Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide
- 1. (−)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-inflammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA).
- 2. CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5′ pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca2+ concentrations in cells over-expressing human VR1; (b) [14C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [14C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase.
- 3. Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC50=3.2–3.5μm, and with a maximal effect similar in efficacy to that of capsaicin, i.e. 67–70% of the effect obtained with ionomycin (4μm). CBD (10μm) desensitized VR1 to the action of capsaicin. The effects of maximal doses of the two compounds were not additive.
- 4. (+)-5′-DMH-CBD and (+)-7-hydroxy-5′-DMH-CBD inhibited [14C]-AEA uptake (IC50=10.0 and 7.0μm); the (−)-enantiomers were slightly less active (IC50=14.0 and 12.5μm). CBD and (+)-CBD were also active (IC50=22.0 and 17.0μm).
- 5. CBD (IC50=27.5μm), (+)-CBD (IC50=63.5μm) and (−)-7-hydroxy-CBD (IC50=34μm), but not the other analogues (IC50>100μm), weakly inhibited [14C]-AEA hydrolysis.
- 6. Only the (+)-isomers exhibited high affinity for CB1 and/or CB2 cannabinoid receptors.
- 7. These findings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological effects of CBD and its analogues. In view of the facile high yield synthesis, and the weak affinity for CB1 and CB2 receptors, (−)-5′-DMH-CBD represents a valuable candidate for further investigation as inhibitor of AEA uptake and a possible new therapeutic agent.
Among the bioactive constituents of Cannabis sativa, (−)-cannabidiol (CBD, Figure 1) is one of those with the highest potential for therapeutic use (Mechoulam, 1999). Although the pharmacological properties of the other major Cannabis component, (−)-Δ9-tetrahydrocannabinol (THC), have been more thoroughly investigated (Mechoulam, 1999; Pertwee, 1999, for reviews), THC, unlike CBD, exhibits potent psychotropic effects, which have complicated the full assessment of its therapeutic potential. Little is known of the molecular mechanism(s) of action of CBD, which, unlike THC, has very little affinity for either cannabinoid receptor subtypes identified so far, the CB1 and CB2 receptors (Pertwee, 1997, for review). Recent studies, together with the earlier finding of the anti anxiety (Guimaraes et al., 1994), neuro-protective and anti-convulsive activity of CBD and some of its analogues (Consroe et al., 1981;Martin et al., 1987), indicate that CBD may also exert cyto-protective effects by inhibiting the release of inflammatory cytokines from blood cells (Srivastava et al., 1998; Malfait et al., 2000), thus producing an anti-inflammatory action, for example against rheumatoid arthritis (Malfait et al., 2000). These effects of CBD may be due to its anti-oxidant properties (Hampson et al., 1998), to its direct interaction with cytochrome p450-enzymes (Bornheim & Correia, 1989) and other enzymes of the ‘arachidonate cascade’ (Burstein et al., 1985), or to an action at a specific receptor. Recent studies have investigated whether CBD interacts with proteins of the ‘endocannabinoid signalling system’ other than the CB1/CB2 receptors. These proteins are: (i) fatty acid amide hydrolase (FAAH) (Cravatt et al., 1996), the intracellular enzyme catalysing the hydrolysis of the endogenous cannabinoid ligand, anandamide (arachidonoylethanolamide, AEA) (Ueda et al., 2000, for review); and (ii) the ‘anandamide membrane transporter’ (AMT) (Di Marzo et al., 1994), which facilitates the transport of AEA across the cell membrane and, subsequently, its intracellular degradation (Hillard & Jarrahian, 2000, for review). It was found that CBD inhibits both AEA hydrolysis by FAAH-containing membrane preparations (Watanabe et al., 1996), and AEA uptake by RBL–2H3 cells via the AMT (Rakhshan et al., 2000). Although these effects were observed at high μmconcentrations, these findings raised the possibility that some of the pharmacological actions of CBD might be due to inhibition of AEA degradation, with subsequent enhancement of the endogenous levels of this mediator, for which neuroprotective (Hansen et al., 1998) and anti-inflammatory (Di Marzo et al., 2000a) properties have been previously suggested.
