CB2 cannabinoid receptor agonists attenuate TNF-alpha-induced human vascular smooth muscle cell proliferation and migration.

BACKGROUND AND PURPOSE: Vascular smooth muscle proliferation and migration triggered by inflammatory stimuli are involved in the development and progression of atherosclerosis and restenosis. Cannabinoids may modulate cell proliferation in various cell types through cannabinoid 2 (CB2) receptors. Here, we investigated the effects of CB2 receptor agonists on TNF-alpha-induced proliferation, migration and signal transduction in human coronary artery smooth muscle cells (HCASMCs). EXPERIMENTAL APPROACH: HCASMCs were stimulated with TNF-alpha. Smooth muscle proliferation was determined by the extent of BrdU incorporation and the migration was assayed by modified Boyden chamber. CB2 and/or CB1 receptor expressions were determined by immunofluorescence staining, western blotting, RT-PCR, real-time PCR and flow cytometry. KEY RESULTS: Low levels of CB2 and CB1 receptors were detectable in HCASMCs compared to the high levels of CB2 receptors expressed in THP-1 monocytes. TNF-alpha triggered up to approximately 80% increase (depending on the method used) in CB2 receptor mRNA and/or protein expression in HCASMCs, and induced Ras, p38 MAPK, ERK 1/2, SAPK/JNK and Akt activation, while increasing proliferation and migration. The CB2 agonists, JWH-133 and HU-308, dose-dependently attenuated these effects of TNF-alpha. CONCLUSIONS AND IMPLICATIONS: Since the above-mentioned TNF-alpha-induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that CB2 agonists may offer a novel approach in the treatment of these pathologies by decreasing vascular smooth muscle proliferation and migration.

Targeting the cannabinoid CB2 receptor: modelling and structural determinants of CB2 selective ligands.

Recent developments indicate that CB2 receptor ligands have the potential to become therapeutically important. To explore this potential, it is necessary to develop compounds with high affinity for the CB2 receptor and little affinity for the CB1 receptor. This review will discuss structure-activity relations at both receptors for classical cannabinoids and cannabimimetic indoles. Examples of CB2 selective ligands from both classes of compounds are presented and the structural features leading to selectivity are described. Two approaches, receptor mutations and molecular modelling, have been employed to investigate the interaction of ligands with both cannabinoid receptors. These results obtained from these techniques are discussed.

CB2 receptor ligands.

The CB(1) receptor is found principally in the central nervous system and is responsible for the overt physiological effects of cannabinoids. In contrast, the CB(2) receptor is expressed primarily in the immune system and is responsible for few, if any, obvious behavioral effects. Although many cannabinoid receptor ligands show little, or at best modest, selectivity for either receptor, a number of synthetic compounds are known which have significant selectivity for the CB(2) receptor. These include cannabimimetic indoles, such as 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH-015) and 1-(2,3-dichlorobenzoyl)-2-methyl-3-(2-[1-morpho-lino]ethyl)-5-methoxyindole (L768242), both of which have good affinity for the CB(2) receptor, but weak affinity for the CB(1) receptor. Efforts have been made to develop structure-activity relationships (SAR) at CB(2) for cannabimimetic indoles, but with limited success. Several derivatives of traditional dibenzopyran based cannabinoids have also been found to have significant selectivity for the CB(2) receptor. These include 1-methoxy-Delta(8)-THC derivatives, 1-methoxy-Delta(8)-THC-DMH (L759633), 1-methoxy-Delta(9(11))-THC-DMH (L759656), and 1-methoxy-3-(1',1'-dimethylhexyl)-Delta(8)-THC (JWH-229), plus a number of 1-deoxy-Delta(8)-THC analogues. In particular, 1-deoxy-3-(1',1'-dimethylbutyl)-Delta(8)-THC (JWH-133) shows two hundred-fold selectivity for the CB(2) receptor. Very recently several compounds belonging to other structural groups have also shown selectivity for the CB(2) receptor. This review will describe the current status of the results of these studies and discuss the SAR for these classes of ligands.

Recent developments in the medicinal chemistry of cannabimimetic indoles, pyrroles and indenes.

