RESEARCH ANALYSIS

and

UTILIZATION SYSTEM

Structure-Activity

Relationships

of the

Cannabinoids

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration
Structure-Activity

Relationships

of the Cannabinoids

Editors:

Rao S. Rapaka, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

Alexandros Makriyannis, Ph.D.

School of Pharmacy and Institute of Materials Science

University of Connecticut

and

F. Bitter National Magnet Laboratory

Massachusetts Institute of Technology

NIDA Research Monograph 79

A RAUS Review Report

1987

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

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For sale by the Superintendent of Documents, U.S. Government Printing Office

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NIDA Research Monographs are prepared by the research divisions of the

National Institute on Drug Abuse and published by its Office of Science. The

primary objective of the series is to provide critical reviews of research problem

areas and techniques, the content of state-of-the-art conferences, and

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dissemination to the scientific and professional community.

Editorial Advisors

MARTIN W. ADLER, Ph.D.

Temple University School of Medicine

Philadelphia, Pennsylvania

SYDNEY ARCHER, Ph.D.

Rensselaer Polytechnic lnstitute

Troy, New York

RICHARD E. BELLEVILLE, Ph.D.

NB Associates. Health Sciences

Rockville, Maryland

KARST J. BESTEMAN

Alcohol and Drug Problems Association

of North America

Washington, D.C.

GILBERT J. BOTVIN, Ph.D.

Cornell University Medical College

New York, New York

JOSEPH V. BRADY, Ph.D.

The Johns Hopkins University School of

Medicine

Baltimore, Maryland

THEODORE J. CICERO, Ph.D.

Washington University School of

Medicine

St. Louis, Missouri

MARY L. JACOBSON

National Federation of Parents for

Drug-Free Youth

Omaha, Nebraska

REESE T. JONES, M.D.

Langley Porter Neuropsychiatric Institute

San Francisco, California

DENISE KANDEL, Ph.D.

College of Physicians and Surgeons of

Columbia University

New York, New York

HERBERT KLEBER, M.D.

Yale University School of Medicine

New Haven, Connecticut

RICHARD RUSSO

New Jersey State Department of Health

Trenton, New Jersey

NIDA Research Monograph Series

CHARLES R. SCHUSTER, Ph.D.

Director, NIDA

JEAN PAUL SMITH, Ph.D.

Acting Associate Director for Science, NIDA

Acting Editor

Parklawn Building, 5600 Fishers Lane, Rockville, Maryland 20857
Structure-Activity Relationships

of the Cannabinoids
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from the RAUS

review on structure-activity relationships of the cannabinoids which

took place on October 30 and 31, 1986, at Bethesda, Maryland. The

review meeting was sponsored by the Office of Science and the

Division of Preclinical Research, National Institute on Drug Abuse.

COPYRIGHT STATUS

The National Institute on Drug Abuse has obtained permission from

the copyright holders to reproduce certain previously published

material as noted in the text. Further reproduction of this

copyrighted material is permitted only as part of a reprinting of

the entire publication or chapter. For any other use, the copyright

holder's permission is required. All other material in this volume

except quoted passages from copyrighted sources is in the public

domain and may be used or reproduced without permission from the

Institute or the authors. Citation of the source is appreciated.

Opinions expressed in this volume are those of the authors and do

not necessarily reflect the opinions or official policy of the

National Institute on Drug Abuse or any other part of the U. S.

Department of Health and Human Services.

The U. S. Government does not endorse or favor any specific

commercial product or company. Trade, proprietary, or company names

appearing in this publication are used only because they are

considered essential in the context of the studies reported herein.

DHHS Publication No. (ADM)90-1534

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1987, Reprinted 1990

NIDA Research Monographs are indexed in the Index Medicus. They are

selectively included in the coverage of American Statistics Index,

BioSciences Information Service, Chemical Abstracts, Current

Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

iv
Preface

The Research Analysis and Utilization System (RAUS) is designed to

serve four functions:

Collect and systematically classify the findings of all

intramural and extramural research supported by the

National Institute on Drug Abuse (NIDA);

Evaluate the findings in selected areas of particular

interest and formulate a state-of-the-art review by a

panel of scientific peers;

Disseminate findings to researchers in the field and

to administrators, planners, instructors, and other

interested persons;

Provide a feedback mechanism to NIDA staff and planners

so that the administration and monitoring of the NIDA

research program reflect the very latest knowledge

gleaned from research in the field.

Since the number of research topics that can be intensively reviewed

annually is limited, four subject areas are chosen each year to

undergo a thorough examination. Distinguished scientists are

invited to participate. Each scientist is provided reports from

NIDA-funded research and asked to add information derived from the

literature and his or her own research and to prepare a

comprehensive, state-of-the-art review paper on an assigned topic.

These papers, together with a summary of the discussions which take

place at the review meeting, make up a RAUS Review Report in the

NIDA Research Monograph series.

In recent years a large number of reviews and books have appeared

summarizing research advances in the chemistry, pharmacology,

toxicology, metabolism, and distribution of cannabinoids and their

synthetic analogs. Yet the volume of research on structure-activity

relationships of cannabinoids appears to have decreased. In order

to evaluate NIDA's current research program on cannabinoids, to

rekindle interest in this critical area, and to focus attention on

profitable avenues for future research, NIDA found it timely to

bring together experts on the structure-activity relationships of

cannabinoids for a comprehensive review.

v
Many cannabinoid analogs have been designed. Some have served as

excellent probes for understanding the mechanism of action of

cannabinoids and have generated optimism that in the near future

potent therapeutic drugs that lack "cannabimimetic" activity can be

developed. In this monograph, while emphasis is placed on

structure-activity relationships and molecular mechanisms of action

of cannabinoids. chapters added to broaden the subject area will

increase the usefulness of this volume as a reference text for

researchers in the chemistry and biology of the cannabinoids.

Drs. Rao S. Rapaka, Alexandros Makriyannis, and Sumner Burstein

served as the scientific moderators of the RAUS review meeting. The

introduction and the concluding overview chapter, by Drs.

Makriyannis and Rapaka, provide a brief summary of the presentations

and the discussions. Jacqueline P. Ludford, Chief, Research

Analysis Branch, Office of Science, is the RAUS coordinator for NIDA.

vi
Contents

Preface

Introduction

Rao S. Rapaka and Alexandros Makriyannis

Structure-Activity Relationships in Cannabinoids: An Overview

Raj K. Razdan

Stereochemical Requirements for Cannabimimetic Activity

Raphael Mechoulam; Naphtali Lander; Morris Srebnik;

Aviva Breuer; Mark Segal; Jeffery J. Feigenbaum;

Tobjorn U. C. Jarbe; and Paul Consroe

Structure-Activity Relationships of Tricyclic and

Nonclassical Bicyclic Cannabinoids

Larry S. Melvin and M. Ross Johnson

Structure-Anticonvulsant Activity Relationships of

Cannabidiol Analogs

Arnold R. Martin; Paul Consroe; Vinayak V. Kane;

Vibhakar Shah; Vishwakarma Singh; Naftali Lander;

Raphael Mechoulam; and Morris Srebnik

Anticonvulsant and Neurotoxic Effects of Tetrahydrocannabinol

Stereoisomers

Paul Consroe and Raphael Mechoulam

Different Cannabinoids Exhibit Different

Electrophysiological Properties

Stuart A. Turkanis and Ralph Karler

Molecular Determinants for Cannabinoid Activity:

Refinement of a Molecular Reactivity Template

Patricia H. Reggio

Different Cannabinoids Exhibit Different Pharmacological

and Toxicological Properties

Ralph Karler and Stuart A. Turkanis

Pharmacological Evaluation of Agonistic and Antagonistic

Activity of Cannabinoids

Billy R. Martin;

Thomas J. Martin

David R. Compton; Patrick J. Little;

; and Patrick M. Beardsley

vii

V

1

3

15

31

48

59

67

82

96

108
Interactions of Cannabinoids With Membranes. The Role of

Cannabinoid Stereochemistry and Absolute Configuration and

the Orientation of Delta-9-THC in the Membrane Bilayer

Alexandros Makriyannis; Ali Banijamali;

Cornelis Van Der Schyf; and Harold Jarrell 123

The High Affinity Cannabinoid Binding Site in Brain:

Regulation by Guanine Nucleotides and Isolation of an

Endogenous Inhibitor

Jeffrey S. Nye and Solomon H. Snyder 134

Regulation of Adenylate Cyclase in a Cultured Neuronal

Cell Line by Marijuana Constituents, Metabolites of

Delta-9-tetrahydrocannabinol, and Synthetic Analogs Having

Psychoactivity

Allyn C. Howlett 148

Inhibitory and Stimulatory Effects of Cannabinoids on

Eicosanoid Synthesis

Sumner H. Burstein 158

Pharmacokinetics and Disposition of Cannabinoids

C. Nora Chiang and Rao S. Rapaka 173

Drug Abuse in India: Progress in Research

B. N. Dhawan 189

The Medicinal Chemistry of Cannabinoids: An Overview

Alexandros Makriyannis and Rao S. Rapaka 204

List of NIDA Research Monographs 211

viii
Introduction

Rao S. Rapaka, Ph.D., and Alexandros Makriyannis, Ph.D.

Although a number of reviews on cannabinoids are held each year,

reviews devoted entirely to structure-activity relationships of the

cannabinoids are less frequent. Moreover, in recent years a

slackening of interest appears to have occurred, both among academic

scientists and scientists from drug houses, in undertaking studies

of SAR and development of drugs based on the cannabinoid molecular

skeleton. A number of related areas in the medicinal chemistry of

the cannabinoids, such as development of new drugs, resolution of

cannabimimetic activity from other bioactivities such as

anticonvulsant action, and studies of receptors, require special

attention. In order to evaluate the progress and to revitalize

research in SAR and related medicinal chemistry aspects of

cannabinoids, the National Institute on Drug Abuse felt that the

time had come for a review and a publication on the

structure-activity relationships of the cannabinoids.

The stereochemical requirements for cannablmimetic activity are one

major issue. Dr. Mechoulam discusses the structure-activity

relationships (SAR) with emphasis on stereochemical requirements.

Dr. Mechoulam presents biological data to show that (-) enantiomers

of cannabinoids are more active than the (+) enantiomers. Dr.

Razdan, who was unable to attend the review, contributed an overview

chapter on structure-activity relationshlp in cannabinoids, with

special emphasis on SAR and drug development, and on SAR and the

mechanism of action of cannabinoids. Dr. Arnold Martin discusses

SAR of cannabinoids regarding anticonvulsant activity along with

recent information on pinyl and carnyl analogs, simplified

derivatives lacking the 4-isopropenyl group, and a variety of 1'-1'

dialkylhexyl and 1'-1'-dialkeylheptyl analogs. Comparisons of

anticonvulsant potency of delta-6-tetrahydrocannabinol,

delta-6-THC-1', 1'-dimethylheptyl and related analogs are presented

by Dr. Consroe. In addition, Dr. Consroe presents evidence for

stereoselectivity for anticonvulsant and neurotoxic activities.

Drs. Melvin and Johnson review SAR of tricyclic and nonclassical

bicyclic cannabinoids. Their studies demonstrate that some of the

analogs are potent analgesics and exhibit enantiomeric potency.

More importantly, their studies show that it is possible to achieve

extreme biological potency in structures different from classical

1
cannabinoid ring systems. This, in turn, gives the researchers

optimism for further development of new analogs and potent analgesic

compounds. Electrophysiological properties of cannabinoids,

delta-9-THC, 11-hydroxy-delta-9--THC (the principal metabolite of

THC) and cannabidiol are discussed by Dr. Turkanis. His studies

demonstrate that different cannabinoids exhibit markedly different

electrophysiological properties. An interesting presentation on the

molecular parameters that determine the cannabimimetic activity is

given by Dr. Reggio. One of the hypotheses presented by Dr. Reggio

is that the basis of cannabimimetic activity is related to molecular

properties conferred by a lone pair of electrons on the phenolic

oxygen and to the orientation of the carboxyclic ring.

Another major point of focus is the molecular mechanisms of

cannabinoid activity. Several researchers are currently involved in

answering the question of "receptor" versus "membrane." Dr. Billy

Martin has reviewed the area, and outlines the different points of

view. Dr. Nye presents findings on the high affinity cannabinoid

binding sites in the brain and on the isolation of an endogenous

inhibitor of cannabinoid binding. This endogenous compound has been

partlally characterized and the role of this compound is not clear

at this time. Dr. Makriyannis presents evidence to support the

hypothesis that the cannabinoids exert their physiologic effects, at

least in part, by interacting with the phospholipid component of the

membrane. The dynamic and conformational changes in the bilayer

that accompany the incorporation of cannabinoids has been studied

using solid state NMR. Dr. Makriyannis shows that cannabinoid

analogs orient in the membrane in such a manner that the phenolic

hydroxyl group has an important role in determining the geometry of

cannabinoid-membrane interaction. Dr. Burstein gives additional

evidence for cannabinoid action at the membrane level. He also

reports an important discovery that major cannabinoid metabolites

antagonize the stimulatory action of THC by inhibiting a specific

step in prostaglandin synthesis. A different view, which favors a

specific protein site for cannabinoid action, is presented by Dr.

Howlett.

Dr. Karler describes the problems involved in the bioassay of

cannabinoids and points out the dramatic differences in cannabinoid

activity among different species. Drs. Chiang and Rapaka make the

monograph more comprehensive with their chapter on pharmacoklnetics

and disposition of cannabinoids. In addition, Dr. B.N. Dhawan of

the Central Drug Research Institute, Lucknow, has contributed a

chapter on drug abuse in India. This is especially timely as it is

expected that, in the near future, there will be more scientific

research collaboration between the two countries.

By bringlng together the contributions of scientists having

multidisciplinary research backgrounds, this monograph represents an

effort to summarize the existing knowledge on the structure-activity

relationships of cannabinoids and to discuss the implications of

this information for further progress toward NIDA's research

objectives.

2
Structure-Activity Relationships

in Cannabinoids: An Overview

Raj K. Razdan, Ph.D.

INTRODUCTION

The most important pharmacologically active member of this family of

compounds is (-) delta-9-THC,6a,10a-trans-tetrahydrocannabinol (THC) which is

the main active constituent of marijuana. Delta-9-THC is an optically active

resinous material which is very lipid soluble and water insoluble, a property

shared by numerous cannabinoids, which makes their pharmacological testing

difficult. Both in man and animals effects produced by ingesting marijuana or

delta-9-THC are unique. The gross effects produced cannot be classified

pharmacologically as being primarily due to a stimulant, sedative, tranquilizer, or

hallucinogen, although they share some properties with each of these. In general,

the effects produced by delta-9-THC are dose dependent. It is not a narcotic

and compared to many drugs, such as the opiates, barbiturates, etc., it does not

produce physical dependence.

Cannabinoids is a term used for the typical C21 group of compounds present in

Cannabis saliva L. and includes their analogs and transformation products. Two

different numbering systems, dibenzopyran and monoterpenoid, are generally

used for naming the cannabinoids, as shown in figure 1.

BACKGROUND

The first systematic study of structure-activity relationships (SAR) in

cannabinoids was reported by Roger Adams in the early 1940s (Adams 1942). It

was based on the characteristic dog ataxia test as developed and quantitated with

estimates of error by Loewe (1947). At about the same period, Todd and others

(Todd 1946; Avison et al. 1949) used the Gayer areflexia test (Gayer 1928) for

suppression of rabbit corneal reflex to determine the potency of various extracts

of cannabis and synthetic cannabinoids. They also reported SAR of cannabinoids

based on this test. Both groups assumed that the activity in these tests was a

quantitative measure of the behavioral or psychotropic activity of marijuana in

man, although no controlled experiments to support this assumption were

reported. Since that time, clinical evaluation of a number of natural and

synthetic cannabinoids has shown that there is good correlation between the

relative potency in the dog ataxia test and the psychoactive component of these

compounds in man. The usefulness of the rabbit areflexia test has, however,

been found to be limited and controversial; and this test is not presently used for

screening cannabinoids (Razdan 1986).

3
FIGURE 1. Dibenzopyran and monoterpenoid numbering systems generally used

for naming cannabinoids.

When pure delta-9-THC and other cannabinoids became available in the early

1960s, several other qualitative tests were developed to determine the "cannabislike" activity of various compounds, for example, the cataleptic reaction in mice

and the effects on motor activity and gross behavior in mice and rats. It was

only after the first study was reported in 1968 on the effects of cannabinoids in

monkeys (Scheckel et al. 1968) that the monkey behavioral test procedures were

standardized and the SAR of cannabinoids were developed. This was largely due

to the efforts of Grunfeld, Edery, and Mechoulam (Grunfeld and Edery 1969;

Mechoulam et al. 1970; Edery et al. 1971, 1972). This test, which provides good

correlation between potencies of various cannabinoids in monkeys and man,

complements the dog ataxia test. Loewe's procedure for the dog ataxia test has

been further refined and standardized by Martin, Dewey, and Harris (Martin et

al. 1975, 1984a). The SAR based on both of these tests are very similar and

provide a good foundation for the SAR of cannabinoids. However, it should be

emphasized that these SAR are mainly reflective of the behavioral activity, rather

than the other pharmacological properties, of the cannabinoids. From time to

time, attempts have been made to introduce newer models for the bioassay of

cannabinoids. These include the mouse ring immobility assay, the genetically

unique tetrahydrocannabinol-seizure susceptible (THC-SS) rabbit, and

spontaneous activity and body temperature in mice. Recently, the drug

discrimination procedure has been used to study cannabinoids in rats and pigeons

(Razdan 1986).

4
SAR IN MAN

On the basis of comparative data, delta-9-THC by smoking is considered to be

2.6 to 3 times more potent, and i.v.-administered delta-9-THC is about 10 times

more potent than delta-9-THC ingested p.o. (Isbell et al. 1967; Hollister and

Gillespie 1973). By either smoking or i.v., the effects of delta-9-THC appear

within seconds or minutes, whereas with p.o. doses, the onset of symptoms is

delayed from 30 minutes to 2 hours. The clinical syndrome, however, is very

similar in all cases. Based on the various cannabinoids which have been

evaluated clinically, the following SAR in man are suggested (Razdan 1986).

1. It appears that a benzopyran ring (figure 2) is a definite requirement for

activity, since the ring-opened compound cannabidiol (CBD) is inactive.

However, the benzopyran by itself does not confer activity, since

cannabichromene is inactive. Moreover, the oxygen in the benzopyran ring can

be substituted by a nitrogen without loss of activity as in levonantradol (figure

3).

2. Attachment of a nonplanar alicyclic ring (i.e., ring C) to the benzopyran ring

in the 3,4-position is important for activity. However, a planar ring attachment

as in cannabinol reduces activity (nearly inactive).

3. In place of a nonplanar ring attachment, a bulky substituent in the 4-position

of the benzopyran ring can also confer activity to the molecule.

4. A variety of substituents can be introduced in the alicyclic ring C without loss

of activity. Thus, the methyl in g-position as present in delta-9-THC is not

essential and can be replaced by a hydroxyl (levonantradol), a hydroxymethyl

(11-hydroxy-delta-9-THC), or a ketone (nabilone) without loss of activity. Even

the presence of two different substituents in the alicyclic ring, such as a methyl

group at 9-position and a double bond in the ring (e.g., delta-9, delta-8, delta6a-THCs), or a hydroxyl in an S-position (8-hydroxy-THCs) retains activity. In

the double-bond isomers, the position of the double bond at g-position appears

optimum for activity (delta-9> delta-8> delta-6a-THCs).

5. The alicyclic ring attachment to the benzopyran ring can be substituted by a

heterocyclic ring without loss of activity.

6. In the aromatic ring, esterification of the phenolic group retains activity.

7. The length of the aromatic side chain can be varied without loss of activity,

but a three-carbon chain seems minimal for activity, and branching of the chain

increases potency. Attachment of the side chain to the aromatic ring can also be

via an oxygen atom (i.e., an ether as in levonantradol) without loss of activity.

It is noteworthy that, within the structural constraints present in the THC ring

system, changing the position of the double bond in the alicyclic ring, the length

and branching of the side chain, or producing hydroxy metabolites at the 11- or

8-positions do not alter qualitatively the THC effects but may strongly affect

potency. Changing the double bond from delta-9- to delta-8- or delta-6areduces potency. Decreasing the length of the side chain by two carbons reduces

potency by 75%. Increasing the length of the side chain with branching enhances

potency several fold. Substitution of a hydroxyl group in the 11-position retains

potency, while hydroxylation in the 8-position reduces potency by 80%.

5
FIGURE 2. Comparison of the structures of the benzopyran and THC ring

systems.

SAR IN ANIMALS

On the basis of recent data available from animals studies, the following

conclusions are drawn regarding the SAR in cannabinoids. It should be

emphasized that the SAR is developed on the basis of cannabis-like effects in

animals, especially in the monkey and the dog. Some natural and synthetic

cannabinoids are shown in figure 3.

1. Essentially, a benzopyran structure with an aromatic hydroxyl group at C-l

position and an alkyl or alkyl ether group at C-3 position is a requirement for

activity. In general, changing the above substitution pattern leads to a major loss

of potency (Avison et al. 1949; Loev et al. 1973). Although a benzopyran ring is

a definite requirement for activity, the benzopyran by itself does not confer

activity. This is based on the observation that the ring-opened compound CBD

is inactive in both the monkey and the dog and does not generalize to delta-9-

THC in the drug discrimination test in the rat. Similarly, cannabichromene,

which has an intact benzopyran ring, is inactive.

2. The position and the environment around the aromatic hydroxyl group are

very important for activity, viz., (a) the OH at position C-1 is in itself necessary

for activity. Thus, the abnormal compounds where the hydroxyl and the side

chain have been interchanged (i.e., side chain at C-1 and OH at C-3) are all

inactive. Similarly, removal of OH at C-1 eliminates activity. (b) Esterification

of the OH at C-1 retains activity. Etherification of the OH at C-1 eliminates

activity. Replacement of the OH by NH, retains activity, and replacement by

SH eliminates activity. (c) Since C-10 in the alicyclic ring C is in close

proximity to the OH group, a methyl substituent at C-10 significantly alters the

activity in the case of a planar five-membered C-ring.

3. Substitution in the aromatic ring by electronegative groups like carboxyl,

carbomethoxyl, and acetyl groups eliminates activity, whereas alkyl or hydroxyl

groups at C-2 position retain, and in C-4 position reduce, activity.

6
FIGURE 3. Some natural and synthetic cannabinoids.

7
8
4. A minimum length of the aromatic side chain is necessary to elicit activity,

and the branching of the alkyl side chain increases potency. Thus, 1’,2’-

dimethylheptyl or 1’, 1’-dimethylheptyl gives the most potent compounds.

Similarly, p-fluorophenyl alkyl and side chains as shown below retain good

activity:

—O—CH(CH3)(CH2)3-Ph,—CH2CH=CHCOC5H11,and—CH2CH2CH(OH)C2H5

5. On ring B, removal of the gem dimethyl group at C-6 decreases activity.

Replacement of one of the geminal methyl groups at C-6 by a hydroxymethyl

group retains activity (12 beta-hydroxy-delta-9-THC is as active as delta-9-

THC). Replacement of pyran O by N and ring expansion of ring B by one

carbon can retain activity.

6. In the alicyclic ring C, compounds with the double bond in delta-9-, delta-8-,

or delta-6a,10a-position are active. It is noteworthy that compounds with a

double bond in the delta-10,10a-position are also active, but only a few examples

are available at present. A 6a,10a-trans junction increases, and a cis junction

decreases, activity. The natural THCs are active in the 10a R and 6a R series

only. A methyl at C-9 increases and is optimum for activity, but metabolism to

the 11-hydroxy-methyl is not a prerequisite for THC activity, since g-nor-delta8-THC is active. Compounds are active even when no double bonds are present

in the ring, e.g., the HHCs. In these compounds, an equatorial group at C-9

increases activity, and an axial group decreases activity. Furthermore, the

activity is retained even when a diverse group is present, such as an epoxide, a

ketone, or an alcohol.

7. The C-ring can be substituted by a variety of nitrogen- and sulfur-containing

rings without loss of activity. With the nitrogen and sulfur analogs, the most

active central nervous system (CNS) agents are obtained when the heteroatom is

in a phenethyl orientation, e.g., inserted in place of C-7 or C-9.

8. Planarity of the C-ring is not a necessary criterion for activity.

9. In both carbocyclic and heterocyclic analogs, opening the pyran ring generally

decreases activity.

SAR AND DRUG DEVELOPMENT

It is noteworthy that the active constituent of marijuana, delta-9-THC, and a

closely related synthetic analog, nabilone, have been recently approved for

marketing in the United States as an antinauseant in cancer chemotherapy

treatment. This may mark the beginning of therapeutic drug development from

this field. It is quite obvious that the future development of clinically useful

drugs from this field will undoubtedly depend on the success achieved by

synthesis in introducing structural changes in the THC molecule, which will lead

to selectivity of pharmacological action. This presupposes the availability of

reliable animal models for screening this class of drugs. The shortcomings in this

area of pharmacological evaluation are numerous.

There is the difficulty of initial screening, i.e., the selection of compounds prior

to the more expensive and more sophisticated behavioral tests in dogs and

monkeys. Generally, the activity cage rotorod, the hypothermia, and the popcorn

tests in mice and rats are used for initial screening. What relevance these tests

9
have in identifying potential therapeutic candidates is presently not clear.

Primarily, these tests only give an indication that the compound is "cannabis-like"

and will show a similar profile. There is need for the development of other

rodent models, which will provide a lead to selectivity of pharmacological action.

For more advanced screening, the presently available pharmacological models for

cannabinoids, such as the dog ataxia and the monkey behavioral tests, do provide

a good indication of the psychoactive component of these compounds, since there

is a good correlation in extrapolating from animal data to humans. But a

problem arises in quantifying the extent of separation of psychoactive and other

CNS side effects from therapeutic effects.

Unless progress is made in this direction, one would be left with no recourse but

to rely on the importance of early studies in humans. The extent of separation

of undesirable CNS side effects from clinically useful effects could only be

meaningfully gauged in human studies. This point is well illustrated by the

results obtained in the clinical evaluation of analgesics. Nabitan and

levonantradol, two synthetic nitrogen analogs of THC, were both developed as

analgesics in cancer and postoperative pain, respectively, but the incidence of

side effects was higher than the animal data indicated (Razdan and Howes 1983).

In drug development from this field, the above clinical experience highlights the

point that, so far, very few cannabinoids have been studied in the clinic, and the

baseline data generated are not sufficient to provide proper guidelines for

extrapolation of animal data to humans in terms of dosages and side effect to

therapeutic ratios. When more compounds have been clinically evaluated, the

interpretation of animal data will become more meaningful.

It appears that further modifications in the structures of nabitan and

levonantradol will be necessary to develop useful analgesics from this field. It

should be emphasized that the potential exists for development of novel

analgesics from this field, as cannabinoids act at different receptors than the

opiates (analgesic action is not antagonized by naloxone), do not have the

physical dependence liability and respiratory depression properties of the strong

opiates, and are orally active with a long duration of action.

Anticonvulsants are one area where animal studies have provided a good

indication for a clinical candidate from this field (Razdan and Howes 1983 and

references cited therein). Of all the natural constituents of marijuana studied for

antiepileptic activity, CBD was proposed as the agent of choice. This is

primarily because in animal screens it was found to be devoid of marijuana-like

CNS effects, a finding that was confirmed in humans. In the only clinical study

(Razdan and Howes 1983 and references cited therein), CBD was reported to be

effective. This bears out the animal model data. Further confirmation of these

results and more clinical studies will no doubt be required before CBD’s place in

the treatment of epilepsy can be judged.

There are a few other instances (Adams et al. 1942; Hartley et al. 1978; Leite et

al. 1982) where animal models have indicated a clear separation or dissociation of

cannabinoid (psychotropic effects) from other therapeutic effects. However, it

remains to be seen if they can be substantiated in the clinic.

10
SAR AND MECHANISM OF ACTION OF CANNABINOIDS

Recent SAR studies have been directed toward elucidation of the mechanism of

action of cannabinoids. It is well known that in spite of extensive knowledge of

the pharmacological actions of cannabinoids, the mechanisms involved in

producing these effects have not been established (Dewey 1986; Martin 1986).

The effects of cannabinoids on the CNS are numerous and include CNS

depression, ataxia, psychoactive effects, hypothermia, analgesia, cardiovascular

effects, etc. They are known to alter neurotransmitter functionality, enzyme

activity, prostaglandin synthesis, membrane perturbation, etc. It is therefore

highly likely that multiple mechanisms are involved in producing the cannabinoid

behavioral syndrome.

Currently, two mechanisms are proposed which may account for the central

effects of cannabinoids, namely, general membrane perturbation, and direct

interaction with a specific cannabinoid receptor. If cannabinoids are producing

their effects solely by perturbation of membranes, there should not be a specific

antagonist but there may be a correlation between activity and membrane (lipid)

solubility. On the other hand, if they are interacting with a specific receptor and

binding sites, then there should be a strict SAR as well as a specific antagonist.

Strict structural requirements for cannabinoid behavioral activity are supported

by SAR studies. Stereospecificity has been demonstrated for delta-9-THC since

(-)-trans-delta-9-THC is many times more potent than the (+)-isomer in a

variety of tests (Mechoulam and Edery 1973; Jones et al. 1974; Martin et al.

1981). In the 3’-hydroxy-delta-9-THC series, the S-isomer is much more active

than the R-isomer (Martin et al. 1984b). Furthermore, greater potency is found

in cannabinoids with the trans ring junction (6a,10a) as compared to the cis

junction; the cis isomers are much less active (Martin et al. 1981).

Recent studies on the evaluation of cannabinoids for antagonistic properties have

shown that 10-N-ethylamino-CBD is a partial antagonist of delta-9-THCinduced antinociception but does not produce an antagonism of delta-9-THC’s

effects on spontaneous activity and rectal temperature in mice. In addition, it

does not alter the behavioral effects of delta-9-THC in dogs (Jorapur et al. 1985;

Martin et al., this volume). Therefore, the attenuation of delta-9-THC’s effects

by this compound appears to be limited to mouse antinociception only. Another

compound which has shown interesting properties in this regard is the exocyclic

analog, delta-9,11-THC. Pretreatment with delta-9,11-THC in mice did not

attentuate any of the effects of delta-9,11-THC. The study of antagonistic

effects of this compound in the dog was hampered by the fact that it had some

depressant properties in this behavioral test. However, in the monkey test,

pretreatment with delta-9,11-THC attentuated the delta-9-THC-induced ptosis,

sedation, ataxia, and operant behavior. The delta-9-THC-induced operant

suppression was decreased by as much as 25 times. This suggests a lead for a

possible antagonist and points out that the monkey may be a better animal model

suited for characterizing cannabinoid antagonistic activity than other behavioral

tests (Beardsley et al., in press; Martin et al. this volume).

Based on the work discussed above and additional SAR developed recently,

Martin and coworkers have proposed that the pharmacological properties unique

to the cannabinoid class of drugs are produced by the interaction of delta-9-THC

with specific receptors, while cannabinoid-induced CNS depression is a general

phenomenon produced by nonspecific membrane interactions (Martin et al. this

volume). Examination of the pharmacological profile in mice, dogs, and

1 1
monkeys of various cannabinoids, including the recently synthesized novel 11-,

12 beta-, and side-chain functionalized THCs, have led Martin’s group to classify

cannabinoids as follows: Analogs which produce the entire spectrum of

cannabinoid effects are grouped in class 1. CBD and many of its analogs appear

to be devoid of cannabinoid effects, but they are capable of producing weak

CNS depression and are grouped in class 2. Whereas, class 3 compounds are

potent CNS depressants that appear to lack other effects that are unique to

cannabinoids, and analogs in class 4 appear to exert selective CNS depression

(Martin et al. this volume). Therefore, recent studies indicate that structural

modifications in the cannabinoid molecule can lead to compounds which show

either a selective or a variety of pharmacological actions.

CONCLUSION

Despite the extensive knowledge about the pharmacological actions of

cannabinoids, the mechanisms involved in producing these effects have not been

established. Difficulties are encountered in correlating animal activity with

activity in man for this class of compounds, and this makes the task of clinical

evaluation and determination of therapeutic utility very complex. This is borne

out by the fact that the two most promising therapeutic areas for the

cannabinoids, antiemetic and antiglaucoma activities, were discovered

serendipitously without any preclinical pharmacology.

In spite of difficulties and complexity in the evaluation of these compounds,

progress is being made. Lately, several novel behavioral and drug discrimination

tests have been designed and introduced to test these compounds, and a

separation of several specific pharmacological actions has been noted in different

analogs. A basis for the pharmacological action of cannabinoids has been

proposed. Hopefully, with the continued progress in this field and the extended

clinical usage of delta-9-THC and nabilone, other areas of therapeutic use will

become evident, and the potential of this class of novel drugs will be realized.

REFERENCES

Adams, M.D.; Earnhardt, J.T.; Martin, B.R.; Harris, L.S.; Dewey, W.L.; and

Razdan, R.K. A cannabinoid with cardiovascular activity but no overt

behavioral effects. Experientia 33:1204-1205, 1977.

Adams, R. Marijuana. Harvev Lect 37:168-197, 1942.

Avison, A.W.D.; Morrison, A.L.; and Parkes, M.W. Tetrahydrodibenzopyran

derivatives isomeric with tetrahydrocannabinols. J Chem Soc (Lond) 952-

955, 1949.

Beardsley, P.M.; Scimeca, J.A.; and Martin, B.R. Studies on the agonistic activity

of delta-9,11-tetrahydrocannabinol in mice, dogs and rhesus monkeys and its

interactions with delta-9-tetrahydrocannabinol. J Pharmacol Exp Ther, in

press.

Dewey, W.L. Cannabinoid pharmacology. Pharmacol Rev 38:151-178, 1986.

Edery, H.; Grunfeld, Y.; Ben-Zvi, Z.; and Mechoulam, R. Structural

requirements for cannabinoid activity. Ann NY Acad Sci 191:40-53, 1971.

Edery, H.; Grunfeld, Y.; Porath, G.; Ben-Zvi, Z.; Shani, A.; and Mechoulam, R.

Structure-activity relationships in the tetrahydrocannabinol series.

Arzneimittelforsch 22:1995-2003, 1972.

Gayer, H. Pharmakologische Wertbestimmung von Oriontatischem Hashish und

herba. Cannabis, Indicae. Arch Exp Pathol Pharmacol 129:312-318, 1928.

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Grunfeld, Y., and Edery, H. Psychopharmacological activity of the active

constituents of hashish and some related cannabinoids. Psychopharmacologia.

14:200-210, 1969.

Hartley, J.P.R.; Nogrady, S.G.; Seaton, A.; and Graham, J.D.P. Bronchodilator

effect of delta-9-THC. Br J Clin Pharmacol 5:523-524, 1978.

Hollister, L.E., and Gillespie, H.K. delta-8- and delta-9-Tetrahydrocannabinol.

Comparison in man by oral and intravenous administration. Clin Pharmacol

Ther 14:353-357, 1973.

Isbell, H.; Gorodetsky, C.W.; Jasinski, D.; Clauseen, U.; Spulak, F.V.; and Korte,

F. Effects of (-) delta-9-trans-tetrahydrocannabinol in man.

Psychopharmacologia 11:184-188, 1967.

Jones, G.; Pertwee, R.G.; Gill, E.W.; Paton, W.D.M.; Nilsson, I.M.; and Agurell,

S. Relative pharmacological potency in mice of optical isomers of delta-1-

tetrahydrocannabinol. Biochem Pharmacol 23:439-446, 1974.

Jorapur, V.S.; Khalil, Z.H.; Duffley, R.P.; Razdan, R.K.; Martin, B.R.; Harris,

L.S.; and Dewey, W.L. Hashish: Synthesis and central nervous; activity of

some novel analogs of cannabidiol and oxepin derivatives of 9

-tetrahydrocannabinol. J Med Chem 28:783-787, 1985.

Leite, J.R.; Carlini, E.A.; Lander, N.; and Mechoulam, R. Anticonvulsant effects

of the (-) and (+) isomers of cannabidiol and their dimethylheptyl homologs.

Pharmacoloay 24:141-146, 1982.

Loev, B.; Bender, P.E.; Dowalo, F.; Macko, E.; and Fowler, P.J. Cannabinoids.

Structure-activity studies related to 1,2-dimethylheptyl derivatives. J Med

Chem 16:1200-1206, 1973.

Loewe, S. Bioassay by direct potency estimation. Science 106:89-91, 1947.

Martin, B.R. Cellular effects of cannabinoids. Pharmacol Rev 38:45-74, 1986.

Martin, B.R.; Dewey, W.L.; Harris, L.S.; Beckner, J.; Wilson, R.S.; and May, E.L.

Marijuana-like activity of new synthetic tetrahydrocannabinols. Pharmacol

Biochem Behav 3:849-853, 1975.

Martin, B.R.; Balster, R.L.; Razdan, R.K.; Harris, L.S.; and Dewey, W.L.

Behavioral comparison of the stereoisomers of tetrahydrocannabinols. Life

Sci 29:565-574, 1981.

Martin, B.R.; Harris, L.S.; and Dewey, W.L. Pharmacological activity of delta-9-

THC metabolites and analogs of CBD, delta-8-THC, and delta-9-THC. In:

Agurell, S.; Dewey, W.L.; and Willette, R.E., eds. The Cannabinoids;

Chemical, Pharmacologic, and Therapueutic Aspects. Orlando, FL: Academic

Press, Inc., 1984a. pp. 523-544.

Martin, B.R.; Kallman, M.J.; Kaempf, G.F.; Harris, L.S.; Dewey, W.L.; and

Razdan, R.K. Pharmacological potency of R- and S-3’-hydroxy-delta-9-

tetrahydrocannabinoh Additional structural requirements of cannabinoid

activity. Pharmacol Biochem Behav 21:61-65, 1984b.

Mechoulam, R., and Edery, H. Structure activity relationships in the

cannabinoid series. In: Mechoulam, R., ed. Marijuana: Chemistrv,

Pharmacology, Metabolism, and Clinical Effects. New York: Academic

Press, Inc., 1973. pp. 101-136.

Mechoulam, R.; Shani, A.; Edery, H.; and Grunfeld, Y. Chemical basis of

hashish activity. Science 169:611-612, 1970.

Razdan, R.K. Structure-activity relationships in cannabinoids. Pharmacol Rev

38:75-149, 1986.

Razdan, R., and Howes, J.F. Drugs related to tetrahydrocannabinol. Med Res

Rev 3:119-146, 1983.

Scheckel, C.L.; Boff, E.; Dahlen, P.; and Smart, T. Behavioral effects in

monkeys of racemates of biologically active marijuana constituents. Science

160:1467-1469, 1968.

Todd, A.R. Hashish. Experientia 2:55, 1946.

13
ACKNOWLEDGMENT

Professor Billy Martin provided helpful suggestions on the content and

organization of this article.

AUTHOR

Raj K. Razdan, Ph.D.

ORGANIX Inc.

600 West Cummings Park

Woburn, Massachusetts 01801

14
Stereochemical Requirements for

Cannabimimetic Activity

Raphael Mechoulam, Ph.D.; Naphtali Lander, Ph.D.; Morris

Srebnik, Ph.D.; Aviva Breuer, Ph.D.; Mark Segal, Ph.D.;

Jeffery J. Feigenbaum, M.Sc.; Tobjorn U. C. Jarbe, Ph.D.;

and Paul Consroe, Ph.D.

About 15 years ago, we formulated some tentative rules for cannabimimetic

structure-activity relationships (SAR) (Edery et al. 1971; Mechoulam and Edery

1973). These rules were based on work our group had done since 1964, when we

isolated and elucidated the structure of delta-1-tetrahydrocannabinol (delta-1-

THC) (Gaoni and Mechoulam 1964), as well as on research by numerous other

groups. Most of our work was done with rhesus monkeys; much of the work by

the other groups was done with either rodents or dogs. However, gratifyingly,

the results obtained with different species were qualitatively comparable.

Most of the rules have withstood the erosion of time, although exceptions have

been noted and certain refinements have to be made.

The generalisations listed below apply today:

1. A dihydro-benzopyran type structure with a hydroxyl group at the 3’

aromatic position and an alkyl group on the 5’ aromatic position seems to be a

requirement. Opening of the pyran ring generally leads to complete loss of

activity.

However, several major exceptions have been found, such as compounds (1) and

(2) and some of their derivatives (Johnson and Melvin 1986). Hopefully, future

work will throw light on these discrepancies,

2. The aromatic hydroxyl group has to be free or esterified. Blocking of the

hydroxyl group as an ether inactivates the molecule. It is possible that the esters

are actually inactive but undergo hydrolysis in vivo to the free phenols. Thus,

delta-1-THC acetate, when tested in vitro, shows negligible activity in

biochemical reactions in which delta-1-THC is active (Banerjee et al. 1975).

15
3. An all carbon side chain on C-5’ is not an absolute requirement. The side

chain may contain an etheric oxygen (e.g., compound 3) (Loev et al. 1973).

4. 7-Hydroxy THCs, which are major metabolites, are very potent

cannabimimetics. Monohydroxylation on other position of the terpene ring also

usually leads to active derivatives. Dihydroxylation generally causes loss of

activity. Further oxidation of the C-7 hydroxyl group to a carboxyl group causes

inactivation.

5. Hydroxylation on the C-1" of the side chain abolishes activity. Hydroxylation

at the other side chain carbons retains activity with hydroxylation on C-3"

potentiating activity. Some of these hydroxylated compounds have been detected

as major metabolites.

6. Alkylation on the C-4’ aromatic position retains activity; alkylation on the C6’ position eliminates activity. Electronegative groups, such as carbonyl or

carboxyl, at either C-4’ or C-6’ eliminate activity.

7. The double bond isomers of THC show the following order of activity in

humans: delta-1-THC> delta-6-THC> delta-3-THC (Hollister 1974). Delta-5-

THC and delta-7-THC are inactive in animal tests, while delta-2-THC and

delta-4-THC have not been tested yet.

8. The 7-methyl group is not an absolute requirement for activity: 7-nor-delta1-THC (4) and 7-nor-delta-6-THC (5) are active in dogs (Wilson and May 1974).

9. The terpenoid ring may be exchanged by some heterocyclic systems (Pars et

al. 1977; Lee et al. 1983).

10. The 1,1- or 1,2-dimethyl heptyl (DMH) side chain strongly potentiates the

cannabimimetic activity of compounds which have low activity in the n-pentyl

series.

The above rules are, in reality, approximations. In the cannabinoid series, for

example, compounds which are considered inactive in some tests frequently show

low activity in other cannabimimetic tests.

The above SAR findings do not cover stereochemical requirements for

cannabimimetic activity. In the present summary, we will bring up to date the

SAR in this area, with emphasis on as yet unpublished material from our

laboratories.

C-1 EPIMERS

Reduction of the double bond in delta-1- or delta-6-THC leads to the formation

of two C-1 epimers (Gaoni and Mechoulam 1966). The equatorial epimer, in

which the methyl group on C-1 is almost in the plane of the hexahydrocannabinol molecule (compound 6a), is about as active in the rhesus monkey as

delta-6-THC and is about 20 times more active than the axial epimer (7a), in

which the methyl group is below the plane (Mechoulam et al. 1980).

The same relationship is observed when the C-1 substituent is an acetoxymethyl

group: epimer (6b), in which the C-1 substituent is equatorial, is active in

monkeys at doses of 0.5 mg/kg while the axial epimer (7b) is inactive at doses 10

times higher (Mechoulam et al. 1980).

16
The same trend is observed in drug discrimination tests. Rats and pigeons

trained to discriminate between the presence and absence of the effects of delta1-THC (3 and 0.56 mg/kg, respectively) were tested for generalization with the

two epimeric 7-hydroxy-hexahydrocannabinols (6c and 7c). Both epimers

generalized with delta-1-THC in rats as well as in pigeons (table 1) (Jarbe et al.

1986). As expected, the equatorial isomer (6c) was considerably more active than

the axial one (7c). Surprisingly, compound (6c) was only 5 to 7 times more

active in the rat than (7c), while in the pigeon, compound (6c) was 85 times

more active. Apparently, species specificity in this test is very pronounced.

TABLE 1

Discrimination of 7-OH-Hexahydrocannabinols

(6c and 7c) to delta-1-THC

Hrs

Compound Compound

THC (6c) (equat) (7c) (axial)

Rats (i.p.)

0.5

1.5

4.5

0.85 (0.94)

1.07 (0.94)

4.78 (0.79)

0.24 (0.95)

0.44 (0.997)

Pigeons (i.m.)

1.58 (0.98)

2.16 (0.96)

0.5 0.53 (0.92) 0.03 (0.99) 2.60 (0.58)

1.5 0.16 (0.91) 0.02 (0.87) 1.72 (0.97)

4.5 0.25 (0.94)

9.0 1.21 (0.70)

Median dose (mg/kg) effect estimates, ED50, according to logarithmic regression

analysis and within brackets corresponding fits (r) for the regressions are given.

Absence of an estimate (-) means no value in the dose-generalization curve

determination was above 50% drug appropriate responding (% RDP). (Jarbe et

al. 1986)

The above experimental data point out that, in the absence of other molecular

changes, the planarity at the C-1 position determines cannabimimetic activity.

The above tentative rule does not necessarily apply to other biological activities.

Thus, the axial 1-alpha-7-dimethylamino-hexahydrocannabinol (8) causes intense

yawning in monkeys, while the equatorial 1-beta-7-dimethylamino-hexahydrocannabinol (9) is inactive. Neither (8) nor (9) are cannabimimetic (Edery et al.

1984).

SUBSTITUTION ON C-1, C-2 VERSUS SUBSTITUTION ON C-1, C-6

Two groups of THC-type cannabinoids have been synthesized which differ only

in that the chemical groupings in one of them at C-1, C-2 are situated at C-1,

17
C-6 in the other (but retain their stereochemistry). They show almost equivalent

cannabimimetic activity.

Thus, 2-alpha-hydroxy-1-beta-hexahydrocannabinol (10) is essentially inactive,

as is 6-alpha-hydroxy-1-beta- hexahydrocannabinol (11); 2-beta-hydroxy-1-

alpha- and 6-beta-hydroxy-1-alpha- hexahydrocannabinol (12 and 13,

respectively) are also inactive. However, both 2-alpha-hydroxy-1-alphahexahydrocannabinol (14) and 6-alpha-hydroxy-1-alpha-hexahydro-cannabinol

(15) are active at the same dose levels (Mechoulam et al. 1980).

A biochemical rationalization of the above data is not obvious at present.

SUBSTITUTION ON C-6 AND C-5

Several groups have compared the cannabimimetic activity of 6-beta-hydroxydelta-1-THC (16) and 6-alpha-hydroxy-delta-1-THC (17) (for a review, see

Narimatsu et al. 1985). In man, the 6-beta-epimer (16), in which the hydroxyl

group is axial, is more active than (17); but the reverse has been observed in

mice and rabbits, and they are equiactive in rhesus monkeys. Recently, the

alpha-epimer (17) was again found to be less active than the 6-beta-epimer (16)

in a mouse catalepsy test but more potent in a test for hypothermia and

equipotent in barbiturate synergism.

5-beta-Hydroxy-delta-6-THC (18) and 5-alpha-hydroxy-delta-6-THC (19) have

been tested in rhesus monkeys only (Mechoulam et al. 1972). The 5-beta-epimer

(18), in which the hydroxyl group is equatorial, was six to eight times less active

than the 5-alpha-epimer (19), in which the hydroxyl group is axial.

The only conclusion that can be drawn (though somewhat fatuous) is that the

level of cannabimimetic activity of THCs hydroxylated at some allylic positions

seems to depend on the test used.

SUBSTITUTION ON THE SIDE CHAIN

A Pfizer group (Milne et al. 1980; Johnson and Melvin 1986) has found that

diastereoisomer A (20) is at least 10 times more potent than its side chain

counterpart, diastereoisomer B (21). in analgetic tests. As analgetic activity in

this series parallels cannabimimetic activity, one can presume that the same ratio

of activity will be observed in cannabimimetic tests.

Martin et al. (1984) have reported that (3"S)-3"-hydroxy-delta-1-THC (22) is

circa (ca) five times more active than delta-1-THC and is about seven times

more active than the R epimer (23) in THC discrimination in rats. Approximately the same ratio of activity was observed in dog ataxia tests; in

hypothermia, (22) and (23) are surprisingly equiactive. However, Martin and

coworkers pointed out that "calculation of exact potency ratios is complicated

somewhat by the lack of absolute purity of the isomers."

3,4-CIS-THCs

Racemic 3,4-cis-delta-1-THC (24), racemic 3,4-cis-delta-6-THC (25), and (+)-

3,4-cis-delta-1-THC appear to be essentially inactive in several animal tests

(Edery et al. 1971; Uliss et al. 1975; Martin et al. 1981).

18
CANNABINOID ENANTIOMERS

Delta-3-THC

This cannabinoid in its racemic form was synthesized in the early 1940s.

Although attempts were made then to obtain the enantiomers in a pure form,

they were only partially successful. Recently, we were able to synthesize both

(1S)-delta-3-THC (26), [alpha]D - 117° and (1R)-delta-3-THC (27). [alpha]D +

114° with absolute optical purity (Srebnik et al. 1984). These enantiomers have

now been tested in human volunteers (Hollister et al., in press). Subjects

received progressively increasing intravenous doses of (1S)-delta-3-THC (26) and

(lR)-delta-3-THC (27), beginning with 1 mg (in 1 ml ethanol) followed by

progressive doubling of doses until. definite effects were observed.

