I. Introduction: Overview of the Cannabinoid Receptors

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Cannabinoid receptors received their name as those receptors that respond to cannabinoid drugs, such as ⁹-tetrahydrocannabinol (⁹-THC¹; Fig. 1), derived from Cannabis sativa and its biologically active synthetic analogs. As detailed under Section II., synthetic agonists that bind to cannabinoid receptors include ⁹-THC-like analogs and aminoalkylindole compounds typified by R-(+)-WIN55212. Several endogenous ligands for cannabinoid receptors have also been identified, most notably arachidonoylethanolamide (anandamide), 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether (noladin ether) (Section II.). However, because it is not yet clear whether these eicosanoid molecules are the only, or primary, endogenous agonists, we continue to call the receptors cannabinoid receptors rather than prematurely renaming them after an endogenous agonist as is recommended by the NC-IUPHAR. Cannabinoid receptor types are denoted by the abbreviation CB and numbered in the order of their discovery by a subscript (CB₁, CB₂). At present, two cannabinoid receptor types have been determined, the distinction between them being based on differences in their predicted amino acid sequence, their signaling mechanisms, and their tissue distribution. It has also proved possible to develop potent agonists and antagonists with marked selectivity for CB₁ or CB₂ receptors (Section II.) as well as CB₁, CB₂, and CB₁/CB₂ knockout mice (Section VI.).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   The structures of four constituents of cannabis: 9-THC, 8-THC, cannabinol, and cannabidiol.

The CB₁ cannabinoid receptor (2.1:CBD:1:CB1:) has been cloned from rat, mouse, and human tissues and exhibits 97 to 99% amino acid sequence identity across species (Section V.). Its structure is that of a seven-transmembrane domain receptor, consistent with biochemical and cellular determinations of signal transduction via G proteins (Section IV.). CB₁ receptor mRNA and protein are found primarily in brain and neuronal tissue (Section VII.). The CB₂ cannabinoid receptor (2.1:CBD:2:CB2:) exhibits 48% homology with the CB₁ cannabinoid receptor (Section V.). Expressed CB₂ receptor protein binds ⁹-THC-like, aminoalkylindole, and eicosanoid ligands (Section II.) and signals a response (Section IV.), thereby defining this receptor as being of the cannabinoid receptor class. The mouse CB₂ receptor has been cloned and has an 82% sequence identity to the hCB₂ receptor (Section V.). CB₂ receptor mRNA is found primarily in immune tissue and is notably absent from normal nervous tissue (Section VII.). Any novel type(s) of cannabinoid receptor will be defined based on multiple criteria of primary structure homology, pharmacological characteristics in biological systems, and signal transduction mechanisms. Although some preliminary pharmacological evidence for the existence of additional types of cannabinoid receptor has already emerged (Section XI.), other kinds of evidence are still lacking.

The CB₁ cannabinoid receptor has been extensively characterized for biological responses, and information about the structure-activity relationships of ligands for interaction with this receptor is extensive (Section II.). Claimed central nervous system responses to ⁹-THC and other cannabinoid receptor agonists include therapeutically beneficial effects of analgesia, attenuation of the nausea and vomiting in cancer chemotherapy, reduction of intraocular pressure, appetite stimulation in wasting syndromes, relief from muscle spasms/spasticity in multiple sclerosis, and decreased intestinal motility (for reviews, see Pertwee, 2000b; 2001a,b, 2002; Piomelli et al., 2000). Untoward side effects accompanying these therapeutic responses include alterations in cognition and memory, dysphoria/euphoria, and sedation (see Abood and Martin, 1992 for a review). Animal models that distinguish cannabinoid receptor activity include drug discrimination paradigms in rodents, pigeons, and nonhuman primates, a typical static ataxia in dogs, and a tetrad of responses in rodents (hypothermia, analgesia, hypoactivity, and catalepsy; reviewed under Section III.). Nerve-muscle tissue preparations (e.g., mouse vas deferens and guinea pig small intestine) respond to CB₁ cannabinoid receptor agonists with an inhibition of electrically evoked contraction, believed to be the result of diminished release of neurotransmitter (Section III.). CB₂ mRNA has been found primarily in cells of the immune system (Sections VII. and IX.). However, because CB₁ receptor transcripts have also been found in immune cells and tissues, it cannot be assumed that immune responses are solely regulated by the CB₂ cannabinoid receptor. Therapeutic applications or untoward effects of cannabinoid receptor agonists in the immune system remain unclear. CB₁ and CB₂ cannabinoid receptors are both coupled to pertussis toxin-sensitive G_i/o proteins to inhibit adenylyl cyclase activity and to initiate the mitogen-activated protein kinase and immediate early gene signaling pathway(s) (Section IV.). In addition, CB₁ receptors are coupled through G_i/o proteins to various types of potassium and calcium channels (Section IV.).

