Marijuana and Medicine - Chapter 2 - Cannabinoids and Animal Physiology

CANNABINOIDS AND ANIMAL PHYSIOLOGY

CHAPTER 2. CANNABINOIDS AND ANIMAL PHYSIOLOGY	2
Introduction	2
Cannabinoid Receptors	9
The Endogenous Cannabinoid System	13
Sites of Action	21
Cannabinoid Receptors and Brain Functions	25
Chronic Effects of THC	31
Cannabinoids and the Immune System	34
Conclusions and Recommendations	43
References	46

2.1

Chapter 2.
Cannabinoids and Animal Physiology

Introduction

Much has been learned since the publication of the 1982 IOM report on Marijuana and Health.^a Although it was clear then that most of the effects of marijuana were due to its actions on the brain, there was little information about how THC acted on brain cells (neurons), which cells were affected by THC, or even what general areas of the brain were most affected by THC. Too little was known about cannabinoid physiology to offer any scientific insights into the harmful or therapeutic effects of marijuana. That is no longer true. During the last 16 years, there have been major advances in what basic science discloses about the potential medical benefits of cannabinoids, the group of compounds related to THC. Many variants are found in the marijuana plant, and other cannabinoids not found in the plant have been chemically synthesized. Sixteen years ago, it was still a matter of debate as to whether THC acted nonspecifically by affecting the fluidity of cell membranes or whether a specific pathway of action was mediated by a receptor that responded selectively to THC (table 2.1).

Basic science is the wellspring for developing new medications and is particularly important for understanding a drug that has as many effects as marijuana. Even committed advocates of the medical use of marijuana do not claim that all the effects of marijuana are desirable for every medical use. But they do claim that the combination of specific effects of marijuana enhances its medical value. An understanding of those specific effects is what basic science can provide. The multiple effects of marijuana can be singled out and studied with the goals of evaluating the medical value of marijuana and cannabinoids in specific medical conditions, as well as minimizing unwanted side effects. An understanding of the basic mechanisms through which cannabinoids affect physiology permits more strategic development of new drugs and designs for clinical trials that are most likely to yield conclusive results.

Research on cannabinoid biology offers new insights into clinical use, especially given the scarcity of clinical studies that adequately evaluate the medical value of marijuana. For example, despite the scarcity of substantive clinical data, basic science has made it clear that cannabinoids can affect pain transmission and specifically that, cannabinoids interact with the brain's endogenous opioid system, an important system for the medical treatment of pain (see chapter 4).

^{a The field of neuroscience has grown substantially since the publication of the
1982 IOM report. The number of members in the Society for Neuroscience provide a rough
measure of the growth in research and knowledge about the brain: as of the middle of 1998,
there are over 27,000 members, more than triple the number in 1982.

2.2

The cellular machinery that underlies the response of the body and brain to
cannabinoids involves an intricate interplay of different systems. This chapter reviews
the components of that machinery with enough detail to permit the reader to compare what
is known about basic biology with specific indications proposed for marijuana. For some
readers' that will be too much detail. Those readers who do not wish to read the entire
chapter should, nonetheless, be mindful of the following key points in this chapter

· The most far-reaching of the recent advances in cannabinoid biology are the
identification of two types of cannabinoid receptors (CB₁ and CB₂)
and of anandamide, a substance naturally produced by the body that acts at the cannabinoid
receptor, and has effects similar to those of THC. The CB₁ receptor is found
primarily in the brain, and mediates the psychological effects of THC. The CB₂
receptor is associated with the immune system, its role remains unclear.
· The physiological roles of the brain cannabinoid system in humans are the subject of
much active research, and not fully known; however, cannabinoids likely have a natural
role in pain modulation, control of movement, and memory.
· Animal research has shown that the potential for cannabinoid dependence exists, and
cannabinoid withdrawal symptoms can be observed. However, both appear to be mild compared
to dependence and withdrawal seen with other drugs.
· Basic research in cannabinoid biology has revealed a variety of cellular pathways
through which potentially therapeutic drugs could act on the cannabinoid system. In
addition to the known cannabinoids, such drugs might include chemical derivatives of
plant-derived cannabinoids or of endogenous cannabinoids such as anandamide, but would
also include non-cannabinoid drugs that act on the cannabinoid system.

This chapter summarizes the basics of cannabinoid biology - as known today. It thus
provides a scientific basis for interpreting claims founded on anecdotes and for
evaluating the clinical studies of marijuana presented in chapter 4.

2.3

Table 2.1 Landmark Discoveries Since the 1982 IOM Report

Since the Previous IOM Report on
Marijuana in 1982:

A Decade of Landmark Discoveries

Year
Discovery
Primary Investigators

1986
Potent cannabinoid agonists are developed the key to discovering the receptor
M.R. Johnson and L.S. Melvin⁷⁵

1988
First conclusive evidence of specific cannabinoid receptors
A. Howlett and W. Devane³⁶

1990
The cannabinoid brain receptor (CB₁) is cloned, its DNA sequence is
identified, and its location in the brain is determined
L. Matsuda et al,¹⁰⁷ and M. Herkenham et al⁶⁰

1992
Anandamide is discovered - a naturally occurring substance in the brain that acts on
cannabinoid receptors
R. Mechoulam and W. Devane³⁷

1993
A cannabinoid receptor is discovered outside the brain; this receptor (CB₂)
is related to the brain receptor but is distinct
S. Munro¹¹²

1994
The first specific cannabinoid antagonist, SR141716A, is developed
M.Rinaldi-Carmona¹³²

1998
The first cannabinoid antagonist, SR144528, that can distinguish between CB₁
and CB₂ receptors discovered.
M. Rinaldi-Carmona¹³³

2.4

The Value of Animal Studies

Much of the research into the effects of cannabinoids on the brain is based on animal
studies. Many speakers in the public workshops associated with this study argued that
animal studies of marijuana are not relevant to humans. While animal studies are no
substitute for clinical trials, they are a necessary complement. Ultimately, every
biologically active substance exerts its effects at the cellular and molecular level, and
at this level, the evidence has shown remarkable consistency among mammals, even those as
different in body and mind as rats and humans. Animal studies typically provide
information about how drugs work that would not be obtainable in clinical studies. At the
same time, animal studies can never completely inform us about the full range of
psychological and physiological effects of marijuana or cannabinoids on humans.

The Active Constituents of Marijuana

⁹-THC and ⁸-THC are the only compounds in
the marijuana plant that show all the psychoactive effects of marijuana. Because ⁹-THC is much more abundant than ⁸-THC, the psychoactivity of
marijuana has been largely attributed to the effects of ⁹-THC 11-OH-⁹-THC is the primary product of ⁹-THC metabolism by the liver and is about three times
as potent as ⁹-THC.¹²⁸

There have been considerably fewer experiments with cannabinoids other than ⁹-THC although a few studies have
been done to examine whether other cannabinoids modulate the effects of THC or mediate the
non-psychological effects of marijuana. Cannabidiol (CBD) does not have the same
psychoactivity as THC, but it was initially reported to attenuate the psychological
response to THC in humans ^{81, 177} however, later studies reported that CBD did
not attenuate the psychological effects of THC.^{11, 69} One double-blind study of
eight volunteers reported that CBD can block the anxiety induced by high doses of THC (0.5
mg/kg).¹⁷⁷ There are numerous anecdotal reports claiming that marijuana with
relatively higher ratios of THC:CBD is less likely to induce anxiety in the user than
marijuana with low THC:CBD ratios; but, taken together, the results published thus far are
inconclusive.

The most significant effects of cannabidiol (CBD) seem to be its interference with drug
metabolism in the liver, including ⁹-THC
metabolism.^{14, 114} CBD exerts this effect by inactivating cytochrome P450s,
which are the most important class of enzymes that metabolize drugs. Like many P450
inactivators, CBD can also induce P450s after repeated doses.¹³ Experiments in
which mice were treated with CBD followed by THC showed that CBD treatment was associated
with a significant

2.5

increase in brain levels of THC and its major metabolites, most likely because of its
effects on decreasing the clearance rate of THC from the body ¹⁵

In mice, THC inhibits the release of luteinizing hormone (LH), the pituitary hormone
that triggers the release of testosterone from the testis in males, this effect is
increased when THC is given together with cannabinol or CBD.¹¹³

Cannabinol is considerably less active than THC in the brain, but studies of immune
cells have shown that it can modulate immune function (see section on Cannabinoids and the
Immune System). In mice, cannabinol lowers body temperature and increases sleep duration.¹⁷⁵

The Pharmacological Toolbox

A researcher needs certain key tools in order to understand how a drug acts on the
brain. To appreciate the importance of these tools, one must first understand some basic
principles of drug action. All recent studies have indicated that the behavioral effects
of THC are receptor-mediated.²⁷ Neurons in the brain are activated when a
compound binds to its receptor, which is a protein typically located on the cell surface.
Thus, THC will exert its effects only after binding to its receptor. In general, a given
receptor will accept only particular classes of compounds and will be unaffected by other
compounds.

Compounds that activate receptors are called agonists. Binding to a receptor
triggers an event or a series of events in the cell that results in a change in the cell's
activity, its gene regulation, or the signals that it sends to neighboring cells (figure
2. 1). This agonist-induced process is called signal transduction.

Another tool for drug research, which only recently became available for cannabinoid
research, are the receptor antagonists, so-called because they selectively bind to
a receptor that would have otherwise been available for binding to some other compound or
drug. Antagonists block the effects of agonists and are tools to identify receptor
functions by showing what happens when a receptor's normal functions are blocked. Agonists
and antagonists are both ligands; that is, they bind to receptors. Hormones,
neurotransmitters, and drugs can all act as ligands. Morphine and naloxone provide a good
example. A large dose of morphine acts as an agonist at opioid receptors in the brain and
interferes with, or even arrests, breathing. Naloxone, a powerful opioid antagonist,
blocks morphine's effects on opiate receptors, thereby allowing an overdose victim to
resume breathing normally. Naloxone itself has no effect on breathing.

Another key tool involves identifying the receptor protein and determining how it
works. That makes it possible to locate where a drug activates its receptor in the brain
-both the general region of the brain and the cell type where the receptor is. The way to
find a receptor for a drug in the brain is to make the receptor "visible" by
attaching a radioactive or fluorescent marker to the drug so that it can be detected.

2.6

Such markers show where in the brain it binds to the receptor, but this is not
necessarily the part of the brain where the drug ultimately has its greatest effects.