During the development of new nonsteroidal anti-inflammatory agents, it was discovered that 1-aminoalkyl-3-aroylindoles have affinity for the cannabinoid brain (CB(1)) receptor. This has led to the development of over 100 cannabimimetic aminoalkylindoles, and the development of SAR for these compounds. Later work demonstrated that the aminoalkyl moiety was not necessary, and could be replaced by a four- to six-membered alkyl chain without loss of affinity. Investigation of these indoles led to the discovery of a CB(2) selective ligand, 3-(1-naphthoyl)-N-propylindole. Subsequent work has provided several additional CB(2) selective indoles. On the basis of a proposed pharmacophore for the cannabimimetic indoles, a series of pyrroles and indenes were developed, some of which are potent cannabinoids. SAR for several series of pyrroles have been developed. Two groups have described cannabimimetic indenes, which have been employed as rigid models for the receptor interactions of cannabimimetic indoles with the CB(1) receptor. There is some evidence that the indoles bind to a somewhat different site on the receptor than traditional cannabinoids, and interact with the receptor primarily by aromatic stacking.

A pyridone analogue of traditional cannabinoids. A new class of selective ligands for the CB(2) receptor.

A pyridone analogue (5) of the potent bicyclic cannabinoid CP 47,497 (6) has been synthesized as a model for one conformational isomer of anandamide and to test the hypothesis that an amide carbonyl may serve as a hydrogen bond acceptor in interactions with the CB(1) cannabinoid receptor. Pyridone 5 was synthesized from 6-bromo-2-methoxypyridine (10) by palladium catalyzed coupling with 1-pentyne to provide 11. Catalytic hydrogenation of 11 and hydrolysis to pyridone 13 followed by N-alkylation gave 1-propyl-6-pentyl-2-pyridone (15). Bromination of 15 gave dibromide 18, which underwent Heck coupling with cyclohex-2-en-1-one to give enone 19. Catalytic hydrogenation of 19 gave ketone 20 which was reduced using NaBH(4) to alcohol 5. Reduction of 20 with K-Selectride gave the axial epimer of 5 (21). Neither alcohol 5 nor 21 have significant affinity for the CB(1) receptor (K(i) > 970 nM), but both have moderately high affinity for the CB(2) receptor (K(i) < 60 nM).

Inhibition of glioma growth in vivo by selective activation of the CB(2) cannabinoid receptor.

The development of new therapeutic strategies is essential for the management of gliomas, one of the most malignant forms of cancer. We have shown previously that the growth of the rat glioma C6 cell line is inhibited by psychoactive cannabinoids (I. Galve-Roperh et al., Nat. Med., 6: 313-319, 2000). These compounds act on the brain and some other organs through the widely expressed CB(1) receptor. By contrast, the other cannabinoid receptor subtype, the CB(2) receptor, shows a much more restricted distribution and is absent from normal brain. Here we show that local administration of the selective CB(2) agonist JWH-133 at 50 microg/day to Rag-2(-/-) mice induced a considerable regression of malignant tumors generated by inoculation of C6 glioma cells. The selective involvement of the CB(2) receptor in this action was evidenced by: (a) the prevention by the CB(2) antagonist SR144528 but not the CB(1) antagonist SR141716; (b) the down-regulation of the CB(2) receptor but not the CB(1) receptor in the tumors; and (c) the absence of typical CB(1)-mediated psychotropic side effects. Cannabinoid receptor expression was subsequently examined in biopsies from human astrocytomas. A full 70% (26 of 37) of the human astrocytomas analyzed expressed significant levels of cannabinoid receptors. Of interest, the extent of CB(2) receptor expression was directly related with tumor malignancy. In addition, the growth of grade IV human astrocytoma cells in Rag-2(-/-) mice was completely blocked by JWH-133 administration at 50 microg/day. Experiments carried out with C6 glioma cells in culture evidenced the internalization of the CB(2) but not the CB(1) receptor upon JWH-133 challenge and showed that selective activation of the CB(2) receptor signaled apoptosis via enhanced ceramide synthesis de novo. These results support a therapeutic approach for the treatment of malignant gliomas devoid of psychotropic side effects.

