirm the importance of the hippocampus. For

example, application of CP-55,940 directly into the dorsal hippocampus dis-

rupted working memory performance in an eight-arm radial maze without pro-

ducing other cannabinoid effects such as anti-nociception, hypomotility,

catalepsy, and hypothermia, believed to be mediated in other brain areas [33].

This dissociation between choice accuracy in the radial maze and other phar-

macological effects supports the notion that the hippocampus plays an integral

role in the cognitive alterations produced by cannabinoids. Similarly, microin-

jections of ∆9-THC into the dorsal and ventral hippocampus disrupted spatial

memory in the eight-arm radial maze, while injections into 11 other brain

regions, including different aspects of the cerebral cortex, amygdala, raphe,

caudate putamen, and mammillary body, were without effect [90].

Interestingly, ∆9-THC administration into the dorsal medial thalamus disrupt-

ed radial-arm maze performance, but it is likely that this was an indirect effect

on menemonic function as an array of abnormal behavior (e.g. increased repet-

itive and pivoting [90]) was reported. Thus a strong case can be made for the

hippocampus as a primary neuroanatomical locus for the exocannabinoids and

endocannabinoid modulation of learning and memory.

Other brain regions linked to mnemonic and attentional tasks such as the

prefrontal (frontal) cortex have also been shown to be sensitive to CB1ago-

nists. For example, ∆9-THC-induced working memory deficits have been asso-

ciated with increased dopaminergic activity in the prefrontal cortex [41], and

later studies have shown that ∆9-THC produces increases of not only

dopamine, but also of glutamate, while decreasing γ-aminobutyric acid

(GABA) release [91]. Also, ∆9-THC and WIN-55,212 have been shown to

increase acetylcholine release in rat frontal cortex in an SR-141716-reversible

manner when given systemically, but not locally [92]. CB1receptors are

expressed in the frontal cortex [78, 84]. It seems likely that these activating

effects of CB1agonists in the frontal cortex may be the result of a disinhibition

mediated via depressed GABAergic activity in other parts of the brain.

Another brain area which is just beginning to be fully appreciated for endo-

cannabinoid modulation of learning processes is the striatum, a brain region

whose role is in habit or procedural learning (e.g. [93]). As with the hip-

pocampus, high levels of CB1receptors as well as anandamide and 2-AG are

Role of the endocannabinoid system in learning and memory 125

expressed in the striatum [78, 84], and as discussed below the endocannabi-

noid system has been shown to play a critical role in synaptic plasticity in this

structure.

Cellular effects/interactions with other transmitter systems

In addition to its activity at the CB1receptor, anandamide has been shown to

have activity at the peripheral cannabinoid (i.e. CB2) receptor, potassium

channels [94], gap junctions [95], and the VR1vanilloid receptor [96, 97].

Nonetheless, endocannabinoid agonist activity at CB1receptors has been the

focus of work to understand their roles in learning processes. Activation of

CB1receptors leads to a cascade of events with consequences that affect the

electrophysiological properties of cells. Well-characterized effects of CB1

receptors include activation of adenylate cyclase activity [98–100], promotion

of mitogen-activated protein kinases [101, 102], inhibition of N- and P/Q-type

voltage-gated calcium channels [81, 103–105], and the opening of A-type and

inwardly rectifying potassium channels through their coupling with Gi/o pro-

teins [105–107]. These effects on ion currents have a hyperpolarizing influ-

ence on cell membranes, inhibiting their excitability. As CB1receptors are

found almost exclusively presynaptically, the main effect of this location is to

inhibit transmitter release (see below).

Interactions with glutamatergic systems

Recent work has focused on the influence of CB1activity on glutamatergic

activity. In rat hippocampal cultures, cannabinoid agonists have been shown to

inhibit presynaptic glutamate release. This effect appears to be the result of

activation of CB1receptors located on presynaptic nerve terminals, which sub-

sequently inhibit N- and Q-type calcium channels via an inhibitory G protein

[108–110]. Given glutamate’s role as the primary excitatory input into the hip-

pocampus and its importance in LTP, it is highly likely that CB1-mediated inhi-

bition of glutamate release is important to the mnemonic effects of cannabi-

noids. However, elucidating the nature of the relationship between endo-

cannabinoids and glutamate in the whole animal represents a difficult chal-

lenge. The ability of glutamate agonists to reverse cannabinoid-induced mem-

ory impairments is limited by the behavioral toxicity associated with this class

of compounds. Additional difficulties arise when the mnemonic effects of

cannabinoids are compared with those of antagonists of glutamatergic recep-

tors. There are several reports suggesting that working-memory (i.e. short

time) systems are largely spared by doses of N-methyl-D-aspartate (NMDA)

antagonists such as phencyclidine and dizocilpine (MK801) that disrupt the

consolidation and retrieval of long-term reference memories [111–115], while

cannabinoids tend to produce the opposite spectrum of effects.

126 S.A. Varvel and A.H. Lichtman

Interactions with GABAergic systems

Cannabinoid receptors also clearly influence hippocampal GABAergic activ-

ity, though the nature of this interaction is far from clear, and may involve

multiple pathways. Several laboratories have reported that CB1receptors in

the hippocampus were located almost exclusively on nerve terminals of

cholecystokinin (CCK)-containing GABAergic interneurons [116–119].

Furthermore, cannabinoid agonists have been shown to inhibit GABA release

in several preparations [116, 119–121]. However, several other lines of evi-

dence have suggested that cannabinoids may also play a facilitating role on

GABAergic transmission by blocking its reuptake. Early studies showed that

∆9-THC could inhibit the uptake of GABA as well as 5-HT, norepinephrine

(NE), and dopamine (DA) in rat brain synaptosomes [122, 123]. In the stri-

atal synaptosome preparations, WIN-55,212-2 inhibited GABA uptake [124].

However, in the hippocampus, ∆9-THC-induced reductions in acetylcholine

turnover were shown to be dependent on septal GABAergic interneurons, as

this reduction in turnover was completely blocked by intra-septal administra-

tion of the GABA antagonist bicuculline [125]. In addition, WIN-55,212-2

has been shown to produce a tonic hyperpolarization of CA1 pyramidal cells

in hippocampal slices, which was reversed by bicuculline [126]. Compelling

evidence has also come to light suggesting that endocannabinoids act as a ret-

rograde signal to inhibit GABA-mediated transmission that follows depolar-

ization of hippocampal pyramidal neurons [121, 127]. In addition, a recent set

of behavioral experiments has demonstrated the importance of GABAergic

transmission in vivo, as shutting down the GABAergic interneuronal system

with the GABA-A antagonist bicuculline completely reversed

∆9-THC-induced deficits in both the Morris water maze working-memory

task and an alternation T-maze task [42].

These results, taken together, suggest a complicated relationship between

endocannabinoid and GABAergic influence in the hippocampus, in which CB1

receptor stimulation may lead to a multitude of effects that depend on the spe-

cific pathway. Undoubtedly, the challenge is now to determine the functional

significance of each of these pathways on cognition.

Interactions with cholinergic systems

It has long been recognized that an important element of the action of cannabi-

noids may be their ability to inhibit cholinergic transmission in the limbic sys-

tem and cortex, and the memory deficits observed with cannabinoids resemble

those seen following administration of cholinergic antagonists [39]. Early

studies revealed that ∆9-THC reduced uptake of choline in the hippocampus,

thereby restricting acetylcholine synthesis [128, 129]. More recently, it has

become clear that cannabinoids presynaptically inhibit the release of acetyl-

choline, possibly though CB1receptors located on the cholinergic nerve ter-

Role of the endocannabinoid system in learning and memory 127

minals. Several cannabinoid agonists have been shown to inhibit electrically

evoked acetylcholine release in hippocampal slices [130–132] and synapto-

somes [133]. Similarly, microdialysis studies in awake rats also showed

cannabinoid-induced decreases in acetylcholine release [134–136]. This effect

on hippocampal acetylcholine release is clearly CB1receptor mediated, as all

the afore-mentioned studies demonstrated that SR-141716 blocks the effect.

Conversely, higher doses of SR-141716 increased the amount of released

acetylcholine in the hippocampus, indicating either inverse agonist activity of

SR-141716 or blockade of a tonic inhibitory influence by endocannabinoids.

In support of the latter possibility, electrically evoked hippocampal (but not

striatal) acetylcholine release was found to be 100% greater in CB1–/– mice

compared to wild-type controls [137]. Behavioral studies also support the

hypothesis that acetylcholine plays a role in cannabinoid-induced memory

impairment. Although an initial study found that the cholinesterase inhibitor

physostigmine failed reverse ∆9-THC-induced deficits in an eight-arm radial

maze [61], subsequent studies demonstrated that low doses of physostigmine

as well as other cholinesterase inhibitors blocked cannabinoid-induced mem-

ory impairment [35, 36].

Role of endocannabinoids in synaptic plasticity

Given that endocannabinergic mechanisms have been strongly implicated in

behavioral paradigms of learning and memory, it is not surprising that a grow-

ing body of work has focused on understanding the role of the endocannabi-

noid system in the electrophysiological correlates of learning, synaptic plas-

ticity. Synaptic plasticity is a network attribute of synapses, referring to their

ability to change in structure and function in response to particular patterns of

activation. These processes are believed to represent the neurobiological basis

of learning in which the experiences of an organism can modify subsequent

responses to stimuli.

Short-term plasticity

Great strides have been made in understanding the physiological role of the

endocannabinoid system with the discovery that endocannabinoids may serve

to mediate a short-term plasticity phenomenon referred to as depolari-

zation-induced suppression of inhibition (DSI) and its corollary depolariza-

tion-induced suppression of excitation (DSE). DSI occurs on GABAergic

synapses and has been studied in hippocampal CA1 pyramidal cells [138, 139]

and in cerebellum [140, 141]. Depolarization of the postsynaptic cell results in

the release of a retrograde messenger which diffuses back across the synapse

and inhibits further GABA release, thus diminishing inhibitory tone for a brief

period of a few seconds. Conversely, DSE involves the short-term inhibition of

128 S.A. Varvel and A.H. Lichtman

glutamate release, and has also been demonstrated in several brain areas

including the VTA [142].

Early reports suggested that the retrograde messenger may have been gluta-

mate, acting on presynaptic metabotropic glutamate receptors [143, 144].

However, Wilson and Nicoll [121] as well as Ohno-Shosaku and colleagues

[127] established that endocannabinoids play a critical role in DSI. Supporting

such a role for endocannabinoids are the findings that DSI in the hippocampus

is completely blocked by CB1antagonists [121, 127] and is absent in CB1–/–

mice [145]. Similarly, the DSI observed in cerebellar Purkinje cells has been

shown to be mediated by CB1receptor activation located on presynaptic neu-

rons [146–148]. Based on these findings it has been hypothesized that endo-

cannabinoids are released from the postsynaptic neuron, and travel retrograde-

ly to presynaptically located CB1receptors where they inhibit GABA release.

Despite the potential importance of these phenomena, their physiological sig-

nificance remains to be established. Hampson and colleagues attempted to

induce DSI using pulse trains that mimic hippocampal cell-firing patterns that

occur in vivo [149]. However, they found that these normal firing patterns of

hippocampal neurons failed to elicit DSI.

Models of long-term synaptic plasticity: LTP

Initial studies examining the effects of cannabinoids on synaptic plasticity

demonstrated that CB1agonists disrupt LTP. LTP refers to the phenomenon in

which brief high-frequency stimulation applied to afferent pathways results in

an increase in the excitatory synaptic potentials of postsynaptic neurons, which

can last from hours to weeks. This phenomenon was first observed in the hip-

pocampus [150] where it has been most extensively characterized, but has

since been demonstrated in many other brain areas.

Nowicky et al. [151] first reported that ∆9-THC significantly impaired the

induction of LTP induced in CA1 region of hippocampal slices using two

tetanizing trains (200 Hz, 0.5-s duration, 5-s intertrial interval (ITI)) delivered

to the stratum radiatum. This effect of ∆9-THC was also produced by HU-210

[152], and was shown to be mediated via the CB1receptor [153]. Terranova et

al. [154] extended these findings to WIN-55212-2 and the endogenous

cannabinoid anandamide, while Stella et al. [155] demonstrated that applica-

tion of 2-AG can also inhibited the induction of hippocampal LTP, all of which

were reversed by SR-141716. Furthermore, it has recently been shown that

AM-404, an inhibitor of the putative anandamide transporter that also inhibits

FAAH, can disrupt induction of LTP in an SR-141716-reversible manner

[156]. Interestingly, CB1–/– mice demonstrated enhanced hippocampal LTP

induced by high-frequency stimulation to the Shaffer collaterals [50]. One

likely mechanism given for this cannabinoid-induced disruption of LTP is that

presynaptic CB1receptors inhibit release of the glutamate necessary to depo-

larize the postsynaptic cell and release NMDA receptors from the magnesium

Role of the endocannabinoid system in learning and memory 129

blockade existent under normal conditions. The opening of these NMDA

receptors and the subsequent influx of calcium triggers calcium-dependent

second-messenger systems that initiate the induction of LTP [157]. While the

studies described above lend support to the hypothesis that endocannabinoids

may serve to diminish induction of LTP, these effects have been shown to

depend on the induction method. In all of the cases mentioned above, LTP was

induced by high-frequency titanic stimulation. However, LTP can also be

observed following a theta burst protocol, which is thought to reflect a more

physiologically relevant process. Lees and Dougalis [156] showed that while

WIN-55,212 will block induction of LTP under both conditioning protocols in

an AM-251-reversible manner, anandamide only prevents high-frequency

stimulation LTP.

In contrast to the clear disruption of LTP observed when CB1agonists are

exogenously administered to whole tissues, more subtle and potentially inter-

esting effects have been reported when the effects of endocannabinoids have

been looked at within their endogenous context. For example Carlson et al.

[158] showed that endocannabinoids released via induction of DSI led to a

facilitation of LTP in targeted cells, but not neighboring ones, by selectively dis-

inhibiting postsynaptic cells (through decreased presynaptic GABA release)

and lowering their thresholds for LTP. It was hypothesized that exogenous

application of cannabinoids may disrupt learning by disrupting the spatial and

temporal selectivity of coding mediated by endocannabinoids. Additionally,

cannabinoids have been shown to promote signaling pathways such as ERK,

which are known to be important for synaptic plasticity and learning [159].

Models of long term synaptic plasticity: long-term depression (LTD)

In contrast to high frequency stimulation that can lead to LTP, low-frequency

stimulation leads to another form of long-term synaptic plasticity, known as

LTD, in which synapse strength is weakened. LTD has been demonstrated in

several brain areas, including the hippocampus, striatum, cerebellum, and var-

ious parts of the cortex (for reviews see [160, 161]). Over the past few years,

endocannabinoids have been demonstrated to be crucial components of LTD

in several brain areas including the striatum, amydala, frontal cortex, and

nucleus accumbens.

LTD in the striatum, a process believed to be important in certain forms of

learning, has been shown to be CB1receptor-dependent. LTD was absent in

CB1–/– mice and greatly reduced in slices preincubated with SR-141716, while

it was potentiated by AM-404 (an inhibitor of both FAAH and the anandamide

transporter) [162]. In addition, postsynaptic loading of anandamide resulted in

reduced presynaptic excitatory transmission. These findings support the notion

that endocannabinoids serve as retrograde messengers to reduce excitatory

cortical inputs to striatal output neurons. A similar facilitative role of endo-

cannabinoids on LTD has been proposed in the amygdala, where LTD induced

130 S.A. Varvel and A.H. Lichtman

by WIN-55212 or amphetamine was blocked by AM-251, and the ampheta-

mine-induced LTD was facilitated by AM-404 [163]. Additionally, LTD in the

neocortex [164] as well as in the nucleus accumbens [165, 166] has been

shown to be dependent on presynaptic activation of CB1receptors.

Interestingly, chronic exposure to ∆9-THC or WIN-552-2 has been shown to

prevent the induction of LTD, suggesting a functional tolerance that may par-

tially underlie putative, long-lasting cognitive deficits associated with chronic

marijuana use [166].

This hypothesized role of endocannabinoids as initiators/mediators of LTD

is also consistent with a series of experiments examining the effects of

cannabinoid agonists and CB1antagonists on synaptic plasticity in the pre-

frontal cortex [167]. Tetanic stimulation induced plasticity in slightly more

than half of the neurons examined, with approximately equal numbers of cells

developing LTP and LTD. However, in the presence of WIN-55212-2 almost

all of the plastic synapses showed LTD, while in the presence of SR-141716

almost all of the plastic synapses developed LTP. The consequences of such a

shift on learning are unclear, though it may suggest that forms of learning

heavily dependent on LTP could be attenuated by endocannabinoids, while

forms of learning heavily dependent on LTD could actually be enhanced.

Other forms of synaptic plasticity

Endocannabinoids have also been shown to modify other forms of synaptic

plasticity. For example, anandamide has been shown to inhibit an interesting

though poorly understood phenomena referred to as long-term transformation

(LTT), where GABAergic inhibition is “transformed” into excitation when

presynaptic tetanic stimulation is paired with postsynaptic depolarization

[168]. This LTT has been shown to play an important role in pairing-induced

LTP, which is also inhibited by anandamide [168].

In addition to changes in presynaptic transmitter release and postsynaptic

sensitivity, synaptic plasticity can be supported by the growth of new synaps-

es, a process that has also been shown to be sensitive to CB1stimulation.

Anandamide, ∆9-THC, and WIN-55212-2 have all been shown to inhibit new

synapse formation induced by forskolin, probably due to CB1receptor-medi-

ated decreases in cAMP [169].

Conclusions on the effects of endocannabinoids on cognition

The weight of evidence clearly demonstrates that the endocannabinoid system

is critically involved with physiological mechanisms underling learning and

memory. It can be stated with a high degree of confidence that CB1receptors

located in brain areas associated with learning and memory, including the hip-

pocampus, frontal cortex, and striatum, are a crucial element of this influence.

Role of the endocannabinoid system in learning and memory 131

CB1receptor activation by exogenous application of cannabinoid agonists con-

sistently results in disturbances of learning and memory. The fact that such dis-

turbances are generally observed at doses lower than those required to elicit

other well-characterized effects (i.e. motor effects, analgesia, hypothermia) are

consistent with the hypothesis that these agents have selective effects on mem-

ory. In general, exogenous administration of cannabinoids inhibits neurotrans-

mitter release in the hippocampus. In vivo studies in which memory duration

is enhanced in SR-141716-treated mice and CB1–/– mice are consistent with the

notion that endocannabinoids are tonically active to dampen memory.

However, the specific actions of endocannabinoids, such as anandamide and

2-AG, in the tonic modulation of the neural pathways that underlie cognition

remains an active area of research. The availability of selective CB1antago-

nists and transgenic mouse models promises to address this question and fur-

ther our understanding of endocannabinoid systems. The possibility of devel-

oping drugs that target the endocannabinoid system to treat cognitive deficits

associated with Alzheimer’s disease, posttraumatic stress syndrome, and drug

abuse is particularly intriguing.

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140 S.A. Varvel and A.H. Lichtman

Cannabinoids and anxiety

Richard E. Musty

Department of Psychology, University of Vermont, Burlington, VT 05405, USA

Introduction

As stated in Ethan Russo’s chapter the first known reference to the use of

Cannabis for the relief of anxiety was about 1500 bce in India. In modern times

the 1860 Report of the Ohio State Medical Committee on Cannabis indica [1]

stated:

“As a calmative and hypnotic, in all forms of nervous inquietude and

cerebral excitement, it will be found an invaluable agent, as it produces

none of those functional derangement or sequences that render many of

the more customary remedies objectionable.”

Anxiolytic effects of CB1receptor antagonists

Musty [2] found that cannabidiol (CBD) inhibited the development of

stress-induced ulcers in rats as compared with diazepam, which produced an

equivalent reduction in the number of stress-induced ulcers. Guimaraes et al.

[3] tested rats in the elevated-plus maze. In the test, rats are placed in a

plus-shaped maze which is elevated above the floor. Two of the maze arms are

enclosed with walls and two are not. Time spent in the enclosed arms is taken

as a measure of anxiety or fear. Both CBD and diazepam decreased the amount

of time spent in the enclosed arms. Since these studies were conducted, Petitet

et al. [4] and Thomas et al. [5] have reported CBD is an antagonist of the CB1

receptor in the micromolar range, suggesting that CBD may have pharmaco-

logical effects an antagonist of the CB1receptor.

Musty et al. [6] found that CBD increased licking for water in the lick-sup-

pression test, which reliably discriminates between anxiolytic drugs and those

that are non-anxiolytic. Equivalent effects were found with the classic anxi-

olytic drug diazepam. In an effort to find more potent effects, they tested two

analogs, 2-pinyl-5-dimethylheptyl resorcinol (PR-DMH) and Mono-methyl

cannabidiol (ME-CBD-2). PR-DMH had anxiolytic activity, but was less

potent than CBD, while ME-CBD-2 had no anxiolytic properties. CBD also

decreased conditioned taste aversion in a dose-related fashion (40 mg/kg gave

the peak effect).

Cannabinoids as Therapeutics

Edited by R. Mechoulam

141

Since the discovery of the synthetic, highly potent CB1receptor antagonist,

SR-141716, by Rinaldi-Carmona et al. [7] several other studies seem to sup-

port the hypothesis that CB1receptor antagonists have anxiolytic properties.

Onaivi et al. [8] used two tests of anxiety in mice, the elevated-plus maze (dis-

cussed above) and the two-compartment black and white box test. When

administered SR-141716 in the elevated-plus maze, mice spent more time in

the open arms, indicating a reduction of anxiety. In the second test, mice were

allowed to choose to spend time in a two-compartment box, one of which was

white and brightly lit, the other of which was black and dimly lit. Time spent

in the dark compartment was taken as an index of anxiety. When administered

SR-141716, mice spent more time in the white, brightly lit compartment, indi-

cating a reduction in anxiety.

In a recent study Rodgers et al. [9], administered the CB1receptor antago-

nist SR-141716A and a reference benzodiazepine chlordiazepoxide (CDP).

They argued that there were “qualitative” differences of cannabinoid ligands

depending on the number of exposures (trials) in the elevated-plus maze. They

tested the effects of SR-141716A (0.1–10.0 mg/kg) and the reference CDP

(15 mg/kg) in mice that had never been exposed to the elevated-plus maze

compared with mice that were given an exposure to the maze undrugged (day

1). Then, 24 h later (day 2), both groups of mice were given the same drug reg-

imen as above. Those mice that had received CDP on day 1 did not exhibit any

anxiolytic response. The authors suggest this indicated an immediate tolerance

to CDP. On the other hand, mice given SR-1411716 on day 1 showed no anx-

iolytic response, but did so on day 2. This result is in contrast to the afore-men-

tioned study in which the effect of SR-141716 was observed on the first expo-

sure to the elevated-plus maze. The authors state: “The apparent experiential-

ly induced ‘sensitization’ to the anxiolytic-like effects of SR-141716 in the

plus-maze contrasts markedly with the widely reported loss of benzodiazepine

efficacy in test-experienced animals.” Further research is required to resolve

these differences.

Cannabinoid receptor agonists

It seems that low doses of cannabinoid receptor agonists are anxiolytic in

mice. In the light/dark box test cited above, 0.3 mg/kg of ∆9-tetrahydro-

cannabinol (∆9-THC) reduced anxiety [10], but in lower doses of 0.03 and

0.1 mg/kg no effect was found. On the other hand, with doses of 5.0 [10] and

4.0 mg/kg, using a THC extract (Deyo R et al., personal communication), anx-

iogenic effects have been observed. Similarly, the potent CB1receptor agonist

HU 210 has been shown to have anxiolytic effects at low doses (4 µg/kg) in the

defensive withdrawal test in a novel environment (associated with fear/anxiety

in rodents), but anxiogenic effects in a familiar environment [11]. These data

seem to show that the context of administration is an important variable in the

effect of CB1agonists.

142 R.E. Musty

CB1receptor-knockout studies

Martin et al. [12] tested CB1-knockout mice and wild-type mice in the

light/dark box test. They found that knockout mice spent more time in the dark

section of the box and less time in the light part of the box compared with the

wild-type mice, indicating that the knockout mice were more anxious, thus

providing strong support that the CB1receptor system is involved in the con-

trol of emotional behaviors such as fear and anxiety.

Rodgers et al. [13] extended the findings of Martin et al. [12]. They tested

CB1-knockout mice and wild-types in the elevated-plus maze under two con-

ditions, low light and high light, the latter having been shown to induce greater

anxiety. They found that there were no differences or indications of anxiogenic

activity in the low-light condition between knockout and wild-type mice.

However, in the high-light condition knockout mice spent significantly more

time the in the closed arms of the maze and less time in the open arms of the

maze, as compared with the wild-type mice. These data provide more con-

vincing evidence that the CB1receptor system is involved in the control of

anxiety.

Anandamide hydrolysis and anxiety

Kathuria et al. [14] hypothesized that that anxiolytic effects of cannabinoids

might be enhanced by endogenous cannabinoids by preventing their inactiva-

tion. Accordingly, this group synthesized several fatty acid amide hydrolase

(FAAH) inhibitors. Two of these (named URB532 and URB597) selectively

inhibited breakdown of anandamide in vitro while an inactive analog did not.

These inhibitors were also tested in the elevated-plus maze. Both increased the

time spent in the open arms of the maze, in a dose-dependent manner. These

effects were blocked by several doses of SR-141716. The researchers also used

the ultrasonic emission test. In this test rat pups are separated from their moth-

er, which causes them to emit ultrasonic distress cries. Normally the mother

uses these signals to locate and retrieve the pups. Anxiolytic drugs selectively

reduce these cries dose-dependently. In this test both FAAH inhibitors reduced

vocalizations in the pups which was reversed by co-administration of

SR-141716. The authors conclude that raising anandamide levels seems to be

important in the regulation of anxiety and suggest a potential new class of

compounds which might be useful in the treatment of anxiety.

Discussion of animal studies

It seems that there is a paradox in the data discussed above. Regarding CB1

antagonists, it seems that the preponderance of the data suggest that these com-

pounds are anxiolytic. Agonists on the other hand seem to have biphasic

Cannabinoids and anxiety 143

effects. Low doses seem to be anxiolytic, while high doses are anxiogenic. In

addition, it seems that the context is important. Further research is needed to

sort out the differences among various studies, but it is clear that both antago-

nists and agonists on the CB1receptor have anxiolytic properties.

Standardization of behavioral testing procedures across laboratories would be

helpful, the problem being that there are many variables which have not been

explored with behavioral methods used to test for anxiolytic properties. Since

it is widely known that activation and inactivation of CB1receptors has a mul-

titude of modulator effects on neurotransmitter systems, it would be advanta-

geous for researchers to examine what changes in neurotransmitter activity

occur in conjunction with the pharmacological effects observed in the types of

studies cited herein.

Human studies

Consroe et al. [15] found that anxiety was reduced in 85% of patients with

multiple sclerosis in a self-report questionnaire. In another self-report study

[16] patients with spinal cord injuries reported similar reductions in anxiety.

In a laboratory setting, when subjects were instructed to smoke marijuana

until they reached their “usual” level of intoxication, regression analysis of a

visual analog scale of the word “anxious” predicted decreased scores on this

scale. These data support the hypothesis that THC has anxiolytic properties at

low doses [17].

In normal volunteers, Zuardi et al. [18] tested the hypothesis that CBD

would antagonize anxiety induced by THC. They used a dose of 0.5 mg/kg

THC for a 68-kg subject, which is a rather large dose, exceeding the dose a

person would take for the intoxicating effect of the drug. Subjects report a

pleasant high at 0.25 mg/kg using the same route of administration without an

increase in anxiety. In a second study Zuardi et al. [19] induced anxiety in nor-

mal subjects by having them prepare a 4-min speech about a topic from a

course they had taken during the year. They were told the speech would be

videotaped for later analysis by a psychologist. The subject began the speech

while viewing his/her image on a video monitor. Anxiety measures were taken

using the Visual Analogue Mood Scale (VAMS), which yields measures on

four factors (anxiety, physical sedation, mental sedation and other feelings,

e.g. interest) at five time points: baseline, immediately before instructions,

immediately before the speech, in the middle of the speech and after the

speech. Heart rate and blood pressure measures were also taken. The subjects

were randomly assigned to one of four drug conditions: CBD (300 mg),

isapirone (5 mg), diazepam (10 mg) or placebo. CBD, diazepam and isapirone

decreased anxiety and systolic blood pressure. Neither CBD nor isapirone had

effects on physical sedation, mental sedation, or other feelings, but diazepam

induced feelings of physical sedation.

144 R.E. Musty

Crippa et al. [20] investigated CBD’s effects on regional cerebral blood in

normal postgraduate students. In addition, they administered the VAMS. Each

subject was tested twice, 1 week apart. On week 1, half the subjects were given

a single dose of 400 mg of CBD in corn oil (in a capsule) and the other sub-

jects received a placebo capsule of corn oil only. On week 2 this procedure was

reversed. During the regional cerebral blood scanning procedure subjects were

resting for 30 min before the VAMS was administered. At the 30-min mark an

intravenous cannula was inserted to administer the radioactive tracer material

and the VAMS was given again. The cannula was removed and the scan was

performed. The VAMS was given again at 60 and 75 min after drug ingestion.

Anxiety decreased significantly by 60 and 75 min, when orally administered

doses of CBD are known to be at peak blood levels. Tracer uptake in the CBD

condition increased relative to placebo in the left parahippocampal gyrus and

the left fusiform gyrus compared with placebo. Tracer uptake decreased in the

CBD relative to placebo in the left amygdala-hippocampal complex and uncus,

the hypothalamus and left superior portion of the posterior cingulate gyrus.

It seems clear that CBD decreased anxiety, which is often observed in peo-

ple undergoing SPECT (single-photon emission computed tomography) or

PET (positron emission tomography) scanning as measured by the VAMS. The

brain area which showed increased activity in relation to placebo was the left

parahippocampal gyrus. Deactivation of this area of the brain has been associ-

ated with panic attacks induced by lactate, anxiety induced by combat-related

images and autobiographical memory scripts. It seems that anxiety is associ-

ated with reduced parahippocampal activity, consistent with the findings that

CBD increases activity in this brain area. Because activity in the CBD condi-

tion decreased relative to the placebo, these data fit well since there are a lot

of data linking amygdala activation in a large variety of anxiety states.

Similarly, the hypothalamus is involved in various anxiety states: imaging

studies in particular have shown increases in hypothalamic activity in anxiety

induced in normal volunteers and panic patients, again consistent with the anx-

iolytic effect of CBD. In regard to the posterior cingulate gyrus, increased

brain activity is associated with viewing anxiety-provoking videos, which pro-

voked obsessions in obsessive patients. Patients with obsessive-compulsive

disorder (OCD), if untreated, have increased metabolism in the brain area,

which decreases with treatment and symptom remission, although there are

some conflicting data (see [20] for references relating to all of the above dis-

cussion). While these data might be considered preliminary, they provide the

first evidence of brain systems that are affected in humans. There seems to be

quite strong convergence between animal research and human research, sug-

gesting strongly that CBD is a true anxiolytic. Given the fact that this drug has

no psychoactivity in terms of intoxication and is very safe, it seems important

to pursue the potential of CBD, with further behavioral pharmacological stud-

ies, mechanistic studies employing neuropharmacological methods and in

clinical studies.

Cannabinoids and anxiety 145

Discussion

The data discussed in this review show there is converging evidence that the

CB1receptor system is involved in the control of anxiety. Many studies have

shown that both antagonists and agonists of the CB1receptor can produce anx-

iolytic effects both in animals and humans. Particularly strong evidence is the

fact that CB1-knockout mice are more anxious than wild-type mice The fact

that anandamide hydrolysis inhibitors are anxiolytic and that they lead to an

increase in anandamide levels in the brain is further support for the role of this

system in the control of anxiety. Finally, the observations that CBD increases

or decreases regional cerebral blood flow in areas of the brain predicted to be

involved in various anxiety states provide strong supportive evidence that at

least this cannabinoid is active in brain areas known to be involved in anxiety.

At present, four cannabininoids are available for clinical trials: SR-141716

(Rimonbant), the GW Pharmaceuticals extract (Sativex®; a 1:1 ratio of

∆9-THC/CBD), ∆9-THC (Marinol) and Nabilone (Cesamet). It would seem

reasonable to consider testing these compounds in specific anxiety states,

which are refractory to traditional anxiolytics and related drugs.

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Cannabinoids and anxiety 147

Cannabinoid targets for pain therapeutics

Susan M. Huang1and J. Michael Walker2

1Department of Neuroscience, Brown University, Providence, RI 02912, USA

2Department of Psychology, Indiana University, Bloomington, IN 47405, USA

Introduction

Historical accounts and anecdotal reports show that among the most common

medicinal uses of cannabis is pain relief [1]. Such accounts date back to the

ancient Chinese physician Hoa-Gho thousands of years ago for surgical anes-

thesia, and to ancient Israel in 315–392 AD, likely for the control of pain in

child birth [2]. The use of cannabis as medicine was so popular that there were

at least 28 pharmaceutical preparations available in the United States prior to

the passing of the Marijuana Tax Act in 1937 [3], which thwarted the legal use

and development of cannabis-based medicine.

The isolation, structural elucidation and chemical synthesis of the active

ingredient in marijuana, ∆9-tetrahydrocannabinol (∆9-THC), by Gaoni and

Mechoulam [4] was a monumental step which revealed the molecular basis for

the behavioral and physiological influences of cannabis reported throughout

history. The subsequent recognition and cloning of specific cannabinoid CB1

and CB2receptors (CB1R and CB2R) in the nervous system and the periphery

[5–7] led to the realization of the likely importance of a cannabinoid neuro-

modulatory system in the body. More recently, the identification of several

endogenous cannabinoids, beginning with anandamide [8], expanded the

ever-more complex story of regulation of various physiological functions by

the cannabinoid system.