Because drugs injected into animals must be dissolved in a water-based solution, it is
easier to deliver water-soluble molecules than to deliver fat-soluble (lipophilic)
molecules such as THC. THC is so lipophilic that it can stick to glass and plastic
syringes used for injection. Because it is lipophilic, it readily enters cell membranes
and thus can cross the blood brain barrier easily. (This barrier insulates the brain from
many blood-borne substances.) Early cannabinoid research was hindered by the lack of
potent cannabinoid ligands (THC binds to its cannabinoid receptors rather weakly) and
because they were not readily water-soluble. The synthetic agonist, CP 55,940, which is
more water-soluble than THC, became the first useful research tool for studying
cannabinoid receptors because of its high potency, and the ability to label it with a
radioactive molecule, which enabled researchers to trace its activity.

2.7

Figure 2.1 Diagram of neuron with synapse

How receptors work: Individual nerve cells, or neurons, both send and receive cellular
signals to and from neighboring neurons, but for the purposes of this diagram only one
activity is indicated for each cell. Neurotransmitter molecules (shown as black dots) are
released from the neuron terminal and move across the gap between the "sending"
and "receiving" neurons. A signal is transmitted to the receiving neuron when
the neurotransmitters has bound to the receptor on its surface. The effects of a
transmitted signal include:

·Changing the cell's permeability to ions such as calcium and potassium.

·Turning a particular gene on or off.

·Sending a signal to another neuron.

·Increasing or decreasing the responsiveness of the cell to other cellular signals.

Those effects can lead to cognitive, behavioral, or physiological changes, depending on
which neuronal system is activated.

The expanded view of the synapse illustrates a variety of ligands, that is,
molecules that bind to receptors. Anandamide is a substance produced by the body that
binds to and activates cannabinoid receptors; it is an endogenous agonist. THC can
also bind to and activate cannabinoid receptors, but is not naturally found in the body;
it is an exogenous agonist. SR141617A binds to, but does not activate cannabinoid
receptors. In this way, it prevents agonists, such as anandamide and THC, from activating
cannabinoid receptors by binding to the receptors without activating them; SR141617A is an
antagonist, but it is not normally produced in the body. Endogenous antagonists,
that is, those normally produced in the body, might also exist, but none have been
identified.

2.8

Cannabinoid Receptors

The cannabinoid receptor is a typical member of the largest known family of receptors:
the G-protein-coupled receptors with their distinctive pattern in which the receptor
molecule spans the cell membrane seven times (figure 2.2). For excellent recent reviews of
cannabinoid receptor biology, see Childers and Breivogel,²⁷ Abood and Martin,¹
Felder and Glass,⁴³ and Pertwee.¹²⁴ Cannabinoid receptor ligands
bind reversibly (they bind to the receptor briefly and then dissociate) and
stereoselectively (when there are molecules that are mirror images of each other, only one
version activates the receptor). Thus far, two cannabinoid receptor subtypes (CB₁
and CB₂) have been identified, of which only CB₁ is found in the
brain.

The cell responds in a variety of ways when a ligand binds to the cannabinoid receptor
(figure 2.3). The first step is activation of G-proteins, the first components of the
signal-transduction pathway. That leads to changes in several intercellular components -
such as cyclic AMP and calcium and potassium ions - which ultimately produce the changes
in cell functions. The final result of cannabinoid receptor stimulation depends on the
particular type of cell, the particular ligand, and the other molecules t hat might be
competing for receptor binding sites. Different agonists vary in binding potency,
which determines the effective dose of the drug, and efficacy, which determines the
maximal strength of the signal that they transmit to the cell. The potency and efficacy of
THC are both relatively lower than those of some synthetic cannabinoids; in fact,
synthetic compounds are generally more potent and efficacious than endogenous agonists.

CB₁ receptors are extraordinarily abundant in the brain. They are more
abundant than most other G-protein-coupled receptors and ten times more abundant than mu
opioid receptors, the receptors responsible for the effects of morphine.¹⁴⁸

2.9

Figure 2.2 Cannabinoid receptors

Receptors are proteins, and proteins are made up of strings of amino acids. Each circle
in the diagram represents one amino acid. The shaded bar represents the cell membrane,
which like all cell membranes in animals, is largely composed of phospholipids. Like many
receptors, the cannabinoid receptors span the cell membrane; some sections of the receptor
protein are outside the cell membrane (extrucellular), some are inside (intracellular).
THC, anandamide, and other known cannabinoid receptor agonists bind to the extracellular
portion of the receptor, thereby activating the signal pathway inside the cell.

The CB₁ molecule is larger than CB₂. The receptor molecules are
most similar in four of the seven regions where they are embedded in the cell membrane
(known as the transmembrane regions). The intracellular loops of the two cannabinoid
receptor sub-types are quite different, which might affect the cellular response to the
ligand, because these loops are known to mediate G-protein signaling - that is, the next
step in the cell signaling pathway after the receptor. Receptor homology between the two
receptor sub-types is 44 percent for the full length protein, and 68 percent within the
seven transmembrane regions. The ligand binding sites are typically defined by the
extracellular loops and the transmembrane regions.

2.10

Figure 2.3 How cannabinoids affected neuron signals

Figure legend. Intracellular events that happen when cannabinoid agonists bind to
receptors. Cannabinoid receptors are embedded in the cell membrane where they are coupled
to G-proteins (G) and the enzyme, adenylyl cyclase (AC). Receptors are activated when they
bind with ligands such as anandamide or THC in this case. This triggers a variety of
reactions including inhibition ((-)) of AC which decreases the production of cAMP and
cellular activities dependent on cAMP, opening potassium (K+) channels which decreases
cell firing, and closing calcium (Ca2+) channels which decreases the release of
neurotransmitters. These changes can influence cellular communication.

2.11

The cannabinoid receptor in the brain is a protein referred to as CB₁. The
peripheral receptor (outside the nervous system), CB₂, is most abundant on
cells of the immune system and is not generally found in the brain.^{43, 124}
Although no other receptor subtypes have been identified, there is a genetic variant known
as CB₁A (such variants are somewhat different proteins that have been produced
by the same genes via alternative processing). In some cases, proteins produced via
alternative splicing have different effects on cells. It is not yet known whether there
are any functional differences between the two, but the structural differences raise the
possibility.

CB₁ and CB₂ are similar, but not as similar as members of many
other receptor families are to each other. On the basis of a comparison of the sequence of
amino acids that make up the receptor protein, the similarity of the CB₁ and CB₂
receptors is 44 percent (figure 2.2). The differences between the two receptors indicate
that it should be possible to design therapeutic drugs that would act only on one or the
other receptor and thus would activate or attenuate (block) the appropriate cannabinoid
receptors. This offers a powerful method for producing biologically selective effects. In
spite of the difference between the receptor subtypes, most cannabinoid compounds bind
with similar affinity^b to both CB₁ and CB₂
receptors. One exception is the plant-derived compound, cannabinol, which shows greater
binding affinity for CB₂ than for CB₁,¹¹² although
another research group has failed to substantiate that observation.129 Other exceptions
include the synthetic compound, WIN 55,212-2, which shows greater affinity for CB₂
than CB₁, and the endogenous ligands, anandamide and 2-AG, which show greater
affinity for CB₁ than CB₂.⁴³ The search for compounds
that bind to only one or the other of the cannabinoid receptor types has been under way
for several years and has yielded a number of compounds that are useful research tools and
have potential for medical use.

Cannabinoid receptors have been studied most in vertebrates, such as rats and mice.
However, they are also found in invertebrates, such as leeches and mollusks.¹⁵⁶
The evolutionary history of vertebrates and invertebrates diverged more than 500 million
years ago, so cannabinoid receptors appear to have been conserved throughout evolution at
least this long. This suggests that they serve an important and basic function in animal
physiology. In general, cannabinoid receptor molecules are similar among different
species.¹²⁴ Thus, cannabinoid receptors likely fill many similar functions in a
broad range of animals, including humans.

^{b Affinity is a measure of how avidly a drug binds to a receptor. The higher
the affinity of a drug, the higher its potency; that is, lower doses are needed to produce
its effects.

2.12

The Endogenous Cannabinoid System

For any drug for which there is a receptor, the logical question is, "Why does
this receptor exist?" The short answer is that there is probably an endogenous
agonist (that is, a compound that is naturally produced in the brain) that acts on that
receptor. The long answer begins with a search for such compounds in the area of the body
that produce the receptors and ends with a determination of the natural function of those
compounds. So far, the search has yielded several endogenous compounds that bind
selectively to cannabinoid receptors. The best studied of them are anandamide³⁷
and arachidonyl glycerol (2-AG).¹⁰⁸ However, their physiological roles are not
yet known.

Initially, the search for an endogenous cannabinoid was based on the premise that its
chemical structure would be similar to that of THC; that was reasonable, in that it was
really a search for another "key" that would fit into the cannabinoid receptor
"keyhole," thereby activating the cellular message system. One of the intriguing
discoveries in cannabinoid biology was how chemically different THC and anandamide are. A
similar search for endogenous opioids (endorphins) also revealed that their chemical
structure is very different from the plant-derived opioids, opium and morphine.

Further research has uncovered a variety of compounds with quite different chemical
structures that can activate cannabinoid receptors (table 2.2 and figure 2.4) It is not
yet known exactly how anandamide and THC bind to cannabinoid receptors. Knowing this
should permit more precise design of drugs that selectively activate the endogenous
cannabinoid systems.

Anandamide

The first endogenous cannabinoid to be discovered was arachidonylethanolamine, named
anandamide from the Sanskrit word ananda, meaning "bliss."³⁷
Compared with THC, anandamide has only moderate affinity for CB₁, and is
rapidly metabolized by amidases (enzymes that remove amide groups). Despite its short
duration of action, anandamide shares most of the pharmacological effects of THC^37,
152 Rapid degradation of active molecules is a feature of neurotransmitter systems
that allows them control of signal timing by regulating the abundance of signaling
molecules. It creates problems for interpreting the results of many experiments and might
explain why in vivo studies with anandamide injected into the brain have yielded
conflicting results.

Anandamide appears to have both central (in the brain) and peripheral (in the rest of
the body) effects. The precise neuroanatomical localization of anandamide and the enzymes
that synthesize it are not yet known. This information will provide

2.13

essential clues to the natural role of anandamide and an understanding of the brain
circuits in which it is a neurotransmitter. The importance of knowing specific brain
circuits that involve anandamide (and other endogenous cannabinoid ligands) is that such
circuits are the pivotal elements for regulating specific brain functions, such as mood,
memory, and cognition. Anandamide has been found in numerous regions of the human brain:
hippocampus (and parahippocampic cortex), striatum, and cerebellum; but it has not been
precisely identified with specific neuronal circuits. CB₁ receptors are
abundant in these regions, and this further implies a physiological role for endogenous
cannabinoids in the brain functions controlled by these areas. But, substantial
concentrations of anandamide are also found in the thalamus, an area of the brain that has
relatively few CB₁ receptors.¹²⁴

Anandamide has also been found outside the brain. It has been found in spleen, which
also has high concentrations of CB₂ receptors; and small amounts have been
detected in heart.⁴⁴

In general, the affinity of anandamide for cannabinoid receptors is only one fourth to
one-half that of THC (see table 2.3). The differences depend on the cells or tissue that
are tested and on the experimental conditions, such as the binding assay used (reviewed by
Pertwee¹²⁴).