Many pharmacological activities of CBD have been established only in vivo, hence some of them may be due to CBD metabolites. The metabolism of CBD is well established. The primary step is hydroxylation on C-7, leading to (−)-7-hydroxy-CBD, followed by further oxidation to (−)-7-carboxy-CBD (Agurell et al., 1986). Although the metabolism of the dimethyl-heptyl homologue of CBD and of the (+) enantiomer of CBD has not been investigated, it is reasonable to assume that it follows the same pathways. Hence we prepared these CBD metabolites, their DMH homologues and some of the respective metabolites in the unnatural (+) series. In particular, in the present study we have examined whether the stereochemistry and the presence of certain chemical groups on the C-5′ and C-1 of CBD affect its capability of influencing AEA inactivation via the AMT and FAAH. Furthermore, we have addressed the question of the possible molecular transducer of CBD by studying the possibility that this natural compound, its (+)-enantiomer and some of its synthetic analogues, interact with another proposed target for AEA, i.e. the vanilloid receptor type 1 (VR1) for capsaicin (Holzer, 1991, Figure 1). This protein is a ligand-, heat- and proton-activated non-specific cation channel acting as a molecular integrator of nociceptive stimuli (Tominaga et al., 1998). Recently, it was discovered that AEA is a full, albeit weak, VR1 agonist (Zygmunt et al., 1999; Smart et al., 2000) and that synthetic capsaicin analogues can interact with either CB1 receptors or the AMT, or both (Di Marzo et al., 1998). Thus, there appears to be some overlap between the ligand recognition properties of VR1 and CB1 receptors and, in particular, of VR1 and the AMT (de petrocellis et al., 2000;Szallasi & Di Marzo, 2000). Although VR1, via the release of inflammatory and algesic peptides, is involved in inflammatory hyperalgesia (Davis et al., 2000; Caterina et al., 2000), the stimulation of this receptor by capsaicin and some of its analogues leads to rapid desensitization, with subsequent paradoxical analgesic and anti-inflammatory effects (Holzer, 1991; Szallasi & Blumberg, 1999). As a consequence of this tachyphylactic effect, capsaicin, like CBD, has been used to treat arthritis (Lorton et al., 2000) and convulsions (Dib & Falchi, 1996).
We report data suggesting that VR1 is a possible molecular target for CBD, and that inhibitors of the AMT can be developed by chemical modification of this natural product.
The synthesis of some of the compounds assayed in this study will be described separately. CBD, whose structure and stereochemistry were described many years ago (Mechoulam & Shvo, 1963; Mechoulam & Gaoni, 1967), was isolated from hashish. (−)-5′-DMH-CBD, (+)-CBD and (+)-5′-DMH-CBD were prepared as described previously (Baek et al., 1985; Leite et al., 1982). The synthesis of the CBD metabolite, (−)-7-hydroxy-CBD was recently reported (Tchilibon & Mechoulam, 2000). [14C]-AEA (5mCimmol−1) was synthesized from [14C]-ethanolamine and arachidonoyl chloride as described (Devane et al., 1992b). Capsaicin, ionomycin and capsazepine were purchased from Sigma.
Cytosolic calcium concentration ([Ca2+]i) assay
where Fmin and Fmax are the fluorescence intensities of fluo-3 without or with maximal [Ca2+], and F is the fluorescence intensity with an intermediate [Ca2+]. Average FEM/FEX was 200 and this value was increased by 60±7% in the presence of 4μm ionomycin.
VR1 receptor binding assays
The affinity of CBD and (+)-CBD for human VR1 receptors was assessed by means of displacement assays carried out with membranes (50μg tube−1) from HEK-hVR1 cells, prepared as described previously (Rosset al., 2001), and the high affinity VR1 ligand [3H]-resiniferatoxin (48Cimmol−1, NEN-Dupont), using the incubation conditions described previously (Ross et al., 2001). Under these conditions the Kd and Bmaxfor [3H]-resiniferatoxin were 0.5nm and 1.39pmolmg−1 protein. The Ki for the displacement of 1nm [3H]-resiniferatoxin by increasing concentrations of CBD and (+)-CBD was calculated from the IC50 values (obtained by GraphPad Software) using the Cheng–Prusoff equation. Specific binding was calculated with 1μm resiniferatoxin (Alexis Biochemicals) and was 78.1±3.7%.
