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

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).

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.

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

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

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.

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.

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.

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

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.

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.

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.

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

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.

Table 2.2 Compounds that bind to cannabinoid receptors

Compounds That Bind to Cannabinoid Receptorsd
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

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.

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

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

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

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.

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),¹⁴⁴ in 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.

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

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

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.

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

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.

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.

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

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.

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.)

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.

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

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

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.

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.

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

* 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.

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).

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.

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.

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.

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.

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

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

Table 2.8 Historical comparisons between cannabinoids and opiates

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

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).

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Chapter 3 ==>>