The molecular structure of anandamide is relatively simple, and it can be formed from
arachidonic acid and ethanolamine. Arachidonic acid is a common precursor of a group of
biologically active molecules known as eicosanoids, including prostaglandins.^c
Although anandamide can be synthesized in a variety of ways, the physiologically relevant
pathway seems to be through enzymatic cleavage of N-arackidonyl-phosphatidyl-ethanolamine
(NAPE), which yields anandamide and phosphatidic acid (reviewed by Childers and Breivogel²⁷).

Anandamide can be inactivated in the brain via two mechanisms. In one, anandamide is
enzymatically cleaved to yield arachidonic acid and ethanolamine- the reverse of what was
initially proposed as its primary mode of synthesis. In the other, it is inactivated
through neuronal uptake-i.e.., by being transported into the neuron, which prevents its
continuing activation of neighboring neurons.

^{c Eicosanoids all contain a chain of 20 carbon atoms, and are named after
eikosi, the Greek word for twenty.

2.14

Table 2.2 Compounds that bind to cannabinoid receptors

Compounds That Bind to Cannabinoid Receptors^d

Compound
Properties

Agonists (Receptor activators)

Plant-derived compounds

⁹-THC
Main psychoactive cannabinoid in the marijuana plant; largely responsible for
psychological and physiological effects. (Except in discussions of the different forms of
THC, THC is used as a synonym for ⁹-THC

⁸-THC
Slightly less potent than ⁹-THC
and much less abundant in the marijuana plant, but otherwise similar.

11-OH-⁹-THC
Bioactive compound formed when the body breaks down ⁹-THC. Presumed to be responsible for some of the
effects of marijuana.

Cannabinoid agonists found in animals

anandamide

(arachidonyl-

ethanolamide)
Found in animals ranging from mollusks to mammals. Appears to be primary endogenous
cannabinoid agonist in mammals. Chemical structure very different from plant cannabinoids,
and related to prostaglandins.

2-AG

(arachidonyl glycerol)
Endogenous agonist. Structurally similar to anandamide. More abundant but less potent
than anandamide.

THC analogues

Dronabinol
Synthetic THC. Marketed in the US under the name Marinol® for nausea associated with
chemotherapy and for AIDS-related wasting.

Nabilone
THC analogue. Marketed in the UK under the name Cesamet® for the same indications as
dronabionol.

CP 55,940
Synthetic cannabinoid; THC analogue; that is, it is structurally similar to THC

Levonantradol
THC analogue.

HU-210
THC analogue, 100-800 fold greater potency than THC.⁹⁷

Chemical structure unlike THC or anandamide

WIN-55,212
Chemical structure different from known cannabinoids, but binds to both cannabinoid
receptors. Chemically related to cyclo-oxygenase inhibitors, which include
anti-inflammatory drugs.

Antagonists (Receptor Blockers)

SR 141716A
Synthetic CB₁ antagonist, developed in 1994.¹³²

SR 144528
Synthetic CB₂ antagonist; developed in 1997.¹³³

^d Sources: Mechoulam et al. 1998 ¹⁰⁹, Felder and
Glass 1998⁴³; BMA 1997¹⁷

2.15

Figure 2.4 Chemical structures of compounds that bind to cannabinoid receptors

Figure Legend. Selected cannabinoid agonists, or molecules that bind to and activate
cannabinoid receptors. THC is the primary psychoactive molecule found in marijuana. CP
55,940 is a THC-analogue; that is, its chemical structure is related to THC. Anandamide
and 2-arachidonyl glycerol (2-AG) are endogenous molecules, meaning they are naturally
produced in the body. Although the chemical structure of WIN 55,212 is very different from
either THC or anandamide, it is also a cannabinoid agonists.

2.16

Table 2.3 Comparison of cannabinoid receptor agonists

Potency can be measured in a variety of ways, from behavioral to physiological to
cellular. This table shows potency in terms of receptor binding, which is the most broadly
applicable to the many possible actions of cannabinoids. For example, anandamide binds to
the cannabinoid receptor only about half as avidly as does THC. Measures of potency might
include effects on activity (behavioral) or hypothermia (physiological).

The apparently low potency of 2-AG may, however, be misleading. A study published late
in 1998, reports that 2-AG is found with two other, closely related compounds that, by
themselves, are biologically inactive, but in the presence of those two compounds, 2-AG is
only three times less active than THC.⁹ Further, 2-AG is much more abundant
than anandamide, although the biological significance of this remains to be determined.

Receptor Binding in Brain Tissue¹²⁴

Compound
Potency Relative to THC

CP 55,940
59

⁹-THC
1

Anandamide
0.47

2-AG
0.08

2.17

Other endogenous agonists

Several other endogenous compounds that are chemically related to anandamide and that
bind to cannabinoid receptors have been discovered, one of which is 2-AG.¹⁰⁸
2-AG is closely related to anandamide and is even more abundant in the brain. At time of
this writing, all known endogenous cannabinoid receptor agonists (including anandamide)
are eicosanoids, which are arachidonic acid metabolites. Arachidonic acid (a free fatty
acid) is released via hydrolysis of membrane phospholipids.

Other, non-eicosanoid, compounds that bind cannabinoid receptors have recently been
isolated from brain tissue, but they have not been identified, and their biological
effects are under investigation. This is a fast-moving field of research, and no review
over six months old can be fully up-to-date.

The endogenous compounds that bind to cannabinoid receptors probably perform a broad
range of natural functions in the brain. This neural signaling system is rich and complex,
and has many subtle variations' many of which await discovery. In the next few years, much
more will likely by known about these naturally occurring cannabinoids.

Some effects of cannabinoid agonists are receptor-independent. For example, both THC
and CBD can be neuroprotective through their antioxidative activity; that is, they can
reduce the toxic forms of oxygen that are released when cells are under stress.⁵⁴
Other likely examples of receptor-independent cannabinoid activity are modulation of
activation of membrane-bound enzymes (e.g., ATPase), arachidonic acid release, and
perturbation of membrane lipids. An important caution in interpreting those reports is
that concentrations of THC or CBD used in cellular studies, such as these, are generally
much higher than the concentrations of THC or CBD in the body that would likely be
achieved by smoking marijuana.

Novel targets for therapeutic drugs

Drugs that alter the natural biology of anandamide, or other endogenous cannabinoids,
might have therapeutic uses (table 2.4). For example, drugs that selectively inhibit
neuronal uptake of anandamide would increase the brain's own natural cannabinoids and
mimic some of the effects of THC. A number of important psychotherapeutic drugs act by
inhibiting neurotransmitter uptake. For example, antidepressants like fluoxetine
(Prozac®) inhibit serotonin uptake, and are known as selective serotonin re-uptake
inhibitors, or SSRI's. Another way to alter levels of endogenous cannabinoids would be to
develop drugs that act on the enzymes involved in anandamide synthesis. Some
anti-hypertensive drugs work by inhibiting

2.18

enzymes involved in the synthesis of endogenous hypertensive agents. Fore example,
anti-converting enzyme (ACE) inhibitors are used in hypertensive patients to interfere
with the conversion of angiotensin I, which is inactive, to the active hormone,
angiotensin II

2.19

Table 2.4. Cellular processes that can be targeted for drug development

TABLE LEGEND. Endogenous cannabinoids are part of a cellular signaling system. This
table lists categories of natural processes that regulate such systems, and shows the
results of altering those processes.

Cellular Processes That Can Be Targeted for Drug
Development

Drug action
.
Biological Result

Block synthesis
Synthesis of bioactive compounds is a continuous process and is one means by which
concentrations of that compound are regulated.
Weaker signal, due to decreased agonist concentration

Inhibit degradation
Chemical breakdown is one method the body uses to inactivate endogenous substances.
Stronger signal, due to increased agonist concentration increased.

Facilitate neuronal uptake
Neuronal uptake is one of the natural ways in which a receptor agonist is inactivated.
Stronger signal, due to increased amount of time during which agonist is
present in the synapse where it can stimulate the receptor

2.20

Sites of Action

Cannabinoid receptors are particularly abundant in some areas of the brain. The normal
biology and behavior associated with these brain areas are consistent with the behavioral
effects produced by cannabinoids (table 2.5 and figure 2.5). The highest receptor density
is found in cells of the basal ganglia that project locally and to other brain regions.
These cells include the substantia nigra pars reticulata, entopeduncular nucleus,
and globus pallidus, regions that are generally involved in coordinating body
movements. Patients with Parkinson and Huntington disease tend to have impaired functions
in these regions.

CB₁ receptors are also abundant in the putamen, part of the relay system
within the basal ganglia that regulates body movements, the cerebellum, which coordinates
body movements; the hippocampus, which is involved in learning, memory, and response to
stress; and the cerebral cortex, which is concerned with the integration of higher
cognitive functions.

CB₁ receptors are found on various parts of neurons, including the axon,
cell bodies, terminals, and dendrites.^{57, 165} Dendrites are generally the
"receiving" part of a neuron, and receptors on axons or cell bodies generally
modulate other signals. Axon terminals are the "sending" part of the neuron.

Cannabinoids like the inhibitory neurotransmitter -aminobutyric acid (GABA) -tend to inhibit neurotransmission,
although the results are somewhat variable. In some cases, cannabinoids diminish the
effects of the inhibitory neurotransmitter, -aminobutyric acid (GABA),¹⁴⁴in other cases, cannabinoids can
augment the effects of GABA.¹²⁰ The effect of activating a receptor depends on
where it is found on the neuron: if cannabinoid receptors are presynaptic (on the
"sending" side of the synapse) and inhibit the release Of GABA, cannabinoids
would diminish GABA effects; the net effect would be stimulation. However, if cannabinoid
receptors are postsynaptic (on the "receiving" side of the synapse) and on the
same cell as GABA receptors, they will probably mimic the effects of GABA; in that case,
the net effect would be inhibition.^{120, 144, 160}

CB₁ is the predominant brain cannabinoid receptor. CB₂ receptors
have not generally been found in the brain, but there is one isolated report suggesting
some in mouse cerebellum.¹⁵⁰ CB₂ is found primarily on cells of the
immune system. CB₁ receptors are also found in immune cells, but CB₂
is considerably more abundant there (table 2.6) (reviewed by Kaminski⁸⁰ in
1998).