Cannabimimetic effects with the (1S) epimer were noted at doses of 8 mg or

higher. No effects were noted with the (1R) epimer. (1S)-delta-3-THC (26) is

thus estimated to have a potency from one-third to one-sixth that of delta-1-

THC.

The behavioral cannabimimetic effects observed with (lS)-delta-3-THC (26)

were indistinguishable from those caused by delta-1-THC. As with delta-1-

THC, an increase in the pulse rate was also noted. These experiments are the

first ones with THC enantiomers in human subjects.

(-)-(3R,4R)-THCs versus (+)-(3S,4S)-THCs

The natural delta-1-THC and delta-6-THC have a (3R,4R) configuration and a

negative rotation. The synthetic route, which was developed by our laboratory

for delta-1-THC and delta-6-THC nearly 20 years ago (Mechoulam et al. 1967),

makes possible also the synthesis of the unnatural (3S,4S) enantiomers as the

starting material; verbenol (28) is available in both enantiomeric forms. This

sequence has been widely used for the preparation of (+) cannabinoids and

apparently is the only one still practical for the synthesis of (+)-delta-1-THC.

(+)-Verbenol on conden- sation with olivetol leads to (29), which on ring opening

leads to (+)-(3S,4S)-delta-6-THC (30), which can be converted with ease into

(+)-(3S,4S)-delta-1-THC (31).

In several tests for cannabimimetic activity (Edery et al. 1971; Jones et al. 1974;

Martin 1986), (+)-delta-1-THC (31) was ca 13 to 230 times less active than the

(-)-isomer. These results indicate pharmacologic enantiomeric preference rather

than absolute stereoselectivity. Indeed, Martin pointed out that, "while

cannabinoid SAR supports the concept of a specific cannabinoid receptor, a

disconcerting element is the apparent lack of greater stereoselectivity in some

animal models." However, because the starting material (+) verbenol was not

necessarily stereochemically pure, this conclusion is tentative at best.

Recently in our laboratories, we were able to prepare a pair of crystalline

enantiomeric cannabinoids (compounds 32 and 33) which could be recrystallized

to absolute enantiomeric purity. Their synthesis is based on myrtenol, which can

be obtained in both enantiomeric forms. The primary allylic group is first

blocked as a pivalate ester (reaction a) (compound 34). If the blocking group is

the more mundane acetate, the penultimate reaction (reaction e) proceeds in an

undesirable fashion leading to the benzofuran (36). Oxidation of (34) leads to

the crystalline ketone (35) which can be highly purified by crystallization. The

synthetic sequence then follows the standard procedure described above, leading

ultimately to the crystalline (+)-(3S,4S)-7-hydroxy-delta-6-THC-DMH (33). The

19
configuration at C-3 and C-4 depends on the absolute configuration of

myrtenol (-) mrytenol leads to the (-) enantiomer (32); (+) myrtenol leads to the

(+) enantiomer (33).

Compounds (32) and (33) were tested in several independent cannabimimetic tests

in Jerusalem, in Uppsala, and in Tucson. In all tests, the (-) enantiomer (32)

exhibited potent cannabimimetic activity; the (+) enantiomer was inactive.

In Jerusalem, the compounds were examined in the mouse ring test. The (-)

enantiomer (32) was ca 100 times more active than natural delta-6-THC: the

ED50 of delta-6-THC was 5 mg/kg; the ED50 of (32) was 0.05 mg/kg. The (+)

enantiomer (33) was inactive up to 20 mg/kg. In Tucson, the same type of

results were observed in the rotorod neurotoxicity test in rats (see table 2). The

(-)-enan-tiomer (32) was ca 260 times more potent than natural (-)-delta-6-THC;

the (+)-enantiomer (33) was inactive at doses ca 2,000 times higher than those of

the ED50 of the (-)-enantiomer. In Uppsala, the compounds were tested in

generalization tests with rats and pigeons. (-)-7-OH-delta-6-THC-DMH (32)

was ca 87 times more active than natural delta-1-THC in the rat and ca 73 times

more active in the pigeon (see table 3). The (+)-enantiomer (33) was inactive at

doses ca 1,000 times and ca 4,500 times (for rats and pigeons, respectively)

higher than those of the ED50 of the (-) enantiomer (32).

TABLE 2

Dose-Response Parameters of THC Stereoisomers

in Rotorod (ROT) Neurotoxicity Tests in Ratsa

Drug

ROT-TD5 0

(95% CL)

(-)-7-OH-delta-6-THC-DMH (32) 0.007

(0.005-0.009)

(+)-7-OH-delta-6-THC-DMH (33)

(-)-delta-6-THC

Not activeb

1.85

(0.98--3.45)

(+)-delta-6-THC 21.66

(19.81-23.68)

(-)-delta-6-THC-DMH 0.034

(0.031-0.040)

(+)-delta-6-THC-DMH 0.53

(0.37-0.76)

a

 Median toxic (TD50) dose and 95% confidence limits (CL) are in mg/kg.

b

 At 1.0, 7.0, and 15.0 mg/kg; higher doses were not tested due to insufficient

quantity of drug.

20
TABLE 3

Generalization Tests with Various Doses of

(-)-delta-1-THC, (-)-7-OH-delta-6-THC-DMH, and

(+)-7-OH-delta-6-THC-DMH in Rats and Pigeons

Trained to Discriminate Between the Presence and

Absence of the Effects Induced by delta-1-THC

Dose Interval

Range Range

Compound n (mg/kg) (hr) (mg/kg)

(-)-delta-1-THC (R) 14 0.30-5.6 0.5-6.5 0.85

(-)-delta-1-THC (P) 7 0.10-0.56 0.5-9.0 0.16

(-)-7-OH-delta-6-

THC-DMH (32) (R) 1 1 0.003-0.03 0.5-6.5 0.0098

(-)-7-OH-delta-6-

THC-DMH (32) (P) 5-7 0.0001-0.0056 0.5-9.0 0.0022

(+)-7-OH-delta-6-

THC-DMH (33) (R) 4-9 3.0- 10.0 0.5-6.5 >10.0

(+)-7-OH-delta-6-

THC-DMH (33) (P) 4 3.0-10.0 0.5-9.0 >10.0

r

0.94

0.91

0.99

0.99

-

-

The animals, rats (R) and pigeons (P), were trained to discriminate between

(-)-delta-1-THC (3 mg/kg, rats; and 0.56 mg/kg, pigeons) and vehicle

administered 0.5-hr i.p. (rats) or 0.5-hr i.m. (pigeons) prior to session onset. The

ED50 values are based on the % RDP (percentage of responding to drug, THC,

associated position) during the test probe yielding the highest % RDP, i.e., the

lowest ED50 value. n = the number of observations on which data points are

based. ED50 values refer to logarithmic regression analysis, and r refers to the

correlation coefficient for the regression. The solubilization vehicle consisted of

5% (v/v) of propylene glycol, 2% Tween-80 (v/v), and 93% saline. In some

cases, 4% Tween-80 and 91% saline were used.

The above results show that (-)-7-hydroxy-delta-6-THC-DMH (32) is one of the

most active cannabimimetic substances prepared so far and that, apparently,

cannabimimetic activity resides exclusively in the (-)-(3R,4R)-enantiomer. We

assume that the results with the enantiomers (32) and (33) are also valid for the

enantiomers of delta-1-THC, i.e., that our results are of a general nature. This

assumption is based on the well-documented increase of cannabimimetic activity

on the exchange of the pentyl chain with a DMH chain and on hydroxylation of

the C-7 methyl group, which are the two features present in (32) and (33) and

absent in delta-1-THC.

The results of animal tests of the types described above with delta-1-THC, delta6-THC, and other cannabinoids have been shown to parallel activity in man

21
TABLE 4

(+) 7-Hydroxy- -THC-DMH (33) and Morphine: Comparative Doses Producing 50 Percent

of the Maximum Possible Analgesic Effect

MPE50 (mg/kg sc) at Time of Estimated Peak Activity of Compounda,b

Compound Mouse (Sabra Strain) Bat (Sabra Strain)

Pawc Acetic Acidd HoteTail f

Tail f Hote

Clamp Writhing Plate Clamp Clamp Plate

(+) 7-OH- -THC- 0.9 0.2 0.1 0.8 4.9 0.3

DMH (33)g

Morphine HCLh

4.2 1.3 1.6 4.3 5.5 2.0

aAll morphine injected animals observed 1 hr postinjection.

bAll (+) 7-OH- -DMH injected animals observed 2 hr postinjection.

cHaffner et al. (1929); an adaptation

d

Sophia et al. (1973)

- 30 sec cutoff.

- 20 min observation.

eWoolfe and MacDonald (1944)

fBianchi and Franceschini (1954)

- 30 sec cutoff.

- 30 sec cutoff.

gDissolved in 5% EtOH, 5% emulphor 620, and 90% ddH2O, with 5x10-4M CuCl2

 added subsequently.

hDissolved in 0.9% saline.
(Hollister 1974). Hence, we assume that the results with (32) and (33) likewise

indicate parallel activity in man. If this correct, cannabimimetic activity in man

has a strict stereochemical requirement, which indicates a probable interaction

with a chiral biological system (enzyme, receptor site, etc.) and not just an

unspecific action due to the high lipid solubility of the cannabinoids.

ANALGETIC ACTION OF THE (+) ENANTIOMER (33)

As mentioned above, the cannabimimetic SAR does not necessarily parallel the

SAR of cannabinoids for other biological or therapeutic effects. Indeed, we have

found now that enantiomer (33), which (as noted above) does not cause

cannabimimetic effects, is a potent analgetic particularly in the presence of

cupric ions. In table 4, we present the results of several analgetic tests. In all

tests, (33) shows activity at (or above) the potency level of morphine. However,

as previously noted with delta-1-THC and other cannabinoids, the dose-response

curve is not sharp (as with morphine) but flat. Surprisingly, the level of

analgetic potency is retained over a period of 3 to 4 days. In some tests, activity

actually falls beyond a certain concentration, the dose-response curve assuming

an inverted U shape.

The above results show that it is possible to achieve complete dissociation

between cannabimimetic effect and analgetic action.

These preliminary results may be of considerable therapeutic value because

cannabinoids generally lack many of the side effects of opiates, such as high

addiction liability and respiratory depression.

SUMMARY

The SAR of cannabimimetic activity in the cannabinoid series are reviewed with

emphasis on the stereochemical requirements. Some new results are presented.

The most important are that a, in humans, (-)-(1S)-delta-3-THC is much more

active than (+)-(1R)-delta-3-THC; and b, with the 7-OH-delta-6-THC DMH

enantiomers (32) and (33), the activity in several animal species resides

completely in the (-)-(3R, 4R) enantiomer (32). the difference between the two

enantiomers being up to several thousand times. The (3R,4R)-enantiomer (32) is

much more active than delta-1- or delta-6-THC in animal tests, the exact level

of activity depending on the test employed. The cannabimimetically inactive (+)-

(3S,4S) enantiomer (33) was shown to be a potent analgetic in several animal

tests. Thus, a complete dissociation between the cannabimimetic and the

analgetic effects in a cannabinoid has been achieved, apparently for the first

time.

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neurotransmitter uptake in rat brain synaptosomes. J Pharmacol Exp Ther

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Bianchi, C., and Franceschini, J. Experimental observations on Haffner’s method

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Edery, H.; Grunfeld, Y.; Ben-Zvi Z.; and Mechoulam, R. Structural

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Edery, H.; Porath, G.; Mechoulam, R.; Lander, N.; Srebnik, M.; and Lewis, N.

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Gaoni, R., and Mechoulam, R. Isolation, structure and partial synthesis of an

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Gaoni, Y., and Mechoulam, R. The isomerization of cannabidiol to

tetrahydrocannabinols. Tetrahedron 22:1481-1488, 1966.

Haffner, F. Experimentelle Prufung schmerzstillender Mittel. Deutsch Med

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Hollister, L.E. Structure-activity relationships in man of cannabis constituents

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Hollister, L.E.; Gillespie, H.K.; Mechoulam, R.; and Srebnik, M. Human

pharmacology of 1S and 1R enantiomers of delta-3-tetrahydrocannabinol.

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Jarbe, T.U.C.; Hiltunen, A.J.; Lander, N.; and Mechoulam, R. Cannabimimetic

activity (delta-1-THC cue) of cannabidiol monomethylether and two

stereoisomeric hexahydrocannabinols in rats and pigeons. Pharmacol Biochem

Behav 25:393-399, 1986.

Johnson, M.R., and Melvin. L.S. The discovery of nonclassical cannabinoid

analgetics. In: Mechoulam, R., ed. Cannabinoids as Therapeutic Agents.

New York: CRC Press, 1986. pp. 121-145.

Jones, G.; Paton, W.D.M.; Nilsson, I.M.; and Agurell, S. Relative

pharmacological potency in mice of optical isomers of delta-1-

tetrahydrocannabinol. Biochem Pharmacol 23:439-446, 1974.

Lee, C.M.; Zaugg, H.E.; Michaels, R.J.; Dren, A.T.; Plotnikoff, N.P.; and Young,

P.R. New azacannabinoids highly active in the central nervous system. J

Med Chem 26:278-280, 1983.

Loev, B.; Bender, P.E.; Dowalo, F.; Macko, E.; and Fowler, P.J. Cannabinoids.

Structure activity studies related to 1,2-dimethyl heptyl derivatives. J Med

Chem 16:1200-1206, 1973.

Martin, B.R. Cellular effects of cannabinoids. Pharmacol Rev 38:45-74, 1986.

Martin, B.R.; Balster, R.L.; Razdan, R.K.; Harris, L.S.; and Dewey, W.L.

Behavioral comparisons of the stereoisomers of tetrahydrocannabinols. Life

Sci 29:565-574, 1981.

Martin, B.R.; Kallman, M.J.; Kaempf, G.F.; Harris, L.S.; Dewey, W.L.; and

Razdan, R.K. Pharmacological potency of R- and S-3’-hydroxy-delta-9-

tetrahydrocannabinol: Additional structural requirement for cannabinoid

activity. Pharmacol Biochem Behav 21:61-65, 1984.

Mechoulam, R., and Edery, H. Structure activity relationships in the

cannabinoid series. In: Mechoulam, R., ed. Marijuana Chemistry,

Metabolism, Pharmacology and Clinical Effects. New York: Academic Press,

1973. pp. 101-136.

Mechoulam, R.; Braun, P.; and Gaoni, Y. A stereospecific synthesis of (-)-delta1- and (-)-delta-6-tetrahydrocannabinols. J Am Chem Soc 89:4552-4554,

1967.

Mechoulam, R.; Lander, N.; Varkony, T.H.; Kimmel, I.; Becker, O.; Ben-Zvi, Z.;

Edery, H.; and Porath, G. Stereochemical requirements for cannabinoid

activity. J Med Chem 23:1068-1072, 1980.

Mechoulam, R.; Varconi, H.; Ben-Zvi, Z.; Edery, H.; and Grunfeld, Y.

Synthesis and biological activity of five tetrahydrocannabinol metabolites. J

Am Chem Soc 94:7930-7931, 1972.

Milne, G.M.; Koe, B.K.; and Johnson, M.R. Stereospecific and potent analgetic

activity for nantradol - A structurally novel, cannabinoid related analgetic.

In: Harris, L.S., ed. Problems of Drug Dependence, 1979. National Institute

on Drug Abuse Research Monograph 27. DHEW Pub. No. (ADM) 80-901.

Washington, D.C.: Supt. of Docs., U.S. Govt. Print. Off., 1980. pp. 84-92.

24
Narimatsu, S.; Matsubara, K.; Shimonishi, T.; Watanabe, K.; Yamamoto, I.; and

Yoshimura, H. Pharmacological activities in the mouse of delta-9-

tetrahydro-cannabinol metabolites oxidized at the 8-position. Chem Pharm

Bull 33:392-395, 1985.

Pars, H.G.; Razdan, R.K.; and Howes, J.F. Potential therapeutic agents derived

from the cannabinoid nucleus. In: Simmonds, A.B., ed. Advances in Drug

Research. Vol II. New York: Academic Press, 1977. pp. 97-189.

Sofia, R.D.; Nalepa, S.; Harakel, J.; and Vassar, H.B. Anti-edema and analgesic

properties of delta-9-tetrahydrocannabinol (THC). J Pharmacol Exp Ther

186:646-655, 1973.

Srebnik, M.; Lander, N.; Breuer, A.; and Mechoulam, R. Base catalysed double

bond isomerizations of cannabinoids: Structural and stereochemical aspects.

J Chem Soc Perkin Trans I 2881-2886, 1984.

Uliss, D.B.; Razdan, R.K.; Dalzell, H.C.; and Handrick, G.R. Hashish: Synthesis

of dl-delta-1,6-cis-tetrahydrocannabinol. Tetrahedron Lett 4369-4372, 1975.

Wilson, R.S., and May, E.L. 9-Nor-delta-8-tetrahydrocannabinol, a cannabinoid

of metabolic interest. J Med Chem 17:475-476, 1974.

Wilson, R.S.; May, E.L.; Martin, B.R.; and Dewey, W.L. 9-Nor-9-hydroxyhexahydrocannabinols, synthesis, some behavioral and analgesic properties. J Med

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Woolfe, G., and MacDonald, A. The evaluation of the analgesic action of

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ACKNOWLEDGMENTS

Supported in Tucson and Jerusalem by NIH grant 5 R01 NS-15441 and in

Uppsala by grants from the Swedish Medical Research Council (5757) and the

Swedish Council for the Planning and Coordination of Research (84/208:2).

AUTHORS

Raphael Mechoulam, Ph.D.

Naphtali Lander, Ph.D.

Morris Srebnik, Ph.D.

Aviva Breuer, Ph.D.

Mark Segal, Ph.D.

Jeffery J. Feigenbaum, M.Sc.

Department of Natural Products, Medical Faculty

Hebrew University of Jerusalem

Jerusalem 91 120, Israel

Tobjorn U.C. Jarbe, Ph.D.

Department of Psychology

University of Uppsala, Box 227

S-751 04 Uppsala, Sweden

Paul Consroe, Ph.D.

Department of Psychology/Toxicology

College of Pharmacy, University of Arizona

Tucson, Arizona 85721, U.S.A.

25
26
27
28
29
30
Structure-Activity Relationships of

Tricyclic and Nonclassical Bicyclic

Cannabinoids

Lawrence S. Melvin, Ph.D., and M. Ross Johnson, Ph.D.

INTRODUCTION AND SUMMARY

The usefulness of marihuana has contributed to folklore and medicine for many

centuries (Li 1974; Lemberger 1980). However, a good medicinal chemical rationale

for the mechanism of action and structure-activity relationships (SAR) of

tetrahydrocannabinol ( -THC, 1) has not been forthcoming. Our interest in

attempting to dissect out the many therapeutic indications reported for THC led

us to question the chemical dogma of cannabinoids that the tricyclic pyrancontaining ring system of -THC was an absolute necessity for biological activity.

After the reported (Wilson et al. 1976) enhanced analgetic activity of 9-nor-9

hydroxyhexahydrocannabinol (HHC, 2), we became especially interested in locating

and defining the pharmacophore for analgesia in the -THC and HHC structures.

Based on the hypothesis that analgetic activity is a dissociable feature of the

cannabinoid molecule, we examined modifications of the side chain, the phenolic

moiety, and, most significantly, structures that lack the benzopyran ring present in

THC and HHC. In our initial studies, we found that a new grouping, the 1-methyl-4-

phenylbutyloxy C-3 side chain (3l), elaborates a unique lipophilic region.

Replacement of the phenol substituent produced several derivatives which retain

analgetic activity in the codeine potency range. Introduction of a weakly basic

nitrogen at C-5 and deletion of the axial methyl group in the B ring, two structural

changes forbidden by traditional cannabinoid SAR, resulted in a unique family of

benzoquinolines with potent analgetic activity. The prototype of this series,

nantradol (4), exhibits potent and enantiospecific analgetic and antiemetic activity.

These early studies supported our three-point receptor model and led to the

synthesis of nonclassical cannabinoids.

31
Synthesis of the first AC-bicyclic cannabinoid followed from our observation that the

pyran ring of HHC was not a requirement in this structural class for expression of

biological activity. Together, this observation and speculation about the necessity of

the lipophilic side chain, phenol and alcohol for biological activity was confirmed

with synthesis of 3-Z-[4-(1,1-dimethylheptyl)-2-hydroxyphenyl]cyclohexanol,

CP-47,497 (5). This compound was shown to possess a biological profile and potency

similar to HHC. Such activity and potency was highly dependent on the side chain but

was not as influenced by substitution in the cyclohexanol ring. Further structural

elaboration and development of SAR around CP-47,497 led to a more potent ACbicyclic derivative, (-)-3-Z-[4-(1,1-dimethylheptyl)-2-hydroxyphenyl]-4-E-(3-hydroxypropyl)cyclohexanol. The degree of importance of the three hydroxyl groups in this

structure was explored and only the phenolic hydroxyl was found to be an absolute

necessity. The two alcohol groups were seen to be modifiers of activity and potency.

Structural optimization of this derivative of CP-47,497 was achieved by incorporation

of the hydroxypropyl chain as a new fused ring and thus the synthesis of the first

ACD-tricyclic cannabinoid, (-)-1,2,3,4,5,5a ,6,7,8,8a -decahydro-4 [4-(1,1-dimethylheptyl)-2-hydroxyphenyl]-6 hydroxymethylnaphthalen-2 ol. This rigid molecule

again exhibits an increase in potency and significantly shows total enantiospecificity

in favor of the levorotatory enantiomer. The combination of potency, stereospecificity, enantiospecificity, and well-defined SAR leads us to conclude that the

cannabinoidsexertat least their analgetic effect by interaction with a discreet, and as

yet unknown, receptor site.

ABC-TRICYCLICS

Analgesia and the Side Chain

Adams and his co-workers (1949) had demonstrated quite early that structural

alterations of the C-3 side chain of synthetic 6a,10a -tetrahydrocannabinols markedly

altered biologic potency (Mechoulam et al. 1976). However, since many of the

biologic effects measured in these early studies were nonanalgetic, we set out to

carefully determine specific structure-analgetic activity relationships of side chains in

the HHC series. Our search led us ultimately to examine four distinct classes of side

chains (alkyl, aralkyl, alkoxy, and aralkoxy) that are summarized in table 1 along with

their analgetic activity (Johnson et al. 1981A, 1981 B).

Initially, we examined the effect of the branched and extended alkyl derivatives 36

and 3c, side chains previously used in the and -THC (Loev et al. 1973)

and in the nabilone (Starkand Archer 1975) and nabitan (Pars et al. 1976) series. The

use of a side chain branched at the benzylic position, such as 3b and 3c, considerably

increase biologic activity relative to the n-amyl side chain. This increase in potency

may be due, in part, to a lessening of metabolic inactivation through benzylic

oxidation. Side chain c, with dimethyl substitution at the benzylic position, has the

32
TABLE 1

Analgetic Activity of HHC Side Chain Derivatives

R= Compound Analgetic MPE50

(mg/kg)*

(-)- -THC

-CH2CH2CH2CH2CH3

-CH-CH-CH2CH2CH2CH2CH3

CH3 CH3

-C-CH2CH2CH2CH2CH2CH3

CH3 CH3

-CH2CH2C6H5

-CH-CH2CH2C6H5

CH3

-CH-CH2CH2CH2H5

CH3

-CH-CH2CH2CH2-4-C6H5N

CH3

-CH-CH2CH2CH2C6H5

CH3

-O-c-C6H11

-O-CH-CH2CH2CH2CH2CH3

CH3

-

3a

3b

3c

3d

3e

3f

3g

3h

3i

3j

9.1

0.63

0.08

0.06

>10**

4.66

0.06

0.22

0.35

<10†

0.17

-O-CH-CH2CH2C6H5 3k 0.23

CH3

-O-CH-CH2CH2CH2C6H5 3l 0.07

CH3

-CH-CH2CH2OC6H5 3m 0.23

CH3

-CH-CH2CH2CH2-O-C6H5 3n 0.38

CH3

-CH-CH2-O-CH2CH2C6H5 3o 1.28

CH3

Morphine - 0.9

*Phenylbenzoquinone writhing in mice (PBQ), 0.3 hr after dose, s.c. **27%

protection at 10 mg/kg, s.c. †75% protection at 10 mg/kg, s.c.

33
inside chain b. As can be seen in table 1, both 3b and 3c exhibit analgetic activity

considerably greater than that of HHC and -THC.

Having demonstrated that it was possible to increase the analgetic potency of HHC

by simple alkyl substitution in the side chain, we next turned our attention to aralkylsubstituted derivatives in order to determine the effect of a pendant phenyl ring on

analgetic activity (previously studied in the nabitan series, cf. Winn et al. 1976). The

unbranched phenethyl side chain (3d). which has approximately the same extended

chain length as HHC, was analgetically less active. However, the branched three-,

four-, and five-carbon homologs exhibited potent analgetic activity with a maximum

effect at four linear carbon atoms (3f ). The heteroaromatic 4-pyridyl derivative 3g,

while somewhat less potent than 3f , retained analgetic activity. Molecular models

show that 3f and 3g have approximately the same extended side chain length as 3b

and 3c.

Previous studies in the -THC series have shown that an oxygen atom directly

attached to the phenolic ring has a variable effect on biologic activity. Thus, while

unbranched ether side chains were reported to decrease activity (Edery et al. 1972),

branched ether side chains exhibited a small increase in activity relative to their

carbon isomers (Loev et al. 1973). Furthermore, simultaneous changes in the C-ring

and side chain have been shown to produce nonpredictive SAR (Loev et al. 1973).

Therefore, we reinvestigated this parameter to specifically assess the effect of a C-3

oxygen atom on analgetic activity in the HHC series. While the cyclohexyl derivative

3i retains some analgetic activity, the 2-heptyloxy derivative 3j possesses potent

analgetic activity (two to three times that of morphine).

TABLE 2

Compound

Morphine

Comparative Analgetic Effect of HHC Side Chains

MPE50 (mg/kg, s.c.)c

Mouse Mouse Mouse Rat Rat

PBQ Writhinga Tail Flickb Hot Plateb Tail Pinchc Flinch Jumpc

0.9 5.72 4.23 4.77 10.3

3a 0.63 9.1 34.4 70.5 36.4

3b 0.06 3.0 0.32-0.56 N.T. 0.39

3f 0.06 0.25 0.55 0.49 0.47

3j 0.17 N.T. 0.56-1.0 0.65 N.T.

3l 0.07 0.33 0.46 0.38 0.38

3n 0.38 0.8 10 N.T. 2.7

a

 Values at 0.3 hr postdose. bValues at 1 hr postdose. cValues at 2 hr postdose.

Introduction of oxygen in the aralkyl derivative 3e yielded 3k. which is 20 times more

potent than 3e. Increasing the chain length by one carbon yielded 3l, which is fully as

potent as 3f and 3c. Since the extended chain length of 31 is greater than that of 3c

and 3f, this finding indicates that the oxygen atom itself and not just the chain length

affects analgetic activity. However, when the oxygen atom is not directly attached to

the phenolic ring of HHC, it generally behaves as a carbon unit. Thus, 3n and 3o more

closely resemble 3h than 3I. However, 3m does resemble its isomer 3k rather than 3f.

34
We selected a potent member of each side chain class to more fully evaluate

analgesia across a range of stringent assays. Our results, reported in table 2, with 3a,

3b, 3f, 3j, 3I, and 3n confirm their ability to exhibit antinociceptive activity in tests

traditionally responsive only to the opiates.

These studies demonstrated that the C-3 side chain of HHC can be modified in a

structure-dependent fashion to yield derivatives that produce potent analgesia in

animals, previously characteristic only of the opiate analgetic class. In addition, we

have shown a unique effect for the placement of an oxygen atom directly attached

to the aromatic ring of HHC.

The Importance of the Phenol

Previous studies dealing with modification of only the phenolic portion of

cannabinoids have been limited to derivatives of -THC, -THC, and -THC

(Loev et al. 1973; Edery et al. 1972; Kurth et al. 1976; Matsumoto et al. 1977). These

studies have suggested, with some ambiguities, that optimal activity is associated

with only the phenol. With the potent prototype 31 in hand, we began a study of the

aromatic ring of HHC to determine the optimum structural requirements for

analgesia. initially, we studied modifications of 31 which replaced the phenol with

potential bioisosteric equivalents (Johnson et al. 19818; Melvin et al. 1980).

TABLE 3

Analgetic Activity of Phenol Replacements

Analqetic MPE50 (mg/kg)a

X

Subcutaneousb Oralc

OH 0.07 0.11

H 30.5 30.2

CH3 >56 38.2

CH20H >56 24.6

COOH >56 -

NH2 11.3 32.5

NHSO2CH3 >56 -

NHCOCH3 >56 -

Cl >56 -

SH >56 -

Morphine 0.91 -

Codeine 6.7 21.3

-THC 5.93 7.98

aPhenylbenzoquinone writhing in mice (PBQ). b

0.3 hr postdose. c1 hr postdose.

35
Retaining the phenolic functionality (X = OH) appears to be a prerequisite for

optimum analgetic activity (table 3). Further, although analgesia in the range of

codeine is obtained for the amino substituent (X= NH2), substituents possessing

acidic hydrogens (X=COOH, NHSO2CH3, SH) do not mimic the phenol. Complete

removal of the phenol (X = H) yields a weakly active compound, as does replacement

by methyl and hydroxymethyl groups. The oral test data suggest that these latter

two groups are metabolically interconvertible, and it is equally likely that some

hydroxylation occurs at C-1 of the X = H analog yielding the active phenol derivative

3l .

C-5 and C-6 Positions

Since nuclear modification of the cannabinoids has been largely unsuccessful to date

(Mechoulam et al. 1976), we were impressed by the finding of potent analgesia in

animals for both the phenanthrene (Y = CH2) and the phenanthridine (Y = NH) (table

4) (Althuis et al. 1980; Milne et al. 1978).

TABLE 4

Analgetic Activity of C-5 and C-6 Modifications

R1 R2

CH3 CH3

CH3 CH3

MPE50 (mg/kg, s.c.)

Y Mouse PBQa Mouse Tailb Rat Tailc

Writhing Flick Pinch

CH2

0.78 1.3 1.1

O 0.07 0.33 0.38

CH3

H NH 0.4 0.7 1.0

aValues at 0.3 hr postdose. bValues at 1 hr postdose. cValues at 2 hr postdose.

The phenanthrene prototype is one to four times more potent than morphine in the

three tests shown in table 4. Although peak activity is generally observed 1 to 2

hours after dibenzo(b,d)pyrans, such as THC, this is not true for the phenanthrenes.

As shown in table 5, the peak activity of this compound occurs about 4 hours after

administration, and this activity remains near the maximum during the period of 2 to

8 hours after administration and drops off at 24 hours. It is further striking to note

that even 24 hours after dose, the phenanthrene has a tail-flick MPE50 less than that

of morphine 1 hour after administration.

The phenanthridine, nantradol (4), culminated our initial efforts to design a potent,

nonopiate analgetic. This is a significant finding since previous attempts utilizing a

cannabinoid-based phenanthridine nucleus resulted in compounds lacking biologic

36
TABLE 5

Duration of Phenanthrene Analgetic Activity

Mouse Tail Rat Tail

Flick Pinch

MPE50 (mg/kg, s.c.)

Time (hr)

3.3 - 0.5

1.3 1.1 2.0

1.5 0.74 4.0

- 0.66 6.0

3.5 0.88 8.0

2.2 - 24.0

activity (Hoops et al. 1968; Biel 1970). Nantradol has a number of structural and

pharmacologic features which distinguish it from both the opiates and the

cannabinoids (Mast et al. 1979; Johnson and Milne 1980A). The most notable

structural differences are the presence of a weakly basic nitrogen in place of a pyran

oxygen, the deletion of an axial methyl group, and the introduction of an oxygencontaining C-3 side chain. Nantradol is considerably more potent (20- to 100-fold)

than -THC as an analgetic and possesses two to seven times greater potency than

morphine across a full battery of analgetic tests (table 4). Like the opiates, nantradol

also possesses antidiarrheal and antitussive activity; however, unlike the opiates,

nantradol does not bind to the opiate receptor and its analgetic activity is not

blocked by naloxone in vivo (Milne et al. 1980A).

Phenanthridine Stereospecificity

Nantradol's substantial potency suggested to us that it might be acting specifically to

produce its analgetic effects. Therefore, we sought to determine if nantradol's

actions were stereospecific (Milne et al. 1980B; Johnson and Milne 1980B).

Nantradol has a total of five asymmetric centers; however, owing to its defined

stereochemistry at positions 6, 6a, 9, and 10a, it is an approximately equal mixture of

only 4 of the possible 32 isomers and has been studied in both animals and man as a

50: 50 mixture of two pairs of racemic diastereomers, both of which possess the trans6a,10a stereochemistry and have -oriented substituents at positions 6 and 9 (figure

1). The first step toward identifying the major active isomer was the separation of

nantradol into its component diastereomers, herein designated A and B.

Diastereomer A was found to be at least 10 times more potent than its C-3 side chain

counterpart diastereomer B in analgetic tests (table 6). These results clearly

demonstrate that isomer A contains the majority of nantradol’s analgetic activity.

Resolution of isomer A into its two optically active enantiomers demonstrated that it

37
TABLE 6

Analgetic Activity of Nantradol Stereoisomers

MPE50 (mg/kg, s.c.)

Compound Mouse PBQ

Writhing*

Nantradol 0.4

Diastereoisomer A 0.2

Diastereoisomer B 1.7

Levonantradol 0.07

Dextronantradol 6.5

* 1 Hr after dose. †2 Hr after dose.

Mouse Tail

Flick*

0.7

0.2

2.4

0.2

>10.0

Rat tail

Pinch†

1.0

0.7

14.2

0.2

> 10.0

Rat Flinch

Jump†

1.4

0.3

2.4

0.3

> 10.0

is the levorotatory isomer, levonantradol, that possesses the largest portion of the

analgetic activity of nantradol, being four and two times more potent than

nantradol and diastereomer A, respectively. Importantly, the enantiomer

dextronantradol is at least 100 times less active than levonantradol.

Figure 1

NANTRADOL ISOMERS

38
AC-BICYCLICS

Previously reported work and studies in our laboratories suggested that the

dimethyldihydropyran ring portion of HHC was not required for potent biological

activity. Unexpectedly, substitution of carbon (phenanthrenes) or nitrogen

(phenanthridines) for the oxygen of the pyran ring in HHC yielded compounds that

were of similar analgesic potency to HHC. Additionally, we showed in the work

leading to levonantradol that the geminal dimethyl group on the pyran ring of HHC

was not necessary for analgesic activity.

Thus, the dihydropyran moiety appeared to serve no other purpose than to firmly

anchor the phenolic ring and cyclohexyl ring in an active conformation. This

hypothesis led us to further postulate that HHC interacts at a receptor site by a threepoint contact (Milne and Johnson 1981). the three binding sites being the equatorial

alcohol, phenol, and C-3 side chain.

As a result of these considerations, we prepared (Melvin et al. 1982, 1984A) a novel

AC-bicyclic cannabinoid, 5 (CP-47,497), which is lacking the dihydropyran ring of

HHC. As table 7 shows, prototype 5 retains all the analgetic activity and potency of

HHC and is equipotent with morphine (Johnson et al. 1982; Milne et al. 1983). In

addition, 5 still exhibits many of the motor disruption effects in common with HHC

and the cannabinoids (Weissman et al. 1982). We believe these striking results can be

rationalized by considering the minimum energy conformation of 5. For the

calculated minimum energy conformation of 5 and HHC, the dihedral angle between

the C-OH (phenol) and C-OH (alcohol) bond is + 54.0° and + 41.3°, respectively. The

distance between the oxygen atoms in these calculated conformations was found to

be 5.0Å for 5 and 4.8Å for HHC. Thus, we propose that 5 readily assumes a

conformation quite similar to the rigid HHC molecule and is recognized by a

biological system as being equivalent to a tricyclic cannabinoid.

TABLE 7

Compound

Analgetic Activity Test Results of CP-47,497 (5)

MPE5O (mg/kg)a

PBQb RTCc HPd TFe FJf

5 1.0 4.7 8.1 4.4 4.2

HHC 0.63g7.0 34.4 9.1 36.4

Ag-THC 5.9 29.1 >178h55g,i 83.1

Morphine 1.8 4.8 4.2 5.7 10.3

a

The dose to produce 50% of the maximum possible effect (subcutaneous). bMouse

phenylbenzoquinone-induced writhing, 1 hr postdose. cRat tail clamp, 2 hr postdose.

dMouse hot plate, 1 hr postdose. eMouse tail flick, 1 hr postdose. fRat flinch jump, 2

hr postdose. g20 min postdose. h

Less than 50% response at this dose. i>178 at 1 hr

postdose.

39
We developed side chain SAR for 5 as compiled in tables 8 and 9. These tables

illustrate the optimum carbon side chains are 1,1-dimethylheptyl and 1,1-

dimethyloctyl, while the optimum alkoxy substituents are 2-octyloxy and 2-nonyloxy.

Table 10 compares carbocyclic ring size with analgetic activity, showing the optimum

sizes to be cyclohexyl and cycloheptyl (Melvin et al. 1984B). In addition, tables 8

through 10 record the effect on side chain and ring size of varying the

stereochemistry of the alcohol in 5 as well as the oxidation state (ketone vs. alcohol)

at the same carbon atom. Thus, analgetic potency varies with the alcohol position 5

as equatorial OH>axial OH ketone.

TABLE 8

PBQa Writhing Results for Alkyl Side Chains

MPE50 (mg/kg)b

n/R Equatorial OH Axial OH =O

0 IAc

IA IA

1 IA IA IA

2 IA IA IA

3 IA IA IA

4 11.9 > 56d34.8

5 1.00 3.8 4.5

6 1.20 3.93 1.06

7 15.2 IA IA

8 IA IA IA

9 IA IA IA

–e IA IA IA

aMouse phenylbenzoquinone-induced writhing, 1 hr postdose. b

The dose to produce

50% of the maximum possible effect (subcutaneous). cIA, less than 20% inhibition at

56 mg/kg. d35% inhibition at 56 mg/kg. eThese compounds have the entire alkyl side

chain repiaced by H.

Substitution at the C-4 position of the cyclohexane ring of 5 maximized analgetic

potency in this series. The hydroxy propyl analog (-)-6 (CP-55,940) is shown in table

11 to be significantly more potent than morphine and to have at least a 200-fold

ratio of stereospecificity (Melvin et al. 1983A). Table 12 compares the effect of simple

n-alkyl versus n-hydroxyalkyl substitution at the C-4 position of 5. Analgetic potency

with alkyl substitution was maximized for n-propyl, while n-hydroxypropyl or nhydroxybutyl were the optimum hydroxyalkyl substitution.

40
TABLE 9

PBQa Writhing Results for Alkoxy Side Chains

MPE50 (mg/kg)b

n R

1

/R2

4 CH3

5 CH3

6 CH3

2 C6H5

3 C6H5

4 C6H5

3

f

C6HS

Equatorial OH

22.3

5.04

9.3

~56e

38.0

IA

IA

Axial OH

>56c

>56

IAd

IA

>56

IA

IA

=O

>56

>56

>56

IA

>56

IA

IA

aMouse phenylbenzoquinone-induced writhing, 1 hr postdose. b

The dose to produce

50% of the maximum possible effect (subcutaneous). cLess than 50% inhibition at 56

mg/kg. dlA, less than 20% inhibition at 56 mg/kg. e57% inhibition at 56 mg/kg.

f

These compounds lack the CHCH3 in the side chain of the general structure.

41
TABLE 10

Analgetic Test Results with Varying Ring Size

MPE50(mg/kg)a

n R PBQb RTCc HPd

TFe FJf

0 ~56g NTh NT N T NT

1 1.00 4.7 8.1 4 4 4.9

2

3

0

1

2

1.44 7.66 39.4 12.2 3.5

4.61 NT >56i~32j NT

>56i NT N T NT NT

3.8 12.3 >56i55.7 ~17.8k

1.53 5.64 <32l7.54 5.36

3 39.2 NT N T NT NT

0 IA m NT NT NT NT

1 4.5 26.1 >32n15.3 11.5

2 1.23 4.36 >32n11.7 10.8

3 ~56° NT NT NT NT

a

The dose to produce 50% of the maximum possible effect (subcutaneous). bMouse

phenylbenzoquinone-induced writhing, 1 hr postdose. cRat tail clamp, 2 hr postdose.

dMouse hot plate, 1 hr postdose. eMouse tail flick, 1 hr postdose. fRat flinch jump, 2

hr postdose. g51% inhibition at 56 mg/kg. hNT = Not tested. iLess than 50%

inhibition at 56 mg/kg. j54% inhibition at 32 mg/kg. k58.7% inhibition at 17.8

mg/kg. l65.6% inhibition at 32 mg/kg. mIA, less than 20% inhibition at 56 mg/kg.

n

Less than 50% inhibition at 32 mg/kg. °57.5% inhibition at 56 mg/kg.

42
TABLE 11

Analgetic Activity and Stereospecificity of 6

Compound PBQa

(-)-6 0.07

MPE50 (mg/kg)

TFb HPc RTCd FJe

0.4 0.7 0.13 0.50

( +)-6 14.6

(±)-6 0.29 0.55 1.11 0.46 0.63

(±)-5 1.0 4.4 8.1 4.7 4.9

HHC 0.63f9.1 34.4 7.0 36.4

Morphine 1.8 5.7 4.2 4.8 10.3

aMouse phenylbenzoquinone-induced writhing, 1 hr postdose. bMouse tail flick, 1 hr

postdose. cMouse hot plate, 1 hr postdose. dRat tail clamp, 2 hr postdose. eRat flinch

jump, 2 hr postdose. f0.3 hr postdose.

TABLE 12

Analgetic Activity for Analogs of (±)-5

R PBQ MPE50

(mg/kg)*

H

CH3

CH2CH3

CH2CH2CH3

CH2CH2CH2CH3

CH2CH2CH2CH2CH3

CH2OH

CH2CH2OH

CH2CH2CH2OH

CH2CH2CH2CH2OH

CH2CH2CH2CH2CH2OH

1.00

1.02

1.75

0.54

0.83

50

1.77

2.58

0.29

0.23

0.55

*Phenylbenzoquinone writhing in mice, 1 hr post subcutaneous dose.

43
ACD-TRICYCLICS

The successful modification of HHC and subsequent derivatization of 5 giving the

more potent hydroxypropyl analog 6 encouraged us to further pursue this new lead.

Assuming we had found the optimum substitution at C-4 from our studies with 5, we

next chose to explore potential stereochemical requirements of this new primary

alcohol group. To this end, we prepared a more complex ring system, 7 (CP-55,244),

that incorporates a rigidly positioned hydroxypropyl moiety. From table 13, which

displays the analgetic test results obtained with rigid decalin 7, it is seen that this

compound is 2 to 10 times more potent than less rigid structure 6. A suggestion that

the axial hydroxymethyl group of 7 is optimally oriented comes from the lessened

(52x) potency observed for the equatorial isomer (± )-8 . Even more striking, (-)-7

now exhibits virtually complete stereospecificty of action versus (+)-7. This fact

supports our hypothesis that compounds such as 6 and 7 interact with a specific

receptor system.

TABLE 13

Analgetic Effect of (-)-7 vs. (-)-6 and Stereospecificity of (-)-7

MPE50 (mg/kg)a

Compound PBQb RTCc Hpd TFe

(-)-7 0.02 0.06 0.07 0.03

FJf

0.11

(+)-7 >100 - - - -

(±)-7 0.054 0.22 0.24 0.15

(±)-8 2.86 6.83 - -

-

-

(±)-5 1.0 4.7 8.1 4.4 4.9

(-)-6 0.07 0.13 0.7 0.4 0.5

HHC 0.63g7.0 34.4 9.1 36.4

Morphine 1.8 4.8 4.2 5.7 10.3

a

The dose to produce 50% of the maximum possible effect. bMouse

phenylbenroquinone-induced writhing, 1 hr postdose. cRat tail clamp, 2 hr postdose.

dMouse hot plate, 1 hr postdose. eMouse tail flick, 1 hr postdose. fRat flinch jump, 2

hr postdose. g0.3 hr postdose.

4 4
DISCUSSION

We have detailed the SAR for side chains of ABC-tricyclic cannabinoids showing

optimum effects from both alkyl and alkoxy substitution. The necessity for phenolic

functionality at C-1 of HHC was confirmed. Nitrogen and carbon were successfully

substituted for the dihydropyran oxygen of HHC. Structural dissection of HHC led to

the synthesis of AC-bicyclic cannabinoids and demonstration that the rigid B-ring

(dihydropyran ring) is not required for biological activity. The biological activity of

AC-bicyclic cannabinoids supports our hypothesis that cannabinoids have three sites

of receptor recognition (side chain, phenol, and alcohol). The synthesis of ACD- and

ABCE-multicyclic cannabinoids demonstrates that there is structurally unexplored

space proximate to the HHC molecule.

The extreme potency and stereospecificity of these synthetic, nonclassical

cannabinoids, and their retained activity at the extremes of structural elaboration,

lead us to believe that these compounds are exerting their effects by acting at an as

yet unknown receptor site.

The current studies demonstrate the ability to achieve extreme biological potency in

structures that diverge significantly from the classical cannabinoid ring system. This

and the clear relationship between the stereochemistry and biological activity of

these compounds provides support for the premise that they interact in very specific

and important ways with biological systems. However, it seems likely that further

improvements in selectivity between the desired biological activity and the typical

cannabinoid side effects will be necessary before the full therapeutic potential of

these compounds is fully realized. It is possible that the next round of discovery in

this area, possibly leading to greater selectivity, will be sparked by the identification

of a novel site of cannabinoid action involving a distinct neurotransmitter system.

Toward this end, the potent and stereochemically defined compounds described

herein could provide important tools to facilitate this effort.

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Johnson, M.R.; Melvin, L.S.; and Milne, G.M. Prototype cannabinoid analgetics,

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therapeutants: Further studies in the search for non-addicting drugs to relieve

pain and discomfort. Abstracts of Papers, 186th Meeting of the American

Chemical Society, Washington, D.C., Aug. 1983, American Chemical Society,

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Johnson, M.R.; Eggler, J.F.; Melvin, L.S.; and Milne, G.M., Cannabinoid-derived

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Chem 1313-1318:1976.

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Matsumoto, K.; Stark, P.; and Meister, R.G. Cannabinoids. 1. 1-Amino- and 1-

mercapto-7,8,9,10-tetrahydro-6H-dibenzo(b,d)pyrans. J Med Chem 20:17-30,

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and biochemistry of cannabis. Chem Rev 76:75-112, 1976.

Melvin, L.S.; Johnson, M.R.; and Milne, G.M. Synthesis and analgetic activity of

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National Meeting of the American Chemical Society, Las Vegas, NV, August 1980,

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AUTHORS

Lawrence S. Melvin, Ph.D.

M. Ross Johnson, Ph.D.

Pfizer Central Research

Groton, Connecticut 06340

47
Structure-Anticonvulsant Activity

Relationships of Cannabidiol

Analogs

Arnold R. Martin, Ph.D.; Paul Consroe, Ph.D.; Vinayak V.

Kane, Ph.D.; Vibhakar Shah, M.S.; Vishwakarma Singh, Ph.D.;

Naftali Lander, Ph.D.; Raphael Mechoulam, Ph.D.; and Morris

Srebnik, Ph.D.

ABSTRACT

Cannabidiol (CBD) exhibits anticonvulsant activity in

experimental animals and in man. As part of a structureactivity study, analogs were prepared wherein the terpene unit,

the aryl unit, and/or the side chain were modified. Thus,

several pinenyl and carenyl derivatives, aryl ethers and

acetates, and a variety of 1",1"-dialkylhexyl and 1",1"-

dialkylheptyl analogs were synthesized. The compounds were

evaluated for anti-convulsant activity in seizure susceptible

(AGS) rats and for neurotoxicity in the rat rotorod (ROT) test.

Comparisons of stereoisomers of CBD and several analogs revealed

a general lack of stereoselectivity for anticonvulsant and other

CNS properties of this class of compounds.

INTRODUCTION

(-)-Cannabidiol [CBD, (-)-la] is a neutral major cannabinoid of

cannabis with potential therapeutic utility. In humans, the

drug has antiepileptic efficacy (Cunha et al. 198O) and possibly

antidystonic activity (Consroe and Snider 1986) and exhibits no

significant side effects. It also has no cannabimimetic

activity, i.e., subjective effects in common with cannabis or

its neutral major psychoactive ingredient, (-)-delta-9-tetrahydrocannabinol [(-)-delta-9-THC (2), also known as delta-1-

THC (Razdan 1986)]. In laboratory animals, (-)-CBD has

selectivity of anticonvulsant action relative to neurotoxic

effects (Consroe and Snider 1986) and is inactive in models that

predict cannabimimetic activity (Razdan 1986). This dichotomy

of action between (-)-CBD and (-)-delta-1-THC has led to many

investigations of the structure-activity relationships (SAR) in

cannabinoids (Razdan 1986). However, most of these studies have

been carried out with THC analogs and with laboratory animal

models of cannabimimetic activity; thus, virtually all knowledge

of cannabinoid SAR is based on the psychoactive component of

48
cannabis-like effect (Razdan 1986). On the other hand, some

studies evaluating the anticonvulsant effects of CBD

stereoisomers (Leite et al. 1982) and putative metabolites

(Carlini et al. 1975) in mice have been reported. We have been

carrying out an interdisciplinary

the antiepileptic potential and SAR of new synthetic CBD analogs

research project to evaluate

in rats. Some of our results have been published (Consroe et

al. 1981, 1985), and the present report provides an overview of

these findings and of new data on the anticonvulsant and

differential neurotoxic activities of

MATERIALS AND METHODS

additional CBD analogs.