As to endogenous cannabinoid receptor agonists (endocannabinoids), it is likely that anandamide and 2-arachidonoylglycerol both function as neurotransmitters or neuromodulators and that one of their roles may be to serve as retrograde synaptic messengers (Section VIII.). Thus, there is evidence that they are synthesized by neurons "on demand", that they can undergo depolarization-induced release from neurons, and that after their release, they are rapidly removed from the extracellular space by a membrane transport process yet to be fully characterized (Di Marzo et al., 1998; Maccarrone et al., 1998; Di Marzo, 1999; Piomelli et al., 1999; Hillard and Jarrahian, 2000). Once within the cell, anandamide is hydrolyzed to arachidonic acid and ethanolamine by the microsomal enzyme, fatty acid amide hydrolase (FAAH) (Di Marzo et al., 1998; Maccarrone et al., 1998; Di Marzo, 1999; Ueda et al., 2000). 2-Arachidonoylglycerol can also be hydrolyzed enzymically, both by FAAH and by other hydrolases yet to be characterized (Di Marzo et al., 1998; Di Marzo, 1999; Khanolkar and Makriyannis, 1999). Mechanisms underlying the release and fate of noladin ether remain to be identified.

This review summarizes the main features of the structure, pharmacology, and function of cannabinoid receptors that provide the basis for the classification of these receptors. Because it does not set out to be a comprehensive review of the literature, readers seeking more detail should refer to the many relevant reviews in the field (Table 1).

	II. Classification of Ligands That Bind to Cannabinoid Receptors

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A. Cannabinoid Receptor Agonists

1. Classical Cannabinoids. This group of cannabinoids consists of ABC-tricyclic dibenzopyran derivatives that are either compounds occurring naturally in the plant, C. sativa, or synthetic analogs of these compounds. The most investigated of the classical cannabinoids have been ⁹-THC (Fig. 1), ⁸-THC (Fig. 1), 11-hydroxy-⁸-THC-dimethylheptyl (HU-210) (Fig. 2), and desacetyl-L-nantradol (Fig. 2). Of these, ⁹-THC is the main psychotropic constituent of cannabis. ⁸-THC is also a psychotropic plant cannabinoid, whereas HU-210 and desacetyl-L-nantradol are synthetic cannabinoids. All these cannabinoids have been demonstrated to elicit cannabimimetic responses both in vivo and in vitro (Johnson and Melvin, 1986; Howlett et al., 1988; Martin et al., 1991; Martin et al., 1995; Pertwee, 1999).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   The structures of the synthetic classical cannabinoid receptor agonists, HU-210 and desacetyl-L-nantradol, and of HU-211, the (+)-enantiomer of HU-210.

• A dihydrobenzopyran-type structure with a hydroxyl group at the C-1 aromatic position and an alkyl group on the C-3 aromatic position seems to be a requirement. Opening of the pyran ring generally leads to complete loss of activity if both phenolic groups are present and are not substituted. Thus, ()-cannabidiol (Fig. 1) has markedly less affinity for CB₁ or CB₂ receptors than ⁸- or ⁹-THC (Tables 2 and 3).
• The aromatic hydroxyl group has to be free or esterified for significant CB₁ activity. Blocking of the hydroxyl group as an ether inactivates the molecule. It is possible that the esters are actually inactive but undergo hydrolysis to the free phenols in vivo. Thus, ⁹-THC acetate, when tested in vitro, shows negligible activity in biochemical reactions in which ⁹-THC is active (Banerjee et al., 1975).
• The length of the chain on C-3 is of major importance. Some activity may be noted with propyl or butyl substitution; ⁹-THC has a pentyl group. A 1',1'-dimethylheptyl or 1',2'-dimethyl heptyl side chain strongly potentiates the cannabimimetic activity of compounds that have low activity in the n-pentyl series. An all carbon side chain on C-3 is not an absolute requirement. The side chain may contain an etheric oxygen (Loev et al., 1973).
• 11-Hydroxy THCs, which are major metabolites of classical cannabinoids, are potent cannabimimetics. Monohydroxylation on other positions of the terpene ring also usually leads to active derivatives. Dihydroxylation generally causes loss of activity. Further oxidation of the C-11 hydroxyl group to a carboxyl group causes inactivation.
• Hydroxylation of C-1 of the side chain on C-3 abolishes activity. Hydroxylation at the other C-3 side chain carbons retains activity, with hydroxylation on C-3 of the side chain potentiating activity. Some of these hydroxylated compounds have been detected as major metabolites.
• Alkylation of the C-2 aromatic position retains activity; alkylation on the C-4 position eliminates activity. Electronegative groups, such as carbonyl or carboxyl, at either C-2 or C-4 eliminate activity.
• The methyl group on C-9 is not an absolute requirement for activity; 9-nor-⁹-THC and 9-nor-⁸-THC are active in the dog static ataxia test (Martin et al., 1975).
• The double bond in the terpene ring is not essential for activity (Mechoulam and Edery, 1973; Mechoulam et al., 1980), and, indeed, this ring may be exchanged by some heterocyclic systems (Pars et al., 1977; Lee et al., 1983).