As can be appreciated in the next section, the presence of cannabinoid systems in key
brain regions is strongly tied to the functions and pathology associated with those
regions. The clinical value of cannabinoid systems is best understood in the context of
the biology of these brain regions.

2.21

Table 2.5 Brain regions in which cannabinoid receptors are abundant^e

Brain Region
Functions Associated with Region

Brain regions in which cannabinoid receptors are abundant

Basal ganglia

Substantia nigra pars reticulate

Entopeduncular nucleus

Globus pallidus

Putamen

Movement control

Cerebellum
Body-movement coordination

Hippocampus
Learning and memory, stress

Cerebral cortex, especially cingulate, frontal, and parietal regions
Higher cognitive functions

Nucleus accumbens
Reward center

Brain regions in which cannabinoid brain receptors are moderately
concentrated

Hypothalamus
Body housekeeping functions (body-temperature regulation, salt and water balance,
reproductive function)

Amygdala
Emotional response, fear

Spinal cord
Peripheral sensation, including pain

Brain Stem
Sleep and arousal, temperature regulation, motor control

Central gray
Analgesia

Nucleus of the solitary tract
Visceral sensation, nausea and vomiting

^e Based on reviews by Pertwee 1997 ¹²⁴ and Herkenham 1995⁵⁷
This table will be accompanied by a figure.

2.22

Figure 2.5. Location of brain regions in which cannabinoid receptors are abundant.

See table 2.5 for summary of functions associated with those regions.

2.23

Table 2.6 Summary table of cannabinoid receptors

.
CB₁
CB₂

Effects of various cannabinoids

⁹-THC
Agonist
Weak antagonist

Anandamide
Agonist
Agonist

Cannabinol (CBN)
Weak agonist
Agonist; greater affinity for CB₂ than for CB₁

Cannabidiol (CBD)
Does not bind to receptor
Does not bind to receptor

Receptor distribution

Areas of greatest abundance
Brain
Immune system, especially B cells and natural killer cells

2.24

Cannabinoid Receptors and Brain Functions

Motor effects

Marijuana affects psychomotor performance in humans. The effects depend both on the
nature of the task and the experience with marijuana. In general, effects are clearest in
steadiness (body sway and hand steadiness) and in motor tasks that require attention. The
results of testing Cannabinoids in rodents are much clearer.

Cannabinoids clearly affect movement in rodents, but the effects depend on the dose:
low doses stimulate and higher doses inhibit locomotion.^{111, 159} Cannabinoids
mainly inhibit the transmission of neural signals, and they inhibit movement through their
actions on the basal ganglia and cerebellum, where cannabinoid receptors are particularly
abundant (figure 2.6a and 2.6b). Cannabinoid receptors are also found in the neurons that
project from the striatum and subthalamic nucleus, which inhibit and stimulate movement,
respectively.^{58, 101}

Cannabinoids decrease both the inhibitory and stimulatory inputs to the substantia
nigra, and therefore might provide dual regulation of movement at this nucleus. In the
substantia nigra, Cannabinoids decrease transmission from both the striatum and the
subthalamic nucleus.141 The globus pallidus has been implicated in mediating the
cataleptic effects of large doses of Cannabinoids in rats.¹²⁶ (Catalepsy is a
condition of diminished responsiveness usually characterized by trancelike states and waxy
rigidity of the muscles.) Several other brain regions - the cortex, the cerebellum, and
the neural pathway from cortex to striatum - are also involved in the control of movement
and contain abundant cannabinoid receptors.^{52, 59, 101} They are, therefore,
possible additional sites that might underlie the effects of Cannabinoids on movement.

2.25

Figure 2. 6a & b Diagrams showing motor regions of the brain

Figure 2.6. Basal ganglia are a group of three brain regions, or nuclei - caudate,
putamen, and globus pallidus. Figure 2.6a is a 3-dimensional view showing the
location of those nuclei in the brain. Figure 2.6b shows those structures in a vertical
cross-sectional view The major output pathways of the basal ganglia arise from the globus
pallidus and pars reticula of the substantia nigra. Their main target is the
thalamus.

2.26

Memory effects

One of the primary effects of marijuana in humans is disruption of short-term memory.⁶⁸
That is consistent with the abundance of CB₁ receptors in the hippocampus, the
brain region most closely associated with memory. The effects of THC resemble a temporary
hippocampal lesion.⁶³ Deadwyler and colleagues have demonstrated that
cannabinoids decrease neuronal activity in the hippocampus and its inputs ^{23, 24, 83}
In vitro, several cannabinoid ligands and endogenous cannabinoids can block the cellular
processes associated with memory formation.^{29, 30, 116, 157, 163} Furthermore,
cannabinoid agonists inhibit release of several neurotransmitters: acetylcholine from the
hippocampus,^49,50,51 norepinephrine from human and guinea pig (but not rat or
mouse) hippocampal slices,¹⁴³ and glutamate in cultured hippocampal cells.¹⁴⁴
Cholinergic and noradrenergic neurons project into the hippocampus, but circuits within
the hippocampus are glutamatergic.^f Thus, cannabinoids could block
transmission both into and within the hippocampus by blocking presynaptic neurotransmitter
release.

Pain

After nausea and vomiting, chronic pain was the condition cited most often to the IOM
study team as a medical use for marijuana. Recent research presented below has shown
intriguing parallels with anecdotal reports of the modulating effects of cannabinoids on
pain - both the effects of cannabinoids acting alone and the effects of their interaction
with opioids.

Behavioral Studies

Cannabinoids reduce reactivity to acute painful stimuli in laboratory animals. In
rodents, cannabinoids reduced the responsiveness to pain induced through various stimuli,
including thermal, mechanical, and chemical stimuli.^{12, 19, 46, 72, 96, 154, 174}
Cannabinoids were comparable with opiates in potency and efficacy in these expeniments. ^12,
72

Cannabinoids are also effective in rodent models of chronic pain. Herzberg and
coworkers found that cannabinoids can block allodynia and hyperalgesia

^{fNeurons are often defined by the primary neurotransmitter released at their
terminals. Thus, cholinergic neurons release acetylcholine, noradrenergic neurons
release noradrenalin (also known as norepinephrine), and glutamergic neurons release
glutamate.

2.27

associated with neuropathic pain in rats.¹¹⁷ (Allodynia refers to pain
elicited by stimuli that are normally innocuous; hyperalgesia refers to abnormally
increased reactivity to pain.) This is an important advance, because chronic pain
frequently results in a series of neural changes that increase suffering due to allodynia,
hyperalgesia, and spontaneous pain; furthermore' some chronic pain syndromes are not
amenable to therapy, even with the most powerful narcotic analgesics.¹⁰

Pain perception is controlled mainly by neurotransmitter systems within the central
nervous system, and cannabinoids clearly play a role in the control of pain in those
systems.⁴⁵ However, pain-relieving and pain-preventing mechanisms also occur in
peripheral tissues, and endogenous cannabinoids appear to play a role in peripheral
tissues. Thus, the different cannabinoid receptor subtypes might act synergistically.
Experiments in which pain is induced by injecting dilute formalin into a mouse's paw have
shown that anandamide and palmitylethanolamide (PEA) can block peripheral pain.^{22, 73
22} Anandamide acts primarily at the CB₁ receptor, whereas PEA has been
proposed as a possible CB₂ agonist, in short, there might be a biochemical
basis for their independent effects. When injected together, the analgesic effect is
stronger than that of either alone. That suggests an important strategy for the
development of a new class of analgesic drug: a mixture of CB₁ and CB₂
agonists. Because there are few, if any, CB₂ receptors in the brain, it might
be possible to develop drugs that enhance the peripheral analgesic effect while minimizing
the psychological effects.

2.28

Neural sites of altered responsiveness to painful stimuli

The brain and spinal cord mediate cannabinoid analgesia. A number of brain areas
participate in cannabinoid analgesia and support the role of descending pathways (neural
pathways that project from the brain to the spinal cord).^{103, 105} Although more
work is needed to produce a comprehensive map of the sites of cannabinoid analgesia, it is
clear that the effects are limited to particular areas, most of which have an established
role in pain.

Specific sites where cannabinoids act to affect pain processing include the
periaqueductal gray,¹⁰⁴ the rostral ventral medulla, ^{105, 110} and
the thalamic nucleus submedius,¹⁰² the thalamic ventroposterolateral nucleus,¹⁰²
dorsal horn of the spinal cord,^{64, 65} and peripheral sensory nerves.^{64,
65, 66, 131} Those nuclei also participate in opiate analgesia. Although similar to
opiate analgesia, cannabinoid analgesia is not mediated by opioid receptors; morphine and
cannabinoids sometimes act synergistically, and opioid antagonists generally have no
effect on cannabinoid induced analgesia.¹⁷¹ However, a kappa-receptor
antagonist has been shown to attenuate spinal, but not supraspinal, cannabinoid analgesia.^{153,
170, 171} (Kappa opioid receptors constitute one of the three major types of opioid
receptors; the other two types are mu and delta receptors.)

2.29

Neurophysiology and neurochemistry of cannabinoid analgesia

Because of the marked effects of cannabinoids on motor function, behavioral studies in
animals alone cannot provide sufficient grounds for the conclusion that cannabinoids
depress pain perception. Motor behavior is typically used to measure responses to pain,
but this behavior is itself affected by cannabinoids. Thus, experimental results include
an unmeasured combination of cannabinoid effects on motor and pain systems. The effects on
specific neural systems, however, can be measured at the neurophysiological and
neurochemical levels. Cannabinoids decrease the response of immediate-early genes (genes
that are activated in the early or immediate stage of response to a broad range of
cellular stimuli) to noxious stimuli in spinal cord, decrease response of pain neurons in
the spinal cord, and decrease the responsiveness of pain neurons in the ventral
posterolateral nucleus of the thalamus.^{67, 102} Those changes are mediated by
cannabinoid receptors, selective for pain neurons, and unrelated to changes in skin
temperature or depth of anesthesia, and they follow the time course of the changes in
behavioral responses to painful stimuli, but not the time course of motor changes.⁶⁷
Cannabinoids also modulate the responses of on-cells and off-cells in the rostral ventral
medulla in a manner that is very similar to that of morphine.^{55, 110} These
cells control pain transmission at the level of the spinal cord.