The audiogenic seizure (AGS-susceptible) rat model of epilepsy

(Dailey and Jobe 1985) was used to assess anticonvulsant

activity, and the rat rotorod (ROT) paradigm (Dunham and Miya

1957) was used to assess differential neurotoxicity (i.e.,

reflecting mainly sedation and/or incoordination), of CBD

analogs. For AGS tests, male and female, 150 to 225-gm,

genetically epilepsy prone rats (from the Uaz:AGS-SD colony of

the University of Arizona, or from the GEPR-3 colony at the

University of Illinois) were used (Consroe et al. 1981, 1985;

Dailey and Jobe 1985). For ROT tests, male and female, 150 to

225-gm rats (from the Uaz:AGS-SD colony or the Hsd:(SD)BR colony

of the Harlan Sprague Dawley Company, Indianapolis, IN) were

used. As detailed previously (Consroe et al. 1981, 1985) CBD

analogs (suspended in a vehicle of 10% polysorbate 80 and 90%

distilled water) were injected intravenously (iv) and rats were

tested 15 minutes later (a peak-effect time for both tests). In

the AGS test, responses to sound were recorded as the presence

or absence of seizure (i.e.,

generalized flexion).

generalized clonus or clonus and

In the ROT test, effects were measured as

the ability or inability (i.e., neurotoxicity) of trained rats

to remain on a revolving drum (rotorod) for 60 seconds.

Separate groups of ten to fifteen rats were used for each dose

of drug tested and, typically, three to six doses of each analog

were used for each AGS and ROT test. For appropriate doseresponse data, median effective anticonvulsant (ED50) and

neurotoxic (TD50) doses and regression line parameters were

calculated by computer-assisted probit analysis (Dixon 1977).

Subsequent calculations of 95% confidence limits (CL) and

comparisons between slopes and median doses (i.e., ED50 and TD50

potency comparisons) were carried out using the method of

Litchfield and Wilcoxon (1949). which we have incorporated into

49
a BASIC program for use on a microcomputer. Protective indexes

(PI = ROT-TD50/AGS-ED50) also were calculated where possible.

Cannabidiol analogs were prepared by condensation of the

appropriate terpene alcohol (+)-p-mentha-2,8-dien-1-ol (-)-Pmentha-2,8-dien-3-ol, (-)-2-caren-4-ol, (+)-verbenol, (-)-

verbenol: -(+)-8-acetoxyverbanol, (-)-8-acetoxyverbanol, (+)-8-

pivaloxyverbanol, or (-)-8-pivaloxyverbanol with a 5-

substituted resorcinol according to the methods of Petrazilka et

al. (1969) and Razdan et al. (1974). The synthesis of 5-

(1',1',1'-trialkyl)resorcinols have been described previously

(Singh et al. 1981). Olivetol (5-n-amylresorcinol), (-)-

limonene, (+)- and (-)- -(-pinene, (-)-myrtenol, and (+)-trans-pmentha-2,8-dien-1-0l were obtained from commercial sources and

were used as received.

(-)-cis-p-Mentha-2,8-dien-1-ol was prepared from (-)-limonene by

the method employed by Rickards and Watson (1980) for the

synthesis of (+)-cis-p-mentha-2,8-dien-1-ol from (+)-limonene.

(-)-p-Mentha-1,8-dien-3-ol (Leite et al. 1982) was obtained as a

1:3 mixture of cis- and trans-isomers from the sodium borohydride-cerium trichloride reduction (Luchie et al. 1982) of pmentha-1,8-dien-3-ol (Dauben et al. 1969). (-)-trans-4-Caren-3-

ol was obtained by the reaction of

(-)-3 ,4 -epoxycarane

sodium phenylselenide on

(Cocker and Grayson 1969), followed by

oxidation of the phenylselenide and elimination of the

selenoxide (Uzarewicz and Zientek 1977). (+)- and (-)-Verbenol

were obtained as mixtures of cis- and trans-isomers from (+)-

and (-)- -pinene (Whitham 1961)) respectively. (+)-Myrtenol

was prepared by selenium dioxide oxidation of (-)- -pinene

followed by reduction with sodium borohydride. (+)- and (-)-

Myrtenols were acylated with acetic anhydride or pivaloyl

chloride in pyridine to obtain the corresponding acetate or

pivalate, and the latter were subjected to allylic oxidation

with chromium trioxide/pyridine (Dauben et al. 1969) and then

reduction with dibutyllithfum aluminium hydride (DiBAH) to give

the corresponding 8-acetoxy- and 8-pivaloxy-verbenols.

The optical purity of (+)-CBD obtained from either (-)-p-mentha1,8-dien-3-ol or (-)-p-mentha-2,8-dien-1-ol was calculated to be

83% based on observed D values of + 80± 1

0

 for the two

chemically pure samples. Optical purities for the cannabidiol

analogs derived from (+)-3-carene and (+)- and (-)- -pinene

were not determined, but were estimated to be in the range of

80% to 95% based on the optical purities of the starting

materials. Structures of the final compounds were confirmed by

proton nmr and elemental analysis, which agreed within ± 0.4% of

the theoretical values. The cannabidiols prepared along with

data for their anticonvulsant and neurotoxic activities are

provided in table 1.

RESULTS AND DISCUSSION

Dose-response data for standard compounds phenytoin (PHT),

50
TABLE 1

Preliminary and Completed Dose-Response Data of

Standard Anticonvulsants and CBD Analogs in ROT and AGS Testsa

Compd. No. ROT AGS PI

Phenobarbital

Carbamazepine

Phenytoin

28.9 10.8 2.7

(26.0-32.2) (8.5-13.8)

33.9 12.8

(9.7-16.9)

2.7

(32.6-35.4)

23.9 14.7

(22.3-25.6) (11.1-19.6)

1.6

(-)-CBD: Aryl Ring Modificationsb

Compd. No. R1 R2 R3 ROT AGS PI

(-)-1a H H H 30.7 14.9 2.1

(26.1-36.1) (10.9-20.1)

lb AC AC H 47.9 11.5 4.2

(43.2-53.2) (9.5-13.9)

1c H AC AC O/10 at O/l2 at ---

31 mg/Kg 15 mg/Kg

1d

CH3 CH3 H O/10 at O/10 at ---

31 mg/Kg 15 mg/Kg

1e

CH3 H H O/10 at 2/10 at ---

31 mg/Kg 15 mg/Kg

(-)-CBD: Side Chain Modificationsc

Compd. R1 R2 R3 R4 R5

ROT AGS PI

No.

(-)-3a CH3 CH3 H H

3b CH3 C2H5

H H

C 5 H 1 1 2.1 (2.3-6.3)

0.6

C5H11 6/10 at ---

31 mg/Kg 15 mg/Kg

51
3c CH3 CH2CH=CH2 H H C5H11 O/10 at O/10 at ---

31 mg/Kg 15 mg/Kg

(-)-3d CH3 CH3 H H C4H9 6/10 at 6/10 at ---

3e CH3 C2H5

H H C4H9

31 mg/kg 15 mg/Kg

9/10 at 4/10 at ---

31 mg/Kg 15 mg/KG

3f CH3 CH2CH=CH2 H H C4H9

O/10 at 1/10 at ---

31 mg/Kg 15 mg/Kg

3gH H CH3 CH3 C5H11 1O/1O at 4/10 at ---

31 mg/Kg 15 mg/Kg

(+)-CBDsc

Compd. No. R ROT AGS PI

(+)-1a

(+)-3a

C5H11 39.7 16.2 2.5

(36.6-42.9) (14.6-18.0)

1 ,1-DMH 10/10 at 9/10 at ---

31 mg/Kg 4 mg/Kg

(-)-Pinenyl CBDs

Compd. No. R1 R2 R3 ROT

(-)-4a

CH3 H C5H11 8/10 at

(-)-4b

31 mg/Kg

CH3 H 1,1-DMH 9.2

(6.8-12.3)

(-)-4c CH2OAC H 1,1-DMH 2.6

(1.8-3.7)

(-)-4d CH20AC AC 1,2-DMH 4/4 at

(-)-4e

31 mg/Kg

CH20Pv H 1,1-DMH O/6 at

31 mg/Kg

AGS PI

7/10 at ---

15 mg/Kg

5.2

(3.6-7.4)

1.8

2.5

(1.4-4.3)

1.0

4/10 at ---

15 mg/Kg

5/10 at ---

15 mg/Kg

52
(+)-Pinenyl CBDs

Cmpd. No. R1 R2 R3 ROT AGS PI

(+)-4a CH3 H C5H11 5/10 at 1/10 at ---

31 mg/Kg 15 mg/Kg

(+)-4b CH3 H 1,1-DMH 1.9 0.7

(1.6-2.2) (O.5-1.O)

2.7

(+)-4c

CH20Ac AC 1,2-DMH 9/10 at 4/10 at ---

4 mg/Kg 4 mg/Kg

(+)-4e CH20Pv H 1,1-DMH 4/10 at 8/10 at ---

4 mg/Kg 4 mg/KG

(+)-4f

CH20Ac AC 1,1-DMH 1O/1O at 9/9 at ---

4 mg/Kg 4 mg/Kg

(+)-Carenyl CBDs

Compd. No. R ROT AGS PI

(+)-5a

(+)-5b

C5H11 99.8 18.1 5.5

1,1-DMH

(90.4-110.1) (16.2-20.3)

3.7 3.4

(3.3-4.1) (3.0-3.9)

1.1

a

Median toxic (TD50) and effective (ED50) doses and 95%

confidence limits (in parentheses) are in mg/Kg, iv

PI = protective index = ROT-TD50/AGS-ED50. Ratios refer to the

number of rats protected against AGS or number of rats toxic/

number of rats tested (at the iv dose listed). Chemical

abbreviations are: AC = acetyl; DMH = dimethylheptyl; and

Pv = pivaloyl.

b

Data taken, in part, from Consroe et al. (1981).

c

Data taken, in part, from Consroe et al. (1985).

53
phenobarbital (PB), and carbamazepine (CBZ); (-)-CBD [(-)-

1a] and a few selected analogs; plus single dose comparisons

[for most compounds equivalent to the calculated AGS-ED50 and

ROT-TD50 of (-)-1a] are presented in table 1. In the AGS test,

peak-effect times were observed 15 minutes after (-)-1a, CBD

analogs, and PHT; 30 minutes after PB; and 45 minutes after CBZ

injections. Vehicle administration neither protected against

AGS nor caused ROT toxicity.

In an earlier study (Consroe et al. 1981), it was observed that

certain aryl modifications in (-)-1a markedly reduced both antiAGS activity and ROT neurotoxicity. For example, 1d, the

dimethylether of (-)-1a, is inactive in the AGS and ROT tests at

15 and 31 mg/Kg, respectively, and the monomethyl ether (1e) is

marginally active in the AGS test and inactive in the ROT test

at the same doses. These earlier data are repeated in table 1.

The monoacetate of 3'-acetyl CBD (1c), obtained by photo-Fries

rearrangement of CBD diacetate (1b), was similarly inactive in

both tests. It was therefore concluded (Consroe et al. 1981)

that both phenolic hydroxyl groups of CBD must be free for antiAGS and ROT activities to occur. Weak anti-AGS activity of 1e

could be due to inefficient demethylation to 1a. Intramolecular

hydrogen bonding between the free phenolic group and the 3'-

acetyl group of 1c may be responsible for its lack of activity.

The lack of anti-AGS activity in abn-CBD analogs (Consroe et al.

1981) indicates that the positions of the hydroxyl groups on the

aromatic unit are important.

CBD diacetate (1b), in contrast to the ethers, was found to be

equivalent to 1a in anti-AGS potency in a single dose comparison

(Consroe et al. 1981). It was tentatively concluded that 1b is

efficiently hydrolyzed by esterases in vivo to la which is

responsible for the anti-AGS activity. The neurotoxicity of 1b,

on the other hand, was significantly lower than that associated

with 1a. These preliminary findings prompted us to perform

complete dose-response and time course studies on 1b. Doseresponse data (table 1) reveal an ED50 of 11.5 mg/Kg and a TD50

of 47.9 mg/Kg for 1b, as compared with 30.7 mg/Kg and 14.9

mg/Kg, respectively, for 1a. Thus, the protective index (PI) of

1b is two times greater than the PI of 1a. Comparative time

course studies of la and lb at equiactive doses indicate that

the anti-AGS effects of 1a have a more rapid onset and a longer

duration than those of 1b. The ROT neurotoxic effects of the

two agents, on the other hand, follow a similar time course.

The slightly greater anti-AGS potency of 1b and its

significantly reduced ROT neurotoxicity as compared with 1a,

together with the time course differences, suggest that the

sites of action of the two effects may be different. If,

indeed, 1b must be converted to 1a in vivo in order to be

active, then this conversion must- be very efficient.

Acetylation of the phenolic groups of 1a could serve to increase

the lipophilicity of the compound, thereby facilitating its

penetration into the central nervous system. At the same time,

the phenolic groups are protected from inactivation by

54
conjugating enzymes.

We have recently investigated the effects of structural

modification of the side chain of CBD on anti-AGS and ROT

potencies (Consroe et al. 1985). The compounds investigated

were the branched chain compounds 3a-3g. For anti-AGS activity,

the order of potency (-)-3a 7 3b = 1a >3c was observed in the

1",1"-dialkylheptyl series, while it was 1a> 3e > 3d = 3f in

the 1",1"-dialkylhexyl series. In the ROT neurotoxicfty test,

the rank order of potencies was (-)-3a > (-)-1a 7 3b 7 3c in the

1",1"-dialkylheptyl series and 3e > 3d = 1a in the 1",1"-

dialkylhexyl series. Thus, optimal anti-AGS potency, together

with maximal neurotoxicity, was observed with the 1", 1"-

dimethylheptyl analog (-)-3a. The 1" ,1"-dimethylhexyl analog

(-)-3d, on the other hand, is significantly less potent than

(-)-3a. Leite et al.

anticonvulsant and CNS

(1982) observed similar enhancemeent in

depressant potencies of 1" ,1"-

dimethylheptyl analogs of CBD in mice. In contrast, the 1"-

methyl-1"-ethylheptyl analog 3b exhibited anti-AGS activity

comparable to that of 1a, but was considerably less neurotoxic.

Additional studies on this compound (which, like side chain

analogs 3c-, 3e, and 3f, is a diastereoisomeric mixture due to

asymmetry at the 1"-carbon) appear to be warranted. The 1"-

methyl-1"-ethylhexyl analog 3e, on the other hand, is

significantly more neurotoxic than (-)-1a, but significantly

less potent in the AGS test. The basis for the remarkable

differences in the side chain analogs differing by a single

carbon atom remain to be explained. Significant neurotoxicity

and some anti-AGS activity are retained in the 1",2"-

dimethylheptyl analog 3g.

Early comparisons of CBD analogs derived from (+)- and

(-)- -pinene suggested that the anti-AGS effects of CBD and its

derivatives might be stereoselective. Thus, (-)-4a (which is

stereochemically related to (-)-1a) was significantly more

potent than its enantiomer (+)-4a in the AGS test when each was

administered in a dose of 15 mg/Kg (Consroe et al. 1981).

However, Leite et al. (1982) found that (-)-1a and (+)-1a were

essentially equiactive as anticonvulsants (protection against

pentylenetetrazole seizures) and as CNS depressants

(prolongation of phenobarbital sleeping time) in mice. A

parallel lack of stereoselectivity was observed for the highly

potent 1" ,1"-dimethylheptyl enantiomers (-)-3a and (+)-3a. A

general lack of stereoselectivity for the anti-AGS and ROT

neurotoxic effects of CBD and its derivatives was recently

confirmed (Consroe et al. 1985). Thus, (-)-1a is marginally

more potent than (+)-1a in both the AGS and ROT tests, while

(+)-4b is somewhat more active than (-)-4b in the two tests (see

table 1). . There are insufficient data with which to

quantitatively compare (-)-3a and (+)-3a, but it is evident that

both are highly active in the AGS and ROT tests. Similarly, the

enantiomeric pair (-)-4d and (+)-4d appear to exhibit comparable

anti-AGS and ROT neurotoxic potencies. On the other hand, (+)-

4e is apparently more potent than (-)-4e. These two enantiomers

55
seem to be worthy of further study since single dose data

suggest that each will exhibit a favorable protective index.

The general lack of stereoselectivity for the anti-AGS and ROT

neurotoxic activity of la and its analogs stands in stark

contrast to the situation with THC-like compounds (see Mechoulam

et al., this volume). Thus, (-)-delta-6-THC analogs that are

stereochemically related to (-)-delta-1-THC (2) are markedly

more potent than their (+)-enantiomers in both AGS and ROT

tests. These findings suggest that the mechanisms of anticonvulsant and central nervous system depressant actions of CBDs

and THCs may be different.

The CBD analog derived from (+)-delta-3-carene, i.e., (+)-5a, is

of interest because of its high protective index. Thus,

although (+)-5a has an ED50 of 18.1 mg/Kg in the AGS test and is

therefore comparable to (+)-1a (to which it is stereochemically

related) in potency, it has a TD50 of approximately 100 mg/Kg.

This separation of activities is, unfortunately, not maintained

in the 1",1"-dimethylheptyl derivative (+)-5b, which has a PI of

1.1.

Solid state (X-ray crystallographic, Jones et al. 1977).

solution (Tamir et al. 1980; Kane et al. 1984), and theoretical

(Kane et al. 1984) studies all indicate that the 3-olivetyl and

4-isopropenyl groups of CBD prefer a dfequatorfal conformation.

These functionalities in the highly ri gfd pinenyl derivatives 4,

on the other hand, are held in a diaxial conformation. The less

rigid carenyl derivatives 5 no doubt also prefer a diaxial

conformation. Since the energy difference between the

diequatorial and diaxial conformations of CBD may be slight, it

is possible that the "active form" of the molecule may be its

energetically less favored diaxial conformer.

The following structure-activity generalizations have emerged

from these studies: The aryl unit and the side chain of CBD are

highly sensitive to structural modification. Most changes in

the aryl unit, e.g., blocking or changing the positions of the

phenolic groups, greatly decreased anti-AGS activity. Optimal

anti-AGS and ROT neurotoxic effects were observed in 1"-1"-

dimethylheptyl derivatives. On the other hand, the terpene unit

appears to be relatively insensitive to structural changes.

Derivatives of -pinene and delta-3-carene are as potent as

(-)-1a and there is a general lack of stereoselectivity

associated with the terpene unit.

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Carlini, E.A.; Mechoulam, R.; and Lander, N. Anticonvulsant

activity of four oxygenated cannabidiol derivatives. Res

Commun Chem Pathol Pharmacol 12:1-12, 1975.

Cocker, W., and Grayson, D.H. A convenient preparation of (-)-

3,4-epoxycarane. Tetrahedron Lett 4451-4452, 1969.

Consroe, P.; Martin, A.; and Mecholam, R. Anticonvulsant

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effects of cannabidiol stereoisomers and analoos in rats. In:

Harvey, D.J., ed. Marijuana '84. Proceedings of the Oxford

Symposium on Cannabis. Oxford: IRL Press, 1985. pp. 705-712.

Consroe. P.; Martin, A.; and Singh. V. Antiepileptic potential

cannabidiol analogs. J Clin Pharmacol 21:428s-436s, 1981.

Consroe, P., and Snider, S.R. Therapeutic potential of

cannabinoids in neurological disorders. In: Mechoulam, R.,

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Pimentel, C.; Gagliardi, R.; Sanvito, W.L.; Lander, N.; and

Mechoulam. R. Chronic administration of cannabidiol to

healthy volunteers and epileptic patients. Pharmacology

21:175-185, 1980.

Dailey, J.W., and Jobe, P.C. Anticonvulsant drugs and the

genetically epilepsy-prone rat. Fed Proc 44:2640-2644, 1985.

Dauben, W.G.; Lorber, M.; and Fullerton, D.S. Alylic oxidation

of oleffns with chromium trioxide•pyridine complex. J Org

Chem 34:3587-3592, 1969.

Dixon, W.J. BMD Biomedical Computer Programs. Berkeley:

University of California Press, 1977. pp. 439-450.

Dominiani, S.J.; Ryan, C.W.; and De Armitt, C.N. Synthesis of

5-(tert-alkyl)resorcinols. J Org Chem 42:344-346, 1977.

Dunham. N.W.. and Miva. T.S. A note on a simple apparatus for

detecting neurological deficit in rats and mice. J Am Pharm

Assoc 46:208-209, 1957.

Jones, P.G.: Falvello. L.; Kennard, O.; Sheldrick, G.M.; and

Mechoulam, R. Cannabidiol. Acta Cryst B33:3211-3214, 1977.

Kane, V.V.; Martin, A.R.; Jaime, C.; and Osawa, E. Dynamic

nuclear magnetic resonance and empirical force field studies

of cannabidiol. Tetrahedron 40:2919-2927, 1984.

Leite, J.R.; Carlini, E.A.; Lander, N.; and Mecholam, R.

Anticonvulsant effects of the (-)- and (+)-isomers of

cannabidiol and their dimethylheptyl homologs. Pharmacology

24:141-146, 1982.

Litchfield J.T., and Wilcoxon, F.

calculating dose-effect experiments.

A Simplified method of

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96:99-133, 1949.

Luchie, J.-L.; Rodriguez-Hahn, L.; and Crabbe, P. Reduction of

natural enones in the presence of cerium trichloride. J Chem

Soc Chem Comm 601-602, 1982.

Petrizilka, T.; Haeflinger, W.; and Sikemeier, C. Synthese von

Haschisch-Inhaltstoffen. Helv Chim Acta, 52:1102-1134, 1969.

Razdan, R.K. Structure-activity relationships in cannabinoids.

Pharmacol Rev 38:75-149, 1986.

Razdan, R.K.; Dalzell, H.C.; and Handrick, G.R. Hashish. A

simple one-step synthesis of (-)-delta-1-tetrahydro-cannabinol

(THC) from p-mentha-2,8-dien-1-ol and olivetol. J Am Chem Soc

96:5860-5865, 1974.

Rickards, R.W., and Watson, W.P. Conversion of (+)-R-limonene

into (+)-(IS,4R)-p-mentha-2,8-dien-1-ol, an intermediate in

the synthesis of tetrahydrocannabinoids. Aust J Chem

33:451-454, 1980.

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Singh, V.; Kane, V.V.; and Martin, A.R. A new synthesis of 5-

(1',1',1'-trialkylmethyl)resorcinols. Synth Comm 11:429-437,

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ACKNOWLEDGMENTS

This research was supported by a grant (No. NS15441) from the

National Institute of Neurological and Communicative Disorders

and Stroke. The National Institute on Drug Abuse provided a

generous supply of (-)-CBD. Dr. Phillip Jobe (Department of

Basic Sciences, University of Illinois College of Medicine,

Peoria, Illinois) supplied GEPR-3 rats that were used in some of

our experiments.

AUTHORS

Paul Consroe, Ph.D.

Vinayak V. Kane, Ph.D.

Arnold R. Martin, Ph.D.

Vibhakar Shah, M.S.

Vishwakarma Singh, Ph.D.

Departments of Pharmacology &

Toxicology and Pharmaceutical

Sciences

College of Pharmacy

University of Arizona

Tucson, Arizona 85721, U.S.A.

Naftali Lauder, Ph.D.

Raphael Mechoulam, Ph.D.

Morris Srebnik, Ph.D.

Department of Natural

Products

Faculty of Medicine

Hebrew University

Jerusalem 91120, Israel

58
Anticonvulsant and Neurotoxic

Effects of Tetrahydrocannabinol

Stereoisomers

Paul Consroe, Ph.D., and Raphael Mechoulam, Ph.D.

ABSTRACT

Enantiomers of delta-6-tetrahydrocannabinol (THC),

delta-6-THC-1", 1"-dimethylheptyl (DMHP), and 7-OHdelta-6-THC-l", 1"-DMHP were assessed for their ability

to block audiogenic seizures in genetically epilepsyprone rats. The stereoisomers were evaluated also for

their ability to produce differential neurotoxicity in

the rat rotorod (ROT) paradigm. Potency comparisons

among the compounds revealed modest to profound

stereoselectivity for anticonvulsant and neurotoxic

activities, a general increase in both activities with

the DMHP and 7-OH modifications of delta-6-THC, and some

favorable separation between anticonvulsant and

neurotoxic activities with selected THC analogs.

INTRODUCTION

Two important generalizations of tetrahydrocannabinol

activity are that DMHP side chain analogs are much more

potent than are their n-pentyl side chain homologs

(Razdan 1986), and that (+)-optical isomers are

substantially less active than are their (-)-enantiomers

(Dewey et al. 1984). These structure-activity

relationships (SAR) are based largely on studies

beginning over four decades ago with analogs of the

unnatural racemic delta-3-THC (also known as delta-6-

alpha-10-alpha-THC) and on studies beginning over two

decades ago with derivatives of (-)-delta-1-THC (also

known as (-)-trans-delta-9- THC) and (-)-delta-6-THC

(also known as (-)-trans-delta-8-THC), the latter being

the major and minor psychoactive principles of cannabis,

respectively (Mechoulam and Edery 1973). The activities

of the natural THCs (each having an n-pentyl side chain)

and a few DMHP analogs of delta-3-THC have been

determined in humans --most commonly as a function of

their cannabimimetic activity, i.e., cannabis-like

subjective effects such as the euphoric "high" (Pradhan

59
1984; Razdan 1986). The (+)-THC isomers and a wider

range of DMHP analogs have been evaluated in laboratory

animals -- most commonly in dog, monkey, and/or rodent

behavioral paradigms (Dewey et al. 1984; Pradhan 1984).

The relative potencies of cannabinoids in these animal

tests are thought to be largely reflective of their

potential to produce cannabimimetic effects in humans

(Razdan 1986).

Unfortunately, many investigations carried out to date

have used impure optical isomers, such as the oftenstudied 1", 2" -DMHP homologs of (-)-delta-6-THC (four

possible isomers) and racemic delta-3-THC (eight

possible isomers). Depending upon the proportion of

isomers, the racemic mixtures may show significant

variability in their potencies and activities (Pradhan

1984). Interestingly, substantial variability in

activity was cited as the reason for the termination

(Hardman et al. 1971, p. 296) of an otherwise promising

(and the only) clinical trial of DMHP analogs of THC in

epileptic patients (Davis and Ramsey 1949).

In light of the above considerations, we sought to

evaluate the stereoselectivity relationships of six

stereoisomers of THC. The compounds were assessed for

anticonvulsant and (rotorod) n eurotoxic activities in

the rat --activities which pertain to a potential

therapeutic application of cannabinoids.

MATERIALS AND METHODS

The cannabinoids employed, along with their structural

formulae, are illustrated in figure 1. (+)-Delta-6-THC,

and (+)- and (-)- stereoisomers of delta-6-THC-l", 1"-

DMHP were circa 97% optically pure, and 7-OH-delta-6-

THC-l",l"-DMHP (also known as 11-OH-trans-delta-8-THC1',1'- DMHP) was 99.9% optically pure. These

cannabinoids were synthesized by Mechoulam et al. (this

volume). (-)- Delta-6-THC was 96.7% optically pure and

was obtained from the National Institute on Drug Abuse.

The audiogenic seizure- (AGS-) susceptible rat model of

epilepsy (Dailey and Jobe 1985) was used to assess

anticonvulsant activity, and the rat ROT paradigm

(Dunham and Miya 1957) was used to assess differential

neurotoxicity (i.e., reflecting mainly sedation and/or

incoordination), of the THC compounds. Male, 150-200

gm, genetically epilepsy-prone rats (Dailey and Jobe

1985) were used for the AGS tests; and male, 150-200 gm

rats (Hsd: (SD)BR; Harlan Sprague Dawley Company,

Indianapolis, IN) were used for the ROT tests. As

detailed previously (Consroe and Man 1973; Consroe et

al. 1985), cannabinoids were prepared in a vehicle of

10% polysorbate 80 and 90% distilled water, and were

injected intravenously (i.v.); and rats were tested 15

60
minutes (for DMHP analogs) or 30 minutes (for the parent

THC isomers) later, the respective peak-effect times for

both tests. In the AGS test, responses to sound were

recorded as the presence or absence of seizure (i.e.,

generalized clonic convulsions). In the ROT test,

effects were measured as the ability or inability (i.e.,

neurotoxicity) of trained rats to remain on a revolving

rotorod for 60 seconds.

FIGURE 1

Separate groups of 10 to 15 rats were used for each

dose, and 3 to 6 doses of each cannabinoid were used for

each AGS and ROT test. Median effective anticonvulsant

(ED50) and neurotoxic (TD50) doses and regression line

parameters were calculated by computer-assisted probit

analysis (Dixon 1977). Subsequent calculations of 95%

confidence limits (CL) and comparisons between

and median doses (i.e.,

slopes

ED50 and TD50 potency

comparisons) were carried out by using the equations of

Litchfield and Wilcoxon (1949). We have incorporated

61
these equations into a GW BASIC program for use on a

microcomputer operating under MS-DOS. Finally, the

protective index (PI) of compounds was calculated by the

usual method (i.e., PI = TD50/ED50).

RESULTS AND DISCUSSION

Table 1 presents the dose-effect parameters of the THC

stereoisomers. Comparisons among these data reveal

several salient findings.

TABLE 1

Dose-Response Parameters of Delta-6-THC Stereoisomers in

Audiogenic Seizure (AGS) and Rotorod (ROT) Neurotoxicity

Tests in Rats1

Drug ROT-TD50 AGS-ED50

(95% CL) (95% CL) PI

(-)-7-OH-THC-1",1"-DMHP 0.007 0.004 1.75

(0.005-0.009)(0.002-0.007)

(+)-7-OH-THC-l", 1"-DMHP Not active2

Not active3

(-)-THC41.85 4.70 0.39

(0.98-3.45) (3.11-7.09)

(+)-THC 21.66 15.23 1.42

(1g.81-23.68)(12.42-18.68)

(-)-THC-l", 1"-DMHP 0.035 0.041 0.85

(0.031-0.040) (0.037-0.046)

(+)-THC-1", 1"-DMHP 0.53 0.89 0.60

(0.37-0.76) (0.73-1.1)

1

Median toxic (TD50) and effective (ED50) doses and 95%

confidence limits (CL) are in mg/kg, i.v. PI =

protective index = ROT-TD50/AGS-ED50.

2

At 1.0, 7.0, and 15.0 mg/kg, i.v.; higher doses were

not tested due to insufficient quantity of drug.

3

At 0.1, 1.0, and 4.0 mg/kg, i.v.; higher doses were not

tested due to insufficient quantity of drug.

4

Data taken from Consroe and Man (1973).

First, the (-)-enantiomers are significantly (P < .05)

more potent (or substantially more active) than are

62
their respective (+)-enantiomers in both anticonvulsant

(AGS) and neurotoxicity (ROT) tests. For anticonvulsant

activity, (-)-delta-6-THC is 3 times more potent than

(+)-delta-6-THC, and (-)-delta-6-THC- 1",1"-DMHP is 22

times more potent than (+)-delta-6-THC-1",1"- DMHP.

Also, (-)-7-OH-delta-6-THC-1",1"-DMHP is extremely

potent and its (+)-enantiomer is not active in doses up

to 1,000 times higher. For neurotoxic activity, (-)-

delta-6-THC is 12 times more potent than (+)-delta-6-

THC, and (-)-delta-6-THC-1",1"-DMHP is 15 times more

potent than (+)-delta-6-THC-1",1"-DMHP. Again, (-)-7-

OH-delta-6-THC-1",1"-DMHP is extremely potent and its

(+)- enantiomer is not active in doses up to 2,143 times

higher.

Although neither these nor any other pairs of optically

pure THC stereoisomers have been evaluated for

anticonvulsant or ROT neurotoxicity effects, the

comparative activities (or in some cases, dose-response

potencies) of the enantiomers of delta-1-THC (Dewey et

al. 1984) and of delta-6-THC (Consroe et al. 1982; Dewey

et al. 1984) have been ascertained in several other

laboratory animal paradigms. In all cases, the (-)-

isomers were more active than their respective (+)-

isomers; and for the enantiomers of delta-6-THC,

differences in activity ranged from about fourfold to

fiftyfold depending upon the species and the

pharmacological test system used. While it is clear

there is a stereoselectivity of action for the THCs,

such is not the case for cannabidiol (CBD), a

noncannabimimetic cannabinoid of cannabis. In a

previous study (Consroe et al. 1985), we found that the

natural (-)-CBD was equipotent to the unnatural (+)-CBD

in both AGS and ROT tests. These findings suggest that

stereoselective receptor mechanisms may be important for

many effects of the THCs, including their anticonvulsant

and neurotoxic activities. However, it is unlikely that

such receptor mechanisms underlie the latter effects of

CBD.

Second, the 1", 1"-DMHP and 7-OH modifications of THC

greatly increase potencies in both anticonvulsant and

neurotoxic paradigms (table 1). (-)-Delta-6-THC-l",l"-

DMHP is 115 times more potent in the AGS test and 53

times more potent in the ROT test than is (-)-delta-6-

THC. (-)-7-OH-delta-6-THC-1",1''-DMHP is 1,175 times

more potent in the AGS test and 264 times more potent in

the ROT test than is (-)-delta-6-THC. Additionally,

(+)-delta- 6-THC-1", 1"-DMHP is 17 times more potent in

the AGS test and 41 times more potent in the ROT test

than is (+)-delta-6-THC. However, the "inactivity" of

(+)-7-OH-delta-6-THC-1",1"-DMHP is an enigma. Because

of limited quantity of the latter drug, we were unable

to evaluate doses higher than 4 mg/kg in the AGS test

and 15 mg/kg in the ROT test. Nevertheless, these doses

6 3
are not too different from the AGS-ED50 (15.23 mg/kg)

and ROT-TD50 (21.66 mg/kg) of (+)-delta-6-THC.

Aside from this apparent exception, the present results

are generally congruent with many previous data showing

potency increases with 1 ",2"-DMHP and 7-OH modifications

of the THCs (Mechoulam and Edery 1973; Pradhan 1984;

Razdan 1986). Additionally, several 1",2"-DMHP analogs

of (+/-)-delta-3-THC (i.e., compounds existing as a

mixture of 4 to 8 possible isomers) have been tested for

anticonvulsant and ROT neurotoxic activities in rodents

and their potencies compared to that of (-)-delta-1-THC

(Karler et al. 1974; Plotnikoff et al. 1975). Depending

on the analog, species, seizure test, and other

experimental conditions, potency increases ranging

from about twofold to sixteenfold were reported.

Finally, a perusal of the PIs of cannabinoids evaluated

in the present study (table 1) show that only (-)-7-OHdelta-6-THC-l", 1"-DMHP (PI = 1.75) and (+)-delta-6-THC

(PI = 1.42) have a favorable separation between

anticonvulsant and (ROT) neurotoxic activities.

In a previous study (Consroe et al. 1985), we determined

the i.v. potencies of three major antiepileptic drugs in

our rat AGS and ROT tests. A collation of these data

indicates that the PIs of the above THC analogs are

similar to that of phenytoin (PI = 1.6), but are lower

than those of phenobarbital (PI = 2.7) and carbamazepine

(PI = 2.7). The clinical efficacy of these classic

antiepileptic drugs is unquestionably good; but the

therapeutic usefulness of each of these agents,

especially of phenytoin, is limited in many patients by

sedation, incoordination, and/or other neurologic side

effects (Livanainen and Savolainen 1983; Keranen and

Sivenius 1983). Our comparative, albeit limited,

preclinical data suggest that the THC analogs might have

this same type of therapeutic limitation. Additionally,

(-)-7-OH-delta-6-THC-1", 1"-DMHP has recently been found

to be extremely potent (and, in fact, the most potent

cannabinoid ever tested) in the drug discrimination

learning (DDL) test in rats and pigeons (Mechoulam et

al., this volume). As this test (Jarbe et al. 1981;

Weissman 1981) is a qualitative and possibly a

quantitative predictor of cannabimimetic activity, (-)-

7-OH-delta-6-THC-1",1"-DMHP would appear to be a poor

candidate for therapeutic application. On the other

hand, (+)-delta-6-THC is essentially inactive in the DDL

paradigm (Jarbe et al. 1981) and, thus, would be

predicted to have minimal, if any, cannabimimetic

activity. While the presently determined PI of (+)-

delta-6-THC is certainly not optimum, other (+)-THC

analogs might have better selectivity of anticonvulsant

activity relative to neurotoxic action. Further

evaluation of (+)-THC analogs, such as (+)-7-OH-delta-6-

64
THC-1", 1"-DMHP , would appear to be an important

endeavor for future research in this area.

REFERENCES

Consroe, P., and Man, D.P. Effects of delta-8- and

delta-9-tetrahydrocannabinol on experimentally

induced seizures. Life Sci 13:429-439, 1973.

Consroe, P.; Martin, A.R.; and Fish, B.S. Use of a

potential rabbit model for structure-behavioral

activity studies of cannabinoids. J Med Chem

25:596-599, 1982.

Consroe, P.; Martin, A.; and Mechoulam, R.

Anticonvulsant effects of cannabidiol stereoisomers

and analogs in rats. In: Harvey, D.J., ed.

Marihuana '84. Proceedings of the Oxford Symposium

on Cannabis. Oxford:

712.

IRL Press, 1985. pp. 705-

Dailey, J.W., and Jobe, P.C. Anticonvulsant drugs and

the genetically epilepsy-prone rat. Fed Proc

44:2640-2644, 1985.

Davis, J.P., and Ramsey, H.H. Anti-epileptic

action of marijuana-active substances. Fed Proc

8:284, 1949.

Dewey, W.L.; Martin, B.R.; and May, E.L. Cannabinoid

stereoisomers: Pharmacological effects. In: Smith

D.F., ed. CRC Handbook of Stereoisomers: Drugs in

Psychopharmacology. Boca Raton: CRC Press, 1984.

pp. 317-326.

Dixon; W.J, BMD Biomedical Computer Programs.

Berkelev:

pp. 439-450.

University of California Press. 1977.

Dunham, N.W., and Miya, T.S. A note on a simple

apparatus for detecting neurological deficit in

rats and mice. J Am Pharm Assoc 46:208-209, 1957.

Hardman, H.P.; Domino, E.R.; and Seevers, M.H.

General pharmacological actions of some synthetic

tetrahydrocannabinol derivatives. Pharmacol Rev

23:295-315, 1971.

Jarbe, T.U.C.; Swedberg, M.D.B.; and Mechoulam, R.

A repeated tests procedure to assess onset and

duration of the cue properties of (-)-delta-9-THC,

(-)-delta-8-THC-DMH and (+)- delta-8-THC.

Psychopharmacology 75:152-157, 1981.

Karler, R.; Cely, W.; and Turkanis, S.A. Anticonvulsant properties of delta-9-

tetrahydrocannabinol and other cannabinoids. Life

Sci 15:931-947, 1974.

Keranen, T., and Sivenius, J. Side effects of

carbamazepine, valproate and clonazepam during

long-term treatment of epilepsy. Acta Neurol Scand

(Suppl 97) 68:69-80, 1983.

Litchfield, J.T., and Wilcoxon, F. A simplified

method of evaluating dose-effect experiments. J

Pharmacol Exp Ther 96:99-133, 1949.

65
Livanainen, M., and Savolainen, H. Side effects

of phenobarbital and phenytoin during long-term

treatment of epilepsy. Acta Neurol Stand (Suppl

97) 68:49-67, 1983.

Mechoulam, R., and Edery, H. Structure-activity

relationships in the cannabinoid series. In:

Mechoulam, R., ed. Marihuana Chemistry, Metabolism

and Clinical Effects. New York:

1973. pp. 101-136.

Academic Press,

Plotnikoff, N.P.; Zaugg, H.E.; Petersen, A.C.;

Arendsen, D.L; and Anderson, R.F. New benzopyrans:

Anticonvulsant activities. Life Sci 17:97-104,

1975.

Pradhan, S.N . Pharmacology of some synthetic tetrahydrocannabinols. Neurosci Biobehav Rev 8:369-385,

1984.

Razdan, R.K.Structure-activity relationships in

cannabinnoids. Pharmacol Rev 38:75-149, 1986.

Weissman, J.A. On the definition of cannabinoids:

Botanical? Chemical? Pharmacological?

Pharmacol 21:159s-165s, 1981.

J Clin

ACKNOWLEDGMENTS

This research was supported by grant NS15441 from the

National Institute of Neurological and Communicative

Disorders and Stroke. GEPR-3 rats were supplied by Dr.

Phillip Jobe (Department of Basic Sciences, University

of Illinois College of Medicine, Peoria, IL).

AUTHORS

Paul Consroe, Ph.D.

Department of Pharmacology

and Toxicology

College of Pharmacy

University of Arizona

Tucson, Arizona 85721 U.S.A.

Raphael Mechoulam, Ph.D.

Department of Natural

Products

Faculty of Medicine

The Hebrew University

of Jerusalem

Jerusalem 91120 ISRAEL

66
Different Cannabinoids Exhibit

Different Electrophysiological

Properties

Stuart A. Turkanis, Ph.D., and Ralph Karler, Ph.D.

INTRODUCTION

Delta-9-tetrahydrocannabinol (THC) is a nonselective, centrally active drug that

produces a wide variety of pharmacological effects (Truitt and Braude 1974;

Paton 1975); for example, in conscious animals, this cannabinoid causes a

complex mixture of central depressant and excitatory responses ranging from

sedation, anticonvulsant effects, and motor toxicity to overt convulsions (Karler

and Turkanis 1981). Fifteen years ago there were few marijuana-related

electrophysiological investigations, and those mainly involved marijuana extracts

(Boyd et al. 1971, 1974; Pirch et al. 1972; Truitt and Braude 1974; Paton 1975);

consequently, we began a series of electrophysiological investigations of the

pharmacological properties of THC. Our approach was multifarious: 1) to

evaluate the cannabinoid’s responses at various levels of organization ranging

from the cerebral cortex to the neuromuscular junction, 2) to assess the drug’s

effects on pools of neurons as well as on single excitable cells, 3) to use

conscious animals when possible in order to avoid the confounding problems of

anesthesia, and 4) to evaluate the active form of the cannabinoid by using in

vitro test preparations. The goals of our research were twofold: to describe the

electrophysiological effects of THC, and to uncover possible synaptic mechanisms

of action for these effects.

Another important feature of our research has been the electrophysiological

evaluation of the properties of cannabidiol (CBD), a drug that was seldom

studied prior to our work because it was considered to be pharmacologically

inactive (Hollister 1973). The data presented here represent an extensive

comparison of the properties of the psychoactive THC with the nonpsychoactive

CBD (Perez-Reyes et al. 1973). In addition, it is well documented that THC is

metabolized in vivo to various pharmacologically active metabolites (Burstein

1973); therefore, in order to understand the pharmacology of the parent drug, it

is necessary to determine the properties of its active metabolites. For this reason,

we comparatively evaluated the effects of THC’s principal metabolite, 11-

hydroxy-THC, and its hydroxylated metabolites. The findings demonstrate that

different cannabinoids can produce markedly different neuropharmacological

responses.

67
METHODS

The procedures for the study of the electrophysiological preparations and

descriptions of the experimental designs of the various cannabinoid studies have

been published in detail elsewhere: electrically produced transcallosal cortical

evoked responses in rats (Turkanis and Karler 1981a), photically evoked cortical

after discharges in rats (Turkanis et al. 1977), cobalt-caused epilepsy in rats (Chiu

et al. 1979), iron-caused epilepsy in rats (Turkanis and Karler 1982), electrically

induced limbic afterdischarges in rats (Smiley et al. 1979), cat spinal-cord

preparation (Turkanis and Karler 1983, 1984a, 1986), isolated bullfrog ganglia

(Turkanis and Karler 1975), and isolated frog sartorius neuromuscular junctions

(Turkanis and Karler, in press).

For parenteral preparations, the cannabinoids were dispersed in isotonic sodiumchloride solution with the aid of Tween 80 and ultrasound (Turkanis et al. 1974).

For bathing solutions, the cannabinoids were dispersed in Ringer solutions with

the nonionic detergent Pluronic F68 and ultrasound (Turkanis and Karler 1975,

in press). Pentylenetetrazol (PTZ) was dissolved in isotonic sodium-chloride

solution. Vehicle and drug preparations were administered intraperitoneally (i.p.)

for the cortical and limbic studies and intravenously (i.v.) for the spinal-cord

studies.

RESULTS

The effects of THC, CBD, and PTZ on the amplitude of the initial phase of

transcallosal cortical evoked responses in rats were investigated (Turkanis and

Karler 1981a). Specifically, THC in the lower dosage range (0. 1-1.0 mg/kg)

markedly increased the evoked response amplitude, whereas higher dosages (1.2-

6.0 mg/kg) elicited only depression. Our reference convulsant, PTZ, in

subconvulsant doses (10-20 mg/kg), like THC, enhanced potential amplitude.

Both THC and PTZ also increased electrically caused cortical evoked potentials in

the monkey (Boyd et al. 1971). In contrast, CBD over a wide dosage range (0.l80 mg/kg) exerted only depressant effects. In summary, the findings

demonstrate qualitative differences in the pharmacological properties of the two

cannabinoids.

We also compared the effects of THC, CBD, and PTZ on photically evoked

cortical afterdischarges in rats (Turkanis et al. 1977); THC (5-10 mg/kg), but not

CBD, produced a PTZ-like increase of cortical potentials. In contrast to the

results with electrically caused responses, high THC doses (50-100 mg/kg) did

not affect photic potentials; furthermore, neither THC nor CBD displayed any

depressant properties in this test system.

The results of our limbic afterdischarge investigations also demonstrate that THC

and CBD exhibit different neuropharmacological properties (Smiley et al. 1979).

THC in a dosage range of 0.3 to 3.0 mg/kg elicited only a threshold increase--a

depressant response; doses of 5 to 15 mg/kg, however, caused an even greater

rise in threshold, but also a prolongation of the afterdischarge. This prolongation

of the response is an electrophysiological manifestation of the drug’s excitatory

properties. In contrast to THC, CBD (0.3-200 mg/kg) only depressed the

afterdischarge; that is, it increased the threshold, shortened the duration, and

reduced the amplitude. In summary, low THC doses and all CBD doses yielded

depression, whereas relatively high THC doses produced concurrent excitation

and depression of the limbic afterdischarge.

68
The effects of cannabinoid on iron-epileptic rats are complex: THC (1-20 mg/

kg) elicited excitation by producing behavioral signs of central nervous system

(CNS) stimulation, generalized bursts of epileptiform potentials, and frank

convulsions (table 1 and figure 1); in addition, it produced depression by causing

motor toxicity and reduction of focal epileptic activity (table 1 and figures 1 and

2).

TABLE 1

Influences of Cannabinoids on Conscious Iron-Epileptic

Rats with Chronically Implanted Cortical Electrodes

Treatment Dosage

Range

Focal Precipita- Bursts CNS Motor

Epileptic tion of of Epilep-Excita- Toxicity

Activity Convul- tiform tion

sions Potentials

(mg/kg,

i.p.)

THC

11 -HydroxyTHC

 8-BetahydroxyTHC

CBD

PTZ

l-20 - Yes

l-20 - No

0.5-20 0 Yes

l-100 0 No

5-20 0 Yes

Yes

Yes

Yes

No

Yes

Yes

Yes

No

No

No

Yes

Yes

No

No*

No

- = decrease; 0 = no change. Each drug was studied in at least six different

experiments. Behavioral signs of CNS excitation were vocalization, hissing, and

jumping. For additional details, refer to figures 1 and 2 and to Turkanis and

Karler 1982.

*The median-effective motor-toxic dose of CBD in nonepileptic control rats is

173 mg/kg, i.p. (Smiley et al. 1979).

11-Hydroxy-THC (1-20 mg/kg) exerted THC-like effects with one major

exception; that is, it did not precipitate convulsions (table 1 and figure 2). The

properties of 8-beta-hydroxy-THC, another THC metabolite, were different

compared with those of either the parent compound or the 11-hydroxy

metabolite. In doses of 0.5 to 20 mg/kg, it exerted only excitation, as evidenced

by bursts of epileptiform potentials and convulsions (table 1 and figures 1 and 2).