Influence of the N-1 alkyl chain length of cannabimimetic indoles upon CB(1) and CB(2) receptor binding.

The N-1 alkyl side chain of the aminoalkylindole analogues (AAI) has been implicated as one of a three-point interaction with the cannabinoid CB(1) receptor. In this study, the morpholinoethyl of WIN 55,212-2 was replaced with carbon chains of varying lengths ranging from a methyl to heptyl group. Additional groups were added to the naphthoyl and the C2 positions of the molecule. These structural changes revealed that high affinity binding to the CB(1) and CB(2) receptors requires an alkyl chain length of at least three carbons with optimum binding to both receptors occurring with a five carbon side chain. An alkyl chain of 3-6 carbons is sufficient for high affinity binding; however, extension of the chain to a heptyl group results in a dramatic decrease in binding at both receptors. The unique structure of the cannabimimetic indoles provides a useful tool to define the ligand-receptor interaction at both cannabinoid receptors and to refine proposed pharmacophore models.

The search for selective ligands for the CB2 receptor.

Following the identification of the CB2 receptor several groups explored the development of selective ligands for this receptor which occurs principally in the periphery. This led to the discovery that two cannabimimetic indoles, 1-(2, 3-dichlorobenzoyl)-2-methyl-3-(2-[1-morpholino]ethyl)-5-methoxyind ole (L768242) and 2-methyl-1-propyl-3-(1-naphthoyl)indole (JWH-015) have high affinity for the CB2 receptor with low affinity for the CB1 receptor. Shortly thereafter two 1-methoxy-delta8-THC analogues, 1-methoxy-delta8-THC-DMH (L759633) and 1-methoxy-delta9(11)-THC-DMH (L759656), were also found to have high affinity for the CB2 receptor and very little affinity for the CB1 receptor. Almost simultaneously two 1-deoxy-delta8-THC analogues, 1-deoxy-11-hydroxy-delta8-THC-DMH (JWH-051) and 1-deoxy-delta8-THC-DMH (JWH-057) were reported to have high affinity for the CB1 receptor, but even greater affinity for the CB2 receptor. These discoveries gave rise to a concerted effort by Huffman and co-workers to explore the structure-activity relationships (SAR) at CB2 of cannabimimetic indoles and 1-deoxy-delta8-THC analogues. These efforts have resulted in the synthesis and pharmacological evaluation of a number of derivatives of 3-(1-naphthoyl)indoles and 1-deoxy-delta8-THC analogues with various side chains. This review will describe the current status of the results of these studies and discuss the SAR for both these classes of ligands.

Cannabinoids control spasticity and tremor in a multiple sclerosis model.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) is an autoimmune model of multiple sclerosis. Although both these diseases are typified by relapsing-remitting paralytic episodes, after CREAE induction by sensitization to myelin antigens Biozzi ABH mice also develop spasticity and tremor. These symptoms also occur during multiple sclerosis and are difficult to control. This has prompted some patients to find alternative medicines, and to perceive benefit from cannabis use. Although this benefit has been backed up by small clinical studies, mainly with non-quantifiable outcomes, the value of cannabis use in multiple sclerosis remains anecdotal. Here we show that cannabinoid (CB) receptor agonism using R(+)-WIN 55,212, delta9-tetrahydrocannabinol, methanandamide and JWH-133 (ref. 8) quantitatively ameliorated both tremor and spasticity in diseased mice. The exacerbation of these signs after antagonism of the CB1 and CB2 receptors, notably the CB1 receptor, using SR141716A and SR144528 (ref. 8) indicate that the endogenous cannabinoid system may be tonically active in the control of tremor and spasticity. This provides a rationale for patients' indications of the therapeutic potential of cannabis in the control of the symptoms of multiple sclerosis, and provides a means of evaluating more selective cannabinoids in the future.

Synthesis and pharmacology of a hybrid cannabinoid.

