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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.

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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.

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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.

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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.

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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.

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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

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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.

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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 affinityb 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.

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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 THC37, 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

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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.

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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

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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.

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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

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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

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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

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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

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