In contrast, CBD, even in doses up to 100 mg/kg, was devoid of all of THC’s

effects. In addition, pretreatment with CBD markedly altered the responses to

THC in the following ways: Focal depression was partially blocked, generalized

epileptiform activity was enhanced, and convulsions were abolished. In general,

the cannabinoids exhibited a wide spectrum of CNS effects in iron-epileptic rats,

functional significance of the cannabinoid-caused generalized but the

pharmacological profile of each agent was demonstrably different. The bursts of

functional significance of the cannabinoid-caused generalized epileptiform

69
FIGURE 1. Influences of cannabinoids on electrocorticogram of conscious ironepileptic rats. The iron-caused epileptic focus was located in the parietal cortex;

recordings were obtained from screw electrodes chronically implanted on the focus

and other cortical sites. The position of the iron focus was identical to that of the

cobalt focus (figure 3 and table 2). A1 depicts recording 15 min after vehicle;

A2, recording 15 min after THC. B illustrates generalized bursts of epileptijorm

potentials jrom three different rats: B1 and B2 represent potentials observed 45-

60 min after THC: B3 and B4, potentials 15-20 min after 8-beta-hydroxy-THC;

B5 and B6, potentials 10-15 min after PTZ (adapted from Turkanis and Karler

1982).

potentials is unknown, but these potentials look like the electrical manifestation

of a seizure. In support of such a possibility, the convulsant control PTZ

produced seizurelike discharges that are indistinguishable from those caused by

THC and its metabolites (table 1 and figure 1). In addition, THC and its

metabolites also evoked these generalized seizurelike discharges in nonepileptic

control rats.

70
FIGURE 2. Influences of cannabinoids on iron-induced focal epileptic potentials

in conscious rats. Throughout each experiment, focal electrocorticograms and

integrals of the electrocorticogram voltage were recorded. The data shown are

maximum changes in the integrals of the electrocorticographic voltage over a 5-

min period, which are expressed as a percentage of the internal vehicle control

values obtained in each experiment. Percentage oj control focal activity is plotted

against the cumulative dose. Each dose-effect curve represents the results

obtained from an experiment with one rat. For additional details, refer to

figure 1 and to Turkanis and Karler 1982. Copyright 1982 by Pergamon Press

(New York).

The effects of THC, its 11-hydroxy metabolite, and CBD were assessed in

cobalt-epileptic rats. As shown in table 2 and in figure 3, THC (0.5-100 mg/kg)

increased the frequency of focal epileptic potentials, elicited generalized bursts of

epileptiform potentials, and precipitated or exacerbated convulsions.

11-Hydroxy-THC (1.0-20 mg/kg) exerted only one of the three THC excitatory

responses, namely, bursts of epileptiform potentials. Again, CBD (0.5-200

mg/kg) only produced depression-suppression of the spontaneous convulsions.

Each of the drugs, therefore, produced characteristic effects in this test system.

The five investigations with conscious animals described above involved pools of

cortical and limbic neurons, and the results demonstrated that the cannabinoids

can exert a complex mixture of excitatory and depressant electrophysiological

effects. The next step in our research program was to uncover possible synaptic

sites and mechanisms of cannabinoid actions. Initial studies with cat spinal

monosynaptic reflexes indicated that cannabinoid effects at this level were

comparable to those in higher centers. Specifically, THC in low doses (0.05-

0.15 mg/kg, i.v.) increased the reflex itself and posttetanic potentiation (PTP),

whereas higher doses of THC (0.2-0.75 mg/kg, i.v.) and all doses of CBD

71
TABLE 2

Influences of Cannabinoids on Conscious

Cobalt-Epileptic Rats with Chronically

Implanted Cortical Electrodes

Treatment Dosage Range Frequency Effect on Bursts of

(mg/kg, i.p.) of Focal Convulsions EpileptiEpileptic form

Potentials Potentials

THC 0.5-100 + + Yes

11-HydroxyTHC 1 0-20 0 0 Yes

CBD 0.5-200 0 No

+ = increase; - = decrease; 0 = no change. THC and CBD were studied in eight

epileptic rats, and 11-hydroxy-THC was studied in three animals due to a limited

drug supply. The production of generalized bursts of epileptiform cortical

potentials by THC and its metabolite also occurred in control rats. For additional

details, refer to figure 3 and to Chiu et al. 1979.

FIGURE 3. Influence of THC and CBD on frequency of spontaneously firing

focal epileptic potentials recorded from a cobalt-induced focus in the left parietal

cortex. Each point represents the results from a different conscious rat and is the

maximum frequency expressed as a percentage of predrug vehicle control. For

additional details, refer to table 2 and to Turkanis and Karler 1981b. Copyright

1981 by The American College of Clinical Pharmacology (New York).

72
(0.05-14 mg/kg, i.v.) were only depressant (Tramposch et al. 1981; Turkanis and

Karler 1984a). In this context, intracellular recording techniques were used on

single spinal motoneurons in order to define the cannabinoids’ synaptic

pharmacology.

FIGURE 4. Excitatory effects of THC on electrophysiological responses of single

spinal motoneurons in spinal cats. Each trace represents the electronically

obtained average of 16 consecutive potentials. A and B are from two different

cats. A depicts THC-caused increase of EPSP amplitude: AI, control response

20 min after vehicle; A2, response 20 min after drug. The increase in amplitude

is at least partially attributable to an increase in membrane resistance. EPSPs

were recorded jrom a triceps surae motoneuron. B depicts THC-caused limited

depression of IPSP amplitude: B2, control response 20 min after vehicle: Bs,

response 20 min after drug; B3, response 30 min after drug. The figure

illustrates the limited efficacy of THC in reducing the amplitude of lPSPs

because the effect of 10 mg/kg (B3) is no greater than that of 0.1 mg/kg (B2).

For additional details, refer to table 3. (Adapted from Turkanis and Karler

1983.)

THC effects on the parameters of single spinal motoneurons are complicated: The drug (0.01-0.1 mg/kg) produced excitation by increasing excitatory

postsynaptic potentials (EPSPs) and by decreasing inhibitory postsynaptic

potentials (IPSPs) (table 3 and figure 4).

THC’s responses on the two synaptic potentials appear to be unrelated to

alterations of the afferent input because up to 10 mg/kg, i.v., of the drug had no

measurable effects on compound-nerve action potentials recorded from the dorsal

roots (Turkanis and Karler 1984b). EPSP enhancement, however, is at least

partially attributable to an increase in membrane resistance (table 3). In addition

to excitation, THC produced depression by raising the firing threshold and

reducing soma action-potential amplitude (table 3); thus, in single neurons, as in

conscious animals and pools of neurons, the cannabinoid exerted dual effects.

11-Hydroxy-THC’s effects were similar to those of the parent compound, except

73
TABLE 3

Influences of Cannabinoids on the Afferent

Input and on Single Spinal-Motoneuron

Parameters in Spinal Cats

Treatment EPSP Membrane IPSP Firing Nerve Soma

Ampli- Resis- Ampli- Thresh- Action Action

tude tance tude old Potential Potential

THC + + - + 0 -

11-HydroxyTHC + + 0 + 0 -

CBD - 0 0 + 0 -

+ = increase; - = decrease; 0 = no change. All of the effects occurred between

0.01 and 0.1 mg/kg, i.v., except for the reduction of the soma action potentials;

the latter was affected by 0.2-0.5 mg/kg 11-hydroxy-THC and 1-5 mg/kg THC

and CBD. Compound nerve-action potentials (afferent input) were recorded

extracellularly from dorsal roots; the other measurements were made with

intracellular microelectrodes from motoneuron somas. Measurement of excitatory

EPSPs, IPSPs, membrane resistance, and firing threshold were made from single

spinal motoneurons with intracellular glass microelectrodes filled with 3M

potassium-acetate solution. EPSPs were evoked by stimulating the triceps surae

or posterior-biceps semitendinosus nerves, and IPSPs were evoked by stimulating

the quadriceps nerve. Membrane resistance was determined by passing sufficient

current to produce small depolarizations. The threshold for the production of the

soma potential was the voltage required to fire the cell consistently. For

additional details, refer to figures 4 and 5 and to Turkanis and Karler 1983,

1984a, 1984b, 1986.

that the metabolite did not depress IPSPs (table 3). Again, CBD (0.1-5 mg/kg)

was only a depressant; that is, it raised the firing threshold, depressed the

amplitude of soma action potentials, and reduced the EPSP amplitude (table 3

and figure 5). In relation to mechanisms and sites of drug action, the

cannabinoids’ effects on the firing threshold and amplitude of soma action

potentials and on membrane resistance indicate that changes in postsynaptic ionic

conductances are involved.

Subsequent cannabinoid investigations were carried out with isolated synaptic

preparations. The goals of these studies were twofold: to determine the active

form of the cannabinoid by avoiding in vivo drug metabolism, and to uncover

possible synaptic mechanisms and sites of drug action. To this end, the effects

of several cannabinoids were evaluated on PTP of extracellularly recorded action

potentials in isolated bullfrog ganglia: Two THC metabolites, 11-hydroxy-THC

74
FIGURE 5. Depressant effects of CBD on electrophysiological responses of

single spinal motoneurons in spinal cats. Traces in A are electronically obtained

averages of 16 consecutive potentials: traces in B and C are of single potentials.

A, B, and C are from three different cats. A depicts CBD-caused depression of

EPSP amplitude; responses measured 30 min after vehicle and 30 min after drug.

B illustrates CBD-elicited increase in firing threshold; responses measured 20 min

after vehicle and 20 min after drug. C shows the CBD-caused depression of the

soma action potential; responses measured 30 min after vehicle and 30 min after

drug. The effects shown in A, B, and C are not the result of changes in

membrane resistance or resting membrane potential. For additional details, refer

to table 3. (Adapted from Turkanis and Karler 1986.)

and 8-alpha, 11-dihydroxy-THC, in low concentrations (01.-0.33 μM) reduced

PTP, whereas THC, in concentrations as high as 25μM, exerted no effect (figure

6).

Drug-induced changes in PTP may also relate to THC’s anticonvulsant as well as

to its convulsant properties, since a reduction in PTP has been reported to be an

electrophysiological mechanism of action for such antiepileptic agents as

75
FIGURE 6. Influences of cannabinoids on PTP at isolated bullfrog paravertebral

ganglia X. PTP was defined as an increase in the amplitude of the extracellucellularly recorded ganglionic potentials following a period of tetanic stimulation

and is expressed as the ratio of posttetanic to pretetanic potential amplitudes.

Bracketed o = the mean and standard deviation of four control values; values

60 min after drug treatment. Raising the THC concentration above 25 μM

reduced the amplitude of the pretetanic potential, thereby invalidating calculations

of PTP (Turkanis and Karler 1975). Copyright 1975 by Pergamon Press (New

York).

phenytoin (Esplin 1957). Our research demonstrates that THC in vivo, depending

upon the dose, can either increase or decrease PTP of cat spinal monosynaptic

reflexes; therefore, similar effects in higher centers may contribute to the drug’s

convulsant and anticonvulsant properties. Our CBD results with spinal reflexes

substantiate a role for PTP because the drug only depresses PTP and is only

anticonvulsant.

The cannabinoids’ effects on IPSPs of spinal motoneurons may also provide a

possible explanation for THC’s convulsant properties. In this instance, THC, like

the convulsant strychnine (Kuno and Weakly 1972), reduces the amplitude of

spinal motoneuron IPSPs. The 11-hydroxy-THC data are consistent with

involvement of inhibitory potentials because the metabolite, unlike THC, does

not affect IPSPs and is not a convulsant. On the other hand, THC, unlike

76
strychnine, precipitates convulsions only in special circumstances; that is, in

cobalt- and iron-epileptic rats (tables 1 and 2), in epileptic beagles (Feeney

1979), and in a special strain of rabbits (Martin and Consroe 1976). The reason

for the restricted convulsant activity of THC may be due to its limited efficacy

in reducing inhibitory potentials. In short, the cannabinoids’ anticonvulsant and

convulsant activities are probably associated with several pharmacological

properties of the drugs as well as related to the susceptibility of the subjects.

Intracellular recordings from such single cells as spinal motoneurons and

neuromuscular junctions may provide information about possible electrophysiological mechanisms and sites of the cannabinoids’ central actions. First,

the synapse is likely to be the major site of drug action: As demonstrated in in

vivo experiments, synapses are affected by relatively low THC doses (0.01-0.1

mg/kg, i.v.), whereas doses up to 10 mg/kg, i.v., failed to modify compound

action potentials of axons (table 3). Second, cannabinoids can exert both preand post-synaptic effects; for example, THC caused excitation presynaptically by

enhancing neurotransmitter release and postsynaptically by increasing membrane

resistance. In addition, THC produced depression presynaptically by reducing

neurotransmitter release and postsynaptically by decreasing neurotransmitter

response. Lastly, cannabinoid-induced effects on the motoneuron soma, that is,

changes in membrane resistance, firing threshold, and action-potential amplitude,

indicate that the drugs alter postsynaptic ionic conductances. Depression of

neurotransmitter responses may be due to drug-receptor interactions or to

changes in postsynaptic ionic conductances. Nevertheless, if these drug responses

occur in the CNS, they suggest possible mechanisms and synaptic sites of action

for the cannabinoids’ central excitatory and depressant properties, and the

consistent pattern of the drugs’ effects at every level of organization studied--

from the cerebral cortex to the neuromuscular junction--validate these

conclusions.

It is possible that a higher THC concentration might reduce PTP, but raising the

concentration above 25 μM markedly reduced the amplitude of the pretetanic

potential and invalidated the calculation of PTP. These findings support those of

Brady and Carbone (1973), who found that 11-hydroxy-THC, but not the parent

compound, depressed the action potentials of squid giant axons. Like THC’s

metabolites, CBD (33-100 μM) was pharmacologically active; that is, it reduced

PTP. It is noteworthy that the isolated ganglion, like the squid axon, only

displayed depression in response to the cannabinoids.

The purpose of the next study was to determine whether THC affects neurotransmitter release in an electrophysiologically functional system; specifically,

a preparation in which nerve-action potentials release neurotransmitter. To

accomplish this aim, we evaluated the influences of THC on neurotransmitter

release by determining electrophysiologically the mean quantum content of the

endplate potential (m) (Katz 1969). A neuromuscular preparation was selected

because it is one of the limited number of preparations in which m can be

measured, while the rationale for using a cholinergic system was that

cannabinoids have been reported to affect cholinergic pathways (Drew and Miller

1974); therefore, intracellular recording techniques were used with isolated frog

sartorius neuromuscular junctions to describe the effects on transmitter release

(Turkanis and Karler, in press). The results were again complex: The frequency

of miniature endplate potentials (m.e.p.ps) was increased by THC, decreased by

CBD, and unaffected by 11-hydroxy-THC, whereas m.e.p.ps amplitude was

depressed by all three cannabinoids. In addition, m was first increased and then

decreased by THC and its 11-hydroxy metabolite, but CBD produced only

77
depression. Changes in m and m.e.p.ps frequency indicate presynaptic sites of

drug action, and reduction of m.e.p.ps amplitude suggests a postsynaptic site.

The pre- and post-synaptic effects described here are possible mechanisms of

action for the drug-caused amplitude changes of evoked and synaptic potentials

in the CNS.

DISCUSSION

At every level of organization studied--from pools of cortical neurons to

individual spinal motoneurons and neuromuscular junctions--the cannabinoids

produce excitation and/or depression. This pattern of drug response suggests that

the observed effects on single neurons may account for the mechanisms of action

of the cannabinoids in conscious animals; for example, if the effects on single

spinal motoneurons are applicable to higher centers, then they may contribute to

the cannabinoids’ anticonvulsant activity. In fact, THC and CBD raise thresholds

for electrically induced minimal convulsions in conscious animals (Karler and

Turkanis 1981b), and the drugs raise the threshold for electrically induced

afterdischarge (seizure discharges) in pools of limbic neurons (table 3). Both

effects reflect the drugs’ anticonvulsant activity, and both effects may be the

direct result of an increased firing threshold in individual neurons.

The results of comparative electrophysiological investigations of THC with its

11-hydroxy metabolite can be classified, as shown in table 4, into three

categories: In the first, both drugs produced similar responses; that is, the two

drugs were either equipotent or 11-hydroxy-THC was more potent than the

parent cannabinoid; for example, dose-effect relations for depression of cobaltinduced focal epileptic activity were identical for both drugs; in contrast, the 11-

hydroxy metabolite was 10 times more potent than THC in the production of

generalized cortical bursts of epileptiform potentials in cobalt-epileptic rats. In

the second category, only THC has pharmacological activity (table 4). Finally, in

two in vitro test systems, only 11-hydroxy-THC has activity (table 4). In this

case, the interpretation of the findings is not complicated by drug metabolism,

and the data suggest that the 11-hydroxy derivative may account for some of the

in vivo responses to the parent drug. In brief, the results indicate that there are

both quantitative and qualitative differences between THC and its principal

metabolite and that, depending upon the effect, the metabolite may contribute to,

be dissociated from, or account for THC’s activity.

The data also demonstrate marked differences in the neuropharmacological

properties of two major marijuana constituents, that is, THC and CBD. These

findings confirm our previously drawn conclusions (Turkanis and Karler

1981b): First, THC is a nonselective, centrally active drug producing both

excitation and depression; second, CBD is a relatively selective central depressant.

In fact, CBD appears to be devoid of all of THC’s central stimulatory properties,

including psychotoxicity. In general, the results of the electrophysiological

studies of the cannabinoids repeatedly illustrate that different cannabinoids

possess different properties.

78
TABLE 4

Comparison of the Electrophysiological Effects

of THC and its 11-Hydroxy Metabolite

I. Responses produced by both cannabinoids:

A. Cortical bursts of epileptiform potentials in cobalt- and

iron-epileptic rats,

B. Depression of cortical focal epileptic activity in iron-epileptic rats,

C. Enhancement of spinal motoneuron EPSPs,

D. Increase in motoneuron membrane resistance,

E. Depression of motoneuron action potential,

F. Depression of m.e.p.ps amplitude, and

G. Increase and subsequent decrease of mean quantum content of

endplate potentials.

II. Responses produced only by THC:

A. Depression of spinal motoneuron IPSP, and

B. Enhancement of m.e.p.ps frequency.

III. Responses produced only by 11-hydroxy-THC:

A. Depression of PTP at isolated bullfrog ganglia, and

B. Depression of action potentials of isolated squid giant axons

(Brady and Carbone 1973).

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ACKNOWLEDGMENT

This research was supported by research grant DA-00346 from the National

Institute on Drug Abuse.

AUTHORS

Stuart A. Turkanis, Ph.D.

Ralph Karler, Ph.D.

Department of Pharmacology

University of Utah School of Medicine

Salt Lake City, Utah 84132

81
Molecular Determinants for

Cannabinoid Activity: Refinement

of a Molecular Reactivity Template

Patricia H. Reggio, Ph.D.

INTRODUCTION

Numerous studies of structure-activity relationships (SARs) in the cannabinoid

series have been published; e.g., see review by Sofia (1978) and some more recent

work by Martin et al. (1984a, 1984b) and Narimatsu et al. (1985). An

observation which emerges from a review of all of these studies is that some

cannabinoids can be structurally dissimilar as in delta-9-tetrahydrocannabinol

(delta-9-THC) and Abbott 40656 (Rosell et al. 1979) and yet can have similar

activities; whereas others, which show only slight structural differences as in 10-

alpha-OH-delta-8-THC and 10-beta-OH-delta-8-THC (Edery et al. 1971). can

exhibit dramatic activity differences (figure 1). This observation emphasizes that

a common basis for the activity of these compounds must be sought in more than

their mere three-dimensional structures.

Traditionally, cannabinoid SARs have been focused almost entirely on the

independent contribution of certain structural groups ("functional groups") of the

molecules. These SARs have been compiled into extensive "lists of requirements"

(Razdan 1984). Certain regions or conformations of the molecules have been

proposed to be important. These regions or conformations include a methyl

group in the plane of the aromatic ring, a free phenol, and a free C-4(C-5’)

aromatic position (Edery et al. 1971); the phenolic hydroxyl group (Uliss et al.

1975); the side chain (Osgood et al. 1978); nonplanarity (Binder et al. 1979); and

planarity of the molecule (Burstein et al. 1983). Stereoselectivity in the activity

of the tetrahydrocannabinols has been reported (Dewey et al. 1984). This type of

approach in SAR studies often assumes the following: that functional groups

must react directly with specific sites in the receptor, that modification of one

group does not affect the reactivity of another, and that geometric and

stereometric factors, such as distance and spatial relationships between functional

groups, are all important. Such focus on isolated aspects of the cannabinoids

ignores the fact that the molecular properties that are directly responsible for the

molecular interactions (that lead to the pharmacological effect) are encoded in the

entire molecular structure. To date, no characterization of the entire molecule

and correlation of this characterization with activity has been attempted in the

cannabinoid field.

The importance of quantum chemical methods in describing the physical and

chemical properties of molecules is evident in every facet of modern chemical

82
FIGURE 1. Cannabinoids (a) which are structurally dissimilar yet exhibit similar

activities, and (b) which are structurally similar yet exhibit different activities.

From Reggio and Mazurek 1987. Copyright 1986 by Elsevier Science Publishers

B.V. (Amsterdam).

research and is very well documented. In the study of biological systems,

experimental data must be obtained in a variety of forms and from many

different sources. The value of quantum chemical approaches to the study of

biological systems is in an ability to analyze, interpret, and rationalize all

experimental observations on molecular processes in a unified language based on

the primary principles of physics. Considerable evidence exists proving the

relative success of theoretical approaches. These approaches have elucidated

specific mechanisms of drug action, have provided a basis for rational drug

design, and have led to conclusions about the nature of drug-receptor

interactions (Dearden 1983; Vida and Gordon 1984). We have used theoretical

approaches to successfully describe the molecular determinants for binding of

methylenedioxytryptamines at SHT/LSD receptors (Reggio et al. 1981) and have

also used theoretical approaches to explain the role that tautormerism plays in the

activation of the histamine-H2 receptor by 2-methyl and 4-methylhistamine

(Reggio and Mazurek 1987). The basic assumption in any of these theoretical

studies of drug action is that the interaction between drugs and their biological

targets is dependent on the same molecular parameters that determine chemical

interactions and reactions. Molecular Reactivity Characteristics (MRCs) such as

molecular electrostatic potentials (MEP), polarizabilities, and proton affinities are

molecular properties that determine specific chemical reactivity. In theoretical

studies of structure-activity relationships, such MRCs are used to establish how

the information necessary for receptor recognition and activation is encoded in

the molecular structure of the drug.

83
Despite the extensive application of theoretical methods in the study of many

pharmacological systems, very little attention has been focused on the

cannabinoids to date. Those theoretical studies on cannabinoids which have been

published have not focused on the calculation of molecular reactivity

characteristics. Most of these studies have concentrated on the calculation of

conformation in an attempt to predict the most probable geometry (lowest energy

conformer) and its relation to activity. The structures and energies of delta-9-

THC and three other cannabinoids obtained by Westheimer calculations and

extended Huckel molecular orbital calculations have been reported (Archer et al.

1970). Theoretical studies of cannabinoids with analgetic and anticonvulsant

activity have also been reported (Johnson et al. 1982; Tamir et al. 1980). The

work presented here extends beyond the considerations which have prevailed in

theoretical studies of the cannabinoids. The aim of this work is to reveal

elements of molecular reactivity that are related to psychopharmacological

activity.

A major step in identifying the MRCs necessary for cannabinoid activity was to

select a template molecule (a molecule with demonstrated activity whose MRCs

can be used as a basis for comparison with those of other cannabinoids). Since

(-)trans-delta-9-THC (delta-9-THC) has been reported to be the major

psychoactive component of cannabis (Gaoni and Mechoulam 1964, 1971) and

since studies indicate that it can exert behavioral effects without metabolic

activation (Carney et al. 1979), delta-9-THC was chosen as the template molecule

for this study. Figure 2 illustrates the numbering systems which are commonly

employed in the literature for delta-9-THC.

FIGURE 2. An illustration of the two numbering systems which are commonly

employed for the cannabinoids. From Reggio and Mazurek 1987. Copyright 1986

by Elsevier Science Publishers B.V. (Amsterdam).

84
Our first working hypothesis deals with two components of the delta-9-THC

structure which confer reactivating characteristics upon the molecule that are

crucial to activity. These components are the lone pairs of electrons of the

phenol oxygen (these lone pairs generate reactivity properties dependent on the

orientation of the OH bond relative to the carbocyclic ring, Ring A), and the

orientation of the carbocyclic ring, Ring A (this ring and its orientation generate

hydrophobic properties). The spatial arrangements of these reactivity

characteristics are the components of the template to which the characteristics of

other cannabinoids can subsequently be compared.

Template for Cannabinoid Activity

Recently, we reported the characterization of the template molecule delta-9-THC

(Reggio and Mazurek, 1987). Since the molecular reactivity characteristics of

delta-9-THC are to be used as a basis of comparison with other cannabinoids,

the essential features of the template are presented here. The reader should refer

to the original paper (Reggio and Mazurek 1987), however, for all calculational

details.

The lowest energy conformer of delta-9-THC as obtained by conformational

analysis is shown in figure 3. It is clear from this figure that delta-9-THC is a

nonplanar molecule. The carbocyclic ring (Ring A) of delta-9-THC exists in a

half-chair conformation. Ring B assumes a conformation such that the axial C6

methyl group is on the same side of the molecule as H

H10a than is the other methyl group. In this optimized

10a and is much closer to

structure, the phenolic

hydrogen points away from the carbocyclic ring (dihedral angle, C2-C1-0-H = -

l

o

). Figure 4 shows a different perspective of the molecule. Here, delta-9-THC

is shown in the direction parallel to the vector from C2 to C10b.From this

perspective, the conformation of the carbocyclic ring (Ring A) causes the top

part of the ring to move to the left, thus permitting no protrusion of Ring A into

the bottom face of the molecule. It is part of our working hypothesis that this

orientation is necessary for cannabinoid activity. The conformation of the

carbocyclic ring may allow for nondirectional hydrophobic interactions.

The lone pairs of electrons of the phenol oxygen generate directional reactivity

properties dependent on the orientation of the OH bond relative to the

carbocyclic ring. Studies of molecular energy as a function of rotation about the

C1-O axis reveal that two minimum energy conformations of the phenolic OH

exist. In Conformation I, the phenolic OH is slightly bent out of the plane of

the aromatic ring with the hydrogen pointing away from Ring A (C2-C1-O-H

dihedral angle, = -1°). In Conformation II, the phenolic hydrogen is below the

plane of the aromatic ring and is pointing toward Ring A ( = 155°). The

conformation of the rest of the molecule remains essentially unchanged.

The molecular electrostatic potential (MEP) is a molecular reactivity characteristic

that determines specific chemical reactivity. It is an important determinant of

drug action. The electrostatic potential field extends into space and influences

nearby molecules. Unless the fields of a drug and its receptor are complementary, they will not attract or bind to one another. Indeed, electrostatic forces

appear to guide an incoming drug into its receptor site and align so that shorter

range binding forces can take effect (Tucker 1984). Therefore, a very direct

indication of the nature and extent of electrostatic drug-receptor interactions can

be obtained by mapping the potential generated by a molecule.

85
FIGURE 3. Conformation of delta-9-THC (with propyl side chain) as

determined by MM2. From Reggio and Mazurek 1987. Copyright 1986 by

Elsevier Science Publishers B.V. (Amsterdam).

FIGURE 4. Conformation of delta-9-THC as determined by MM2: here the

perspective of Ring A is viewed in the direction parallel to the vector from C2 to

C10b . From Reggio and Mazurek 1987. Copyright 1986 by Elsevier Science

Publishers B.V. (Amsterdam).

86
The calculated electrostatic potential patterns generated by the bottom face of

delta-9-THC in each of its minimum energy conformers are shown in figures 5

and 6. These patterns are composed of lines of equipotential energy in kcal/mole

that would be felt by a test positive charge placed at any specified point on the

map. Figures 5 and 6 illustrate the regions of negative potential in planes

parallel to the benzene ring at distances of 1.5 Ås below the plane of the ring.

Because delta-9-THC is a nonplanar molecule. the MEPs differ depending upon

whether the molecule is viewed from above or below. We found that the change

in the position of the phenol group from Conformation I to Conformation II

caused a distinguishable difference in the MEPs of the two conformers when

viewed from the bottom face.

At this stage of the investigation, it was impossible to identify which conformation of the phenolic OH of delta-9-THC (I or II) was more relevant at the site of

action. Hence, the MEPs shown in figures 5 and 6 along with the conformation

of Ring A, as illustrated in figure 4, formed the preliminary set of molecular

reactivity characteristics for cannabinoid activity.

Refinement of the Template

In order to refine our template, a comparison of the MRCs of delta-9-THC with

those of another cannabinoid was undertaken. Edery et al. (1971) reported that

in rhesus monkey behavioral tests the replacement of the phenolic hydrogen of

delta-9-THC with a methyl group renders the molecule inactive. This inactive

cannabinoid, O-methyl-delta-9-THC (figure 7), was selected for comparison in

order to assess the importance of the position of the lone pairs of electrons of the

phenol oxygen. We report here the results of our comparison of the properties of

O-methyl-delta-9-THC with those of the delta-9-THC reactivity template.

METHODS OF PROCEDURE

Conformational Study of O-Methyl-Delta-9-THC

The first step in the characterization was the conformational analysis of Omethyl-delta-9-THC. The force field or molecular mechanics method as encoded

in the program MM2 (Allinger and Yuh 1980) was used in this analysis. The

force field available in MM2 has been parameterized for oxygen containing

molecules and has proven satisfactory for the calculation of structures and

energies of oxygen containing compounds (Allinger et al. 1980). The X-ray

structure of delta-9-THC Acid B (Rosenquist and Ottersen 1975) was used to

obtain an input geometry (i.e., bond lengths, bond angles, and dihedral angles for

the fused ring skeleton of delta-9-THC). Necessary atoms were added to this

skeleton at standard bond lengths and bond angles (Mitchell and Cross 1965) to

produce an input geometry for O-methyl-delta-9-THC in the MM2 calculation.

In the calculational scheme for the template molecule, delta-9-THC, molecular

size constraints necessitated reducing the pentyl side chain to propyl (Reggio and

Mazurek 1987). In order to maintain consistency, the side chain in O-methyldelta-9-THC was also reduced to propyl. Such a modification was acceptable

since the focus of this study is on the fused ring structure and the phenol group

and not on the side chain.

Because the position of the methoxy group was very important to our working

hypothesis, two studies of the molecular energy as a function of rotation about

the C1-O axis were conducted. In the first study, the dihedral driver option in

87
FIGURE 5. Molecular electrostatic potential of the bottom face of delta-9-THC

(Conformational I). Contours represent the MEP in a plane 1.5Å below the

aromatic ring. Values are in Kcal/mole. From Reggio and Mazurek 1987.

Copyright 1986 by Elsevier Science Publishers B.V. (Amsterdam).

FIGURE 6. Molecular electrostatic potential of the bottom face of delta-9-THC

(Conformational II). See figure 5 for details. From Reggio and Mazurek 1987.

Copyright 1986 by Elsevier Science Publishers B.V. (Amsterdam).

88
FIGURE 7. O-methyl-delta-9-THC

the MM2 program (Allinger and Yuh 1980) was used. Rotations in 36 steps were

made about the C1-O axis.

In the second study, an SCF calculation was performed for a model fragment of

O-methyl-delta-9-THC (see figure 8). The Gaussian 80 system of programs and

the STO-3G basis set were employed here (Fluder et al. 1980). Initially, all

atoms in the fragment were frozen in their optimized positions. These positions

were determined by MM2 calculations for the full molecule. Rotations of the

methoxy group in 18 steps were made about the C1-O axis.

RESULTS AND DISCUSSION

Conformational Analysis

Figure 9 shows the minimum energy conformer of O-methyl-delta-9-THC as

obtained from the MM2 calculation. Previous studies have predicted that the

cyclohexene ring (Ring A) of delta-9-THC should exist predominantly in a halfchair-like conformation (Archer et al. 1970). The present calculations indicate

that Rng A retains this conformation in O-methyl-delta-9-THC. As a result of

the interaction between the C6 methyl groups and H6a , Ring B assumes a conformation such that the axial C6 methyl group is on the same side of the

molecule as H10a and iS much

substituents on C6 and C6a

closer to H10a than is the other methyl group. The

are staggered with respect to one another (optimized

C10a -C6a -C6 -O5 dihedral angle is 63°). Here, the numbering system employed is

the same as that for delta-9-THC (see figure 2).

The C1 methoxy group is subject to steric interaction with the C10 proton. This

interaction causes the methoxy oxygen to bend slightly out of the plane of the

benzene ring. The methoxy group optimizes at a position away from Ring A

(optimized C2-C1-O-C dihedral angle is -5°).

89
FIGURE 8. Model fragment for O-methyl-delta-9-THC.

FIGURE 9. Conformation of O-methyl-delta-9-THC (with propyl side chain) as

determined by MM2.

90
Optimized bond lengths are within .01 Å of the following values: Csp P2-Csp2

1.40 Å, Csp 2-Csp3 1.51 Å, Csp 3-Csp 3 1.54 Å, Csp2-O 1.37 Å, Csp3-O

1.41 Å, Csp2-H 1.10 Å, and Csp3-H 1.11 Å.

Figure 10 shows a perspective of the molecule viewed in the direction parallel to

the vector from C2 to C10b From this perspective, the conformation of Ring A

causes the top part of the ring to move to the left, thus permitting no protrusion

of Ring A into the bottom face of the molecule. A comparison of figure 10 with

figure 4 reveals that the position of Ring A in O-methyl-delta-9-THC mimics

that of Ring A in delta-9-THC.

The results of the MM2 study of molecular energy as a function of rotation

about the C1-O axis are summarized in figure 11. There are local minima at =

-500 and 85° separated by a 0.8 kcal barrier. The SCF results for the model

fragment of O-methyl-delta-9-THC agreed very well with those above. The

SCF method pinpointed two minimum energy conformers = -5° and = 85°

separated by a 1.0 kcal barrier.

It is clear from figure 11 that a of 155° (which corresponds to Conformation II

of delta-9-THC) does not represent an energy minimum for the molecule. In

fact, in the MM2 study, the conformation with = 155° is 7.3 kcal higher in

energy than the minimum energy conformer of O-methyl-delta-9-THC.

In its minimum energy conformation, the methoxy group of O-methyl-delta-9-

THC essentially adopts Conformation I of the template. In this conformation,

the lone pairs of electrons of the phenol oxygen are directed toward Ring A.

However, the methoxy group cannot mimic Conformation II of the template in

which the lone pairs of electrons of the phenol oxygen are directed away from

Ring A. On the basis of these results, the inactivity of O-methyl-delta-9-THC

can be explained by the fact that the molecule cannot mimic Conformation II of

delta-9-THC. These results seem to indicate, then, that it is Conformation II

which is the more relevant conformation at the site of action.

In an early SAR study, Edery et al. (1971) postulated the need for a free phenol

for cannabinoid activity. Recently, Binder and Franke (1982) have proposed a

picture of delta-9-THC interacting with its hypothetical receptor. In this

picture, the phenolic hydrogen is shown to form a hydrogen bond with the

receptor. Such a hypothetical drawing implies the necessity of a phenolic

hydrogen for activity. It is possible to argue from this viewpoint that O-methyldelta-9-THC is inactive due to its lack of a phenolic hydrogen. Although the

demonstrated activity of some esterified THC would argue against the necessity

of a phenolic hydrogen for activity, there appears to be no definitive compound

yet synthesized which can answer this question. In the next stage of our

investigation, we hope to propose new cannabinoids which could be useful in

delineating an answer.

CONCLUSIONS

The finding that O-methyl-delta-9-THC cannot mimic Conformation II of delta9-THC in which the lone pairs of electrons of the phenol oxygen are directed

away from Ring A has led to a refinement of our template for cannabinoid

activity. This inability of O-methyl-delta-9-THC to mimic Conformation II may

cause the inactivity of the molecule. As a result of this finding, we have

tentatively refined our template for cannabinoid activity to include two aspects.

91
FIGURE 10. Conformation of O-methyl-delta-9-THC (with propyl side chain)

as determined by MM2.

FIGURE 11. Conformation of O-methyl-delta-9-THC as determined by MM2:

here the perspective of Ring A is viewed in the direction parallel to the vector

from C2to C1Ob .

92
1. The first aspect is the orientation of the lone pairs of electrons of the

phenol oxygen such that these lone pairs point away from Ring A

(Conformation II) (these lone pairs generate directional reactivity

properties).

2. The second aspect is the orientation of Ring A such that the ring moves

out of the bottom face of the molecule (this conformation is likely to

allow for nondirectional hydrophobic interactions).

Further refinements of this template will be made in subsequent comparisons of

the template MRCs with those of other cannabinoids.

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tetrahydrocannabinolic acid B. Acta Chem Scand 1329:379-384, 1975.

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94
ACKNOWLEDGMENTS

Dr. Harel Weinstein consulted on this project. This work was supported in part

by grant DA-03934 from the National Institute on Drug Abuse and by a grant of

computer time from from the University Computing Center of the City

University of New York. Data analysis was done on the PROPHET computer

system, a national computational resource sponsored by the NIH through the

Division of Research Resources.

AUTHOR

Patricia H. Reggio, Ph.D.

Department of Chemistry

Kennesaw College

Marietta, Georgia 30061

95
Different Cannabinoids Exhibit

Different Pharmacological and

Toxicological Properties

Ralph Karler, Ph.D., and Stuart A. Turkanis, Ph.D.

INTRODUCTION

By the mid-1970s. the anticonvulsant properties of the cannabinoids were well

documented (Loewe and Goodman 1947; Karler et al. 1973, 1974; Carlini et al.

1975; Consroe et al. 1976). but an analysis of their therapeutic potential as

antiepileptics had not yet been carried out. For this reason, a major goal

originally of our marijuana-related research was to determine the cannabinoids’

antiepileptic potential by the use of conventional laboratory tests for anticonvulsant and toxic properties (Turkanis and Karler 1981). The aim was to define

the relative selectivity of desired-to-undesired effects of these agents. In the

course of these studies, which involved many different cannabinoids, we

observed that there exist among these drugs, not only quantitative differences,

but also qualitative differences. The accretion of these differences ultimately

provides a profile of the pharmacological characteristics of the cannabinoids as a

class of drugs. The data presented below represent examples from our investigations of some properties of the cannabinoids, specifically related to their

antiepileptic potential.

METHODS

The anticonvulsant characteristics were measured in a variety of tests in order to

obtain a spectrum of properties for comparative purposes; the tests included both

electro- and chemo-shock tests. Because a major toxic effect of the clinical

antiepileptics is central depression, neurotoxicity was determined in terms of

motor toxicity, a classical measure of central nervous system (CNS) depression.

The details of the anticonvulsant and motor-toxicity tests have been previously

described (Karler et al. 1974; Turkanis et al. 1974).

The cannabinoids were dispersed in isotonic sodium chloride solution with the

aid of Tween 80 and ultrasound (Turkanis et al. 1974), and all drug and vehicle

preparations were administered intraperitoneally (i.p.). All determinations of

potency were made from dose-effect curves, which were obtained at peak-effect

times; and anticonvulsant potencies were expressed as either the effective-dose 50

(ED50) or the anticonvulsant-dose 50 (AD50), and motor toxic potency as the

toxic-dose 50 (TD50).

96
RESULTS

Figure 1 illustrates the dose-effect curves for anticonvulsant activity in a

maximal electroshock (MES) test and motor toxicity in a bar-walk test.

FIGURE 1. Dose-effect relationships for cannabinoids in MES and bar-walk

tests. The bar-walk test was carried out just prior to the MES test at the peakeffect times: THC and DMHP, 2 hr; CBD, 1 hr. The AD50s and their 95%

confidence limits (mg/kg, i.p.) are 12 (8-19) for DMHP, 92 (74-115) for THC,

and 122 (94-159) for CBD. The TD50s and their 95% confidence limits (mg/kg,

i.p.) are 4.7 (3-7) for DMHP, 82 (66-103) for THC, and 170 (144-200) for

CBD. Each point represents the results obtained jrom about 15 male Charles

River mice (ICR) weighing 15-26 gm. AD50s and TD50s and their 95%

confidence limits were calculated by methods described by Litchfield and

Wilcoxon (1949). THC = delta-9-tetrahydrocannabinol; CBD = cannabidiol;

DMHP = dimethylheptylpyran.

In this study, we compared DMHP, THC, and CBD. At the time, these three

compounds were selected for our initial studies for a variety of reasons: THC

was included because we intended to use it as the prototype cannabinoid; CBD

was included because it lacks the psychotoxicity of THC; and, finally, DMHP

was studied because it was thought to be the compound that had been tried with

97
apparently some success in epileptic children (Davis and Ramsey 1949). DMHP

was later found, like CBD, to be nonpsychoactive in humans (Lemberger et al.

1974). In the upper half of this figure, we compared the dose-response curves

for these three drugs against MES, which is one measure of anticonvulsant

activity. Some points are clear from these data: first, that all three compounds

were anticonvulsant in the MES test; second, that the psychotoxicity of THC is

separable from the anticonvulsant activity because the nonpsychoactive CBD and

DMHP are both anticonvulsant; and, finally, in terms of anticonvulsant potency,

the dose-response curves illustrate that there is a big difference in potency

between DMHP and the other two drugs.

The separation between psychoactivity and anticonvulsant activity encouraged us

to continue this study and to pursue other aspects of the potential toxicity

problem. One property that especially interested us was motor toxicity because

all antiepileptic drugs are motor toxic. The lower half of the figure illustrates

that the cannabinoids are also motor toxic, as measured in a bar-walk test; in

these instances, potency in terms of motor toxicity appeared to be directly

proportional to the anticonvulsant potency; that is, the greater the anticonvulsant

potency, the greater the motor-toxic potency.

The data shown in table 1 represent the continuation of our study of relative

potencies with respect to the anticonvulsant and toxic effects of a large number

of cannabinoids. The drugs included in table I were not selected for their

structural characteristics; rather, they were included in the study because they

were the drugs that were available to us at the time. Nevertheless, the data

shown illustrate that most of these compounds displayed both anticonvulsant and

motor-toxic activity and that the potency relative to THC varied. For example,

in the MES test, the 11-hydroxy derivative is about seven times as potent as is

THC; and, similarly, it is about three or four times as potent in terms of motor

toxicity. The remaining compounds listed in the table illustrate the quantitative

differences in both anticonvulsant activity and motor toxicity, even in such a

limited sample of cannabinoids. The anticonvulsant activity ranged from a

fraction of that of THC, as illustrated by THC Acid B, to many times that of

THC, as in the case of d,1,threo-DMHP. Similar ranges were observed for the

motor-toxic activity in the bar-walk test.

At this point in our studies, one of the frequent criticisms that we received was

that the anticonvulsant dose of THC in the mouse, about 100 mg/kg, is very

high--approximately 100 times that of the psychoactive dose in humans;

therefore, the meaning of the anticonvulsant effects vis-a-vis humans was

questioned. To address this question, we examined the potency of some of the

cannabinoids in three different species: the mouse, the rat, and the frog. The

data are shown in table 2.

Here, we can see a very wide range of potency for these drugs. At the extremes,

the AD50 for both THC and CBD in the mouse is about 100 mg/kg; in the frog,

it is about 0.1 mg/kg. This species variability is not seen for either phenytoin or

phenobarbital. The point of this table is that there are large species differences

in the potency of the cannabinoids, which means that it is not possible to

extrapolate meaningful potency data from animals to humans; therefore, the

anticonvulsant dose in the mouse (or the frog) cannot be equated with a

psychoactive dose in humans. An anticonvulsant dose in humans can only be

determined in humans.

98
TABLE 1

Relative Potencies of Cannabinoids in Anticonvulsant

and Motor Performance Tests in Mice

Drug MES Test Bar-walk Test

THC 1.0 1.0

11-Hydroxy-THC 7.1 3.6

8 Alpha-hydroxy-THC * 0.7

8 Beta-hydroxy-THC 1.0 *

THC Acid A 0.5 0.2

THC Acid B 0.3 0.2

Delta-8-THC

9-Nor-delta-8-THC

1.0

0.8

1.7

1.3

CBN

9-Nor-9 alpha-hydroxyhexahydro CBN

9-Nor-9 beta-hydroxyhexahydro CBN

0.4

0.8

2.2

0.4

0.8

1.7

CBD 0.8 0.5

DMHP 7.7 15

d,l,Threo-DMHP 14

d,l,Erythro-DMHP 4.0

Cannabichromene * 0.4

The relative potency is the ratio of the median-effective THC dose to that of

another cannabinoid. The average median-effective THC dose was 100 mg/kg,

i.p., for anticonvulsant effects in the MES test and 80 mg/kg, i.p., for motor

toxicity measured in a bar-walk test.

*Compound lacks activity within the dosage range tested.

–No data available.

The data shown in figure 2 represent another example of species differences in

response to the cannabinoids. The figure depicts dose-effect curves for the

motor toxicity of THC and CBD in the rat. These two drugs are of similar

motor-toxic potency in the mouse (about 100 mg/kg); but in the rat, the TD50

for THC is about 7 mg/kg, compared with 175 mg/kg of CBD. In the rat,

therefore, THC is about 25 times as toxic as is CBD and about 10 times as toxic

as it is in the mouse. Again, the species differences in response to these two

cannabinoids are striking; and they serve to re-emphasize the inappropriateness

of extrapolating data from one species to another, including humans.

99
TABLE 2

Species Differences in Drug Potencies

in the Maximal Electroshock Test

AD50 (mg/kg)*

Drug Mouse Rat Frog

THC

CBD

Phenytoin

Phenobarbital

100

120

9

12

5

50

5

12

0.1

0.1

10

8

*Values calculated from dose-response data. From Karler and Turkanis 1981.

Copyright 1981 by The American College of Clinical Pharmacology, Inc. (New

York).

FIGURE 2. Dose-effect relationships for THC and CBD in a bar-walk test with

rats. The TD50s and their 95% confidence limits (mg/kg, i.p.) are 6.6 (4.8-9.0)

for THC and 173 (156-192) for CBD. The values were calculated by methods

described by Litchjield and Wilcoxon (1949). Each point represents results jrom

6-8 male Sprague-Dawley rats weighing 250-280 gm. The relative potency for

CBD compared to THC was 0.04 (Smiley et al. 1979).

100
The data in table 3 illustrate the comparative selectivity of the cannabinoids, as

described conventionally in terms of the protective index, that is the ratio of the

TD50 to the ED50. The point of the experiment was to examine the quantitative

relationship between anticonvulsant activity and motor toxicity in order to

determine if these two effects are separable. Motor toxicity is a major side

effect of all antiepileptic drugs. In pharmacological terms, we were raising the

issue here of the selectivity of these compounds as anticonvulsants relative to

their general CNS depressant activity. Clinically, the usefulness of any drug, in

part, depends upon its selectivity for a desired effect.

TABLE 3

Effects of a Single Dose of Cannabinoid in Mice

in a Maximal Electroshock and a Bar-walk Test

Drug

Peak-effect ED50

Time (hr)* (mg/kg)

TD50

(mg/kg)

Protective

Index

(TD50/ED50)

DMHP 2 13 5 0.4

Delta-9-THC 2 100 80 0.8

CBN 1 230 230 1.0

CBD 1 120 180 1.5

11-Hydroxy-THC 1 14 22 1.6

Delta-8-THC 2 83 152 1.8

All experiments were performed at an ambient temperature of approximately

22°C; ED50 and TD50 values were calculated by the method of Litchfield and

Wilcoxon (1949) from conventional dose-response curves based on 3-6 doses with

15-20 animals for each dose; drugs were administered i.p.

*The peak-effect times for the anticonvulsant and toxic effects were similar.

From Karler and Turkanis 1976. Copyright 1976 by Plenum Publishing Corp.

(New York).

In this table are listed the protective index data for DMHP, delta-9-THC,

cannabinol (CBN), CBD, the 11-hydroxy metabolite, and delta-8-THC. For

DMHP, the ED50 against the maximal electroshock is 11.5 mg/kg and the TD50

is 4.6 mg/kg, yielding a protective index of 0.4. Similarly, the protective index

for delta-9-THC is 0.8; for CBN, 1.0; for CBD, 1.5; for 11-hydroxy-THC, 1.6;

and for delta-8-THC, 1.8. For comparative purposes, the protective index for

phenobarbital is 1.5 (Karler and Turkanis 1981). These data clearly illustrate that

101
the anticonvulsant and motor-toxic activities are at least partially separable,

ranging from DMHP and THC, which are more motor toxic than anticonvulsant,

to CBD, 11-hydroxy-THC, and delta-8-THC, which exhibit more anticonvulsant

than toxic activity.

The data in table 4 represent a summary of the results obtained from five

different convulsive threshold tests. All of the anticonvulsant data described

above have been limited to only one convulsive test, that is, the MES test. There

are, however, many such tests, and table 4 contains the comparative results of

THC and CBD from a variety of electro- and chemo-shock tests.

TABLE 4

Anticonvulsant Properties of the Cannabinoids

Convulsive

Threshold Test THC CBD

Minimal

60-Hz-EST

6-HZ-EST

Pentylenetetrazol

- +

+ +

- 0

Maximal

MEST

Pentylenetetrazol

+ +

+ +

+ = anticonvulsant activity; - = proconvulsant activity; 0 = no activity.

CBD is anticonvulsant in four of the five tests; THC, however, has a very

different spectrum of activity because it is anticonvulsant in three tests, but is

proconvulsant in the 60-Hz-EST and the pentylenetetrazol minimal threshold test.