Knowledge of the molecular components of the endocannabinoid system

[9] allowed the development of pharmacological agents that target them [10].

Pharmacological agonists and antagonists have been synthesized for the

cannabinoid CB1R and CB2R. The discovery of endogenous cannabinoids and

studies of their biosynthetic and degradatory pathways also yielded molecular

targets for perturbing the endocannabinoid system. Inhibitors of fatty acid

amide hydrolase (FAAH), the enzyme that degrades several of the endogenous

cannabinoids and inhibitors of the anandamide transporter, have been devel-

oped. These agents are useful research tools for studying the effects of

cannabinoids on pain as well as potential therapeutic drugs. This review focus-

es on studies that evaluate the behavioral and physiological consequences of

modulating the above-mentioned targets, as these are promising avenues for

Cannabinoids as Therapeutics

Edited by R. Mechoulam

149

the development of cannabinoid-based therapeutics for pain. Reviews covering

basic research on the physiological role of endogenous cannabinoids in pain

modulation can be found elsewhere [11].

Effects of direct-acting cannabinoid receptor agonists

Perhaps the earliest published preclinical experiment on the effects of cannabi-

noids on pain was that conducted by Dixon [12], who showed that dogs that

inhaled cannabis smoke failed to react to pin pricks. After the isolation of

∆9-THC, Bicher and Mechoulam [13] and Kosersky and colleagues [14]

demonstrated that this chemical component of cannabis profoundly suppressed

behavioral reactions to acute noxious stimuli and inflammatory pain. It was

noted early on that the potency and efficacy of cannabinoids in acute pain par-

adigms rival that of morphine [15, 16]. However, cannabinoids also produce

profound motor effects such as immobility and catalepsy [17], which raised a

potential confounding factor for studies that assessed pain behavior, because

escape or withdrawal responses to noxious stimuli are integral to the assess-

ment. In part to address this potential confounding factor, experiments were

initiated to determine whether cannabinoids suppress the spinal and thalamic

circuits that give rise to pain sensations. These experiments demonstrated that

cannabinoids selectively suppress noxious stimulus-evoked neuronal activity

in spinal and thalamic nociceptive neurons [18–22]. This effect is observed

with all modalities of noxious stimulation tested (mechanical, thermal, chem-

ical), is mediated by cannabinoid receptors, and correlates with the pain-sup-

pressive behavioral effects of cannabinoids [18–21]. Cannabinoids suppress

C-fiber-evoked responses in spinal dorsal-horn neurons recorded in normal

and inflamed rats [22–24]. Spinal expression of Fos protein, a marker of sus-

tained neuronal activation [25], is also suppressed by cannabinoids in animal

models of persistent pain [26–31].

Brain action of cannabinoid agonists

Intracerebroventricular administration of systemically inactive doses cannabi-

noid agonists suppresses pain with only miniscule amounts reaching the spinal

cord at the time of peak analgesia [32]. When administered in this fashion,

cannabinoids inhibit spinal nociceptive responses by actions on specific cir-

cuits in the brain that serve naturally to modulate pain sensitivity. These areas

include the dorsolateral periaqueductal gray, dorsal raphe n., rostral ventral

medulla, amygdala, lateral posterior and submedius regions of the thalamus,

superior colliculus and noradrenergic nucleus A5 region [33– 35]. It appears

that the descending noradrenergic system is important in mediating the effects

of cannabinoids in the brain [36, 37]. When spinal transection was performed

in rats, there was a marked attenuation of the analgesic effects of systemically

150 S.M. Huang and J.M. Walker

administered cannabinoids [20, 38], suggesting substantial contribution sites

in the brain. Hence it is possible that maximal analgesic effects could be diffi-

cult to attain without penetration into brain sites, either by selective routes of

administration or with drugs that cannot penetrate the blood–brain barrier.

However, the real measure of clinical success often lies in balancing the degree

of pain relief with the extent of the unwanted side effects, which for cannabi-

noids are mainly due to actions in the brain leading to undesirable psy-

chotropic effects.

Spinal action of cannabinoid agonists

Anti-nociceptive effects of cannabinoids are mediated in part at the spinal level,

as inhibition of spinal reflexive responses to noxious stimuli was observed in

spinally transected dogs [39]. Support for spinal mechanisms of cannabinoid

analgesic action is also found in studies that demonstrated analgesia following

intrathecal injections [40–42]. The behavioral data are consistent with the abil-

ity of spinally administered cannabinoids to suppress noxious heat-evoked and

afterdischarge firing [19] and noxious stimulus-evoked Fos protein expression

in spinal dorsal horn neurons [27]. Spinal administration of a CB1R agonist also

inhibits C-fiber- and A-δ-fiber evoked responses of spinal nociceptive neurons

in a CB1-dependent mechanism [24]. Systemic and intrathecally administered

cannabinoids retain a weak but long-lasting anti-nociceptive effect in spinally

transected rats [38, 40], providing compelling evidence for spinal mechanisms

of cannabinoid anti-nociception.

Spinal administration of the ultra-potent cannabinoid HU-210 suppresses

C-fiber-mediated neuronal hyperexcitability in carrageenan-inflamed and

non-inflamed rats [23]; these effects were blocked by a CB1R antagonist.

Similar to the results with HU-210, spinal administration of anandamide also

produced CB1R-mediated effects in carrageenan-inflamed rats, but inconsis-

tent effects were observed in non-inflamed rats [43]. Although not established

following inflammation, upregulation of CB1Rs is observed in the spinal cord

following nerve injury, suggesting that regulation of spinal CB1Rs may con-

tribute to the therapeutic efficacy of cannabinoids in pathological pain states

[44]. The observation that cannabinoids act spinally to inhibit pain implies that

epidural cannabinoids may be effective in treating certain types of pain.

Peripheral action of cannabinoid agonists

Richardson and colleagues observed that peripheral administration of the

cannabinoid agonist anandamide suppressed thermal hyperalgesia and edema

in the carrageenan model of inflammation in a CB1R-dependent manner [45].

The same dose administered to the non-inflamed contralateral paw was inac-

tive, indicating that the compound did not produce its effects by absorption

Cannabinoid targets for pain therapeutics 151

into the systemic circulation. This finding indicates that CB1R agonists acting

in the periphery are sufficient to inhibit pain. Intraplantar administration of the

mixed CB1/CB2agonist WIN-55,212-2 also attenuated the development of car-

rageenan-evoked mechanical hyperalgesia, allodynia and spinal Fos protein

expression in a CB1R- and CB2R-dependent manner [30].

Effects of cannabinoid CB2-specific agonists

CB2Rs are either absent or expressed in low levels by neural tissues [7, 46] but

are present on immune cells and hence may have implications for pain. This

distribution has led to the evaluation and validation of the CB2R as a target for

novel pharmacotherapies for pain, an attractive possibility because CB2R ago-

nists lack psychotropic side effects.

CB2R agonists are anti-nociceptive in models of acute [47] and persistent

[29, 48–51] pain. Hanusˇ and colleagues [49] demonstrated that the selective

CB2agonist HU-308 produced marked decreases in pain behavior in rats

receiving hindpaw injections of dilute formalin with no change in motor func-

tion, a side effect often seen with CB1R agonists which may predict psy-

choactivity in humans. Another CB2R agonist, AM-1241, has also been shown

to induce CB2R-mediated analgesia in acute pain paradigms while failing to

elicit centrally mediated side effects such as hypothermia, catalepsy and

hypoactivity [47]. In inflammatory pain models, AM-1241 also induces

CB2R-mediated suppression of carrageenan- and capsaicin-evoked thermal

and mechanical hyperalgesia and allodynia [30, 50, 52].

Substances released by immune cells such as histamine, serotonin

(5-hydroxytryptamine), eicosanoids, interleukin 1, tumor necrosis factor-α,

and nerve growth factor sensitize nociceptors [53–55]. Therefore, it is plausi-

ble that activation of CB2Rs on immune cells could suppress pain.

Cannabinoid agonists have been shown to inhibit the release of inflammatory

mediators from monocytic cells [56] and mast cells [57]. Direct effects on

CB2Rs localized to primary afferents have been postulated [58–62], though the

basis for such effects is unclear in light of the paucity or lack of CB2R expres-

sion in neural tissues. It would appear from these findings that CB2R selective

agonists are a promising target for drugs to treat pain and inflammation.

Effects of cannabinoid agonists in humans

The human trials of cannabis and ∆9-THC are few in number and typically

small in subject size. There are marked differences between studies in dose

regimens and drug preparations, with some using smoked marijuana and oth-

ers using ∆9-THC by oral or intravenous routes. Some studies used healthy

volunteers, whereas others used patients with clinical pain. Therefore, it is

important to note that: (1) some negative results may have arisen from admin-

152 S.M. Huang and J.M. Walker

istration of doses that are ineffective; (2) the oral route of administration adds

variability owing to the unpredictable absorption of ∆9-THC; (3) smoked mar-

ijuana contains additional constituents that modify its actions; and (4) studies

of experimental pain in healthy subjects provide information on the spectrum

of the effects of cannabinoids in humans and initial indications with regard to

the feasibility of their use in humans, while clinical trials on specific patho-

logical pain syndromes are more relevant in assessing the effectiveness of

cannabinoids on particular pain conditions.

Studies of cannabinoid agonists on experimental pain in humans

Several investigators have studied the effects of cannabinoids on pain percep-

tion in humans by administering controlled painful stimuli to healthy volun-

teers. One such study [63] found that an oral dose of 5 mg of ∆9-THC given to

healthy volunteers decreased their ability to distinguish between various inten-

sities of painful heat stimuli with a time course that was distinguishable from

the effects on memory and psycholinguistic measures. This effect is consistent

with a pain-suppressive effect of the compound. Using sensory decision theo-

ry, they separated this effect from response bias, which refers to the tendency

to respond either positively or negatively and is influenced by non-sensory fac-

tors such as the subject’s culture, temperament and mood. Another study that

used sensory decision theory reached the opposite conclusions [64], but in this

study the large amount of ∆9-THC that was consumed by the volunteers (an

average of 19.4 marijuana cigarettes per day for high consumption and 13.1 for

moderate users) almost certainly produced drug tolerance, which develops rap-

idly with cannabinoids, and may have confounded the results, making the data

very difficult to interpret.

Raft and colleagues [65] demonstrated in healthy subjects that intravenous-

ly administered ∆9-THC (0.022 and 0.044 mg/kg) increased pain threshold

(the lowest intensity of stimulation that gives rise to pain) but not pain toler-

ance (the intensity at which pain becomes unbearable) to mechanical and elec-

trical stimulation. Hill and colleagues [66] also measured pain thresholds and

tolerance. In this study, healthy volunteers inhaled marijuana smoke.

Marijuana smoking lowered the pain threshold as well as pain tolerance. A

drawback of this study is the inability to state the dose with any accuracy, a

possible basis for the fact that it is at variance with the results of Raft and col-

leagues [65].

A recent study employing topical administration of the cannabinoid agonist

HU-210 has demonstrated its effectiveness in reducing the magnitude of pain

produced by capsaicin as well as mechanical and thermal hyperalgesia and

allodynia in human volunteers [67]. This was a particularly intriguing finding

because topical application led to reduced pain sensation with no observable

psychotropic effects.

Cannabinoid targets for pain therapeutics 153

Studies of cannabinoid agonists on clinical pain in humans

The studies discussed in this section are the most compelling, because the sub-

ject population was drawn from patients suffering from significant chronic

clinical pain. Chronic pain differs from acute pain due to neural changes that

occur with prolonged noxious stimulation. These changes lower the threshold

for pain (allodynia) and heighten the painfulness of noxious stimulation

(hyperalgesia). The mechanisms underlying different classes of pain (e.g.

inflammatory pain versus neuropathic or nerve injury pain) differ.

Consequently, different analgesics exhibit different degrees of efficacy in

chronic pain of different etiologies. For example, morphine is an excellent

analgesic for inflammatory pain, whereas it frequently lacks efficacy in neu-

ropathic pain [68]. Therefore, studies of different types of clinical pain are nec-

essary precursors to drawing sound conclusions about the efficacy of cannabi-

noids for pain pharmacotherapy.

Positive results of cannabinoids have been found in the studies of cancer

pain conducted by Noyes and colleagues [69, 70]. The patients in the study

(n= 36) reported continuous pain of moderate intensity. In a double-blind ran-

dom pattern, patients received on successive days placebo, 10 and 20 mg of

∆9-THC, and 60 and 120 mg of codeine. Pain ratings by the patients were used

to estimate pain-relief and pain-reduction scores. The results indicated that

20 mg of ∆9-THC was roughly equivalent to 120 mg of codeine. Five of the 36

patients experienced adverse reactions to ∆9-THC, one following 10 mg of

∆9-THC, four following 20 mg. The effectiveness of cannabinoids on cancer

pain has also been observed in animal models [71].

Neuropathic pain is a potential target for cannabinoid pharmacotherapies,

which has been validated in preclinical as well as clinical studies. A

double-blind study evaluated the effect of intramuscular administration of var-

ious doses of the ∆9-THC analog levonantradol in moderate to severe postop-

erative or trauma pain. Levonantradol provided pain relief at all four doses

studied (1.5–3.0 mg) compared to placebo. More than half of the patients

reported side effects, the most frequent being drowsiness with other symptoms

such as dizziness, mild hallucinations and nervousness occurring less fre-

quently. Recently, ∆9-THC was evaluated in multiple sclerosis patients with

central neuropathic pain in a double-blind, placebo-controlled crossover

design [72]. Orally administered ∆9-THC (10 mg daily for 3 weeks) lowered

median spontaneous pain-intensity scores and increased the median pain-relief

scores relative to placebo treatment. The modest but clear therapeutic effect

was associated with improvements on the SF-36 quality-of-life scale with no

change in the functional ability of the multiple sclerosis patients. During the

first week of treatment, adverse side effects of ∆9-THC treatment (dizziness,

light-headedness) were more frequent with ∆9-THC than placebo, but the

adverse effects decreased over the therapeutic course, possibly due to tolerance

[72]. In agreement with positive results from clinical data, a substantial num-

ber of preclinical studies have found cannabinoids to be effective in models of

154 S.M. Huang and J.M. Walker

neuropathic or nerve-injury pain [44, 51, 73–80], sometimes with increased

potency compared to effects in naive animals.

Data from the clinical studies tend to agree with centuries of anecdotal data

showing the effectiveness of systemically administered cannabinoids against

clinical pain. However, the unfortunate fact is that for the compounds tested to

date, maximal analgesia could not be obtained from systemic administration of

direct-acting CB1cannabinoid agonists at doses that do not elicit psychotrop-

ic effects. While typical cannabinoids are relatively safe (no deaths linked to

overdose) and do not appear to have serious toxicity problems, psychotropic

effects have limited the dosing, preventing cannabinoid agonists from reach-

ing their full potential for use in the clinic. More work on developing agonists

that lack psychotropic side effects would be beneficial, and may be fruitful

based upon recent studies described below.

Development of non-psychotropic cannabinoids

One possible strategy that has emerged from the study of the cannabinoid sys-

tem at the cellular and molecular level is the exploitation of differential signal

transduction mechanisms that can be coupled to the cannabinoid receptor.

Several studies reported that a single cannabinoid agonist could elicit different

degrees of signal amplification across various regions of the brain [81, 82], and

the various types of G-protein subunits that exist throughout the brain were

activated to varying degrees [83]. Moreover, different cannabinoid agonists

were found to evoke different levels of activation of a single G protein subtype

[84, 85], and the G protein subtype selectivity is conferred by distinct intra-

cellular domains of the receptor [86]. Hence, it is conceivable that an agonist

could be developed that would activate cannabinoid receptors and signal trans-

duction pathways associated with pain suppression but not those associated

with psychotropic effects and motor dysfunction. This possibility is further

supported by the separation of neural circuits that mediate cannabinoid motor

dysfunction (basal ganglia, cerebellum) from those that mediate analgesia

(periaqueductal gray, rostral ventral medulla, spinal cord, peripheral nerve)

[87–89], as it allows for the compartmentalization of distinct G protein sub-

types and second messengers to particular regions of the brain, and hence

physiological functions. If the circuits mediating cannabinoid agonist-induced

pain-suppressive effects rely principally on second messengers different from

those responsible for psychotropic effects, then it may be possible to develop

drugs that preferentially activate these signalling mechanisms and achieve a

pain-suppression-specific cannabinoid agonist. Further investigation into this

avenue of drug development is necessary to determine its practical feasibility.

Examples of results from recent work aimed at developing cannabinoids

lacking psychotropic side effects are the ∆9-THC and cannabidiol acid deriva-

tives ajulemic acid (CT-3) and HU-320. These compounds were reported to

produce anti-inflammatory effects with a reduced side-effect profile [90–92],

Cannabinoid targets for pain therapeutics 155

perhaps because they possess either modest (ajulemic acid) or virtually no

(HU-320) affinity for either CB1receptors or CB2receptors. While ajulemic

acid produces its effects via CB1receptors (unpublished findings from the

authors’ laboratory), HU-320 produces its effects by an unknown mechanism

that is unlikely to be the CB1R. At sufficient doses, CT-3 produces catalepsy

[92] similar to that observed with ∆9-THC. However, extensive dose studies

would be required to determine whether there is a greater dose separation

between its anti-nociceptive/anti-inflammatory effects and its psychomotor

side effects than that observed with typical cannabinoids. In a recent clinical

trial of patients suffering from neuropathic pain, ajulemic acid possessed some

efficacy [93]. While many questions about these and similar compounds are

awaiting further research, this appears to be an important line of inquiry.

Development of inhibitors of FAAH

Shortly after the isolation of the first endocannabinoid anandamide [5], the

enzyme responsible for anandamide hydrolysis, FAAH, was described [94]

and cloned [95]. In addition to anandamide two other endocannabinoids,

2-arachidonoyl glycerol (2-AG) and N-arachidonoyldopamine (NADA), also

appear to be susceptible to degradation by FAAH [96, 97].

Immunohistochemical studies show that FAAH is present in the ventral poste-

rior lateral nucleus of the thalamus [98–100], the termination zone of the

spinothalamic tract, which carries pain input from the periphery. FAAH is also

found in Lissauer’s tract, which comprises primary afferent fibers entering the

spinal cord, and in small neurons in the superficial dorsal horn, which is the

termination zone of nociceptive primary afferents. These observations demon-

strate that a mechanism capable of inactivating anandamide, 2-AG and NADA

is present in regions of the central nervous system related to nociceptive pro-

cessing and thus suggest a role for these ligands in pain modulation.

FAAH-knockout mice exhibit enhanced analgesic effects of exogenously

administered anandamide in a CB1receptor-dependent manner, suggesting

that the lack of this degradatory enzyme prolonged the action of anandamide

[101]. Moreover, these animals exhibit tonic CB1R-mediated analgesia in both

acute and chronic pain paradigms concurrent with a marked elevation of

endogenous anandamide levels [101, 102]. The results indicate that enhanced

activity of endogenous cannabinoid(s) from the lack of FAAH in the trans-

genic animals caused blunted sensitivity to pain.

The studies discussed above suggest that inhibitors of FAAH would

enhance the action of endogenous cannabinoids, thereby inhibiting pain. In

fact, pharmacological agents that inhibit FAAH, such as phenylmethylsulfonyl

fluoride (PMSF), palmitylsulfonyl fluoride (AM-374), methyl arachidonyl flu-

orophosphonate (MAFP) and arachidonoyl serotonin (AA-5-HT) produce a

number of effects [94, 103–105]. However, as FAAH inhibitors, these agents

have potential for improvement either in potency (often requiring micromolar

156 S.M. Huang and J.M. Walker

concentrations in in vitro assays) or specificity (often also acting on the CB1R

or other hydrolases). More recently, efforts from Boger’s and Cravatt’s groups

have resulted in the development of ultra-potent (effective at low nanomolar

concentrations) inhibitors of FAAH [106–108]. Some of these compounds,

such as URB532, URB597 and BMS-1, also affected other hydrolases, where-

as OL-135 was highly selective for FAAH and lacked activity at cannabinoid

receptors [109]. OL-135 suppressed pain in rodents in a CB1-dependent man-

ner concurrent with elevated levels of endocannabinoids [109]. These results

are encouraging for the development of a clinically effective agent for pain

based on selective inhibition of FAAH.

Development of inhibitors of cellular transport of endogenous

cannabinoids

Another approach for manipulating the endocannabinoid system is with

inhibitors of the putative transport mechanism for the endocannabinoid anan-

damide. Blocking cellular transport would be expected to cause increased

anandamide levels to occur in the vicinity of cannabinoid receptors, a similar

consequence as that caused by inhibition of FAAH. Several transport

inhibitors have been synthesized, beginning with the compound AM-404

[110], followed by VDM11 [111], OMDM-1, OMDM-2 [112], UCM707

[113] and UCM719 [114]. These compounds provided good separation in

potencies for anandamide uptake (low micromolar range) versus FAAH inhi-

bition, but most also bind to CB1Rs at doses similar to those required for inhi-

bition of anandamide uptake. The best separation of these effects was found

with UCM707 and OMDM-1, which offered a 5–6-fold separation in dose.

Following similar rationales as those for FAAH inhibitors, it is likely that fur-

ther development of inhibitors of the cellular transport mechanisms for endo-

cannabinoids may be fruitful for clinical pain relief.

Synergism between cannabinoid and opioid agonists

The first indication of the existence of synergistic (greater than additive)

pain-suppressive effects from co-administration of cannabinoid and opioid

agonists came from a study by Ghosh and Bhattacharya [115] when they found

that cannabis enhanced the analgesic effect of morphine in the rat. Further

study into this phenomenon, with major contributions from Welch’s group,

have provided information on the particular cannabinoid agonists, the dosages,

and the routes of administration required for the synergy to occur [41,

116–118]. An isobolographic analysis has been reported in rats, which plots

the theoretical ED50 for the drug combinations codeine/∆9-THC and mor-

phine/∆9-THC [119]. These synergistic effects of cannabinoid and opioid ago-

nists appeared to be receptor-mediated. Moreover, not only do low or inactive

Cannabinoid targets for pain therapeutics 157

doses of cannabinoids enhance opioid analgesia, inactive doses of opioids can

also enhance cannabinoid analgesia [120, 121].

The synergistic pain-suppressive effects of cannabinoid and opioid agonists

observed in the animal studies may provide the basis for a promising approach

in clinical pain management. The synergism would allow lower doses of opi-

oids and cannabinoids to be administered for attainment of a given degree of

pain suppression. As moderate to high doses of most cannabinoid or opioid

agonists alone often cause unpleasant psychotropic or physiologic side effects

in humans, the use of lower doses of these drugs may improve their clinical

utility in patients. Additionally, the reduced exposure to these drugs may slow

the development of tolerance. It is also possible that the inclusion of a cannabi-

noid agonist in the regime could prevent the development of opioid tolerance,

as the results of an animal study by Cichewicz and Welch [122] seemed to

indicate. As opioids are emetic substances and cannabinoids are anti-emetic,

the combination may improve the side-effect profile of both drugs. Hence it

appears that the use of carefully titrated doses of a combination of cannabinoid

and opioid agonists may provide a better quality of pain management for many

conditions.

Summary and conclusions

A large body of literature from numerous preclinical studies as well as some

clinical studies has demonstrated the ability of cannabinoids to suppress pain.

The realization of the clinical potential of cannabinoids still requires more

work in the areas of target refinement, drug selectivity and drug delivery.

Promising approaches include development of CB1or CB2agonists, inhibitors

of endocannabinoid degradation or transport mechanisms, combination dosing

of cannabinoids with other analgesics such as opioids, and new delivery sys-

tems such as transdermal patches. Highly potent and selective pharmacologi-

cal agents that focus on normalization of pain threshold and sensitivity with-

out causing psychotropic side effects in humans are much desired. Along with

drug development, further characterization of the cannabinoid system is

important in providing insights that are integral to identifying potential thera-

peutic approaches.

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Potential use of cannabimimetics in the treatment of

cancer

Luciano De Petrocellis1, Maurizio Bifulco2, Alessia Ligresti3and Vincenzo

Di Marzo3

1Istituto di Cibernetica “Eduardo Caianiello”, Consiglio Nazionale delle Ricerche, Via Campi

Flegrei 34, Comprensorio Olivetti, Fabbricato 70, 80078 Pozzuoli (Napoli), Italy

2Istituto di Endocrinologia ed Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, and

Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, via Ponte Don Melillo,

84084 Fisciano (SA), Italy

3Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34,

Comprensorio Olivetti, Fabbricato 70, 80078 Pozzuoli (Napoli), Italy

Introduction

The medicinal use of Cannabis sativa preparations has a millennial history [1]

and is currently being critically re-evaluated [2]. The hemp plant Cannabis sati-

va produces about 66 compounds known as cannabinoids, and the exact chem-

ical structure of the major psychotropic principal, (–)-∆9-tetrahydrocannabinol

(∆9-THC) [3], was only identified in 1964, after decades of attempts and fail-

ures. ∆9-THC is highly hydrophobic and was initially thought to work by inter-

acting directly with biomembranes. A few pharmaceutical items, such as

Marinol®and Dronabinol®, both based on ∆9-THC, and Cesamet®, which

instead is based on a synthetic ∆9-THC analog, nabilone, have been prescribed

in the USA as anti-emetics and appetite-stimulants to cancer or AIDS patients

even before the molecular mode of action of ∆9-THC was revealed [4]. It took

the development of more-potent and enantiomerically pure ∆9-THC analogs to

understand that psychotropic cannabinoids act via specific sites of action to pro-

duce their typical effects. The long-standing issue of the mechanism of action

of ∆9-THC was solved with the discovery of cannabinoid receptors [5], and then

of the endocannabinoids, endogenous agonists at cannabinoid receptors [6].

Two such receptor types have been cloned and characterized in mammalian tis-

sues; they are coupled to Gi/o proteins, through which they inhibit the adenylate

cyclases, stimulate mitogen-activated protein kinases, and modulate the activi-

ty of Ca2+ and K+channels to transduce the binding of agonists into biological

responses (see [7] for a review). CB1receptors are expressed in several brain

regions, with very high concentrations in the basal ganglia, hippocampus, cere-

bellum and cortex, and mediate the typical psychotropic effects of Cannabis,

marijuana and ∆9-THC. Lower, albeit functionally active, amounts of CB1

receptors are also found in peripheral neurons and various extra-neural sites

Cannabinoids as Therapeutics

Edited by R. Mechoulam

165

such as the testis, eye and vascular endothelium, as well as in many epithelial

cells. CB2receptors are mostly confined to immune tissues and seem to under-

lie the immune-suppressant actions of ∆9-THC [6]. Both CB1and CB2recep-

tors are expressed from the early stages of fertilized oocyte development [8],

and CB1expression in the developing brain is significantly different from that

observed in the adult brain [9]. Several other plant cannabinoids, with little or

no psychoactive action, have been identified and their possible therapeutic

actions investigated. In particular, cannabidiol [10] appears as a promising ther-

apeutic tool, even though its sites of action are not yet well understood.

The endogenous cannabinoid receptor ligands (endocannabinoids) identi-

fied so far are all derivatives (amides, esters, ethers) of long-chain polyunsat-

urated fatty acids, and exhibit varying selectivity for the two cannabinoid

receptors [7] as well as for other molecular targets [5]. The two best-studied

endocannabinoids are anandamide (N-arachidonoylethanolamine) and

2-arachidonoyl glycerol (2-AG) [11–13], and appear to be ubiquitous in mam-

malian tissues. Endocannabinoids, with their receptors [14, 15] and specific

processes of ligand synthesis [16, 17], cellular uptake [16, 18] and degradation

[19, 20], constitute the so-called endocannabinoid system.

The previous knowledge of ∆9-THC pharmacology [21] and, most impor-

tantly, recent studies carried out by using multiple pharmacological, biochem-

ical, analytical and genetic approaches [22], have revealed several possible

functions of endocannabinoid signaling under both physiological and patho-

logical conditions. Endocannabinoids have been proposed to act as retrograde

messengers [23] being released from the post-synaptic cell following its depo-

larization, to then act back on CB1on pre-synaptic neurons to inhibit neuro-

trasmitter release. Due to their chemical nature as lipophilic compounds, and

their peculiar biosynthetic mechanisms, endocannabinoids appear to act as

local mediators in an autocrine and/or paracrine manner, and their modulatory

activities on proteins and nuclear factors involved in cell proliferation, differ-

entiation and apoptosis suggest that the endocannabinoid signaling system is

involved, inter alia, in the control of cell survival, proliferation, transformation

or death [24].

The anti-neoplastic activity of ∆9-THC and its analogs was first observed in

the early 1970s, when neither cannabinoid receptors nor endocannabinoids

had yet been discovered. Although these observations were of potential inter-

est, no in-depth investigations were performed on this topic until 7 years ago,

when the effects of plant, synthetic and endogenous cannabinoids on cancer

cell proliferation and apoptosis started to be revisited. By contrast, the benefi-

cial effects of cannabinoids on some cancer-related disorders, such as emesis,

nausea, depression, muscle tension, insomnia, chronic pain and appetite sup-

pression, have been in part exploited pharmaceutically, since oral ∆9-THC

(dronabinol®, marinol®) can be prescribed legally in the USA for the treatment

of nausea and emesis and as an appetite-stimulating drug for cancer patients

undergoing chemotherapy and patients with AIDS, respectively. The results of

a large body of recent studies now suggest that targeting the endocannabinoid

166 L. De Petrocellis et al.

system might provide a significant contribution to both palliative and curative

cancer therapies.

Cannabinoid receptor stimulation causes inhibition of cancer growth

through multiple intracellular mechanisms and pathways

Based on Cannabis perturbation of the immune response, in vivo studies carried

out in animals in the late 1990s investigated the possibility that marijuana smok-

ing and long-term ∆9-THC treatment may favor tumor growth. These studies,

however, often produced opposing outcomes. For example, the enhancement of

lung carcinoma was seen [25], and more recently it has been demonstrated that

the treatment of glioma and lung carcinoma cell lines with nanomolar concen-

trations of ∆9-THC, comparable with those detected in the serum of patients

after ∆9-THC administration, leads to accelerated cancer cell proliferation

dependent on metalloprotease and epidermal growth factor receptor (EGFR)

activity [26]. While in this study the involvement of cannabinoid receptors was

not investigated, in another recent work low concentrations of ∆9-THC also

stimulated the proliferation of prostate carcinoma cells in vitro, in a

CB1-/CB2-mediated manner and androgen receptor-dependent manner [27].

However, Munson and coworkers showed about 30 years ago that ∆9-THC

inhibits lung adenocarcinoma cell growth in vitro and after oral administration

in mice [28], and a recent 2-year chronic administration study with high ∆9-THC

doses revealed a reduction of the spontaneous onset of hormone-dependent

tumors in particular [29]. Experiments carried out in vitro seem to go more often

in the direction of an anti-proliferative property of CB1and CB2receptor ago-

nists (Fig. 1). For example, it was found that 4–5-day treatment of human breast

cancer cell (HBCC) lines with sub-micromolar concentrations of endocannabi-

noids results in complete blockade of their proliferation [30]. CB1activation

blocks the cell cycle at the G0/G1–S transition via the inhibition of adenylate

cyclase and the cAMP/protein kinase A pathway. Protein kinase A phosphory-

lates and inhibits Raf1, and therefore anandamide, by preventing the inhibition

of Raf1, induces the sustained activation of the Raf1/mitogen-activated protein

kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cas-

cade [31]. These signaling events result in the inhibition of the expression of the

long form of the receptor for endogenous prolactin [30], a hormone that HBCCs

in culture use as an autocrine growth factor. In fact, agents activating the CB1

receptor via the same mechanism also counteract the proliferation of human

prostate cancer cells when induced by exogenous prolactin [32]. Indeed, both

human breast and prostate cancer cells express high levels of CB1receptors that

had never been detected previously in the corresponding healthy tissues. HBCCs

also respond to nerve growth factor (NGF) by proliferating more rapidly, and

2-day treatment of HBCCs with CB1receptor agonists suppresses the levels of

trk proteins, one of the two known types of NGF receptor, thus resulting in the

inhibition of NGF-induced proliferation [32].

Potential use of cannabimimetics in the treatment of cancer 167

Substances that activate CB1receptors might also exert more general

anti-tumor as well as anti-angiogenic effects by interfering with the expression

of other growth and mitogenic factors (Fig. 2). CB1receptor activation also

induces cell-cycle arrest at the G0/G1–S transition in thyroid epithelioma cells

(KiMol Cells) obtained from the transformation of rat thyroid epithelial cells

with the K-ras oncogene. Furthermore, repeated intra-tumor administration of

a very low and non-psychotropic dose of a metabolically stable and more

potent CB1receptor ligand, met-fluoro-anandamide, inhibits the growth of

tumors induced in nude mice by injection of these cells [33]. This effect is

accompanied by a strong reduction of the activity of the K-ras oncogene pro-

tein product, p21ras. It was also shown that the expression of CB1receptors is

regulated in healthy and transformed thyroid cells (as well as in tumors derived

from these latter cells) in opposite ways following treatment with met-fluo-

168 L. De Petrocellis et al.

Figure 1. Possible ways for endocannabinoids to inhibit cancer growth. Stimulation of cannabinoid

receptors by endocannabinoids can interfere with tumor growth in two possible ways: (1) by stopping

the cancer cell cycle (anti-proliferative or anti-mitogenic effect) via inhibition of the activity of p21ras

or of the expression of growth factors and/or of their receptors; (2) by promoting cancer cell apopto-

sis. The intracellular pathways mediating these two effects are schematically depicted in Figure 2.