Endogenous cannabinoids modulate pain

Endogenous cannabinoids can modulate pain sensitivity, through both central and
peripheral mechanisms. For example, animal studies have shown that pain sensitivity can be
increased when endogenous cannabinoids are blocked from acting at CB₁ receptors
^{22, 62, 110, 130, 158} Administration of cannabinoid antagonists in either the
spinal cord ¹³⁰ or paw ²² increase the sensitivity of animals to
pain. In addition, there is evidence that cannabinoids also act at the site of injury to
reduce peripheral inflammation.¹³¹

Current data suggest the endogenous cannabinoid analgesic system might offer protection
against the long-lasting central hyperalgesia and allodynia that sometimes follow skin or
nerve injuries ^{130, 158} These results raise the possibility that therapeutic
interventions that alter the levels of endogenous cannabinoids might be useful for
managing pain in humans.

2.30

Chronic Effects of THC

Most substances of abuse produce tolerance, physical dependence, and withdrawal
symptoms. Tolerance is the most common response to repetitive use of the same drug (not
necessarily a drug of abuse) and is the condition in which, after repeated exposure to a
drug, increasing doses are needed to achieve the same effect. Physical dependence develops
as a result of tolerance (adaptation) produced by a resetting of homeostatic mechanisms in
response to repeated drug use. It is important to reiterate that the phenomena of
tolerance, dependence, and withdrawal are not associated uniquely with drugs of abuse.
Many medications that are not addicting can produce these types of effects; examples of
such medications include clonidine, propranolol, and tricyclic antidepressants. The
following sections discuss what is known about the biological mechanisms that underlie on
tolerance, reward, and dependence; clinical studies about those topics are discussed in
chapter 3.

Tolerance

Chronic administration of cannabinoids to animals results in tolerance to many of the
acute effects of THC, including memory disruption,³⁴ decreased locomotion,^2,
119 hypothermia,^{42, 125} neuroendocrine effects,¹³⁴ and
analgesia.⁴ Tolerance also develops to the cardiovascular and psychological
effects of THC and marijuana in humans (see also discussion in chapter 3).^{55, 56, 76}

Tolerance to cannabinoids appears to result from both pharmacokinetic (how the drug is
absorbed, distributed, metabolized, and excreted) and pharmacodynamic (how the drug
interacts with target cells) changes. Chronic treatment with the cannabinoid agonist, CP
55,940, increases the activity of the microsomal cytochrome P450 oxidative system.³¹
Because this is the system through which drugs are metabolized in the liver, this suggests
pharrnacokinetic tolerance. Chronic cannabinoid treatments also produce changes in brain
cannabinoid receptors and cannabinoid receptor mRNA levels, indicating that
pharmacodynamic effects are important, as well.

Most studies have found that brain cannabinoid receptor levels usually decrease after
prolonged exposure to agonists,^{42, 119, 136, 138} although some studies have
reported increases ¹³⁷ or no changes² in receptor binding in brain.
Differences among studies may be due to the particular agonist tested, the assay used,
brain region examined, or treatment time. For example, the THC analogue, levonantradol,
produces a greater desensitization of adenylyl cyclase inhibition than THC in cultured
neuroblastoma cells,⁴⁰ which may be explained by the efficacy differences
between these two agonists ^{18, 147} Furthermore, a time course study revealed
differences in the rates and magnitudes of receptor down-regulation across brain

2.31

regions.¹⁶ These findings suggest that tolerance to different effects of
cannabinoids develops at different rates

Chronic treatment with THC also produces variable effects on cannabinoid-mediated
signal transduction systems. Chronic THC treatment produces significant desensitization of
cannabinoid-activated G-proteins in a number of rat brain regions.¹⁴⁷ Moreover,
the time course of this desensitization varies across brain regions.¹⁶

It is difficult to extend the findings of these short-term animal studies to human
marijuana use In order to simulate long-term use, the doses used in animal studies are
higher than normally achieved by smoking marijuana. For example, the average human will
feel "high" after a 0.06 mg/kg injection of THC,¹¹⁸ compared to 10-20
mg/kg/day used in many chronic studies in rats. At the same time, doses of marijuana
needed to observe behavioral changes in rats (usually changes in locomotor behavior) are
substantially higher than doses at which people feel "high." In addition,
pharmacokinetics of THC distribution in the body are dramatically different between rats
and humans, as well as being highly dependent on the THC delivery system - that is,
whether it is inhaled, injected, or swallowed. Nevertheless, it is likely that some of the
same biochemical adaptations to chronic cannabinoid administration occur in both
laboratory animals and humans, but the magnitude of the effects in humans may be smaller
in proportion to the respective doses used.

Reward and dependence

Experimental animals that are given the opportunity to self-administer cannabinoids
generally do not choose to do so, which has led to the conclusion that they are not
reinforcing and rewarding.³⁸ However, behaviora^l95 and brain
stimulation⁹⁴ studies have shown that THC can be rewarding to animals. The
behavioral study used a "place-preference" test, in which an animal is given
repeated doses of a drug in one place, and is then given a choice between a place where it
did not receive the drug and one where it did; the animals chose the place where they
received the THC. These rewarding effects are highly dose-dependent. In all models
studied, cannabinoids are only rewarding at mid-range; doses that are too low are not
rewarding, doses that are too high can be aversive. Mice will self-administer the
cannabinoid agonist, WIN 55,212, but only at low doses.¹⁰⁶ This effect is
specifically mediated by CB₁ receptors, and indicates that stimulation of those
receptors is rewarding to the mice. Antagonism of cannabinoid receptors is also rewarding
in rats; in conditioned place-preference tests, animals show a preference for the place
they receive the cannabinoid antagonist, SR141716A, at both low and high doses.¹⁴⁰
Cannabinoids increase dopamine levels in the mesolimbic dopamine system of rats, a pathway
associated with reinforcement.^{25, 39, 161} However, the

2.32

mechanism by which THC increases dopamine levels appears to be different from that of
other abused drugs ^{51 g} (see chapter 3 for further discussion of
reinforcement).

Physical dependence on cannabinoids has only been observed under experimental
conditions of "precipitated withdrawal", in which animals are first treated
chronically with cannabinoids and then given the CB₁ antagonist, SR141716A.^3,
166 The addition of the antagonist accentuates any withdrawal effect by competing
with the agonist at receptor sites; that is, the antagonist helps to clear agonists off
and keep them off receptor sites. This suggests that, under normal cannabis use, the long
half-life and slow elimination from the body of THC, and the residual bioactivity of its
metabolite, 11-OH -THC, may prevent significant abstinence symptoms. The precipitated
withdrawal effects produced by SR141716A have some of the characteristics of opiate
withdrawal, but are not affected by opioid antagonists and affect motor systems
differently. An earlier study with monkeys also suggested that abrupt cessation of chronic
THC is associated with withdrawal symptoms,⁸ Monkeys in that study were trained
to work for food after which they were given THC on a daily basis; when the investigators
stopped administering THC, the animals stopped working for food.

A study in rats indicated that the behavioral cannabinoid withdrawal syndrome
correlates with stimulation of central amygdaloid corticotropin-releasing hormone release,
consistent with the consequences of withdrawal from other abused drugs.¹³⁵
However, the withdrawal syndrome for cannabinoids and the corresponding increase in
corticotropin-releasing hormone are only observed following administration of the CB₁
antagonist, SR 141716A, to cannabinoidtolerant animals;^{3, 166} The implications
of data based on precipitated withdrawal in animals for human cannabinoid abuse have not
been established.¹⁶⁶ Furthermore, acute administration of THC also produces
increases in corticotropin-releasing hormone and adrenocorticotropin release, both of
which are stress-related hormones.⁷¹ This set of withdrawal studies may explain
the generally aversive effects of cannabinoids in animals, and may indicate that the
increase in corticotropin-releasing hormone is merely a rebound effect. Thus, while
cannabinoids appear to be conforming to some of the neurobiological effects of other drugs
abused by humans, the underlying mechanisms of these actions and their significance in
determining the reinforcement and dependence liability of cannabinoids in humans remain
undetermined.

^g These increases in dopamine are due to increases in the firing rate
of dopamine cells in the ventral tegmental area by ⁹-THC⁴⁷. However, these increases in firing rate in the
ventral tegmental area could not be explained by increases in the firing of the A10
dopamine cell group, where other abused drugs have been shown to act⁵¹.

2.33

Cannabinoids and the Immune System

The human body protects itself from invaders such as bacteria and viruses through the
elaborate and dynamic network of organs and cells referred to as the immune system (see
box on Cells of the Immune System).

Cannabinoids, especially THC, can modulate the function of immune cells in various ways
- in some cases enhancing, and in others diminishing the immune response ⁸⁵
(summarized in table 2.7). However, the natural function of the cannabinoids in the immune
system is not known. Immune cells respond to cannabinoids in a variety of ways, depending
upon experimental factors such as drug concentration, timing of drug delivery to
leukocytes in relation to antigen stimulation, and the type of cell function analyzed.
Although the chronic effects of cannabinoids on the immune system have not been studied,
based on acute exposure studies in experimental animals it appears that the concentrations
of THC which modulate immunological responses are higher than those required for
psychoactivity.

2.34

Table 2.7 Effects of Cannabinoids on the Immune System

Drug Tested
Cell Types Tested or Type Drug of Animal Experiment
Drug Concen- tration ^a
Result
Reference

THC

2-AG

11-OH-THC

CBN
Lymphocytes and Splenocytes in vitro
0.1-30 µM
Higher doses suppress T cell proliferation
Luo, 1992;

Pross,1992*

Klein, 1985%;

Specter,1990&

Lee, 1995*

Herring, 1998

THC

2-AG

Anandamide
Lymphocytes and Splenocytes
0.1-25 µM
Lower doses increase T cell proliferation in vitro
Luo,1992; Lee,1995* Pross,1992*

Splenocytes in vitro
1-25 µM
Little or no effect on T cell proliferation
Lee,1995* Devane,1992

THC, 11-OH-THC AG-2
Splenocytes in vitro
3-30 µM
Decrease B cell proliferation
Klein,1985% Lee,1995*

THC

CP 55,940

WIN 55,212-2
Lymphocytes in vitro
0.1-100nM

[0.0001-0.1 µM]
Increase B cell proliferation
Derocq, 1995

THC
Mice were injected with drug
>5mg/kg
Antibody production was suppressed
Baczynsky, 1983 Schatz,1993

HU-210
>0.05 mg/kg
Titishov,1989

THC

11-OH-THC

CBD

CP55,940

CBN
Splenocytes in vitro
1-30µM
Antibody production was suppressed
Klein,1990 Baczynasky,1983 Kaminski,1994 Kaminski,1992 Herring,1998

THC
Rodents were injected with drug
3mg/kg/day for 25days

40mg/kg/day for 2 days
Repeated low doses or a high dose of THC suppress the activity of natural killer cells
Patel,1985 Klein,1987

THC 1l-OH-THC
Natural killer cells in vitro
0.1-32 µM
Doses of >=10 µM suppress natural killer cell cytolytic activity, doses <10 µM
produced no effect
Klein,1987 Luo,1989

THC
Peritoneal macrophages and monocytes
3-30 µM
Variable doses of THC suppress macophage functions in vitro
Lopez-Cepero, 1986

Specter,1991 Tang,1992

2.35

THC

CBD
Mice injected with drug; in one case, in vitro tests done on spleens
>5mg/kg for 4 days or 50 mg/kg every 5 days for up to 8 weeks
THC suppresses normal immune response, interferons failed to increase when exposed to
cytokine inducer, while CBD had no suppressive effect
Cabral,1986 Blanchard,1986

THC

CBD
Peripheral blood mononuclear cells in vitro
<0.1 µM
Increased interferon production
Warzl, 1991

30 µM
Decreases interferon production
.