A pentacyclic hybrid cannabinoid (4) has been synthesized, which combines structural elements of traditional cannabinoids and cannabmimetic indoles. Cannabinoid 4 contains a 1-pentylindole structure fused to the 2,3-positions of the partially reduced hydroxydibenzopyran system of THC. The successful approach to 4 employed 9-benzoyl-5,7-dimethoxy-1,2,3,4-tetrahydrocarbazole (17) as the starting material. Dehydrogenation to carbazole 18, followed by demethylation and condensation with trans-p-menthadienol gave N-benzoyl hybrid cannabinoid 22, N-alkylation of which afforded target cannabinoid 4. The hybrid cannabinoid had affinity for the CB1 receptor approximately equal to that of delta8-THC (Ki = 19.3+/-3 nM), and shows comparable potency in vivo.

Retrospective evaluation of urinary tract infection in 42 dogs with hyperadrenocorticism or diabetes mellitus or both.

A retrospective study was performed to determine the proportion of dogs with hyperadrenocorticism or diabetes mellitus or both that had urinary tract infection (UTI) and to describe clinical and laboratory findings. Dogs with these endocrine disorders were included if results of quantitative urine culture were available and dogs were not receiving antimicrobials. Dogs with positive urine cultures were considered to have UTI and dogs with negative urine cultures were used as controls. Information including history, clinical signs, physical examination findings, and results of laboratory tests and urine culture was extracted from all records. Findings in dogs with UTI were compared with control dogs. There were 101 dogs with hyperadrenocorticism or diabetes mellitus or both that met inclusion criteria; 42 (41.6%) had UTI and 59 (58.4%) did not. UTI was present in 46% of dogs with hyperadrenocorticism, 37% of dogs with diabetes mellitus, and 50% of dogs with both endocrine disorders. There was no association between endocrine group and occurrence of UTI. Escherichia coli was the most common bacteria isolated, and cultures from 29 dogs (69%) showed growth of this organism. Of dogs with UTI, <5% had stranguria, pollakiuria, or discolored urine, whereas 60% had pyuria and 69% had bacteriuria. We conclude that UTIs are common in dogs with hyperadrenocorticism, diabetes mellitus, or both diseases. Clinical signs of UTI, however, are uncommon and results of urinalysis may be normal. Therefore, it is appropriate to recommend urine culture as part of the evaluation of dogs with these endocrine disorders.

The third transmembrane helix of the cannabinoid receptor plays a role in the selectivity of aminoalkylindoles for CB2, peripheral cannabinoid receptor.

Two subtypes of the human cannabinoid receptor have been identified. The CB1 receptor is primarily distributed in the central nervous system, whereas the CB2 receptor is associated with peripheral tissue, including the spleen. These two subtypes are also distinguished by their ligand-binding profiles. The goal of this study was to identify critical residues in transmembrane region III (TM3) of the receptors that contribute to subtype specificity in ligand binding. For this purpose, a chimeric cannabinoid receptor [CB1/2(TM3)] was generated in which the TM3 of CB1 was replaced with the corresponding region of CB2. These receptors were stably expressed in Chinese hamster ovary cells for evaluation. The binding affinities of CB1/2(TM3) and the wild-type CB1 receptor to several prototype ligands were similar with one notable exception: the chimeric receptor exhibited a 4-fold enhancement in binding affinity to WIN 55,212-2 (K(d) = 4.8 nM) relative to that observed with CB1 (K(d) = 21.7 nM). Two additional aminoalkylindoles, JWH 015 and JWH 018, also bound the chimeric receptor (K(i) = 1.0 microM and 1.4 nM, respectively) with higher affinity compared with the wild-type CB1 (K(i) = 5.2 microM and 9.8 nM, respectively). Furthermore, the increase in binding affinities of the aminoalkylindoles were reflected in the EC(50) values for the ligand-induced inhibition of intracellular cAMP levels mediated by the chimeric receptor. This pattern mirrors the selectivity of WIN 55,212-2 binding to CB2 compared with CB1. Site-specific mutagenesis of the most notable amino acid changes in the chimeric receptor, Gly195 to Ser and Ala198 to Met, revealed that the enhancement in WIN 55,212-2 binding is contributed to by the Ser but not by the Met residue. The data indicate that the amino acid differences in TM3 between CB1 and CB2 play a critical role in subtype selectivity for this class of compounds.