Furthermore, some endocannabinoids, like anandamide or N-arachidonoyldopamine [115], can also

cause cancer cell apoptosis by both activating CB1receptors and gating vanilloid TRPV1 receptors.

The latter effect causes a strong Ca2+ influx that can then lead to apoptosis and in some cases also to

cell toxicity. AC, adenylate cyclase; AEA, N-arachidonoylethanolamine (anandamide); CBR,

cannabinoid receptor; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein

kinase kinase; PKA, protein kinase A; TRPV1, transient potential receptor vanilloid type 1 channel.

ro-anandamide, by being suppressed or enhanced in healthy or cancer cells,

respectively. Thus, the degree of CB1receptor expression determines the

extent of the responsiveness of normal or transformed FRTL-5 cells to

(endo)cannabinoids [33].

Inhibition of growth factor receptor signaling following CB1receptor acti-

vation has been shown also in pheochromocytoma [34], skin carcinoma [35]

and prostate carcinoma cells [36] and is likely to be a general mechanism

underlying the anti-proliferative actions by (endo)cannabinoids (Fig. 2).

However, another molecule involved in cancer cell proliferation by governing

cyclin-dependent kinase 2 activity, and whose over-expression can block the

cell cycle in the G1phase during the G1–S transition, is the p27 protein, which

is also under the negative control of the ras oncogene. Accordingly, met-fluo-

Potential use of cannabimimetics in the treatment of cancer 169

Figure 2. Mechanisms for cannabinoid receptor-mediated inhibition of cancer growth and spreading.

The different intracellular signaling pathways implicated in cannabinoid receptor-mediated inhibition

of cancer cell proliferation and metastasis, stimulation of cancer cell apoptosis and inhibition of

endothelial cell proliferation and migration (and hence of angiogenesis) are shown. Under certain con-

ditions, however, ∆9-THC and anandamide have been found to stimulate either EGFR or androgen

receptor expression, and to lead to enhanced proliferation in vitro of glioma and prostate carcinoma

cells, respectively [26, 27]. AEA, N-arachidonoylethanolamine (anandamide); ANG-2, angiopro-

tein-2; EGFR, epithelial growth factor receptor; ERK, extracellular signal-regulated kinase; MMP-2,

matrix metalloproteinase-2; NGF, nerve growth factor; p27(kip1), cyclin-dependent kinase inhibitor;

PIGF, placental growth factor; PKA, protein kinase A; PKB, protein kinase B; PRL, long form of the

prolactin receptor; Trk, high-affinity neurotrophin receptor; VEGF, vascular endothelial growth fac-

tor; VEGFR-1, vascular endothelial growth factor receptor-1. Adapted from [116].

ro-anandamide treatment of rat thyroid epitheliomas in vitro and in vivo, with

subsequent inhibition of p21ras, leads to sensibly increased levels of p27, an

effect that may contribute to the anti-proliferative actions of (endo)cannabi-

noids in this model [37].

Finally, as shown by using various biochemical and pharmacological

approaches, the stimulation of CB2, or of both CB1and CB2, receptors can also

lead to significant counteraction of tumor growth via various mechanisms [38,

39]. In fact, inhibition of cell mitosis was shown not to be the only mechanism

through which cannabinoids block solid tumor growth, particularly when CB2

or other non-CB1receptors (see below) are involved. ∆9-THC was found to

induce the programmed death (apoptosis) of glioma and prostate cancer cells

[40, 41]. The increased ceramide levels observed in glioma cells after cannabi-

noid action would drive the prolonged activation of the Raf1/MEK/ERK sig-

naling cascade [42] and Akt inhibition [43], as well as induction of cyclooxy-

genase-2 (COX-2) expression [44]. While the relation between ERK activation

and cell fate is complex and depends on many factors [45], the involvement of

oxidative stress [46] and stress-activated protein kinases [47] cannot be ruled out

during (endo)cannabinoid-induced apoptosis (Fig. 2). At any rate, these effects

of CB2receptor stimulation result in glioma cell apoptosis in vitro, and in pow-

erful inhibition of glioma growth in vivo [38, 42], although some of the tumors

developed in vivo can be significantly less sensitive to ∆9-THC than others [42].

Cannabinoid receptor stimulation leads to inhibition of tumor

angiogenesis and metastasis

Expression of various oncogenes, particulary ras, can lead to a marked induc-

tion of a potent paracrine stimulator of angiogenesis, the vascular endothelial

growth factor (VEGF) [48] (Fig. 2). The enhanced expression of VEGF is

associated with a large number of human tumor types, including human thy-

roid tumors and cancer cells. The observation that met-fluoro-anandamide is

able to block p21ras activity [33], and that endocannabinoids can inhibit the

expression of several growth factors and/or their receptors (see above), sug-

gested that CB1receptor stimulation could also interfere with VEGF and

VEGF receptors. Indeed, in rat thyroid epitheliomas, met-fluoro-anandamide

was found to inhibit the growth of already established tumors, in part by reduc-

ing the expression of VEGF and of the VEGF receptor Flt-1, which plays a

crucial role in mediating VEGF-induced neo-angiogenesis and endothelial cell

proliferation [37]. The expression of both VEGF and Flt-1 was suppressed not

only in the tumor in vivo but also in tumor cells in vitro, indicating that the

blockage of VEGF signaling may have a direct effect on tumor growth and

metastasis, not only by blocking neo-angiogenesis but also by disrupting

VEGF/VEGF receptor autocrine pathways [49].

It has been reported that CB1and CB2cannabinoid receptors are expressed

in normal epidermis and in mouse skin tumors, and in this case both receptors

170 L. De Petrocellis et al.

are functional in the regression of skin carcinomas, which may also rely on the

inhibition of tumor angiogenesis. The blood vessels developed by cannabi-

noid-treated carcinomas were in fact small, and the expression of pro-angio-

genic factors was depressed [35]. Once more, ras activation seems to be cru-

cial in mouse skin carcinoma initiation and angiogenesis in which VEGF plays

a pivotal role [50].

Also in a mouse model of glioma, local administration of a CB2

receptor-selective agonist inhibits angiogenesis of malignant gliomas as deter-

mined by immunohistochemical and functional analyses [51]. In vitro and in

vivo studies have shown a direct inhibition by cannabinoids of vascular hyper-

plasia characteristic of actively growing tumors into a pattern of small, differ-

entiated and impermeable blood capillaries. This is once more associated with

a decreased expression of VEGF and other vascular pro-angiogenic factors.

Furthermore the activation of cannabinoid receptors inhibited endothelial cell

migration and survival. Interestingly, the expression and activity of matrix

metalloproteinase-2, a proteolytic enzyme that allows tissue remodelling dur-

ing angiogenesis and metastasis, was also decreased by cannabinoids [51].

More recently, ∆9-THC was also shown to reduce the expression in gliomas of

the VEGF receptor, VEGFR-2, both in vitro and in vivo, and via blockade of

ceramide biosynthesis [119].

Finally, it was observed that the CB1-mediated anti-proliferative effect of

met-fluoro-anandamide on thyroid cancer cells was much more efficacious on

metastasis-derived cells than on the primary cancer line, possibly due to an

upregulation of CB1receptors in the former cells (see below). Accordingly, in

the Lewis lung carcinoma model of metastatic spreading, met-fluoro-anan-

damide was found to interfere efficaciously with the formation of lung

metastatic nodules by acting at CB1receptors [37]. The mechanism through

which stimulation of CB1receptors can lead to inhibition of the cellular

processes involved in cancer cell metastatic spreading, including cell motility

and adhesion, are currently under investigation in our laboratories. However,

preliminary data on the inhibition of the migration of SW 480 colon carcino-

ma cells by anandamide and the selective CB2receptor agonist JWH133, via

CB1- and CB2-receptor-mediated mechanisms, respectively, have been pub-

lished [52]. Furthermore, experiments carried out by using human prostate

cancer cells, also showed that 2-AG can inhibit invasion in vitro only in andro-

gen-independent cells and via inhibition of cAMP- and protein kinase A-medi-

ated signaling [120].

The endocannabinoid system attempts to provide protection from the

growth and spread of cancer

The increasing expression of cannabinoid receptors in cancer cells and tissues

observed with their increasing degree of malignancy and invasiveness, for

example in astrocytomas and transformed thyroid cells [33, 38], might suggest

Potential use of cannabimimetics in the treatment of cancer 171

a possible role of the endocannabinoid system in the tonic suppression of the

growth and spread of some tumors. In support of this hypothesis it was found

that alterations also of anandamide or 2-AG levels occur in many tumors as

compared to the corresponding healthy tissues ([53–55], see also [56]). In par-

ticular, an enhancement of endocannabinoid levels was observed in human

breast cancers and prostate carcinomas ([55], see also [56]), in human pituitary

tumors [53] and in human colorectal carcinomas [57]. In addition, human col-

orectal cancer Caco-2 cells loose their capability to respond to cannabinoid

receptor agonists, and make less endocannabinoids, when they differentiate

into non-malignant cells [57]. As cells from these tumors all respond to

cannabinoids with inhibition of proliferation, or by entering apoptosis, it was

suggested that endocannabinoids are endogenously over-produced in malig-

nant tissues and cells, and cannabinoid receptors (and other endocannabinoid

molecular targets) are subsequently over-stimulated, in the attempt to counter-

act cancer growth and spread. So far, evidence for this hypothesis has been

obtained mostly in vitro. Anandamide and its congeners are produced by

HBCCs, and a substance that elevates the levels of anandamide in these cells

also inhibits cell proliferation [58], suggesting that synthetic compounds that

selectively inhibit endocannabinoid degradation might also be used to inhibit

cancer growth. In fact, two selective and specific inhibitors of endocannabi-

noid inactivation were recently found to inhibit Caco-2 cell proliferation in a

CB1-receptor-mediated manner and through elevation of cell endocannabinoid

levels [57]. Palmitoylethanolamide, an anandamide congener that is synthe-

sized in higher amounts than anandamide in all tumor cells analysed so far,

inhibits the degradation of endocannabinoids in HBBCs, and may act as an

endogenous enhancer of the tumor-suppressing activity of this endocannabi-

noid, whether this is exerted via CB1[59] or other ([60] and see below) recep-

tors. Data from our laboratories indicate that this endogenous control of can-

cer growth by endocannabinoids can also occur in vivo, as two selective

inhibitors of anandamide and 2-AG inactivation induce a strong CB1-mediat-

ed inhibition of rat thyroid epithelioma growth in athymic mice by enhancing

the tumor levels of these two compounds [117]. Furthermore, it was recently

shown that inhibition of 2-AG degradation and biosynthesis can inhibit and

enhance, respectively, the invasion of human prostate cancer cells in vitro

[120].

Non-CB1/-CB2receptors are also involved in (endo)cannabinoid

anti-tumor actions

An ever-increasing number of reports (reviewed recently by Pertwee [61]) sug-

gests that endocannabinoids might exert their biological effects also through

non-CB1/-CB2receptors. Regarding the inhibition of cancer growth, experi-

ments carried out in our laboratories have shown that, rather than favoring can-

cer cell proliferation, the CB1-selective antagonist SR-141716A causes a

172 L. De Petrocellis et al.

slight, albeit significant, anti-proliferative effect both in vitro and in vivo [30,

32, 117]. This might suggest that non-CB1-receptor-mediated anti-tumor

effects of endocannabinoids might be unmasked when CB1receptors are

blocked. In recent studies [62, 118] the apoptotic effect of anandamide on

glioma cells was suggested to be mediated by another proposed target for this

compound, the transient receptor potential (TRP) vanilloid type 1 (TRPV1)

channel, also known as the VR1 receptor. This protein is a member of the large

family of TRP non-selective cation channels, and is activated by heat, protons,

plant toxins such as capsaicin and resiniferatoxin, and by some other endoge-

nous arachidonate-derived metabolites (see [63, 64] for reviews). However, the

mechanism through which anandamide induces apoptosis in TRPV1- and

CB1-expressing cells – that is, whether it involves both CB1and TRPV1 recep-

tors, or only TRPV1 receptors with CB1receptors instead playing a protective

role against TRPV1-induced apoptosis – is still controversial, and might

depend on the experimental conditions used for the experiments [65–68]. In

HBCCs, arvanil, a synthetic substance that activates both CB1and TRPV1,

exhibits a more potent anti-proliferative activity in vitro than ‘pure’ agonists of

either receptor class, and this action can be attenuated by both CB1and TRPV1

antagonists [65]. In glioma cells, anandamide causes apoptosis by acting via

both CB1and TRPV1 receptors [66] or only via TRPV1, depending on the cell

culture conditions [62]. In uterine cervix cancer cells in vitro, however, possi-

bly due to aberrant over-expression of TRPV1, anandamide induces apoptosis

only via activation of these receptors, and activation of CB1receptors again

counteracts this effect [67]. Therefore, the actual role of TRPV1 channels in

anandamide-induced inhibition of cancer cell growth (Fig. 1) is still not fully

understood. Furthermore, it remains to be established whether the slight

anti-cancer effect of SR-141716A is due to endocannabinoids being re-direct-

ed towards TRPV1, to counteract the protective action against apoptosis exert-

ed by CB1under certain conditions, or to other, entirely unrelated and

as-yet-identified, mechanisms of action of the CB1antagonist. Vanilloid com-

pounds themselves have been shown to inhibit the proliferation and induce

apoptosis of both cancer and non-transformed cells in vitro by acting via both

TRPV1-and non-TRPV1-mediated mechanisms [68– 70], and palmi-

toylethanolamide was found to enhance the antiproliferative effect on HBCCs

not only of anandamide and other CB1agonists, but also of vanilloids [60].

Therefore, one could envisage the use of this compound, co-administered with

either met-fluoro-anandamide or capsaicin derivatives, to lower the threshold

of the anti-tumor effects of these compounds to doses that do not exhibit either

undesired psychotropic activity or toxicity to healthy cells, respectively.

Plant cannabinoids such as cannabidiol, on the one hand, and ∆9-THC and

cannabinol, on the other, were shown to activate TRPV1 receptors [71] and the

ANKTM1 channel (another member of the TRP family of proteins [72]),

respectively. In fact, Cannabis components with little or no activity on

cannabinoid CB1and CB2receptors have been shown in the past to exhibit

anti-neoplastic activity in vitro [73]. Cannabidiol has anti-tumor effects on

Potential use of cannabimimetics in the treatment of cancer 173

human glioma cell lines [74–76], and inhibits the growth of human glioma

cells subcutaneously implanted in nude mice [76] as well as of human acute

myeloid leukaemia [77], in both cases through induction of apoptosis.

Cannabigerol inhibits the growth of human oral epitheloid carcinoma cells

[78]. The synthetic ajulemic acid (CT3), inhibits glioma cells in vitro and in

vivo [79] and induces apoptosis in human T lymphocytes [80], although the

lack of activity of this compound at CB1receptors is still a matter of contro-

versy. The molecular mechanisms for the anti-cancer effects of most of these

compounds are not yet understood, and are currently under investigation in our

laboratories. TRPV1 receptors, however, do not seem to be involved in the

inhibitory effect of cannabidiol against glioma [76].

Substances targeting the endocannabinoid system in cancer therapy –

pros and cons

The anti-tumor potential of substances that modulate the activity of cannabi-

noid receptors or the levels of endocannabinoids, as well as of other possible

targets for the anti-cancer action of these compounds, are still largely unex-

plored. It seems that cannabinoids selectively affect tumor cells but not their

non-transformed counterparts and might even protect the latter from cell death

(see above). For example, cannabinoids induce apoptosis of glioma cells in

culture [38, 42, 74, 81], but, by contrast, they protect glial cells from apopto-

sis [82, 83], possibly due to a differential ability to synthesize ceramide [81].

Indeed, a general protective role of the endocannabinoid system is emerging

from several recent studies (for a recent review see [84]). Cannabinoids appear

to be well tolerated in animal studies and do not produce the generalized toxic

effects in normal tissues that are a major limitation of most conventional

agents used in chemotherapy, the median lethal dose of ∆9-THC in animals

being of several grams per kilogram of body weight [85]. However, together

with obvious social, political and legal considerations, the therapeutic applica-

tion of agonists selective for CB1receptors might be limited by the undesired

psychotropic side effects expected from the stimulation of these receptors in

the brain (even though some ‘central’ actions of CB1activation may be,

instead, desirable, as discussed below). On the other hand, although devoid of

psychotropic actions, the administration of compounds selective for the CB2

receptor, as in the treatment of gliomas, skin carcinomas and lymphomas,

might cause the immune-suppressive effects typical of ∆9-THC, and this would

then play against the organism’s own defense against tumor growth. Yet selec-

tive CB2receptor agonists appear to be nowadays very efficacious against

some types of pain [86], which could represent an additional benefit of

anti-cancer drugs derived from these compounds. The limitations at least of

CB1-selective agonists might be overcome by the use of metabolically stable

endocannabinoid analogs in combination with a non-psychotropic substance

like palmitoylethanolamide, which might lower the threshold of concentra-

174 L. De Petrocellis et al.

tions necessary to observe the tumor-suppressing effect [60]. Another

approach for the development of new anti-cancer drugs would be the use of

selective inhibitors of endocannabinoid degradation. These substances would

be devoid of most psychotropic effects as they would preferentially act in those

tissues where the levels of endocannabinoids are pathologically altered [87].

Another possible difficulty for the exploitation of (endo)cannabinoids

against cancer growth is their very low solubility in water and their poor bio-

vailability when given orally. It seems that Cannabis is more efficient when

smoked, but this administration route presents all the very well known and

unwelcome consequences [88]. A very promising alternative has been recent-

ly proposed by GW Pharmaceuticals with Sativex®,a Cannabis extract con-

taining ∆9-THC and cannabidiol that is sprayed sublingually and is now under-

going clinical trials [89]. Alternatively, the use of water-soluble cannabinoids

such as O-1057 might solve the solubility problems [90, 91], but to date the

intra-tumor application of low doses of cannabinoids seems to represent the

most viable option for those types of tumor that can be treated in this way, as

it results, in animal models, in few if any undesired ‘central’ effects. The safe-

ty and efficacy of such type of administration of ∆9-THC to treat glioma in

humans is currently being assessed in a pre-clinical study in Spain [45].

Probably the greatest advantage offered by the use of cannabimimetics over

other conventional anti-cancer agents might reside in their beneficial effects on

some serious cancer-related disorders in humans, as follows. (1) Cannabinoids

are anti-emetics in animal models of vomiting [92]. Marinol®and Cesamet®are

approved to treat nausea and emesis associated with cancer chemoterapy [93,

94]. Modern anti-emetics are selective serotonin 5-HT3-receptor antagonists,

and, although cannabinoids can block 5-HT3-receptors [95] they have a very

distinct pharmacological profile. Hence, further studies should be performed to

establish the mechanism of action and what types of cancer chemotherapies are

suitable to cannabinoid anti-emetic treatment [96–99]. It is interesting to note

that the potent TRPV1 agonist resiniferatoxin antagonizes cisplatin-induced

emesis in dogs [100]. (2) ∆9-THC and other cannabinoids reinforce appetite and

increase food intake, seemingly via inhibition of anorexic signals [101–103].

Anorexia and cachexia are, in fact, primary problems in cancer patients.

However, a recent phase III trial has questioned the efficacy of oral ∆9-THC

appetite-stimulating effects in advanced cancer [104]. (3) The neuromodulato-

ry actions of endocannabinoids in the central, sensory and autonomic nervous

systems result, mostly via CB1receptors, in the regulation of pain perception

[105]. Cannabinoids produce spinal, supra-spinal and central analgesia by sup-

pressing the activity of nociceptive circuits [106]. Peripheral CB2might medi-

ate local analgesia [107] and might be important for cancer pain [108]. At the

moment, cannabinoids seem to be no more potent than codeine (for a review

see [109]), but clinical trials on their use for the treatment of cancer pain are in

progress. Interestingly, the synergic actions of cannabinoid CB1receptor ago-

nist with opioid receptor agonists produces, in animal models, analgesic actions

stronger and longer-lasting (through the avoidance of the development of mor-

Potential use of cannabimimetics in the treatment of cancer 175

phine tolerance) than those obtained with each agonist alone [110, 111]. (4)

Finally, studies in animals have shown that cannabinoids or agents that enhance

the endogenous levels of endocannabinoids exert anti-anxiolytic effects [112,

113] and mediate the extinction of aversive memories [114], with potential ben-

eficial psychological effects on cancer patients.

In conclusion, the use of cannabinoid receptor agonists and/or substances

inhibiting endocannabinoid degradation might not only retard the growth of

tumors via multiple mechanisms, but also alleviate simultaneously the weight

loss, nausea and pain that so badly affect the life of women and men suffering

from cancer. As multiple and different approaches are more and more likely to

solve successfully the issue of cancer treatment, one can only hope that the fur-

ther pre-clinical and clinical research that is certainly needed to substantiate

this hypothesis will provide ever more encouraging results in this direction.

Acknowledgements

The authors thank the Associazione Italiana per la Ricerca sul Cancro and GW Pharmaceuticals, UK,

for supporting their studies on this subject.

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Cannabinoids: effects on vomiting and nausea in

animal models

Linda A. Parker, Cheryl L. Limebeer and Magdalena Kwiatkowska

Department of Psychology, Wilfrid Laurier University, Waterloo, Ontario, Canada N2L 3C5

Introduction

The development of chemotherapy treatment has prolonged the lives of many

cancer patients. However, use of these powerful drugs presents a serious chal-

lenge to both clinicians and patients. Significant side effects of cancer

chemotherapy include nausea and vomiting which may last for several days.

These symptoms come to be dreaded by patients, often interfering with suc-

cessful completion of treatment. The emetic reflex is conventionally consid-

ered to include vomiting, retching and the more subjective sensation of nausea.

However, the organization of the reflex is very complex, because although

nausea, retching and vomiting usually occur in a temporal sequence, they can

be separated experimentally [1].

Vomiting is a widespread protective reflex that serves to expel accidentally

ingested toxins from the upper gastrointestinal tract. The sensation of nausea

serves as a warning (as does pain), and usually results in the cessation of inges-

tion and an associative aversion to the ingestant in the future. The act of vom-

iting is often followed by a feeling of well-being which may serve to reinforce

that behavior [1]. In the case of chemotherapy patients, however, the vomiting

reflex does not remove the perceived toxin; therefore, in contrast to the

removal of an ingested toxin from the gut, vomiting is not self-limiting [1].

Chemotherapy patients experience three separate types of emetic episode:

(1) acute nausea and/or vomiting occurs within minutes to hours of receiving a

dose of a toxic chemotherapy drug, (2) delayed nausea and/or vomiting that has

been arbitrarily defined as emesis begins or persists more than 24 h after

chemotherapy, and (3) anticipatory nausea and/or vomiting (ANV) occurs when

the patient is re-exposed to cues associated with the toxin. ANV occurs in near-

ly half of patients treated, frequently during later cycles of chemotherapy [2].

The more intense the initial acute emetic episode, the worse the resultant ANV.

A major advance in the control of emesis was the finding that blockade of one

subtype of the 5-hydroxytryptamine (5-HT) receptor, the 5-HT3receptor, could

suppress the acute emetic response (retching and vomiting) induced by cisplatin

in the ferret and the shrew [3–7]. In clinical trials with humans, treatment with

Cannabinoids as Therapeutics

Edited by R. Mechoulam

183

5-HT3antagonists often combined with the corticosteroid, dexamethasone, dur-

ing the first chemotherapy treatment has reduced the incidence of acute vomit-

ing by 70–90% [1, 8–14]. If acute vomiting is prevented, the incidence of

delayed and anticipatory vomiting is reduced [2, 8–11, 20]. However, the 5-HT3

antagonists are less effective at suppressing acute nausea than they are at sup-

pressing acute vomiting [1, 9, 10, 14, 20] and they are ineffective in reducing

instances of delayed nausea/vomiting [13, 15–20] and ANV [1, 10, 11, 14,

20–22] when they do occur. Therefore, it is likely that another system may be

involved in chemotherapy-induced nausea, delayed nausea/vomiting and ANV.

Two such systems include the neurokinin 1 (NK1) tachykinin receptors for sub-

stance P (e.g. [16, 17, 23]) and the endocannabinoid system [24–39]. The effect

of cannabinoids on nausea and vomiting is the subject of this review.

Cannabinoids as anti-emetics

The marijuana plant has been used for several centuries for a number of ther-

apeutic results, including nausea and vomiting [40]; however, it was only

recently that Gaoni and Mechoulam [41] isolated the major psychotropic com-

ponent, ∆9-tetrahydrocannabinol (∆9-THC). Twenty-five years later, the spe-

cific brain receptors for this compound, cannabinoid1(CB1) and cannabinoid2

(CB2), were identified (for review see [42]) and cloned [43]. Therefore, it was

only natural to start the search for an endogenous ligand for the cannabinoid

receptor, which was discovered 2 years later [44]. This ligand was the

ethanolamide of arachidonic acid, and called anandamide. A second type of

endocannabioid was discovered in 1995 [45], also a derivative of arachidonic

acid, but its ester, 2-aracidonoyl glycerol (2-AG). Both anandamide and 2-AG

are rapidly inactivated after their formation and release by the enzyme fatty

acid amide hydrolase (FAAH) [46].

The anti-emetic effects of cannabinoids appear to be mediated by action at

the CB1receptor. CB1receptors are found in the gastrointestinal tract and its

enteric nervous system [47] as well as within the emetic system of the brain

[34, 35] in the dorsal vagal complex, consisting of the area postrema (AP),

nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus

(DMNX) in the brainstem of rats, ferrets and the least shrew [33, 34]. Recent

reviews on the gastrointestinal effects of cannabinoids have concluded that

cannabinoid agonists act mainly via peripheral CB1receptors to decrease

intestinal motility [47], but act centrally to attenuate emesis [34, 35]. The dor-

sal vagal complex is involved in the nausea and/or vomiting reactions induced

by either vagal gastrointestinal activation or several humoral cytotoxic agents.

It is considered the starting point of a final common pathway for the induction

of emesis in vomiting species. CB1receptors in the NTS are activated by

∆9-THC and this activation is blocked by the selective CB1antagonists

SR-141716 [28] and AM-251 [35]. Indeed, c-Fos expression induced by cis-

platin in the DMNX, specific subnuclei of the NTS and AP is significantly

184 L.A. Parker et al.

reduced by ∆9-THC [34, 35]. Endogenous cannabinoid ligands, such as anan-

damide, as well as synthetic cannabinoids, such as WIN-55,212-2, also act on

these receptors [33].

Recent findings indicate that the cannabinoid system interacts with the sero-

tonergic system in the control of emesis. The dorsal vagal complex not only

contains CB1receptors, but is also densely populated with 5-HT3receptors [48,

49], potentially a site of anti-emetic effects of 5-HT3antagonists. Anandamide

has also been reported to interact with serotonin [50]. Cannabinoid receptors

are co-expressed with serotonin 5-HT3receptors in some neurons in the central

nervous system [51] and inhibitory functional interactions have been reported

between cannabinoid CB1and 5-HT3receptors [52, 53]. Additionally, canabi-

noids reduced the ability of 5-HT3agonists to produce emesis [28] and this

effect was prevented by pretreatment with the selective cannabinoid CB1recep-

tor antagonist SR-141716. Cannabinoids may act at CB1presynaptic receptors

to inhibit release of newly synthesized serotonin [28, 54, 55].

Anti-emetic effects of cannabinoids in human clinical trials

The potential for marijuana to suppress nausea and vomiting produced by

chemotherapy is of considerable therapeutic interest. Indeed, three canna-

bis-based medicines are available: dronabinol, nabilone and levonantradol.

Tramer et al. [56] present a thorough systematic review of 30 clinical trial com-

parisons of cannabis (oral nabilone, dronabinol (THC) and intramuscular levo-

nantradol) with a placebo or other anti-emetics (predominantly dopamine antag-

onists). Tramer et al. [56] conclude that the cannabinoids were superior to the

conventional dopaminergic antagonists in the treatment of nausea and vomiting.

There has been only one [57] comparison of cannabis with a 5-HT3antago-

nist using the short-acting emetic agent syrup of ipecac. Human participants

compared the effectiveness of a single dose of ondansetron (8 mg) with one of

two doses of smoked marijuana (8.4 and 16.9 mg ∆9-THC) in attenuating nau-

sea and vomiting produced by syrup of ipecac. Unlike cisplatin, ipecac pro-

duces short-lasting nausea and vomiting with a fast onset. They report that

within the limited dose range tested, ondansetron was considerably more

effective than smoked marijuana in attenuating vomiting and nausea and nau-

sea produced by the ipecac. There have been no clinical trials comparing the

relative efficacy of marijuana and 5-HT3antagonists in suppressing long-last-

ing nausea and vomiting produced by chemotherapy treatment.

Cannabinoids produce psychotropic side effects, which partially accounts

for their lack of popularity in clinical use [58]. Patients who have not had any

experience with cannabis often find the psychotropic effects unpleasant and

disturbing. Most importantly, the development of 5-HT3antagonist anti-emet-

ic drugs, with few side effects, has limited clinical use of cannabis-based med-

icines. The 5-HT3antagonist anti-emetic agents are highly effective at pre-

venting chemotherapy-induced vomiting, but are much less effective at inhibit-

Cannabinoids: effects on vomiting and nausea in animal models 185

ing nausea, as well as delayed nausea and vomiting and ANV when they do

occur. There is some evidence that cannabis-based medicines may be effective

in treating these intractable symptoms. Abrahamov et al. [59] evaluated the

anti-emetic effectiveness of ∆8-THC, a close but less psychoactive relative of

∆9-THC, in children receiving chemotherapy treatment. Two hours before the

start of each cancer treatment and every 6 h thereafter for 24 h, the children

were given ∆8-THC as oil drops on the tongue or in a bite of food. After a total

of 480 treatments, the only side effects reported were slight irritability in two

of the youngest children (3.5 and 4 years old); both acute and delayed nausea

and vomiting were controlled.

Furthermore, Tramer et al. [56] conclude that many patients have a strong

preference for smoked marijuana over the synthetic cannabinoids delivered

orally. This could be for various reasons: (1) possible advantages of self-titra-

tion with the smoked marijuana, (2) the difficulty of swallowing the pills while

experiencing emesis, (3) faster speed of onset for the inhaled or injected

∆9-THC than oral delivery, or (4) a combination of the action of other cannabi-

noids with ∆9-THC that are found in marijuana. Although many marijuana

users have claimed that smoked marijuana is a more effective anti-emetic than

oral ∆9-THC, no controlled studies have yet been published that evaluate this

possibility. Most of the evidence is based upon anecdotal testimonials, such as

that of the late Stephen Jay Gould [60]: “I was miserable and came to dread

the frequent treatments with an almost perverse intensity. …Absolutely noth-

ing in the available arsenal of medications worked at all. Marijuana, on the

other hand, worked like a charm.”

Smoking marijuana may represent a more efficient and rapid route of

administration. However, it is also possible that as marijuana contains over 60

other compounds, some of these additional consitutents may contribute to the

anti-emetic/anti-nausea effect. Another major cannabinoid found in marijuana

is cannabidiol (CBD); however, unlike ∆9-THC, CBD does not produce psy-

chomimetic effects [61]. CBD, unlike ∆9-THC, does not bind to the known

cannabinoid receptors. It may act by blocking the reuptake of anandamide (an

endogenous cannabinoid), or by inhibiting enzymatic hydrolysis of anan-

damide, or bind with some as-yet-unknown cannabinoid receptor [61–63]. In

mice, CBD is a highly effective anti-inflammatory agent [63], as well as a neu-

roprotective antioxidant [64]. In shrews, CBD inhibits cisplatin-induced [32]

and lithium-induced [31] emesis and in rats CBD inhibits nausea [38]. These

effects are described more fully below.

Effects of cannabinoids on emesis in animals

In order to understand the pathways involved in the response to anti-cancer

therapies to develop appropriate drug therapies, animal models have been

developed. Since rats and mice do not vomit in response to a toxin challenge,

it was necessary to develop other animal models of emesis. As indicated in

186 L.A. Parker et al.

Table 1, there is considerable evidence that cannabinoids attenuate vomiting in

emetic species. Cannabinoids have been shown to reduce vomiting in cats [30],

pigeons [65, 66], ferrets [33–35], least shrews, Cryptotis parva [24–29] and

the house musk shrew, Suncus murinus [31, 32].