• The stereochemistry at 6a,10a in the natural active cannabinoids is trans (6aR,10aR). A few cis isomers have been tested and have shown very low activity. However, cis compounds have not been studied over a wide range of tests. (6aS,10aS) THCs are either completely inactive or show very low activity both in animal tests and in binding assays. Thus, although the 6aR,10aR analog HU-210 is a highly potent cannabinoid, its 6aS,10aS enantiomer (HU-211), when well purified, has been shown to be less active by more than three orders of magnitude (Järbe et al., 1989; Howlett et al., 1990; Mechoulam et al., 1991; Felder et al., 1992; Pertwee et al., 1992). With ⁸- and ⁹-THC, the picture is less clear. In the original publications, the synthetic (+)-enantiomers of these cannabinoids were apparently not completely separated from the corresponding ()-enantiomers, such that activity was determined to be about 5 to 10% of the () compounds (Mechoulam et al., 1992). For ⁹-THC, careful purification led to a (+)-enantiomer with activity less than 1% of the ()-enantiomer (Herkenham et al., 1990; Matsuda et al., 1990; Felder et al., 1992; Pertwee, 1997).
• Reduction of ⁹-THC leads to hexahydrocannabinol epimers that are both active, the equatorial epimer being considerably more active than the axial one (Mechoulam and Edery, 1973; Mechoulam et al., 1980). The same relationship is observed with the 11-hydroxyhexahydrocannabinols (Mechoulam et al., 1991). Thus, it seems that an equatorial substitution (i.e., one in which the C-9 methyl or hydroxymethyl group is in the plane of the cyclohexane ring) is preferable to an axial one.
• Several hydroxylated metabolites of ⁹-THC and ⁸-THC are known in both epimeric forms. For example, 8- and 8-hydroxy-⁹-THC and 7- and 7-hydroxy-⁸-THC have been identified as relatively minor metabolites, and slight differences in activity between the epimers in each pair have been observed (Mechoulam and Edery, 1973; Razdan, 1986).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   The structures of the CB2-selective cannabinoid receptor agonists, HU-308, L-759633, L-759656, JWH-133, JWH-139, and JWH-051.