Cannabimimetic indoles, pyrroles and indenes.

In the course of efforts to develop new nonsteroidal antiinflammatory agents, it was discovered that 1-aminoalkyl-3-aroylindoles have affinity for the cannabinoid brain (CB1) receptor. This led to the synthesis of well over 100 cannabimimetic aminoalkylindoles by the group at Sterling Winthrop, and to the development of structure-activity relationships (SAR) for these compounds. These SAR require a heterocyclic aminoethyl group attached to the indole nitrogen, and a 1-naphthoyl group at C-3 for significant receptor affinity. Other workers subsequently demonstrated that an aminoalkyl group was not necessary for cannabinoid activity, but that an N-alkyl group of four to six carbons was sufficient. This led to the discovery that 1-propyl-3-(1-naphthoyl)indole is a selective ligand for the peripheral cannabinoid (CB2) receptor, and to the development of a series of cannabimimetic pyrroles. Comprehensive SAR for this group of cannabinoids have been developed. Two groups have described cannabimimetic indenes, which have been employed as rigid models for the receptor interactions of cannabimimetic indoles with the CB1 receptor. There is some evidence that the indoles interact at a somewhat different site on the receptor than traditional cannabinoids.

Manipulation of the tetrahydrocannabinol side chain delineates agonists, partial agonists, and antagonists.

Structure-activity relation studies have established that the alkyl side chain in tetrahydrocannabinol (THC) plays a crucial role in the activation of the cannabinoid receptor. Unfortunately, the flexible nature of this side chain has hampered efforts to elucidate the precise nature of the interaction of THC with its receptors. Therefore, a series of analogs with structurally restrained side chains of varying length was synthesized and evaluated for pharmacological potency in mice and for receptor affinity. The introduction of cis double bonds inserted rigid angles, whereas triple bonds developed regions of planarity. Receptor affinity for the acetylenic and saturated side chains were the same, whereas double bond substitution increased affinity 10-fold. Moreover, the relationship between receptor affinity and potency was 10-fold less than that of Delta(8)-THC in the case of some acetylenic derivatives, whereas changing the triple bond to a double bond restored the potency/affinity ratio. Additionally, an acetylene at C2-C3 in the octyl and nonyl side chains favored antinociception by as much as 70-fold. Surprisingly, several high-affinity acetylenic derivatives, especially those with cyano substitutions at the terminus of the side chain, were partial agonists or were inactive. Some of these low-efficacy, high-affinity ligands elicited antagonistic activity. The finding that manipulations of the side chain produces high- affinity ligands with either antagonist, partial agonist, or full agonist effects reveals a critical structural feature for receptor activation.

3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC and related compounds: synthesis of selective ligands for the CB2 receptor.

The synthesis and pharmacology of 15 1-deoxy-delta8-THC analogues, several of which have high affinity for the CB2 receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-delta8-THC (5), 1-deoxy-delta8-THC (6), 1-deoxy-3-butyl-delta8-THC (7), 1-deoxy-3-hexyl-delta8-THC (8) and a series of 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 0-4, 6, 7, where n = the number of carbon atoms in the side chain-2). Three derivatives (17-19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB1 and CB2 receptors were determined employing previously described procedures. Five of the 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 1-5) have high affinity (Ki = < 20 nM) for the CB2 receptor. Four of them (2, n = 1-4) also have little affinity for the CB1 receptor (Ki = > 295 nM). 3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC (2, n = 2) has very high affinity for the CB2 receptor (Ki = 3.4 +/- 1.0 nM) and little affinity for the CB1 receptor (Ki = 677 +/- 132 nM).

Differential blockade of the antinociceptive effects of centrally administered cannabinoids by SR141716A.