Cannabinoids: effects on vomiting and nausea in animal models 187

Table 1. Effect of cannabinoid on emesis across species

Species Emetogen Cannabinoid Effect on

emesis

Cat Cisplatin (7.5 mg/kg, iv) Nabilone (0.025–0.1 mg/kg, iv) ↓[30]

N-Methyllevonantradol ↓[30]

(0.003–0.02 mg/kg, iv)

Dog Cisplatin (3 mg/kg, iv) Nabilone (0.1 mg/kg, iv) – [67]

Apomorphine ∆9-THC (0.003–0.3 mg/kg, iv) – [68]

(0.05–5 mg/kg, iv)

Pigeon Cisplatin (10 mg/kg, iv) ∆9-THC (5.0 mg/kg) with CuCl2↓[65]

HU-211 (2.5 mg/kg) with CuCl2↓[65]

Cisplatin (7.5 mg/kg, iv) HU-210 (0.012 –0.05 mg/kg, sc) ↓[66]

Emetine (20 mg/kg, sc) HU-210 (0.012–0.05 mg/kg, sc) ↓[66]

Ferret Morphine (1 mg/kg, sc) WIN55,212–2 (0.03–0.13 mg/kg, sc) ↓[33]

Morphine-6-glucuronide ∆9-THC (1 mg/kg, ip) ↓[34]

(M6G; 0.05 mg/kg, sc) WIN-55,212-2 (1 mg/kg, ip) ↓[34]

Methanandamide (3 mg/kg, ip) ↓[34]

Cisplatin (10 mg/kg iv) ∆9-THC (0.1 –1.0 mg/kg ip) ↓[35]

C. parva SR-141716A (20 mg/kg, ip) CP-55,940 (1 mg/kg, ip) ↓[24]

(least WIN-55,212–2 (10 mg/kg, ip) ↓[26]

shrew) ∆9-THC (20 mg/kg, ip) ↓[24]

Cisplatin (20 mg/kg, ip) ∆9-THC (1–10 mg/kg, ip) ↓[25]

WIN-55,212-2 (1–5 mg/kg, ip) ↓[25]

CP-55,940 (0.025–0.3 mg/kg) ↓[29]

2-AG (2.5–10 mg/kg, ip) CP-55,940 (0.05– 0.1 mg/kg, ip) ↓[27]

WIN-55,212–2 (1–5 mg/kg, ip) ↓[27]

∆9-THC (2.5–5 mg/kg, ip) ↓[27]

CBD (10–20 mg/kg, ip) – [27]

Anandamide (5 mg/kg, ip) ↓[27]

Methanandamide (10 mg/kg, ip) ↓[27]

SR-141716A (2.5–5 mg/kg, ip) ↓[27]

5-HTP (100 mg/kg, ip) ∆9-THC (5–20 mg/kg, ip) ↓[28]

5-HT (5 mg/kg, ip) ∆9-THC (20 mg/kg, ip) ↓[28]

2-methylserotonin (5-HT3∆9-THC (20 mg/kg, ip) ↓[28]

agonist; 5 mg/kg, ip) ∆9-THC (20 mg/kg, ip) ↓[28]

S. murinus Cisplatin (20 mg/kg, ip) ∆9-THC (2.5–10 mg/kg, ip) ↓[32]

(house CBD (5–10 mg/kg, ip) ↓[32]

musk shrew) LiCl2(390 mg/kg, ip) ∆9-THC (3–20 mg/kg, ip) ↓[31]

CBD (5–10 mg/kg, ip) ↓[31]

?, reduced; –, no effect; iv, intravenous; sc, subcutaneous; ip, intraperitoneal.

Cats and dogs

Although the two studies that evaluated the potential of nabilone [(0.1 mg/kg,

administered intravenously (iv)] to antagonize cisplatin-induced emesis [67]

and apomorphine-induced emesis [68] in dogs failed to find an anti-emetic

effect of the cannabinoid, studies with cats have shown more promising

results. McCarthy and Borison [30] reported that the synthetic cannabinoids

methyllevonantradol (0.003–0.02 mg/kg, iv) and nabilone (0.025–0.1 mg/kg,

iv) protected cats against cisplatin-induced vomiting and reduced the number

of vomiting episodes among cats not protected in a dose-dependent manner.

Pigeons

An early study [65] with pigeons demonstrated that the non-psychotropic syn-

thetic cannabinoid, HU-211, was more effective than ∆9-THC in suppressing

cisplatin-induced vomiting. The anti-emetic effect of HU-211 was U-shaped

over a narrow dose range, with maximal efficacy at 2.5 and 3 mg/kg, adminis-

tered subcutaneously (sc). More recently, the potent psychoactive cannabinoid,

HU-210 (0.0125–0.05 mg/kg, sc) has also been reported to suppress cis-

platin-induced vomiting in the pigeon [66].

Ferrets

One of the most widely used animal models of emesis is the ferret. In this

model, morphine-induced emesis [33] was suppressed by the synthetic

cannabinoid agonist WIN-55,212-2 (0.03–0.13 mg/kg, sc) and this effect was

reversed by the selective CB1receptor antagonist, AM-251. Van Sickle et al.

[34] report that the emesis produced by morphine-6-glucuronide (M6G; sc) in

ferrets was also inhibited by ∆9-THC [1 mg/kg, administered intraperitoneally

(ip)], WIN-55,212-2 (1 mg/kg, ip) and methanandamide (3 mg/kg, ip), and that

this anti-emetic effect was also reversed by AM-251 (5 mg/kg, ip). Although

AM-251 did not produce emetic episodes on its own, it did potentiate the eme-

togenic effects of M6G [34]. More recently, ∆9-THC (0.05–1 mg/kg, ip)

dose-dependently inhibited the emetic actions of cisplatin [35]. Furthermore,

∆9-THC applied to the surface of the brain stem also inhibited emesis induced

by intragastric hypertonic saline [35].

Shrews

Insectivores, such as the shrew, are the closest extant relatives to primates as

well as the oldest group of eutherians. Insectivores have sensitive emetic

reflexes. Shrews are smaller than carnivores (such as cats, dogs and ferrets)

188 L.A. Parker et al.

that have typically been used to evaluate the anti-emetic properties of drugs

and, therefore, are easier to maintain in a laboratory.

Considerable recent work by Darmani and colleagues [24–29] has evaluated

the potential of different groups of cannabinoids to inhibit emesis induced by

toxins in C. parva (the least shrew), which weighs 4–6 g. Cisplatin-induced

emesis was inhibited by WIN-55,212-2 (1–5 mg/kg, ip) and ∆9-THC

(1–10 mg/kg, ip) in a dose-dependent manner with similar potency [25, 26].

However, the synthetic cannabinoid CP-55,940 (0.025–0.3 mg/kg, ip) more

potently antagonized cisplatin-induced vomiting than WIN-55,212-2 or

∆9-THC and also had a higher affinity for the CB1receptor [29]. The synthetic

HU-210 has a higher affinity for the CB1receptor than CP-55,940, but has not

been systematically evaluated for its anti-emetic capacity in the shrew model.

Emesis induced by the precursor to serotonin, 5-hydroxytryptophan (5-HTP)

(100 mg/kg, ip) was suppressed by ∆9-THC [28] at doses of 5–20 mg/kg, ip;

however, a dose of 20 mg/kg, ip, of ∆9-THC was required to suppress the eme-

sis produced by 5-HT (5 mg/kg, ip) and the selective 5-HT3agonist

2-methylserotonin (5 mg/kg, ip). Therefore, cannabinoids appear to suppress

the emetic reaction to drugs that activate the serotonin system in the least shrew.

Since cannabinoid agonists prevent emesis, Darmani [24] predicted that

blockade of the CB1receptor would induce vomiting. Indeed, at a dose of

10 mg/kg, ip, or 40 mg/kg, sc, SR-141716 induced vomiting in the least shrew;

on the other hand, the CB2receptor antagonist, SR-144528, did not produce

vomiting at any dose tested. WIN55,212-2 (minimal dose 10 mg/kg, ip) was

more effective than ∆9-THC (minimum dose 20 mg/kg, ip) in preventing

SR-141716-induced vomiting [24, 29], which was also prevented by

CP-55,940 (1 mg/kg, ip). The selective CB1receptor antagonist SR-141716

blocked the anti-emetic activity of cannabinoids at low doses [24–29] and pro-

duced vomiting on its own at higher doses in least shrews [24], suggesting that

the endogenous cannabinoid system plays a role in the regulation of emesis.

Darmani [27] has also shown that the endocannabinoid 2-AG is a potent

emetogenic agent, whereas anandamide may cause weak anti-emetic effects

[27, 35]. The emetic effects of 2-AG were inhibited by CP-55,940

(0.05–0.1 mg/kg, ip), WIN-55,212-2 (1–5 mg/kg, ip), ∆9-THC (2.5–5 mg/kg,

ip), anandamide (5 mg/kg, ip), methanandamide (10 mg/kg, ip) and SR-141716

(2.5–5 mg/kg, ip), but not by CBD (10–20 mg/kg, ip). These results suggest

that endogenous cannabinoids may also play a role in promoting emesis.

Over the past number of years, the S. murinus (house musk shrew) has been

used as a model for emesis research [4, 6, 7]. These shrews weigh 30–60 g in

contrast to the 4–6 g of the least shrew. Like rats, Suncus will avoid a flavor

paired with lithium chloride [69]; however, unlike rats, these animals vomit in

response to toxins, even though they possess similar neural circuitry in the

emetic regions of the brain as rats [70]. In a series of experiments, Parker et al.

[31] evaluated the emetogenic potential of lithium in Suncus and the ability of

two principal cannabinoids found in marijuana, the psychoactive ∆9-THC and

the nonpsychoactive CBD, to reverse lithium-induced emesis. A prior study

Cannabinoids: effects on vomiting and nausea in animal models 189

[27] indicated that, unlike other cannabinoids tested including ∆9-THC, CBD

(10 and 20 mg/kg, ip) did not reverse retching and vomiting induced by the

endogenous cannabinoid, 2-AG, in the much smaller least shrew. It has also

been reported to be ineffective at inhibiting gastric motility in mice [71].

However, we had previously shown that CBD did prevent the establishment of

lithium-induced conditioned gaping in rats [38], a putative rat model of nausea

(reviewed below). We [31] found that ∆9-THC produced a dose-dependent

suppression of lithium-induced vomiting with higher doses producing greater

suppression than lower doses. CBD, however, produced a biphasic effect with

lower doses (5 and 10 mg/kg, ip), producing suppression and higher doses (20

and 40 mg/kg) producing enhancement of lithium-induced vomiting. The

anti-emetic properties of CBD are of clinical relevance, because CBD does not

produce the psychomimetic effects that are produced by ∆9-THC. The sup-

pression of lithium-induced vomiting by ∆9-THC, but not by CBD, was

reversed by pretreatment with SR-141716, confirming previous findings that

CBD does not act at the CB1receptor [61].

Cannabinoid and serotonin systems interact centrally and both systems are

involved in the control of emesis. There have been no studies with humans that

have systematically evaluated the relative effectiveness of 5-HT3receptor

antagonists and cannabinoid agonists to suppress acute and/or delayed nausea

and vomiting produced by chemotherapeutic agents, such as cisplatin. Using

the Suncus model of emesis, Kwiatkowska et al. [32] found that ip-injected

ondansetron and ∆9-THC both dose-dependently suppressed cisplatin-induced

vomiting and retching; however, the minimally effective dose of ondansetron

(0.2 mg/kg, ip) was considerably lower than the minimally effective dose of

∆9-THC (2.5 mg/kg, ip). A combined pretreatment of doses of the two drugs

that were ineffective alone (0.02 mg/kg ondansetron and 1.25 mg/kg ∆9-THC)

completely suppressed cisplatin-induced vomiting and retching. These results

suggest that a combination of lower doses of ondansetron and ∆9-THC may be

an effective alternative treatment for the acute phase of chemotherapy-induced

vomiting that may have fewer side effects than higher doses of either agent

alone. The anti-emetic effects of ondansetron were not affected by pretreat-

ment with SR-141716, suggesting that ondansetron does not act at the CB1

receptor. Finally, as we saw with lithium-induced vomiting [31], low doses

(5–10 mg/kg, ip) of the nonpsychoactive cannabinoid, CBD, effectively sup-

pressed cisplatin-induced vomiting, but high doses of CBD (20–40 mg/kg, ip)

potentiated vomiting.

The anti-emetic effect of low doses of CBD against lithium- and cis-

platin-induced emesis in Suncus is inconsistent with the failure of a dose of

10 mg/kg to suppress emesis produced by 2-AG in the least shrew [27]. The

difference may be related to the mechanism of action of the emetogens, the

difference in body mass of the two species (the least shrew is 10 times small-

er than the Suncus), or the biphasic effects of CBD on toxin-induced emesis.

Our [32] results suggest that both primary cannabinoids found in cannabis, the

190 L.A. Parker et al.

psychoactive ∆9-THC and the nonpsychoactive CBD, effectively suppress

lithium- and cisplatin-acute vomiting in Suncus at appropriate doses.

Conclusion

Until recently, there were few experimental studies that evaluated the

anti-emetic properties of cannabinoids, probably for two reasons: (1) the

mechanism of action of cannabinoids has only recently been discovered and

(2) animal models of emesis relied on large animals, since rodents do not

vomit. The recent work using the shrew provides the opportunity to evaluate

the potential anti-emetic properties of drugs using smaller animal models.

Conditioned gaping in shrews: a model for ANV

ANV often develops over the course of repeated chemotherapy sessions [1, 2,

10, 11, 14, 20–22]. For instance, Nesse et al. [21] described the case of a

patient who had severe nausea and vomiting with each treatment. After his

third treatment, the patient became nauseated as soon as he walked into the

clinic building and noticed a “chemical smell”, that of isopropyl alcohol. He

experienced the same nausea when returning for routine follow-up visits, even

though he knew he would not receive treatment. The nausea gradually disap-

peared over repeated follow-up visits. Nesse et al. [21] reported that about

44% of the patients being treated for lymphoma demonstrated such anticipa-

tory nausea. He suggested that “this syndrome of pretreatment nausea can be

understood as a classically conditioned response” ([21], p. 33); certain odors

have become aversive because of their association with chemotherapy-induced

illness. Indeed, Bernstein [72] has clearly demonstrated that the flavor of foods

also becomes aversive to patients receiving chemotherapy treatment.

Since it is best understood as a classically conditioned response [21, 73],

control over ANV could be exerted at the time of conditioning or at the time

of re-exposure to the conditioned stimulus (CS). If an anti-emetic drug is pre-

sented at the time of conditioning, then a reduction in ANV would be the result

of an attenuated unconditioned response (UCR); that is, reduced nausea and

vomiting produced by the toxin at the time of conditioning thereby attenuating

the establishment of the conditioned response (CR). Indeed, when adminis-

tered during the chemotherapy session, the 5-HT3antagonist granisetron has

been reported to reduce the incidence of ANV in repeat cycle chemotherapy

treatment [2]. On the other hand, if a drug is delivered prior to re-exposure to

cues previously paired with the toxin-induced nausea and vomiting, then sup-

pressed ANV would be the result of attenuation of the expression of the CR

(conditioned nausea and/or vomiting); the 5-HT3antagonists are ineffective at

this stage [1, 10, 11, 14, 20].

Cannabinoids: effects on vomiting and nausea in animal models 191

Anecdotal evidence suggests that ∆9-THC alleviates ANV in chemothera-

py patients [40, 59, 60]. Although there has been considerable experimental

investigation of unconditioned vomiting in response to toxins, there have

been relatively few reports of conditioned emetic reactions elicited by

re-exposure to a toxin-paired cue (ANV). Conditioned gaping and retching

has been observed to occur in coyotes, wolves and hawks upon re-exposure

to cues previously paired with lithium-induced toxicosis [74, 75] and ferrets

have been reported to display conditional emetic reactions during exposure to

a chamber previously paired with lithium-induced toxicosis [76]. We [77]

have presented a model of ANV based on the emetic reactions of S. murinus.

Following two pairings of a novel distinctive contextual cue with the emetic

effects of an injection of lithium chloride, the context acquired the potential

to elicit gaping in the absence of the toxin. The expression of this conditioned

reaction was completely suppressed by pretreatment with ∆9-THC at a dose

that did not suppress general activity. This provides the first experimental evi-

dence in support of anecdotal reports that ∆9-THC suppresses ANV.

Conditioned gaping in rats: a model of nausea

Nausea has been reported to be the most unpleasant and distressing aspect of

chemotherapy, superceding even vomiting and retching [14]. Nausea is less

effectively reduced by the 5-HT3receptor antagonists than is acute vomiting

[1, 9, 10, 14, 20]. Even when the cisplatin-induced emetic response is blocked

in the ferret by administration of a 5-HT3receptor antagonist, c-fos activation

still occurs in the area postrema, suggesting that an action here may be respon-

sible for some of the other effects of cytotoxic drugs, such as nausea or

reduced food intake [12]. Grundy and colleagues [78–80] have demonstrated

that in rats the gastric afferents respond in the same manner to physical and

chemical (intragastric copper sulfate and cisplatin) stimulation that precedes

vomiting in ferrets (presumably resulting in nausea that precedes vomiting).

Furthermore, 5-HT3antagonists that block vomiting in ferrets also disrupt this

preceding neural afferent reaction in rats. That is, in the rat the detection mech-

anism of nausea is present, but the vomiting response is absent [81].

Nauseogenic doses of cholecystokinin and lithium chloride induce specific

patterns of brainstem and forebrain c-fos expression in ferrets that are similar

to c-fos expression patterns in rats [82]. In a classic review paper, Borrison and

Wang [83] suggest that the rats’ inability to vomit can be explained as a spe-

cies-adaptive neurological deficit and that, in response to emetic stimuli, the

rat displays autonomic and behavioral signs corresponding to the presence of

nausea, called the prodromata (salivation, papillary dilation, tachypnoea and

tachycardia).

Over a number of years, our laboratory has provided considerable evidence

that conditioned nausea in rats may be displayed as a conditioned rejection

reaction [84–89] using the Taste Reactivity (TR) test [90]. When rats are intra-

192 L.A. Parker et al.

orally infused with a bitter-tasting quinine solution, they display a distinctive

pattern of rejection reactions (including gaping, chin rubbing and paw tread-

ing). Interestingly, following pairing with a toxin, sweet solutions come to

elicit the rejection pattern of reactions. Therefore, conditioned rejection reac-

tions provide an alternative measure of flavor aversion learning to that of a

consumption test.

The consumption test is much less selective to emetic drugs than the TR

test, because most psychoactive drugs (with rewarding or emetic properties)

produce taste avoidance [84–87, 91]. In fact, rats will simultaneously avoid an

amphetamine-paired flavor while demonstrating a preference for the amphet-

amine-paired place [92]. Considerable evidence indicates that not all taste

avoidance is accompanied by conditioned aversion (rejection reactions) to the

taste when assessed using the TR test. Stimuli that produce taste avoidance in

the absence of nausea do not produce an aversion to the taste. Lower-intestin-

al discomfort [93], footshock [93], lesions of the lateral hypothalamus [94] as

well as reinforcing drugs [84–87] produce taste avoidance but do not produce

rejection reactions in the TR test. Conditioned rejection and conditioned

avoidance are mediated by qualitatively, not quantitatively, different process-

es. Zalaquett and Parker [95] demonstrated that when the doses of lithium and

amphetamine are adjusted such that rats develop stronger avoidance of the

amphetamine-paired flavour, they still only reject the less-avoided

lithium-paired flavour. That is, only drugs with emetic effects produce condi-

tioned rejection reactions.

An early theory that attempted to explain paradoxical reports that reinforc-

ing drugs produce taste avoidance suggested that any novel change in physio-

logical state (be it hedonic or aversive) signals danger to the rat, a species that

cannot vomit [96]. A flavor paired with this change in state comes to signal

danger, resulting in subsequent avoidance of that taste. On the other hand,

shrews do not avoid a flavor paired with the rewarding drugs, amphetamine or

morphine; in fact, they develop a preference for a flavor paired with these

drugs [97]. Presumably, the emetic shrew does not need to be as wary as the

non-emetic rat about changes in state following ingestion, because it can

vomit.

The most reliable conditioned rejection reaction in the rat is that of gaping

[37, 87]. If conditioned gaping reflects nausea in rats, then anti-nausea drugs

should interfere with this reaction. Limebeer and Parker [88] demonstrated that

when administered prior to a saccharin/lithium pairing, the 5-HT3antagonist,

ondansetron, prevented the establishment of conditioned gaping in rats, but not

the establishment of conditioned taste avoidance. Presumably, ondansetron

interfered with lithium-induced nausea preventing the conditioned gaping; on

the other hand, it did not prevent the establishment of conditioned taste avoid-

ance, suggesting that nausea was not necessary to suppress saccharin con-

sumption. Ondansetron also interfered with the expression of previously estab-

lished conditioned gaping, but not taste avoidance. Since ondansetron did not

modify unconditioned gaping elicited by bitter quinine solution, the effect was

Cannabinoids: effects on vomiting and nausea in animal models 193

specific to nausea-induced gaping. In fact, ondansetron suppressed conditioned

gaping elicited by exposure to a lithium-paired saccharin solution even during

a drinking test, but it did not modify the amount consumed. Therefore, the

effects of ondansetron on conditioned gaping, but not conditioned taste avoid-

ance, cannot simply be attributed to differences in the delivery of the taste when

using the two measures (e.g. [98]); conditioned gaping is suppressed by

ondansetron whether the taste is presented by bottle or by intraoral infusion.

Subsequently, Limebeer and Parker [89] demonstrated a very similar pattern

following pretreatment with the 5-HT1A autoreceptor antagonist, 8-hydroxy-2-

di-N-propylaminotetraline (8-OH-DPAT), that also reduces serotonin availabil-

ity and serves as an anti-emetic agent in animal models. Most recently,

Limebeer et al. [99] report that lesions of the dorsal and median raphe that

reduce forebrain serotonin availability interfere with the establishment and the

expression of conditioned gaping consistent with reports that reduced serotonin

availability interferes with nausea [99]. Since rats are incapable of vomiting, we

have argued that the gape represents an incipient vomiting response. As is evi-

dent in Figure 1, the orofacial characteristics of the rat gape are very similar to

those of the shrew just before it vomits [87].

Using the conditioned gaping measure of nausea in rats, we have demon-

strated that a low dose (0.5 mg/kg, ip) of ∆9-THC interferes with the estab-

lishment and the expression of cyclophosphamide-induced conditioned gaping

[36]. Most recently, we found that CBD (5 mg/kg, ip), as well as its synthetic

dimethylheptyl homolog (5 mg/kg, ip), suppressed the establishment and the

expression of lithium-induced conditioned gaping, but not taste avoidance

[38]. Finally, the potent agonist HU-210 (0.001–0.01 mg/kg) also suppressed

lithium-induced conditioned gaping [37, 39] and this suppression was reversed

by the CB1antagonist SR-141716, suggesting that the effect of HU-210 was

mediated by its action at CB1receptors [39]. Although SR-141716 did not pro-

194 L.A. Parker et al.

Figure 1. The rat gape is topographically similar to the shrew gape just before it vomits.

duce conditioned rejection on its own, it potentiated the ability of lithium to

produce conditioned gaping. This same pattern has been reported in the eme-

sis literature. Van Sickle et al. [34] reported that although the CB1antagonist

AM-251 did not produce vomiting on its own, it potentiated the ability of an

emetic stimulus to produce vomiting in the ferret.

Conclusions

Since the discovery of the mechanism of action of cannabinoids, considerable

research using smaller animals (ferrets and shrews) confirms prior experimen-

tal reports [30, 65, 66] that cannabinoids serve as effective anti-emetics. In the

ferret and shrew models, the site of action has been identified in the emetic

area of the brainstem, the dorsal vagal complex [28, 29, 34, 35]. The shrew

model, in particular, is cost-effective for the evaluation of the anti-emetic prop-

erties of agents. It is clear that many cannabinoids act on the CB1receptors to

produce their anti-emetic properties; however, it is not known how the nonpsy-

choactive cannabinoid, CBD, which does not act at the CB1receptor, produces

anti-emetic effects within a limited dose range in S. murinus [31, 32].

Research has also supported anecdotal reports that cannabis may attenuate

ANV. Using S. murinus,∆9-THC effectively prevented conditioned gaping

elicited by re-exposure to a lithium-paired chamber [77]. Further work on this

model may reveal optimal agents for alleviating ANV, which is resistant to

treatment with 5-HT3antagonists [1, 10, 11, 14, 20].

Finally, the conditioned gaping response appears to selectively reflect nau-

sea in the rat [84–89], which is not capable of vomiting in response to a toxin.

Rats display this response to flavors previously paired with emetic agents and

this response is prevented by anti-emetic pretreatments [88, 89]. Cannabinoids

suppress the establishment of this conditioned gaping when administered prior

to a taste-toxin pairing [36–39]. Cannabinoids also suppress the expression of

previously established conditioned gaping, again suggesting that they may

serve to suppress ANV. Not only psychoactive cannabinoids, but also the

non-psychoactive CBD, suppresses the establishment and the expression of

lithium-induced conditioned gaping in rats [38].

The endogenous cannabinoid system clearly plays a role in the regulation of

nausea and vomiting. The CB1antagonists SR-141716 and AM-251 respec-

tively potentiated the strength of toxin-induced conditioned gaping in rats [37,

39] and toxin-induced vomiting in ferrets [34]. However, the finding that the

endogenous cannabinoid 2-AG serves as an emetogenic agent in the least

shrew [27] suggests that the endocannabinoid system may contribute to the

control of nausea and vomiting in a complex manner.

Acknowledgements

The authors would like to thank Marion Corrick for care of the shrews. This research was supported

by research grants to L.P. from the Canadian Institutes of Health Research and the Natural Sciences

and Engineering Research Council of Canada.

Cannabinoids: effects on vomiting and nausea in animal models 195

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The skeleton: stone bones and stoned heads?

Itai A. Bab

Bone Laboratory, The Hebrew University of Jerusalem, Jerusalem 91120, Israel

Introduction

In vertebrates, bone mass is maintained constant between the end of linear

skeletal growth, when the peak bone mass is established, and gonadal failure,

when accelerated bone loss begins. The bone mass is preserved by a continu-

ous destruction/formation process termed bone remodeling [1]. This destruc-

tion/formation cycle occurs at the same time in multiple foci that in humans

encompass approximately 5% of trabecular, endosteal, and Haversian system

surfaces. A cycle consists initially of a relatively rapid (i.e. a few weeks) resorp-

tion of pre-existing bone by a bone-specific, bone marrow hematopoietic cell

type, the osteoclast, derived from the monocyte/macrophage lineage [2]. It is

then followed by a slower (i.e. a few months) step of de novo bone formation

by another bone-specific cell type, the osteoblast [3], which belongs to the

stromal cell lineage of bone marrow [4]. Although different foci present differ-

ent phases of the cycle, the overall net effect is that of a balance between bone

destruction and formation. The physiologic importance of bone remodeling is

best illustrated in osteoporosis, the most frequent degenerative disease in devel-

oped countries, which results from impaired remodeling balance that leads to

bone loss and increased fracture risk mainly in females but also in males.

The synchronized occurrence of multiple remodeling sites has long been

viewed as suggestive of a complex, local, autocrine/paracrine [5] as well as

endocrine regulation. Indeed, experiments in knockout (KO) and transgenic

mice have demonstrated paracrine regulation of osteoclast differentiation and

activity by factors such as receptor activator of NFκB (RANK) ligand, osteo-

protegerin, macrophage colony-stimulating factor (M-CSF) and interleukin 6,

which are derived from neighboring stromal cells, including osteoblasts and

osteoblast precursors [6–11]. The most convincing evidence for local osteoblast

regulation is by bone morphogenetic proteins [12]. Systemically, ablation of

gonadal hormones in females and males has been repeatedly demonstrated to

favor bone loss in humans, rats, and mice [13, 14]. In addition, parathyroid

hormone [15, 16], calcitonin [17], insulin-like growth factor I [18], and the

osteogenic growth peptide [19] have been shown to regulate bone formation.

More recently, it has been demonstrated that bone remodeling is also subject to

a potent central control consisting of hypothalamic leptin and neuropeptide Y

Cannabinoids as Therapeutics

Edited by R. Mechoulam

201

202 I.A. Bab

signaling [20, 21] as well as downstream noradrenergic signaling by osteobal-

stic β2 adrenergic receptors [22]. It thus appears that the critical systems in the

control of bone remodeling are gonadal and central nervous system-derived,

and apply tonic inhibition of osteocalst and osteoblast function, respectively.

A couple of recent striking findings led us to study the involvement of the

endocannabinoid system in the regulation of bone remodeling. One is that, as

in the case of bone formation and bone mass, the central production of at least

one major endocannabinoid, 2-arachidonoyl glycerol (2-AG), is subject to

negative regulation by leptin [23]. The other observation is that traumatic

head injury stimulates both bone formation [24, 25] and central 2-AG pro-

duction [26].

Strategy

Our approach, designed to elucidate the regulatory role of the endocannabi-

noid system in bone, consisted of in vitro experiments in bone cells followed

by in vivo skeletal phenotyping of cannabinoid receptor (CB)-deficient mice.

After demonstrating a low bone mass (LBM) in these mice we assessed the

prevention of bone loss in estrogen-deficient mice by CB agonists.

The initial experiments demonstrated CB mRNA in bone cells in vitro, con-

firmed by immunostaining in vivo. We then further used the in vitro system to

demonstarte the regulation of osteoblast and osteoclast differentiation and

activity by cannabinoid ligands. The relevance of the in vitro findings to the in

vivo scenario was established by analyzing the cortical and trabecular bone of

CB-KO and ovariectomized mice using micro-computed tomography and his-

tomorphometry.

Expression of cannabinoid receptors in bone

Undifferentiated osteoblast progenitors, such as mouse bone marrow-derived

stromal and MC3T3 E1 preosteoblastic cells [27, 28], exhibit very low levels

of the neuronal CB1and peripheral CB2mRNA cannabinoid receptors [29],

detectable only by ultrasensitive methods. When these cells are grown for 2–4

weeks in so-called osteogenic medium, which contains vitamin C, β-glyc-

erophosphate, and dexamethasone [30], CB1mRNA remains at the same lev-

els. However, CB2mRNA expression increases progressively in parallel to the

expression of the osteoblastic marker genes which encode tissue non-specific

alkaline phosphatase (TNSALP) [31], parathyroid hormone receptor 1

(PTHRc1) [32], and particularly the osteoblastic master regulatory gene,

RUNX2 [33]. CB2, but not CB1mRNA transcripts are also present in high

abundance in monocytic cells undergoing osteoclast differentiation induced by

RANK ligand and M-CSF [34]. In vivo,CB

2protein is present in trabecular

osteoblasts and their decedents, the osteocytes [33], as well as in osteoclasts.

Cannabinoid ligands regulate bone cell differentiation and activity

CB2activation has different effects in early preosteoblasts and in more mature

osteoblastic cells. In bone marrow derived partially differentiated osteoblasts,

with limited CB2expression, the specific CB2agonist HU-308 [35] but not the

specific CB1agonist noladin ether [36], triggers a Giprotein-mediated mito-

genic effect. This response leads to a dose-response expansion of the pre-

osteoblastic pool. In more mature osteoblastic cells, represented by the

MC3T3 E1 cell line, HU-308 also stimulates osteoblast-differentiated func-

tions such as alkaline phosphatase activity and matrix mineraliztion. Thus,

CB2signaling has multiple regulatory osteogenic anabolic functions along the

osteoblast differentiation pathway. CB2activation has an opposite effect on

osteocalstogenesis, namely, it inhibits osteoclasts differentiation.

Cannabinoid receptor signalling regulates bone mass in vivo

Although only the CB2receptor is demonstrable in bone, both CB1- and

CB2-deficient mice have LBM. However, the underlying mechanisms of these

LBMs appear to be different inasmuch as a pronounced low trabecular bone

density is already found in the CB1-KO mice at a young age. At the same age

the skeleton of CB2-KO mice is almost normal, with severe bone loss report-

ed only in nearly 1-year-old animals [38]. This age-related difference suggests

that CB1is mainly involved in the establishment of peak bone mass, which

develops during infancy, sexual maturation, and young adulthood [39, 40].

CB2appears to be an important regulator of bone remodeling and maintenance

of constant bone mass in later life. Further support for this notion is derived

from the different pathogenic processes that lead to the LBM in the CB1- and

CB2-deficient mouse lines. Consistent with their early LBM, CB1-KO mice

have increased bone resorption associated with decreased bone formation [38].

Reminiscent of human postmenopausal osteoporosis [41], CB2-KO mice have

a high turnover LBM characterized by increases in both bone resorption and

formation which are at a net negative balance [38].

Because CB2is only peripherally expressed, CB2-specific ligands could

provide an opportunity to augment bone mass while avoiding the cannabinoid

psychotropic activity. Indeed, HU-308 attenuates bone loss induced by estro-

gen depletion in ovariectomized animals with significant respective bone

anti-resorptive and anabolic activities in the trabecular and cortical skeletal

compartments [38].

Summary

Although the CB2receptor was cloned more than a decade ago, its physiolog-

ical role remained elusive. Using a combined approach encompassing mole-

The skeleton: stone bones and stoned heads? 203

cular and cellular biology, pharmacology, and genetic analyses in mice, we

were able to show a role for CB2signaling in regulating bone mass.

Furthermore, attenuation of the deleterious effects of ovariectomy on bone by

a peripherally selective CB2cannabinoid receptor agonist have major implica-

tions for osteoporosis, offering new molecular targets for the diagnosis and

treatment of this disease.

CB1is apparently not involved in age-related bone loss commonly diagnosed

in humans, but may have an important role in early skeletal development.

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Cannabinoids and drugs of abuse

Daniela Parolaro and Tiziana Rubino

Center of Neuroscience, University of Insubria, Via A. da Giussano 10, 20152 Busto Arsizio (VA),

Italy

Introduction

Derivatives of Cannabis sativa, such as marijuana and hashish, are the most

widely consumed illicit drug: almost half of all 18-year olds in the USA and in

most European countries admit to having tried it at least once, and 10% of that

age group are regular users. There have been many subjective accounts of the

cannabis ‘high’. A typical ‘high’is preceded initially by a transient stage of tin-

gling sensations felt in the body and head accompanied by a feeling of dizzi-

ness or light-headedness. The ‘high’ is a complex experience, characterized by

a quickening of mental association and a sharpened sense of humor, sometimes

described as a state of “fatuous euphoria”. As reported by Atha and Bianchard

[1] in a survey of 1333 young British cannabis users the most common benefit

reported were relaxation and relief from stress, insight/personal development

and euphoria, but 21% of the users also described some adverse effects, includ-

ing impaired memory, paranoia and amotivation/laziness. As with other intox-

icant drugs, little is known about the brain mechanisms that underlie the

cannabis high. The intoxicant effects are clearly mediated via CB1receptors. In

a well-controlled study in 63 healthy cannabis users [2] who received either a

CB1receptor antagonist (Rimonabant) or placebo and smoked either a

∆9-tetrahydrocannabinol (∆9-THC)-containing or placebo marijuana cigarette,

Rimonabant blocked the acute psychological effects of the active cigarettes.