This was the first evidence that we had to indicate that THC exerts not only

depressant effects but also excitatory effects. Subsequently, we have observed

THC’s excitatory properties in many other experimental situations. For example,

THC can precipitate frank convulsions in cobalt- and iron-epileptic rats (Chiu et

al. 1979; Turkanis and Karler 1982); THC enhances electrically induced limbic

seizure activity in the rat (Smiley et al. 1979); and THC increases the rate of

kindling to both electrical and chemical stimuli (Karler et al. 1984). In striking

contrast, in all of these test situations, CBD displays only anticonvulsant or

depressant properties. We have also observed THC excitatory effects at every

level of organization of the nervous system, from the intact animal to the synapse

(Turkanis and Karler 1981). In none of these tests has CBD shown any

excitatory activity. On these grounds, we have concluded that the excitatory

properties of the cannabinoids are completely separable from the depressant

properties.

102
The data shown in figures 3 and 4 represent the influence of chronic THC or

CBD treatment on their anticonvulsant properties. All of the anticonvulsant data

described above were derived from acute experiments. In contrast, the data in

figure 3 were obtained from animals treated for 22 days with a single daily dose

of THC. The top of the figure shows the results from the 6-HZ-EST test. On

day 1, THC raised this convulsive threshold about 20 V, which reflects its

anticonvulsant activity in this test. During the course of the 22 days, tolerance

developed because the anticonvulsant activity decreased. In the next test, the 60-

Hz-EST, THC is proconvulsant because on day 1 it decreased seizure threshold

by 2 mA, but again tolerance appeared to develop with repeated daily treatment.

Finally, in the third test, the maximal EST, THC acutely is anticonvulsant

because it elevated the threshold on day 1, but again tolerance developed to this

effect. The two curves at the bottom of the figure illustrate that the tolerance in

these instances is a true tolerance because there were no significant changes in

either the brain- or the plasma-THC concentrations with chronic treatment.

Figure 4 presents the results of chronic CBD treatment in the identical

experimental paradigm as that used for the previous THC study. But the results

are very different. In the two seizure-threshold tests shown at the top of the

figure, that is, the 6-HZ- and 60-Hz-EST tests, the anticonvulsant activity of

CBD progressively increased with chronic treatment, just the opposite response

from the tolerance observed with THC.

An enhanced response to a given dose of drug administered chronically has been

termed by some investigators as "reverse tolerance," and it is a phenomenon that

has been repeatedly described for CNS locomotor stimulants, such as cocaine,

amphetamine, and morphine (Post and Kopanda 1975; Segal and Mandell 1974;

Shuster et al. 1975). This may be the first report of "reverse tolerance"

developing to depressant effects, in this case, anticonvulsant effects. The

instances of "reverse tolerance" again clearly distinguish CBD from THC. The

results of the maximal EST indicate that tolerance develops to the other two

effects. With respect to this test, CBD resembles THC because tolerance

developed to both of the drugs.

Although antiepileptics are taken chronically, studies of chronic effects of these

drugs are conspicuously absent from the literature. For this reason, we included

phenytoin in our cannabinoid study and found that in one of the tests, tolerance

developed; in another test, there was no change in pharmacodynamics; and in the

third test, "reverse tolerance” developed (Karler et al. 1982). The "reverse

tolerance" in this case is of special interest because it is clinically recognized that

there exists a time lag of a few weeks before the antiepileptic activity of

phenytoin completely manifests itself. Currently, there is no explanation for the

time lag, but our animal data suggest that the lag may be due to the time

required to develop "reverse tolerance." In any event, the phenomenon of

"reverse tolerance" may add a new dimension to the study of antiepileptic drugs,

as well as to the study of cannabinoids.

DISCUSSION

The data presented were selected to provide an overview of what we consider to

be some of the more important pharmacological characteristics of the

cannabinoids that have emerged from our whole-animal studies of their

anticonvulsant properties. We have seen, for example, as have others, that the

anticonvulsant activity is separable from the psychoactivity. Without such a

separation, these drugs would have no clinical potential as antiepileptics. We also

103
FIGURE 3. Influence of repeated THC treatment on ESTs. In each test, 60 mice

were given 10 mg/kg THC i.p. once daily, and another 60 mice, vehicle. The

points indicate the differences in EST between drug- and vehicle-treated groups.

—— = period of daily treatment: ----- = period oj withdrawal. Brain and

plasma values represent data from 6-8 mice. On day 1 the vehicle-control values

were 23 (21-24) V for the 6-Hz-EST, 5.9 (5.7-6.1) mA Jor the 60-Hz-EST and

7.5 (7.0-8.0) mA for the MEST and, as determined by a relative potency test of

Litchfield and Wilcoxon (1949) (P < 0.05), were significantly different from the

drug-treated groups. From Karler et al. 1982. Copyright 1982 by SpringerVerlag (Heidelberg).

104
FIGURE 4. Influence of repeated CBD treatment on the ESTs and on brain and

plasma-drug concentrations. On day 1 vehicle-control values were 21.5 (20-23) V

for the 6-HZ-EST, 5.4 (5.1-5.8) mA for the 60-Hz-EST and 7.9 (7.6-8.3) mA

for the MEST, and on day 1 only the 6-HZ-EST and MEST vehicle-control values

were significantly different from those obtained from CBD-treated groups as

determined by a relative potency test. See figure 3 for additional information.

From Karler et al. 1982. Copyright 1982 by Springer-Verlag (Heidelberg).

observed that among the different cannabinoids, there are differences in potency,

there are differences in the protective index, and there are species differences in

response to them. None of these observations is too surprising because such

differences are general characteristics of drugs. Detailed structure-activity

studies would probably yield cannabinoids with more selectivity as

anticonvulsants than those that we have studied. The "reverse tolerance" to CBD

was a surprise. What this means vis-a-vis an antiepileptic drug is not clear, but

the observation that a similar response to chronic phenytoin treatment raises the

possibility that the clinical efficacy of phenytoin may, in part, be due to "reverse

tolerance." Although "reverse tolerance" was not observed for THC in the

105
anticonvulsant study, the CBD data illustrate that "reverse tolerance" can occur to

cannabinoid effects; therefore, the toxicological or pharmacological evaluation of

any cannabinoid should involve the assessment of effects in chronically treated,

as well as acutely treated, animals.

In the course of our studies, we have tried to emphasize that anticonvulsant

activity is not a single entity; rather, it is a complex group of effects. That point

is clearly seen in the case of THC which is anticonvulsant in some tests and

proconvulsant in others. This resembles the clinical response generally to

antiepileptic drugs. Phenytoin, for example, is effective in the treatment of

grand mal epilepsy; however, it can exacerbate absence seizures. The reverse is

true for ethosuximide. In animal studies, these drugs resemble THC because they

all exhibit both anticonvulsant and proconvulsant properties. In retrospect, what

is interesting about CBD as an anticonvulsant is that we have not yet seen any

indication of an excitatory effect on CNS function. In this sense, it may be

unique as an anticonvulsant. Despite a number of favorable properties of CBD,

its future as an antiepileptic--at least in this country--is not very promising.

Although the anticonvulsant drug-screening program sponsored by the National

Institute of Neurological and Communicative Disorders and Stroke (NINCDS)

screened CBD and found it to have anticonvulsant activity, it has not been

recommended for clinical trial, and it is the NINCDS recommendation that

determines which new antiepileptics will be clinically tested in this country. The

drug industry in the past has been reluctant to develop new antiepileptics because

the potential market is too small to justify the drug developmental expense.

Even now that the NINCDS is providing much of the developmental costs, what

drug company would be willing to bear the marketing cost of a nonpatentable,

marginally profitable drug such as CBD? It is for this reason that we are

pessimistic about the future of CBD as an antiepileptic. It may remain just as

we found it: a very interesting laboratory drug.

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12:1-15, 1975.

Chiu, P.; Olsen, D.M.; Borys, H.K.; Karler, R.; and Turkanis, S.A. The

influence of cannabidiol and delta-9-tetrahydrocannabinol on cobalt epilepsy

in rats. Epilepsia 20:365-375, 1979.

Consroe, P.; Jones, B.; Laird, H., II; and Reinking, J. Anticonvulsant-convulsant

effects of delta-9-tetrahydrocannabinol. In: Cohen, S., and Stillman, R.C.,

eds. The Therapeutic Potential of Marihuana. New York: Plenum

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Karler, R.; Cely, W.; and Turkanis, S.A. The anticonvulsant activity of

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tetrahydrocannabinol and other cannabinoids. Life Sci 15:931-947, 1974.

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Karler, R.; Borys, H.K.; and Turkanis, S.A. Influence of 22-day treatment on

the anticonvulsant properties of cannabinoids. Naunym Schmiedebergs Arch

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Sharp, C., ed. Mechanisms of Tolerance and Dependence: 1984. National

Institute on Drug Abuse Research Monograph 54. DHHS Pub. No. (ADM)

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pp. 212-222.

Lemberger, L.; McMahon, R.; Archer, R.; Matsumoto, K.; and Rowe, H.

Pharmacologic effects and physiologic disposition of delta-6-alpha, 10-alphadimethylheptyltetrahydrocannabinol (DMHP) in man. Clin Pharmacol Ther

15:380-386, 1974.

Litchfield, J.T., Jr., and Wilcoxon, F.A. A simplified method of evaluating

dose-effect experiments. J Pharmacol Exp Ther 95:99-113, 1949.

Loewe, S., and Goodman, L.S. Anticonvulsant action of marihuana-active

substances. Fed Proc 6:352, 1947.

Post, R.M., and Kopanda, R.T. Cocaine, kindling and reverse tolerance. Lancet

1:409-410, 1975.

Segal, D.S., and Mandell, A.J. Long-term administration of d-amphetamine:

Progressive augmentation of motor activity and stereotypy. Pharmacol

Biochem Behav 2:249-255, 1974.

Shuster, L.; Webster, G.W.; and Yu, G. Increased running response to morphine

in morphine-treated mice. J Pharmacol Exp Ther 192:64-72, 1975.

Smiley, K.A.; Turkanis, S.A.; Borys, H.K.; Olsen, D.M.; and Karler, R. An

electrophysiological analysis of the anticonvulsant action of cannabidiol on

limbic seizures in the conscious rat. Epilespsia 20:351-363, 1979.

Turkanis, S.A., and Karler, R. Electrophysiologic properties of the cannabinoids.

J Clin Pharmacol 21:449S-463S, 1981.

Turkanis, S.A., and Karler, R. Central excitatory properties of delta-9-

tetrahydrocannabinol and its metabolites in iron-induced epileptic rats.

Neuropharmacology 21:7-13, 1982.

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of cannabidiol. Res Commun Chem Pathol Pharmacol 8:231-246, 1974.

ACKNOWLEDGMENT

This research was supported by research grant DA-00346 from the National

Institute on Drug Abuse.

AUTHORS

Ralph Karler, Ph.D.

Stuart A. Turkanis, Ph.D.

Department of Pharmacology

University of Utah School of Medicine

Salt Lake City, Utah 84132

107
Pharmacological Evaluation of

Agonistic and Antagonistic Activity

of Cannabinoids

Billy R. Martin, Ph.D.; David R. Compton, Ph.D.;

Patrick J. Little, B.S.; Thomas J. Martin, B.S.; and

Patrick M. Beardsley, Ph.D.

INTRODUCTION

The currently proposed mechanisms by which the cannabinoids could be

producing their central effects involve either general membrane perturbation or

direct interaction with a specific cannabinoid receptor. The difficulty in defining

the mechanism by which -THC alters central function could be because the

cannabinoid behavioral syndrome represents a composite of several independent

effects, rather than a single event. Therefore, it is possible that both mechanisms

play an important role in the total behavioral effect of the cannabinoids. The

observed behavioral syndrome may be a composite of multiple independent

effects, each of which could be a function of either a nonspecific cannabinoid

action or, alternatively, of a specific cannabinoid effect which might be modified

(at higher drug doses) by nonspecific interactions. It is our objective to determine

whether either of these mechanisms is responsible for -THC’s effects. If

cannabinoids are interacting with specific binding sites, then there should be a

strict structure-activity relationship (SAR) as well as a specific antagonist.

Alternately, if the cannabinoids are producing their effects solely by perturbation

of membranes, there should not be a specific antagonist, but there may be a

correlation between activity and membrane (lipid) solubility as measured by

partition coefficients. The partial development of a SAR for several cannabinoid

behavioral effects has been established by evaluating the agonistic and antagonistic

activity of newly synthesized analogs in a variety of behavioral test systems.

These tests include spontaneous activity, rectal temperature and analgesia (mouse),

blood pressure, heart rate, and respiratory rate (rat), static ataxia (dog), and

conditioned operant behavior (monkey) (Martin et al. 1975, 1984a, 1984b;

Beardsley et al., in press). Additionally, attempts have been made to distinguish

those cannabinoid structural features associated with only a portion of the

behavioral syndrome normally observed with THC as an effort to determine if a

unique SAR exists for each behavioral measure, which would support the notion

that the cannabinoid syndrome is composed of multiple behavioral effects (Martin

1986). We propose that the pharmacological properties unique to the cannabinoid

class of drugs are produced by the interaction of -THC with specific receptors,

while cannabinoid-induced central nervous system (CNS) depression is a general

phenomenon produced by nonspecific membrane interactions.

108
METHODS

Cannabinoids

The analogs 1-24, 32, and 33 were synthesized by Dr. Raj Razdan and 25,

29, and 31 by Dr. Everette May. Compounds 26-28 were obtained from the

National Institute on Drug Abuse and compound 30 was provided by the National

Cancer Institute. The procedure of Olson et al. (1973) was used to prepare

micellar suspensions of cannabinoids suitable for injection. The cannabinoids (10

mg) were dissolved in 100 μl of a 1:1 mixture of 95% ethanol and emulphor (a

polyoxyethylated vegetable oil manufactured by GAF) by sonification. This

solution was then diluted with ethanol:emulphor:saline (1:1:18) to provide the

desired concentration, and injected intravenously (i.v.), unless stated otherwise.

Spontaneous Activity, Rectal Temperature, and Antinociceptive

Activity in Mice

Analogs are evaluated initially in these three tests in order to assess their general

pharmacological activity. To conserve limited supplies of drugs, these

measurements are recorded sequentially in the same animal as described

previously (Martin et al. 1984a, 1984b; Jorapur et al. 1985). Male ICR mice

(22-30 g) are housed in the laboratory for 24 hr before treatment. The ambient

temperature of the laboratory, which varies from 22 to 24 °C from day to day, is

recorded at the beginning and end of each experiment. Rectal temperature is

determined by a thermistor probe (inserted 25 mm) and a telethermometer (Yellow

Springs Instrument Co., Yellow Springs, OH) just prior to vehicle or drug

administration. Following the initial temperature determinations, control tail-flick

reaction times are determined for each animal as described below. The mice are

then injected i.v. with either vehicle or drug (0.1 ml/10 g of body weight) and

immediately placed in individual photocell activity chambers. After the animals

have acclimated for 5 min, interruptions of the photocell beams are recorded for a

10-min session. The mice are removed from the activity chambers, and tail-flick

response is determined 5 min later. Rectal temperature is measured 60 min after

drug administration. The spontaneous activity of the treated animals is expressed

as percent of control, and the ED50s(and confidence limits) are determined by the

method of Litchfield and Wilcoxon (1949). The difference between rectal

temperature before and 60 min after treatment is calculated for each mouse.

The antinociceptive activity procedure is essentially the same as that described by

D'Amour and Smith (1941) and Dewey et al. (1970). A mouse's tail is placed on

a notched block so as to occlude a slit over a photocell detector. Heat is applied by

a 100-watt lamp mounted in a reflector. The apparatus is arranged so that when

the operator turns on the lamp a timer is activated. When the mouse flicks its tail,

the light falls on the photocell and automatically stops the timer. The intensity of

the lamp is adjusted to give a normal reaction time between 2 and 4 sec. A control

reaction time is taken for each animal prior to treatment. The mice then receive an

i.v. injection of the drug and are tested at the appropriate time. A maximum

10-sec cut-off (latency) time is employed in order to prevent damage to the

animal’s tail. The antinociceptive response is expressed as a percent of the

maximum possible increase in latency. Percent response is calculated from the

equation: % R = (T - Tc/10 - Tc) x 100 where T is the mean test reaction time, and

Tc is the control reaction time. The ED50 (and confidence limits) are determined

by the method of Litchfield and Wilcoxon (1949).

109
Static Ataxia in Dogs

The ability of cannabinoids to produce static ataxia (an effect unique to

psychoactive cannabiioids) and other characteristic behavioral effects is examined

in mongrel dogs of either sex (10-18 kg). On the test day, the animals are moved

from their home cages to the observation room, and behavior is evaluated for 5 to

10 min prior to drug administration. The normal behavior of each animal (which

includes spontaneous activity, gait, tail tuck, hyperreflexia, etc.) is recorded.

Each animal is then injected i.v. with either the cannabiioid or the vehicle (1 ml/kg

of body weight), and their behavior is rated at 5-min intervals according to the

static ataxia scale as described previously (Walton et al. 1938; Martin et al. 1975)

and as depicted in table 1. Typical test sessions consist of four animals that

receive either vehicle, -THC (0.2 mg/kg which typically produces a rating of

3-4), or one of two other cannabinoid treatments. Only one of the experimenters

is knowledgeable to the treatment. Behavior is scored by three observers who are

"blind" with regard to treatment.

TABLE 1

Quantitation of the Behavioral Effects Produced by Cannabinoids in Dogs

Score Behavioral Effects

0

1

2

3

4

5

6

No effect

Slight depression of activity, slight static ataxia seen only after

the dog has been standing in one position for 3 to 5 min.

Walks with a prance-like placement of feet, exaggerated reflex

to a swinging hand, and static ataxia after standing in one

position for 2 to 3 min.

Tail is often tucked, some loss of tone in hind legs as

evidenced by a semisquatting position, static ataxia more

pronounced and seen after dog stands in one position for 1 to 2

min, and nodding may be observed 30 to 60 min after

injection.

Marked static ataxia, sways forward and backward and/or side

to side, and almost falls after standing in one position for 1

min.

Cannot stand for longer than 30 sec without almost falling and

often plunges about.

Lies prostrate on the floor.

110
Conditioned Operant Responding in Monkeys

Rhesus monkeys are trained on a multiple timeout 3-min fixed-ratio x schedule to

lever press for food pellets as described by Beardsley et al. (in press). During drug

testing, the animals receive cumulative dosing of cannabinoids, as described in table

5, and the dose at which the behavioral sign appears is recorded. The vehicle did

not produce any of the indicated behavioral signs nor did it suppress conditioned

responding.

Protocol for Determining Antagonistic Activity

Analogs with little or no agonistic activity in these tests are evaluated for antagonistic

activity. Antagonism in spontaneous activity and rectal temperature measures is

studied in one group of mice, whereas antinociceptive activity is evaluated in a

separate group of animals. A subthreshold dose of the analog (or vehicle) is

administered i.v. 10 min prior to an i.v. administration of -THC (ED50 dose for

producing hypoactivity) or vehicle. Spontaneous activity and rectal temperature are

measured (described above) in the following treatment groups: analog plus -THC

(A), analog (B), -THC (C), and vehicle (D). Antagonism of hypothermia is

calculated from the following equation:

%ADhypothermia . = {1 - [(A -B) ÷ (C -D)]} x 100 where A through D represent the

differences between preinjection and postinjection (60 min) rectal temperatures. The

antagonism of hypoactivity is calculated from the following equation:

%ADhypoactivity = {1-[1-(A ÷ B)] ÷ [1- (C ÷D)]) x 100 where A through D

represent the spontaneous activity (counts per 10-min period). To measure

antagonism in the tail-flick procedure, the analog is administered i.v. 10 min prior to

an i.v. dose of -THC (ED80 dose for producing antinociception). The mice are

tested 5 min after the second injection. Two injections of vehicle into the tail vein

have no effect on the tail-flick response. The results are expressed as percent

antagonism of the THC antinociceptive effect. The AD50 values (and confidence

limits) are determined by the probit analysis method of Litchfield and Wilcoxon

(1949).

Antagonistic activity of analogs is evaluated in dogs by pretreating i.v., with either

vehicle or analog, 10 min prior to the i.v. administration of either vehicle or -THC

(0.2 mg/kg). In this way, each test session consists of the following treatments:

vehicle, analog, -THC, and analog + -THC. Behavioral activity is evaluated

for 60 min after the -THC injection. The maximum effect of -THC is usually

observed between 15 and 30 min after administration. Analogs are evaluated at the

highest possible dose in duplicate or triplicate. Analogs with antagonistic activity are

evaluated at two other doses (in duplicate) so that AD50 values can be calculated.

Dogs are retested at weekly intervals, but never more than four times total.

Development of tolerance has not been observed when low doses of cannabinoids

are given as infrequently as once a week.

RESULTS

Antinociceptive Studies

Cannabinoids have previously been considered to be weak antinociceptive agents

(Buxbaum 1972; Dewey et al. 1972; Chesher et al. 1973; Sofia et al. 1973; Noyes et

al. 1975; Wilson et al. 1976; Bloom et al. 1977; Gilbert 1981; Sofia and Barry

111
1983). However, i.v. administration of -THC demonstrates that it has

antinociceptive properties comparable to those of morphine sulfate (Martin 1985a).

SAR studies demonstrate that the structural requirements for cannabinoid-induced

antinociceptive activity, as measured in the tail-flick procedure, are quite similar to

those necessary for inducing hypoactivity and hypothermia (Martin 1985b). These

findings, coupled with the observation that i.v. administration of -THC (ED50 =

1.9 mg/kg) results in consistent and potent antinociceptive activity, suggest that the

tail-flick procedure is a reasonable test for assessing agonistic and antagonistic

activity of cannabinoids.

Evaluation of Agonistic Activity of CBD Analogs

The behavioral profiles and structures of CBD analogs evaluated for

pharmacological activity in mice are presented in table 2. Interest in these

10-N-substituted analogs is derived from early suggestions that CBD itself exhibited

some antagonistic properties (see Martin 1986 for a recent review). The ethylamino

(1) and propylamino (2) analogs are as potent as -THC in decreasing spontaneous

activity and rectal temperature, but exhibit only slight antinociceptive activity

(Jorapur et al. 1985). Additionally, 1 lacks specific cannabinoid effects in the dog

static-ataxia test, although it does produce CNS depresson at i.v. doses of 1 to 2

mg/kg. An interesting aspect of these two analogs' activity profile is their high

toxicity (LD50 = 5 mg/kg), whereas the LD50 of CBD is >100 mg/kg. However,

other lo-substituted CBD analogs (3-10) have been prepared, and they are

relatively inactive in all mouse tests. The N-methyl- N-propargyl (7) and

morpholino (10) CBD analogs are diacetyl derivatives which are weakly active (

-THC) in producing hypoactivity, but inactive in the other two tests. Due to

concerns regarding the penetrability of N-mustards into the CNS, analog 3 was also

evaluated following intraventricular (i.v.t.) administration, and doses up to 150

μg/mouse ( 5 mg/kg) do not alter spontaneous activity or tail-flick latency in mice,

but they do produce a 3.8 °C drop in rectal temperature. An identical i.v.t. dose of

-THC produces a 5.5 °C decrease in temperature, as well as a 68% decrease in

spontaneous activity and a 55% increase latency in the tail-flick response. Only the

ethylamino analog 1 has been evaluated in the dog static-ataxia test, and it failed to

produce the classical cannabinoid syndrome. However, it did produce CNS

depression at i.v. doses of 1 to 2 mg/kg, which was consistent with its effects in

mice. Therefore, it appears that this analog may be prototypic of a cannabinoid with

potent CNS depressant properties, but without antinociceptive or static-ataxia

properties. The lack of agonistic properties in the other CBD analogs make them

suitable for testing as antagonists.

Evaluation of Agonistic Activity of -THC Analogs

The importance of the C-11 position has been well documented. The general

consensus is that hydroxylation at this position increases biological activity, whereas

removal of the C-11 position decreases potency (Martin et al. 1975). Numerous

substitutions have been made at C-11 of -THC (11-21) and the results are

summarized in table 3. Alkylamino (11-14) or thioacetyl (16) substitutions have

more or less resulted in loss of agonistic activity in the mouse tests with the

exception of the N-methyl analog (11) which was as potent as -THC in

depressing spontaneous activity. The N-mustard (13, acetate of 14) was

administered i.v.t. as described for analog 3 and found to be inactive up to doses of

150 μg/mouse. The alkyl analogs (17-21) are primarily additions of a bulky

substituent at C-11 which dramatically attenuate behavioral activity.

112
TABLE 2

Phamacological Activity of CBD and Structurally Related Analogs in Micea

R

Pharmacological Profile

S.A. b

R.T. cT.F. d

CBD 0.08

1 -NHC2H5

1.15

2 -NHC3H7·HCl 1.10

3 -N(CH2CH2Cl)2 N.T.e

4 -N3 <0.20

5 -SH <0.02

6 -Br f

< 0.08

7 -N(CH3)CH2C=CHf

0.25

8 -N[CH(CH3)2]2

f <0.05

9 -NHCO(CH2)3NH2

0.08

10 -cN(CH2CH2)2O

f

0.2

0.03 <0.03

0.44 ~0.30

1.00 ~0.40

N.T. N.T.

<0.05 <0.20

0.05 <0.20

0.15 <0.20

0.03 <0.06

0.03 <0.06

0.11 <0.08

0.02 <0.06

a

 Pharmacological potency of i.v. analogs relative to that of i.v. -THC.

b

 Spontaneous activity — the ED50 of -THC (2.3 mg/kg) was divided by

that of the analog. Less than (<) indicates the analog did not produce a 50%

effect at the highest dose tested. Therefore, the ED50 was divided by the

highest dose tested. cRectal temperature — the dose of -THC (3.0

mg/kg) that produced a 1.6 °C decrease in rectal temperat

dose of the analog that produced a comparable effect. d

ure divided by the

Tail-flick response

— the ED50 of -THC (1.9 mg/kg) was divided by that of the analog.

Less than (<) indicates the analog did not produce a 50% effect at the

highest dose tested. e Not tested. fDiacetyl derivatives previously reported

(Jorapur et al. 1985).

113
TABLE 3

Pharmacological Activity of -THC and Structurally Related Analogs in Micea

R

b

Pharmacological Profile

S.A. R.T. T.F.

-THC 0.23 0.98 0.28

11

12

13

14

15

16

17

18

19

20

21

"

"

"

22

23

24

25

26

27

28

R l

-CH2NHCH3

c

0.21

-CH2NHCH2CH3 <0.20

-CH2N(CH2CH2Cl)2 acetate <0.20

-CH2N(CH2CH2Cl)2 < 0.07

-SH N.T.

-SCOCH3 < 0.20

-C6H5

c

0.03

-CH2C6H5

c

0.07

-CH(CH3,)2

c 0.09

-CH2CH2CH2CH3

C

0.07

-CH(OH)CH2CH3

c

0.06

R2

-NH2

0.82

-NHCH2CH3 0.92

-SCOCH3 <0.20

R3 and/or Side Chain

-O2C(CH2)3N(CH2CH2)2O 0.22

-O1.1

Side chain = -(CH2)5N

+

(CH3)3

Side chain = -(CH2)5Br 1.1

Side chain = -(CH2)5OAc 0.79

0.04

< 0.05

0.08

0.03

N.T

0.06

0.08

0.04

0.02

0.27

0.03

0.94 < 0.30

0.82 < 0.60

0.06 < 0.20

0.34 0.11

1.13 N.T.

2.44

~1.0

N.T.

~ 0.20

< 0.20

0.06

N.T

< 0.20

N.T.

"

"

"

a

 Potency of analogs relative to -THC as described in legend of table 2.

b

 R1

 and R2 = -CH3, R3 = -OH unless indicated. c

 Martin et al. 1984a.

114
Substitutions have also been made at position 12 of -THC, which is comparable

to position 10 of CBD. Both 12ß-amino- -THC (22) and the corresponding

ethylamino derivative (23) effectively reduce spontaneous activity and rectal

temperature as potently as -THC, but have little or no effect on tail-flick

response. In contrast, introduction of a thioacetyl group (24 ) reduces activity

compared to -THC. Analogs 22 and 23 were similar to the CBD analogs 1 and

2 in that they too were quite toxic (lethal at 6-10 mg/kg). The pharmacological

effects of 1-morpholinobutyryl- -THC (MB- -THC, 25) were compared to the

values obtained for -THC tested during the same session, and the data suggest

MB- -THC is somewhat less potent (0.3-0.1) than -THC. The values

obtained with -THC were similar to those previously reported, and the potency

range for the effects of MB- -THC is similar to that obtained for -THC

relative to -THC (Martin et al. 1984a). These data would seem to support the

notion that MB- -THC is metabolized rapidly by liver enzymes in vivo to

-THC, which is probably responsible for the observed behavioral effects (Zitko

et al. 1972).

The possibility that -THC exerts some of its effects by interacting with a

specific receptor has recently gained partial support from the studies of Nye et al.

(1985) who defined a stereoselective, saturable, high affinity site for tritiated

5'-trimethylammonium- -THC phenolate (5'-TMA, 26) binding. Therefore, in

order to determine the relevance of this binding site to the behavioral effects of the

cannabinoids, 5'-TMA has been evaluated and compared to -THC in our

cannabinoid assays. Pharmacological comparison reveals that both -THC and

5'-TMA induce hypothermia (2.6° and 3.0°, respectively) at 5 mg/kg, and decrease

spontaneous activity (ED50 = 10 mg/kg and 2.1 mg/kg, respectively) in the mouse

after i.v. administration. -THC and 5'-TMA also induce hypothermia (4.4° and

5.0°, respectively at 100 μg), as well as hypoactivity (ED50 = 46 μg and 133 μg,

respectively) after i.v.t. administration in the mouse. In the do both drugs, as

well as -THC, produce mild sedation. However, only - and -THC

produce static ataxia (at 0.4 mg/kg and 0.2 mg/kg i.v., respectively), while

5'-TMA is inactive at doses up to 1.0 mg/kg (i.v.). Although 5'-TMA exhibits

CNS depressant properties, it appears to lack the specific behavioral effects that

are typically produced by psychoactive cannabiioids.

The 5'-bromo- (27) and 5'-acetyl- (28) -THC analogs are similar in potency to

to both 5'-TMA and -THC in producing hypoactivity. However, 27 is more

potent in decreasing rectal temperature (2.8 °C at 1 mg/kg). Analog 28 does

produce hypothermia (maximum effect of 2.8 °C at 30 mg/kg), but not in a

dose-related fashion (a 1.5 °C drop at doses of 1,3, and 10 mg/kg).

Evaluation of Agonistic Activity of -THC Analogs

The behavioral data demonstrate that 1-O-succinyl- -THC (29) possesses a

pharmacological profile similar to that of -THC. However, the analog is less

potent than -THC in depressing spontaneous activity and in producing

hypothermia and antinociception. While 29 appears to retain cannabinoid activity,

one cannot rule out the possibility that the pharmacological activity is due to in

vivo hydrolysis to -THC. These data support the contention that substitutions

at the phenolic hydroxyl of -THC reduce pharmacological activity.

115
Alkylating agents of - and -THC have been prepared for the purpose of acting

as receptor probes. The N-mustard -THC-1-bis(2-chloroethyl)carbamate (30 )

was found to be inactive if administered intraperitoneally (i.p.) or orally (p.o.),

which suggests that this ester is not rapidly hydrolyzed in vivo to -THC.

However, 30 is active after either i.v. or i.v.t. administration. Following i.v.

administration (table 4), 30 is at most only 0.06 times as potent as -THC. In

contrast, i.v.t. administration of the 30 produces effects on rectal temperature

equivalent to that of -THC and is 0.2 times as potent as -THC in producing

hypoactivity (the tail-flick latency was not tested). These data suggest that the

ability of 30, and possibly other N-mustards, to enter the CNS may be limited.

TABLE 4

Pharmacological Activity of -THC and Structurally Related Analogs in Micea

Rb

Pharmacological Profile

S.A. R.T. T.F.

-THCc

1.00 1.00 1.00

29 R3 = succinyl 0.15 0.30 0.34

30 R3 = -02CN(CH2CH2Cl)2 0.06 0.01 <0.01

31 R2 = -(CH2)5Br 1.21 2.80 19.0

32 R2 = -(CH2)2CHOH-C2H3

d

S-3'-OH 6.20 1.00 N.T.

R-3'-OH 0.90 0.92 N.T.

R/S-3'-OH 2.50 0.89 N.T.

33 -THC 0.28 0.07 0.09

a

 Potency of i.v. analogs relative to i.v. -THC on the spontaneous

activity (S.A.), rectal temperature (R.T.), and tail-flick (T.F.) response

measures; calculated as described in the legend of table 2. b R1 = -CH3, R2

= -C5H11, and R3 = -OH unless indicated otherwise. c The ED50 is 2.3 and

1.9 mg/kg for S.A and T.F., respectively, and a 1.6 °C drop occurs at 3

mg/kg. d Based on previously reported values (Martin et al. 1984a).

116
The 5'-bromo- -THC analog (31) is similar to the 5'-bromo- -THC analog

(27) in potency and profile. Analog 31 (table 4) is equipotent to -THC in

reducing spontaneous activity and is roughly 3-fold more otent in producing

hypothermia. Interestingly, 31 is 19-fold more potent than -THC in the mouse

antinociception test. Tests are currently under way to determine if 27 , the

5'-bromo- -THC analog, also shares this characteristic.

Introduction of a hydroxyl group at the 3' position of the side chain (32 ) results in

an R/S 3'-OH racemic mixture that retains cannabinoid-like activity (Martin et al.

1984a). Final resolution of each of the compounds results in a S-3'-OH mixture

contaminated by 9% of the R isomer, and in an R-3'-OH mixture contaminated by

15% of the S isomer. Therefore, it is possible that the pharmacological effects

observed by one of the "pure" isomers is partially (or totally) due to the opposite

isomer. However, the pharmacological profiles of the racemate and each of the

isomers suggest that the S isomer is 6-fold more potent at reducing spontaneous

activity than -THC. The R isomer also produces hypoactivity, but the result

could be because 15% of the administered compound is actually the S isomer. In

contrast, the ability of both the R and the S isomer, as well as the racemate, to

produce hypothermia is nearly identical, and in fact is equipotent with -THC.

These data suggest that there is a specific side chain stereospecificity required for

one effect (hypoactivity) that is not required for another (hypothermia). In fact, it

is possible that the S, but not the R, isomer produces hypoactivity, while both

isomers are equipotent in producing hypothermia.

Evaluation of Cannabinoids for Antagonistic Properties

Since 10-N-ethylamino-CBD (1) is a partial antagonist of -THC-induced

antinociception (Jorapur et al. 1985), this compound has been tested further for

antagonistic activity. Although 1 lacks specific cannabinoid effects in the dog

static-ataxia test, pretreatment with 0.5 mg/kg (i.v.) of 1 does not alter the

behavioral effects of -THC in dogs. Compound 1 also does not produce an

antagonism of -THC's effects on spontaneous activity and rectal temperature.

Therefore, the attenuation of -THC's effects by 1 appears to be limited to

mouse antinociception. Additionally, 2 also produces a partial antagonism (38%

at 3 mg/kg) of -THC-induced antinociception, similar to 1. However,

compound 2 also does not block -THC’s effects on spontaneous activity and

rectal temperature, and therefore is not being tested further. The evaluation of the

10-bromo-CBD diacetate analog (6) for antagonistic activity indicates no alteration

of -THC-induced antinociception at doses up to 30 mg/kg.

The 12ß-amino- -THC analog (22) does not alter -THC-induced hypoactivity

and hypothermia as expected due to its agonistic activity. However, 22 does

produce a slight attenuation in -THC's antinociceptive activity. Doses of 22 of

0.3 to 3.0 mg/kg antagonized -THC’s effects by 25%.

Assessment of the antagonistic activity of the N-mustard -THC-1-bis

(2-chloroethyl) carbamate (30) following i.v. administration reveals that this

compound is not an antagonist. High doses of 30 (100 mg/kg) attenuate the

effects of -THC administered 24 hr later, but this blockade appears to be due to

desensitization, rather than antagonism, since -THC itself produces a similar

effect. However, it is possible that lower doses of 30, or a different experimental

protocol, might reveal antagonistic properties.

117
TABLE 5

- and -THC's Effects on Conditioned Operant Responding in Monkeys

Cumulative Dose (mg/kg)a

Operant

Test Monkey Ptosis Ataxia Sedation Suppression

Vehb

-THcd

-THCe

-THCf

INT g

-THC d

639

M661

M572

824

639 0.250 0.25

M661 0.125 0.50

M572 0.125 0.50

824 0.250 1.00

0.50

1.00

1.00

0.25

0.050

0.250

0.125

0.005

639

M661

1.000

639

M661

M572

824

1.000

0.500

639

M661

M575

824

1.50

1.50

1.00

1.00

0.500

0.250

0.125

1.00

639 0.25

M661 0.25

M572 0.25

824 0.25

1.00

1.00

1.00

0.50

0.25 0.125

0.50 0.500

0.50 0.500

0.25 0.005

a

 Cumulative dose equals the summation of all previous doses. bVehicle

consists of emulphor: ethanol: saline in a ratio of 1:1:18, respectively. c

The effect was not observed. d-f Total cumulative dose of 1.0 mg/kg of

-THCd, 1.0 mg/kg of -THCe

,

 and 5.0 mg/kg of -THCf. 

g

 A

cumulative dose of 1.0 mg/kg -THC preceded infusions of -THC.

All monkeys received a maximum cumulative dose of 10 mg/kg of -THC

within the normal 1-hr experimental session. Monkeys 639 and M661

received additional 0.5 mg/kg infusions of -THC after the session to

provide a total cumulative dose of -THC of 1.5 and 2.0 mg/kg,

respectively.

118
TABLE 6

Selective Pharmacological Profiles of Cannabinoids Analogs

Class 1 - All Cannabinoid Effects

-THC

-THC

11-OH- -THC

S-3'-OH- -THC

Class 2 - Weak CNS Depressant Devoid of other Cannabinoid Effects

10-Azido-CBD (4)

10-Sulfhydryl-CBD (5)

10-Bromo-CBD diacetate(6)

10-Morpholino-CBD diacetate (10)

10-(N-Methyl-N-propargylamino)CBD (7 )

10-(N,N-Diisopropylamino)CBD (8)

10-[(4-Aminobutyryl)amino]CBD (9 )

CBD

-THC (33)

11-N-Ethylamino- -THC (12)

11-N-Bis(2-chloroethyl)- -THC ( 13 & 14)

11-Thiol- -THC (15)

11-Thioacetyl- -THC (16)

9-nor-9-Phenyl- -THC (17)

9-nor-9-Benzyl- -THC (18)

9-nor-9-Isopropyld -THC (19)

9-nor-9-nButyl- -THC (20)

Class 3 -Potent CNS Depressant Devoid of other Cannabinoid Effects

10-N-Ethylamino-CBD (1)

10-N-Propylamino-CBD (2)

12ß-amino- -THC (22)

12ß-ethylamino- -THC (23)

-THC (33) (dogs only

5'-trimethylammonium-

)

-THC phenolate (26)

Class 4- Selective CNS Depressant Devoid of other Cannabinoid Effects

10-N,N-Bis-chloroethyl-CBD (3) - hypothermia, i.v.t.

11-Methylamino- -THC (11) - hypoactivity

9-nor-9-nButyl- -THC (20) - hypothermia

119
Agonistic Effects of -THC in the Mouse, Dog and Monkey

The exocyclic analog (33) , -THC, was investigated extensively as a result of

the suggestions of Binder and his colleagues (Binder et al. 1979; Binder and

Franke 1982; Semjonow and Binder 1985) that this analog should lack

psychoactive properties and would be useful for delineating nonspecific and

specific cannabinoid binding in in vitro assays. The pharmacolocal profile of

-THC in mice is similar to that of -THC, even though -THC is less

potent in all tests (table 4). It appears to be approximately 4, 15, and 11 times less

active than -THC in depressing spontaneous activity, reducing body

temperature, and producing antinociception in mice, respectively (Beardsley et al.,

in press).

Antagonistic Effects of -THC

The effects of -THC pretreatment on the actions of -THC in mice, dogs,

and monkeys have also been studied (Beardsley et al., in press). -THC

pretreatment did not attenuate any of the effects of -THC in mice. Evaluation of

-THC for cannabinoid antagonistic properties in the dog is hampered by the

fact that it has some depressant properties in this behavioral test. However,

pretreatment with either 0.5 or 1.0 mg/kg of -THC 15 min before -THC

does not result in an attenuation of the effects of the latter. One of the most

intriuing observations in the monkey tests is the finding that pretreatment with

-THC attenuates -THC-induced ptosis, sedation, ataxia. and operant

behavior in monkeys (table 5). The importance of this observation is that

-THC produces more than a modest reduction in the effects of -THC in

most cases. -THC-induced operant suppression is decreased by as much as 25

times. Although it may be premature to describe this interaction in monkeys as

antagonism, two exciting leads emerge. This exocyclic compound suggests

further possible alterations in the cannabinoid structure for developing an

antagonist, and implies that the monkey may be better suited for characterizing

cannabinoid antagonistic activity than other behavioral tests.

SUMMARY

The behavioral effects of cannabinoids are dependent upon numerous variables

which include species differences, dose, experimental conditions, etc. The

complexity of the cannabinoid behavioral syndrome certainly complicates attempts

to establish biochemical correlates. It is for this reason that attempts are being

made to develop analogs that exhibit selective behavioral effects. The

pharmacological profile of the analogs summarized in table 6 suggests a tentative

classification for the cannabinoids. While there are numerous analogs which

produce the entire spectrum of cannabinoid effects, only those discussed within

this article are grouped in class 1. CBD and many of its analogs appear to be

devoid of cannabinoid effects, but they are capable of producing CNS depression

at large doses (class 2). It is our working hypothesis that the compounds in class

2 may be altering membrane peturbation in a nonspecific fashion. This latter effect

may or may not be related to the psychoactive cannabinoids. Class 3 compounds

are potent CNS depressants that appear to lack other effects that are unique to

cannabinoids. These analogs are particularly interesting in that they may be

altering membrane function in a specific fashion in contrast to nonspecific effects

120
exerted by analogs in class 2. Analogs in class 4 appear to exert selective CNS

depression. While the mechanism by which these analogs are producing their

effects may be proven to be different from those postulated, it does appear that a

classification of cannabinoid effects is possible through structural alterations in the

cannabinoid molecule.

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ACKNOWLEDGMENTS

The research described herein was supported by a research grant (DA-03672) and

a training grant (DA-07027) from the National Institute on Drug Abuse.

AUTHORS

Billy R. Martin, Ph.D

David R. Compton, Ph.D

Patrick J. Little, B.S.

Thomas J. Martin, B.S.

Patrick M. Beardsley, Ph.D

Department of Pharmacology and Toxicology, Box 613

Medical College of Virginia/Virginia Commonwealth University

Richmond, Virginia 23298-0001

122
Interactions of Cannabinoids With Membranes.

The Role of Cannabinoid Stereochemistry and

Absolute Configuration and the Orientation of

Delta-9-THC in the Membrane Bilayer

Alexandros Makriyannis, Ph.D.; Ali Banijamali, Ph.D.;

Cornelis Van Der Schyf, Ph.D.; and Harold Jarrell, Ph.D.

INTRODUCTION

Cannabinoid Membrane Effects

Many pharmacological actions of the cannabinoids, such as psychotropic effects,

bronchodilatation, increased heart rate, reduced intraocular pressure, analgesia,

alteration in body temperature, and anticonvulsant activity, can be related to

their known effects on a variety of cellular membranes. Indeed, cannabinoids

are known to affect a variety of membrane functions (Martin 1986; Mechoulam

et al. 1985; Hillard et al. 1985, 1986). Among these we can list their effects on

numerous membrane enzymes (e.g., adenylate cyclase; Na+, K

+

-, Mg2+-, Ca2+-

ATPases; NADH dehydrogenase; LPC acyltransferase; phospholipase A2;

monoamine oxidase; cholesterol esterase; 5'-nucleotidase), brain synaptosomes,

blood platelets, human fibroblasts, neuroblastoma cells, rabbit neutrophils

(Naccache et al. 1982), excitable membranes (Martin 1986), and membrane

receptors (e.g., beta-adrenergic, cholinergic, opioid, dopaminergic) (Bloom 1984;

Bloom and Hillard 1985). The membrane functions affected by the cannabinoids

can be related to a wide variety of membrane-bound proteins. A common

feature with all of these effects is that the cannabinoids do not seem to interact

directly with the protein catalytic sites and do not compete with those ligands

known to interact with these sites.

Cannabiniod Activity Through Interactions with Phospholipids

It is generally accepted that the lipid microenvironment surrounding membraneassociated enzymes (Sandermann 1978), receptors, or other functional proteins

(Loh and Law 1980) can influence protein function. On this basis, the view was

advanced that cannabinoids may exert their effects by interacting with the lipid

component of the membrane. The following evidence is offered in support of

this view: (I) Cannabinoids are highly lipophilic molecules and partition

favorably in membrane lipids. (2) Cannabinoids have been shown to affect the

thermotropic properties of phospholipids. They thus decrease the phase

transition temperature of pure (Bruggemann and Melchior 1983; Makriyannis et

al. 1987) and brain membrane (Bach and Sela 1981) phospholipids. Also, they

eliminate the discontinuity in the Arrhenious plot for microsomal O-demethylase

(Bach et al. 1977), an effect attributed to a change in the phase transition

properties of the lipids associated with the enzyme. (3) Cannabinoids affect the

"microviscosity" of model and biological membranes as determined by electron

123
spin resonance (ESR) (Lawrence and Gill 1975), fluorescence (Hillard et al.

1986), and NMR methods (Makriyannis et al. 1987; Kriwacki 1986). The lipid

hypothesis is bolstered by the fact that some of these studies were carried out

using cannabinoid concentrations considered to be "physiological" (Hillard et al.

1985).

The Role of Drug Geometry in Drug: Phospholipid Interactions

Traditionally, the potency of drugs that are believed to act on the lipid

component of the membrane has been correlated with their lipid solubility and

no consideration was made for variations in their conformational properties or

their absolute configurations. This assumption was shown to be true with the

low molecular weight general anesthetics (Kendig et al. 1973), but does not seem

to hold with the larger membrane-active drugs. It was thus reported that

enantiomeric pairs of barbiturates showed marked differences in potency

(Wahlstrom et al. 1970). This has also been well documented in the case of

anesthetic sterioids where small structural variations in the steroid molecule,

having no effect on its lipid solubility, produce dramatic changes in anesthetic

activity (Atkinson et al. 1965). When tested for their effectiveness in inhibiting

the function of a membrane-bound protein (Makriyannis and Fesik 1980) and for

their abilities to perturb model phospholipid membranes (Makriyannis and Fesik

1983; Makriyannis et al. 1986), the anesthetic steroids showed variations that

paralleled those of their respective potencies. We explained the observed

differences in steroid membrane interactions on the basis of observed

conformational differences between the drug molecules (Fesik and Makriyannis

1985).

Interestingly, an analogous effect was demonstrated by Bloch and coworkers, who

found that small variations in sterol structure influence in a dramatic manner

their effects on model membranes, including solute permeation, microviscosity,

and steroid mobility in the membrane (Lala et al. 1978; Dahl et al. 1980), as well

as the growth rates of microorganisms (Lala et al. 1978).

Analogous evidence is also available for cannabinoids, pointing to the role of

structure and geometry in their interactions with phospholipids. Earlier work

using ESR (Lawrence and Gill 1975) showed that cannabinoid analogs with only

slight structural differences, or having the same structure but differing in

absolute configuration, exhibited relatively large differences in their effects on

model membrane preparations. As reported in a recent publication, fluorescence

polarization was used to study the effects of cannabinoids on synaptosomal

plasma membranes (Hillard et al. 1985). The study found significant changes in

membrane "fluidity" when "physiological" concentrations of cannabinoids were

used. Another study compared the thermotropic properties of model membrane

preparations containing each of the two delta-9-THC enantiomers and found

differences between them (Burstein et al. 1986). We would like here to report on

more detailed work from our laboratories using solid-state 2H-NMR

spectroscopy.

RESULTS AND DISCUSSION

Model Membranes and the Use of Solid-State NMR Techniques

Because of their demonstrable applicability to the natural systems, studies with

model membranes have contributed a great deal toward understanding the

properties of biological membranes. Model membranes have also been used

124
successfully to obtain information on drug:membrane interactions. Many of these

studies have made use of fluorescence, E.S.R., and high resolution NMR

techniques. However, the availability of solid-state NMR has opened new

horizons in this field of research. During the past few years, these methods have

led to previously unavailable molecular information on the conformational and

dynamical properties of model and biological membranes (semisolids) (Griffin

1981; Davis 1983). The use of solid-state NMR has also been extended to study

drug:membrane interactions and showed it to be the method of choice (Boulanger

et al. 1981; Makriyannis et al. 1986). The model membranes used for the solidstate NMR experiments are multilamellar bilayer phospholipid dispersions that

have proven to be excellent models, sharing to a surprisingly large degree many

of the properties of the lipid component in biological membranes (Davis 1983).

The Role of Cannabinoid Conformation and Absolute Configuration During its

Interaction with Membranes as Studied by Solid-State 2H-NMR

In systems undergoing anisotropic motions, as is the case with phospholipid

multilamellar bilayers, the deuterium nucleus gives rise to a "powder spectrum" in

which the doublet with the maximum intensity is the 2H quadrupolar splitting

( ) and equal to:

= (3/4) (e2qQ/h) SCD (Davis 1983)

where e

2

qQ/h) is the static quadrupole coupling constant which for, paraffinic

C-D bonds, is approximately 170 KHz. SCD is the order parameter which can be

used to describe the amount of motional averaging of the C-D bond vector with

respect to a fixed symmetry axis and has been used as a measure of membrane

"fluidity."