We evaluated delta-9 tetrahydrocannabinol (Delta9-THC), delta-8 tetrahydrocannabinol (Delta8-THC), CP55,940 (CP55), 1-deoxy-11-hydroxy-Delta8-THC-dimethylheptyl (deoxy-HU210, a CB2-selective cannabinoid that also binds the CB1 receptor) and the endogenous cannabinoid anandamide (ANA) via i.c.v. and/or intrathecal (i.t.) routes of administration, alone and in combination with SR141716A (SR), a CB1 antagonist, using the tail-flick test. Our studies were performed in order better to characterize potential diversity in interactions of the cannabinoids with the cannabinoid (CB1) receptor. When SR was administered i.c.v. or i.p. before Delta9-THC, Delta8-THC or CP55 (i.c.v. or i.t.), SR was a potent antagonist and the blockade was complete (AD50

Structure-activity relationships of indole- and pyrrole-derived cannabinoids.

Early molecular modeling studies with Delta9-tetrahydrocannabinol (Delta9-THC) reported that three discrete regions which interact with brain cannabinoid (CB1) receptors corresponded to the C-9 position of the cyclohexene ring, the phenolic hydroxyl and the carbon side chain at the C3 position. Although the location of these attachment points for aminoalkylindoles is less clear, the naphthalene ring, the carbonyl group and the morpholinoethyl group have been suggested as probable sites. In this study, a series of indole- and pyrrole-derived cannabinoids was developed, in which the morpholinoethyl group was replaced with another cyclic structure or with a carbon chain that more directly corresponded to the side chain of Delta9-THC and were tested for CB1 binding affinity and in a battery of in vivo tests, including hypomobility, antinociception, hypothermia and catalepsy in mice and discriminative stimulus effects in rats. Receptor affinity and potency of these novel cannabinoids were related to the length of the carbon chain. Short side chains resulted in inactive compounds, whereas chains with 4 to 6 carbons produced optimal in vitro and in vivo activity. Pyrrole-derived cannabinoids were consistently less potent than were the corresponding indole derivatives and showed pronounced separation of activity, in that potencies for hypomobility and antinociception were severalfold higher than potencies for hypothermia and ring immobility. These results suggest that, whereas the site of the morpholinoethyl group in these cannabinoids seems crucial for attachment to CB1 receptors, the exact structural constraints on this part of the molecule are not as strict as previously thought.

Synthesis of a tetracyclic, conformationally constrained analogue of delta8-THC.

A tetracyclic, conformationally constrained analogue of delta8-THC (2) has been synthesized in which a two carbon bridge exists between C2 and C2'. Two conceptually related syntheses of 2 are described, both of which employ 5,7-dimethoxy-4-oxo-1,2,3,4-tetrahydronaphthoic acid (11) as starting material. This substrate was converted to 5,7dimethoxy-2-propyl-1,2,3,4-tetrahydronaphthalene (7) and its 4-keto derivative (18). Demethylation of 11 and 18 provided the corresponding resorcinols, which were condensed with trans-p-menthadienol to afford cannabinoid 2, and a keto derivative (20). LiA1H4/A1C1(3) reduction of 20 provided 2. Cannabinoid 2 has relatively low affinity for the cannabinoid brain receptor (Ki = 703+/-98 nM).

Synthesis and pharmacology of the isomeric methylheptyl-delta8-tetrahydrocannabinols.

The synthesis of the 3-heptyl, and the eleven isomeric 3-methylheptyl-delta8-tetrahydrocannabinols (3-7, R and S methyl epimers, and 8) has been carried out. The synthetic approach entailed the synthesis of substituted resorcinols, which were subjected to acid catalyzed condensation with trans-para-menthadienol to provide the delta8-THC analogue. The 1'-, 2'- and 3'-methylheptyl analogues (3-5) are considerably more potent than delta8-THC. The 4'-, 5'- and 6'-methylheptyl isomers (6-8) are approximately equal in potency to delta8-THC.

Enantioselective synthesis and pharmacology of 11-hydroxy-(1'S,2'R)-dimethylheptyl-delta 8-THC.

An enantioselective synthesis of the (1'S,2'R)-dimethylheptyl cannabinoid side chain has been developed and employed in the synthesis of 11-hydroxy-(1'S,2'R)-dimethylheptyl-delta 8-THC (3). Pharmacology, in vivo and in vitro, indicate (3) to be one of the most potent traditional cannabinoids known.