Moreover, self ratings of cannabis intoxication correlated most markedly with

increased blood flow in the right frontal region as demonstrated using positron

emission tomography (PET) to measure changes in cerebral blood flow.

The potential ability of cannabis derivatives to produce dependence in

humans is still a controversial issue. Earlier clinical literature (for reviews see

[3–5]) suggested that tolerance also occurs after repeated administration of

∆9-THC in humans, although many of these studies were poorly controlled.

But for many years cannabis was not considered to be a drug of addiction.

Withdrawal of the drug did not lead to any obvious physical withdrawal syn-

drome either in people or in animals, and animals failed to self-administer the

drug, a behavior usually associated with drugs of addiction.

Attitudes have changed markedly in recent years. According to the

Diagnostic and Statistical manual of Mental Disorders (DSM IV) [5a] criteria

Cannabinoids as Therapeutics

Edited by R. Mechoulam

207

(American Psychiatric Association, 1994) for ‘substance dependence’ and

‘substance abuse’, surprisingly a high proportion of regular cannabis users

appear to fall into these categories. Recent studies [6] indicated that almost

one-third of regular cannabis users fell within the definition of ‘substance

abuse’ or ‘substance dependence’. Moreover, carefully controlled studies have

also shown that a reliable and clinically significant withdrawal syndrome does

occur in human cannabis users when the drug is withdrawn. The symptoms

include craving for cannabis, decreased appetite, sleep difficulty and weight

loss, and may sometimes be accompanied by anger, aggression, increased irri-

tability, restlessness and strange dreams [7].

The existence of abuse liability of cannabinoids in animals is much more

clearly observable. Processes involved in substance abuse are neurobiological-

ly and behaviorally complex. Tolerance and withdrawal syndrome represent

adaptive responses to the prolonged exposure of neurons to drugs, but the main

factor common to all drugs of abuse is their ability to induce drug-seeking

behavior, which is due to the positive reinforcing effects of the drugs. Several

behavioral models have been used to evaluate tolerance and withdrawal, as

well as the rewarding effects of cannabinoids, which will be briefly summa-

rized here together their proposed molecular basis.

Tolerance

Chronic administration of natural or synthetic cannabinoid agonists in differ-

ent animal species induces tolerance to most of their pharmacological effects

(see [8, 9] for review). Although some papers have reported that pharmacoki-

netic events take place during the development of cannabinoid tolerance [10,

11], there is general agreement that this phenomenon is pharmacodynamic in

nature. The best-known events that occur after development of cannabinoid

tolerance are receptor downregulation and uncoupling from the G protein sys-

tem, which ends in receptor desensitization (see [8] for review). Besides these

alterations, other cellular adaptations are present in the brain of cannabi-

noid-tolerant rats, such as modulation of effector proteins. Specifically, it has

been shown that increased activation of the cAMP pathway (i.e. cAMP accu-

mulation and protein kinase A activity) [12–15], together with adaptations in

the extracellular-signal-regulated kinase (ERK) cascade [16], were observed in

some cerebral regions of chronic ∆9-THC-treated animals. An elegant demon-

stration of the involvement of the Ras/ERK pathway in development of

cannabinoid tolerance comes from studies in Ras-GRF1-knockout mice [16],

a useful model where cannabinoid-induced ERK activation is lost. These ani-

mals did not develop tolerance to ∆9-THC’s analgesic and hypolocomotor

effects, suggesting that the ERK cascade could play a pivotal role in the induc-

tion of synaptic plasticity due to chronic cannabinoid exposure. Finally, recent

work reported that the pyrazolopyrimidine (PP1), the Src family tyrosine

kinase inhibitor, reversed ∆9-THC-induced tolerance, supporting a role for Src

208 D. Parolaro and T. Rubino

tyrosine kinase in phosphorylation events in ∆9-THC-tolerant mice [15]. Taken

together, these recent data seem to indicate an outstanding role in cannabinoid

tolerance for some protein kinases (protein kinase A, ERK, Src tyrosine

kinase), suggesting that cannabinoid tolerance could be depicted as

activity-dependent synaptic plasticity. Whether and how these kinases could

contribute to CB1receptor downregulation or desensitization remains to be

determined. In line with this view, large-scale analysis of gene-expression

changes during acute and chronic exposure to ∆9-THC in rat hippocampus [17]

revealed that the altered genes were predominantly associated with membrane

repair and synaptic structures, indicating that they are involved in transcription

or proteosomal processes, possibly reflecting a change in neuronal capacity to

deal with the ubiquitous consequences of chronic cannabinoid receptor activa-

tion over long time periods.

Finally, it cannot be ruled out that prolonged activation of cannabinoid recep-

tors also leads to decreased endocannabinoid content and signalling in the stria-

tum and to increased anandamide formation in the limbic forebrain [18], areas

involved in the tonic control of movements and in reinforcement processes.

Physical dependence

Although the presence of spontaneous withdrawal after chronic cannabinoid

treatment is also controversial in animals, there are no doubts that administra-

tion of the CB1-selective antagonist SR-141716A precipitates a pronounced

withdrawal syndrome in animals that have been chronically treated with

cannabinoids (see [8, 9] for review). Biochemical indicies of adaptive changes

have been demonstrated during cannabinoid withdrawal and they include com-

pensatory changes in the cAMP pathway in the cerebellum [12–14, 19], which

appears to be a key area in the modulation of somatic expression of cannabinoid

abstinence syndrome. These findings directly demonstrated that, in analogy

with other addictive drugs, the activation of the cAMP pathway is a crucial phe-

nomenon at the onset of ∆9-THC-withdrawing behaviors. Interestingly, a key

structure in controlling this process could be the cerebellum, a region not previ-

ously associated with drug abuse, and whose participation in cognitive networks

is actually a most exciting field of investigation. Moreover, activation of corti-

cotropin-releasing factor [20] and a decrease in mesolimbic dopamine trans-

mission [21, 22] have also been observed in withdrawn rats, strengthening the

evidence that cannabinoids share with other drugs of abuse those neurochemi-

cal properties that are regarded as the biological substrate of drug addiction.

Behavioral sensitization

Behavioral sensitization represents another adaptive neurobiological alter-

ation that occurs after repeated exposure to drugs and plays a role in drug

Cannabinoids and drugs of abuse 209

addiction, particularly in drug-seeking behavior that persists long after the

discontinuation of drug use [23]. Rats repeatedly treated with ∆9-THC for

several days (3/5 days) and then challenged with ∆9-THC 2/3 weeks after the

last ∆9-THC injection show a greater behavioral activation than rats repeated-

ly treated with vehicle [24, 25]. The molecular underpinnings of this phe-

nomenon are still not well understood, but they involve altered CB1receptor

functionality in the striatum and cerebellum of sensitized rats [26]. Moreover,

in the cerebellum the cAMP pathway and the ERK cascade seem to lose their

responsiveness to cannabinoids ([26] and T. Rubino et al., unpublished

results). Preliminary data obtained in our laboratory indicate differential

responsiveness of specific transcription factors in selected brain areas (stria-

tum, prefrontal cortex and hippocampus) of pre-exposed rats, supporting the

working hypothesis that relapse can be viewed as a certain kind of memory

(addiction memory) since the brain obviously remembers the prior adminis-

tration of the drug and induces craving.

Drug discrimination

Drug discrimination is a behavioral procedure based on the ability of a drug

to induce a specific set of interoceptive stimulus conditions perceived by the

animals that might be predictive of the subjective reports of perceptions/feel-

ings induced by the same drug in humans. As a result, studies of the subjec-

tive effects of new drugs in both humans and animals have been relatively

good predictors of either or not a drug will be abused. Since animals do not

easily self-administer cannabinoids, the drug-discrimination procedure has

long been the primary animal model available for evaluating the potential

abuse liability of cannabinoids [27]. Cannabinoid drugs show a pharmaco-

logical specificity in this behavioral procedure. Thus, in animals trained to

discriminate injections of ∆9-THC from injections of saline, only drugs that

possess the ability to activate CB1cannabinoid receptors fully generalize to

the ∆9-THC training stimulus (see [9] for review). Moreover, the discrimina-

tive stimulus effects of ∆9-THC and other synthetic CB1agonists can be com-

pletely blocked by pre-treatment with the selective CB1receptor antagonist

SR-141716A [28], further demonstrating that the cannabinoid discrimination

is mediated by CB1receptors [29, 30]. In contrast, anandamide and stable

analogs of this endocannabinoid do not fully substitute for ∆9-THC in mon-

keys and rats [31–33], or has done so only at doses that severely decrease

food-mantained responding [32]. The fast reuptake and rapid metabolism of

anandamide by the fatty acid amide hydrolase enzyme is a likely explanation

for why anandamide, which is a partial agonist of CB1receptors, just like

∆9-THC, usually fails to produce ∆9-THC-like discriminative-stimulus

effects. Anandamide has been shown to have cannabinoid-like discriminative

stimulus effects under some situations. Recently Jarbe et al. [33] demonstrat-

ed that methanadamide was successfully used as a training stimulus in rats,

210 D. Parolaro and T. Rubino

and ∆9-THC produced complete generalization. Anandamide was able to pro-

duce generalization to the methanandamide but not to the ∆9-THC training

stimulus that could be related to the different affinities of ∆9-THC and

methanadamide for CB1receptors, resulting in a discriminative stimulus for

methanadamide with an intensity and a quality closer to the anandamide

stimulus as compared to the ∆9-THC stimulus. It could be also the case that

anandamide and methanadamide but not ∆9-THC possess affinity for a sub-

population of receptors other than CB1. Unfortunately the ability of

SR-141716A to block the generalization to anandamide was not tested.

Among non-cannabinoid drugs, only the benzodiazepine diazepam has been

found to produce partial generalization to cannabinoid training stimulus that

was SR-141716A-insensitive, suggesting that this effect is mediated by an

interaction through the GABAergic system [34].

Self-administration

Drug self-administration behavior has been one of the most direct and pro-

ductive approaches for studying the rewarding properties of abused drugs.

Using this methodology, it has been possible to study neuropharmacological

mechanisms involved in such behaviors and preclinically evaluate therapeutic

strategies for treatment of drug abuse. Since 1970, all attempts to obtain a

robust procedure for ∆9-THC self-administration have failed and this has been

fundamental to claims of a differential status for cannabinoids with respect to

major abused drugs. Within the last few years, however, reinforcing effects of

some synthetic CB1cannabinoid agonists have been reported using intra-

venous self-administration procedures in rats and mice [35–37], although the

experimental procedures employed in each of these studies limit the general-

ity of the findings. Persistent intravenous self-administration of ∆9-THC itself

was first demonstrated in squirrel monkeys by Tanda et al. [38]. However,

monkeys in this study had a history of cocaine self-administration, raising the

possibility that persistent neurobiological adaptations might subsequently

predispose animals to self-administer ∆9-THC. This problem was successful-

ly overcome by Justinova et al. [39], who demonstrated that

∆9-THC-self-administration behavior was initiated and subsequently main-

tained at very high rates in monkeys with no history of exposure to other

drugs, showing that this drug possesses reinforcing properties of its own that

are not dependent on prior self-administration of other drugs. Thus

self-administration of ∆9-THC by squirrel monkeys provides a reliable animal

model of human marijuana abuse, suitable for studying the relative abuse lia-

bility of other natural and synthetic cannabinoids and for developing new

therapeutic strategies for the treatment or prevention of marijuana abuse in

human.

Cannabinoids and drugs of abuse 211

Conditioned place preference

The conditioned place preference procedure is a classical procedure that pro-

vides an indication of drug-related reward/aversion effects in animals.

Previous studies into the reinforcing properties of cannabinoids have produced

conflicting evidence with respect to the generation of place preference. Some

studies have shown that ∆9-THC can produce place preference [40, 41], where-

as others reported place aversion [12, 42–46] or no effect [41]. The discrep-

ancies in results have been interpreted as being due to differences in appara-

tus, experimental design and the subjects used. Positive place preferences,

when found, are usually highly dose-dependent, often occurring at only a sin-

gle dose either in mice or in rats using ∆9-THC as well as synthetic cannabi-

noid compounds [40, 41, 47, 48]. Indeed, place preference was obtained with

a low dose of ∆9-THC in mice (1 mg/kg) when they received a previous prim-

ing ∆9-THC exposure in the home cage before the conditioning sessions [41].

Place aversion properties are often produced by ∆9-THC and synthetic

cannabinoids either in rats or in mice using similar dose ranges and standard

place preference procedures [40, 43–46]. These apparently conflicting results

could be explained by the possible dysphoric/anxiogenic consequences of the

first cannabinoid exposure that could mask the development of positive place

preference [41]. Discrepant results are also present for the CB1receptor antag-

onist SR-141716A: while some papers reported a positive place preference in

rats [44, 49] some others failed to demonstrate either place preference or place

aversion [45, 47]. These opposite results do not allow us to precisely indicate

a role for endocannabinoid tone as a physiological system to suppress reward

or to induce aversion.

Neurochemical correlates of cannabinoid rewarding properties

The mesolimbic dopaminergic system is part of a brain reward circuit that has

been long thought to play a major role in mediating reinforcing/rewarding

effects of drugs of abuse [50]. Many drugs abused by humans share the com-

mon property of selectively increasing dopamine release in the nucleus accum-

bens, the major terminal area of the mesolimbic dopamine system, but this has

been a matter of debate with regard to ∆9-THC and other cannabinoids. It is

now well-accepted that cannabinoids are able to increase dopamine levels in

the shell compared with the core of the nucleus accumbens, likely through an

opioid receptor-mediated mechanism or a direct activation of dopaminergic

neurotransmission in the nucleus accumbens (see [9] for review). Moreover,

cannabinoids might exert part of their reinforcing effects through the endoge-

nous opioid system [51, 52]. For example, ∆9-THC-induced conditioned place

preference is suppressed in µ-opioid receptor-knockout mice [48] and

∆9-THC-induced self-administration can be blocked by µ-opioid receptor

antagonists [36, 37]. The neurochemical mechanism of the interaction between

212 D. Parolaro and T. Rubino

the endocannabinoid and opioid systems has not been elucidated, but might

involve cannabinoid-induced synthesis and release of endogenous opioids or

converging signal transduction pathways if the receptors are co-expressed [52].

Cannabinoid system and drug addiction

Animal models of drug reward provide evidence that endogenous cannabi-

noids play a role in determining the rewarding effects not only of cannabis but

also of other psychoactive drugs, such as ethanol, cocaine, morphine, nicotine

and amphetamine. Plenty of published works report the involvement of

cannabinoid processes in positive reinforcement activated by both natural

rewards and drugs of abuse. For example, in CB1-knockout mice nicotine was

not able to induce place preference as it does in wild-type mice [53], and

administration of SR-141716 in the rat decreased nicotine self-administration

[54]. These results suggest that activation of the endogenous cannabinoid sys-

tem may participate in the motivational effect of nicotine; thus SR-141716

may be effective as an aid for smoking cessation.

Results on morphine-conditioned place preference in CB1-knockout mice

had controversial effect: in one study [55] morphine induced conditioned place

preference in wild-type mice but failed to produce any response in knockout

mice, indicating the inability of morphine to induce rewarding effects in the

absence of CB1cannabinoid receptors. In a more recent work [56] CB1recep-

tor-knockout mice developed a strong place preference to morphine, similar to

that in wild-type Swiss-Webster mice, thus not supporting a contribution of the

brain cannabinoid system to morphine reward. A possible explanation for this

discrepancy could rely in the slightly more intensive conditioning paradigm

and differences in the nature of conditioning chambers used for the experiment

in the last paper. However, self-administration studies support the idea that the

CB1cannabinoid receptor is essential for the modulation of morphine’s

rewarding effects. Cossu et al. [57] found that morphine did not induce intra-

venous self-administration in mutant CB1receptor-knockout mice, whereas it

was significantly self-administered by the corresponding wild-type mice.

Approaches involving the CB1antagonist SR-141716 gave more compelling

results. Recently it was shown that SR-141716A pretreatment dose-depend-

ently reduced operant heroin self-administration by male Wistar rats under a

fixed-ratio schedule of reinforcement, and significantly lowered the breaking

point of responding for heroin under a progressive-ratio schedule of reinforce-

ment [58]. In the same line Solinas et al. [59] reported that SR-141716A

markedly decreased heroin self-administration under the progressive-ratio

schedule. In contrast, SR-141716A had no effect on heroin self-administration

under the fixed-ratio schedule at heroin doses of 50 or 100 µg/kg per injection,

but produced small decreases in self-administration at lower doses (25 and

12.5 µg/kg per injection). These data demonstrate that the cannabinoid CB1

receptor antagonist SR-141716 produces a clear attenuation, but not a com-

Cannabinoids and drugs of abuse 213

plete blockade, of the reinforcing effects of heroin, suggesting a facilitatory

modulation of opioid reinforcement by endogenous cannabinoid activity that

is unmasked by CB1receptor blockade. All these lines of evidence provide

support for the potential efficacy of cannabinoid CB1antagonists in the pre-

vention and treatment of opioid addiction.

Evidence for endocannabinoid involvement in the rewarding effects of

ethanol also exists (see [60] for review). Here we only cite the latest papers.

Voluntary ethanol intake was significantly lower in CB1–/– versus CB1+/+ young

male mice [61–63]. Moreover, administration of the cannabinoid CB1receptor

antagonist SR-141716 significantly reduced ethanol intake in CB1wild-type

(+/+) mice [61] and rats [54]. The role of endocannabinoids and CB1receptor

in alcohol-drinking behavior is now unequivocal; thus SR-141716 may be

effective in reduction of alcohol consumption. Surprisingly the combination of

the synthetic cannabinoid agonist CP-55,940 with MDMA (methylene-

dioxymethamphetamine; ecstasy) in rat reduced the number of drug-associated

lever pressings compared to the single drugs [64] and pre-treatment with

SR141716A significantly increased MDMA self administration. At first glance

these data seem to suggest that the endocannabinoid system might have nega-

tive effects rather than the positive ones shown in the above cited studies. The

nature of this interaction remains unclear due to the lack of studies on dopamine

levels in mesolimbic structures that could add further insight on the neuro-

chemical correlates of MDMA’s reinforcing properties.

Finally, particularly relevant seems to be the role of endocannabinoid tone

in relapse to drugs of abuse. This aspect of drug addiction assumes a striking

interest in the human context. In fact, detoxification from drug addiction has

been a medical problem for as long as drugs have been abused, due to relapse

occurring even after prolonged drug-free periods. Several reinstatement mod-

els are currently available to investigate major factors contributing to relapse

and have been used to study the involvement of the cannabinoid system. In

recent work Fattore et al. [65] reported that the CB1receptor antagonist

SR-141716A prevented heroin-induced reinstatement of heroin-seeking

behavior but did not show any effect per se, suggesting that CB1receptor

blockage alters the reinforcing consequences of heroin administration.

Moreover, in animals with a history of heroin self-administration, cannabinoid

primings elicit relapse to heroin-seeking behavior following an extended

drug-free period. Similar results were also obtained by De Vries et al. [66]: the

potent cannabinoid agonist HU-210 reinstated heroin seeking, whereas

SR-141716A attenuated both heroin-primed and cue-induced heroin seeking

following a 3-week extinction period. The same group [67] found very similar

results also in animals withdrawn from cocaine self-administration: HU-210

provoked relapse to drug seeking after a prolonged withdrawal period, while

blockade of CB1receptor attenuated the relapse induced by re-exposure to

cocaine-associated cues or cocaine itself. The CB1cannabinoid antagonist was

also used on alcohol-deprivation effects (i.e. the temporary increase in alcohol

intake after a period of alcohol withdrawal) in Sardinian alcohol-preferring

214 D. Parolaro and T. Rubino

(sP) rats [68]. As expected, alcohol-deprived rats virtually doubled voluntary

alcohol intake during the first hour of re-access. Acute administration of

SR-141716 completely abolished the alcohol-deprivation effect. These results

suggest that the cannabinoid CB1receptor is part of the neural substrate medi-

ating the alcohol-deprivation effect and that SR-141716 may possess

anti-relapse properties.

Taken together, these results seem to indicate that SR-141716 could specif-

ically counteract reward-related behaviors, whatever the specific factors

involved in the action of each reinforcer, and that cannabinoid CB1receptors

could be crucially involved in the neurobiological events evoked by appetitive

reinforcers. However this does not necessarily mean that a permanent endoge-

nous cannabinoid tone exists to ensure the organism a basal hedonic level.

Thus it can be postulated that cannabinoid-related processes are elicited and

maintained by pleasant reinforcers. This suggests that the activation of reward

system could be under the permissive control of some complex CB1-related

cannabinoid processes which are required for the perception of the incentive

value of positive reinforcements. Is the recent finding that Rimonabant reduces

food intake in obesity and tobacco consumption in more than 500 adults

underlying the relevance of therapeutic modulation of the endocannabinoid

system?

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218 D. Parolaro and T. Rubino

Cannabinoids in appetite and obesity

Francis Barth and Murielle Rinaldi-Carmona

Sanofi-aventis, 371, rue du Professeur Joseph Blayac, 34184 Montpellier Cedex 04, France

Introduction

In contrast to most drugs, many of the pharmacological activities of cannabi-

noids were described in humans before being investigated by pharmacologists

in laboratory animals. This peculiarity is of course due to the use of Cannabis

sativa preparations for recreational purposes from ancient civilizations up to

modern times.

The effects of cannabinoids on appetite makes no exceptions: the fact that

cannabis can stimulate appetite has been observed since AD 300 [1]. Recent

experiments using potent synthetic or natural endogenous cannabinoid ago-

nists, as well as transgenic animals in which the cannabinoid system has been

disrupted confirm the role of the cannabinoid system as a modulator of food

intake.

Particularly interesting are the opposite effects of the newly developed

cannabinoid antagonists. These compounds have been shown to decrease food

intake and to regulate body-weight gain, and are expected to provide a new

therapeutic approach to treat obesity, a condition that affects up to 27% of the

US population and is now considered by the World Health Organization as a

global epidemic that poses a serious threat to world health, particularly in ado-

lescents [2].

However, the mechanism by which the cannabinoid system modulates food

intake is far from fully understood, and its elucidation is the subject of much

research at the moment.

Cannabinoid agonists and appetite

A large anecdotal and descriptive literature suggest that smoking cannabis

stimulates hunger, and selectively increases the appetite for sweet and palat-

able food, which smokers sometimes refer to as ‘the munchies’. Starting in the

1970s, a series of well-controlled scientific studies was conducted to better

characterize this effect [3].

Cannabinoids as Therapeutics

Edited by R. Mechoulam

219

An increased desire for sweet food (marshmallows) was indeed noted in

subjects after smoking marijuana [4]. Other studies have shown that

∆9-tetrahydrocannabinol (∆9-THC)-induced appetite stimulation was depend-

ent on the route of administration [5], the dose used [6], the social environment

[7] and the satiety status [8]. Higher appetite stimulation was observed with

the suppository versus oral intake or inhalation, with low-∆9-THC versus

high-∆9-THC cigarettes, in smoking in social groups versus isolated use and

finally in fed versus fasted individuals.

Based on these findings, dronabinol (Marinol®), an oral formulation of ∆9-

THC, was approved by the US Food and Drug Administration (FDA) in 1992 for

the treatment of AIDS-related anorexia. This wasting syndrome, which occurs in

about 18% of AIDS patients, is of particular concern in that it may exacerbate

the primary illness, decrease quality of life and also decrease survival.

Several clinical studies involving patients with anorexia related to AIDS,

cancer or Alzheimer’s disease demonstrated the efficacy of dronabinol [9, 10].

In a double-blind, placebo-controlled 6-week study involving 139 patients

with AIDS-related anorexia, dronabinol (5 mg/day) was associated with

increased appetite (38% above baseline versus 8% for placebo), improvement

in mood (+10 versus –2%) and decreased nausea (–20 versus –7%). Body

weight was stable in dronabinol patients, while placebo recipients had a mean

loss of 0.4 kg [11]. These effects were later confirmed in a long-term, open-

label follow-up study with 94 patients treated for up to 12 months [12].

Dronabinol therapy was generally well tolerated during long-term use, with

most adverse events being associated with the known central effects of

cannabinoids (euphoria, dizziness, confusion). No significant changes in

hematology or blood-chemistry parameters due to the drug were noted.

The use of dronabinol or other cannabinoids, including smoked marijuana,

to treat cancer or AIDS-related anorexia remains, however, controversial due

to the psychotropic properties of these drugs, and also due to the reluctance to

treat immuno-deficient patients with a cannabinoid drug, which may by itself

negatively affect the immune system. This fear may however be unfounded, at

least for dronabinol, if one considers a study that compared the use of dron-

abinol and megestrol acetate for the treatment of the HIV wasting syndrome in

which no difference in the CD4 T-lymphocyte count due to the drugs during

the 12-week study was noted [13]. Another study involving 67 patients with

the HIV-1 infection concluded that smoked or oral ∆9-THC did not produce

any harmful effects on viral loads. The findings of this short-term (21-day)

study should however be confirmed in additional long-term trials [14].

In laboratory animals, treatment with ∆9-THC or synthetic cannabinoid ago-

nists has shown inconsistent effects on food intake [1], with an increase

[15–17], or decrease [18] of food intake, or without any effect [19]. The spe-

cies, route of administration and dose used clearly influence the outcome of

the experiments. In particular, the decreased locomotor performance induced

by high doses of cannabinoids may often be responsible for the decreased food

intake observed in the high-dose experiments.

220 F. Barth and M. Rinaldi-Carmona

Of particular interest are the experiments conducted with anandamide and

2-arachidonoyl glycerol (2-AG), the endogenous ligands for cannabinoid

receptors. Subcutaneous injection of anandamide (0.5–10 mg/kg) induced sig-

nificant overeating in pre-satiated male rats [20]. Subsequent studies have

shown that very low doses of anandamide [0.001 mg/kg, administered

intraperitoneally (i.p.)] also increased food intake in female mice while high-

er doses were not active in this paradigm [21]. Direct injection of anandamide

(50 ng in 0.5 µl) into the ventromedial hypothalamus also initiated food intake

in rats [22]. Similarly, injection of 2-AG into the nucleus accumbens shell, a

limbic forebrain area strongly linked to eating motivation, potently and

dose-dependently stimulated feeding in rats [23]. This effect was blocked by

pre-treatment with the cannabinoid antagonist SR-141716 (Fig. 1), indicating

the involvement of the cannabinoid CB1receptor.

These experiments clearly demonstrated that activation of central cannabi-

noid receptors by endocannabinoids stimulates eating behavior, and provided

important evidence for the involvement of a central cannabinoid system in the

normal control of feeding.

Interestingly, the selectivity for sweet or palatable food observed in humans

was also observed in animal experiments. The potent synthetic cannabinoid

agonist CP-55,940 (0.01–0.05 mg/kg i.p.) was shown to increase motivation

for beer and for a sucrose solution in rats [24]. ∆9-THC (0.5–2.5 mg/kg i.p.)

also selectively increased the consumption of a high-fat or high-fat sweetened

diet versus standard chow in free-feeding Lewis rats [16].

Recent studies also suggested that the endocannabinoid 2-AG present in

milk may play a vital role in the initiation of milk suckling, and hence in

growth and development during the early stages of mouse life [25, 26].

Cannabinoid antagonists and obesity

The first potent and selective CB1cannabinoid antagonist, SR-141716, was

described in 1994 [27]. This compound was able to reverse the hyperphagia

Cannabinoids in appetite and obesity 221

Figure 1. Selected cannabinoid CB1 antagonists acting on food intake.

induced by cannabinoid agonists such as ∆9-THC [28], anandamide [20] and

2-AG [23].

More interestingly, SR-141716 also produced changes in ingestive behav-

iors when administered alone. This was first shown by Arnone et al. [29], who

described the effects of SR-141716 [0.3–3 mg/kg, taken per os (p.o.)] on spon-

taneous sucrose feeding (sucrose pellets versus standard chow) and sponta-

neous or neuropeptide Y (NPY)-induced sucrose drinking in rats. SR-141716

markedly and dose-dependently reduced the consumption of sucrose, with

only a marginal effect on regular chow and water consumption. Moreover, it

also decreased ethanol consumption in C57BL/6 mice, a strain known for its

genetic predisposition for ethanol consumption, without affecting water intake

[29]. In another study, marmosets were given the choice between standard

food or a sweet cane-sugar mixture; administration of SR-141716 (1–3 mg/kg

p.o.) significantly and dose-dependently reduced the amount of sweet food

ingested during the 6-h test period, with no effect on the standard food intake

[30]. This preferential effect on palatable food intake compared with standard

food suggested that an endogenous cannabinoid tone may modulate the appet-

itive value of food. Similar results were obtained by Colombo et al. [31], who

described an overall decrease in food intake and body weight in rats treated

with SR-141716 (2.5–10 mg/kg i.p.) for 14 days. Using a self-administration

protocol it was also shown that SR-141716 reduces sucrose intake by acting on

both the appetitive and the consummatory aspects of ingestive behavior in rats

[32].

These results suggested a potential for cannabinoid antagonists in the treat-

ment of eating disorders and, in particular, obesity. Therefore, the effect of

chronic administration of SR-141716 in pharmacological models more rele-

vant to obesity was investigated. One of these model is the diet-induced obese

(DIO) mice, in which the animals are made obese simply through eating of a

high-fat diet. Daily administration of SR-141716 (10 mg/kg/day p.o.) for 5

weeks produced a transient decrease in food intake with a sustained reduction

in body weight. At the end of the treatment period, a significant 20% weight

loss was obtained, together with a 50% reduction of adiposity. Moreover raised

plasma leptin, insulin and free fatty acid levels were decreased to values found

in lean (non-DIO) mice [33].

Similar results were obtained with AM-251, a close analogue of SR-141716

(Fig. 1). DIO mice were treated with AM-251(3–30 mg/kg/day p.o.) using a

chronic, interrupted dosing schedule (2 weeks on treatment, 2 weeks off and a

further 2 weeks on treatment). A significant reduction of body weight togeth-

er with a reduction of adipose tissue mass was observed. While the anti-obesi-

ty effect was lost during the off-treatment period, it was recovered during the

second treatment period, suggesting that chronic treatment of obese individu-

als with CB1antagonists is a viable pharmacological approach [34].

Other obesity models involve genetically obese animals; these include the

ob/ob mice, which have an inherited lack of leptin, db/db mice, which have a

defective leptin receptor, and Zucker (fa/fa) rats, which lack the leptin recep-

222 F. Barth and M. Rinaldi-Carmona

tor. In both ob/ob and db/db obese mice with unrestricted access to food, acute

administration of SR-141716 (3 mg/kg i.p.) produced a significant reduction

of food intake [35]. In Zucker (fa/fa) rats, SR-141716 induced a transient

decrease in food intake and body-weight gain when administered orally for 4

weeks [36].

These encouraging results have stimulated the search for cannabinoid

antagonists. Both publications and patent literature suggest that new cannabi-

noid antagonists with chemical structures distinct from that of SR-141716 may

exhibit anti-obesity activity in animal models. The pyrazole derivative

SR-147778 (Fig. 1) was able to reduce sucrose consumption in mice and rats,

and food intake in fasted and non-deprived rats [37]. A new imidazole deriva-

tive acting as a potent cannabinoid CB1inverse agonist (Fig. 1, compound 1)

was recently shown to be effective in reducing body weight in DIO rats fol-

lowing 14 days treatment (3–10 mg/kg p.o.) [38] . Similar compounds are

claimed in a Bayer patent application which describes significant reduction in

food intake in normal rats and weight loss following chronic treatment in

Zucker obese rats [39].

Following the results obtained in animal models of obesity,

Sanofi-Synthelabo advanced SR-141716 (rimonabant) into clinical trials

involving obese patients. Rimonabant was tested in obese male subjects with

a body mass index (BMI = weight/height2) of >27 kg/m2in a double-blind

cross-over study (20 mg once a day versus placebo). The patients were treated

for 7 days with a 28-days wash out. Rimonabant had no effect on taste and spit

tests, yet induced a significant decrease in hunger (visual analogue scale),

caloric intake and weight [40]. Moreover, in a subsequent study involving 287

obese patients (BMI values of between 29 and 41 kg/m2) treated for 16 weeks

with either placebo or rimonabant (5, 10 and 20 mg once daily), rimonabant

was able to significantly reduce body weight as well as waist circumference.

In the 20-mg group the mean decrease in body weight was 3.8 kg (versus

0.9 kg in the placebo group) and the mean decrease in waist circumference was

3.9 cm (versus 1.1 cm in the placebo group). The observed decrease in weight

did not reach a plateau during the 4-month duration of the study and the toler-

ance of SR-141716 was excellent [41].

Rimonabant is currently being evaluated in multi-centre, randomized, dou-

ble-blind phase III studies in order to assess its efficacy and long-term safety.

The results of one of these studies, the RIO-Lipids trial, which enrolled 1036

overweight or obese patients (BMI values between 27 and 40 kg/m2) with

untreated dyslipidemia [high triglycerides and/or low high-density lipoprotein

(HDL)-cholesterol] were presented at the 2004 American College of

Cardiology annual meeting [42]. Patients were randomized to receive either a

daily, fixed dose of rimonabant (5 or 20 mg) or placebo along with a mild

hypocaloric diet for 1 year. Patients treated for 1 year with rimonabant

20 mg/day lost 8.6 kg (versus 2.3 kg in the placebo group). Over 72% of

patients treated for one year with rimonabant (20 mg/day) lost over 5% of their

body weight (versus 27.6% in the placebo group). Moreover, 44.3% of the

Cannabinoids in appetite and obesity 223

patients in the 20-mg rimonabant group lost more than 10% of their body

weight (versus 10.3% in the placebo group). In addition to weight loss, the

study was designed to assess a number of important associated cardiovascular

risk factors. Study findings for rimonabant 20 mg include:

• a waist-circumference reduction of 9.1 cm in patients treated for 1 year

(completers);

• an average increase in HDL-cholesterol (+23%) and a reduction in triglyc-

erides (–15%) in completers;

• reduction of C-reactive protein (CRP), an important inflammatory marker

predictive of cardiovascular risk (–27% versus –11% in the placebo group);

• and improved insulin sensitivity (oral glucose tolerance test) [42, 43].