THC

CBD
Splenocytes and T cells in vitro
10 µM
Both THC and CBD suppress IL-2 secretion and the number of IL-2 transcripts
Condie,1996

THC
Phorbol myristate acetate differentiated macrophage in vitro
10-20 µM
Increase in tumor necrosis factor production and IL-I supernatant bioactiviy
Shivers, 1994

THC
Endotoxin-activated macrophages in vitro
10-30 µM
Increase processing and release of IL-I rather than cellular production of the IL-I
Zhu, 1994

THC
Peritoneal macrophages in vitro
10-30 µM
Increased IL-I bioactivity
Klein, 1990

THC
Mice were injected with drug and either sublethal or lethal dose of Legionalla
pneumophilia
8mg/kg given before and after bacteria infection
Cytokine-mediated septic shock and death occurs with exposure to sublethal dose of the
bacteria
Klein, 1993 and 1994

Newton, 1994

< 5 mg/kg doses. or one 8 mg/kg or 4 mg/kg dose given before bacteria infection
Survival occurs, but with greater susceptiblity to infection when challenged with
bacteria and death when challenged with a lethal dose of bacteria

THC
Immuno-deficient mice injected with drug and herpes simplex virus
100mg/kg before and after virus infection
Two high doses of THC potentiates the effects of herpes simplex and enhances the
progression of death
Specter, 1991

100 mg/kg before virus infection
A single dose did not promote death

* cell density dependent;

* mitogen dependent;

% % serum dependent;

& dependent on timing of drug exposure relative to mitogen exposure.

^a Drug concentrations are given in the standard format of molarity (M). A
one molar solution is the molecular weight of the compound (in grams) dissolved in 1 liter
of water or other solvent. The molecular weight of THC is 314 so a 1 molar solution would
be 314 grams of THC dissolved in 1 liter of solution, a 10 µM solution would be 3.14 mg
THC/liter.

A 1-10 µM concentration will generally elicit a physiologically relevant response in
immune cell cultures. Higher doses are often suspected of not being biologically
meaningful, because they are a much larger dose than would ever be achieved in the body.
The doses listed in this table are, for the most part, very high. See text for further
discussion.

2.36

Cells of the Immune System

The various organs of the immune system are positioned throughout the body and include
bone marrow, thymus, lymph nodes and spleen. The cells of the immune system consist of
white blood cells, or leukocytes, which are formed in the bone marrow from stem cells
so-called because a great variety of cells descend from them (see below). There are two
kinds of leukocytes: Lymphocytes and phagocytes. Lymphocytes consist of B cells, T
cells,^h and natural killer cells (NK), and the major phagocytes include
monocytes, macrophages and neutrophils. Phagocytes have many important roles in the
immune response, but most significantly they initiate these responses by engulfing and
digesting foreign substances (e.g., bacteria, viruses, foreign proteins), or antigens,
that enter the body. Once digested, the antigen is exposed to specialized Iymphocytes
(i.e., B cells and T cells) so that antibodies and effector T cells can be produced to
help destroy any remaining antigens in the body. Antibodies are proteins produced
by B cells that bind to antigens and promote antigen destruction. effector T cells include
killer T cells which attack and kill antigen laden cells, and helper T cells, which
secrete special proteins called cytokines that promote antigen elimination. Natural killer
cells are specialized Iymphocytes that are also activated by antigen to either kill
infected targets or secrete immunoregulatory cytokines.

^h The B and T refer to where the cells mature, either in the bone marrow (B)
or thymus (T).

2.37

The CB₂ receptor gene, which is not expressed in the brain, is particularly
abundant in immune tissues, with an expression level 10-100 times higher than that of CB₁
In spleen and tonsils, the CB₂ mRNAⁱ content is equivalent to that
of CB₁ mRNA in the brain.⁴⁸ The rank order, from high to low, of CB₂
mRNA levels in immune cells is B-cells > natural killer cells >> monocytes >
polymorphonuclear neutrophil cells > T8 cells in T4 cells. In tonsils, the CB₂
receptors appear to be restricted to B-lymphocyte-enriched areas. In contrast, CB₁
receptors are mainly expressed in the central nervous system and, to a lower extent, in
several peripheral tissues such as adrenal gland, heart, lung, prostate, uterus, ovary,
testis, bone marrow, thymus and tonsils.

Cannabinoid Receptors and Intracellular Action in Immune Cells

CB₂ appears to be the predominant gene expressed in resting leukocytes.^78,
112 The level of CB₁ gene activity is normally low in resting cells but
increases with cell activation.³² Thus the CB₁ receptor might be
important only when immune responses are stimulated, but the physiological relevance of
this observation remains to be determined. Some of the cannabinoid effects observed in
immune systems, especially at high drug concentrations, are likely mediated through
non-receptor mechanisms, but these have not yet been identified.⁴

Ligand binding to either the CB₁ or CB₂ receptors inhibits
adenylate cyclase, an enzyme that is responsible for cAMP production, and is, thus, an
integral aspect of intracellular signal transduction (see figure 2.3).^{53, 79, 91,
122, 139, 151, 167} Increases in intracellular cAMP concentrations lead to immune
enhancement, while decreases lead to an inhibition of immune responses.⁷⁷
Cannabinoids inhibit the rise in intracellular cAMP that normally results from leukocyte
activation, and this might be the pathway through which cannabinoids suppress immune cell
functions.^{28, 74, 167} In addition, cannabinoids activate other molecular
pathways such as the nuclear factor-kB pathway and therefore these signals might be
modified in drug treated immune cells.^{33, 74}

ⁱ After a gene is transcribed it is often spliced and modified into
mRNA, or message RNA. The CB-2 mRNA is the gene "message" that moves from the
cell nucleus into the cytoplasm where it will be translated into the receptor protein.

2.38

T and B Cells

When stimulated by antigen, lymphocytes (see box on Cells of the Immune System) first
proliferate and then mature or differentiate to become potent effector cells, such as B
cells that release antibodies or T cells that release cytokines. The normal T cell
proliferation that is seen when human lymphocytes and mouse splenocytes (spleen cells) are
exposed to antigens and mitogens^j can be inhibited by THC, 11-OH-THC,
cannabinol, and 2-AG, as well as synthetic cannabinoid agonists such as CP 55,940, WIN
55,212 and HU-210 ^{61, 89, 93, 99, 127, 155} In contrast, one study testing
anandamide revealed little or no effect on T-cell proliferation.⁹³

However, these drug effects are variable, and depend on experimental conditions such as
the experimental drug dose used, mitogen used, percent of serum in the culture, and timing
of cannabinoid drug exposure. In general, lower doses of cannabinoids increase
proliferation while higher doses suppress proliferation. Doses that are effective in
suppressing immune function are typically greater than 10 µM in cell culture studies and
greater than 5 mg/kg in whole animal studies.⁸⁵ By comparison, at 0.05 mg/kg,
people will experience the full psychoactive effects of THC; however, because of their
high metabolic rates, small rodents frequently require drug doses that are 100-fold higher
than doses needed for humans to achieve comparable drug effects. Thus, the immune effects
of doses of cannabinoids higher than those ever experienced by humans, should be
interpreted with caution.^{89, 93, 93, 127, 155}

As with T cells, B cell proliferation can be suppressed by various cannabinoids, such
as THC, 11-OH-THC and 2-AG, but B cell proliferation is more inhibited at lower drug
concentrations than T cell proliferation.^{89, 93} Conversely, low doses of THC,
CP 55,940 and WIN 55,212-2 increase B-cell proliferation in cultured human cells exposed
to mitogen.³⁵ This effect possibly involves the CB₂ receptor,
because the effect appears to be the same when the CB₁ receptor was blocked by
the antagonist, SR-141716A (which does not block the CB₂ receptor). The reason
for the differences in cell responsiveness to cannabinoids is probably due to differences
in cell type and source; for example, B cells collected from mouse spleen might respond to
cannabinoids somewhat differently than B cells from human tonsils.

^jMitogens are substances that stimulate cell division (mitosis) and
cell transformation.

2.39

Natural Killer Cells

Repeated injections of relatively low doses of THC (3 mg/kg/day ^{121 k}) or
two injections of a high dose (40 mg/kg⁸⁶) suppress the ability of natural
killer (NK) cells to destroy foreign cells in rats and mice. THC can also suppress natural
killer cell cytolytic activity in cell cultures; 11-OH-THC, is even more potent.⁸⁶
In contrast, THC doses below 10µM had no effect on natural killer cell activity in mouse
cell cultures.⁹⁸

Macrophages

Macrophages perform various functions including phagocytosis (ingestion and destruction
of foreign substances), cytolysis, antigen presentation to lymphocytes, and production of
a variety of active proteins involved in destroying microorganisms, tissue repair and
modulation of immune cells. Those functions can be suppressed by THC doses similar to
those capable of modulating lymphocyte functions (see above).^{88, 109}

Cytokines

Cytokines are proteins produced by immune cells. When released from the producing cell
they can alter the function of other cells they come in contact with. In a sense, they are
like hormones. Thus, cannabinoids can either increase or decrease cytokine production
depending upon experimental conditions.

Certain cytokines, such as interferon- and interleukin-2 (IL-2) are produced by T helper-1 (Th1) cells. These
cytokines help to activate cell-mediated immunity and the killer cells that eliminate
microbes from the body (see Box on cells of the immune system). When injected into mice,
THC suppresses the production of those cytokines that modulate the host response to
infection (see below).¹¹⁵ Cannabinoids also modulate interferons induced by
viral infection,²¹ as well as other interferon inducers.⁸⁵
Furthermore, in human cell cultures, interferon production can be increased by low
concentrations, but decreased by high concentrations of either THC or cannabidiol. 6 In
addition to Th1 cytokines, cannabinoids also modulate the production of cytokines such as
interleukin-1 (IL-1), tumor necrosis factor (TNF), and interleukin-6 (IL-6). ^{145, 176}
At 8 mg/kg, THC can increase the in vivo mobilization of serum acute phase
cytokines including IL-1, TNF, and IL-6.⁹⁰ Finally, although these studies
suggest that cannabinoids can induce an increase in cytokines, other studies suggest that
they can also suppress cytokine production.⁸⁵ The different results might be
due to different cell culture conditions or because different cell lines were studied.