2. Nonclassical Cannabinoids. During the course of their extensive SAR studies on the analgesic activity of classical cannabinoids, researchers at Pfizer synthesized new analogs lacking the dihydropyran ring of THC. CP47497 (Fig. 4) represents the prototypical compound of this series of AC-bicyclic and ACD-tricyclic cannabinoid analogs (Melvin et al., 1984; Melvin et al., 1993). Further developments ultimately led to the bicyclic analog, CP55940 (Fig. 4), which has become one of the major cannabinoid agonists. Less lipophilic than THC, [³H]CP55940 has allowed the discovery and characterization of the CB₁ cannabinoid receptor (Devane et al., 1988), and it is still the most used radiolabeled cannabinoid ligand. It binds to CB₁ and CB₂ receptors with similar affinity (Table 2) and displays high activity in vivo as well, being 10 to 50 times more potent than ⁹-THC in the mouse tetrad model (Johnson and Melvin, 1986; Little et al., 1988). CP55940 behaves as a full agonist for both receptor types, its maximal effects in CB₁ and CB₂ receptor assay systems often matching or exceeding the maximal effects of several other cannabinoid receptor agonists (Pacheco et al., 1993; Slipetz et al., 1995; Burkey et al., 1997; Griffin et al., 1998; MacLennan et al., 1998; Pertwee, 1999). One potent ACD-tricyclic nonclassical cannabinoid is CP55244 (Fig. 4), which also displays signs of high affinity and high relative intrinsic activity, at least for CB₁ receptors (Howlett et al., 1988; Little et al., 1988; Herkenham et al., 1990; Gérard et al., 1991; Griffin et al., 1998). Indeed, CP55244 seems to have even higher CB₁ affinity and relative intrinsic activity than CP55940. It seems likely that other nonclassical cannabinoids share the ability of CP55940 to interact with CB₂ receptors; however, this remains to be established. Like classical cannabinoids, nonclassical cannabinoids with chiral centers exhibit significant stereoselectivity, those compounds with the same absolute stereochemistry as ()-⁹-THC at 6a and 10a (6aR,10aR) exhibiting the greater pharmacological activity (Little et al., 1988; Herkenham et al., 1990; Melvin et al., 1993).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   The structures of the ()-enantiomers of three nonclassical cannabinoid receptor agonists: CP55940, CP47497, and CP55244.

3. Aminoalkylindoles. Until the early 1990s, all the compounds known to act as cannabimimetics were structurally derived from THC. The situation changed when Sterling Winthrop researchers reported a new family of aminoalkylindoles possessing cannabimimetic properties. This discovery resulted from the development of structurally constrained analogs of pravadoline (Bell et al., 1991; Pacheco et al., 1991), a series of compounds with reduced ability to behave as nonsteroidal anti-inflammatory agents that inhibit cyclooxygenase but increased ability to bind to the CB₁ receptor (D'Ambra et al., 1992; Eissenstat et al., 1995). R-(+)-WIN55212 (Fig. 5) is the most highly studied, commercially available compound of the series. It displays high affinity for both cannabinoid receptors, with moderate selectivity in favor of the CB₂ receptor (Table 2), and exhibits high relative intrinsic activity at both CB₁ and CB₂ receptors (Bouaboula et al., 1997; Griffin et al., 1998; Tao and Abood, 1998; Pertwee, 1999). In vivo, it produces the full spectrum of pharmacological effects of THC and substitutes totally for other cannabinoids in discriminative stimulus tests, whereas its S-()-enantiomer, WIN55212-3, lacks activity both in vivo and in vitro (Martin et al., 1991; Compton et al., 1992a; Pacheco et al., 1993; Slipetz et al., 1995; Wiley et al., 1995b; Pertwee, 1997; Pertwee, 1999). A [³H]R-(+)-WIN55212 assay has been developed, which has been used to characterize and map cannabinoid receptors in rat brain (Jansen et al., 1992; Kuster et al., 1993). There is evidence that R-(+)-WIN55212 binds differently to the CB₁ receptor than classical or nonclassical cannabinoids, albeit in a manner that still permits displacement by R-(+)-WIN55212 of other known types of cannabinoid from CB₁ binding sites (Petitet et al., 1996; Song and Bonner, 1996; Pertwee, 1997; Chin et al., 1998; Tao and Abood, 1998; see also Section V.).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   The structures of three aminoalkylindole cannabinoid receptor agonists: R-(+)-WIN55212, JWH-015, and L-768242.

4. Eicosanoids. The prototypic member of the eicosanoid group of cannabinoid receptor agonists is anandamide, which belongs to the 20:4, n-6 series of fatty acid amides (Fig. 6). This is the first of five endogenous cannabinoid receptor agonists to have been discovered in mammalian brain and certain other tissues (Devane et al., 1992b), the other compounds being homo--linolenoylethanolamide and docosatetraenoylethanolamide (Hanus et al., 1993), 2-arachidonoylglycerol (Mechoulam et al., 1995; Sugiura et al., 1995), and noladin ether (Fig. 6) (Hanus et al., 2001). Of these endocannabinoids, the most investigated to date have been anandamide and 2-arachidonoylglycerol.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   The structures of five endogenous cannabinoids.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   The structures of the CB1-selective synthetic cannabinoid receptor agonists, methanandamide, ACEA, ACPA, and O-1812.