In its liquid crystalline phase, the 2H spectrum of DPPC labeled in a single site

has the powder pattern features observed in the 2H spectra of solids. However,

the quadrupolar splitting is reduced due to motional averaging, including axial

diffusion of the chains and gauche:trans isomerization in the methylene chain

segments.

To compare the effects of cannabinoid analogs on membranes, we used DPPC

model membranes containing 0.15M cholesterol into which 0.15M cannabinoid

was incorporated. From earlier studies with anesthetic steroids (Makriyannis,

unpublished), we have found that model membranes of such composition are

very suitable for discriminating between membrane-acting drug analogs and that

the relative effects of these molecules on the model membrane parallel their

effects on biological membranes.

We compared the effects of (-)-delta-9-THC to those of its inactive (-)-delta9,11-THC isomer on the model membrane by obtaining solid-state 2H-NMR

spectra as a function of temperature for each of the two preparations as well as

spectra for the membrane preparation without cannabinoid. We also compared in

a similar manner the effects of the two delta-9-THC enantiomers.

Figure 1 shows representative spectra from the model membrane preparations

with (-)-delta-9-THC, (-)-delta-9,11-THC, and no drug, obtained at two

different temperatures. The spectra are of the liquid crystalline type and allow

us to extract directly values for the residual 2H quadrupolar splittings ( ). As

125
expected, the splittings decrease as the temperature is increased, reflecting the

higher ratio of gauche:trans chain conformers in the chain at higher temperatures.

In figure 2, we compare the values for the above three preparations as a

function of temperature, while figure 3 compares the effects of the two delta-9-

THC enantiomers on the same membrane preparation. As can be seen, the

presence of each of the three cannabinoids in the membrane results in a decrease

in the respective 2H quadrupolar splittings. Also, membrane preparations

containing the pharmacologically active (-)-delta-9-THC analog give 2H spectra

with consistently smaller splittings than those from its inactive or less active

isomers, (-)-delta-9,11-THC or (+)-delta-9-THC. This is evidence that, although

all three cannabinoids increase the ratio of gauche:trans conformers in the

membrane chain, the more active analog produces the larger increase in

gauche:trans methylene ratio and the smaller order parameter (SCD). This, in

turn, may be interpreted to mean that the active (-)-delta-9-THC analog

"perturbs" the model membrane more effectively than its two less active isomers.

The above data show that the interaction of cannabinoids with the phospholipid

component of the membrane is governed by specific stereoelectronic requirements. Our results also provide support for the view that at least some of the

cannabinoid effects result from their interactions with the lipid component of the

membrane. The above observations on the stereoselectivity of cannabinoid:

phospholipid interactions are understandable if the defined geometric parameters

of the phospholipid bilayer are considered. These include the natural asymmetry

of the lecithin and cholesterol molecules as well as the conformational

preferences and dynamic properties of the lipid hydrocarbon chains and/or polar

head groups.

126
FIGURE 1. Solid-state 2H-NMR spectra due to the 2(7’,7’-C2H) segment of

DPPC obtained using a 90°x - -90° quadrupolar echo sequence at 45.3 MHz

with a 90° pulse width of 2.1-2.4 μs. To obtain each spectrum, 8,000-16,000

ethos were signal averaged. (A) DPPC + 0.15m cholesterol + 0.15M (-)-delta-9-

THC; (B) DPPC + 0.15M cholesterol + 0.15M (-)- delta-9,11-THC; (C) DPPC +

0.15M cholesterol.

FIGURE 2. Temperature dependence of the 2H quadrupolar splittings in the 2HNMR spectra of the 2(7’,7’-C2H2

) segment DPPC. DPPC + 0.15M

cholesterol + 0.15M (-)-delta-9-THC: ( )DPPC 0.15M cholesterol + 0.15M

(-)-delta-9'11-THC; ( ) DPPC 0.15M cholesterol.

127
FIGURE 3. Temperature dependence of the 2H quadrupolar splittings in the 2HNMR spectra of the 2(7’, 7’-C2H2

) segment of DPPC. ( ) DPPC + 0.15M

cholesterol + 0.15M (-)-delta-9-THC; ( ) DPPC + 0.15M cholesterol + 0.15M

(+)-delta-9-THC.

Since the drug concentrations used in our experiments are much higher than

"pharmacological," the results presented here cannot be viewed as a quantitative

description of the membrane perturbation produced by cannabinoids in vivo.

However, the data give us a qualitative description of the type of conformational

and dynamical changes produced by cannabinoids on the membrane bilayer.

Moreoever, such experiments provide us with a method for comparing the effects

of structurally related cannabinoid analogs on membranes.

The Orientation of (-)-delta-9-THC in DPPC Bilayers

In our effort to understand the geometric parameters involved in the interaction

of cannabinoids with membrane bilayers. we have made use of solid-state 2HNMR to study the orientation of (-)-delta-9-THC in the membrane bilayer.

This experiment required the strategic introduction of 2H labels in different

positions of the tricyclic cannabinoid ring system. To that effect, we synthesized

(-)delta-9-THC with specific 2H labels in each of the 2, 4, 8 alpha, 8 beta, 11-

CH3, 10, and 10a positions (Banijamali et al. 1987). The labeled drug molecule

was then introduced in DPPC bilayers. 2H spectra were generally obtained for

membrane preparations containing (-)-delta-9-THC with one 2H label or with up

to three 2H labels if these could be unambiguosly assigned in the solid-state

spectra. Spectra from the 2H-labeled THC in the membrane in the liquid

crystalline phase were axially symmetrical or nearly so and had different

quadrupolar splittings ( ) depending on the position of the 2H label in the

molecule (figure 4). The va ue of these splittings was then used to calculate the l

orientation of (-)-delta-9-THC in DPPC bilayers.

128
FIGURE 4. Below: Representative solid-state 2H-NMR spectrum of 2,4-d2

-(-)-

delta-9-THC (.3M) in hydrated DPPC bilayers at 42°C;

(

2H2

) = 20.8 KHZ. The central doublet ( 

(

2H4

 ) = 43.0 KHz,

=6.6 KHz) is due to the

phenolic deuteron. Above: Firs& derivative of the same spectrum from which 2H

splittings could be obtained more accurately.

Briefly, the principle of this determination is as follows (Taylor 1981):

Deuterons attached to a rigid structure will give quadrupolar splittings described

by the equation:

= 3/4 e

2

qQ (3cos 2

a - 1) [ 3cos 2

- 1]

h 2 2

where is the angle between the individual C-2H bond and the rotation axis.

The last term, which is known as the molecular order parameter (Smol), defines

the anisotropic motion of the entire molecule. The angles between the rotation

axis and the C-2H bonds cannot be calculated directly because Smol is not known.

However, Smol is expected to be identical for all 2H atoms attached to the

cannabinoid ring system in which overall tumbling is expected to dominate the

dynamics of this part of the molecule. Thus, the ratios (R ) between two

quadrupolar splittings for two different deuterium atoms (

2Hi

 and 2Hj) are equal

to:

R = 3cos2

a

3cos 2

Our calculations showed that (-)-delta-9-THC orients with its axis of rotation

almost in the plane of the aromatic ring. However, the molecule does not orient

with its long axis parallel to the bilayer chains, as is the case with cholesterol,

but almost orthogonal to the chains. Details of the above calculations will be

given elsewhere.

129
The above-described orientation allows the cannabinoid phenolic hydroxy group

to direct itself toward the polar side of the bilayer, indicating that this group

serves as the orienting anchor in the molecule. We refer to this cannabinoid:

bilayer interaction as "amphipathic interaction" in which its primary incentive is

to direct the polar and hydrophobic components of the cannabinoid molecule

toward the respective components in the amphipathic bilayer. This anchoring

effect of the THC phenolic OH on the membrane is intriguing and deserves more

extensive investigation.

Indeed, the requirement of a free OH for activity in THC is well established.

Also, differences in activities have been reported between cannabinoid isomers,

differing only in the presence and/or orientation of hydroxy and keto groups in

the C ring. Since it is reasonable to assume that these hydrogen bonding groups

may also be involved in the orientation of the cannabinoid molecule, one may

speculate that cannabinoid orientation in the membrane may have an important

effect on its biological activity. The drug orientation in the amphipathic

membrane may, thus, play an important (although not unique) role in

determining the nature of the cannabinoid:membrane interaction and its ability to

produce "productive membrane perturbations." We are currently investigating the

above hypothesis.

FIGURE 5. The orientation of (-)-delta-9-THC in DPPC bilayers as determined

from solid-state 2H-NMR data.

We combined our results on the orientation of delta-9-THC in the bilayer with

previously obtained data on the conformation of delta-9-THC (Kriwacki 1986) to

model its interaction with a DPPC bilayer using computer graphics. The

parameters for modeling the phospholipid bilayer were obtained from available

crystallographic data. As can be seen, the THC molecule inserts between the two

fatty acid chains of the bilayer while maintaining its phenolic hydroxy group at

the level of the bilayer interphase. Also, it can be noted that the plane of the C

ring in delta-9-THC deviates from those of the A and B rings. This conformational property of delta-9-THC may explain its ability to perturb membranes.

130
FIGURE 6. Modeling of (-)-delta-9-THC in a DPPC bilayer from experimental

data using a E&S PS 300 system and ChemX graphics software (developed and

distributed by Chemical Design Ltd., Oxford, England).

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ACKNOWLEDGMENT

This research was supported by grant number 3801 from the National Institute on

Drug Abuse.

AUTHORS

Alexandros Makriyannis, Ph.D.

School of Pharmacy and Institute of Materials Science

University of Connecticut

Storrs, Connecticut 06268

and

F. Bitter National Magnet Laboratory

Massachusetts Institute of Technology

Cambridge, Massachusetts 02139

Ali Banijamali, Ph.D.

Cornelis Van der Schyf, Ph.D.

School of Pharmacy and Institute of Materials Science

University of Connecticut

Storrs, Connecticut 06268

Harold Jarrell, Ph.D.

Division of Biological Sciences

National Research Council of Canada

Ottawa, Canada K1A 0R6

133
The High Affinity Cannabinoid Binding Site

in Brain: Regulation by Guanine Nucleotides

and Isolation of an Endogenous Inhibitor

Jeffrey S. Nye and Solomon H. Snyder, M.D.

INTRODUCTION

The molecular mechanism by which delta-9-tetrahydrocannabinol (delta-9-THC)

produces its characteristic psychotropic effect in man and animals remains a

mystery. The very high potency of cannabinoids and their stereochemical and

structural requirements for eliciting characteristic psychotropic effects suggest

that a neuronal receptor exists for delta-9-THC. Neuronal receptors have been

identified for a wide range of psychotropic agents; these sites have been

characterized by studying the binding of radiolabeled analogues of high potency

drugs.

The biochemical labeling of a drug receptor helps illuminate the drug’s

mechanism of action. The identification of receptor subtypes in different organs

or regions of the brain has helped clarify the pharmacological effects of certain

drugs. Sometimes the site of action of a drug may in fact be the receptor for a

well known neurotransmitter, as was the case for the antipsychotic drugs which

act at dopamine and serotonin receptors. Alternatively, a drug receptor may

physiologically interact with a previously unknown neurotransmitter, as in the

case of opiate receptors and the enkephalins.

DEVELOPMENT AND CHARACTERIZATION OF THE RADIOLIGAND

[

3H]5'-TRIMETHYLAMMONIUM-DELTA-8-THC ([3H]TMA)

To identify a receptor for cannabinoids, we developed a cationic analogue of

THC which was water-soluble and biologically active. Because of the extensive

membrane partioning of cannabinoids, studies with 3H-delta-9-THC have not

revealed saturable binding sites. TMA (figure 1) (Seltzman et al. 1985) possesses

a quaternary ammonium moiety at the 5’ position. Pharmacological studies reveal

that modification of the 5’ position does not result in loss of psychotropic activity

(Ohlsson et al. 1979). A tritiated version of TMA was prepared from

5'dimethylammonium delta-8-THC and [

3H]CH3

I.

Cannabimimetic Activity of TMA

To evaluate the biological activity of TMA, we studied its effects on the fieldstimulated contractions of the guinea pig ileum (Nye et al. 1985, table 1). The

ileum serves as an excellent model of cannabinoid action since its contractions

134
FIGURE 1. 5’-Trimethylammonium delta-8-tetrahydrocannabinol (TMA)

are inhibited by cannabinoids in a stereospecific manner which accurately

parallels their psychotropic effects (Rosell et al. 1979). Active cannabinoids

suppress the contractions of the ileum when stimulated by low voltage fields or

serotonin application, but not those elicited by acetylcholine. Contractions are

suppressed by an inhibition of release of acetylcholine from the presynaptic

nerves.

TMA and 5’-dimethylammonium delta-8-THC inhibit contractions of the ileum

with IC50s of 1000 and 900 nM, respectively. TMA also inhibits serotonininduced contractions, but not those elicited by acetylcholine. These studies

suggest that TMA is an active cannabinoid. Because of its permanent positive

charge, TMA may not cross the blood-brain barrier and, thus, be inactive when

administered i.v.

Binding of [

3H]TMA to Neuronal Membranes

Binding of [

3H]TMA to homogenates of membranes from whole rat brain is

saturable (Nye et al. 1985). Typically, we use 75 pM [

3H]TMA with 5 to 10 ug

protein of brain membranes. Inhibition of binding by unlabeled TMA reveals a

half-maximal inhibition at 100 nM TMA (figure 2). The dissociation constant

(KD) is 89 nM, calculated from Scatchard analysis of the data. The maximal

number of binding sites is calculated as 1.1 nmol mg protein. The Hill

coefficient is 1.1, indicating a single class of binding sites and the absence of

significant cooperative interactions.

Detailed kinetic analysis of the binding of [

3H]TMA to brain membranes

indicates very slow association and dissociation rates (figure 3). The association

rate constant (k1) is 4.5 x 109 M-1h

-l

. Dissociation of radioligand was initiated

with 10 uM unlabeled TMA yielding a dissociation rate constant (k-1) of

0.24 h

-1. The dissociation constant (KD) calculated as the quotient of the rate

constants (k-1/k1) is 5.7 x 10-11 M, which is more than 1000-fold lower than the

KD determined above, calculated from inhibition of equilibrium binding. This

discrepancy may reflect a nonspecific interaction of [

3H] TMA with membranes,

resulting in a low free concentraction of TMA.

135
FIGURE 2. [3H]TMA binding to rat cortical membrane homogenates. Triplicate tubes containing [ 3H]TMA

(75 PM) were incubated with crude homogenates (20 ug protein) at 37°C for 3 hr with varying concentrations

of unlabelled TMA. Nonspecific binding in the presence of 10 uM TMA is subtracted and the result is

compared to binding in the absence of unlabelled TMA. Data presented are from a representative experiment

which was repeated four times with results varying less than 15%. Inset: Scatchard analysis of [3H]TMA

binding. Bmax, maximum binding. From Nye et al. 1985. Copyright 1985 by American Society for Pharmacology

and Experimental Therapeutics (Baltimore).
FIGURE 3. Kinetic analysis of [

3H]TMA binding. Left: [3H]TMA (100 PM)

was incubated with crude rat brain membrane homogenates at 37°C for varying

periods of time before rapid filtration and washing. Right: [

3H]TMA (100 PM)

and crude membrane homogenates were preincubated for 4 hr. Ten micromolar

TMA was added to aliquots of the mixture to initiate dissociation for varying

times before rapid filtration and washing. Fraction specific binding is calculated

as the total binding minus the binding in the presence of 10 uM TMA divided by

the maximal specific binding (at 4 hr). The rate constants are calculated

assuming first-order dissociation (r=.99) and pseudo first-order association

kinetics (r=.96).

Cannabinoid Specificity of [3H]TMA Binding

The binding of [

3H]TMA is not inhibited at concentrations less than 1 uM by a

wide range of drugs and compounds spanning numerous drug-receptor classes,

active peptides, and highly hydrophobic compounds (legend to table 1).

However, cannabinoids show high affinity for the [

3H]TMA site. Delta-9-THC,

the major naturally occurring psychotropic cannabinoid, inhibits [

3H]TMA

binding with an inhibitory constant (Ki) of 27.3±5.0 nM, and delta-8-THC has a

Ki of 32.9±0.8 nM. These concentrations represent the calculated concentration

of compound in the assay volume. Because of extensive nonspecific membrane

partitioning, the actual free concentrations are substantially lower. Similar

concentrations of delta-9-THC are found in the blood of human subjects and

rhesus monkeys after threshold doses (Agurell et al. 1985).

Binding studies of a series of cannabinoid analogues reveal a well-defined

structure-potency relationship for the [

3H]TMA site in brain (Nye et al. 1985,

table 1). Taken together, the potencies for all of the cannabinoids at the

[

3H]TMA site do not correlate with their potencies observed in the psychotropic

test or in any other known behavioral assay. However, among groups of

137
TABLE 1

Comparison of potencies for cannabinoids

a

Inhibitory constant (Ki) for [

3H]TMA site. Compounds with negligible effects

at 1 uM are: amphetamine, atropine, capsaicin, captopril, carbamazepine, chloral

hydrate, cholesterol, choline, cocaine, cortisone, cyclohexyladenosine, diazepam,

dihydro-ergotamine, doxepin, 17-beta-estradiol, GABA, haloperidol, imipramine,

2-isobutyl-3-methoxypyrazine, lysergic acid diethylamide, mazindol,

meprobamate, mianserin, muscimol, naloxone, norepinephrine, nitrendipine,

nortriptyline, orcinol, ouabain, paraldehyde, pargyline, phencyclidine,

phenobarbitol, phentolamine, phenytoin, phorbol dibutyrate, picrotoxin,

progesterone, resorcinol, SKF525a. strychnine, testosterone, thiothixene,

thyroxine, tyrosine, valproic acid, verapamil, and zopiclone. bPotency of

cannabinoids on guinea-pig ileum. c

Threshold for rhesus psychotropic effects

from Mechoulam and Edery (1973) and references therein. Codes are: H, high,

<.1 mg/kg; M, medium, .1-.5 mg/kg; L, low, .5-2 mg/kg; I,inactive, >2 mg/kg.

dDifferent from levo isomer (P<.05). eDifferent from levo isomer (P<.1). f

ntNot tested. j

See Lee et al. 1983. From Nye et al. 1985. Copyright 1985 by

American Society for Pharmacology and Experimental Therapeutics (Baltimore).

138
cannabinoids which are active in eliciting a psychotropic response, the order of

affinity for the [

3H]TMA site parallels the order of potency in behavioral studies

and in inhibiting contractions of the ileum. The features of [

3H]TMA

pharmacology which parallel behavioral potencies are as follows:

1. The [

3H]TMA site shows a stereochemical preference for the natural isomer

of THC. Thus, for both delta-8- and delta-9-THC, the levo stereoisomer is

significantly more potent than the dextro isomer.

2. The pentyl side chain is essential for [

3H]TMA binding. Substitution of the

side chain with a carboxylic acid abolishes potency, while the addition of 1’ and

2’ methyl groups enhances affinity for the [

3H]TMA site. The order of

inhibitory potencies for side-chain hydroxylated analogues of delta-9-THC

accurately reflects their order of potency in both psychotropic tests and

physiological assays.

3. The phenolic hydroxyl (position 1) must be unobstructed for significant

[

3H]TMA binding potency. Thus, desacetyl-levonantradol is 10-fold more potent

than levonantradol (figure 4). This deacetylation occurs in vivo and is essential

for psychotropic activity (McIlhenny et al. 1981). Cannabinol acetate is also

significantly weaker than cannabinol.

4. The addition of a carboxylic acid moiety inactivates delta-9-THC in inhibiting

[

3H]TMA binding and in behavioral assays. The addition of a hydrox to the 11

position of delta-9- or delta-8-THC does not impair affinity for the[

3H]TMA

site, whereas 8-hydroxyl derivatives have significantly reduced affinity.

A number of natural cannabinoids and synthetic analogues display significant

affinity for the [

3H]TMA site despite their apparent inactivity in numerous tests

for psychotropic efficacy. Thus cannabinol, cannabidiol, cannabigerol,

cannabicyclol and delta-9,11-THC lack psychoactivity but show affinities for the

[

3H]TMA site of 34.4, 73.1 8.3, 73.1, and 13.6 nM, respectively. These

discrepancies suggest that [

3H]TMA may not be labeling the site of action of

delta-9-THC for producing psychotropic effects.

An alternative possibility suggested by analogy to other drug-receptor systems is

that structural isomers that lack bioactivity may be antagonists. Although the

pharmacological data are incomplete for many of the inactive cannabinoids, a

growing body of evidence indicates that antagonists do exist. The best studied is

cannabidiol, which antagonizes the effects of delta-9-THC in humans, rodents

(Karniol and Carlini 1973; Karniol et al. 1974) and seizure-sensitive animals

(Consroe et al. 1982, this volume). More recently, Martin et al. (this volume)

have demonstrated that delta-9, 11-THC suppresses the psychoactivity of delta9-THC in rhesus monkeys. Burstein et al. (this volume) have shown that 9-

catboxy,11-nor-delta-9-THC reduces the catalepsy in mice produced by THC.

Since most measures of the psychotropic effects of cannabinoids in man and

monkey are not quantitative, it is difficult to observe the shifts in dose-response

relationships that result from antagonism. Moreover, several of these compounds

may be weak agonists or partial agonists. This is supported by the observation

that cannabinol in high doses elicits psychotropic effects indistinguishable from

delta-9-THC in humans (Perez-Reyes et al. 1973).

139
FIGURE 4. Inhibition of [

3H]TMA binding by nantradol isomers. [3H]TMA

(100 pM) was incubated with crude rat brain membranes in the presence of

varying concentrations of nantradol isomers under standard assay conditions.

Nonspecific binding is determined in incubations with 10 uM TMA and is

subtracted from all data.

Localization and Physical Properties of the High Affinity Cannabinoid Site

[

3H]TMA binding is found in brain as well as a number of peripheral organs.

The density of binding sites is greatest in brain compared to other organs, and

the highest binding levels occur in the hippocampus. The relative potencies for

several cannabinoids at the [

3H]TMA site are similar in all brain regions. Of

peripheral organs, the kidney shows the highest density of sites, while lung and

muscle show the lowest densities.

The [

3H]TMA site is an integral membrane component which is fully extracted

by detergents, such as CHAPS and Triton X-100. Molecular sieve chromatography of the [

3H]TMA binding site in CHAPS-extracted membranes reveals a

bimodal peak of activity with an Mr of 60,000 daltons. This molecular size

probably includes an undetermined number of bound detergent molecules.

REGULATION OF THE HIGH AFFINITY CANNABINOID SITE BY PURINE

NUCLEOTIDES

We evaluated the effect on [

3H]TMA binding of guanine and adenine nucleotides

(figure 5). The specific binding of [

3H]TMA is enhanced about 3-fold by the

nonhydrolyzable analogue GTP-gamma-S. This enhancement occurs between 0.1

and 10 micromolar; at higher concentrations the enhancement is abolished, with

small inhibition observed at >200 uM. GTP does not produce an increase in

[

3H]TMA binding; instead, it shows a similar inhibition at high concentrations.

GTP is probably hydrolyzed to GDP and GMP by nonspecific hydrolases during

140
FIGURE 5. Regulation of [3H]TMA binding by purine nucleotides. [

3H]TMA

was incubated with washed crude brain membranes under standard assay

conditions in the presence of added nucleotides. Control binding is defined as the

specific [3H]TMA binding measured in the absence of added nucleotides minus

nonspecific binding (10 uM TMA). Each data point is the average of duplicate

assays and is representative of three experiments.

141
the long incubations required for measuring [

3H]TMA binding. The nonhydrolyzable analogue GDP-beta-S produces a similar enhancement of binding,

but at somewhat higher concentrations. The ED200 (concentration that doubles

binding) is about 1 uM for GTP-gamma-S and about 10 uM for GDP-beta-S.

GMP is without either stimulatory or inhibitory effect. These concentrations are

in the relevant range for an effect which is mediated by a GTP binding protein

(Lefkowitz et al. 1983).

Adenine nucleotides fail to display high potency stimulation of specific [

3H]TMA

binding. ATP inhibits binding, but only at high concentrations (IC50 = 100 uM)

(figure 5). The nonhydrolyzable analogue of ATP, ADP-imidophosphate (AMPPNP), inhibits [

3H]TMA binding with an IC50 between 5 and 10 uM. These

concentrations of AMP-PNP are sufficient to inhibit ATP-dependent processes,

such as adenylate cyclase or GDP-phosphorylation. Forskolin, a stimulator of

adenylate cyclase, has no effect on [

3H]TMA binding at the concentrations tested.

The enhancement of [

3H]TMA binding by nonhydrolyzable GTP and GDP

analogues suggests that the site may be regulated by a guanine nucleotide binding

protein or G-protein. G-proteins are coupled to a large number of

neurotransmitter and hormone receptors, and they regulate a variety of

intracellular processes, including adenylate cyclase and phospholipase C (Stryer

1986). Paradoxically, both GTP and GDP analogues produce an apparent

increase in [

3H]TMA binding. In the case of another receptor linked to a Gprotein, the beta-adrenergic receptor, the affinity of agonists is decreased by

GTP analogues leading to a reduction in binding. Antagonists, on the other

hand, are unaffected by GTP analogues (Lefkowitz et al. 1976).

Recent work by Howlett et al. (1986) suggests that cannabinoids act via a Gprotein in the neuroblastoma cell line N18tg2. Psychotropically active

annabinoids inhibit the adenylate cyclase of these cells at submicromolar

concentrations (Howlett and Fleming 1984; Howlett, this volume). Interestingly,

stimulation of adenyl cyclase via a number of different receptors is inhibited by

cannabinoids. Moreover, Howlett et al. (1986) have demonstrated that the

inhibition is reversed by ADP-ribosylation with pertussis toxin, suggesting that

the cannabinoids are acting via the inhibitory G-protein or Gi.

ISOLATION OF AN ENDOGENOUS INHIBITOR OF CANNABINOID

BINDING

The presence of a neuronal receptor for a psychoactive drug implies that some

endogenous neural mechanism is perturbed by that drug. One possible

neurochemical mechanism is the interaction of a neurotransmitter or

neuromodulator and its receptor. Accordingly, we decided to search in brain

extracts for an endogenous inhibitor of [

3H]TMA binding.

Extracts of rat brain were prepared according to the method of Bennett et al.

(1978). Such extracts preserve peptides from proteolysis and precipitate most

proteins. These preparations inhibit [

3H]TMA binding in a dose-dependent

manner when added to standard assays. Because inhibitory activity was stable to

acid and acetonitrile, we partially purified the active component using a

combination of conventional chromatography and HPLC. The extracts were

loaded on a C18 reverse-phase Sep Pak (Waters), washed with 0.1% trifluoracetic

acid, and eluted with 80% acetonitrile 0.1% aqueous TFA. This material was

chromatographed on a Biogel P10 column (Biorad) and eluted with 10% formic

acid 0.1% 2-mercaptoethanol. The fractions were evaporated and assayed for

142
inhibition of [

3H]TMA binding (figure 6). A single peak of inhibitory activity is

observed in fractions corresponding to the void volume of the column. HPLC

gel filtration chromatography of the pooled fractions on a TSK column (Biorad)

reveals a single peak of [

3H]TMA inhibitory activity corresponding to a

calculated molecular size of 15-20,000 daltons (figure 7).

FIGURE 6. Gel filtration chromatography of the endogenous inhibitor of

[

3H]TMA binding. Rat brain extracts. prepared as described in the text, were

loaded onto a Biogel P10 (Biorad) column (1x25 cm) equilibrated in 10% formic

acid/0.1% 2-mercapto-ethanol running at 5 ml/hr. Fractions (0.35 mL) were

evaporated and resuspended in 25 mM TES buffer (pH 6.8) before being added to

standard [3H]TMA binding assays in the presence of 75 pM [3H]TMA. Fractions

26-32 correspond to the void volume of the column. Assays were performed in

singlicate and represent two similar experiments.

Partially purified fractions of the endogenous inhibitor reduce [

3H]TMA binding

(figure 8) in a concentration-dependent manner. Although the preparation tested

is only partially purified, it shows an inhibitory potency (IC50) of 1.5-2.0 x 10-4

g protein/L. Assuming that a 15-20,000 dalton protein accounts for the

inhibitory activity, the maximal molar concentration of the preparation at the

IC50 is 10 nM. When fully purified, the actual IC50 of the endogenous inhibitor

of [

3H]TMA binding may therefore be substantially lower than 10 nM. Many

possibilities exist for the role of the endogenous inhibitor of [

3H]TMA binding.

It is enticing to speculate that this protein is a neurotransmitter or

neuromodulator whose normal functioning is perturbed by cannabinoids. Thus,

further characterization of the endogenous inhibitor may lead to additional

insights into the effects of cannabinoids upon the brain. Alternatively, the page

endogenous inhibitor may be an intracellular regulatory protein which interacts

with the cannabinoid site to alter certain cellular processes.

143
FIGURE 7. Molecular sizing of the endogenous inhibitor of [3H]TMA binding by

high pressure liquid chromatography. Partially purified rat brain extracts (see

text) were loaded onto a Biosil TSK column triplet (TSK-400, TSK 250, and

TSK-125) connected in series. The columns were eluted with 32% acetonitrile

0.1% aqueous trifluoroacetic acid at a flow rate of mL/min. Fractions (0.5

mL) were evaporated, resuspended in 0.1 N HCl, and added to standard

[

3H]TMA binding assays. The data presented (upper tracing) are from single

determinations on each fraction. Equivalent quantities of HCl reduced binding to

60% to 70% of maximal levels. The eluate was monitored for absorbance at 280

nm (lower tracing). Bovine serum albumin (a, Mr=66,000), alpha-MSH (b,

Mr=1572), and 2-mercaptoethanol (c, Mr=78) were used as standards to calibrate

the column.

144
FIGURE 8. Inhibitory potency of a partially purified preparation of the

endogenous inhibitor of [3H]TMA binding. Dilutions of partially purified

extracts obtained from gel filtration chromatography were added to triplicate

standard [3H]TMA binding assays with crude bruin membranes. Maximal

binding is defined as the total binding minus the binding in the presence of 10 uM

TMA. The abscissa represents the logarithm of the concentration of added protein

in the final volume of the [3H]TMA binding assay. Protein values were

determined from protein assay (BCA, Pierce) of the partially purified endogenous

inhibitor preparation.

MODEL OF CANNABINOID ACTIONS

One of the most perplexing phenomena in studying the cellular actions of

cannabinoids is their profound effects on a host of enzymes, transport

mechanisms, and neurochemical processes (Martin 1986). One possible

explanation for these diverse effects is that cannabinoids modulate the

metabolism of intracellular second messengers, such as cyclic AMP or the

phosphoinositide phospholipids (PIP,). These intracellular mediators in turn

regulate the activity of multiple cellular processes by stimulating the

phosphorylation of proteins, by the release of calcium ion stores, and by other

mechanisms.

A mechanism for the regulation of intracellular second messengers by

cannabinoids is suggested by the finding that the high affinity cannabinoid site

as labelled by [

3H]TMA appears to be coupled to a G-protein. Moreover, the

effect of cannabinoids on adenylate cyclase is mediated via Gi (Howlett, this

volume). Since G-proteins are the regulatory proteins which modulate the

activities of adenylate cyclase, phospholipase, and other second messengermetabolizing enzymes, an effect by cannabinoids upon these critical proteins

would in turn affect multiple cellular processes. Although the evidence is

incomplete, the hypothesis that all of the cellular actions of cannabinoids are

145
mediated via G-proteins offers a unified explanation for the diverse and multiple

effects of cannabinoids in Vitro and in vivo.

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29:307-313, 1986.

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and delta-9-tetrahydrocannabinol. Psychopharmologia 33:53-70, 1973.

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hydrochloride: Pharmacokinetics and behavioral effects after acute and

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Mechoulam, R., and Edery, H. Structure-activity relationship in the cannabinoid

series. In: Mechoulam, R., ed. Marijuana: Chemistry, Pharmacology,

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Nye, J.S.; Seltzman, H.H.; Pitt, C.G.; and Snyder, S.H. High-affinity

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234:784-791, 1985.

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S.; and Mechoulam, R. Synthesis and psychotropic activity of side-chain

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16:21-33, 1979.

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Perez-Reyes, M.; Timmons, M.C.; Davis, K.H.; and Wall, E.M. A comparison of

the pharmacological activity in man of intravenously administered delta-9-

tetrahydrocannabinol, cannabinol and cannabidiol. Experientia 29:1368- 1369,

1973.

Rosell, S.; Bjorkroth, U.; Agurell, S.; Leander, K.; Ohlsson, A.; Martin, B.; and

Mechoulam, R. Relation between effects of cannabinoid derivatives on the

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ACKNOWLEDGMENTS

This work was supported by National Institute on Drug Abuse grant DA-00266,

Medical Scientist Training Program grant GM-07309 (to J.S.N.), and NIDA

Research Scientist Award DA-00074 (to S.H.S.). Susan Voglmaier and Adele

Snowman assisted in the binding experiments and Patrice Obermiller assisted with

the HPLC. Pamela Sklar provided helpful discussions. The nantradol compounds

were a gift of Pfizer Central Research (Groton).

AUTHORS

Jeffrey S. Nye, A.M.

Solomon H. Snyder, M.D.

Department of Neuroscience and Pharmacology

The Johns Hopkins University School of Medicine

725 N. Wolfe Street

Baltimore, Maryland 21205

147
Regulation of Adenylate Cyclase in a Cultured

Neuronal Cell Line by Marijuana Constituents,

Metabolites of Delta-9-tetrahydrocannabinol,

and Synthetic Analogs Having Psychoactivity

Allyn C. Howlett, Ph.D.

The psychoactive properties of cannabinoid drugs have been described in

man and investigated in a variety of animal models. However, in vitro

studies have failed to elucidate the mechanism of cannabinoid action at the

neuronal level. The activities of a number of enzymes have been reported

to be modified by cannabinoid compounds (Martin 1986). However, in many

studies, problems arise in correlating these cellular events with changes in

animal behavior. This may be because the drug concentrations required for

alterations in enzyme activity are greater than would be expected at the

site of action in the brain. In other studies, cannabinoid drugs having little

or no psychoactivity produce a cellular response similar to that of

psychoactive cannabinoid drugs. These difficulties in defining a mechanism

of action for cannabimimetic drugs have been discussed by Martin (1986) in

a recent review on the cellular effects of cannabinoid drugs. Studies in my

laboratory have determined that cannabimimetic drugs inhibit cyclic AMP

accumulation in neuronal cells (Howlett 1984; Howlett and Fleming 1984).

These studies will be described in this chapter. It is proposed that the

cannabimimetic inhibition of adenylate cyclase is a receptor-mediated

cellular response that may be one mechanism of action for these drugs.

A neuroblastoma cell culture system is used for these studies as a model for

neurons. The N18TG2 cell line was cloned from the cultured C1300 tumor

(Augusti-Tocco and Sato 1969; Gilman and Minna 1973; Schubert et al.

1969). This tumor arose spontaneously in the region of the spinal cord of the

A/J mouse (Augusti-Tocco and Sato 1969) and, therefore, may have been

derived from sympathetic ganglial cells. N18TG2 cells have retained a

typical neuronal morphology in culture (Augusti-Tocco and Sato 1969;

Schubert et al. 1969), and they exhibit electrophysiological responses to

neurotransmitters such as acetylcholine (Chalazonitis et al. 1977). These

cells also synthesize the neuromodulator, vasoactive intestinal peptide (VIP)

(Brick et al. 1985). The advantage of using cultured neuroblastoma cells

rather than brain cells for biochemical studies of adenylate cyclase is that

plasma membranes may be isolated from large quantities of cells that are

genetically and phenotypically identical.

A number of neuromodulators interact with receptors that regulate the

synthesis of cyclic AMP in neuronal cells. Cyclic AMP diffuses into the

neuron as the "second messenger" that activates cyclic AMP-dependent

148
protein kinase. Protein kinase modifies the activity of key enzymes

involved in such functions as neurotransmitter synthesis or membrane

excitability (Bloom 1975; Daly 1977; Greengard 1976). The regulation of

adenylate cydase by neuromodulator receptors is complex and has been

reviewed by Gilman (1984). According to our current understanding,

stimulatory receptors interact with a regulatory protein complex, Gs, and

inhibitory receptors interact with a homologous complex, Gi (figure 1). Both

regulatory proteins are heterotrimers comprising , , and subunits.

Although the and subunits of Gs and Gi are thought to be nearly

identical, the a subunits differ structurally and functionally. Stimulatory

and inhibitory neuromodulator-receptor interactions promote the binding of

GTP onto the and subunits, respectively. This causes dissociation of

the or from the and proteins. The subunit is then free to

activate the catalytic protein of adenylate cyclase. The free ai subunit may

inhibit the catalytic protein directly. However, inhibition of adenylate

cyclase may also occur as the free proteins form a new equilibrium with

proteins and thereby prevent the interaction of with the catalytic

subunit.

INHIBITION OF ADENYLATE CYCLASE BY CANNABINOID COMPOUNDS

My laboratory provided the first evidence that cannabimimetic drugs

decreased cyclic AMP accumulation in neuronal cells (Howlett 1984).

Tetrahydrocannabinol (THC) and -THC decreased both basal and

prostacydin- (prostaglandin I2-) stimulated cyclic AMP accumulation in

neuroblastoma cells. Prostanoid-stimulated adenylate cyclase in a

membrane preparation from these cells was inhibited by cannabimimetic

compounds, indicating that this enzyme complex was the target for the

effect of -THC on cellular cyclic AMP concentrations. The

cannabimimetic drugs caused a decrease in Vmax of the enzyme, with no

alteration in the Km for substrate (Howlett 1985). Adenylate cyclase

inhibition was apparent immediately upon addition of -THC and the

effects of the drug were readily reversible after the membranes were

sedimented and resuspended (Howlett 1985). The inhibition was shown to be

concentration-dependent over a nM range for both -THC and -THC.

The activity of cyclic nudeotide phosphodiesterase, responsible for the

metabolic breakdown of cyclic AMP, was unaltered by these agents (Howlett

1984).

The cannabimimetic inhibition of adenylate cyclase was not observed

universally in all cell types. The response was not observed in either the

soluble adenylate cyclase from rat sperm or the membrane-bound adenylate

cyclase from C6 glioma or S49 lymphoma cells (Howlett et al. 1986). This

cellular selectivity provides evidence for the existence of specific receptors

for the cannabimimetic drugs. If these drugs were influencing enzyme

activity by intercalating into lipid membranes and altering membrane

fluidity, then one would have expected universal inhibition of adenylate

cyclase in all cell types. This was not the case.

The inhibition of adenylate cyclase by -THC in neuroblastoma plasma

membranes was not due to an effect on stimulatory receptor regulation of

the enzyme (Howlett and Fleming 1984). Inhibition by -THC was not

competitive with prostaglandin E1

 or prostacyclin. Furthermore, noncompetitive inhibition was also observed when the peptide hormones

149
FIGURE 1. Hypothetical model of the regulation of adenylate cyclase by stimulatory and inhibitory neuromodulators. Rs

and Ri represent one of several pharmacologically distinct receptors for stimulatoy and inhibitory neuromodulators,

respectively, and Hs and Hi indicate occupancy by their respective agonist ligands. The G-protein complexes, Gs and Gi are

denoted as their subunit constituents, and respectively. The catalytic protein is designated C. Details

concerning the interaction of these protein subunits are found in the text.
secretin and VIP stimulated the enzyme. Additional evidence that the

THC was not acting at the level of the receptors that stimulate adenylate

cyclase was provided by the observation that inhibition also occurred when

forskolin was used to activate the enzyme. Forskolin is believed to act

directly at the catalytic subunit (Seamon and Daly 1981).

The cannabimimetic inhibition of adenylate cyclase appeared to resemble

the regulation resulting from inhibitory neuromodulator receptors that act

via Gi (Howlett 1985). The effects of cannabimimetic drugs were related to

the ability of the enzyme to be regulated by divalent cations and guanine

nucleotides. The inhibition was greatest at micromolar Mg2 + or Mn2 +

concentrations and was attenuated by millimolar concentrations of Mn2+.

Half-maximal inhibition of adenylate cyclase was observeo at 800 nM GTP

for both cannabimimetic and muscarinic cholinergic agents. This

concentration is higher than that required for stimulation of the enzyme by

hormones and is typical of the high GTP concentrations required by

inhibitory hormones and neuromodulators in other systems (Cooper 1982).

These observations illustrate the similarities between the enzyme inhibition

by cannabimimetic drugs and by muscarinic cholinergic drugs. It is inferred

that the cannabimimetic drugs must act via regulatory mechanisms similar

to those operating for hormonal inhibition of adenylate cyclase.

Receptor-mediated inhibition of adenylate cyclase in most eukaryotic

systems requires the presence of the guanine nucleotide-binding protein

complex, Gi. Further studies confirmed that cannabimimetic inhibition of

adenylate cyclase also required the presence of Gi (Howlett et al. 1986). Ci

can be functionally inactivated as the result of an ADP-ribosylation

modification catalyzed by pertussis toxin. Therefore, pertussis toxin can be

used as a tool to identify Gi-mediated events in the cell. Pertussis toxin

treatment of intact N18TG2 cells abolished the cannabimimetic regulation

of cellular cyclic AMP content. The adenylate cyclase response to -THC

was also attenuated in membranes isolated from cells that had been treated

with pertussis toxin. The action of pertussis toxin for in vitro modification

of neuroblastoma Gi requires NAD as substrate for the ADP-ribosylation.

The toxin was able to catalyze the labeling of a neuroblastoma membrane

protein in vitro using (

32P)NAD under conditions similar to those by which

attenuation of the cannabimimetic inhibition of adenylate cyclase could be

demonstrated. Protein labeling and the loss of the response to -THC

followed the same dose-response curve for pertussis toxin. This strengthens

the argument that cannabimimetic drugs act via receptors that interact

with the inhibitory GTP-binding protein complex.

THE PHARMACOLOGY OF CANNABIMIMETIC INHIBITION OF

ADENYLATE CYCLASE

Inhibition of adenylate cyclase in N18TG2 cells is known to be regulated by

muscarinic cholinergic, -adrenergic, and -opioid receptors. Thus, it was

possible that -THC may be acting via one of these pharmacological

receptor types. When this was tested, it was found that inhibition by

THC was neither synergistic nor additive with muscarinic cholinergic or

adrenergic agents when each was present at maximally inhibitory

concentrations (Howlett and Fleming 1984). Furthermore, the response to

-THC was not blocked by the antagonists to these receptors, atropine and

yohim bine, respectively.

151
The opioid drugs are another pharmacological class of neuromodulatory

compounds that inhibit adenylate cyclase in N18TG2 cells. Because of the

interaction of cannabimimetic and opioid drugs in animal models of

tolerance, the possible involvement of opioid receptors was examined (see

Devane et al. 1986 and references contained therein). -Opioid receptors

were found on neuroblastoma membranes using (

3H)-D-ala 2

-D-ieu5-

enkephalin. No μ- or -opioid receptors were detected. The -opioid

receptor binding affinity and capacity were unaltered by cannabimimetic

drugs. Opioid drugs as well as -THC and the nantradol series of analgesic

cannabimimetic analogs decreased cyclic AMP accumulation in intact cells

and inhibited adenylate cyclase in membrane preparations. Opioid

antagonists blocked the response to the opioid agonists but not to the

cannabimimetic drugs. The interaction between cannabimimetic drugs and

the opioid agonist etorphine showed no antagonistic, synergistic, or additive

effects at maximal concentrations. These results suggest that the opioid

and cannabimimetic drugs operate via distinct, noninteractive receptors

that can be coupled to the same effector, adenylate cyclase.

Other neuronal cell lines having a -opioid response were also examined

(Devane et al. 1986). The cannabimimetic inhibition of cyclic AMP

accumulation in NC108-15 neuroblastoma x glioma hybrid cells was not as

great as the response in N18TG2 cells. N4TG1 neuroblastoma cells, which

exhibit a prominent inhibition of adenylate cyclase in response to opioid

drugs, did not respond to cannabimimetic drugs under any conditions tested.

Thus, the cannabimimetic response does not correlate with the efficacy of

the -opioid response.

STRUCTURE-ACTIVITY RELATIONSHIPS AMONG THE CANNABINOID

DRUGS

Given that the cannabimimetic response in neuroblastoma cells is mediated

by Gi and does not result from drug interaction with -opioid, muscarinic

cholinergic, or -adrenergic receptors that inhibit adenylate cyclase, the

hypothesis can be proposed that these drugs act via a "cannabinoid"

receptor. The classical means to describe a receptor is to define its

pharmacological spectrum of activity. This was performed using

constituents of marijuana extracts and metabolites of these compounds

(Howlett 1987). The concentration of -THC exhibiting half-maximal

inhibition was less than 500 nM (figure 2A). -THC was less active,

cannabinol was only partially active, and cannabidiol was inactive. Other

constituents of marijuana that have no psychoactivity (cannabigerol,

cannabichromene, olivetol) and compounds having a reduced length of the

C3 alkyl side chain were not active as inhibitors of adenylate cyclase (table

1). These inactive compounds did not behave as antagonists to the effects

of -THC.

The nantradol class of cannabinoid analogs were potent inhibitors of

adenylate cyclase (figure 2B). Desacetyllevonantradol was more potent than

nantradol. The nonpsychoactive isomer dextronantradol was a poor inhibitor

of adenylate cyclase.

The metabolities of -THC and -THC hydroxylated at the C11-position

were more potent than the parent drugs (table 1). However, hydroxylation

at the C8-position of the terpenoid ring resulted in loss of activity of

152
THC. Compounds hydroxylated along the C3 alkyl side chain were equally

efficacious but less potent than -THC.

These structure-activity relationships are consistent with cannabinoid

pharmacology reported for psychological effects in humans and for

behavioral effects reported in a variety of animal models. Potency

estimates have suggested that the psychological high experience in humans

is greater for -THC than for -THC (Hollister 1974; Hollister and

Gillespie 1973). Neither cannabinol nor cannabidiol produced responses in

human subjects at the oral dosages tested (Hollister 1973, 1974). However,

when infused intravenously, cannabinol (but not cannabidiol) produced a

psychological high and cardioacceleration at ten-fold the dose of -THC

(Hollister 1973; Perez-Reyes et al. 1973a). 11-OH-metabolites of -THC

and -THC were up to twice as active as the parent compounds in

producing a psychological high (Hollister 1974; Lemberger et al. 1973). The

ability of these compounds to inhibit adenylate cyclase parallel these

responses.

FIGURE 2. Inhibition of adenylate cyclase by cannabinoid and nantradol

compounds. Forskolin was present at 1.0 μM to activate adenylate cyclase.

Other compounds present at the indicated concentmtfons were cannabinol

(CBN), cannabidiol (CBD), levonantradol ( -Nan), dextronantradol (d-Nan),

and desacetyllevonantradol (Da- -Nan).

In animal studies, -THC was less potent than -THC in such tests as dog

ataxia (Martin et al. 1981; Wilson et al. 1976), monkey behavior (Edery and

Crunfeld 1971), mouse hypothermia (Martin et al. 1981), and hot-plate

analgesia (Uliss et al. 1975; Wilson and May 1975) and in genetically THCseizure prone rabbits (Consroe and Fish 1981). Cannabidiol (Consroe and

Fish 1981; Edery and Crunfeld 1971; Pertwee 1972; Uliss et al. 1975),

cannabinol (Edery and Grunfeld 1971), and cannabichromene (Edery and

Grunfeld 1971) were inactive in a number of these models. The THC-seizure

prone rabbits responded to cannabinol at high doses (Consroe and Fish 1981).

The 11-OH metabolites of -THC and -THC were more potent than the

153
parent compounds in the dog ataxia (Wilson et al. 1976), mouse immobility

(Gill et al. 1973), hot-plate analgesia (Wilson and May 1975), and THCseizure prone rabbit models (Consroe and Fish 1981) and in the drug

discrimination studies using rats or pigeons (Jarbe and McMillan 1980).

Animal studies of the hydroxylated metabolites at the 8-position are

inconsistent; although in each study, 8-OH metabolites are either inactive or

less potent than -THC (Ben-Zvi et al. 1971; Wilson and May 1975; PerezReyes et al. 1973b; Jarbe and McMillan 1980). The data reported here for

inhibition of adenylate cyclase are consistent with this pharmacological

pattern.

TABLE 1

Pharmacological Parameters of Marijuana Constituents and Metabolites

Compound Efficacya

Potency

Kinh

-THC

-THC

Cannabinol

Cannabidiol

Cannabichromene

Cannabigerol

Olivetol

-THC

11-OH- -THC

11-OH- -THC

11-OH-cannabinol

8 OH- -THC

8 OH- -THC

8 , 11-diOH- -THC

2 ́-OH- -THC

3 ́-OH- -THC

4 ́-OH- -THC

5 ́-OH- -THC

Tri Nor- -THCcarboxylic acid

Penta Nor- -THCcarboxylic acid

1.0 430

0.9 560

0.5 1400

O

b

- -

0 - -

0 - -

0 - -

0.6 290

1.0 100

1.0 260

1.0 320

0 - -

0 - -

0 - -

1.0 2800

1.0 840

1.0 1800

1580

0 - -

0 - -

a

Relative to -THC equal to 1.

b

An efficacy of 0 was assigned to all compounds exhibiting a

lack of inhibition at concentrations up to 50 μM .