These robust data were replicated in another phase III study (RIO-Europe)

involving 1507 obese patients with or without comorbidities [44].

Rimonabant, which was well tolerated, could therefore become an important

agent in the management of cardiovascular risk in obese patients.

Mechanism of action of cannabinoid ligands on food intake and energy

balance

The mechanisms that control food-intake and energy homeostasis in mammals

are particularly complex. Leptin and insulin, two hormones secreted by

adipocytes and the pancreas cells respectively, are key elements of this process.

Leptin and insulin are released from peripheral organs and interact with specif-

ic brain receptors in the hypothalamus in order to adapt food intake to body fat

mass. However, it would appear that more than 16 other hypothalamic neu-

ropeptides are involved in the precise control of energy homeostasis and act to

either stimulate or inhibit food intake [45]. Moreover, monoamine neurotrans-

mitters such as noradrenaline, serotonin and dopamine have also been shown to

affect food intake, with noradrenaline and serotonin uptake being the pharma-

cological mechanism of sibutramine, one of the only two prescription drugs

approved by the FDA for the treatment of obesity. How the endogenous and syn-

thetic cannabinoids interact with all these systems remains largely unknown.

The involvement of cannabinoid CB1receptors in both the agonist-induced

hyperphagia and antagonist-induced food-intake reduction has been demon-

strated by several authors.

The involvement of cannabinoid CB1receptors in food consumption

induced by anandamide and 2-AG was confirmed by its sensitivity to the spe-

cific CB1antagonist SR-141716, which reversed for example the hyperphagia

induced by anandamide [20] and 2-AG [23]. On the other hand, the CB2antag-

onist SR-144528 [46] did not affect the ∆9-THC-induced feeding in pre-satiat-

ed rats, excluding the participation of the CB2subtype in this effect [28]. The

involvement of CB1receptors in the food-intake reduction induced by the

224 F. Barth and M. Rinaldi-Carmona

antagonist SR-141716 was confirmed by using CB1receptor-knockout mice.

While SR-141716 significantly reduced food intake in wild-type mice, it was

totally ineffective in mice lacking the CB1receptor [35, 47]. Moreover, it was

shown that following temporary food restriction, the knockout mice eat less

than their wild-type littermates. These results suggest the existence of an

endogenous cannabinoid orexigenic tone which, when disrupted, may lead to

decreased food consumption.

Interaction between cannabinoids and hormones controlling energy

homeostasis

The link between cannabinoids and the anorexigenic hormone leptin was first

suggested by Di Marzo et al. in 2001 [35]. Acute leptin treatment of normal

mice and ob/ob mice (mutant mice lacking the leptin gene) not only decreased

food intake but also resulted in decreased levels of anandamide and 2-AG in

the hypothalamus. On the other hand, defective leptin signaling was associat-

ed with elevated hypothalamic but not cerebellar levels of endocannabinoids in

obese db/db and ob/ob mice and Zucker rats [35]. These findings suggest a

direct link between endogenous cannabinoids and the leptin-controlled energy

homeostasis. Moreover, interactions between the cannabinoid system and

other neuropeptides involved in the control of food intake have also been sug-

gested in the literature. A cross-talk between the orexin-1 and the CB1recep-

tors was recently described [48], and a synergistic interaction between CB1

and melanocortin MC4 systems in feeding behavior has also been reported

[49]. Moreover, the co-expression of hypothalamic CB1mRNA with corti-

cotropin-releasing hormone (CRH), cocaine-amphetamine-regulated transcript

(CART) and melanin-concentrating hormone (MCH) may also be indicative of

possible interactions between the cannabinoid system and these peptides.

Cannabinoids and motivational processes

Ingestive behaviors, however, are not only linked to energy homeostasis con-

trol, as the brain-reward system, a complex neural network activated by pleas-

urable stimuli also mediates the incentive or hedonic value of food. The asso-

ciation of the cannabinoid system with the motivational processes is indicated

by several lines of evidence. The preference for palatable sweet-food intake

induced by administration of cannabinoid agonists in both humans and labo-

ratory animals is most likely indicative of the involvement of an effect on the

brain-reward systems. Moreover, the CB1antagonist SR-141716 not only

selectively decreased the intake of sweet or palatable food, but also decreased

both alcohol [29, 32] and nicotine self-administration in rats [50]. It also

decreased heroin self-administration in rats [51] and was shown to reduce the

reinforcing value of the median forebrain bundle (MFB) electrical stimulation

Cannabinoids in appetite and obesity 225

[52]. In addition, cannabinoids also interact with known opioid reward path-

ways, as indicated by the synergistic action of cannabinoid and opioid antag-

onists on food intake [53, 54]. These results suggest that the central cannabi-

noid system may act to amplify certain motivation indices and that blockade

of its interaction with the reward system may contribute to the effects of CB1

antagonists on food intake.

Peripheral mechanisms and effects on metabolic processes

Recently, a third, purely peripheral, mechanism of action was also proposed by

several authors to explain some of the effects of SR-141716 on body weight. A

direct effect of SR-141716 on the activation of metabolic processes was pro-

posed by Ravinet-Trillou et al. [33]. This was supported by the fact that during

the course of a 5-week study using DIO mice the drug produced only a tran-

sient effect on food intake, yet a sustained effect on body weight. Moreover,

SR-141716-treated mice had an accelerated weight loss versus control during

a 24-h fast and the weight loss was increased in SR-141716-treated mice com-

pared with pair-fed animals [28]. Chronic treatment with SR-141716 also

reduced the respiratory quotient of obese Zucker rats from 0.9 to 0.7, a change

consistent with an increase in fat oxidation [55] and it was shown that meta-

bolic factors contribute to the lean phenotype in adult mice with a disrupted

CB1gene (CB1–/– mice) [56]. A direct action of SR-141716 on adipocytes may

be responsible for this peripheral effect, as CB1mRNA was detected in mouse

and rat adipocytes [56, 57]. Interestingly, SR-141716 was shown to increase

adiponectin mRNA expression in adipose tissue of obese fa/fa rats and in cul-

tured mouse adipocytes. Adiponectin (Acrp 30) is a plasma protein exclusive-

ly expressed and secreted by adipose tissue which has been shown to increase

fatty acid oxidation and improve insulin sensitivity and so reduce body weight.

This effect of SR-141716 was abolished in CB1-/- mice, indicating the involve-

ment of CB1receptor [57]. All these results clearly underline that cannabinoid

antagonists such as SR-141716 could exert a ‘peripheral’ metabolic action in

addition to their known ‘central’ effect on food intake.

Other recent studies demonstrated that sensory deafferentation which

destroyed the sensory terminals innervating the gut abolished both the hyper-

phagic effects of cannabinoid agonists and the hypophagic effect of

SR-141716. These results suggest that CB1receptors, located in the gastroin-

testinal tract, may also participate in the modulation of feeding induced by

cannabinoid agonists and antagonists [58].

Conclusion

Once considered as purely anecdotal, the effects of cannabinoids on appetite

are now the subject of intense interest in the scientific community. The natural

226 F. Barth and M. Rinaldi-Carmona

cannabinoid agonist ∆9-THC is indeed already used as a therapeutic agent to

treat anorexia and cachexia associated with severe illness such as AIDS or can-

cer. New synthetic CB1agonists may be of therapeutic value for this thera-

peutic application if they are able to associate higher potency with a reduction

of the side effects linked to the psychotropic effects of ∆9-THC.

On the other hand, the opposite, anorectic effect of cannabinoid antagonists

offers an important potential for the treatment of obesity, a condition which

has become a major health problem in developed countries. In this respect, the

results obtained in clinical studies with rimonabant (SR-141716), the first

cannabinoid antagonist, are extremely encouraging. Moreover, based on pre-

liminary pharmacological studies, CB1antagonists may also be useful for the

treatment of alcoholism, and in smoking cessation. Clinical studies with

rimonabant have already shown increased tobacco-smoking abstinence as well

as prevention of the secondary weight gain often associated with smoking ces-

sation [42, 59]. In any case, more research is needed to fully understand the

complex role of the cannabinoid system in the regulation of feeding and

body-weight control, a biological function which is particularly complex and

involves interactions with many different biochemical messengers.

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230 F. Barth and M. Rinaldi-Carmona

The development of Sativex®– a natural

cannabis-based medicine

Geoffrey W. Guy and Colin G. Stott

GW Pharmaceuticals plc, Porton Down Science Park, Salisbury, Wiltshire SP4 OJQ, UK

History of the development

Cannabis has been used medicinally for 4000 years [1–4] in a variety of cultures

and was re-introduced into British medicine in 1842 by W. O’Shaughnessy [5].

It remained in the British pharmacopaeia until 1932, when cannabis, extract of

cannabis and tincture of cannabis were among 400 medicines removed, though

all three remained in the British Pharmaceutical Codex of 1949 [5].

However, following the 1961 UN Single Convention on Narcotic Drugs,

cannabis and cannabis derivatives became scheduled products and were sub-

ject to special measures of control and parties could ban their use altogether.

Following the 1971 UN Convention on Psychotropic Substances, the UK

enacted the Misuse of Drugs Act 1971. Cannabinol and its derivatives, includ-

ing ∆9-tetrahydrocannabinol (∆9-THC), appeared in Schedule I to the

Convention, and their regular medical use was prohibited. The introduction of

the Misuse of Drugs Regulations in the UK in 1973 listed cannabis and

cannabis products in Schedule 4 (now Schedule I in current legislation), there-

by prohibiting medical use altogether [5].

Early research

Although the medicinal properties of cannabis had been well documented for

a number of years, the constituent(s) responsible for therapeutic efficacy had,

until recently, not been identified. The discovery, isolation (and subsequent

synthesis) of the principal cannabinoid present in cannabis, ∆9-THC, by

Raphael Mechoulam and Yehiel Gaoni in 1964 [6] ensured that interest in

cannabinoid chemistry remained and led to an expansion of cannabinoid

research.

Despite the scheduling and prohibition of cannabis and the ban on medical

use of cannabis-based products in the 1970s, research into the pharmacology

and toxicology of ∆9-THC continued through the 1970s and 1980s, mainly by

the National Institute of Health (NIH) in the USA.

Cannabinoids as Therapeutics

Edited by R. Mechoulam

231

However, much of the work concentrated solely on ∆9-THC (NTP program,

NIH) [7]. In many cases, the investigation of the pharmacokinetics of cannabis

components involved the delivery of smoked marijuana, and the measurement

of ∆9-THC levels and its primary metabolite, 11-hydroxy-tetrahydrocannabi-

nol (11-OH-THC).

Recent research and development of a cannabis-based medicine

In January 1997, the White House Office of National Drug Control Policy

(ONDCP) asked the Institute of Medicine (IOM) to conduct a review of the

scientific evidence to assess the potential health benefits and risks of marijua-

na and its constituent cannabinoids. That review began in August 1997 and

resulted in the report published in 1999 [8]. Reports were also published in

August 1997 by the US NIH [9] and in December 1997 by the American

Medical Association (AMA) [10].

In parallel with the timing of the IOM review, a number of expert bodies in

the UK were asked to review the medical and scientific evidence for and against

the use of cannabis as a medicine. The British Medical Association (BMA) pub-

lished a report on the topic in 1997 [11]. The UK Department of Health com-

missioned three literature reviews on cannabis, at the request of the Advisory

Council on the Misuse of Drugs (ACMD); and these were reviewed by the

House of Lords Select Committee on Science and Technology in 1998. The

authors of the report all gave evidence to the House of Lords inquiry [12–14].

Dr Geoffrey Guy was also invited to submit evidence to the House of Lords

enquiry, and subsequently GW Pharmaceuticals Ltd was founded in the UK in

early 1998. As GW’s Executive Chairman, Dr Guy successfully floated the

company (GW Pharmaceuticals plc) on the Alternative Investment Market

(AIM) of the UK Stock Exchange in June 2001. The first UK Home Office

licenses received by GW were to cultivate, possess and supply cannabis for

research purposes were received in June 1998 and cultivation began in August

1998.

In November 1998, the House of Lords Select Committee on Science and

Technology published its report Cannabis: The Scientific and Medical

Evidence [15], which recommended that clinical trials of cannabis medicines

should be carried out as a matter of urgency. The Committee warmly wel-

comed GW’s research programme.

September 1999 saw the start of GW’s first phase I clinical trials in healthy

volunteers and in March 2000 GW received authorization from the Medicines

Control Agency (MCA; now the Medicines and Healthcare Products

Regulatory Agency, MHRA) to start phase II clinical trials in patients.

In March 2001, the same House of Lords Select Committee published a fol-

low-up report, Therapeutic Uses of Cannabis [16], which confirmed the UK

Government’s intention to permit the prescription of cannabis-based medi-

cines (CBMs) subject to the approval of the MHRA.

232 G.W. Guy and C.G. Stott

GW entered into its pivotal phase III clinical trials programme in March

2001. The initial phase III studies involved patients with multiple sclerosis

(MS), neuropathic pain and cancer pain. The results of the first four phase III

studies were reported in November 2002, and six of the trials have now been

completed, yielding positive results, and a further three are due to report in 2005.

In March 2003 GW submitted an application to the MHRA for its first prod-

uct, Sativex®.

In May 2003 GW entered into an exclusive UK marketing agreement for

Sativex®with the German pharmaceutical company Bayer AG. This agree-

ment was extended in November 2003, to add the Canadian market.

In May 2004 GW submitted a New Drug Submission for Sativex®to the

Canadian regulatory authorities, Health Canada.

The endogenous cannabinoid system

The discovery and chemical synthesis of ∆9-THC initiated the modern era of

cannabis research because it enabled investigation of the effects and mode of

action of individual cannabinoids in laboratory models [17]. The production of

synthetic analogues of ∆9-THC enabled structure – activity relationships of

∆9-THC to be established. Further, pharmacological investigation of ∆9-THC

indicated that it might exert its effects by interacting with a specific receptor

protein in the brain [18, 19]. The conclusion from this work was that the

so-called cannabinoid receptor was a G-protein-coupled receptor. Once a CB

receptor agonist, CP-55,940, was synthesized, radiolabelled binding studies

were performed [20], and the distribution of CP-55,940-binding sites were

found to be similar to those coded for by cDNA for another G-protein-coupled

receptor, SKR6, a receptor without a known ligand (an orphan receptor).

Further investigation using cannabinoid-binding assays revealed that SKR6

was indeed a cannabinoid receptor identified in rat brain [21]. Soon afterwards

a human G-protein receptor was identified that had an amino acid sequence

98% identical to the SKR6 receptor in rat brain.

In 1993, a second G-protein-coupled cannabinoid receptor sequence (CX5)

was identified among cDNAs from the human promyelocytic leukaemic cell

line HL60 [22].

Munro et al. [22] suggested that the brain receptor be referred to as CB1and

that the second receptor, which is expressed by cells of the immune system, be

referred to as CB2.

It has since become widely accepted that CB1receptors are widely distrib-

uted but are particularly abundant in some areas of the brain, including those

concerned with movement and postural control, pain and sensory perception,

memory, cognition and emotion, and autonomic and endocrine functions [23,

24]. They are also prevalent in the gut, testes and uterus. The role of the sec-

ond type of receptor, CB2receptor, is still under investigation but it is believed

to mediate the immunological effects of cannabinoids [23, 24].

The development of Sativex®– a natural cannabis-based medicine 233

In the meantime, Mechoulam and Devane isolated and elucidated the struc-

ture of a brain constituent that bound to the cannabinoid receptor [25]: arachi-

donylethanolamide (AEA, anandamide). During subsequent investigation of

several lipid fractions collected from rat brain, it was discovered that the frac-

tions also contained materials that bound to cannabinoid receptors [26].

Characterization of these fractions revealed that some contained polyunsatu-

rated acid ethanolamides (similar to AEA), but others contained a distinct lipid

component, 2-arachidonoyl glycerol (2-AG).

AEA is found to be a partial agonist at CB1receptors; whereas 2-AG binds

to CB1and CB2with similar affinities, and is a full agonist at CB1. 2-AG occurs

in concentrations in the brain that are 170 times higher than those of AEA [26].

The role of these endogenous cannabinoids (so-called endocannabinoids) is

currently unclear, and others have subsequently been identified: noladin ether

[27], virodhamine [28], N-arachidonoyl-dopamine (NADA) [29] and arachi-

donoyl-serine (ARA-S) [30]. The identification of AEA and 2-AG has led to a

resurgence of interest in the field of cannabinoid medicine, especially within

the pharmaceutical industry, as they may represent potential molecular targets

for the treatment of a number of disorders.

Cannabinoid receptor ligands

In the wake of widespread availability of synthetic CB receptor-specific lig-

ands, research into the identification of potential sites of action of cannabi-

noids has increased around the world. However, until recently, the lack of sig-

nificant available quantities of pure cannabinoids other than ∆9-THC and

cannabidiol (CBD) has been a constant source of frustration for researchers.

To date, of the synthetic research receptor ligands, only SR-141716A (CB1

receptor antagonist) has shown sufficient potential to be developed into a phar-

maceutical product (Rimonabant). A number of other synthetic cannabinoids

have been developed into pharmaceuticals including Marinol®, Synhexyl,

Nabilone and Levonantradol. However, regulatory approval of these products

varies between territories and, as a result, they are not currently widely used or

accepted.

Classification of cannabinoids

The existence of the various types of cannabinoid molecule available and their

source has led to the proposal of four distinct classes of cannabinoids:

1. phytocannabinoids: those which occur naturally in the plant;

2. endocannabinoids: those that occur naturally in the body (AEA, 2-AG, etc.);

3. synthetic cannabinoids: cannabinomimetic compounds resulting from chem-

ical synthesis (e.g. dronabinol, nabilone, HU-210, CP-55,940, SR-141716A);

234 G.W. Guy and C.G. Stott

4. fatty acid amide hydrolase (FAAH) inhibitors: compounds that affect AEA

production, release, metabolism and re-uptake.

Production of cannabis-based medicines

Cannabis-based medicines may be produced according to the regulatory

requirements in a variety of ways:

• isolation and purification of individual molecules from plant sources;

• chemical synthesis of required molecular components;

• extraction of required plant components;

• selective delivery of required components.

Rationale for the development of a cannabis-based medicine as a

whole-plant extract

The cannabinoids that are currently of most interest and have received the most

scientific interest to date are the principal components of cannabis, ∆9-THC and

CBD. Both have important pharmacology [31, 32]. ∆9-THC has analgesic,

anti-spasmodic, anti-tremor, anti-inflammatory, appetite-stimulant and anti-

emetic properties; CBD has anti-inflammatory, anti-convulsant, anti-psychotic,

anti-oxidant, neuroprotective and immunomodulatory effects. CBD is not intox-

icating and indeed it has been postulated that the presence of CBD in cannabis

may alleviate some of the potentially unwanted side effects of ∆9-THC.

It is postulated that the beneficial therapeutic effects of cannabis result from

the interaction of different cannabinoids [31]. This may explain why

cannabis-based medicines made from whole-plant extracts may be more effec-

tive than single cannabinoid products, as the extracts consist of multiple

cannabinoids in defined, specific ratios. Different ratios of cannabinoids may

be effective in treating different diseases or conditions across a number of ther-

apeutic areas.

Although research has focused primarily on the two principal cannabinoids,

∆9-THC and CBD, it is possible that other components within the plant are also

important, which is why GW Pharmaceuticals’ medicines are made from

whole-plant extracts. McPartland and Russo [31] cite a number of literature

reports, which support this theory. Mechoulam et al. [33] suggested that other

compounds present in herbal cannabis might influence ∆9-THC activity. Carlini

et al. [34] determined that cannabis extracts produced effects “two or four times

greater than that expected from their THC content.” Similarly, Fairbairn and

Pickens [35] detected the presence of unidentified “powerful synergists” in

cannabis extracts causing 330% greater activity in mice than ∆9-THC alone.

Other compounds in cannabis may ameliorate the side effects of ∆9-THC

[31]. Whole cannabis causes fewer psychological side effects than synthetic

The development of Sativex®– a natural cannabis-based medicine 235

∆9-THC, seen as symptoms of dysphoria, depersonalization, anxiety, panic

reactions and paranoia [36].

It is possible that the observed difference in side-effect profiles may also be

due, in part, to differences in routes of administration: orally administered

∆9-THC undergoes ‘first-pass metabolism’ in the small intestine and liver, to

11-OH-THC; and the metabolite has been reported to be psychoactive, albeit

on the basis of limited evidence [37]. Inhaled ∆9-THC undergoes little

first-pass metabolism, so less 11-OH-THC is formed [38, 39]. The effect of the

route of administration on tolerability has been known for years. Walton, in

1938, remarked that “smoking cannabis is a satisfactory expedient in combat-

ing fatigue, headache and exhaustion, whereas the oral ingestion of cannabis

results chiefly in a narcotic effect which may cause serious alarm” [40].

The other classes of compounds present in cannabis also have their own

pharmacology (e.g. terpenoids, flavonoids) [31, 32]. The potential for interac-

tion and synergy between compounds within the plant may play a role in the

therapeutic potential of cannabis as a medicine. This may explain why a

cannabis-based medicine using extracts containing multiple cannabinoids, in

defined ratios, and other non-cannabinoid fractions, may provide better thera-

peutic success and be better tolerated than the single synthetic cannabinoid

medicines currently available.

CBD, as a non-psychoactive cannabinoid, is currently the cannabinoid of

considerable interest. CBD, along with ∆9-THC, has been demonstrated to

have a wide range of pharmacological activity, with the potential to be devel-

oped for a number of therapeutic areas [41]. It is likely that other cannabinoids,

present in small amounts in Cannabis sativa L., may also have interesting

pharmacological properties, for example tetrahydrocannabivarin (THC-V),

cannabichromene (CBC) and cannabigerol (CBG) [31, 32, 39].

Regulatory requirements

The pharmaceutical development of cannabis-based medicines is well docu-

mented [42, 43]. For cannabinoids to be made into pharmaceuticals, licensed

by the regulatory bodies around the world, they must reach strict requirements

laid down in terms of the product’s quality, safety and efficacy and increas-

ingly the healthcare industry requirement of cost-effectiveness. Such standards

are achieved by adhering to the industry and regulatory standards of Good

Laboratory Practice (GLP), Good Manufacturing Practice (GMP) and Good

Clinical Practice (GCP), according to the guidance documents provided by the

International Conference on Harmonisation [44]. All requirements are now

implemented through European Union and national legislation. In the case of

plant-based medicines they must also adhere to Good Agricultural Practice

(GAP) standards.

As a result, quality control is required throughout the whole of the manu-

facturing chain, including the production of raw materials. For pharmaceuti-

236 G.W. Guy and C.G. Stott

cals produced from plants, the regulatory authorities have produced their own

guidelines on the production of botanical drug products (BDPs) [45]. As

botanical pharmaceuticals have more than a single chemical entity present,

their control is paramount, and hence detailed characterization and specifica-

tion is required.

Breeding of cannabis plants for generation of cannabis extracts

Cannabis is in most cases a dioecious plant; that is to say, the species produces

separate male (staminate) and female (pistillate) plants [46].

Analysis of the various parts of the plant confirms that the major source of

cannabinoids is the female flower. Cannabinoids are not detected in the roots.

The richest sources of the principal cannabinoids ∆9-THC and CBD are the

leaves and flowers and hence these plant components are selected for the pro-

duction of ∆9-THC- and CBD-based medicines.

In the wild, Cannabis is a short-day-length plant. This means that the plant

grows vegetatively through the long days of summer. Only when the day

length falls, signalling the end of summer, does the female plant start to flower

and hence the cannabinoids are produced. As an annual herb in the field, nor-

mally only one crop per year would be produced.

It is during the last few weeks of life that the female plant is most active in

the production of cannabinoids and terpenes. The plant will produce variable

inflorescences, these being complex clusters of flowers and bracts. Each

flower consists of a furled specialized single leaf – the calyx – within which is

housed the ovary. Each calyx is covered in minute sticky organelles – the

stalked glandular trichomes. When viewed through a hand lens, each trichome

resembles a golf ball (the resin head, also known as the glandular head) sitting

on a tee (the trichome’s stalk; Fig. 1)

The particular day length that induces flowering is termed the ‘critical day

length’.This will differ according to the geographical and genetic origin of the

plant in question. Thus, flowering in response to exposure to a defined amount

of light may be achieved through selective breeding.

Cannabinoid content varies in different varieties but the high cannabinoid

content of modern varieties is purely due to plant breeding.

However, by growing under glass in controlled conditions, a succession of

crops can be planned to meet production requirements. To be suitable for

long-term commercial use, plants must have selected characteristics. Plants that

are selectively bred for their characteristics are termed chemovars. In order to

be commercially useful, they must possess the following characteristics:

• high rate of cannabinoid production;

• high yield of cannabinoid per unit area;

• high level of purity of the desired cannabinoid (purity as used here defines

the consistency of cannabinoid content as a ratio);

The development of Sativex®– a natural cannabis-based medicine 237

• high inflorescence-to-leaf ratio (the harvest index);

• natural resistance to pests and diseases;

• sturdy growth capable of bulk plant handling;

• ease of harvesting;

• minimal production of anthers on female plants.

The production of uniform high-quality botanical raw material (BRM) of

defined composition is dependent upon the bulk production of cloned plants;

that is to say, all plants are derived from cuttings taken from a few select moth-

er plants. Being genetically identical, all the cloned plants have the potential

to replicate exactly the characteristics of the mother plant.

BRM is obtained from distinct varieties of C. sativa plant hybrids to maxi-

mize the output of specific cannabinoids. The chemovars used are the result of

an extensive breeding programme spanning more than 15 years.

GW’s cannabis-based medicines are pharmaceutically formulated

whole-plant extracts of chemovars of C. sativa produced by selective breeding

to give a high content of defined cannabinoids, optimum habit and early flow-

ering. A wide range of chemovars of C. sativa has been selectively bred by

GW Pharmaceuticals. Each of these chemovars has a different cannabinoid

profile, and the chemovars have been specifically bred to produce the required

238 G.W. Guy and C.G. Stott

Figure 1. A glandular trichome from C. sativa L. (left) alongside a non-glandular trichome (right). The

head on the glandular trichome is the main site of cannabinoid biosynthesis.

level of specified cannabinoids. From this range, two separate chemovars, one

that produces ∆9-THC as the principal cannabinoid and one that produces

CBD as the principal cannabinoid, have been selected for production of

Sativex®.

Cultivation of chemovars for generation of cannabis extracts

Crops are produced from cuttings, which ensures that the genotype is fixed,

giving a constant ratio of cannabinoid content. Cannabinoid content may be

selectively bred to produce defined ratios of principal and other minor

cannabinoids. By further careful, selective breeding, it is possible to cultivate

chemovars which produce minor cannabinoids (CBC, CBG, THC-V, etc.) in

greater amounts than have been observed to date in wild-type cannabis plants

or in varieties produces by recreational growers. The pharmacology of the

minor cannabinoids has yet to be clearly established, but may yet provide a

whole new range of therapeutic options for both patient and clinician.

Mother plants

Potter [46] has described the use of “mother plants” to maintain the genotype

for each subsequent generation of plants (rooted cuttings, termed “clones”).

Once potted up and grown in continuous bright light [75 W/m2PAR (photo-

synthetically active radiation)] at 25 °C in optimized compost, a rooted cutting

will reach a height of 2 m in 12 weeks. This plant is then capable of being

heavily pruned; the removed branches being cut up to produce up to 80 cut-

tings per mother plant. If well kept, over the next 10–15 weeks the trimmed

mother plant will regrow to produce at least two more flushes of cuttings. The

vigour of the mother plant then wanes, and the plant is destroyed to make way

for younger mothers.

Clones

Branches of the mother plant are removed where there are sufficient numbers

of axial buds developing, these being the new growths that eventually develop

into mature plants. Each branch is then cut into sections, each supporting only

one axial bud. The cutting is then placed in rooting powder and immediately

transferred into a very moist peat plug. In the correct environment, roots begin

to appear after 7 days, and the cuttings allowed to acclimatize to their sur-

roundings before they are potted up.

Rooted cuttings are transferred into large pots, filled with a proprietary

growing media, which contains sufficient fertilizer to stimulate vegetative

growth and flower production.

The development of Sativex®– a natural cannabis-based medicine 239

For the first 3 weeks after potting, plants are grown in continuous bright

light. With no night-time breaks during this period the plant grows to around

50 cm and establishes a healthy root system.

After 3 weeks the lighting is switched to a 12-h light/12-h dark cycle.

Having established themselves in a 24-h daylight environment in subtropical

temperatures, the plants suddenly detect the change in light exposure, as if they

had experienced the immediate arrival of the autumn equinox. For a short-day

plant (i.e. late summer/autumn flowering) like cannabis, the response is dra-

matic. The GW chemovars flower within 5 days of the photoperiod switch.

The inflorescences (flowers) increase in size over the next 6 weeks, becoming

white with myriad receptive stigmas. The unfertilized stigmas then start to

senesce to an orange/brown colour. After 8 weeks in flower, the bulk of stig-

mas have senesced and the rate of cannabinoid biosynthesis in the selected

varieties slows rapidly. At this point, the crop is harvested.

Mother plants, seedlings and mature clones are produced under glass, which

allows a very high degree of control of growing conditions to be exercised. The

controls significantly exceed the controls possible for field-grown crops. In

particular:

• proprietary compost is used, warranted free of artificial pesticides and her-

bicides by the supplier;

• the compost contains sufficient fertilizer to ensure optimum vegetative

growth and eventual flowering;

• stringent hygiene conditions reduce ingressive pests and diseases – adven-

titious infestation is controlled biologically with predatory mites;

• fresh potable water, rather than stored or untreated water, is used for the

irrigation of the plants; this reduces the potential for contamination with

water-borne organisms;

• during growing, the plants are inspected regularly, and plants showing male

characteristics are removed to avoid fertilization of plants;

• growing conditions are strictly controlled via computer technology to

ensure that optimal cultivation conditions are maintained at all times in

terms of light, temperature, humidity, airflow, etc.

Drying

At harvest, the entire plant is cut and dried in a temperature- and humidity-con-

trolled environment until it meets the specification for loss on drying. Leaves

and flowers are stripped from the larger stems to provide the BRM, which is

stored in suitable containers protected from light under controlled conditions.

Drying the crop as quickly as possible reduces the cannabinoid losses, and

this is achieved by keeping the plants in a stream of dehumidified air. Plants

are crisp to the touch in less than 7 days.

240 G.W. Guy and C.G. Stott

As part of GAP and GMP, the BRM must conform to a specification. The

specification for BRM includes tests for identification, extraneous matter and

identification and assay for cannabinoids and cannabinoic acids, confirmatory

thin-layer chromatography (TLC) and loss on drying. Additionally, BRM is

tested for aflatoxins and microbial bioburden. The growing parameters

employed have been selected to minimize the conditions that would be expect-

ed to result in microbial and fungal spoilage.

Extraction

Cannabinoids are present in the plant as the corresponding carboxylic acid and

it is necessary to decarboxylate material before extraction. The conditions for

efficient decarboxylation have been optimized to maximize decarboxylation

and minimize oxidation. The process is time- and temperature-dependent and

a criterion of not less than 95% efficiency was adopted for BRM used in sub-

sequent manufacture of botanical drug substance (BDS; whole-plant extract).

Development work has shown that efficient extraction can be carried out

using patented extraction technology. The conditions of the extraction have

been carefully assessed during development and are essential to ensure the

optimum conditions and hence the correct composition of the extract pro-

duced. The extraction produces a whole-plant extract, from which the BDS is

prepared.

The whole-plant extract is subject to further processing (covered by intel-

lectual proprietary rights) to remove unwanted materials from the extract. The

exact content of the BDS is defined by a specific BDS specification. BDS is

transferred to sealed, stainless steel containers and stored at –20 ± 5 °C to

maintain stability.

A schematic diagram of the process flow from cultivation to final process-

ing and quality-control release of the pharmaceutical product is detailed in

Figure 2.

BDS content

Using any defined BRM, a corresponding BDS may be created using the above

GW proprietary process. The contents of the BDS will depend on the geneti-

cally defined content of the BRM, and the technology used to extract the active

constituents. Thus, BDSs may be produced which have defined levels of prin-

cipal cannabinoids, other cannabinoids and other non-cannabinoid con-

stituents. Thus a series of individual BDSs may be described.

Each BDS contains a cannabinoid fraction and a non-cannabinoid fraction.

GW describes its BDSs individually as each BDS generated has a unique com-

position. The two BDSs used to generate Sativex®are Tetranabinex®,an

extract of a chemically and genetically characterized cannabis plant, contain-

The development of Sativex®– a natural cannabis-based medicine 241

ing ∆9-THC as the principal cannabinoid, and Nabidiolex®, an extract of a

chemically and genetically characterized cannabis plant containing CBD as

the principal cannabinoid. Other BDSs may be generated from extracts high in

CBC, CBG, THC-V, cannabidivarin (CBD-V), etc.

Cannabinoid fraction

In addition to the principal cannabinoids present, each BDS contains other

cannabinoids that may contribute to the activity of the whole extract.

Non-cannabinoid fraction

Each BDS also contains a non-cannabinoid fraction, which contains terpenes,

sterols, fatty acids, anti-oxidants and flavonoids.