• Monosubstitution of the amide is a requirement for activity. Substitution by an alkyl, fluoroalkyl, or hydroxyalkyl increases activity, with a two- or three-carbon chain being optimal. Branching of the chain (methyl is optimal) retains activity.
• Substitution of the hydroxyl in anandamide by a methyl ether, phenyl ether, or forming a phosphate derivative of anandamide decreases activity, whereas introduction of an amino or a carboxyl group eliminates activity.
• Highest potencies are observed when structural changes are carried out in both the arachidonoyl and ethanolamide moieties of anandamide.
• The introduction of an alkyl substituent (methyl is optimal) on the carbon  to the carbonyl or on the carbon adjacent to the nitrogen increases metabolic stability.
• The SAR of the end pentyl chain (C-16 to C-20) in anandamide is very similar to that of classical cannabinoids; however, by branching the chain, the effect on pharmacological measures is not as dramatic in the anandamide series as in the classical series.
• As a requirement for activity in the 20:x, n-6 series, x has to be three or four; however, activity is strongly reduced when n-6 is changed to n-3.
• Activity is retained by increasing the chain length of anandamide by two methylenes (i.e., 22:4 and n-6) but is dramatically reduced or eliminated if the chain length is decreased by two methylenes.

B. Cannabinoid Receptor Antagonists/Inverse Agonists

1. Diarylpyrazoles. The prototypic members of this series of compounds are the Sanofi compounds SR141716A, a potent CB₁-selective ligand, and SR144528, a potent CB₂-selective ligand (Fig. 8). These ligands readily prevent or reverse effects mediated respectively by CB₁ and CB₂ receptors (Rinaldi-Carmona et al., 1994, 1998). There are many reports that, by themselves, SR141716A and SR144528 can act on CB₁ or CB₂ receptors to produce effects that are converse to those produced by cannabinoid receptor agonists (Pertwee, 1999). Although these effects of the arylpyrazole antagonists may be attributable to the inhibition of endogenously produced agonists in the biological preparation, there is evidence that SR141716A and SR144528 can evoke inverse agonist responses (Bouaboula et al., 1997; MacLennan et al., 1998; Pan et al., 1998; Rinaldi-Carmona et al., 1998; Portier et al., 1999; Ross et al., 1999a; Coutts et al., 2000; Sim-Selley et al., 2001). This notion rests on the ability of the CB₁ and CB₂ receptors to exhibit signal transduction activity in the absence of endogenous or exogenous agonists (constitutive activity). As such, arylpyrazoles can behave as "inverse agonists" to reduce the constitutive activity of these signal transduction pathways. In some experiments, SR141716A has been found to be more potent in blocking the actions of CB₁ receptor agonists than in eliciting inverse cannabimimetic responses by itself (Gessa et al., 1997, 1998a; Schlicker et al., 1997; Acquas et al., 2000; Sim-Selley et al., 2001). Sim-Selley et al. (2001) have obtained evidence that this may be because SR141716A binds with relatively low affinity to a site on the CB₁ receptor that is distinct from the agonist binding site for which it has higher affinity. Their data also suggest that it is this lower affinity site that is responsible for the inverse agonist properties of SR141716A.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   The structures of the cannabinoid receptor antagonists/inverse agonists, SR141716A, AM251, AM281, SR144528, and LY320135.

• Disubstitution of the amide nitrogen of SR141716A strongly decreases CB₁ affinity (Lan et al., 1999b).
• Replacement of the amide function by ketone, alcohol, or ether also greatly decreases CB₁ binding affinity (Wiley et al., 2001). Interestingly, some of the ether or alkylamide derivatives display partial agonist activity in mice in vivo. The highly hindered endo-fenchyl amide was used to design the CB₂ receptor antagonist SR144528 (Rinaldi-Carmona et al., 1998).
• Although the 2,4-dichlorophenyl substituent at the 1-position of the pyrazole ring seems to be optimal (Barth and Rinaldi-Carmona, 1999), its replacement by a 1-(5-isothiocyanato)-pentyl group decreases CB₁ affinity only by a factor 4 (Howlett et al., 2000). The phenyl group has been replaced by a 4-methylbenzyl group in SR144528 (Rinaldi-Carmona et al., 1998).
• In the 3-position of the pyrazole ring of SR141716A, replacement of the N-aminopiperidine substituent by the related 5- or 7-membered rings or by cyclohexyl does not alter CB₁ binding affinity, whereas replacement by aminomorpholine or linear alkyl chains leads to a reduction in CB₁ affinity (Lan et al., 1999b; Wiley et al., 2001).
• Compounds with methyl, bromine, or iodine in the 4-position of the pyrazole ring are approximately equipotent, whereas replacement of methyl with hydrogen at this position results in a 12-fold decrease in CB₁ affinity (Wiley et al., 2001). Methyl has been replaced by hydrogen at the 4-position of the pyrazole ring in SR144528.
• In the 5-position of the pyrazole ring, replacement of the 4-chloro substituent of the phenyl group by other halogen or alkyl groups does not alter CB₁ binding affinity (Thomas et al., 1998; Lan et al., 1999b). However, replacement by nitro or amino groups or displacement from the 4-(para) position to the 2-position of the phenyl group leads to poor CB₁ receptor ligands, and replacement of the aromatic ring by alkyl groups abolishes CB₁ affinity (Lan et al., 1999b).
• A particularly potent compound in the SR141716A series is AM251 (Fig. 8). This contains a para-iodophenyl group at the 5-position, a piperidinyl carboxamide at the 3-position, and a 2,4-dichlorophenyl group at the 1-position of the pyrazole ring (Lan et al., 1999b).