^k While 3 mg/kg would be a high dose for humans (see table 3.1); in
rodents, it is a low dose for immunological effects, and a moderate dose for behavioral
effects.

2.40

Antibody Production

Antibody production is an important measure of humoral immune function as contrasted
with cellular (cell-mediated immunity). Antibody production can be suppressed in mice
injected with relatively low doses of THC (>5 mg/kg) or HU210 (>0.05 mg/kg) and in
mouse spleen cell cultures exposed to a variety of cannabinoids, including THC, 11-OH-THC,
cannabinol, cannabidiol, CP 55,940, or HU-210.^{5, 6, 61, 78, 79, 84, 85, 142, 164}
However, the inhibition of antibody response by cannabinoids was only observed when
antibody-forming cells were exposed to T cell-dependent antigens (the responses require
functional T cells and macrophages as accessory cells). Conversely, antibody responses to
several T cell-independent antigens were not inhibited by THC, suggesting that the B cell
is relatively insensitive to inhibition by cannabinoids.¹⁴²

Resistance To Infection In Animals Exposed To Cannabinoids

Bacterial infections evaluated in mice demonstrated that THC can suppress resistance to
infection, although the effect depends upon the dose and timing of drug administration.
Mice pre-treated with THC (8 mg/kg) one day prior to infection with a sublethal dose of
the pneumonia-causing bacteria, Legionella pneumophilia, and then treated again one
day after the infection with THC, developed symptoms of cytokine-mediated septic shock and
died; control mice that were not pre-treated with THC became immune to repeated infection
and survived the bacterial challenge.⁹⁰ If only one injection of THC was given
or doses less than 5 mg/kg were used, all of the mice survived the initial infection, but
failed to survive a subsequent challenge with a lethal dose of the bacteria; hence these
mice failed to develop immune memory in response to the initial sublethal infection.⁸⁷
Note that these are very high doses, and are considerably higher than doses experienced by
marijuana users (see figure 3.1).¹¹⁵ In rats, doses of 4.0 mg/kg THC are
aversive⁹⁵

Few studies have been done to evaluate the effect of THC on viral infections, and this
is an area that needs further study.²⁰ Compared to healthy animals, THC might
have greater immunosuppressive effects in animals whose immune systems are severely
weakened. For example, a very high dose of THC (100 mg/kg) given twice, two days before
and after herpes simplex virus infection, was shown to be a co-factor with herpes simplex
virus in enhancing the progression to death in an immunodeficient mouse model infected
with a leukemia virus.⁸⁵ However, THC given as a single dose (100 mg/kg) two
days before herpes simplex virus infection did not promote the progression to death in
these animals. Hence, whether THC is immunosuppressive likely depends on the timing of THC
exposure relative to an infection.

2.41

Anti-inflammatory Effects

As discussed above, cannabinoid drugs can modulate the production of cytokines, which
are central to inflammatory processes in the body. In addition, several studies have shown
directly that cannabinoids can be anti-inflammatory. For example, in rats with autoimmune
encephalomyelitis (an experimental model used to study multiple sclerosis), cannabinoids
were shown to attenuate the signs and the symptoms of central nervous system damage.^100,
172 (Some believe that nerve damage associated with multiple sclerosis is caused by
an inflammatory reaction.) Likewise, the cannabinoid, HU-211, was shown to suppress brain
inflammation that resulted from closed head injury ¹⁴⁶ or infectious meningitis
⁷ in studies on rats. HU211 is a synthetic cannabinoid that does not bind to
cannabinoid receptors, and is not psychoactive,⁷ thus, without direct evidence,
the effects of marijuana cannot be assumed to include those of HU-211. CT-3, another
atypical cannabinoid, suppresses acute and chronic joint inflammation in animals.¹⁷⁸
It is a nonpsychoactive, synthetic derivative of 11-THC-oic acid (a breakdown product of
THC), and does not appear to bind to cannabinoid receptors. ¹²⁹
Cannabichromene, a cannabinoid found in marijuana, has also been reported to have
anti-inflammatory properties.¹⁷³ No mechanism of action for possible
anti-inflammatory effects of cannabinoids has been identified and the effects of these
atypical cannabinoids and effects of marijuana are not yet established.

It is interesting to note that two reports of cannabinoid-induced analgesia are based
on the ability of the endogenous cannabinoids, anandamide and PEA, to reduce pain
associated with local inflammation that was experimentally induced by subcutaneous
injections of dilute formalin.^{22, 73} Both THC and anandamide can increase serum
levels of ACTH and corticosterone in animals.¹⁶⁹ Those hormones are involved in
regulating many responses in the body, including those to inflammation. The possible link
between experimental cannabinoid-induced analgesia and reported anti-inflammatory effects
of cannabinoids is important for potential therapeutic uses of cannabinoid drugs, but has
not yet been established.

Conclusions regarding effects on immune system

Based on cell culture and animal studies, cannabinoids have been established as
immunomodulators - that is, they increase some immune responses and decrease others. The
variable responses depend upon experimental factors such as drug dose, timing of delivery,
and type of immune cell examined.

Cannabinoids affect multiple cellular targets within the immune system and a variety of
effector functions. Many of the effects noted above appear to occur at

2.42

concentrations of > 5 µM in vitro and > 5 mg/kg in vivo.^l
By comparison, a 5 mg injection of THC into a person (about 0.06 mg/kg) is enough to
produce strong psychoactive effects. It should be emphasized, however, that little is
known about the effects of chronic low dose exposure to cannabinoids on the immune system.

Another issue in need of further clarification involves the potential usefulness of
cannabinoids as therapeutic agents in inflammatory diseases. Glucocorticoids have
historically been used for these diseases, but non-psychotropic cannabinoids potentially
have fewer side effects and might thus offer an improvement over glucocorticoids in
treating inflammatory diseases.

Conclusions and Recommendations

Following the progress of the past fifteen years in understanding the effects of
cannabinoids, research over the next decade is likely to reveal even more. It is
interesting to compare how little we know about the cannabinoids with how much we know
about the opiates (table 2.8). This table, in fact, suggests good reason for optimism
about the future of cannabinoid drug development. Now that many of the basic tools of
cannabinoid pharmacology and biology have been developed, one can expect to see rapid
advances that can begin to match what is known for opiate systems in the brain.

Despite the tremendous progress in understanding the pharmacology and neurobiology of
brain cannabinoid systems, this field is still in its early developmental stages. A key
focus for future study is the neurobiology of endogenous cannabinoids. Establishing the
precise brain localization - i.e.,., in which cells and where in those cannabinoids are
found, cellular storage and release mechanisms, and uptake mechanisms will be crucial in
determining the biological role of this system. Technology that will be crucial in
establishing the biological significance of these systems will be broad based and include
such research tools as the transgenic, or gene knockout mice, as have already been
accomplished for various opioid receptor types.²⁶ In 1997, both CB₁
and CB₂ receptor knockout mice were generated by a team of scientists at NIH,
and a group in France has developed another strain of CB₁ in receptor knockout
mice.⁹²

^{lIn vitro studies are those in which animal cells or tissue are removed and
studied outside the animal; in vivo studies are those in which experiments are conducted
in the whole animal.

2.43

Table 2.8 Historical comparisons between cannabinoids and opiates

Comparisons between cannabinoids and opiates

.
Cannabinoids
Opiates

Pharmacological

Discoveries

Discovery of receptor existence
1988 (Howlett and Devane)^{36, 40}
1973 (Pert and Snyder, Terenius, and Simon)^{123, 149, 162}

Identification of receptor antagonist
1994 SR141716A (Rinaldi- Carmona)¹³²
pre- 1973 Naloxone

Discovery of 1st endogenous ligand
1992 Anandamide (Devane And Mechoulam)³⁷
1975 Met- and Leu-enkephalin (Hughes et al)⁷⁰

1st Receptor cloned
1990 (Matsuda) ¹⁰⁷
1992 (Evans et al. and Kieffer et al.)^{41, 82}

Natural functions of cannabinoid / opiate systems
Unknown
Pain, reproduction, mood, movement, and others

There are several research tools that will greatly aid such investigations - in
particular, a greater selection of agonists and antagonists that permit discrimination
between the activation of CB1 versus CB2 receptors; hydrophilic agonists (that can be
delivered to animals or cells more effectively than hydrophobic compounds). In the area of
drug development, future progress should continue to provide more specific agonists and
antagonists for CB1 and CB2 receptors, with varying potential for therapeutic uses.

There are certain areas that will provide keys to a better understanding of the
potential therapeutic value of cannabinoids. For example, basic biology indicates a role
for cannabinoids in pain and control of movement, which is consistent with a possible
therapeutic role in these areas. The evidence is relatively strong for the treatment of
pain, and intriguingly, although less well-established, for movement disorders. The
neuroprotective properties of cannabinoids might prove therapeutically useful, although it
should be noted that this is a new area and other, better studied, neuroprotective drugs
have not yet been shown to be therapeutically useful. Cannabinoid research is clearly
relevant not only to drug abuse, but also to

2.44

understanding basic human biology. Further, it offers the potential for the discovery
and development of new, therapeutically useful drugs.

CONCLUSION: At this point, our knowledge about the biology of marijuana and
cannabinoids allows us to make some general conclusions:

Cannabinoids likely have a natural role in pain modulation, control of movement, and
memory.
The natural role of cannabinoids in immune systems is likely multifaceted and remains
unclear.
The brain develops tolerance to cannabinoids.
Animal research demonstrates the potential for dependence, but this potential is
observed under a narrower range of conditions than with benzodiazepines, opiates cocaine,
or nicotine.
Withdrawal symptoms can be observed in animals, but appear to be mild compared to
opiates or benzodiazepines, such as diazepam (Valium ®).

CONCLUSION: The different cannabinoid receptor types found in the body appear to
play different roles in normal physiology. In addition, some effects of cannabinoids
appear to be independent of those receptors. The variety of mechanisms through which
cannabinoids can influence human physiology underlies the variety of potential therapeutic
uses for drugs that might act selectively on different cannabinoid systems.