242 G.W. Guy and C.G. Stott

Figure 2. Schematic flow diagram of the production of GW Pharmaceuticals’ BDSs. CBDA,

cannabidiolic acid; QC, quality control; THCA, tetrahydrocannabinolic acid.

Characterization, control and specification of BDS

The ranges for the principal cannabinoids and other cannabinoids are defined

in the BDS specification, as are the levels of non-cannabinoid compounds. The

minor cannabinoids and non-cannabinoids are considered to be adjuvants to

the principal cannabinoid rather than impurities. The non-cannabinoid fraction

may be regarded as a diluent, rather than an impurity, making up the difference

between assayed percentage of cannabinoids and 100% of the extract.

For regulatory approval, tight control of the content of the BRM, BDS and

BDP is essential. Even though the pharmaceutical product is a botanical prod-

uct, rather than a new chemical entity, characterization of more than 90% of

the composition of the whole extract is required. GW has achieved this.

Stability

Stability studies are ongoing to assess the stability of Tetranabinex®,

Nabidiolex®and the finished product Sativex®in order to establish a suitable

shelf-life for the product. Such studies include temperature cycling and photo-

stability, in compliance with international regulatory (International

Conference on Harmonisation) conditions. Additionally, studies are being per-

formed to investigate forced degradation.

Profile of a BDS

Typically, a GW Pharmaceuticals BDS contains the following.

• Principal cannabinoids

∆9-THC (>90% of the cannabinoid fraction in THC BDS)

CBD (>85% of the cannabinoid fraction in CBD BDS)

• Minor cannabinoids

Cannabichromene (CBC)

Cannabigerol (CBG)

Cannabinol (CBN)

Tetrahydrocannabivarin (THC-V)

Cannabidivarin (CBD-V)

Tetrahydrocannabinolic acid (THCA)

Cannabidiolic acid (CBDA)

Cannabicyclol (CBL)

Cannabitriol (CBO)

Cannabielsoin (CBE)

Cannabichromivarin (CBC-V)

• Terpenes

Monoterpenes: myrcene, limonene, linalool, α-pinene

Sequiterpenoids: trans-caryophyllene, α-caryophyllene, caryophyllene

The development of Sativex®– a natural cannabis-based medicine 243

oxide, cis-nerolidol, trans-nerolidol

Diterpenoids: phytol

Triterpenoids: squalene

• Fatty acids

Linolenic acid, palmitoleic acid, linoleic acid, palmitic acid, oleic acid,

stearic acid, myristic acid, arachidic acid and behenic acid

• Sterols

β-Sitosterol, Campesterol and Stigmasterol

• Carotenoids

β-Carotene, lutein

• Chlorophylls and related compounds

Phaeophytin

• Vitamins

Vitamin E

• Phenolic compounds

Flavonoids, coumarins, cinnamic acids and psoralens

Finished product – BDP: formulation and filling

The dosage form for Sativex®is a solution, consisting of a vehicle of ethanol,

propylene glycol and peppermint, containing Tetranabinex®and Nabidiolex®

extracts, that is sprayed into the oral cavity, on to the oromucosal surface.

Sativex®contains Tetranabinex®and Nabidiolex®extracts of C. sativa

equivalent to 27 mg/ml ∆9-THC and 25 mg/ml CBD per actuation. The con-

tainer is an amber Type I glass vial, with a sealed pump, designed to deliver a

uniform 100 µl volume. An actuator is used to produce the spray (Fig. 3).

Administration of Sativex®: achieving the therapeutic window

Appropriate delivery of the active components of a cannabis-based medicine

is important in terms of patient acceptability, and achieving optimal and pre-

dictable effect. The rate of delivery of constituents to the site of action is as

important as the amount delivered. Hence, the formulation selected to deliver

cannabinoids is very important. The fact that cannabinoids are extremely

lipophilic compounds limits the number of excipients that may be used to for-

mulate cannabis-based medicines.

Sativex®is self-titrated by patients. Its frequency of use is determined by

the type, severity and frequency of symptoms that patients endure. As patients

vary enormously in terms of the symptoms they exhibit upon presentation to

their physician, the administration of Sativex®is unique in each individual

patient.

The ability of Sativex®to relieve a variety of single primary symptoms

across different patient populations, coupled with its ability to relieve ‘clus-

244 G.W. Guy and C.G. Stott

ters’ of symptoms in individual patients as reported in GW’s clinical pro-

gramme, demonstrates the real strength and potential of Sativex®as a medi-

cine. These beneficial effects are not only due to the pharmacological actions

of the medicine but also due to the flexibility of dosing that the medicine

offers. It accommodates inter-individual variation, but also allows each patient

to establish a dose regimen that provides patient benefits with minimal

unwanted side effects. It allows patients the opportunity to develop their own

dosing regimen, including dosing interval and acceptable dose range, and also

enables them to assess the time course of symptom relief, using their own per-

sonal endpoints as markers of efficacy and tolerability. In this way, the patient

is able to optimize the relief of their symptoms, while minimising and resolv-

ing the occurrence of any side effects that they may experience (i.e. patiens can

target the therapeutic window).

By utilizing this approach, a number of significant clinical benefits of

Sativex®have been reported in GW’s clinical trial programme.

Clinical effects of Sativex®

The clinical effects of Sativex®have undergone investigation in an interna-

tional clinical trials programme, with centres in UK, Romania, Belgium,

The development of Sativex®– a natural cannabis-based medicine 245

Figure 3. Administration of Sativex®.

Ireland and Canada. More than 1400 subjects have participated in the clinical

programme, which has initially targeted MS patients who have symptoms, and

patients with neuropathic pain.

A summary of the programme is presented in Table 1. A total of 13 phase I

studies have been undertaken to investigate the pharmacokinetics of Sativex®

246 G.W. Guy and C.G. Stott

Table 1. GW clinical programme, wave 1

Study number Study population Number Study

enrolled status

Acute studies

Phase II studies

GWN19901A Various symptoms in MS and SCI#34 C

GWN19902 Various symptoms in MS#25 C

GWN19904 Various symptoms in MS, RA and SCI#29 C

GWCRI016 Pain and stiffness caused by RA*58 C

GWQSCBME01 Bladder dysfunction in MS†21 C

Phase III studies

GWMS0106 Spasticity in MS*189 C

GWNP0101 Peripheral neuropathic pain characterized by 125 C

allodynia*

GWMS0001 Multiple symptoms in MS*160 C

GWPS0105 Chronic refractory pain in MS and other 70 C

defects of neurological function*

GWMS0107 Neuropathic pain in MS*66 C

GWBP0101 Pain in brachial plexus avulsion#48 C

GWCA0101 Cancer pain*176 O

GWMS0208 Bladder dysfunction in MS*130‡O

GWSC0101 Neuropathic pain in SCI*120‡O

Long-term extension studies

Phase II

GWN19901A Various symptoms in MS and SCI#29 O

GWN19902 Various symptoms in MS#20 O

GWN19904 Various symptoms in MS, RA and SCI#22 O

GWCRI016 Pain and stiffness caused by RA*35 O

GWQSCBME01 Bladder dysfunction in MS†16 C

Phase III

GWEXT0101 Cancer pain†40 O

GWMS0001 EXT Multiple symptoms in MS†137 O

GWEXT0102 Neuropathic pain and bladder dysfunction in MS†494 O

C, complete; O, ongoing; MS, multiple sclerosis; SCI, spinal cord injury; RA, rheumatoid arthritis.

#Randomized, double-blind, placebo-controlled crossover study.

*Randomized, double-blind, placebo-controlled parallel group study.

†Open-label study.

‡Target recruitment figure.

and other formulations/products of GW’s portfolio. To date, the results from

three pharmacokinetic studies have been published [47–49].

Clinical programme results

Of the 11 efficacy studies completed to date (five phase II; six phase III), all

11 have yielded a range of positive results [50–60]. An additional three phase

III trials commenced in 2002 and are due to complete in 2005.

In all studies all patients remained on the best current therapy available for

their condition. However, they still had sufficient residual symptom-severity

scores for them to seek further treatment (i.e. there was still a high clinical

unmet need despite best available therapy). Sativex®was added to all their

other medications, which were kept stable during the baseline/run-in periods

and throughout the study period. The subsequent improvement in symptoms

that was observed following treatment with Sativex®was in addition to any

benefit they had previously derived from their existing therapy.

Phase II data

In phase II studies the following effects were seen:

• relief of neuropathic pain [50];

• improvement in spasticity [51, 52];

• improvement in muscle spasms [51, 53];

• improvement in bladder-related symptoms [52];

• improvement in sleep, mood and overall sense of well-being [50–52];

• improvement in morning pain in rheumatoid arthritis [54];

• opiate sparing effects

Phase III data

In randomized, double-blind, placebo controlled, phase III studies the follow-

ing effects were seen:

• relief of central neuropathic pain (CNP) in MS [55] (see Fig. 4);

• relief of CNP in brachial plexus avulsion [56] (see Fig. 5);

• relief of chronic refractory pain of neurological origin [57];

• relief of spasticity in MS [58, 59] (see Figs 6 and 7);

• relief of peripheral neuropathic pain [60];

• relief of relief of sleep disturbance and improvement in sleep quality

[55–58, 60] (see Fig. 8);

• improvement in patients quality of life [55, 60].

The development of Sativex®– a natural cannabis-based medicine 247

Figures 4–8 present the primary efficacy data for Sativex®, from a number

of the randomized, double-blind, placebo-controlled, phase III clinical studies

conducted and presented to date.

Figure 9 presents the long-term data from patients who have reported pain

as a symptom. The results encompass data from patients with a variety of pain

syndromes who have completed the randomized studies and have elected to

continue on the medicine long-term.

248 G.W. Guy and C.G. Stott

Figure 4. Relief of central neuropathic pain in MS [55]. BS11, Box Scale 11. Adapted from Rog and

Young [55].

Figure 5. Relief of neuropathic pain in brachial plexus avulsion [56]. BS11, Box Scale 11. Adapted

from Berman et al. [56].

Neuropathic pain in MS

Sativex®has been investigated for its effects on neuropathic pain from a vari-

ety of aetiologies. A study evaluating its effects in CNP in MS was undertak-

en in 2002 [55]. Following a baseline period during which their pain scores

were assessed, 66 patients with CNP were randomized to receive either

Sativex®or placebo for 4 weeks. The primary endpoint of the study was pain

scores as measured on a patient diary card using an 11-point Numerical Rating

The development of Sativex®– a natural cannabis-based medicine 249

Figure 6. Relief of spasticity in MS (clinic assessments) [58]. Placebo was crossed over to Sativex®

in weeks 7–10. Adapted from Wade et al. [58].

Figure 7. Relief of spasticity in MS (diary cards) [58]. Placebo crossed over to Sativex®in weeks

7–10. Adapted from Wade et al. [58].

Scale (range 0–10). A summary of the results is given below, and the primary

endpoint presented in Figure 4.

Sixty-four patients (96.9%) completed the trial. Fourteen patients were

male, mean age 49.2 years (range 26.9–71.4, SD 8.3), mean expanded dis-

ability status scale (EDSS) 5.9 (range 2.0–8.5, SD 1.3) and mean duration of

MS since diagnosis 11.5 years (range 1–36, SD 7.7).

The mean number of daily sprays taken in the final week of treatment was 9.6

of Sativex®(range 2–25, SD 6.1) and 19.1 of placebo (range 1–47, SD 12.9).

Thirty patients (88.2%) on Sativex®and 22 (68.8%) on placebo had at least

one adverse event, none of which were serious. There was a statistically sig-

nificant mean reduction in pain in favour of Sativex®, as measured using the

250 G.W. Guy and C.G. Stott

Figure 9. Sustained relief of neuropathic pain [64]

Figure 8. Relief of sleep disturbance [50, 51, 55–58, 60]

11-point numerical rating scale (NRS; 0 = none, 10 = worst), which was the

primary outcome of the study [–1.25; 95% confidence interval (CI), –2.11,

–0.39; p= 0.005].

There was a statistically significant improvement in mean sleep disturbance

in favour of Sativex®(–1.39; 95% CI, –2.27, –0.50; p= 0.003). A significant

mean reduction in pain with Sativex®compared with placebo was also demon-

strated using the 10-item, 100-point neuropathic pain scale (–6.82; 95% CI,

–13.28, –0.37; p= 0.039). On a seven-point Patient’s Global Impression of

Change (PGIC), those treated with cannabis-based medicine extracts were 3.9

times more likely (95% CI, 1.51, 10.06; p= 0.005) to feel “much” or “very

much” improved than those receiving placebo, and no patient felt “much” or

“very much” worse at the end of either treatment. No significant mean differ-

ences were found between treatment groups prior to treatment.

Neuropathic pain in brachial plexus avulsion

A further study evaluating the effects of Sativex®on CNP was undertaken in

patients with brachial plexus avulsion [56]. Brachial plexus avulsion is a rela-

tively uncommon condition but is characterized by severe, intractable neuro-

pathic pain, which is difficult to treat. Due to the low numbers of patients avail-

able, even at the national treatment centre in the UK, the study was performed

as a crossover study rather than to a parallel group design.

Following a baseline period during which their pain scores were assessed,

48 patients with brachial plexus avulsion were randomized to receive Sativex®,

a formulated ∆9-THC-rich extract (formulated Tetranabinex®), or placebo,

each for a period of 2 weeks. The primary endpoint of the study was pain

scores as measured on a patient diary card using an 11-point NRS (range

0–10). A summary of the results is given below, and the primary endpoint pre-

sented in Figure 5.

Forty-eight patients were enrolled. They all had at least one brachial plexus

root avulsion for at least 18 months. They also had pain of at least 4 on an

11-point NRS at the time of enrolment. The study was a randomized, dou-

ble-blind, crossover design consisting of three 2-week periods following a

run-in period of 7–24 days. Patients continued on all previous stable medica-

tions including analgesics. During each 2-week period subjects received, in

random order, either placebo, formulated Tetranabinex®or Sativex®. These

were given as patient-activated oromucosal 100 µl sprays.

Efficacy endpoints were: 11 point NRSs for pain and sleep, short-form

McGill (McGill Pain Questionnaire), General Health Questionnaire-12

(GHQ-12) and sleep quality and sleep disturbance were all recorded.

The mean number of daily sprays taken in the final week of treatment was

6.93 for Sativex®(range 1.1–22.2, SD 4.79), 7.26 for Tetranabinex®(range

1.2–21.6, SD 5.04) and 9.15 for placebo (range 2.0–35.6, SD 7.30). The

results for the efficacy endpoints are shown in Table 2.

The development of Sativex®– a natural cannabis-based medicine 251

These two studies [55, 56] and a third reported by Sharief [57] demonstrate

that Sativex®has a significant analgesic effect in CNP. A further study yet to

be fully reported also demonstrated a significant improvement in peripheral

neuropathic pain characterized by allodynia [60]. These results are consistent

with a recent report of dronabinol being effective in CNP in MS [61].

Symptoms of MS

In addition to reports of Sativex®being effective in the treatment of neuropath-

ic pain, early studies indicated that it had a broad spectrum of activity across a

variety of other symptoms in MS such as spasm, spasticity and bladder dys-

function [51–53]. In order to test the breadth of effect of the medicine, a study

was undertaken evaluating a range of nominated primary symptoms in MS [58].

Patients chose one of five symptoms (pain, spasm spasticity, tremor or blad-

der dysfunction) as their nominated primary symptom. Despite their existing

treatment prior to study entry, patients were required to have a symptom sever-

ity rated as >50 mm on a 100-mm VAS scale in order to be eligible. Other sec-

ondary impairments/symptoms (if present) were also monitored during the

study.

A total of 160 patients entered a baseline period (14 days maximum); fol-

lowed by a 6-week randomized, double-blind, placebo-controlled

parallel-group comparison of Sativex®with placebo. Patients self-titrated to

symptom resolution or maximum tolerated dose. Existing medication contin-

ued at a constant dose.

Primary efficacy comparisons were made between symptom scores record-

ed during baseline and scores recorded at the end of the 6-week parallel group

period.

Patients then entered weeks 7–10 and all patients were re-titrated on to

Sativex®and received open-label treatment for 4 weeks.

252 G.W. Guy and C.G. Stott

Table 2. Study GWBP0101 efficacy results

Formulated

Baseline Placebo Tetranabinex®Sativex®

Pain NRS Score 6.7 6.7 6.1 (P= 0.002) 6.1 (P= 0.005)

McGill Pain Questionnaire

(total intensity) 17.3 15.5 13.4 (P= 0.04) 13.8 (P= 0.15)

McGill (Part II)

Pain-intensity VAS score 60.9 52.9 43.6 (P= 0.04) 45.1 (P= 0.09)

Sleep-quality NRS 4.8 5.2 6.0 (P= 0.001) 5.9 (P= 0.02)

GHQ-12 13.4 13.5 12.3 (P= 0.18) 10.9 (P= 0.02)

VAS, Visual Analogue Scale; GHQ-12, General Health Questionnaire-12.

The results of the study are presented below and the outcome on the symp-

tom of spasticity is presented in Figures 6 and 7.

Thirty-nine patients (n= 19 for Sativex®,n= 18 for the placebo) who nom-

inated spasticity as their primary impairment showed a statistically significant

improvement in their spasticity VAS scores as assessed at either their clinic

visits or as recorded on their daily diary cards.

When the changes in each of the clinic visit spasticity VAS scores (in

patients with spasticity as a primary impairment) were analysed, there was a

highly statistically significant treatment difference of 22.79 mm in spasticity

in favour of Sativex®(P= 0.001).

When the changes in each of the diary card spasticity VAS scores (in

patients with spasticity as a primary impairment) were analysed, there was a

highly statistically significant treatment difference of 18.41 mm in spasticity

in favour of Sativex®(P= 0.009).

Effect on sleep

The most consistent endpoint in terms of response to Sativex®(measured in all

GW studies except GWMS0106) has been the improvement in sleep quali-

ty/sleep disturbance reported by patients which chronic symptoms, irrespec-

tive of the aetiology. Patients with chronic refractory pain of neurological ori-

gin, CNP (from conditions such as MS and brachial plexus avulsion), periph-

eral neuropathic pain, and other symptoms of MS such as spasm, spasticity

and bladder dysfunction have all reported statistically significant improve-

ments in sleep (Fig. 8).

It is well accepted that sleep quality has a major impact on the quality of life

of patients with chronic conditions. In the above clinical studies, Sativex®has

not only produced statistically and clinically significant improvements in the

patients primary symptoms, but also the ability to gain rest as a result of the

relief of those symptoms. On average across the studies Sativex®has produced

a 40% improvement in sleep quality/disturbance.

However, the effect of Sativex®on sleep is not due to a direct hypnotic

effect of the medicine. The effect of Sativex®on the sleep process was inves-

tigated in a sleep laboratory study [62].

Nicholson et al. have reported the effects of Sativex®and formulated

Tetranabinex®on nocturnal sleep and early-morning behaviour in young adults

[62]. The effects of the medicines on nocturnal sleep, early-morning perform-

ance, memory and sleepiness were studied in eight healthy volunteers.

The study was double-blind and placebo-controlled with a four-way

crossover design. The four treatments were placebo, Sativex®(six sprays,

delivering a total dose of 15 mg of ∆9-THC and 15 mg of CBD), formulated

Tetranabinex®(six sprays, delivering a 15 mg dose of ∆9-THC), and a

“low-dose” Sativex®formulation (six sprays delivering a total dose of 5 mg of

∆9-THC and 5 mg of CBD; i.e. identical to Sativex®formulation, but one-third

The development of Sativex®– a natural cannabis-based medicine 253

of the potency). Electroencephalogram (EEG) recordings made during the

sleep period (11:00 PM to 7:00 AM). Performance, sleep latency and subjective

assessments of sleepiness and mood were measured from 8:30 AM (10 h after

drug administration).

There were no effects of 15 mg of ∆9-THC (Tetranabinex®) on nocturnal

sleep. Low-dose Sativex®(5 mg of ∆9-THC and 5 mg of CBD) and Sativex®

(15 mg of ∆9-THC and 15 mg of CBD), produced a decrease in stage 3 sleep,

but interestingly with Sativex®(15 mg) wakefulness was increased.

The next day, with Tetranabinex®(15 mg of ∆9-THC), memory was

impaired, sleep latency was reduced and the subjects reported increased sleepi-

ness and changes in mood. However, interestingly, when 15 mg of CBD was

added to the 15 mg of ∆9-THC (i.e. following administration of 15 mg of

Sativex®) there was no observed effect on daytime sleep latency and memory.

From this study, at the doses investigated, it appears that ∆9-THC appears to

have sedative properties, while CBD (present in Sativex®) appears to have alert-

ing properties as it increased awake activity during sleep of patients taking

Sativex®and counteracted the residual sedative activity of 15 mg of ∆9-THC.

Thus Sativex®appears to promote sleep without changing the sleep archi-

tecture, but minimizes the residual effects that may be present if a ∆9-THC-rich

medicine (without the presence of CBD) is used.

What do patients want?

In a number of GW’s clinical studies, patients have reported good overall

improvement with Sativex®, as measured using the PGIC. Even small changes

in symptom relief appear to be important to the patients, with a subset of the

patients gaining large and sustained responses (e.g. ≥50% improvement from

baseline).

This is reflected in reports from a number of patient groups. In the MS

Society’s (the UK’s largest charity for people affected by MS) submission to

the UK’s National Institute for Clinical Excellence (NICE), the importance of

small improvements in symptoms and sleep quality has been emphasized.

For example, the following quotes were included in their submission:

“If cannabinoid-based medicines provide even minor symptom relief

they could still have a major impact on people’s quality of life and boost

their self esteem.”

“An ideal treatment for spasticity would be short-acting so that it could

reduce nocturnal spasms and aid sleep, but not compromise functioning

during the daytime. Many of the existing treatments have long-term

effects. Cannabinoid-based medicines have the advantage that they are

short acting – they could therefore allow much better control of symp-

toms.”

254 G.W. Guy and C.G. Stott

“Q: What is it like to have MS?

Person with MS: Get somebody to stay awake for 48 hours, make them

drink loads of coffee so they just can’t sleep, put weights on their

ankles, a pack on their back, make them wear two lots of rubber gloves,

the whole thing. Tell them it’s for the rest of their life, because that’s the

most important thing.”

“…Others obtained pain relief or found that the drug (cannabis) simply

helped them to sleep. Sleepless nights caused by spasms and nocturia

can make the extreme fatigue in MS even worse. The importance of a

good night’s sleep cannot be overestimated. It has a major impact on

Quality of Life.”

Long-term data

The majority of patients (>70%) who participated in the GW randomized stud-

ies elected to receive the drug in long-term, open-label extension studies (>750

patients) [63, 64]. Efficacy with respect to a variety of symptoms has been

maintained over an extended period of time (>1 year). To date, more than 200

patients have remained on treatment for more than 1 year, and a significant

number have remained on treatment for more than 2 years (the maximum is

814 days as of November 2003), with no evidence of tolerance developing.

Dosing has remained steady over the same period, and only minimal levels of

intoxication have been reported using a 0–100 mm VAS scale (scores up to a

maximum of approximately 20 upon initial exposure, diminishing over time).

This, coupled with the low number of serious adverse reactions reported,

demonstrates the tolerability of the product.

The effects observed in the randomized clinical studies have been sustained

over the long-term (Fig. 9).

At baseline, patients in the randomized, placebo-controlled phase had the

following NRS scores: brachial plexus injury, 6.8; neuropathic pain in MS,

6.5; peripheral neuropathic pain, 7.2; spinal cord injury (data not available,

study ongoing).

Safety

GW has now generated more than 800 patient-years of exposure to Sativex®

since the year 2000. By June 2004, more than 200 patients had been exposed

to Sativex®for at least 1 year.

The most common adverse events reported during clinical studies were gen-

erally non-serious in nature and are mainly due to application site reactions

(oral pain, dry mouth, oral mucosal disorder, tooth discolouration, mouth ulcer-

ation, oral discomfort, application-site pain, dysgeusia) or intoxication-like

The development of Sativex®– a natural cannabis-based medicine 255

reactions (fatigue, feeling drunk, lethargy, dizziness, somnolence, disturbance

in attention, memory impairment, euphoric mood, disorientation).

Other common adverse events reported were nausea, vomiting, diarrhoea,

constipation, dyspepsia, weakness and headache.

Intoxication

The long-standing concern regarding the development of cannabis-based

medicines has been the psychoactivity of ∆9-THC. Until now, this has been

perceived as a major barrier to the safety and tolerability of such medicines.

To date, patients have often reported that they are often unable to tolerate the

synthetic cannabinoid medications currently available to them due to their

side effects. The main concern for many patients regarding the use of

cannabis-based medicines is the symptom of intoxication. Patients do not

wish to get high and actively seek to avoid this as it interferes with their daily

life, which in many cases has already been compromised by their symptoms

and/or underlying condition. This is not a situation that is unique to cannabi-

noid medicines, as many other classes of licensed pharmaceuticals may pro-

duce intoxication-like effects (e.g. opioids, benzodiazepines, tricyclic antide-

pressants, etc.). Indeed, many patients suitable for treatment with

cannabis-based medicines are already experiencing polypharmacy with such

products.

The range of intoxication like reactions reported by patients taking Sativex®

in clinical trials has consistently been reported [50–60, 63, 64]. Safety data

have been collected in randomized, double-blind studies and in long-term

open-label extension studies. Safety data from more than 500 patients in the

long-term extension studies are now available, where patients were allowed to

take up to 48 sprays per day (maximum ∆9-THC dose = 130 mg/day). The

most common intoxication like reaction reported is dizziness, reported initial-

ly in approx. 35% of patients. However, this includes patients who are new to

the medication and are titrating their initial dose. In long-term use the inci-

dence of such an event is approximately 25%. All other intoxication-like reac-

tions are reported at incidences of less than 5% (with the exception of somno-

lence, 7%).

However, the most important issue regarding intoxication is not the inci-

dence, but the severity of any intoxication-like reactions. This is where the

composition of the medicine and its delivery become important. Sativex®not

only produces a low incidence of intoxication, but when experienced by

patients it is generally very low in severity. The ability of the patient to

self-titrate with Sativex®makes it easier to target the therapeutic window, and

makes the occurrence of any such side effects much more manageable, as the

dose and dosage interval can be tailored to each patient’s needs as required

according to their daily circumstances.

256 G.W. Guy and C.G. Stott

As can be seen from Figure 10, the maximum severity of intoxication expe-

rienced by patients (measured using a VAS) was only approximately 20 out of

100 mm following initial exposure to Sativex®. This severity occurs early on

in their initial titration period (within the first 2 weeks) and rapidly diminish-

es over time to scores less than 5 out of 100 mm. Figure 10 also shows that the

picture is repeated in placebo patients who were then switched over to

Sativex®. The long-term intoxication data presented in Table 3 also support

this (see also Fig. 11).

So, although a relatively small amount of intoxication may occur initially in

patients who use Sativex®, it subsides over time, and may be easily managed

using patient self-titration, to minimize levels even further.

The development of Sativex®– a natural cannabis-based medicine 257

Table 3. Long-term intoxication produced by Sativex®[64]

Study week No. of Mean VAS SD Median Minimum Maximum

patients score

4 330 4.84 11.69 1 0 75

12 268 3.08 8.33 0 0 62

20 211 2.04 4.75 0 0 35

28 205 2.46 6.26 0 0 42

36 184 2.83 6.77 0 0 45

44 150 3.69 10.54 0 0 77

52 121 2.26 7.29 0 0 50

60 90 1.37 6.02 0 0 53

68 62 1.92 8.94 0 0 69

VAS scale is 0–100 mm, where 0 means no intoxication and 100 is extreme intoxication.

Figure 10. Intoxication produced by Sativex®[58]. BL, baseline; DB, randomized, double-blind peri-

od (weeks 1–6). Placebo crossed over to Sativex®in weeks 7– 10. Adapted from Wade et al. [58].

Dosing

The review of the efficacy and safety information above clearly demonstrates

that there is a therapeutic window for Sativex®between the level at which

patients can receive significant benefit without significant adverse effects, and

the dose which may produce intoxicating effects. There is no evidence of tol-

erance, it can be seen that improvements in symptoms can be maintained while

on a stable dose (Fig. 12).

258 G.W. Guy and C.G. Stott

Figure 11. Long-term intoxication produced by Sativex®[63]. BL, baseline. Adapted from Wade et al.

[63].

Figure 12. Long-term dosing of Sativex®in neuropathic pain [64]

Conclusion

There has been great debate with regard to merits of cannabis-based medicines

with little scientific and clinical evidence to substantiate the anecdotal effica-

cy and safety. The discovery of the endogenous cannabinoid receptors and

endocannabinoids such as AEA, 2-AG, noladin ether and NADA has spawned

resurgence in the search for therapeutic agents to treat severe and chronic con-

ditions.

To date, medicines made from single synthetic cannabinoid molecules have

yet to be widely used, mainly due to their side-effect profiles. The develop-

ment of a new product, Sativex®, made from whole-plant extracts of cannabis,

may change the way cannabis is viewed, its therapeutic potential maximized

and its universal approval as a medicine granted.

Sativex®is produced from botanical raw materials that have been specifi-

cally grown for their defined cannabinoid ratios. It is a blend of defined

extracts, which ensure batch-to-batch reproducibility is attained. The other

components of the extracts, in addition to the principal cannabinoids add to the

benefits of the medicine.

Clinical studies with Sativex®have focused initially on symptom relief in

chronic conditions, such as MS, neuropathic pain and rheumatoid arthritis, but

it may have further potential as a disease-modifying agent in such conditions.

Further clinical studies will be necessary to investigate this.

The clinical efficacy of Sativex®has been demonstrated in the largest pro-

gramme of clinical studies of a cannabis-based medicine ever undertaken.

Positive benefits have been observed in all 11 studies completed to date by

GW. Dosing at levels of 8–15 sprays per day have produced significant

improvements in central and peripheral neuropathic pain and improvement in

a number of symptoms of MS (neuropathic pain, spasm, spasticity and blad-

der dysfunction) have also been reported. Further, the first study of cannabi-

noids in rheumatoid arthritis has demonstrated that Sativex®may have poten-

tial in relieving not only symptoms of rheumatoid arthritis, but it also may

have a modulating effect on the disease process. A characteristic, which

accompanies the symptom relief achieved with Sativex®, is an improvement in

sleep quality.

Sativex®appears to improve symptom relief in the most difficult groups of

patients – i.e. those who have significant residual symptoms even after best

available therapy has been implemented. The benefits it confers are in addition

to any relief patients may previously have attained with other medications.

Patient groups continue to clamour for the approval of a cannabis-based med-

icine and have indicated that even a small reduction in symptoms is of major

importance to patients, their quality of life and their overall sense of well

being.

In addition to its considerable and sustained efficacy, Sativex®, in clinical

studies, has a very acceptable safety and tolerability profile. It is generally well

tolerated, and the flexibility offered to patients ensures they can quickly and

The development of Sativex®– a natural cannabis-based medicine 259

easily self-titrate to optimum benefit. Intoxication is not usually a limiting fac-

tor for the majority of patients, and any low levels of intoxication upon the

patient’s initial exposure to the medicine are further reduced as they become

familiar with the medicine and the process of self-titration. Side effects expe-

rienced are usually mild or moderate in severity, and there have been few with-

drawals from treatment in the clinical studies to date due to undesirable effects.

Most adverse effects resolve without treatment, and some on a reduction of

dosage of the medicine.

Long-term dosing with Sativex®maintains the clinical benefits initially

observed in the acute setting, over prolonged periods. There is no evidence that

tolerance to the beneficial effects develops. In some cases the benefits

achieved with Sativex®have allowed patients to reduce the doses of, or even

stop taking, other medications.

The approval of Sativex®as a pharmaceutical medicine by regulatory

authorities around the world will represent a milestone in modern medicine

and may catalyse a new era of BDPs.