2. Other Chemical Series. The most notable members of these series are the substituted benzofuran, LY320135, and the aminoalkylindole, 6-iodopravadoline (AM630) (Fig. 9). LY320135, developed by Eli Lilly, shares the ability of SR141716A to bind with much higher affinity to CB₁ than CB₂ receptors (Table 2). However, it has less affinity for CB₁ receptors than SR141716A and, at concentrations in the low micromolar range, also binds to muscarinic and 5-HT₂ receptors (Felder et al., 1998). Like SR141716A, LY320135 not only blocks the effects of CB₁ receptor agonists (Felder et al., 1998; Coruzzi et al., 1999; Holland et al., 1999; Molderings et al., 1999; Christopoulos et al., 2001) but also exhibits inverse agonist activity at some signal transduction pathways of the CB₁ receptor (Felder et al., 1998; Christopoulos et al., 2001).

View larger version (17K): [in this window] [in a new window]   Fig. 9.   The structures of the pravadoline analogs, AM630, WIN56098, and WIN54461 (6-bromopravadoline).

View larger version (11K): [in this window] [in a new window]   Fig. 10.   The structures of O-1184 and O-1238.

	III. Bioassay

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A. In Vivo Bioassay Systems

1. Introduction. Cannabinoids produce a complex array of behavioral effects that have been characterized in numerous animal species as well as in humans. Although the diverse behavioral effects of cannabinoids provide ample opportunity for quantitating the pharmacological actions of this class of compounds, they provide a challenge to the elucidation of mechanism of action. A major focus of cannabinoid research has been the identification of pharmacological effects that are receptor-mediated. Until the recent development of a specific CB₁ receptor antagonist, SARs provided the only in vivo approach for implicating receptor mechanisms. A major goal of cannabinoid research is elucidating the mechanisms responsible for the behavioral "high". Of course, the psychotomimetic effects can only be assessed in humans, which imposes severe restrictions on SAR studies. Few cannabinoid analogs have sufficient toxicological histories to qualify for human experimentation. The difficulties with human studies have necessitated close examination of pharmacological effects in several animal species, many of which vary in their response to cannabinoids. However, it has now been established that numerous pharmacological effects are mediated via the cannabinoid receptor. There are several fundamental principles that have guided this undertaking. One of the most critical aspects of the choice is whether the pharmacological measure in animals is representative of cannabinoid effects in humans. Equally important is the characterization of behavioral effects that are unique to cannabinoids (i.e., mediated through cannabinoid receptors). Finally, there are the practical aspects of selecting pharmacological effects that can be quantitated and readily obtained. Using these criteria, several pharmacological effects in vivo can be attributed to the activation of cannabinoid receptors.

2. Dog Static Ataxia. Walton et al. (1937) described the effects of cannabinoids in dogs, which represented one of the first animal models that was highly unique for this class of compounds. These effects include sedation, catalepsy, motor incoordination, and hyperexcitability; however, it is the combination of these effects that causes dogs to weave to and fro while remaining fixed in one spot that led to the somewhat anomalous term "static ataxia". Again, the primary advantage of this model is that these behaviors describe a highly specific profile for cannabinoids that is not confused with that produced by other behaviorally active compounds. These behaviors can also be semiquantitated, and extensive SAR studies have revealed both dramatic changes in potency with modest changes in structure (Walton et al., 1937; Martin et al., 1975; Beardsley et al., 1987) and enantioselectivity (Dewey et al., 1984; Little et al., 1989). The strength of this model is that the results obtained correlate well with psychoactivity. These findings strongly suggest that cannabinoid-induced static ataxia is receptor-mediated. Moreover, the CB₁ receptor antagonist, SR141716A, antagonizes the effects of ⁹-THC in this model, a finding that strongly supports CB₁ involvement (Lichtman et al., 1998).