RECOMMENDATION: Research should continue into the physiological effects of synthetic
and plant-derived cannabinoids and the natural function of cannabinoids found in the body.
Because different cannabinoids appear to have different effects, cannabinoid research
should include, but not be restricted to effects attributable to THC alone.

This chapter has summarized recent progress in understanding the basic biology of
cannabinoids, and provides a foundation for the next two chapters which review studies on
the potential health risks (chapter 3) and benefits of marijuana use (chapter 4).

2.45

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2.59}}}}}

Compounds That Bind to Cannabinoid Receptors^d
*Compound*	*Properties*
Agonists (Receptor activators)
Plant-derived compounds
⁹-THC	Main psychoactive cannabinoid in the marijuana plant; largely responsible for psychological and physiological effects. (Except in discussions of the different forms of THC, THC is used as a synonym for ⁹-THC
⁸-THC	Slightly less potent than ⁹-THC and much less abundant in the marijuana plant, but otherwise similar.
11-OH-⁹-THC	Bioactive compound formed when the body breaks down ⁹-THC. Presumed to be responsible for some of the effects of marijuana.
Cannabinoid agonists found in animals
anandamide (arachidonyl- ethanolamide)	Found in animals ranging from mollusks to mammals. Appears to be primary endogenous cannabinoid agonist in mammals. Chemical structure very different from plant cannabinoids, and related to prostaglandins.
2-AG (arachidonyl glycerol)	Endogenous agonist. Structurally similar to anandamide. More abundant but less potent than anandamide.
THC analogues
Dronabinol	Synthetic THC. Marketed in the US under the name Marinol® for nausea associated with chemotherapy and for AIDS-related wasting.
Nabilone	THC analogue. Marketed in the UK under the name Cesamet® for the same indications as dronabionol.
CP 55,940	Synthetic cannabinoid; THC analogue; that is, it is structurally similar to THC
Levonantradol	THC analogue.
HU-210	THC analogue, 100-800 fold greater potency than THC.⁹⁷
Chemical structure unlike THC or anandamide
WIN-55,212	Chemical structure different from known cannabinoids, but binds to both cannabinoid receptors. Chemically related to cyclo-oxygenase inhibitors, which include anti-inflammatory drugs.
Antagonists (Receptor Blockers)
SR 141716A	Synthetic CB₁ antagonist, developed in 1994.¹³²
SR 144528	Synthetic CB₂ antagonist; developed in 1997.¹³³
^d Sources: Mechoulam et al. 1998 ¹⁰⁹, Felder and Glass 1998⁴³; BMA 1997¹⁷

Cellular Processes That Can Be Targeted for Drug Development
Drug action	.	Biological Result
Block synthesis	Synthesis of bioactive compounds is a continuous process and is one means by which concentrations of that compound are regulated.	Weaker signal, due to decreased agonist concentration
Inhibit degradation	Chemical breakdown is one method the body uses to inactivate endogenous substances.	Stronger signal, due to increased agonist concentration increased.
Facilitate neuronal uptake	Neuronal uptake is one of the natural ways in which a receptor agonist is inactivated.	Stronger signal, due to increased amount of time during which agonist is present in the synapse where it can stimulate the receptor

Since the Previous IOM Report on Marijuana in 1982: A Decade of Landmark Discoveries
*Year*	*Discovery*	*Primary Investigators*
1986	Potent cannabinoid agonists are developed the key to discovering the receptor	M.R. Johnson and L.S. Melvin⁷⁵
1988	First conclusive evidence of specific cannabinoid receptors	A. Howlett and W. Devane³⁶
1990	The cannabinoid brain receptor (CB₁) is cloned, its DNA sequence is identified, and its location in the brain is determined	L. Matsuda et al,¹⁰⁷ and M. Herkenham et al⁶⁰
1992	Anandamide is discovered - a naturally occurring substance in the brain that acts on cannabinoid receptors	R. Mechoulam and W. Devane³⁷
1993	A cannabinoid receptor is discovered outside the brain; this receptor (CB₂) is related to the brain receptor but is distinct	S. Munro¹¹²
1994	The first specific cannabinoid antagonist, SR141716A, is developed	M.Rinaldi-Carmona¹³²
1998	The first cannabinoid antagonist, SR144528, that can distinguish between CB₁ and CB₂ receptors discovered.	M. Rinaldi-Carmona¹³³

Receptor Binding in Brain Tissue¹²⁴
*Compound*	*Potency Relative to THC*
CP 55,940	59
⁹-THC	1
Anandamide	0.47
2-AG	0.08

*Brain Region*	*Functions Associated with Region*
Brain regions in which cannabinoid receptors are abundant
Basal ganglia Substantia nigra pars reticulate Entopeduncular nucleus Globus pallidus Putamen	Movement control
Cerebellum	Body-movement coordination
Hippocampus	Learning and memory, stress
Cerebral cortex, especially cingulate, frontal, and parietal regions	Higher cognitive functions
Nucleus accumbens	Reward center
Brain regions in which cannabinoid brain receptors are moderately concentrated
Hypothalamus	Body housekeeping functions (body-temperature regulation, salt and water balance, reproductive function)
Amygdala	Emotional response, fear
Spinal cord	Peripheral sensation, including pain
Brain Stem	Sleep and arousal, temperature regulation, motor control
Central gray	Analgesia
Nucleus of the solitary tract	Visceral sensation, nausea and vomiting

.	CB₁	CB₂
Effects of various cannabinoids
⁹-THC	Agonist	Weak antagonist
Anandamide	Agonist	Agonist
Cannabinol (CBN)	Weak agonist	Agonist; greater affinity for CB₂ than for CB₁
Cannabidiol (CBD)	Does not bind to receptor	Does not bind to receptor
Receptor distribution
Areas of greatest abundance	Brain	Immune system, especially B cells and natural killer cells

Drug Tested	Cell Types Tested or Type Drug of Animal Experiment	Drug Concen- tration ^a	Result	Reference
THC 2-AG 11-OH-THC CBN	Lymphocytes and Splenocytes in vitro	0.1-30 µM	Higher doses suppress T cell proliferation	Luo, 1992; Pross,1992* Klein, 1985%; Specter,1990& Lee, 1995* Herring, 1998
THC 2-AG Anandamide	Lymphocytes and Splenocytes	0.1-25 µM	Lower doses increase T cell proliferation in vitro	Luo,1992; Lee,1995* Pross,1992*
THC 2-AG Anandamide	Splenocytes in vitro	1-25 µM	Little or no effect on T cell proliferation	Lee,1995* Devane,1992
THC, 11-OH-THC AG-2	Splenocytes in vitro	3-30 µM	Decrease B cell proliferation	Klein,1985% Lee,1995*
THC CP 55,940 WIN 55,212-2	Lymphocytes in vitro	0.1-100nM [0.0001-0.1 µM]	Increase B cell proliferation	Derocq, 1995
THC	Mice were injected with drug	>5mg/kg	Antibody production was suppressed	Baczynsky, 1983 Schatz,1993
HU-210	Mice were injected with drug	>0.05 mg/kg	Antibody production was suppressed	Titishov,1989
THC 11-OH-THC CBD CP55,940 CBN	Splenocytes in vitro	1-30µM	Antibody production was suppressed	Klein,1990 Baczynasky,1983 Kaminski,1994 Kaminski,1992 Herring,1998
THC	Rodents were injected with drug	3mg/kg/day for 25days 40mg/kg/day for 2 days	Repeated low doses or a high dose of THC suppress the activity of natural killer cells	Patel,1985 Klein,1987
THC 1l-OH-THC	Natural killer cells in vitro	0.1-32 µM	Doses of >=10 µM suppress natural killer cell cytolytic activity, doses <10 µM produced no effect	Klein,1987 Luo,1989
THC	Peritoneal macrophages and monocytes	3-30 µM	Variable doses of THC suppress macophage functions in vitro	Lopez-Cepero, 1986 Specter,1991 Tang,1992

THC CBD	Mice injected with drug; in one case, in vitro tests done on spleens	>5mg/kg for 4 days or 50 mg/kg every 5 days for up to 8 weeks	THC suppresses normal immune response, interferons failed to increase when exposed to cytokine inducer, while CBD had no suppressive effect	Cabral,1986 Blanchard,1986
THC CBD	Peripheral blood mononuclear cells in vitro	<0.1 µM	Increased interferon production	Warzl, 1991
THC CBD	Peripheral blood mononuclear cells in vitro	30 µM	Decreases interferon production	.
THC CBD	Splenocytes and T cells in vitro	10 µM	Both THC and CBD suppress IL-2 secretion and the number of IL-2 transcripts	Condie,1996
THC	Phorbol myristate acetate differentiated macrophage in vitro	10-20 µM	Increase in tumor necrosis factor production and IL-I supernatant bioactiviy	Shivers, 1994
THC	Endotoxin-activated macrophages in vitro	10-30 µM	Increase processing and release of IL-I rather than cellular production of the IL-I	Zhu, 1994
THC	Peritoneal macrophages in vitro	10-30 µM	Increased IL-I bioactivity	Klein, 1990
THC	Mice were injected with drug and either sublethal or lethal dose of Legionalla pneumophilia	8mg/kg given before and after bacteria infection	Cytokine-mediated septic shock and death occurs with exposure to sublethal dose of the bacteria	Klein, 1993 and 1994 Newton, 1994
THC		< 5 mg/kg doses. or one 8 mg/kg or 4 mg/kg dose given before bacteria infection	Survival occurs, but with greater susceptiblity to infection when challenged with bacteria and death when challenged with a lethal dose of bacteria	Klein, 1993 and 1994 Newton, 1994
THC	Immuno-deficient mice injected with drug and herpes simplex virus	100mg/kg before and after virus infection	Two high doses of THC potentiates the effects of herpes simplex and enhances the progression of death	Specter, 1991
THC		100 mg/kg before virus infection	A single dose did not promote death	Specter, 1991

Comparisons between cannabinoids and opiates
.	*Cannabinoids*	*Opiates*
Pharmacological Discoveries
Discovery of receptor existence	1988 (Howlett and Devane)^{36, 40}	1973 (Pert and Snyder, Terenius, and Simon)^{123, 149, 162}
Identification of receptor antagonist	1994 SR141716A (Rinaldi- Carmona)¹³²	pre- 1973 Naloxone
Discovery of 1st endogenous ligand	1992 Anandamide (Devane And Mechoulam)³⁷	1975 Met- and Leu-enkephalin (Hughes et al)⁷⁰
1st Receptor cloned	1990 (Matsuda) ¹⁰⁷	1992 (Evans et al. and Kieffer et al.)^{41, 82}
Natural functions of cannabinoid / opiate systems	Unknown	Pain, reproduction, mood, movement, and others