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265

Index

AA-5-HT 156

absent-mindedness 23

ACEA 92

acetylcholine 96

acetylcholinesterase inhibitor 95

2-acyl-glycerol 90

β2 adrenergic receptor 202

adverse effect 258, 260

adverse event 255, 256

AEA membrane transporter (AMT)

ageing 75

ajulemic acid 155

alkaline phosphatase activity 203

allodynia 153, 252

Alzheimer’s disease (AD) 12, 37,

38, 59, 79, 89, 95, 101, 121, 132

AM-251 184, 188, 195

AM-281 32

AM-356 32

AM-374 156

AM-404 157

AM-630 32

AM-1241 32

AM-2233 32

γ-aminobutyric acid (GABA) 16

amphetamine 193

β-amyloid peptide 95

β-amyloid precursor protein 95

amyotrophic lateral sclerosis (ALS)

79, 89, 99–101

analgesia 150

ananda 4

Anandakanda 3

anandamide (N-arachidonoylethanol-

amine, AEA) 17, 26, 67, 68,

113–115, 119, 122, 13, 156, 184,

185, 187, 189, 234, 259

angiogenesis 15, 170

animal models of HD 91

anorexia 12, 220

anti-cancer effect of cannabidiol 58

anti-convulsant effect of cannabidiol

13, 48

anti-emetics 184–186, 195

anti-emetic effect of cannabidiol 57

anti-emetic effect of

tetrahydrocannabinol 57

anti-inflammatory activity of

cannabidiol 55

anti-inflammatory potential of

cannabinoid agonists 86

anti-neoplastic activity of ∆9-THC

166

anti-nociception and cannabidiol 55

antioxidant activity of cannabidiol

13, 54, 242

anti-proliferative effects of

cannabinoids 81

antipsychotic activity of cannabidiol

anti-tumour effect of cannabidiol

28, 58

ANV 184, 186, 192, 195

anxiety and cannabidiol 52

anxiety, agonists, HU210 142

anxiety, agonists, ∆9-THC 142

anxiety, anandamide hydrolysis 143

anxiety, anandamide hydrolysis,

(FAAH) inhibitors 143

anxiety, antagonists (SR-141716)

142

anxiety, anxiolytic effects,

cannabidiol 141

anxiety, anxiolytic effects, mono-

methyl cannabidiol 141

anxiety, anxiolytic effects, 2-pinyl-5-

dimethylheptyl 141

anxiety, behavioral tests, conditioned

taste aversion 141

anxiety, behavioral tests, defensive

withdrawal test 142

anxiety, behavioral tests, elevated-

plus maze 141

anxiety, behavioral tests, lick-

suppression test 141

anxiety, behavioral tests, two-

compartment black and white box

test 142

anxiety, behavioral tests, ultrasonic

emission test 143

anxiety, behavioral tests, Visual

Analogue Mood Scale (VAMS)

144

anxiety, brain imaging 145

anxiety, brain regions, amygdala

145

anxiety, brain regions, cingulate

gyrus 145

anxiety, brain regions,

parahippocampal activity 145

anxiety, CB1-knockout mouse 143

anxiety, clinical trials, Marinol 146

anxiety, clinical trials, Nabilone 146

anxiety, clinical trials, Sativex®146

apomorphine 187

apoptosis 28, 75, 88, 170

appetite and cannabidiol 58, 219

appetite stimulation 13

application site reaction 255

2-arachidonoyl glycerol (2-AG) 26,

156, 184, 189, 190, 202, 259

arachidonoyl serotonin 156

arachidonoyl-serine (ARA-S) 234

asthma 10, 13

ataxia 98

Atharvaveda 2, 12

autoimmune disease 97

aversive memory 13

axonal degeneration 98

Ayurveda 1

Ayurvedic medicine 1

Aβdeposition 95, 96

Aβ-enriched neuritic plaques 96

Aβ-induced neurotoxicity 97

bangue 5

basal ganglia 91, 94, 96, 99

behavioral sensitization 209

bhang, bhanga 2, 3, 4, 6

bladder dysfunction 252, 253, 259

bladder-related symptoms 247

blood-brain-barrier breakdown 83

blood-brain-barrier disruption 86

BMS-1 157

body temperature 81

bone formation 201–203

bone mass 201–204

bone remodeling 201–203

bone resorption 201, 203

botanical drug products (BDP) 237,

243, 260

botanical drug substance (BDS) 241

–243

botanical raw material (BRM) 238,

240, 241, 243, 259

brachial plexus avulsion 247, 251,

253

brachial plexus injury 255

bradykinesia 93, 94

brain damage 79

bronchodilation 13

calcium channel 84

calcium influx 94

calpain 83, 92

cAMP-response element (CRE) 73

cancer 37, 58, 154, 165, 167, 233

cancer cell proliferation 167

cancer pain 233

cannabichromene (CBC) 12, 16, 25,

236, 242, 243

cannabicyclol 25

cannabidiol structure 47

cannabigerol (CBG) 25, 236, 242,

243

266 Index

cannabinoid agonist 190, 219

cannabinoid ratio 259

cannabinoid tolerance 208

Cannabis indica 8

Cannabis sativa L. 236, 238, 244

caryophyllene 16

caspase 3 83, 92, 97

caspase 83

cat 187, 188

catalase 84

CB1agonist noladin ether 203

CB1antagonist 184, 194, 195

CB1deficient mouse 90, 99, 202,

203

CB1receptor 67, 84, 88, 90–92, 99,

184, 185, 189, 194, 195, 233

CB1receptor antagonist 185, 189

CB1–/– mouse 112, 118–125, 129

CB1-knockout mouse 213

CB1-selective antagonist 32

CB2activation 203

CB2agonist HU-308 203

CB2deficient mouse 203

CB2receptor 80, 87, 96–98, 100,

233

CB2receptor antagonist 189

CB2$ signaling 203, 204

CB2-selective cannabinoid receptor

ligand 34

central neuropathic pain (CNP) 247,

249, 251–253

cerebral ischemia 88, 90

charas 4, 6

chemotherapy 15

chemotherapy, ANV 192

chemotherapy, emetic episodes 183

chemovar 237, 239, 240

cholera 15

choreic movements 91

chronic refractory pain 247

chronic relapsing experimental

autoimmune encephalomyelitis

(CREAE) 98

cisplatin 183–185, 187–192

classification of cannabinoids 234

clone 239

closed head injury 80, 83, 90

cocaine 14

complex II deficiency 92

conditioned avoidance 18, 193

conditioned freezing 122

conditioned gaping 192, 193

conditioned rejection 195

conditioned rejection reaction 193

conditioned taste avoidance 193

constipation 12, 23

convulsion 10

copper/zinc superoxide dismutase 1

(SOD-1) 100

CP-55,940 27, 189

Crohn’s disease 15

Cryptotis parva 187

cyclophosphamide 194

ρ-cymene 16

cytotoxic levels of Ca2+ 83

dandruff 16

demyelination 86, 97

depolarization-induced suppression

of inhibition (DSI) 128, 129

dexanabinol (HU-211) 82, 83, 90,

100, 142

diagnostic marker 75

diarrhea 13

dog 187, 188

dopamine 99

dose-response relationships of

cannabidiol, bell-shaped 53–55,

dronabinol 96, 185, 252

drug-discrimination 210

dysentery 11

dyskinetic state 93

dysmenorrhea 10, 13, 16

dystonia and cannabidiol 52, 98

dysuria 10

eczema 10, 15

edema 83

EDSS 250

Index 267

electroencephalogram (EEG) 254

embryo implantation 68

emesis across species, effect of

cannabinoid 187

emesis and cannabidiol 57

emesis 57, 184–191

emetine 187

endocannabinoid 149, 165, 202,234

endocannabinoid degradation 172

endocannabinoid level 99

endocannabinoid system 202

endogenous cannabinoids 25

endonuclease 83

endothelium-derived factor 100

endothelium-derived mediators 87,

endotholin-1 (ET-1) 87, 100

entourage effect 26

epilepsy 12, 48

estrogen depletion 203

ET-1 100

ET-1-induced vasoconstriction 88

ethanol 214

excitotoxicity 79, 81, 88, 90–92,

94, 98–100

experimental autoimmune

encephalomyelitis (EAE) 98

extension study 255

extract of cannabis 231

fatty acid amide hydrolase (FAAH)

68, 69, 96, 149, 156, 184, 235

FAAH activator 69

female disorder 23

ferret 187, 188, 192, 195

fibrillary tangle 95

first-pass metabolism 236

flavonoid 236, 242

flavor aversion learning 193

follicle-stimulating hormone (FSH)

food consumption and cannabidiol

Fos 150

fracture risk 201

G protein 155

GABA 16, 83, 99, 127, 128

GABA transmission 83

ganja 3, 4, 6

gaping 192–195

gastritis 12, 15

General Health Questionnaire-12

(GHQ-12) 251

glaucoma 38, 57

glial cell 80, 86

gliosis 95

glucose deprivation 88

glucose utilization 81

glutamate 99, 126

glutamate release 82

glutamatergic excitotoxicity 88

glutation 84

gonorrhea 10

Good Agricultural Practice (GAP)

236, 241

Good Clinical Practice (GCP) 236

Good Laboratory Practice (GLP)

236

Good Manufacturing Practice

(GMP) 236, 241

granisetron 191

haoma-soma 2

harvest index 238

head trauma 79, 88

hemorrhoids 16

heroin 213

herpes simplex virus 16

hippocampal activity 113

hippocampus 96, 122, 125

HIV/AIDS 15

house musk shrew 187, 189

5-HT3agonist 189

5-HT3antagonist 184, 185, 191,

193, 195

5-HT3receptor antagonist 189, 192

5-HT3receptor 183, 185

5-HTP 187

6-hydroxydopamine 94, 95

5-hydroxytryptamine (5-HT) 187

268 Index

HU-210 93, 95, 142, 153, 189, 194

HU-211 86

HU-308 92

HU-320 155

human sperm 74

huntingtin protein 91

Huntington’s chorea 37

Huntington’s disease (HD) 52, 79,

89, 91, 93, 101

11-hydroxy-tetrahydrocannabinol

(11-OH-THC) 232, 236

hyperalgesia 154

hypoxia 88

IL-1 receptor antagonist 87

IL-1β-deficient mouse 93

immunomodulatory agent 98

implantation 67

impotence 12, 13, 16

incontinence 16

Indian Hemp Drugs Commission 2,

7, 17

inflammation 39, 55, 79, 85, 90, 92,

inflammatory stimuli 93

inhibitors of endocannabinoid

degradation 175

inhibitors of endocannabinoid

inactivation 172

insanity 8, 10

insomnia 10, 12–14

interleukin (IL)-1β85, 93, 95

interleukin (IL)-6 85, 86

interleukin (IL)-10 86

interleukin (IL)-12 86

intoxication 256–258, 260

intoxication-like reaction 256

intraocular pressure and cannabidiol

intraocular pressure and

tetrahydrocannabinol 57

inwardly rectifying K+channels 84

ionic homeostasis 81

ischemia 79

ischemic episodes 100

ischemic stroke 88

isobolographic analysis 157

itch 15

jaundice 12, 13

jaya 4

JWH-051 33

kainate 90

kainate-induced excitotoxicity 80

ketone bodies, production 81

K-ras oncogene 168

L-dopa 93, 94

least shrew 187, 189, 190

leptin 73, 201, 202

levonantradol 185, 234

Leydig cells 74

lice 16

lithium 186, 190–195

lithium chloride 187, 189, 192

long-term depression (LTD) 130,

131

long-term intoxication 257

long-term potentiation (LTP) 120,

126, 129, 130,

low bone mass (LBM) 202

lower urinary tract 16

lower-urinary-tract symptoms 13

lypase 83

Makhzan-al-Adwiya 6

malaria 16, 23

malonate 92

marijuana 184–186, 189

Marinol®146, 234

materia medica 2

matrix mineralization 203

MDMA 214

memantine 95

memory processing and cognition

mesolimbic dopaminergic system

212

metastasis 170

Index 269

met-fluoro-anandamide 168

methanandamide 32, 187, 189

(R)-methanandamide (AM-356) 32

methyllevonantradol 188

2-methylserotonin 187, 189

microglia 87

microglial activation 94

microglial cell 55, 87, 96

microglial cell migration and

cannabidiol 55, 87

microglial cell proliferation and

migration 87

migraine 10, 13, 14

minor cannabinoid 239

miscarrriage 72

mitochondrial complex II 93

mitochondrial dysfunction 79, 85,

90, 92, 93

mitogen activated protein kinase

pathway 97

model ANV, shrew 191, 192

mood and overall sense of well-

being 247

morning pain in rheumatoid arthritis

247

morphine 157, 187, 213

mother plant 239

mRNA expression 202

multiple scelerosis (MS) 13, 16, 37,

38, 79, 86, 89, 97, 99, 101, 144,

233, 247, 250, 252–255, 259

muscle relaxation 13

muscle spam 247

Na+/K+-ATPase 90

Nabidiolex®242–244

nabilone 185, 187, 188, 234

NADA 156, 259

N-arachidonoyl-dopamine 26, 234

nausea, rat model 192–195

nephritis 39

neuralgia 10

neuritic plaque 95, 96

neurodegenerative diseases 37

neurokinin1 (NK1) 184

neuropathic pain 13, 233, 247, 252–

255, 259

neuropathy 37

neuroprotection 13, 54

neurotransmission and cannabidiol

nicotine 213

nigral dopaminergic neurons 93

3-nitropropionic acid 92

3-nitropropionic acid toxicity 92

NMDA receptor 81, 83, 92, 95, 100

NMDA-induced excitotoxicity 90

N-methyl-D-aspartate (NMDA) 14

N-methyllevonantradol 187

NO 85–88, 93, 95, 97, 100

NO donors 74

NO synthase 83, 84, 86

nocturia 16

noladin ether 26, 234, 259

non-cannabinoid fraction 241–243

non-psychotropic cannabinoids 155

noradrenergic signaling 202

nuclear factor-κB (NF-κB) 86

numerical rating scale 249

obesity 219

OL-135 157

oleamide 26

oligodendrocytes 98

OMDM-1 157

ondansetron 185, 190, 193, 194

opiate sparing 247

opium 11

orexigenic action 91

oromucosal cannabis-based

medicine 16

oromucosal spray 251

osteoblast 201–203

osteoblastic marker gene 202

osteoclast 201, 202

osteoclast differentiation 201, 202

osteocyte 202

osteogenic medium 202

osteoporosis 201, 204

ovariectomy 204

270 Index

oxidative cell death 84

oxidative injury 99, 100

oxidative stress 79, 84, 88, 90, 92–

94, 100

pain 37, 39, 149, 252

palmitoylethanolamide 26

Parkinson’s disease (PD) 37, 38, 79,

89, 93, 94, 101

Patient’s Global Impression of

Change (PGIC) 251, 254

peak bone mass 203

periaqueductal gray 150

peripheral cannabinoid receptor

(CB2) 26, 30

peripheral lymphocyte 72

peripheral neuropathic pain 247,

252, 253, 255

peripheral pain 37

peroxidase 84

peroxisome proliferator-activated

receptor γ(PPARγ) 28

phospholipase D (PLD) 68

pigeon 187, 188

α-pinene 15

placebo 247

postmenopausal osteoporosis 203

premature ejaculation 13

progesterone 73

proinflammatory cytokines 93

protein kinase 83

pruritis 39

psoriasis 15

psychotic illnesses and cannabidiol

quality of life 247, 253, 255

rabies 14

Rajanighantu 3

Rajavallabha 5

rat 192, 194

rat model of nausea 190

reactive microglia 86

reactive oxygen species 83–85, 97,

100

reference memory 112, 117

relapse 214

reproductive fluids 72

rheumatic disease 15

rheumatic pain 23

rheumatoid arthritis 13, 15, 259

rigidity 93, 94

riluzole 99

rimonabant 29, 30, 172, 187, 223,

234

S. murinus 192, 195

safety 256

Sanskrit 1, 3

Sativex 146, 239, 241, 243–260

Sativex®, clinical effects 245–247

secondary excitotoxicity 88

self-titration 244, 252, 257, 260

serotonin 99, 185, 190, 194

Sertoli cell 74

sex hormones 68

shrew 188–192, 195

side effect 245, 260

skeletal development 204

sleep, 57, 247

sleep disturbance 247, 251, 253

sleep quality 247, 251, 253, 254

small-molecule antioxidants 84

Soma 2

spasm 252, 253, 254, 259

spasticity 98, 247, 252–254, 259

spasticity in MS 247

spatial tasks 116

spinal cord 97, 98

spinal cord injury 255

spinal motor neurons 99

spontaneous activity and rectal

temperature 38

SR-141716 29, 30, 82, 84, 88, 90,

96, 113, 114, 119, 120, 122–124,

129, 172, 184, 187, 189, 194, 195,

213, 221, 223, 234

SR-144528 92, 189

STAT3 73

Index 271

sterol 242

striatum 93

stroke 80, 87

stromal cell 201

substantia nigra pars compacta 93

substantia nigra pars reticulata 83

Suncus murinus 187

Suncus 189, 190, 191

superoxide dismutase 84, 100

superoxide 84

Sushruta Samhita 2

symptom relief 245

synergism 158

Synhexyl 234

tail-flick 38

taste avoidance 193, 194

taste reactivity (TR) 193

tau protein 95

terpene 237, 242

terpenoid 236

α-terpineol 16

Tetanus 9

∆9-tetrahyrocannabinol (∆9-THC)

10, 23, 67, 100, 111, 113–115,

117, 118, 184, 187–192, 194,

195, 211, 231, 232, 235, 237,

239, 242–244, 253, 254, 256

tetrahydrocannabivarin (THC-V)

236, 242

Tetranabinex®241, 243, 244, 251,

253, 254

therapeutic modulation of the

endocannabinoid system 215

therapeutic window 245, 256, 258

thin-layer chromatography (TLC)

241

tolerability 245, 255, 256, 258

tolerability profile 259

trabecular bone 202

trabecular bone density 203

transcription factor Ikaros 73

transforming growth factor-β86

transgenic mouse 91

transplant rejection 37

traumatic brain injury 30, 88

traumatic head injury 202

traumatic injury 87, 100

tremor 93, 94, 98, 252

trichome 237

TRPV1 173

tuberculosis 12

tumor necrosis factor-α(TNF-α)

13–15, 83, 85, 86, 93, 95, 97

tumor reduction 13

tyrosine hydroxylase 94

UCM707 80, 157

ulcerative colitis 15

Unani Tibbi 6

URB532 157

URB597 157

urinary disorders 12

uterine receptivity 68

uterus 68

vasoconstriction 87

VDM11 80, 157

Vedas 2

veterinary medicine 11

vijaya 4, 5

virodhamine 17, 26, 234

Visual Analogue Scale (VAS) 251–

253, 255, 257

voltage-sensitive ion channel 83

VR1 receptor 173

WIN-55,212–2 29, 185, 187–189

withdrawal syndrome 209

working memory 112–114, 117

Zend-Avesta 2

Zoroastrianism 2

272 Index

Anxiolytics of natural origin I. Originally occurring plants and plants cultivated in the Czech Republic

Article

Full-text available

Mar 2012

Zdenka Navratilova

[Anxiolytics of natural origin I]

Article

Full-text available

Mar 2012

Zdenka Navratilova

Anxiety disorders belong to most frequently encountered mental disorders in general, since up to 25 % of the population suffers by a clinically important anxious disorder at least once in a lifetime. Anxiolytics and antidepressants are particularly used to treat anxiety. and insomnia is treated with sedatives and hypnotics. There are a number of products for the treatment of the anxiety, but in benzodiazepine-group anxiolytics, there is a risk of the tolerance and addiction development. Thus, a great attention is being paid to the investigation of new medicines without these side effects. There are a number of plants, which have been traditionally used for the treatment of anxiety and insomnia and these effects are frequently supported by modern trials. Herbaceous products are frequently used even at the time being, particularly for the treatment of mild problems. Tea mixtures, tinctures, food supplements and registered medicinal products containing extracts from plants are used. Further research of effects of these plants and their components can offer new pharmaceuticals. In the article presented here, there is an outline of main plants used in the treatment of anxiety and insomnia. The first part of the article is aimed at our original plants and plants cultivated in our country (Cannabis sativa, Coriandrum sativum, Crocus sativus, Eschscholzia californica, Ginkgo biloba, Humulus lupulus, Hypericum perforatum, Lavandula angustifolia, Magnolia officinalis and M. obovata, Matricaria recutita, Melissa officinalis, Rhodiola rosea, Scutellaria spp., Tilia americana and Valeriana officinalis), and the second part deals with exotic plants (Atractylodes macrocephala, Bacopa monnieri, Centella asiatica, Citrus spp., Clitoria ternatea, Echium amoenum, Erythrina mulungu, Morinda citrifolia, Myristica fragrans, Ocimum sanctum, Panax ginseng and P. quinquefolius, Passiflora incarnata, Piper methysticum, Sceletium tortuosum, Withania somnifera, Zingiber officinale and Ziziphus jujuba).

Information for Healthcare Professionals: Cannabis and the Cannabinoids (2013)

Research

Full-text available

May 2016

Hanan Abramovici

This research monograph is a peer-reviewed summary of the scientific evidence on the uses and harms of cannabis and cannabinoids for medical purposes.

Marijuana (cannabis): It's illusion and information

Article

Jan 2012
IJPBS

S. Rajsekhar

The present article deals with the pharmacognosy, pharmacology and many other aspects about the plant marijuana that is well known as cannabis. The above plant has been through many legal and medical issues all round the world. The use of this plant in medical purposes has been a long time hot topic in our fraternity. The plant has been blamed to have an addiction causing character. On the other hand, It has also been supported to have a good and accepted pharmacological activity by literature and practical users. Marijuana was first used for medical purpose in Britain in the midnineteenth century by O'Shaughnessy, a surgeon in army. In India the above plant has been used in wide range of medical condition such as rabies, epilepsy, muscle spasms and for pain relief. This article is a sincere effort to bring forward the possible information about the plant, its chemical constituents and uses. The plant has been in talk for long time regarding the use and legal aspect and so detailed information regarding it should be necessary.

Elucidating the structural basis of selectivity for drug targets: Discovery of selective CB2 agonists and Mps1 kinase inhibitors

Thesis

Full-text available

Jan 2014

Ken-Ichi Kusakabe

In drug discovery programs, appropriate tuning of binding selectivity is a primary objective. Recently, considerable effort has been expended in elucidating the molecular mechanism of small molecules with potential interaction partners, such as proteins. Such work has elucidated the structural basis of selectivity among protein families or subtypes for which selectivity is considered to be difficult to gain. The continual challenge in drug development is the designing of a drug with appropriate selectivity and elucidating its molecular mechanism of selectivity for proteins such as G-protein coupled receptors (GPCRs) and kinases, which are difficult to crystallize and possess similar amino acid sequences of the catalytic domain and conformation. Thus, following the discovery of selective CB2 agonists and monopolar spindle 1 (Mps1) inhibitors, this work was aimed at elucidating the structural basis of their selectivity.

Rimonabant effects on anxiety induced by simulated public speaking in healthy humans: A preliminary report

Article

Jan 2014
HUM PSYCHOPHARM CLIN

We investigated the hypothesis that rimonabant, a cannabinoid antagonist/inverse agonist, would increase anxiety in healthy subjects during a simulation of the public speaking test. Participants were randomly allocated to receive oral placebo or 90 mg rimonabant in a double-blind design. Subjective effects were measured by Visual Analogue Mood Scale. Physiological parameters, namely arterial blood pressure and heart rate, also were monitored. Twelve participants received oral placebo and 12 received 90 mg rimonabant. Rimonabant increased self-reported anxiety levels during the anticipatory speech and performance phase compared with placebo. Interestingly, rimonabant did not modulate anxiety prestress and was not associated with sedation, cognitive impairment, discomfort, or blood pressure changes. Cannabinoid-1 antagonism magnifies the responses to an anxiogenic stimulus without interfering with the prestress phase. These data suggest that the endocannabinoid system may work on-demand to counteract the consequences of anxiogenic stimuli in healthy humans. Copyright © 2013 John Wiley & Sons, Ltd.

Cannabidiol: de nieuwe haarlemmerolie of een hype?

Article

Jun 2012

Dit artikel is (deels) gebaseerd op een literatuurstudie die werd uitgevoerd in opdracht van het ministerie van Volksgezondheid,Welzijn en Sport (Niesink & Van Laar, 2012). farmacoloog/toxicoloog en universitair hoofddocent, senior wetenschappelijk medewerker bij het Programma Drug Monitoring, landelijk coordinator van het Drugs Informatie en Monitoring Systeem programmahoofd bij het Programma Drug Monitoring

Working memory- and anxiety-related behavioral effects of repeated nicotine as a stressor: The role of cannabinoid receptors

Article

Full-text available

Feb 2013
BMC NEUROSCI

Tamaki Hayase

Background Like emotional symptoms such as anxiety, modulations in working memory are among the frequently-reported but controversial psychiatric symptoms associated with nicotine (NC) administration. In the present study, repeated NC-induced modulations in working memory, along with concurrently-observed anxiety-related behavioral alterations, were investigated in mice, and compared with the effects of a typical cognition-impairing stressor, immobilization stress (IM). Furthermore, considering the structural and functional contributions of brain cannabinoid (CB) receptors in NC-induced psychiatric symptoms including emotional symptoms, the interactive effects of brain CB receptor ligands (CB ligands) and NC and/or IM on the working memory- and anxiety-related behaviors were examined. Results Statistically significant working memory impairment-like behavioral alterations in the Y-maze test and anxiety-like behavioral alterations in the elevated plus-maze (EPM) test were observed in the groups of mice treated with 0.8 mg/kg NC (subcutaneous (s.c.) 0.8 mg/kg treatment, 4 days) and/or IM (10 min treatment, 4 days). In the group of mice treated with NC plus IM (NC-IM group), an enhancement of the behavioral alterations was observed. Among the CB type 1 (CB1) antagonist AM 251 (AM), the non-selective CB agonist CP 55,940 (CP), and the CB1 partial agonist/antagonist virodhamine (VD), significant recovering effects were provided by AM (0.2-2.5 mg/kg) and VD (5 mg/kg) against the working memory impairment-like behaviors, whereas significant anxiolytic-like effects (recoveries from both attenuated percentage of entries into open arms and attenuated percentage of time spent on open arms) were provided by VD (1–10 mg/kg) and CP (2 mg/kg) against the anxiety-like behaviors. Conclusions Although working memory impairment- and anxiety-like behavioral alterations were commonly induced in the NC, IM, and NC-IM groups and the therapeutic involvement of CB receptors was shown, there were discrepancies in the types of effective CB ligands between the working memory- and anxiety-related behaviors. The differential involvements of CB receptor subtypes and indirectly activated neurotransmitter systems may contribute to these discrepancies.

The Endocannabinoid System and the Therapeutic Potential of Cannabinoids

Chapter

Nov 2007

Billy R. Martin

Much has been written about the history of the medical uses of cannabis (1). In the past two centuries, there have been numerous references to the use of cannabis extracts for a wide range of disorders (2). In the early part of the 20th century, a standardized cannabis elixir was marketed in the United States. Following the introduction of synthetic drugs such as barbiturates and opioids into medicine, interest in cannabis elixir declined. The discovery of the primary active constitutent in marijuana, Δ9-tetrahydrocannabinol (THC), in 1964 (3) rekindled interest in the area. However, the emphasis shifted to synthetic cannabinoids rather than the plant or plant extracts. For example, in the 1970s, clinical studies were conducted in an effort to determine the efficacy of THC as an analgesic (4), antiemetic (5), antidepressant (6,7), appetite stimulant (7), and for treatment of glaucoma (8). These efforts resulted in the approval of THC (dronabinol, Marinol™) for treatment of chemotherapy-induced nausea and vomiting in 1985 and for appetite stimulation in 1992.

Mass Spectrometric Methods for Determination of Cannabinoids in Physiological Specimens

Chapter

Nov 2007

Rodger L. Foltz

This chapter describes the published mass spectrometric (MS) methods that have proven most effective for quantitative measurement of Δ9-tetrahydrocannabinol (THC) and its major metabolites in physiological specimens. Because determination of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCA) in urine continues to be the most frequently used indicator of marijuana use, the first portion of the chapter will discuss methods for measurement of THCA in urine. However, the major portion of the chapter is devoted to the most recent developments for measuring THC and its metabolites in other biological specimens including blood, plasma, meconium, oral fluids, hair, and other tissues. Table 1–7 are designed to facilitate location of references describing analytical methods involving key components for analysis of cannabinoids in various matrices.

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB 1 receptor

Article

Full-text available

Mar 2001

Two types of endogenous cannabinoid-receptor agonists have been identified thus far. They are the ethanolamides of polyunsaturated fatty acids--arachidonoyl ethanolamide (anandamide) is the best known compound in the amide series--and 2-arachidonoyl glycerol, the only known endocannabinoid in the ester series. We report now an example of a third, ether-type endocannabinoid, 2-arachidonyl glyceryl ether (noladin ether), isolated from porcine brain. The structure of noladin ether was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by comparison with a synthetic sample. It binds to the CB(1) cannabinoid receptor (K(i) = 21.2 +/- 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB(2) receptor (K(i) > 3 microM).

Cannabielsoic acids : Isolation and synthesis by a novel oxidative cyclization

Article

Full-text available

Oct 1974
TETRAHEDRON

Two tricyclic dihydrobenzofuran cannabinoids, cannabielsoic acids A (4a) and B (5a) were isolated from hashish. Their structures were elucidated by chemical transformations and from spectral data. Cannabielsoic acid A was synthesized from cannabidiolic acid by an oxidative cyclization in the presence of air under irradiation, or with manganese dioxide.

POSSIBLE ANXIOLYTIC EFFECTS OF CANNABIDIOL

Chapter

Dec 1984

Richard E. Musty

Cannabis. XII. Variations of Cannabinoid Contents in Several Strains of Cannabis sativa L. with Leaf-age, Season and Sex

Article

Jan 1980

A quantitative analysis procedure for tetrahydrocannabinolic acid, cannabichromenic acid, cannabidiolic acid and cannabigerolic acid monomethyl ether was established by gas chromatography and by a combined gas chromatography-preparative thin-layer chromatography method using cholestane as an internal standard. The variations of cannabinoid contents with leaf-age, season and sex were investigated in three kinds of "physiological varieties" of Cannabis sativa L., the Mixican, the Minamioshihara No. 1 and the CBDA strain.

Cannabinoid receptors and their ligands

Article

Oct 2002
EUR NEUROPSYCHOPHARM

Roger G Pertwee

The actions of CBD on anxiety and other effects produced by Δ1-THC in normal subjects

Article

Mar 1982
PSYCHOPHARMACOLOGY

The object of the experiment was to verify whether cannabidiol (CBD) reduces the anxiety provoked by ?9-TCH in normal volunteers, and whether this effect occurs by a general block of the action of ?9-TCH or by a specific anxiolytic effect. Appropriate measurements and scales were utilized and the eight volunteers received, the following treatments in a double-blind procedure: 0.5 mg/kg ?9-TCH, 1 mg/kg CBD, a mixture containing 0.5 mg/kg ?9-TCH and 1 mg/kg CBD and placebo and diazepam (10 mg) as controls. Each volunteer received the treatments in a different sequence. It was verified that CBD blocks the anxiety provoked by ?9-TCH, however this effect also extended to marihuanalike effects and to other subjective alterations induced by ?9-TCH. This antagonism does not appear to be caused by a general block of ?9-TCH effects, since no change was detected in the pulse-rate measurements. Several further effects were observed typical of CBD and of an opposite nature to those of ?9-TCH.

Anandamide suppresses nitric oxide and TNF-?? responses to Theiler's virus or endotoxin in astrocytes

Article

May 1997

ASTROCYTES are an important cell population in the CNS, involved in cytokine homeostasis and participating in a variety of important physiological and pathological processes. In the present study we showed that primary cultures of neonatal mouse cortical astrocytes stimulated with lipopolysaccharide (Balb/c mice strain, LPS: 1 mu g/ml, 18 h) or Theiler's virus, TMEV (SJL/J mice strain, TMEV: 10(5) PFU/well, 24h) released an increased amount of nitrites (NO2-) and tumour necrosis factor-alpha (TNF-alpha) into the culture medium. Exogenous cannabinoids are known to modulate the function of immune cells. Anandamide, an endogenous ligand for the cannabinoid receptor, blocked the release of NO2- and TNF-alpha induced by LPS in a dose-dependent manner. In TMEV-stimulated astrocytes anandamide also suppressed, in a dose-related manner, the stimulatory effects of TMEV on both NO2- and TNF-alpha. It is suggested that anandamide exerts an immunoregulatory role in the CNS. These results could have important implications in the modulation of immunological and inflammatory processes by cannabinoid agents.

Effects of Smoked Cannabis and Oral ∆9Tetrahydrocannabinol on Nausea and Emesis After Cancer Chemotherapy: A Review of State Clinical Trials

Article

Jan 2001

Background. In 1999 the Institute of Medicine (IOM) issued a report entitled Marijuana and Medicine (Joy, Watson and Benson, 1999). It recommended the development of cannabinoid drug delivery systems which might be effective for nausea, vomiting and AIDS wasting syndrome, among other chronic disorders. The report went on to recognize that patients should be allowed to smoke marijua- na if they failed to achieve relief from approved symptoms that could be relieved by cannabinoid drugs with rapid onset. Recommended criteria of the report included: access to marijuana within 24 hours of submission by a physician, supervision that allows for assessment of treatment effectiveness, and an oversight strategy comparable to an institutional review board. In this context a review of previously un- published state-run clinical trials with Cannabis sativa (marijuana and/ or ∆9-tetrahydrocannabinol capsules) to test efficacy in reducing nau- sea and vomiting following cancer chemotherapy is warranted. The impetus for these studies came from individual state legislatures re- sponding to constituents' claims that smoking marijuana reduced or blocked nausea and vomiting. Methods. Technical reports were obtained from 6 states which had

A behavioural model to reveal place preference to Δ9-tetrahydrocannabinol in mice

Article

Jan 2000
PSYCHOPHARMACOLOGY

Rationale: The rewarding properties of Δ9- tetrahydrocannabinol (THC) are difficult to demonstrate in rodents using standard procedures. Objective: To evaluate the motivational responses of THC in the place conditioning paradigm in mice after minimizing the dysphoric effects of the first drug exposure and/or the consequences of its pharmacokinetic properties. Methods: Mice were conditioned to THC (1 or 5 mg/kg) using an unbiased procedure with an elevated number of pairings and long conditioning time. Results: A place aversion was observed with 5 mg/kg THC using a standard protocol. Similar results were obtained when the CB-1 receptor antagonist SR 141716A (1 mg/kg) was administered immediately after each THC conditioning period. However, mice receiving a priming THC injection and conditioned 24 h later showed a place preference with 1 mg/kg THC and no effect with 5 mg/kg THC. Conclusion: THC produces a clear place preference in mice by using a long period of conditioning and avoiding the possible dysphoric consequences of the first drug exposure.

Crack Heads and Roots Daughters: The Therapeutic Use of Cannabis in Jamaica

Article

Jun 2002

Melanie Dreher PhD and FAAN

An ethnographic study of women and drug use in inner city neighborhoods in Kingston, Jamaica, revealed that cannabis is commonly used in conjunction with crack cocaine to minimize the undesirable effects of crack pipe smoking, specifically paranoia and weight loss. According to 33 current or former crack using women, who were followed for a period of nine months, cannabis cigarettes (“spliffs”) constitute the cheapest, most effective and readily available therapy for discontinuing crack consumption. The findings of this research suggest the need to reframe “multiple drug use” within thecultural meanings that attend cannabis in Jamaica as a medicine and a sacrament.

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