3. Overt Behavior in Monkeys. Mechoulam and colleagues (Edery et al., 1971) synthesized a large number of cannabinoid analogs that allowed them to develop the first framework for describing the structural features that were critical for cannabinoid pharmacological activity. Their model was based on the gross observation of overt behavioral effects in monkeys. The cannabinoids produced sedation, ptosis, body sag, etc., which was reasonably selective for cannabinoids and could be rated in a semiquantitative fashion. They described a SAR that also included enantioselectivity (Edery et al., 1971); however, there have been no reports of reversal of these effects by the CB₁ receptor antagonist, SR141716A.

4. Rat Drug Discrimination. Drug discrimination is considered one of the most reliable means of predicting whether test drugs produce subjective effects similar to those of a known drug. Initially, an animal is trained to press a lever for food reward and then subsequently trained to press a specific lever for this reward when under the influence of ⁹-THC and another lever when any other drug is administered. Therefore, on test days, which lever the animal chooses tells the experimenter whether the test compound is perceived as THC-like or not. Much of the early rat drug discrimination literature for the cannabinoids was generated by Järbe's laboratory (Järbe and Ohlin, 1977; Järbe and McMillan, 1979, 1980; Järbe et al., 1989; Järbe and Mathis, 1992). Rats have also been trained to discriminate between CP55940, a potent cannabinoid agonist, and vehicle (Gold et al., 1992). These animals perceived ⁹-THC as being like CP55940. Furthermore, the ⁹-THC-discriminative cue has been shown to be selective for cannabinoids (Barrett et al., 1995).

5. Monkey Drug Discrimination. The above description of drug discrimination in rats applies to monkeys; however, it has been argued that primates may provide a more accurate reflection of cannabinoid behavioral effects in humans. This model has provided reassuring data that novel cannabinoids, such as CP55940 (Gold et al., 1992), R-(+)-WIN55212 (Compton et al., 1992a), and the endogenous ligand anandamide (Wiley et al., 1997), are likely to produce cannabinoid behavioral effects in humans. Establishing this fact is particularly crucial since these compounds are being used widely as cannabinoid probes. As with the rat drug discrimination, SR141716A was shown to block the discriminative properties of ⁹-THC (Wiley et al., 1995c), thereby implicating CB₁ receptors.

6. Mouse Tetrad Model. As mentioned earlier, cannabinoids are known to produce a wide range of pharmacological effects that include hyperstimulation, sedation, catalepsy, and several other depressant properties. Individually, none of these effects can be considered unique for cannabinoids, since all of these properties are shared by numerous classes of centrally active agents. Several years ago, it was discovered that i.v. administration of cannabinoids in mice produced sedation, hypothermia, antinociception, and catalepsy in the same dose range and within the same time frame, so that all four behaviors could be determined in the same animal for each injection (Martin et al., 1987). Compounds active in this composite model also produce effects in models that we traditionally consider to be highly predictive of cannabinoid effects, such as drug discrimination (Compton et al., 1993). Furthermore, the SAR studies in the mouse tetrad model are consistent with affinity for the CB₁ receptor for CP55940 and related analogs (Little et al., 1988; Compton et al., 1992b), enantiomers of dimethylheptyl analogs of THC (Little et al., 1989), aminoalkylindoles (Compton et al., 1992a; Huffman et al., 1994), and endocannabinoids (Adams et al., 1998). It has also been shown that SR141716A is highly effective in blocking the effects of most cannabinoid analogs in the mouse tetrad model (Rinaldi-Carmona et al., 1994; Compton et al., 1996), confirming the involvement of CB₁ receptors. The one exception has been the endocannabinoids (Adams et al., 1998). Although SR141716A fails to block the effects of anandamide, it is capable of blocking the effects of metabolically stable anandamide analogs (Adams et al., 1998). However, some anandamide analogs are effective in the mouse tetrad and apparently bind with little affinity for the CB₁ receptor (Di Marzo et al., 2001a). There are several possible explanations for