Cannabinoid CB1 and CB2 receptor binding assays
These methods have been described previously by Devane et al. (1992a) for CB1, and Bayewitch et al. (1996) for CB2. For CB1 receptor binding assays synaptosomal membranes from rat brains were used. Sabra male rats weighing 250–300g were decapitated and their brains, without the brain stem, were quickly removed. Synaptosomal membranes were prepared from the brains by centrifugations and gradient centrifugation after their homogenization. The synaptosomal proteins thus obtained were used in the binding assay. The CB2 receptor binding assays were performed with transfected cells. COS-7 cells were transfected with plasmids containing CB2 receptor cDNA, and crude membranes were prepared. The high affinity CB1/CB2 receptor ligand, [3H]-HU-243, with a dissociation constant of 45pm, was incubated with synaptosomal membranes (3–4μg), for CB1 assays, or transfected cells, for CB2 assays, for 90min at 30°C with the different cannabidiol derivatives or with the vehicle alone, and then centrifuged at 13,000r.p.m. for 6min. Bound and free radioligand were separated by centrifugation. The data were normalized to 100% of specific binding, which was determined with 50nm unlabelled HU-243. All experiments were repeated 2–3 times and each point performed in triplicate. The Ki values were determined with a GraphPad Prism program version 2.01 (San Diego, CA, U.S.A.) and using the Cheng–Prusoff equation.
Anandamide cellular uptake assay
The effect of compounds on the uptake of [14C]-AEA by rat basophilic leukaemia (RBL–2H3) cells was studied by using 3.6μm (10,000c.p.m.) of [14C]-AEA as described previously (Bisogno et al., 1997). Cells were incubated with [14C]-AEA for 5min at 37°C, in the presence or absence of varying concentrations of the inhibitors. Residual [14C]-AEA in the incubation media after extraction with CHCl3/CH3OH 2:1 (by vol.), determined by scintillation counting of the lyophilized organic phase, was used as a measure of the AEA that was taken up by cells (de petrocellis et al., 2000). Data are expressed as the concentration exerting 50% inhibition of AEA uptake (IC50) calculated by GraphPad.
Fatty acid amide hydrolase assay
The effect of CBD and its analogues on the enzymatic hydrolysis of AEA was studied as described previously (Bisogno et al., 1997), using cell membranes from mouse neuroblastoma (N18TG2) cells, incubated with compounds and [14C]-AEA (9μm) in 50mm Tris-HCl, pH9, for 30min at 37°C. [14C]-Ethanolamine produced from [14C]-AEA hydrolysis was measured by scintillation counting of the aqueous phase after extraction of the incubation mixture with 2 volumes of CHCl3/CH3OH 2:1 (by vol.). Data are expressed as the concentration exerting 50% inhibition of AEA uptake (IC50), calculated by GraphPad.
Means were compared by means of analysis of variance followed by Bonferroni’s test (ANOVA, StatMost™ software, DataMost Corp.).