The parallels in cannabinoid structure-activity relationships exhibited by the

154
neuronal cells compared to the human and animal behaviors is intriguing.

These findings suggest that certain behavioral effects of cannabimimetic

drugs may be the result of an interaction of these drugs with a

pharmacologically distinct receptor associated with neuronal adenylate

cyclase. At least one possible mechanism of action for the cannabimimetic

compounds may involve regulation of cyclic AMP synthesis in populations of

neurons associated with modification of such behaviors.

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ACKNOWLEDGMENTS

This work was supported by National Institute on Drug Abuse grant

DA03690. Cannabinoid compounds were supplied by the National Institute

on Drug Abuse and nantradol compounds were a gift from Pfizer Inc.

AUTHOR

Allyn C. Howlett, Ph.D.

Department of Pharmacology

St. Louis University School of Medicine

1402 S. Grand Boulevard

St. Louis, Missouri 63104

157
Inhibitory and Stimulatory Effects

of Cannabinoids on Eicosanoid

Synthesis

Sumner H. Burstein, Ph.D.

BACKGROUND

Introduction

More than a decade ago, we suggested that some of the actions of

tetrahydrocannabinol (THC) were mediated by changes in

prostaglandin synthesis. This was based on a comparison of the

literature from both fields and the results of pilot experiments

in our laboratory (Burstein and Raz 1972). Much evidence in

support of this hypothesis has since been reported both by us and

by others. At this point, it appears that such a mechanism is

still quite viable and can provide a rational framework for new

experiments aimed at developing a mechanism of action for THC (for

a review, see Martin 1986).

Role of Prostaglandins in the Brain

Arachidonic acid, the precursor of all eicosanoids, is a major

lipid component of brain, and the ability of this organ to

synthesize eicosanoids is well established (Chiu and Richardson

1985). In addition, peripherally administered prostaglandins are

known to produce potent effects on central nervous system (CNS)

function (Curro et al. 1982) and can cross the blood-brain barrier

quite readily (Bito et al. 1976). Moreover, evidence for the

existence of receptors in the brain for several of the

prostaglandins has been reported, although their functional

significance is not clear at present (Malet et al. 1982; Shimizu

et al. 1982).

Of particular interest to this discussion are reports that both

centrally and peripherally injected prostaglandins can produce

behavioral changes in rodents (Poddubiuk 1976; Hollingsworth and

Patrick 1984; Laychock et al. 1980). Sedation and the inhibition

of locomotor activities are prostaglandin-induced responses which

have been known for some time (Gilmore and Shaikh 1972). The

molecular mechanisms accompanying these effects are not well

understood. However, there is some evidence suggesting that

prostaglandins regulate neurotransmitter levels and/or calcium

transport in neuronal tissue (Bergstrom et al. 1973).

158
In Vitro Evidence for the Hypothesis

The data reported thus far from our laboratory using a cell

culture model are strongly supportive for a role for prostaglandin

E2 (PGE2) as a mediator of THC action. The structure-activity

relationships (SAR) (Burstein et al. 1983a, 1984a), the development of tolerance (Burstein et al. 1985), and the existence of

stereospecificity (Burstein et al. 1986a) give a profile of

activity similar to that for the psychotropic effects of THC in

vivo. The properties of the other primary cannabinoids (CBD,CBN,

etc.) in our model do not show a good correlation with psychoactivity (Burstein et al. 1983). However, these substances do

have other activities which may be mediated by eicosanoids (Martin

1986). Clearly, any model has its limitations; however, this does

not prevent its utilization as part of a broader approach to

establishing a mechanism.

The experiments we have reported thus far using subcellular

preparations from mouse brain demonstrate that THC can stimulate

lipid turnover by increasing phospholipase activities (Hunter et

al. 1986). We studied phospholipase A2 (PLA2) and phospholipase C

(PLC) activity in synaptosomes, myelin and mitochondrial fractions

using both exogenous and endogenous substrates containing radiolabelled arachidonate. Submicromolar levels of THC produced

significant increases in PLA2 activity in synaptosomes. Diacylglyceride (DAG) levels were also increased by THC, probably due to

its inhibitory action on diglyceride lipase combined with its

stimulatory action on phosphatidyl inositol (PI) hydrolysis by

PLC.

Hillard and Bloom (1983) have suggested that there may be a role

for prostaglandins in the effects of cannabinoids on adenylate

cyclase activity, which they have observed. They reported that

aspirin, indomethacin, and mepacrine all inhibited THC-induced

stimulation of adenylate cyclase in homogenates of mouse cerebral

cortex. It is well known that PGE2 is an effective stimulator of

cyclase in a variety of systems and that CAMP has an essential

role in catecholamine-mediated neurotransmission.

Using an experimental design very similar to ours, White and

Tansik (1980) reported stimulation of human platelet PLA2 activity

by THC and cannabidiol (CBD). The released arachidonate was

converted primarily into lipoxygenase products. A similar finding

was reported by Laychock et al. (1986) in which rat pancreatic

islet preparations were exposed to THC. Again, when arachidonic

acid levels were increased, it resulted in increased synthesis of

12-HETE which enhanced insulin secretion.

The synthesis of F-type prostaglandins by rat brain synaptosomes

was reported to be inhibited by THC (ID50, 750 μM) (Raffel et al.

1976). The high doses needed for this effect cast some doubt on

its relevance to any in vivo action of THC. In a symposium

report, Howes and Osgood (1976) described a THC-induced inhibition

of PGE1 synthesis in rat striatal synaptosomes with an ID50 of

approximately 0.5 uM.

159
In Vivo Evidence for the Hypothesis

Some time ago, Jordan and Castracane (1976) reported that THC

produced a 50% rise in PGE levels in rat uterine venous blood.

The animals were given a dose of 40 mg/kg following estradiol

stimulation, and samples of blood were taken by means of a uterine

vein cannula. They raised the possibility that some of the

actions of THC were due to its ability to increase PGE levels.

Dalterio et al. (1981) found that a complex response was produced

by cannabinoid exposure in mice when pituitary and testicular

levels of PGE and PGF were monitored. Significant increases or

decreases could be observed depending on subtle experimental

conditions. They concluded that, "prostaglandins may mediate some

effects of cannabinoids on male reproductive function in mice."

We have provided evidence for a role for prostaglandins in the

hypotensive action of THC in the dog (Burstein et al. 1982b). A

THC-induced decrease in mean arterial blood pressure could be

profoundly inhibited by the prior administration of aspirin. We

postulated that the hypotensive effect of THC could be mediated by

the release of prostacyclin in the cardiovascular system.

Fairbairn and Pickens (1979, 1980) presented convincing evidence

that the cataleptic response to THC in mice is mediated by PGE2.

Animals treated with any of several prostaglandin synthetase

inhibitors showed a reduced response to THC which could be

restored by intraperitoneal injection of PGE2. Moreover, mice

placed on an essential fatty acid deficient diet showed little

response to THC. However, the administration of arachidonic acid

resulted in a rapid restoration of the cataleptic effect. These

findings raise the possibility that the CNS effects of THC in

humans may be mediated by eicosanoids.

A study in humans was recently reported (Laviolette and Belanger

1986) in which cannabis-induced bronchodilation was measured in a

group of 11 volunteers. A second exposure to marijuana smoke was

monitored in which the subjects were heavily dosed with aspirin

over a 30-hour pretreatment period. No inhibition of bronchodilation was observed, prompting the authors to conclude that

eicosanoids were not mediators of this effect of marijuana.

In the next section, we describe recent findings on the in vitro

antagonism of THC by its major metabolite, THC-7-oic acid-

(Burstein et al. 1986b). In subsequent experiments, mice were

given oral doses of this acid (40 mg/kg), and a 50% reduction in

the cataleptic effect of THC (50 mg/kg) was observed by us (see

table 7). The most likely explanation for this result is that the

metabolite blocks the conversion of arachidonic acid into PGE2

based on our in vitro findings. Thus, THC is able to both

stimulate and inhibit prostaglandin production via its metabolite

depending on experimental parameters.

This possibility for dual effects could explain reports where

there is an apparent reduction of prostaglandin synthesis

160
following THC exposure. Ayalon et al. (1977) reported a decrease

in rat ovarian PGE content after THC; Green and Podos (1974)

similarly found that the arachidonic-induced rise in rabbit

intraocular pressure was inhibited by intravenous THC.

Hypothalamic "PGE2-like material" in THC-treated rats was reduced

in experiments reported by Coupar and Taylor (1982). The tissue

extracts were monitored by a bioassay procedure using the rat

gastric fundus strip which is not a particular specific assay for

PGE2. The same authors have subsequently reported (1983) that

intestinal "PGE2" is also reduced by THC.

In summary, a number of in vitro studies have demonstrated that

cannabinoids are potent stimulators of eicosanoid synthesis. The

data obtained from our cell culture model suggest that this effect

is due to the action of the cannabinoids on the phospholipases

which control the levels of free arachidonic acid needed for

prostaglandin production. In certain instances, some cannabinoids

may inhibit prostaglandin synthesis, indicating the possibility of

complex interactions. The in vivo support for the "prostaglandin

hypothesis" is less complete at this time. Most convincing are

the reports by Fairbairn and Pickens (1979, 1980) which strongly

suggest that PGE2 is an important mediator in THC-induced

catalepsy in mice. While this may or may not be related to THC

effects on the human psyche, it does demonstrate a role for

prostaglandins in a CNS action of THC.

RECENT FINDINGS

In Vivo Studies

Encouraging data in support of a role for prostaglandins in the

actions of THC were obtained by us some time ago (Burstein et al.

1982b). In collaboration with members of the Pharmacology

Department, we were able to demonstrate inhibition of THC-induced

hypotension in dogs by the prior administration of aspirin. Such

an effect is consistent with a mechanism in which THC stimulates

prostacyclin synthesis in the cardiovascular system. Prostacyclin

is a potent vasodilator and could well mediate the observed

hypotensive effect of THC.

Evidence for a role for prostaglandins in the CNS actions of THC

has been more difficult to obtain. Recently (Hunter et al.,

unpublished data), we have shown a 29% rise in mouse brain

prostaglandins (table 1) following oral administration of -THC

(50 mg/kg). Prior administration of probenecid (120 mg/kg) was

necessary, presumably to slow the rapid clearance of PGE2 from the

brain; however, this had no effect on arachidonate metabolism

(table 2). We also utilized a double isotope labelling procedure

to reduce the difficulties in the analysis of the brain extracts

(table 3). This approach reduced the variability of results

caused by losses during the extraction and chromatographic

separations.

161
TABLE 1

Effect of -Tetrahydrocannabinol on [

14C]-Arachidonate

Metabolism in the Mouse Braina

Metabolite Controlb

Treatedb (%) P(n=4)

Phospholipids 47.2 ± 5.8 39.3 ± 9.1 - 17 > .05

Prostaglandins 23.7 ± 13 30.5 ± 3.8 + 29 < .Ol

Arachidonate 10.3 ± 2.9 10.5 ± 2.1 + 1.7 > .05

Diglycerides 9.90 ± 3.9 12.8 ± 3.3 + 29 > .05

Triglycerides 8.00 ± 4.3 6.10 ± 3.6 - 24 > .05

a

 Values are expressed as percentage of total radioactivity

recovered from tlc ± S.D. P was determined by the Student's t

test.

b

-Tetrahydrocannabinol (50 mg/kg) administered orally in sesame

oil to the treated group. Control and treated groups received

probenecid (120 mg/kg) i.p.

TABLE 2

Effect of Probenecid (120 mg/kg) on [14C]-Arachidonate

Metabolism in the Mouse Braina

Metabolite Control Probenecid Treated P(n=8)

Phospholipids 44.0 ± 12 46.4 ± 6.6 > .05

Prostaglandins 18.9 ± 6.9 18.8 ± 5.5 > .05

Diglycerides 15.8 ± 4.6 14.8 ± 3.0 > .05

Triglycerides 8.5 ± 2.4 8.0 ± 2.9 > .05

a

 As in table 1.

Cell Culture Experiments

Our initial observations on the stimulatory effect of cannabinoids

on prostaglandin synthesis were made using cells in culture

(Burstein and Hunter 1978). More recently, we have described this

effect in some detail and attempted to develop it into an in vitro

model for the CNS actions of THC (Burstein et al. 1982a). Using

such model systems, we plan to investigate the biochemical basis

for the effects of THC on arachidonate turnover and metabolism.

162
TABLE 3

Effect of -Tetrahydrocannabinol on [

14C/3

H]Arachidonate

Metabolism in the Mouse Brain

Metabolite Isotope Ratioa

Controlb

Treatedb

(%) P(n=4)

"Phospholipids"

PGF2

TXB2

PGE2

HETEs

PGA2 and HETEs

Arachidonate

Neutral lipids

0.52 ± 0.15 0.69 ± 0.04

1.19 ± 0.22 1.16 ± 0.05

1.23 ± 0.33 1.32 ± 0.09

0.99 ± 0.21 1.24 ± 0.18

0.35 ± 0.37 0.38 ± 0.18

1.26 ± 0.20 1.58 ± 0.34

1.24 ± 0.21 1.08 ± 0.23

0.70 ± 0.35 1.02 ± 0.36

+ 34

-2.5

+ 7.5

+ 25

+ 7.2

+ 25

- 13

+ 46

< 0.0005

> 0.05

> 0.05

< 0.05

> 0.05

> 0.05

> 0.05

> 0.05

a

 Values are given as the ratios of the percentage of total

radioactivity recovered from tlc for each isotope ± S.D.

b

 As in table 1.

For a variety of reasons, we have primarily utilized the WI-38

human lung fibroblast cell line for our studies. This cell line

is the most THC-sensitive of those we have tested, which included

mouse neuroblastoma cells, mouse glioma cells, human skin

fibroblasts, and HeLa cells. Moreover, it produces predominantly

PGE2 in response to a variety of agonists, including the

cannabinoids. We have also demonstrated that mouse peritoneal

macrophages are a suitable cell type for studying cannabinoid

effects on arachidonate metabolism by lipoxygenases (Burstein et

al. 1984a).

Dose-response relationships were reported for both primary cannabinoids and several metabolites of -THC in which we measured the

release of free arachidonic acid and the levels of PGE2 in WI-38

cells (Burstein et al. 1983). Responses were obtained starting at

around 0.8 μM and a good correlation was found between PGE2

synthesis and arachidonate release, suggesting that, as expected,

the two events are related. Of particular interest was the

finding that, within the THC series, a good SAR could be seen when

PGE2 levels were compared with literature reports on the

163
subjective "high" in humans and in the monkey behavioral assay. A

similar SAR was demonstrated with mouse peritoneal cells showing

that this was not a special feature of WI-38 fibroblasts (Burstein

et al. 1984a).

A wide range of agents were shown to be effective in blocking the

stimulatory actions of THC in cell culture, including phospholipase inhibitors (such as mepacrine and dexamethasone) and

cyclooxygenase inhibitors (such as aspirin, indomethacin, and

naproxen). Lysolecithin acyltransferase inhibitors, such as

p-hydroxymercuribenzoate, were shown to greatly augment the

stimulatory effects of THC, presumably by preventing released

arachidonate from being incorporated into phospholipids that would

render it unavailable for conversion to eicosanoids (Hunter et al.

1984).

Tolerance to the stimulatory action of THC could also be readily

observed in our model system (Burstein et al. 1985). Three

successive 30-minute exposures with 24-hour intervals in THC-free

media reduced the response to control levels. The effect could

also be seen when arachidonate release was monitored, and it was

reversed by allowing a 5-day recovery period.

We have recently examined the stereospecificity of these effects

again using WI-38 cells as the model Burstein et al. 1986a).

Three pairs of enantiomers ( -THC, -THC, and -dimethylheptylTHC) were studied and all three showed statistically significant

twofold to threefold greater potency for the (-) natural isomer in

stimulating PGE2 synthesis. Interestingly, we did not observe a

significant effect when arachidonate release was measured. A

possible explanation is that PGE2 is derived from a single

stereoselective lipid pool; the free arachidonate we measured was

released from several pools, some of which were not

stereoselective.

As an offshoot of our research, we tested the possibility of using

the lipid mobilizing properties of the cannabinoids to provide

symptomatic relief for certain of the lipid storage diseases, such

as Niemann-Pick. Cells derived from Niemann-Pick patients when

treated with cannabinoids showed a favorable decrease in

sphingomyelin content (Burstein et al. 1984b). Cannabidiol (which

is nonpsychoactive) was particularly effective, thus raising the

possibility of its use in managing patients with such problems.

Brain Subcellular Experiments

Synaptosomes, myelin, and mitochondria were prepared from whole

mouse brain, and cannabinoid effects on their lipases were studied

(Hunter et al. 1986). PLA2 activity was increased in the presence

of THC and its metabolites and the effect exhibited structural

specificity (table 4). Synaptosomal PLC-mediated hydrolysis of

phosphatidyl inositol (PI) was also stimulated by THC (table 5),

giving rise to increased levels of DAG. This is of interest since

DAG has a potent effect on C-kinase activity which has been linked

to neurotransmitter action. In addition, THC inhibited DAG lipase

activity, which could amplify the PLC effect.

164
Metabolite Studies

As part of our long-standing general interest in the metabolites

of THC, we tested several of the available examples for interactions with THC in our model system. The simplicity of the model

allowed us to examine a number of metabolites over a range of

doses wi th relative ease (table 6). It became immediately

apparent that the carboxyl-containing metabolites, in particular,

-THC-7-oic acid, were antagonists to the stimulatory action of

THC on PGE2 synthesis (Burstein et al. 1986a).

A kinetic analysis of the inhibition suggested that it was

noncompetitive. The structure of -THC-7-oic-acid is similar to

many of the nonsteroidal antiinflammatory agents, prompting us to

test it for cyclooxygenase inhibition. The metabolite showed

potent inhibitory action in several different preparations with

cyclooxygenase activity. Moreover, it had no effect on the

release of arachidonate from labelled WI-38 cells, showing that it

has a specific action in antagonizing the effect of THC.

An obvious question was whether this antagonism would manifest

itself in vivo. The cataleptic response to THC in mice has been

suggested to involve prostaglandins as mediators. We established

the procedure as described by Pertwee (1972) in our laboratory and

treated mice with the 7-oic acid (40 mg/kg) orally followed by

-THC (50 mg/kg) orally (Burstein et al., unpublished data). The

cataleptic effect was inhibited by at least 50% by this procedure,

showing that the metabolite can greatly affect this particular CNS

action of THC (table 7).

SUMMARY

Experimental support for a role for prostaglandins in the actions

of THC has been greatly expanded by data from our laboratory

during the last 5 years. The majority of the data has been

obtained using a cell culture model. However, important findings

have also come from in vivo experiments as well as subcellular

preparation studies. We have shown that aspirin can inhibit the

hypotensive action of THC in dogs, which is consistent with a

mechanism in which THC stimulates prostaglandin synthesis in the

cardiovascular system. In vivo treatment of mice with THC was

followed by a significant in the level of PGE2 in whole brain

extracts, suggesting a role for this eicosanoid in the CNS actions

of THC.

We have also shown that our cell culture model displays several of

the characteristics of THC action in vivo. These include

tolerance, stereospecificity, and an SAR which resembles that for

THC and its metabolites in humans and in the rhesus monkey. These

findings would seem to justify the use of this model for further

experiments aimed at elucidating the biochemical events connected

with THC action.

Lipid metabolism in brain preparations such as synaptosomes have

been found by us to be responsive to THC, which generates such

potent mediators as arachidonate and DAG by stimulating the

165
TABLE 4

Effects of Cannabihoids on Arachidonic Acid Formation From

[1-14C] Arachidonylphosphatidylcholine by Synaptosomes

Cannabinoid μM Arachidonic Acid Change (%)

(dpm ± SD)

Vehicle - 302 ± 14 -

-THC 0.32 379 ± 40 25.4

-THC 0.8 370 ± 20

-THC

22.4

1.6 353 ± 15

-THC

16.7

3.2 368 ± 43

-THC

21.9

8.0 423 ± 35 40.1

-THC 16.0 397 ± 38 31.3

7-oxo- -THC 1.6 357 ±

7-oxo- -THC

32 18.3

3.2 303 ± 16

7-oxo- -THC

0.3a

8.0

16.0

371 ± 11

7-oxo- -THC

22.8

351 ± 20 16.1

7-OH- -THC

7-OH- -THC

8.0 464 ± 16 53.8

16.0 503 a 26 66.5

-THC-7-oic acid 8.0 279 ± 15 -7.8

-THC-7-oic acid 16.0 364 ± 24 20.4

6 -OH- -THC ± 15

6 -OH- -THC

8.0 284 -5.9

16.0 360 ± 31 19.0

6 -OH- -THC 8.0 298 ± 15 -1.3a

6 -OH- -THC 16.0 308 ± 17 2. 1a

Synaptosomes (0.22 mg protein) were incubated with [1-14C]arachidonylphosphatidylcholine (35,000 dpm) in 1 ml of 5 mM-CaCl2:0.05 M

Tris-HCl buffer (pH 7.4) for 45 min. Cannabinoids were added in

10 μ1 ethanol. Arachidonic acid levels were obtained by hexane

extraction of the incubate. Values represent mean (dpm) ± SD of

triplicate samples. All values were significantly different from

the vehicle, p < 0.0005 by Student's t test. a

 Not significant.

166
TABLE 5

Synaptosomal Hydrolysis of [1- 14C]Arachidonylphosphatidylinositol

Cannabinoid μM Product Formation

Diacylglyceride (%) Arachidonic acid (%)

Control 10.9 ± 9 (4) 2.5 ± 0.4 (4)

-THC 1.6 25.2 ± 2.3 (3)a2.5 ± 0.6 (3)d

-THC 8 30.4 ± 1.8 (3)b3.2 ± 0.8 (3)d

7-COOH- THC 8 18.2 ± 2.4 (3)c2.2 ± 0.1 (3)c

Synaptosomes (0.1 mg protein per ml) were suspended in 5 mM CaCl2:

S.T.E.M. buffer (pH 7.4) and incubated for 30 min with

[1-14C]arachidonylphosphatidylinositol (25,000 dpm) and either

cannabinoid or vehicle (10 μl). The values are the percentage

mean (dpm) ± SD of the number of determinations given in

a

arentheses. Values were significantly different from control:

p < 0.005; b

 p < 0.0005; c p < 0.01; d Not significant.

appropriate lipases. This showed that the brain responds in a

manner analogous to other tissues with regard to lipid

mobilization.

A surprising and potentially important discovery was made using

the cell culture model we have developed. The major metabolite of

-THC, -THC-7-oic acid, was found to antagonize the stimulatory

action of THC by inhibiting a specific step in the synthesis of

prostaglandins. We were also able to demonstrate antagonism by

this metabolite in vivo using THC-induced catalepsy in the mouse

as the biological parameter. We propose, based on these

observations, that tissue levels of this metabolite will have a

profound influence on THC responses. Future pharmacokinetic

studies should consider this possibility since the acid has been

shown to be present in amounts comparable and even exceeding those

of THC itself. In passing, it is worth noting that THC exhibits

potent antiinflammatory activity in animal models (Sophia et al.

1973a, 1973b) which can now be readily explained by a metabolite

that inhibits prostaglandin synthesis. Finally, all of these

facts are most consistent with a mechanism of action in which THC

stimulates the production of prostaglandins and other

eicosanoids.

167
TABLE 6

Interactions of -THC with Its Metabolites on PGE2

Synthesis in Fibroblasts*

Metabolite

6 -OH- -THC (3.2 μM)

6 -OH- -THC (8.0 μM)

1"-OH- -THC (3.2 μM)

1"-OH- -THC (8.0 μM)

1"-oic Acid (16 μM)

2"-oic Acid (16 μM)

3"-oic Acid (16 μM)

7-oic Acid (8 μM)

7-oic Acid (16 μM)

-THC Concentration (μM)

0.32 0.80 1.6 3.2

0.93 0.93 1.76 0.99

1.15 0.93 1.26 0.89

1.31 1.44 0.93 1.07

2.16 1.89 1.46 1.45

0.96 0.93 1.43 1.56

0.83 0.49 0.51 0.74

1.43 1.14 1.20 0.87

- 0.28 0.28 0.27

- < 0.15 0.13 0.11

*

 The metabolite was added to the cells 30 min prior to the

addition of -THC at the indicated concentrations. The values

shown are the ratios of PGE2 levels in treated over control cells;

the controls consisted of cells to which vehicle (10 μl ethanol)

had been added in place of a metabolite. PGE2 was measured by

RIA. Duplicate determinations were done on three individual

monolayers.

168
TABLE 7

The Inhibition of -THC Induced Catalepsy in Mice

by -THC-7-oic Acid

Control Treated Pretreated

Experiment 1 0.05 ± 0.05 (4) 0.28 ± 0.02 (4) 0.11 ± 0.04 (5)

Experiment 2 0.09 ± 0.04 (3) 0.26 ± 0.03 (3) 0.19 ± 0.10 (5)

Experiment 3 0.08 ± 0.03 (4) 0.42 ± 0.01 (4) 0.19 ± 0.10 (4)

Mean 0.07 ± 0.02 0.32 ± O.O1*0.16 ± 0.05*

Female, CD-1 mice (Charles River) weighing 20 to 25 grams were

given -THC (50 mg/kg) in sesame oil (10 μl) orally. One hour

later the cataleptic effect was measured using the procedure

described by Pertwee (1972). The "control" groups received only

sesame oil and the "pretreated" groups were given -THC-7-oic

acid (40 mg/kg) in oil (50 μl) 1 hour prior to the administration

of -THC. The values are expressed as the means of the fraction

of time the mice remained immobile ± S.D. Values in parentheses

indicate the number of mice in each group. Asterisks denote a p

value of < 0.01 as determined bv a one-wav analvsis of variance

(F = 26.4, DF = 2,33) and a post hoc Newman-Keuls test.

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171
ACKNOWLEDGMENTS

The research from the author's laboratory was supported by grants

DA-02052 and DA-02043 from the National Institute on Drug Abuse.

The author is a recipient of a NIDA Research Scientist Award.

AUTHORS

Sumner Burstein, Ph.D.

Department of Biochemistry

University of Massachusetts Medical School

55 Lake Avenue, North

Worcester, Massachusetts 01605

172
Pharmacokinetics and Disposition

of Cannabinoids

C. Nora Chiang, Ph.D., and Rao S. Rapaka, Ph.D.

INTRODUCTION

Cannabis was used for fiber or medicinal purposes for centuries

(Camp 1936). Recently, it has been widely used for recreational

purposes, due largely to its euphoric effect. Cannabis contains

hundreds of compounds. Among them, a class of compounds unique to

cannabis has been identified as cannabinoids, which have been

associated with cannabis activities. So far, over 60 naturally

occurring cannabinoids have been identified in cannabis (Turner et

al. 1980). In addition, a large number of cannablnoids have been

synthesized for structure-activity studies and for the development

of therapeutic drugs. Due both to concerns about health hazards

related to the social use of marljuana and to the potential use of

cannabinoids for therapeutic purposes, extensive research on their

chemistry, pharmacokinetics and disposition, pharmacology, cellular

effects, and therapeutic effects has been carried out and recently

reviewed (Razdan 1986; Agurell et al. 1986; Dewey 1986; Martin 1986;

Hollister 1986).

As information on the pharmacokinetics and disposition of

cannabinoids is important for understanding their use and abuse,

this chapter summarizes current understanding of the pharmacokinetics and disposition of these substances. The cannabinoids to

be discussed include delta-9-tetrahydrocannablnol (THC), the major

psychoactive component In cannabis; cannabinol (CBN) and cannabidiol

(CBD), which are usually abundant in plants that are rich in THC;

and nabilone, a synthetic cannabinoid approved for therapeutic use.

The major focus will be on THC, as it is the most widely used

cannabinoid and has been the subject of extensive research. In

addition to pharmacokinetics, regulatory requirements for the

approval of drugs by the United States Food and Drug Administration

(FDA) will also be reviewed briefly.

173
PHARMACOKINETICS AND DISPOSITION

TETRAHYDROCANNABINOL

Delta-9-tetrahydrocannabinol, the major psychoactive component of

marijuana, has been shown to have a broad range of cellular,

biological, and behavioral effects (Martin 1986; Dewey 1986). It

was recently approved for therapeutic use in the treatment of nausea

and emesis caused by cancer chemotherapy (Hollister 1986). The

health hazards from the social use of marijuana are a major

concern. In particular, marijuana use has been associated with

decrements in behavior and cognitive performance. For example, safe

operation of motor vehicles or machinery at the workplace when a

person is under the influence of THC is a major concern. There is

an urgent need for the interpretation of a certain concentration of

THC in urine or blood as related to the pharmacological effects,

such as performance impairment. So far, data from controlled

clinical studies indicate that there is a correlation for both the

"high" effect and the performance impairment with plasma THC

concentrations (Chiang and Barnett 1984; Barnett et al. 1985) when a

pharmacokinetlc and a pharmacodynamic analysis are performed on a

series of plasma samples. However, the prediction of the extent of

high effect or impairment from a single plasma level of THC cannot

be established (Hollister et al. 1981) because of the complex nature

of the pharmacokinetics and pharmacodynamics of THC (Agurell et al.

1986; Mason and McBay 1985). Further research is still needed in

these areas.

Pharmacokinetic Parameters

Half-Life. Following an intravenous dose of THC, plasma levels of

THC decline very rapidly over the first few minutes and then decline

at slower rates. A four-exponential equation with a fourcompartment model, proposed by Hunt and Jones (1980), indicates that

there are at least four different composites of tissues into which

THC is distributed with different rates of permeation and different

tissue binding strengths. The averages of the associated

disposition half-lives are 1 minute, 4 minutes, 1 hour, and 19

hours. The half-lives of 1 minute, 4 minutes, and 1 hour are

attributable to the rapid distribution of THC into different tissues

as well as to the rapid metabolism of THC. At approximately 6 hours

after an intravenous dose, a psuedoequilibrium is reached between

plasma and tissues.

After the pseudoequilibrium is reached, THC is then slowly

eliminated. This slow elimination is primarily due to the slow

return of THC from sequestered tissues to blood. The terminal

half-life of THC is approximately 1 day. Hunt and Jones (1980)

suggested a terminal half-life of 19 hours: Ohlsson et al. (1982),

of greater than 20 hours; and Wall et al. (1983), of 25 to 36

hours. There are no significant differences in plasma profiles for

chronic and infrequent users or in half-lives in moderate users

before and after a 2-week exposure of THC (Ohlsson et al. 1982; Hunt

and Jones 1980). The difference between the earlier reported

174
terminal half-life of 28 hours for chronic marijuana users and 57

hours for naive smokers, in studies of Lemberger et al. (1970,

1971a), could be due to relatively nonspecific analytical methods

used for their pioneering studies.

Volume of distribution. The volume of distribution of THC is

estimated to be 10 1/kg (Hunt and Jones 1980: Wall et al. 1983).

which is over 200 time; the plasma volume. This large volume of

distribution is commonly seen for lipophilic drugs, where the

majority of the drug in the body is in tissues and only a small

fraction is in the blood. In blood, THC is preferentially

distributed in plasma and only about 10% in the red blood cells

(Garrett and Hunt 1974; Widman et al. 1974).

Clearance. The average plasma clearance is about 605-980 ml/min

(Hunt and Jones 1980; Ohlsson et al. 1982) and estimated blood

clearance of 1.0 to 1.6 l/min is close to the hepatic blood flow.

This suggests that the rate of metabolism of THC is clearly

dependent on hepatic blood flow to the liver, and enzyme

induction/inhibition appears to have little effect on the metabolic

clearance of THC (Hunt and Jones 1980). Because of the high

metabolic clearance, part of the drug absorbed from the

gastrointestinal tract is metabolized in the liver before it enters

the systemic circulation. As a result, the bioavailability of THC

is low and is in the range of 10% to 20% (Wall et al. 1983).

Distribution

In blood, THC is extensively bound, approximately 97% to 99%, to

plasma proteins and only limited amounts of THC (about 10%) are

present in red blood cells (Garrett and Hunt 1974; Widman et al.

1974).

Because THC is a highly lipophilic compound, it is readily

distributed into tissues from blood circulation. THC concentrations

in brain reach a maximum, about three to six times higher than in

plasma, within 30 minutes (the first sampling time after drug

administration) after a single intravenous dose of THC in mice

(Harvey et al. 1984). THC brain concentrations roughly parallel the

plasma concentrations. This suggests that there is no barrier for

the partition of THC into brain and that an equilibrium between the

brain and plasma is quickly reached.

Earlier studies in animals using labeled THC demonstrated that

tissue levels of THC (in most cases, of THC and metabolites) were

higher in fat, liver, heart, and lung than in brain and blood

(Ryrfeldt et al. 1973; Kreuz and Axelrod 1973; Leighty 1973; Siemens

et al. 1979). A recent study in rabbits, using a more specific

GC-MS method for the assay of unchanged THC, also suggests that much

higher concentrations of THC are found in fat and heart than in

brain (Leuschner et al. 1986). THC concentrations in fat are about

1,000 times those in plasma, while in brain they are only about 3 to

10 times those in plasma. This is consistent with the fact that THC

is a very lipophilic compound and, therefore, accumulates in fatty

tissues. The relatively low THC levels in brain, in contrast to

175
what would be expected from the partition coefficient, are primarily

due to the fact that the brain is well perfused and the blood flow

moves THC in and out of the brain very rapidly.

Animal studies indicate that THC passes through the placenta and

affects fetal development (Lee and Chiang 1985). THC levels in

fetal tissues and blood are shown to be lower than in maternal

tissues for dogs and sheep (Martin et al. 1977; Abrams et al.

1985). In humans, THC is also found in cord blood, and the cord

blood levels are three to six times lower than in maternal blood

(Blackard and Tennes 1984). However, its effects on the fetus are

not yet fully understood. THC is also secreted in milk at a higher

concentration than in plasma, which may be attributed to the high

lipid content in milk (Perez-Reyes 1982).

Metabolism

Metabolism is the major route for the elimination of THC from the

body; only negligible amounts of this substance are excreted as

unchanged THC. So far, approximately 80 metabolites of THC have

been reported from studies in humans, animals, and in vitro liver

preparations (Agurell et al. 1986). In humans, over 20 metabolites

have been identified in urine and feces (Halldin et al. 1982a,

1982b; Widman et al. 1985; Wall and Perez-Reyes 1981). Still, a

significant number of the metabolites, in both urine and feces, have

not been identified.

The metabolism of THC in humans involves allylic oxidation,

epoxldatlon, aliphatlc oxidation, decarboxylation, and conjugation

(Burstein 1985; Harvey 1984; Wldman et al. 1985). Allylic oxidation

at C-8 and C-11 and aliphatic oxidation at the side chain result in

the formation of hydroxylated metabolites. The mono- and di-hydroxy

metabolites are further oxidized to form acid and hydroxy-acid.

Allylic oxidation at C-11 position is the major metabolic pathway,

and the resultant metabolite 11-OH-THC is identified as a

predominant metabolite from in vitro studies using human liver

preparations. This metabolite is also found in significant amounts

in feces (Halldin et al. 1982c; Wall and Perez-reyes 1981).

11-Nor-THC-9-COOH, produced from the oxidation of 11-OH-THC, is a

major metabolite identified in plasma, urine, and feces (Widman et

al. 1985; Wall and Perez-Reyes 1981; Wall et al. 1983). The side

chain is also easily degraded to form decarboxylated metabolites,

and 4',5'-bisnor-THC-11,3'-dioic acid is the second most abundant

metabolite found in urine.

Conjugation with glucuronide acid may occur to THC and to

hydroxylated and carboxylated metabolites. Glucuronic acid

conjugates of 11-nor-THC-9-COOH and THC have been identified in

human urine (Halldin et al. 1982a, 1982b; Widman et al. 1985; Wall

and Perez-Reyes 1981). Epoxide of THC (11-OH-epoxyhexahydrocannabinol) is found in trace amounts in the liver preparation

(Halldin et al. 1982c).

176
Pharmacological activities of metabolites have been studied in

humans. The major metabolite 11-nor-THC-9-COOH is found to be

inactive, while some hydroxylated metabolites are found to be

pharmacologically active (Perez-Reyes et al. 1973; Perez-Reyes

1985). 11-OH-THC, another major metabolite, produces

pharmacological activity similar to THC and follows similar

disposition (Lemberger et al. 1972; 1973; Perez-Reyes et al. 1972;

Perez-Reyes 1985). 8-Beta-OH-THC is less potent and 8-alpha-OH-THC

is essentially inactive in producing THC-like activity (Perez-Reyes

1985).

The activity of side-chain hydroxylated metabolltes has not been

studied well in humans. Pharmacological tests in dogs and mice

indicate that 3'-OH-THC has a pharmacological profile similar to

that of THC and is about 2 to 3 times more potent than THC, while

the dlhydroxy metabolite 3',11-dihydroxy-THC is less potent than THC

(Martin et al. 1984).

Excretion

About 80% to 90% of the dose of THC is excreted as metabolites

within 5 days, 65% in feces and 20% in urine. Only negligible

amounts of THC were recovered as unchanged drug (Hunt and Jones

1980; Wall et al. 1983). Since a significant portion of the

metabolites is excreted in the feces, enterohepatic recirculation

(which may also be a contributing factor for the slow elimination of

a drug) of THC metabolites in humans is also possible. Studies in

dogs indicate that 10% to 15% of the dose of THC is

enterohepatically recirculated (Garrett and Hunt 1977).

Among the metabolltes, 11-nor-9-THC-COOH is a major metabolite

identified in both urine and feces (Wall and Perez-Reyes 1981;

Widman et al. 1985). Metabolites in the urine consist of primary

acidic compounds; while the metabolites in feces consist of both

acidic and neutral compounds. In urine, a total of 20 metabolites

of THC, 2 as glucuronic acid conjugates and 18 as nonconjugated

acids, have been identified. All of the other nonconjugated acidic

metabolltes identified in urine, except for 11-nor-9-THC-COOH, have

their side chains further oxidized or degraded to form various

hydroxylated or carboxylated metabolites (Widman et al. 1985). In

contrast to urine, neutral metabolites appear in significant amounts

in feces: the amount of 11-OH-THC is about the same as that of

11-nor-THC-9-COOH; and 8,11-dihydroxy, 8-alpha-OH, and 8-beta-OH

metabolites are also found in significant amounts. However,

one-third of the metabolites in feces are less well-defined acids

(Wall and Perez-Reyes 1981). Glucuronic acid conjugates of THC and

11-nor-THC-9-COOH are found in urine (Widman et al. 1985). In

general, the glucuronic acid conjugates are labile in the basic

conditions and, hence, it is likely that most of the acidic

metabolites identified could also be partly present in the original

urine or feces as glucuronic acid conjugates which are later readily

hydrolyzed during the experimental procedures.

177
Absorption

THC from various routes of administration is absorbed quite

differently. Smoking marijuana cigarettes is the most common route

for the self-administration of THC in the United States. In cancer

treatment, THC capsules are ingested orally for the antiemetic

effect. Parenteral injectables of THC are used primarily in

research. As THC is practically insoluble in water (Garrett and

Hunt 1974), organic vehicles or surfactants are generally employed

to make parenteral solutions of THC. Commonly used vehicles for the

intravenous administration of THC in humans are alcohol and albumin

solution. For use in animals, many other vehicles--such as

propyleneglycol, polyvinylpyrrolidone, Tween-80, and Emulohor--have

been used for the parenteral preparations. The bioavail

pharmacokinetics of THC from the different preparations

variable. As a result, the time profiles and the magni

pharmacological effects are also different.

t

ability and

are quite

ude of

s lost by

oking study

Smokinq. During the smoking process, part of the dose i

pyrolysis and from side stream smoke. In an in vitro s

(Davis et al. 1984) when cigarettes were consumed as a single puff

to minimize side stream smoke, about 30% of THC was degraded during

smoking. This suggests that the maximum amounts of THC absorbed

from smoking will not exceed 70% of the THC content of cigarettes.

When puff volume and puff duration of a typical marijuana smoker

were simulated, about 16% to 19% of the THC in the cigarettes was

recovered in the smoke condensate (Davis et al. 1984).

THC is readily absorbed from the lungs or respiratory tract into the

systemic circulation. Peak plasma concentrations are rapidly

reached, and the plasma profile is very similar to that of an

intravenous dose (Ohlsson et al. 1982; 1985; Chiang and Barnett

1984). The bioavailability of THC from this route of administration

is quite variable (Barnett et al. 1982; Lindgren et al. 1981;

Ohlsson, et al. 1980; 1982; 1985; Perez-Reyes et al. 1982), as it is

affected not only by the burning characteristics of the cigarettes,

but also by the depth of inhalation, puff duration, and other

factors. Experienced smokers seem to inhale marijuana more

efficiently than nonexperienced ones. The THC bioavailabllity from

actual smoking of a 1% to 2% marijuana cigarette is 16 to 40% for

heavy users and 13% to 14% for light users (Ohlsson et al. 1982).

When cigarettes of different potencies are used, smokers may tend to

titrate the amount of THC inhaled. In one study, subjects were

instructed to smoke cigarettes containing 1.3%, 2.0%, and 2.5% of

THC in their usual fashion (Perez-Reyes et al. 1982). Plasma THC

levels increased with THC potencies, indicating that higher amounts

of THC were absorbed from high potency cigarettes. However, the

actual fractions of THC inhaled were relatively less for high

potency cigarettes. This self-adjustment of THC intake became more

apparent in a study using marijuana cigarettes of 1% and 3.8% THC.

The actual amount of THC inhaled from a 3.8% cigarette was about 1.8

times of that from a 1% cigarette. That indicates that the

fraction of THC inhaled from a 3.8% cigarette was only about half of

that estimated for a 1% cigarette (Perez-Reyes 1985).

178
Oral Administration. The THC oral dosage form that is currently

available in the market is a soft gelatin capsule containing THC in

sesame oil. The absorption of this dosage form is slow, the peak

THC levels are reached at about 2 hours, and the bioavailabillty is

in the range of 10% to 20% (Wall et al. 1983). This low

bioavailabillty of THC is primarily due to the "first-pass effect."

In other words, a large fraction of THC is metabolized to

metabolites in the liver before it reaches the systemic circulation,

thus resulting in a small fraction of the administered dose of THC

getting into the systemic blood. Consequently, the active

metabolites appear in significant amounts in the systemic blood and

therefore may contribute significantly to the effects of THC (Wall

et al. 1983; Hunt and Jones 1980).

Parenteral administration. After an intravenous dose, THC is

distributed instantly in the systemic circulation. Since THC is

highly bound to plasma protein, a vehicle made from plasma protein

(and premixed with THC) may compete with tissue uptake of THC. A

study by Fehr and Kalant (1974) suggested that tissue distribution

could be partly retarded when THC is administered in a vehicle made

from plasma protein as compared with a vehicle made from

polyethylene glycol.

Generally, intramuscular injectables of THC are used in animal

studies. These injetables are commonly made in Tween-80 and in

Emulphor. Studies in monkeys, using radioimmunoassay methods,

suggest that the systemic absorption of THC from the Emulphor

preparation is greater than from Tween-80 preparation (Perlin et al.

1985). Although peak levels attained for these two preparations are

similar, times required to reach peak levels are different: 30

minutes for Tween 80 and about 2.5 hours for Emulphor.

Other routes of administration. THC is absorbed from the ophthalmic

route of administration. Studies in rabbits indicate the absorption

of THC in light mineral oil is slow, and peak levels are reached in

about 1 hour and maintained for approximately 4 to 8 hours. The

bioavailablllty is quite variable, ranging from 6% to 40%. (Chiang

et al. 1983).

Rectal route of administration of THC has been evaluated for several

suppository formulations using Carbowax, Witepsol, sesame oil, cocoa

butter, and Cetomacrogol in monkeys (Perlin et al. 1985). No drug

is detected in plasma, possibly due to the high lipophiliclty of

THC, which might prevent the partitioning of THC out of the vehicles.

Kinetics of the THC Metabolites

THC is rapidly and extensively metabolized. After in intravenous

dose of THC. metabolites appear immediately in plasma, and peak

levels are reached in about 15 to 30 minutes (Hunt and Jones 1980;

Wall et al. 1983). The concentrations of metabolites reach levels

equal to that of THC in about 20 minutes and, at 1 hour, the

metabollte levels increase to about 5 to 10 times that of THC. A

major portion of metabolites in plasma are polar and acidic

179
compounds; neutral metabolites (mostly hydroxy metabolites) account

only for about 10% of total metabolites. A significant fraction of

metabolites in plasma has yet to be identified.

Consistent with the first pass-effect, ratios of concentrations of

metabolites to THC in plasma for an oral dose are much higher than

those for an intravenous dose (Wall et al. 1983). For example, the

concentration levels of 11-nor-THC-9-COOH (a major metabolite that

is devoid of THC activity) in plasma are about 2 to 3 times higher

than that of THC after an intravenous dose and about 6 to 10 times

after an oral dose. Plasma levels of the active metabolite

11-OH-THC are about 10% to 15% of THC levels for an intravenous dose

and about 50% of THC levels for an oral dose. Also found in plasma

are 8-alpha-OH, 8-beta-OH, and 8,11-di-OH metabolites. The levels

of active metabolites 11-OH-THC and 8-beta-OH-THC from an

intravenous dose or smoked dose may not contribute significantly to

THC effects, but they could contribute significantly to THC effects

from an oral dose.

Metabolites are also highly bound to plasma protein (88% to 99%).

The terminal plasma half-life of metabolites from THC administration

is approximately 50 hours, which is longer than that of THC. This

slower elimination rate of THC metabolites is not due to the slower

elimination rate of the metabolites, but rather to their slower

rates of formation, which, in turn, depends on the slower return of

THC from sequestered tissues to form metabolites (Hunt and Jones

1980).

NABILONE

Nabilone, a synthetic cannabinoid, is currently used as an

antiemetic (Herman et al. 1979; Hollister 1986). Following an

intravenous dose of nabilone, plasma levels sharply decline due to

extensive tissue distribution and metabolism. The plasma half-life

is reported to be about 2 hours. Metabolites have not been well

identified for nabilone; however, it is suggested that the long

duration of its action may be due to the active metabolites.

Similarly to THC, 65% of dose of nabilone is excreted in feces and

about 20% in urine within 4 days. Urinary metabolites are primarily

polar, acidic metabolites. The half-life of total metabolites in

plasma is about 35 hours. The oral absorption of nabilone

preparation made from coprecipitation with polyvinylpyrrolidone is

complete and the peak plasma levels are reached in 1 hour (Rubin et

al. 1977).

CANNABIDIOL

Cannabidiol, one of the major cannabinoids found in cannabis, is

shown to be devoid of any psychotomimetic activity in humans

(Perez-Reyes et al. 1973; Perez-Reyes 1985), but it possesses

significant anticonvulsant activity (Martin 1986). Plasma profiles

of CBD following an intravenous dose are quite similar to those of

THC. Plasma levels decline rapidly shortly after intravenous dose

and decline at slower rates later. Similar to THC, plasma

clearances of CBD are 960 to 1560 ml/min and terminal plasma

180
half-life averages 24 hours (range 18 to 33 hours). Volume of

distribution for CBD is estimated to be 30 l/kg (Ohlsson et al.

1986).

CBD, like THC, is extensively metabolized (Burstein 1985).

Hydroxylation is a major metabolic pathway and occurs primarily at

11 position. The 11-OH metabolite is further oxidized to form

dlhydroxy and carboxy metabolites. 9-carboxy-CBD is a major

metabollte identified in plasma. Similar to THC, the majority of

the administered dose of CBD is excreted in feces and a relatively

small fraction is excreted in urine (about 16% of the dose in urine

and 33% in feces over 72 hours). Unlike THC, unchanged CBD is

excreted in large portion in the feces (Wall et al. 1976).

After smoking, CBD is quickly absorbed into the systemic

circulation. The bloavailabilities of CBD averaged 31% (range, 11%

to 45%) (Ohlsson et al. 1986). Coadministration of CBD with THC may

alter THC's effects (Hollister and Gillespie 1975; Karniol et al.

1974); it has minimal or no effects on plasma levels of THC,

however, suggesting that there are no apparent pharmacokinetic

interactions of CBD with THC (Agurell et al. 1981; Hunt et al.

1981).

CANNABINOL

Cannabinol is a major cannabinoid found in cannabis and has about

one-tenth the activity of THC in humans (Perez-Reyes et al. 1973;

Perez-Reyes 1985). Similar to THC, plasma levels of CBN following

an intravenous dose decline rapidly shortly after the dose and then

decline at slower rates (Ohlsson et al. 1984). CBN is also

extensively metabolized (Burstein 1985). 9-COOH-CBN is a major

ity of the dose of CBN is

fraction is found in

ine and 35% in feces over

for CBN are mono-OH-CBN,

).

metabolite identified in plasma. The major

excreted in feces, while a relatively small

urine: About 8% of the dose is found in ur

72 hours. Major metabolites found in feces

9-COOH-CBN, and dl-OH-CBN (Wall et al. 1976

CBN is also quickly absorbed from smoking.