Effect of CBD analogues on human vanilloid VR1 receptors
The effects upon [Ca2+]i in HEK–hVR1 cells of CBD, (+)-CBD, and (−)-7-hydroxy-5′-DMH-CBD (HU-317) are shown in Figure 2. These three compounds all induced an increase in [Ca2+]i and behaved as full agonists as compared to capsaicin, although only the two former compounds exerted this effect with an EC50<10μm and independently of their stereochemistry. In fact, the potency (EC50 3.2±0.4) and efficacy (max. effect 68.5±3.1% of the effect of 4μm ionomycin) of (+)-CBD were indistinguishable from those of CBD (EC50 3.5±0.3, max. effect 64.1±3.9%, means±s.e.mean, n=4). The efficacy of both compounds was almost identical to that of a maximal concentration of capsaicin (70.2±3.5% at 10μm, mean±s.e.mean,n=4), which was however 100 fold more potent (EC50=26±9nm). The other six CBD analogues examined in this study were all inactive or very weakly active on [Ca2+]i. The effect of CBD was abolished by the VR1 receptor antagonist capsazepine (10μm, Figure 2) and could not be observed in wild-type HEK cells (data not shown). This strongly suggests that this effect, like that of capsaicin, was due to stimulation of VR1 receptors. Binding assays for the displacement of [3H]-resiniferatoxin from HEK–hVR1 cell membranes by CBD and (+)-CBD confirmed this hypothesis, and showed that CBD and (+)-CBD compete for the binding of [3H]-resiniferatoxin with Ki values (3.6±0.2 and 3.0±0.3μm, respectively, means±s.d.,n=3) similar to the EC50 values for the effect on [Ca2+]i. Furthermore, capsaicin (0.1μm) and CBD (10μm) exhibited cross-desensitization of their effect on [Ca2+]i, providing evidence consistent with these two compounds acting at the same receptor. A 1h pre-exposure to 0.1μm capsaicin reduced the effect of 10μmCBD from 66.7±3.4 to 11.7±1.5% of the effect of 4μm ionomycin, whereas a 1h pre-exposure to 10μmCBD reduced the effect of 0.1μm capsaicin from 68.1±4.1 to 22.3±3.5% (means±s.e.mean, n=4, P<0.01 by ANOVA). Co-treatment of cells with both capsaicin (10μm) and CBD (10μm) did not produce any additive effect (72.2±4.1% of the effect of ionomycin, mean±s.e.mean, n=4).
Affinity of CBD analogues for cannabinoid CB1 and CB2 receptors
Of the nine compounds tested, the (−) analogues were all weakly active or inactive (Ki>10μm) in binding assays for CB1 and CB2 receptor affinity (Table 1), with the exception of (−)-7-hydroxy-5′-DMH-CBD, which exhibited a weak affinity for CB2 receptors (Ki=0.7μm). By contrast, of the three (+)-analogues tested, (+)-5′-DMH–CBD and (+)-7-hydroxy-5′-DMH–CBD behaved as high affinity CB1 receptor ligands (Ki=17.4 and 2.5nm), and were 10–20 fold less active as CB2 receptor ligands (Ki=211 and 44.0nm).
Effect of CBD analogues on the anandamide membrane transporter
Of the nine analogues tested, only (+)- and (−)-CBD, (+)- and (−)-5′-DMH-CBD and (+)- and (−)-7-hydroxy-5′-DMH–CBD inhibited the uptake of [14C]-AEA from RBL–2H3 cells with IC50 values lower than 25μm (Figure 3 and Table 1). Of these six compounds, the (+)-enantiomers were significantly (P<0.05 by ANOVA) and consistently more active than the (−)-enantiomers, and, in particular, (+)-5′-DMH-CBD and (+)-7-hydroxy-5′-DMH-CBD were as potent as the AMT inhibitor, AM404 (Khanolkar & Makriyannis, 1999) (IC50=10.0, 7.0 and 8.1μm for the two compounds and AM404, respectively). (−)-7-hydroxy-5′-DMH-CBD and (−)-5′-DMH-CBD were also almost as potent as AM404 (IC50=12.5 and 14.0μm). The IC50 of CBD (22.0μm) was higher than that previously reported for this compound in the same cell line (11.4μm; Rakhshan et al., 2000).
Effect of CBD analogues on fatty acid amide hydrolase
Only (+)- and (−)-CBD, and (−)-7-hydroxy-CBD exhibited a IC50<100μm for the inhibition of [14C]-AEA hydrolysis by N18TG2 cell membrane preparations (Figure 4 and Table 1), which express high levels of FAAH. The (−)-enantiomer was significantly more potent than the (+)-enantiomer, CBD being also the most potent compound found (IC50=27.5μm). However, none of the CBD analogues tested can be considered a potent inhibitor of FAAH (i.e. with an IC5020μm). The activity of CBD in this study was higher than that previously reported for anandamide hydrolysis by mouse brain (Watanabe et al., 1996;1998), where however only a very high concentration of the compound (160μm) was used.