CBN average 41% (range, 8% to 77%) (Ohlsson

The bioavailabilities of

et al. 1984, 1985). It

is likely that a very limited pharmacokinetic interaction exits

between CBN and THC, because the coadmlnistration of CBN with THC

results in plasma concentrations of THC slightly higher than, but

not statistically significant from, THC concentrations obtained from

THC administered alone (Agurell et al. 1981).

MEDICINAL ASPECTS OF CANNABIS

Several ancient civilizations used cannabis and related products for

the "treatment" of a number of disease conditions, and the

therapeutic claims were very extensive. As systematic

investigation, isolation, and identification of the active

ingredients proceeded and these ingredients were pharmacologically

investigated, most of the earlier claims were not substantiated.

181
During the early sixties, Goni and Mechoulam (1964) identified

delta-9-THC as the major active component of cannabis. Following

their research, several other active ingredients were identified.

Later, a number of analogs of delta-9-THC, cannabidiol and several

closely related compounds, were synthesized and evaluated for

therapy. Some examples of these synthetic compounds are nabilone,

nabitan, levonantradol, and BRL 4664. Nabilone is currently

available in Canada and Switzerland for the treatment of emetic

effects of cisplatin in cancer chemotherapy. In the United States,

the only two newly approved drugs to date for treatment of

drug-induced emesis are delta-9-THC and nabilone.

Although pharmacological evaluations of several hundred analogs for

various therapeutic purposes are not highly encouraging,

considerable research is being carried out. Some of the therapeutic

aspects that are considered for cannabinoids are:

Antiemetic effects in cancer chemotherapy

Reduction of intraocular pressure in glaucoma

Anticonvulsant effects; and

Miscellaneous minor uses--as an antiasthmatic, analgetic, and

appetite stimulant, and for the treatment of insommia, hypertension,

and anxiety.

It is expected that in the near future at least some synthetic

analogs that are devoid of "cannabimimetic" (psychotropic) effects

may be approved in the United States for therapeutic purposes.

Hence, this is an opportune time to present the regulatory aspects

of drug approval by the Food and Drug Administration.

REGULATORY ASPECTS OF DRUG APPROVAL

In order to understand the extensive procedures involved for FDA

approval, a brief account of the FDA regulatory aspects will be

helpful. Federal regulatory legislation started with the passage of

the 1848 Import Drug Act. This was a result of finding that the

quinine used by American troops in Mexico was adulterated. In 1906,

the original Food and Drug Act was passed, which specified standards

for purity, strength, and quality. In 1938, the Federal Food, Drug

and Cosmetic Act was enacted. This legislation required clearance

for safety of new drugs prior to distribution. Later, following the

thalidomide tragedy in Europe in 1962, the Kefauver-Harris

amendments were passed. These amendments require that, in addition

to the safety of the drugs, their effectiveness must be

demonstrated. For final approval of a drug by FDA for commercial

distribution, the manufacturer must go through the IND and NDA

stages.

182
INVESTIGATIONAL NEW DRUG (IND)

In order for a researcher or a commercial firm to establish the

safety and efficacy of a compound, a "Notice of Claimed

Investigational Exemption for a New Drug" (an IND) must be filed.

The IND contains information on (1) all the components; (2) quantitative composition; (3) methods of preparation; (4) analytical

methodology to assure identity, strength, and quality; (5) results

of preclinical investigations; (6) a detailed clinical protocol of

phase I, II, and III studies; and (7) other related information.

After the sponsor or the applicant is confident from the animal and

human trials that the product is safe and effective, an NDA may be

filed.

NEW DRUG APPLICATIONS (NDA)

In 1985 the Food and Drug Administration published a comprehensive

revision of the Code of Federal Regulations relating to the

procedure for NDA submission and approval (Federal Register, Vol.

50, No. 36, p. 7542). The NDA contains information on each of the

following disciplines: chemistry, clinical medicine, pharmacology,

statistics, biopharmaceutics, and microbiology. The chemistry

section should include:

1. Drug Substance--Names, formulas, physical stability,

manufacturers, methods of manufacture, process controls,

specifications, and analytical methods.

2. Drug Product--components, composition, specifications and

analytical methods for components, manufacturers, methods of

manufacture, packaging procedures, and in-process controls,

specifications and analytical methods for drug product

container--closure system, and stability.

All the material supplied is reviewed by FDA, and only after

approval may the drug be sold in interstate commerce. For

additional information, see Hoiberg and Rapaka (1986).

CONCLUSION

At present, research on the medicinal chemistry of cannabinoids is

being extensively carried out. It is hoped that, in the not too

distant future, this research will lead to the introduction of

useful new therapeutic drugs to alleviate human suffering.

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AUTHORS

Dr. Nora Chiang, Ph.D.

Research Technology Branch

Division of Preclinical Research

National Institute on Drug Abuse

Room 10A-13, 5600 Fishers Lane

Rockville, MD 20857

Rao S. Rapaka, Ph.D.

Research Technology Branch

Division of Preclinical Research

National Institute on Drug Abuse

Room 10A-13, 5600 Fishers Lane

Rockville, MD 20857

188
Drug Abuse in India: Progress

in Research

B. N. Dhawan, M.D.

INTRODUCTION

Intensive studies on drug abuse in India have been undertaken during the last

two decades, but surveys on the use of cannabis and opium have been

undertaken for about 100 years. One of the earliest and most exhaustive studies

on drug dependence was conducted on the abuse of cannabis by the Indian Hemp

Commission (published in 1894). Chopra and Chopra (1935) undertook a detailed

survey of the opium habit in the country. In recent years, there have been

changing trends in usage of drugs, and age-old culturally circumscribed barriers

are being thrown aside.

It is proposed to include work done after 1970 only in the present review due to

constraints of space, etc. These studies can be broadly classified in three major

categories: 1) Epidemiological surveys; 2) Adverse effects of drug abuse; and

3) Experimental studies to investigate mechanisms involved in drug abuse,

particularly in the case of cannabis and methaqualone. Some other miscellaneous

problems have also been investigated.

EPIDEMIOLOGICAL STUDIES

The epidemiological surveys undertaken during this period differ widely in their

sample size, parts of the country covered, type of population, etc. The studies

can be classified in three major groups, depending upon the type of population

surveyed: 1) Rural population; 2) Urban population; and 3) Select categories, like

students, psychiatric patients, etc.

Chopra (1971) has analysed the factors motivating persons to drug abuse and has

identified certain "beneficial" factors which promote drug abuse. The abuse in

most cases starts with such objectives as facilitation of social interaction,

enhancement of sensual pleasures, mystical or religious experience, and quasimedical use (cannabis leaves are used to sharpen appetite, to relieve muscle and

joint pains, etc.). Modern adolescents often use cannabis as part of a purely

exploratory activity without any religious overtones.

Rural Population

These surveys have collected data on the variety of drugs being abused as well as

the type of community structures, etc. According to an ICMR report (Drug

189
Addiction - An Overview) published in 1977, 40% of the rural population

consumed tobacco. The major agents of abuse were alcohol (25.6%). opium

(18.9%), barbiturates (6.2%). and cannabis (2.2%). The rather excessive use of

barbiturates was surprising. The females had a much lower incidence than the

males with all the agents. Several reports of various sample sizes have been

published during the last few years. Their results have been summarised in table

1. In all of these surveys, the incidence has been much lower in the females. In

an earlier survey of rural adolescents of Punjab, Mohan et al. (1978a) had

interviewed 281 male subjects. The incidence of alcohol usage was 32.9% and

another 0.8% were using opium, cannabis, or other agents. The abuse incidence

was higher in illiterate youth. The overall percentage of abusers varied between

15% and 40%. Nandi et al. (1982) have brought out the difference in incidence

between restrictive (where society has a strong disapproval) and permissive

communities. Further, in the restrictive communities, the abuse was almost

totally confined to the psychopaths (antisocial behaviour, from police records) as

opposed to a more uniform distribution in the permissive societies. Mohan et al.

(1980) point out that more than 50% of alcohol abusers start drinking before the

age of 25 years.

TABLE 1

Incidence of Drug Abuse in Rural Communities of India

Investigator Year Sample

size

Incidence

of abuse

No. %

Lal and Singh

Sethi and Trivedi

Mohan et al.

Varma et al.

Nandi et al.

a. Restrictive

community

b. Nonrestrictive

community

1978 443 124 29.3

1979 2,010 427 21.2

1980 3,600 1,309 36.4

1980 1,031 409 39.7

1982 5,423 834 15.4

2,804 45 1.6

2,619 789 30.1

Urban Population

Less data are available about the incidence of drug abuse in the general urban

population, though specific subgroups, like students, have been more extensively

studied. Thacore et al. (1971) investigated the incidence of drug abuse in 2,696

members of 497 families in Lucknow. The survey revealed an incidence of 18.55

per 1,000 population, most of which were cases of alcohol abuse. A significantly

higher incidence was observed in the poor population, in unitary or small

families, and in males; the majority of abusers fell in the age group of 26 to 45

years. Similar findings from other investigators have been reviewed by Sethi

et al. (1984). Among sociodemographic correlates, the incidence is highest in

semi-rural areas, followed by rural and urban areas in decreasing order. It is

190
also common in industrial areas. A study team of the Planning Commission made

a nationwide survey of the alcohol consumption habits and the results have been

summarised in the ICMR report of 1977. It analysed the budgets of 21,197

working class families of 50 different centres. Between 10% and 24% of all

working class families were given to drinking. The incidence was lowest among

factory workers (9.6%) and highest among mine workers (23%). The incidence

was higher in the northwestern part of the country and in those urban centres

where there was a greater trend toward "pseudo-westernisation."

Drug Abuse in Students

A comprehensive survey of incidence of drug abuse among students in various

parts of the country was undertaken by the Department of Social Welfare,

Government of India, in 1976. Their results indicate that pain killers, tobacco,

and alcohol had the highest incidence, followed by cannabis. The relative

incidence as well as the total incidence varied and was higher in bigger cities like

Bombay, Delhi, and Madras. Cannabis had the highest proportion of users at

Varanasi, partly reflecting its widespread use and social acceptance in that region

of the country. Mohan and his colleagues have undertaken a systematic study of

drug abuse in school and college students. Their results, published in a series of

papers, have been summarised in table 2. The data on tobacco have not been

TABLE 2

Incidence of Drug Abuse in University and College Students at Delhi

Agents % Users

School Pre-University* University

Colleges

University

Alcohol 27.9 51.2 24.1 23.5

Amphetamines 4.9 19.1 2.0 3.6

Cannabis 9.8 18.1 4.4 8.4

Barbiturates 3.1 21.7 0.6 3.4

Opium 1.3 11.8 2.0 2.7

Tranquillisers 6.7 17.4 3.2 7.8

LSD - 12.6 - 2.6

Others - 15.3 3.5 12.1

Sample Size 225 117 882 502

Authors Mohan et al. Varma and

1978 Dang 1978

*Data from Chandigarh (ages 13-20 yr)

Mohan and Mohan et al.

Arora 1976 1977

included in this and subsequent tables. The table also includes data on preuniversity students from Chandigarh, where the incidence is much higher. A

similar study of school children from other parts of the country will be useful.

A comparison of findings in students of different age groups indicates only

191
marginal changes after the habit has been acquired at a young age. The

increased use of tranquillisers and other agents as well as exposure to LSD may

be related to the residential character of the university wherein a large number

of students stay in the university hostels. Mohan et al. (1978 ) have found a

significantly higher incidence of drug use among students residing in a hostel. A

major change appears to be a much higher percentage of students becoming

regular users as they move from the school to the university (table 3), as well as

introduction to harder stuff like barbiturates, tranquillisers, etc. There is also a

marked increase in regular use of alcohol among college students. It is possible

that many of these students come from families where other family members also

use alcohol and, as they grow up, it becomes permissible for them also to drink.

Such data, however, are not available. Mohan et al. (1977) have provided

comparative data on the two sexes. A summary of their results in table 4 clearly

indicates that the incidence of both occasional and regular use is much higher in

the male students. As a matter of fact, there was hardly any regular use of drugs

in the female students.

TABLE 3

Frequency of Abuse in Students

Agents % Frequency of Abuse

School Pre-University* University

Colleges

University

O R O R O R O R

Alcohol 24 3.5 37.2 14.0 3.6 20.5 20.1 3.4

Amphetamines 4.9 - 17.3 1.9 1.8 2.0 2.8 0.8

Cannabis 8 1.8 16.3 1.8 3.8 0.6 6.4 2.0

Barbiturates 3.1 - 17.4 4.3 0.6 - 3.0 0.4

Opium 1.3 - 8.1 3.7 1.4 0.7 2.7 -

Tranquillisers 6.7 - 13.1 4.3 2.6 0.6 7.0 0.8

Others - - 23.8 5.0 3.5 - 13.1 2.4

O = Occasional, R = Regular.

*Data from Chandigarh (ages 13-20 yr)

Varma et al. (1977) investigated the incidence of abuse of five specific drugs in

408 college students in Chandigarh. Their results have been summarised in table

5 and the incidence (18.87%) is higher than the rates obtained by Mohan and his

colleagues at Delhi or by Thacore et al. (1971) at Lucknow. One of the reasons

for this could be their differing definition of drug abuse. Varma et al. (1977) and

(1980) have defined it as even occasional intake without medical prescription.

The percentage of regular users is low even in this study. In this study also, the

percentage in female students was much lower.

192
TABLE 4

Sex-wise Distribution of Drug Usage in University Students*

Agents Male students % Female students %

Occasional Regular Occasional Regular

Alcohol 28.9 6.6 10.6

Amphetamines 4.7 1.6 0.8

Cannabis 11.3 3.9 1.2

Barbiturates 5.1 0.8 0.8

Opium 4.7 0.8

LSD 4.3 0.4 0.4

Tranquillisers 9.8 1.6 4.1

Others 13.3 2.3 7.3 0.4

*Sample size was 256 male and 246 female students.

TABLE 5

Incidence of Abuse to Specific Agents in Students in Chandigarh

Agents % Students’ use

Never Occasional Regular

Amphetamines 91.9 5.4 2.7

Barbiturates 96.3 3.7 0

Cannabis 95.1 4.4 0.5

LSD 99.3 0.7 0

Mandrax* 90.9 8.1 1.0

*Mandrax is a combination of methaqualone and diphenhydramine;

its production has now been banned in India.

Agarwal et al. (1980) have analysed the interrelationship between drug abuse,

anomie, alienation, and authoritarianism in 197 postgraduate students at

Chandigarh. Anomie can be defined as deregulation or normlessness; alienation

indicates distance between mind and reality leading to loss of identity and

depersonalization; while authoritarianism represents antidemocratic and fascist

social attitudes. Their results indicate a negative correlation between anomie and

authoritarianism and drug use, particularly in the younger age group, in those

with an urban background and high parental education, and in female students.

A positive correlation under similar conditions was observed between alienation

and drug use. The psychosocial correlates in adolescents have also been

investigated by Mohan and Thomas (1977). Mohan et al. (1978a) have found a

highly significant correlation between the father’s income, education, and place

193

.
of residence as well as the adolescent’s course of study and the drug abuse in

both sexes.

Medical Students and Interns

At least three detailed studies on prevalence and pattern of drug abuse in medical

students have been published in recent years following the survey by Agarwal et

al. in 1975. Singh (1979) has investigated 672 undergraduate students in Punjab,

and 576 students have been studied by Sethi and Manchanda (1977) at Lucknow.

In both studies, the ratio of male to female students involved in drug abuse was

6:1, conforming to the pattern obtained in other studies. Agarwal and Pande

have recently (1982) undertaken a similar study in 270 medical students at

Gorakhpur. The ratio of prevalence in males and females was 3:1. The relative

abuse of different agents in the three studies has been summarised in table 6.

TABLE 6

Incidence of Drug Abuse in Medical Students

Agents % Students’ abusing

Sethi and Manchanda Singh Agarwal and Pande

(1977) (1979) (1982)

Alcohol

Amphetamines

Barbiturates

Cannabis

Nonbarbiturate

sedatives

Tranquillisers

14.2 12.2 11.85

7.6 4.8

0.7 1.5

5.6 4.0 14.45

2.8 3.9 9.25

17.7 12.0 14.81

A rather surprising finding is the high incidence of use of tranquillisers, which

may in part be related to their availability in the hospital. In the sample of

Agarwal and Pande, the greater use of cannabis is related to its widespread use in

that part of the country. The relative frequency of use of various agents has not

been described in Agarwal and Pande, but the data from the other centres appear

to be comparable (table 7).

There have been two recent surveys concerning drug abuse in interns and

resident doctors in medical college. The survey at Jammu (Choudhary et al.

1980) covered 105 doctors, while that at Madras (Ponnudurai et al. 1984) covered

116 interns. In both surveys, the incidence was about sixfold lower in females.

The data in table 8 indicate the marked increase in drug abuse by interns in

comparison with the figures reported for medical students (table 6), particularly

in the case of alcohol and tranquillisers. Choudhary et al. (1980) have reported

that similar results were obtained by Joshi and Bharadwaj in a survey in

194
TABLE 7

Frequency of Drug Abuse in Medical Students

Agents % Frequency of use

Never Occasional Frequent Regular

A B A B A B A B

Alcohol

Amphetamines

Barbiturates

Cannabis

Nonbarbiturate sedatives

Tranquillisers

87.8 85.8 2.4 23.5 0.6 0.7 3.1 -

95.2 92.4 2.5 1.6 2.2 3.3 - 2.7

98.5 99.3 1.0 - 0.5 - - 0.7

96.0 94.4 3.4 5.0 0.6 0.6

96. 1 97.2 3.3 2.8 0.4 - 0.2 -

90.0 82.3 5.4 15.8 4.3 1.0 0.3 0.7

A: Data of Singh (1979), 672 students

B: Data of Sethi and Manchanda (1977), 576 students

Rajasthan in 1978. The increase is largely among those taking it occasionally and

is much less marked in regular users (see tables 9 and 7). The possible reasons

appear to be availability of tranquillisers in the hospital and financial

independence from their parents due to their stipends. To this also must be

added the long hours of work and stress and the urge to get temporary respite

from all this.

TABLE 8

Incidence of Drug Abuse among Interns and House Officers

Agents % Ever used

Choudhary et al. 1980 Ponnudurai et al. 1984

Alcohol 55.2 53.5

Amphetamines 12.6 7.8

Cannabis 16.7 18.1

Hallucinogens - 7.8

Pain killers 12.0 -

Tranquillisers 36.5 14.7

Others 17.2 -

195
TABLE 9

Frequency of Drug Abuse in Interns and House Officers

(Choudhary et al. 1980)

Agents % Frequency of use

Never Occasional Frequent Regular

Alcohol 44.8 44.8 4.2 6.3

Amphetamines 87.6 10.5 1.0 1.0

Analgesics 87.5 5.2 2.1 5.2

Cannabis 83.4 15.6 - 1.0

Tranquillisers 63.5 34.4 - 2.1

Psychiatric Patients

Even though there is no uniform pre-addictive personality, individuals with

personality problems are more likely to look for relief with drugs; therefore,

chronic users may have a higher proportion of insecure personality. There have

been global efforts to establish any possible relationship between drug abuse and

psychiatric disorders. One approach has been to find out the prevalence of drug

abuse in patients coming to psychiatric hospitals. Some interesting observations

have been made in the country during the last few years. Trivedi and Sethi

(1978) investigated 1,000 consecutive newly registered male psychiatric outpatient

cases for drug abuse to find any possible correlation with psychiatric illness. The

pattern of drug abuse in their sample has been summarised in table 10. They

reported an overall incidence of 16.4%. There was a slightly higher incidence

(22.8%) of drug abuse in patients suffering from affective disorders in

comparison to those suffering from schizophrenia (16.5%) or neurosis (15.4%).

The incidence in other diseases was much lower (12.4%). There appeared to be a

possible correlation between abuse of alcohol and cannabis with affective

disorders and neurosis, but further studies are necessary to establish a definitive

relationship and to offer an explanation. Venkoba Rao et al. (1978) have

analysed cases of drug addiction seen in a psychiatric hospital over a 5-year

period. They obtained a sample of 178 cases from 10,023 patients (1.8%)

attending the outpatient clinic and only three of these were female. The relative

distribution in various drug categories has been given in table 10. A majority of

these patients (53.4%) came for the treatment of drug dependence, 39% were

suffering from schizophrenia, and the remaining few had associated affective

disorders or neuroses. More than a third of these cases had a family history of

psychiatric morbidity. Ray and Chandrashekhar (1982) have examined 1,984

consecutive admissions to male psychiatric wards at the National Institute of

Mental Health and Neuro Science, Bangalore, for evidence of alcoholism by

administering to them the Michigan Alcoholism Screening Test (MAST). During

the period, 6.1% of all admissions to that hospital were due to alcohol-related

problems. The results indicated that 19% of the patients were alcoholic and

196
TABLE 10

Incidence of Drug Abuse in Psychiatric Patients

Agents % Incidence

Sethi and Trivedi

1979

Venkoba Rao et al.

1978

Alcohol

Amphetamines

Barbiturates

Cannabis ± alcohol

Opium

Tranquillisers

Multiple

9.3 24.2

1.1 -

0.6 2.2

- 29.2

0.3 3.4

2.1 -

- 11.1

Patient population Outpatient cases Addicted cases

Sample size 1,000 178

another 22% were social drinkers. The remaining patients had not been exposed

to alcohol. The alcoholics had more first-degree relatives afflicted with

alcoholism, sociopathy, and depression.

OTHER CLINICAL STUDIES

There has been considerable interest in abuse of cannabis and methaqualone.

Chopra et al. (1942) were the earliest investigators to draw attention to a possible

relationship between cannabis use, mental diseases, and crime. The problem of

cannabis abuse has been reviewed recently by Sethi et al. (1984). They have also

evaluated 50 pure chronic cannabis users for their physical, mental, cognitive,

and socioeconomic function and compared the results with a matched control

group. On clinical evaluation, most (64%) of the subjects had no psychiatric

problem; indeed, they had significantly low anxiety scores in comparison to

controls on Taylor’s Manifest Anxiety Scale. The other tests also failed to reveal

any impairment. Their earlier study (Sethi et al. 1981) also had failed to detect

any particular identifiable personality trait in chronic cannabis users. Clinical

study of volunteers on the effect of chronic cannabis use did not reveal any

serious harmful effects even though some subjects reported withdrawal effects

(Singh et al. 1981a). Similarly, no differences were observed in cognitive

functions between chronic users of cannabis and nonusers (Ray et al. 1979). The

results indicate that cannabis does not appear to be as injurious in the Indian

setting as has been suggested by reports from Western countries.

197
There are, however, a few reports which indicate that chronic use of cannabis

may not be as harmless as made out by studies on the cognitive functions. There

have been reports of psychoses in chronic cannabis users. In a well-controlled

study, Thacore and Shukla (1974) compared cases of cannabis psychoses with

those of paranoid schizophrenia. Cannabis psychoses were characterised by more

bizarre behaviour, more violence and panic, but an absence of schizophrenic

thought disorders. These cases cleared more rapidly with hospitalisation and

relapsed when drug was resumed. Chopra and Smith (1974) reported that cases

of cannabis psychoses were predominantly instances of toxic delirium, but those

who had schizoid features became overtly schizophrenic during the period of

intoxication. Uppal et al. (1981) have observed a significant decrease in

antipyrine elimination half-life compared to control subjects. It was more

marked than observed even in cigarette smokers (Uppal et al. 1980). A similar

decrease in theophylline clearance has been reported from Western countries

(Jusko et al. 1978).

Thacore et al. (1971) undertook a detailed study of addiction to methaqualone.

The clinical tolerance developed in 4 to 6 months and the withdrawal symptoms

included depression, sleep disturbances, anxiety, and restlessness in addition to an

intense craving for the drug. In most cases, they were first prescribed the drug

by a physician for some ailment. Mahal and Nayar (1978) reported 15 cases of

petrol dependence from Bangalore. All of these were young boys in the second

decade of life, most of them from unsettled families. They suffered from

perceptual distortion and hallucination, in some cases of a wish-fulfilling quality

and having some resemblance with LSD-psychosis. There was no evidence of

tolerance or report of withdrawal symptoms, but there was intense psychological

craving following deprivation.

There seems to have been little effort at innovative treatment of drug addiction

even though institutional care is available at many places. Earlier work in this

area has been reviewed by Wig and Varma (1977). Gangadharan et al. (1982)

investigated the use of clonidine in management of cases of opiate dependence

(morphine or pethidine). In their studies, clonidine controlled the nonpurposive

withdrawal symptoms effectively within 2 to 3 days following abrupt opiate

withdrawal, but the purposive symptoms took 8 to 10 days to be adequately

controlled. The patients could be discharged earlier from the hospital. The drug

merits further studies. Bagadia et al. (1982) have evaluated the usefulness of

disulfiram in management of chronic alcoholics in the Indian settings. They have

concluded that older patients and those brought by relatives or friends did better

and, except for psychotic episodes in 3 out of 75 patients, other side effects were

of mild nature.

Shukla (1980) has investigated the sociocultural aspects of abuse of drugs in male

adults in a rural community, including the use of alcoholic drinks and drugs as a

sociocultural phenomenon. Only 27% of the respondents in the village surveyed

by him were nonusers and about 32% were habitual users of alcohol or cannabis;

the use of opium was rare. This had gone on for generations without posing any

sociocultural crisis or any increase in number of addicts in the village. The use

was more frequent in unskilled labourers, and regular use was more common in

nonfamilial households or in children whose fathers used drugs. Hasan (1975)

investigated social aspects of use of cannabis and concluded that there was no

evidence of increase in dosage or of physical dependence. In most cases, persons

in that rural community used it on occasions of festivity and ceremonial

functions.

198
ANIMAL STUDIES

The major interest in recent years has centered around methaqualone, which was

discovered in India (Gujral et al. 1955, 1956), and cannabis, which has a

widespread use.

Methaqualone

Khanna et al. (1971) have investigated development of tolerance to methaqualone

in rats following once daily oral administration. Maximum reduction of hypnotic

response was obtained in 6 days. There was a cross-tolerance between

phenobarbitone and methaqualone. It appears that induction of microsomal

enzymes in the liver play a role in development of tolerance, since normal

hypnotic response has been restored by SKF 525-A, an inhibitor of liver

microsomal enzyme system. These studies have been subsequently extended by

Kohli et al, (1974). Tolerance was discernible during the first week to

hypothermia and reduction in spontaneous motor activity. Withdrawal resulted in

increased susceptibility to audiogenic seizures, hyperactivity, and precipitious

decrease in body weight. Nath et al. (1980) compared the dependence liability of

methaqualone alone and when used in combination with diphenhydramine (a

combination which is widely abused). Their results indicate a more severe

degree of physical dependence to the combination than to methaqualone alone.

Cannabis

Singh and Das (1977) undertook a detailed study on tolerance to the resin

extracted from Cannabis indica in rats. The resin contained 17% delta-9-

tetrahydrocannabinol (THC) and was administered in a dose of 50 to 200 mg/kg.

The animals developed tolerance to hypothermic, analgesic, and anticonvulsant

activities very rapidly. The potentiating effect on barbiturate hypnosis was also

concomitantly reduced. Tolerance did not develop to either increased or

decreased locomotor activity or to hyperthermia in tolerant rats.

The disappearance of tolerance on stoppage of cannabis was very slow.

Tolerance to hypothermic activity was the first to disappear and that to analgesic

activity was the last. Daily administration of cannabis did not affect rate of

learning or rate of extinction of learned behaviour. This is in agreement with

the clinical results reviewed earlier. Singh et al. (1981) have reported similar

tolerance to petroleum ether extract of flowering tops of Cannabis indica. On

withdrawal, treated animals showed increased sensitivity to audiogenic seizures.

Dixit et al. (1974) found a reduction in the size of testis and a regression of

Leydig cells following chronic administration of cannabis extract in mice.

Investigations have also been directed toward biochemical changes following

chronic administration of cannabis and its constituents. Singh et al. (1980)

allowed rats to inhale marijuana fumes daily for a period of up to 90 days.

Exposure for up to 15 days decreased brain acetylcholine levels but increased the

catecholamine and serotonin levels. After a prolonged administration, the effects

were found to be reversed and associated with a decrease in body weight due to

a decrease in food intake. Ghosh and his colleagues have studied the effects of

acute and chronic administration of cannabis and THC on subcellular

components. Chronic treatment with cannabis or THC was devoid of effect on

dopamine and norepinephrine turnover (Poddar and Ghosh 1976b) and on the

metabolism of other biogenic amines (Poddar et al. 1975). Acute treatment

resulted in either increased mitochondrial (Na+K

+

)- and Mg++ ATPase or a

199
decrease in Na+K

+

 ATPase activity in synaptosomal preparation. The effect

disappeared following chronic treatment (Poddar and Ghosh 1976a). Similarly,

acute treatment led to a marked increase in sialoglyco-proteins and ganglioside

content in both microsomal and synaptosomal fractions, but the effects were not

observed after chronic treatment (Sarkar and Ghosh 1976). Sarkar and Ghosh

(1975) also investigated the effect of THC on lipid constituents of subcellular

fractions in rat brain. Their results have been summarised in table 11. In

contrast to other biochemical parameters, the effect on several lipid components

remained unaltered following chronic administration. It has been suggested that

chronic administration of THC stimulates its own metabolism, leading to its

conversion to hydroxylated polar metabolites which may not interact strongly

with membrane components. This can explain some but not all of the

differences between the effects of acute and chronic administration.

TABLE 11

Effect of Delta-9-Tetrahydrocannabinol on Lipid Constituents of Rat Brain

Subcellular Fractions

Lipid component Subcellular fraction

Mitochondria Microsome Synaptosomes Myelin

A B A B A B A B

1. Total lipids

2. Total cerebroside

3. Total cholesterol

4. Total phospholipids

i Phosphatidylcholine

ii Phosphatidylethanol

iii Phosphatidylserine

iv Sphingomyelin

A = Acute treatment, B = Chronic treatment, - = Decrease, + = Increase,

O = No change.

CONCLUDING REMARKS

The problem of drug abuse in India has taken a different shape and presented

different dimensions than in most of its neighbouring countries. Opium abuse

has not become a major problem due to a well-defined government policy which

has controlled cultivation of opium since 1895. Also, there has been no ban or

control on cannabis. The use of alcohol has undergone a period of rigid controls,

but the laws are more relaxed now. Due to closed borders with Pakistan, the

heroin traffic from Afghanistan has not posed a major problem until very

recently. This situation has also determined the research activity on drug abuse

in the country.

200
Recent studies suggest a change in the traditional pattern of drug usage in two

main directions (Mohan et al. 1978 ). One of these is a movement away from

nonusage of drugs as a cultural norm, even in restrictive societies, beginning at

an earlier age, particularly with respect to alcohol and tobacco. Second, and a

somewhat redeeming feature, is that the social strata involved in such deviation is

the small affluent upper socioeconomic group and the pattern has not undergone

a major change in the rural areas. Such a change had been predicted (Nayar

1972) since the concerned group of urban population tends to confuse

modernisation with westernisation and tends to adopt symbols rather than

substances of Western societies. Fortunately, this does not appear to have

assumed subculture or counterculture overtones or a symbolic protest value

against accepted social norms as has happened in many Western countries.

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AUTHOR

B.N. Dhawan, M.D.

Scientist-in-Director’s Grade

Central Drug Research Institute

Lucknow 226001, India

203
The Medicinal Chemistry of

Cannabinoids: An Overview

Alexandros Makriyannis, Ph.D., and Rao S. Rapaka, Ph.D.

Since the early 194Os, when Roger Adams synthesized the first

cannabinoid analogs and subjected them to pharmacological testing,

cannabinoid research has maintained a sustained progress involving

the synthesis of a large number of analogs with a varied degree of

resemblance to the natural analogs. At the same time, the

pharmacological properties of the principal cannabis constituents

were studied in detail, while less extensive studies were reported

on their synthetic analogs. A good effort was also made to study

biochemical effects of cannabinoids on a variety of biochemical

preparations and the use or potential use of those compounds as

therapeutic agents.

Over four decades later and after the successful synthesis and

testing of several hundred analogs a number of important questions

related to the function of cannabinoids still remain unanswered.

Nevertheless, we have made progress in defining correlations between

structure and function while recognizing that such correlations

should be made in the context of the biological function being

measured. It has also become clear that differences between the

species tested are an important consideration. More recently

testing of the cannabinoids on discrete biochemical systems has

allowed us to better dissect the molecular events involved in the

"cannabinoid effect." Parallel to such tests, we should note that

increased sophistication in behavioral testing has allowed for more

accurate structure-function correlations.

Still, the specific details involved in the cannabinoid molecular

mechanism of action are far from clear. The "receptor" versus

"membrane" site of action arguments have not been resolved, and

there are indications that each of the two hypotheses can provide

partial answers, while no unifying theory has yet been articulated.

The recent informative review by Martin (1986) does a very good job

of outlining these different points of view. The present overview

is not intended to be all inclusive, nor does it seek to define all

the unanswered questions related to cannabinoid research. Instead,

it seeks to highlight some of the important developments in this

interesting multidisciplinary field of research, drawing to a large

extent from the presentations made during the recent NIDA technical

review on structure-activity relationships of cannabinoids.

204
STRUCTURE-FUNCTION CORRELATIONS

Structure-function correlations in cannabinoids have recently

been reviewed in great detail by Razdan (Razdan 1986, Razdan,

this volume; Mechoulam et al., this volume). From the emerging

picture, we can identify the key structural features required

for activity. These should also provide us with insights about

the molecular mechanism of cannabinoid activity.

Phenollc Hydroxyl

First, the presence of a free phenolic group is an absolute

requirement for cannabinoid activity. Phenolic esters of

delta-9-tetrahydrocannabinol (THC) are active in vivo,

presumably after hydrolysis, and are inactive in in vitro

preparations. However, the phenolic ethers are totally

inactive. The role of the phenolic group during its interaction

with the active site could be either as hydrogen bonding donor

or a hydrogen bond acceptor through its lone pair of electrons.

The latter hypothesis has been elaborated by Regglo (this

volume) who points out that the orientation of the phenolic lone

pair is the key molecular determinant and that only the free

phenolic group has the opportunity to align in the proper

"active orientation" while substituted phenols are unable to do

so because of steric considerations. The ability of the

phenolic OH to act as a hydrogen donor with one or more of the

polar membrane sites (either at the phospholipid or the protein

component) has been investigated by Makriyannis and colleagues

and is discussed later in this review.

The role of hydroxyl substitution in cannabinoid molecules

becomes more intriguing in the case of analogs having an

additional one or two hydroxyl groups in the C ring. Several of

these analogs have been synthesized, and their structurefunctions correlations can be clearly visualized. Certainly,

the OH orientations must have an important molecular role during

the cannabinoid interaction with the site(s) of action. Because

of the large differences in activity between individual analogs,

the di- and trihydroxy cannabinoids may prove to be very good

tools for probing the molecular requirements of the drug-active

site interactions.

Ring C Geometry

An important molecular determinant is the geometry of the C

ring. It is now being recognized that compounds where the C

ring is totally coplanar with the aromatic ring, as in the case

of delta 9- and 11-THC and cannabinol, result in inactive

compounds. A certain deviation from this coplanarity between

rings A and C appears to be one off the crucial molecular

requirements (Razdan, this volume; Mechoulam et al., this

volume; Makriyannls et al., this volume). This may be related

to specific geometric requirements for interaction with a

specific "receptor protein." On the other hand, this

"out-of-plane" conformation may be required for the molecule to

produce perturbations at the level of the phosphollpid bilayer

(Makriyannis et al., this volume).

205
Side Chain in Ring A

The role of this molecular fracture is a crucial one, pointing

to some requirement for hydrophobic interactions with the

membrane components. However, these hydrophobic interactions

still allow for the presence of some polar groups such as that

of a phenolic ether oxygen or a hydroxy substituent in certain

positions of the chain. The length of the chain is critical,

with the n-heptyl group being optimal. Also, methyl

substituents (e.g., 1,1- or 1.2-dimethyl) enhance activity not

only by interfering with the drug metabolism but apparently by a

direct effect at the site(s) of action as well.

Absolute Configuration

Although it was early recognized that tetrahydrocannabinol

enantiomers differ in potency, accurate values for enantiomeric

potency ratios were not available. This, to a large extent, was

due to the fact that the enantiomeric starting materials were

not of high enough purity. This situation has been remedied

through careful purification of these compounds. More recently,

starting materials possessing high optical purity have become

available commercially.

Recent work by Mechoulam et al. (this volume) involving the

11-hydroxy-delta-8-THC enantiomers showed a potency difference

of two to three orders of magnitude between the enantiomers.

This difference is much higher than anything reported earlier

and does not appear to be solely due to metabolic factors. In

all likelihood, it is to a large extent due to the abilities of

individual cannabinoids for productive interaction with their

site(s) of action.

Nonclassical Cannabinoids

The term "nonclassical cannabinoids" includes a group of

compounds that were developed by Melvin and Johnson (this

volume). These resemble the tetrahydrocannabinols but do not

incorporate the pyran ring. These analogs, developed as

potential analgesics, possess pharmacolgical profiles resembling

those of the classical cannabinoids. First among these analogs

possessing pharmacological activity is a hydroxyphenyl

cyclohexanol with a 1.1-dimethylheptyl side chain. It

represents the simplest of the cannabinoid structures and

incorporates the required phenolic group and an appropriate side

chain. The presence of a secondary hydroxy group on the

cyclohexane ring enhances activity; as with the "classical

cannabinoids," its orientation is crucial in determining

activity. This compound also shows a modest enantiomeric

potency ratio. The nonclassical blcyclic structure introduces a

hydroxypropyl group on the cyclohexanol ring that results in

dramatic increases both in potency and in enantlomeric potency

ratio. The highest enantiomeric potency ratio is obtained when

the hydroxypropyl group is incorporated into a six-membered ring

to yield the corresponding hydroxymethyl decalin tricyclic

analog.

206
Melvin and Johnson (this volume) synthesized a relatively large

number of such analogs and examined the structural and

stereochemical correlates with activity in detail. Based on

their studies, they formulated a structure for the hypothetical

receptor site. The correlations made here do not necessarily

advance our knowledge about the nature of the cannabinoid site

of action. However, they do provide very interesting molecular

templates, beyond the classical cannabinold structure, which may

serve as important tools in the search for the cannabinoid

active sites.

MOLECULAR MECHANISMS OF CANNABINOID ACTIVITY

The idea of a specific receptor through which cannabinoids

produce some of their effects has traditionally received

favorable attention (Mechoulam et al., this volume; Mechoulam et

al 1985; Martin 1986; Martin et al., this volume). Recently,

evidence for a cannabinoid receptor has been advanced from two

laboratories. In one case, the water soluble nonhydrolyzable

cannabinoid trimethylammonlum delta-8-tetrahydrocannabinol (TMA)

was used by Snyder and his coworkers as a radiolabeled ligand to

test for the presence of "cannabinoid receptors" (Martin et al.,

this volume; Nye and Snyder, this volume). Indeed, the compound

showed some specific binding to rat brain membranes.

Furthermore, delta-9-THC and other cannablnoids competed

successfully with TMA for its binding site. Snyder and

coworkers also demonstrated some enantiomeric selectivity among

the THC antipodes and lack of binding for other non-cannabinoid

structures. There was no good correlation between cannabinoid

"affinity" for the TMA binding receptor and for the abilities of

the cannabinoiids to displace TMA from its binding site.

One shortcoming of the above approach rests with TMA's most

attractive feature, namely its water solubility. Although, this

molecule maintains the key cannabinoid structural features, it

introduces a permanently charged bulky group in the molecule.

This alters one of the key characteristics of the cannabinoid

structure, i.e., its overall hydrophobic properties, especially

at the level of the side chain. The other data favoring a

specific protein site for cannabinoid activity by Howlett (this

volume) comes from experiments with murine neuroblastoma cells

showing that cannabinoids decrease cyclic AMP production. This

response can be attributed to the inhibition of adenylate

cyclase activity. Since adenylate cyclase is regulated by a

number of receptors through the intermediacy of the ubiquitous G

protein, the data suggest that cannabinoids may act either at

the receptor or the G-protein level. After having shown that

cannabinoids do not act at the receptor protein level, Howlett

suggests that the G protein is, indeed, their site of action.

The results from this biochemical system are encouraging because

there are good correlations between adenylate cyclase inhibition

and cannabinoid potency. This hypothesis for the site of action

of cannabinoids is yet to be proved through direct binding

experiments.

207
A different type of evidence pointing to a role played by

membrane lipids in cannabinoid activity comes from the

laboratories of Makriyannis and coworkers (this volume). Using

model membranes, and with the help of solid state nuclear

magnetic resonance and other biophysical techniques, they showed

differences in the manner in which cannabinoid enantiomers and

cannabinoid isomers having different potencies interact with

model membranes. Such data refute the general notion that

cannabinoid phospholipid interactions are "nonspecific" and

totally dependent on the drug's oil/water partitioning

properties. Makriyannis also showed that cannabinoid analogs

orient in the membrane in such a manner that the phenolic

hydroxy group serves as the anchor at the membrane bilayer

polar/nonpolar interphase and has an important role in

determining the geometry of the cannabinoid-membrane amphipathic

interaction. The above studies are also congruent with a

hypothesis involving more specific cannabinoid-lipid

interactions which would, in turn, affect the function(s) of the

neighboring proteins. Although one may expect some common

general features in the interaction of cannabinoids with

membranes, one also expects differences in the molecular details

of these interactions depending on the nature and composition of

the membrane. Such differences may explain the observed

variations in cannabinoid selectivity with different membrane

preparations.

Additional evidence for cannabinoid action at the membrane level

comes from the laboratories of Burstein (this volume). Using a

cell culture model to test cannabinoid activity, Burstein showed

that cannabinoids have marked stimulatory action on eicosanoid

synthesis and that the cannabinoid interaction does not occur at

the level of the protein catalytic site. Also, the model system

shows a fair degree of correlation between structure and

activity relationships, tolerance, and stereospecificity for the

cannabinoids tested. These effects of cannabinoids on the

eicosanoid biochemical systems may be related to their CNS

activity. Indeed, delta-9-THC when administered orally was

found to increase PGE2 content in the mouse brain and also

increased phospholipase activity in brain synaptosomes. A more

recent interesting finding from the same laboratories is that

the 9-carboxylic acid metabolite of delta-9-THC inhibits

delta-9-THC effects on eicosanoid synthesis, apparently through

its action on prostaglandin synthetase. This metabolite may

thus prove useful as a nonsteroidal antiinflammatory agent.

CANNABINOID PHARMACOLOGY, TOXICOLOGY, AND PHYSIOLOGY

Based on extensive pharmacological evidence, Martin et al. (this

volume) pointed out that the cannabinoid behavioral syndrome

represents a composite rather than a single event. To dissect

these different effects, which he classifies in two general

categories as membrane- and receptor-related effects, he used a

variety of behavioral test systems. These include spontaneous

activity, rectal temperature and analgesia in the mouse, static

ataxia in the dog, and drug discrimination and conditioned

208
behavior in monkeys. This approach is very useful and should

contribute towards putting the correlations between cannabinoid

structure and function on more solid ground. In the same vein,

detailed studies by Karler and Turkanis (this volume) have

served to point out the dramatic differences in cannabinoid

activity among different species. These studies are important

because they help to show the possible discrepancies when

correlations are made between cannabinoid structure and function

using pharmacological data from difference species. Also, from

the laboratories of Karler and Turkanis (this volume),

toxicological and electrophysiological data demonstrate the wide

diversity of action between different cannabinoids. Again,

correlations between cannabinoid structure and function should

be made judiciously, taking into account the details of

pharmacodynamics, toxicology, and electrophysiological testing.

Last but not least, the use of cannabinoids as anticonvulsants

is an important therapeutic application. In this regard,

Consroe and Mechoulam (this volume) have made good contributions

towards elucidating the correlations between cannabinold

structure and anticonvulsant properties. The testing is done

with epilepsy-prone rats, and the compounds are tested for their

abilities to prevent audiogenlc seizures (AGS) and to cause

rotorod neurotoxicity (ROT). From these studies, it is clear

that the anticonvulsant SAR for cannabinoids does not overlap

with the corresponding correlations for psychotropic or

analgesic activity. This observation holds a good deal of

promise for the use of cannabinoid analogs as anticonvulsants.

Excellent research is being carried out all over the world on

the SAR of cannabinoids. This research has resulted in the

development of several compounds of therapeutic use and, more

importantly, provided tools for further research on cannabinoid

biochemistry and pharmacology. It is not unrealistic to hope

that in the near future some additional compounds of potential

therapeutic value will be developed.

REFERENCES

Martin, B.R. Cellular effects of cannabinoids. Pharmacological

Reviews 38:45-74, 1986.

Mechoulam. R .; Srebnik. M.; and Burstein. S. Cannabinoid

chemistry; biochemistry and therapeutic applications: An

overview. In: Harvey, D.J., ed. Marihuana '84. Proceedings

of the Oxford Symposium on Cannabis. Oxford: IRL Press,

1985. pp. 1-12.

Razdan, R.K. Chemistry and structure-activity relationships of

cannabinoids: An overview. In: Agurell, S .; Dewey, W.L.:

and Willete, R.E.. eds. The Cannabinoids: Chemical,

Pharmacologic, and Therapeutic Aspects. Orlando: Academic

Press, 1985. pp. 63-78.

Razdan, R.K. Structure-activity relationships in cannabinoids.

Pharmacological Reviews. 38:75-149, 1986.

209
AUTHORS

Alexandros Makrlyannis, Ph.D.

School of Pharmacy and

Institute of Materials Science

University of Connecticut

Storrs, CT 06268

Rao S. Rapaka, Ph.D.

Research Technology Branch

Division of Preclinical Research

National Institute on Drug Abuse

Room 10A-13, 5600 Fishers Lane

Rockville, MD 20857

210
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22 QUASAR: QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIPS OF

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29 DRUG ABUSE DEATHS IN NINE CITIES: A SURVEY REPORT. Louis A.

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30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY PERSPECTIVES. Dan

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31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen, Ph.D.,

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32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz,

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33 BENZODIAZEPINES: A REVIEW OF RESEARCH RESULTS, 1980. Stephen I.

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35 DEMOGRAPHIC TRENDS AND DRUG ABUSE, 1980-1995. Louise G.

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36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A REVIEW OF

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37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE. Travis

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38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettieri, Ph.D.,

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39 YOUNG MEN AND DRUGS IN MANHATTAN: A CAUSAL ANALYSIS.

Richard R. Clayton, Ph.D., and Harwin L. Voss, Ph.D.

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40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES. Herbert

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41 PROBLEMS OF DRUG DEPENDENCE, 1981: PROCEEDINGS OF THE 43RD

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42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS. Richard L.

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44 MARIJUANA EFFECTS ON THE ENDOCRINE AND REPRODUCTIVE SYSTEMS.

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45 CONTEMPORARY RESEARCH IN PAIN AND ANALGESIA, 1983. Roger M.

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46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE TREATMENT. John

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47 PREVENTING ADOLESCENT DRUG ABUSE: INTERVENTION STRATEGIES.

Thomas J. Glynn, Ph.D .; Carl G. Leukefeld, D.S.W.; and

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48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF SMOKING BEHAVIOR.

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50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

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51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES, PROGRESS, AND

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52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND ABUSE

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53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION. John Grabowski,

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57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING CURRENT

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58 PROGRESS IN THE DEVELOPMENT OF COST-EFFECTIVE TREATMENT FOR DRUG

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59 CURRENT RESEARCH ON THE CONSEQUENCES OF MATERNAL DRUG ABUSE.

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60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS. C. Nora Chiang,

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61 COCAINE USE IN AMERICA: EPIOEMIOLOGIC AND CLINICAL PERSPECTIVES.

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62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH. Roger M. Brown,

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63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG CHILDREN AND

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64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed.

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65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH. Barbara A. Ray, Ph.D.,

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66 GENETIC AND BIOLOGICAL MARKERS IN DRUG ABUSE AND ALCOHOLISM.

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68 STRATEGIES FOR RESEARCH ON THE INTERACTIONS OF DRUGS OF ABUSE.

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69 OPIOID PEPTIDES: MEDICINAL CHEMISTRY. Rao S. Rapaka, Ph.D.;

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71 OPIATE RECEPTOR SUBTYPES AND BRAIN FUNCTION. Roger M. Brown,

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72 RELAPSE AND RECOVERY IN DRUG ABUSE. Frank M. Tims, Ph.D., and

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73 URINE TESTING FOR DRUGS OF ABUSE. Richard L. Hawks, Ph.D., and

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74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG ABUSE. Stephen I.

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75 PROGRESS IN OPIOIO RESEARCH. PROCEEDINGS OF THE 1986 INTERNATIONAL NARCOTICS RESEARCH CONFERENCE. John W. Holaday, Ph.D.;

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76 PROBLEMS OF DRUG DEPENDENCE, 1986. PROCEEDINGS OF THE 48TH

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IN PRESS

77 ADOLESCENT DRUG ABUSE: ANALYSES OF TREATMENT RESEARCH.

Elizabeth R. Rahdert, Ph.D. and John Grabowski, Ph.D., eds.

78 THE ROLE OF NEUROPLASTICITY IN THE RESPONSE TO DRUGS

David P. Friedman, Ph.D., and Doris H. Clouet, Ph.D., eds.

216

U.S. GOVERNMENT PRINTING OFFICE: 1990 – 2 7 6 - 2 0 7 / 2 0 8 6 5
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Alcohol, Drug Abuse, and Mental Health Administration

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