In this study we investigated whether CBD, now being considered as a possible therapeutic agent (Straus, 2000), is capable of interacting with the recently cloned vanilloid VR1 receptor. In fact, some of the pharmacological actions of CBD are similar to those of natural (e.g. capsaicin) and synthetic agonists of VR1. Although stimulation of VR1 receptors leads to vasodilation and inflammation, capsaicin and its long chain analogues exert anti-inflammatory effects by rapidly desensitizing VR1 receptors to the action of nociceptive stimuli and causing depletion of sensory vasoactive neuropeptides (Szallasi & Blumberg, 1999). CBD also induces anti-inflammatory effects, a possible explanation for this property being its capability of modulating the release of anti-inflammatory or pro-inflammatory mediators (Srivastava et al., 1998, Malfait et al., 2000). CBD and capsaicin also have in common anti-convulsive and anti-rheumatoid-arthritis effects (Consroe et al., 1981; Dib & Falchi, 1996; Malfait et al., 2000; Lorton et al., 2000). Here we found that CBD, compared to capsaicin, is a full, although weak, agonist of human VR1 at concentrations that might be attained after administration of this compound at the doses often used in vivo(10–50mgkg−1 in men), and lower than those required for CBD to bind to cannabinoid receptors. CBD desensitized VR1 to the action of capsaicin, thus opening the possibility that this cannabinoid exerts an anti-inflammatory action in part by desensitization of sensory nociceptors. Future studies with capsazepine (which antagonizes capsaicin effects in rats (Di Marzo et al., 2001) but not always in mice (Di Marzo et al., 2000b), and VR1 ‘knockout’ mice (Davis et al., 2000; Caterina et al., 2000), should test the involvement of VR1 in the pharmacological actions of CBD.
We found that insertion in CBD of a DMH instead of an n-pentyl chain on the C-5′, or of a carboxyl function instead of the methyl group on the C-1, abolishes the capability of the cannabinoid to induce a VR1-mediated functional response, whereas insertion of both a hydroxy-group on the C-7 and of a 5′-DMH group decreases the potency but not the efficacy of CBD. By contrast, inversion of the stereochemistry does not modify the activity of CBD. These data suggest that the C-1 methyl and the aromatic ‘A’ ring, which is chemically similar to the vanillyl moiety of capsaicin (Figure 1), are more important than the chiral part of CBD for its interaction with VR1. That CBD binds to the same site as capsaicin is suggested by the finding that both compounds displace [3H]-resiniferatoxin from its specific binding sites in membranes from cells over-expressing VR1 receptors (this study and Ross et al., 2001). However, while capsaicin exhibits higher potency than affinity for vanilloid receptors (Szallasi & Blumberg, 1999), CBD is as active in the [3H]-resiniferatoxin binding assay as in the hVR1 functional assay. This suggests that CBD is less capable than capsaicin to induce a VR1-mediated functional response at low concentrations, even though the efficacy of high concentrations of both compounds is the same. It should be noted that the cells used here to assess the functional activity at VR1 express high levels of this receptor, and that the potency and efficacy of CBD in native cells containing lower amounts of vanilloid receptors might be lower than those observed here.
The endocannabinoid AEA is thought to exert anti-inflammatory and neuroprotective actions (Di Marzo et al., 2000a; Hansen et al., 1998). As it was found to inhibit the re-uptake and hydrolysis of AEA in vitro(Rakhshan et al., 2000; Watanabe et al., 1996), it is possible that CBD acts in part by interfering with AEA inactivation, thereby enhancing the putative tonic inhibitory action of AEA on inflammation. The fact that the pharmacological actions of CBD are not influenced by CB1/CB2 receptor antagonists should not be taken as evidence against this hypothesis, since it is now established that AEA also acts upon non-cannabinoid receptor targets, including VR1 receptors and TASK-1 K+ channels (Zygmunt et al., 1999;Maingret et al., 2001). Here we confirmed that CBD inhibits AEA transporter-mediated uptake by cells and enzymatic hydrolysis. We also found that analogues of CBD are inhibitors of the AMT, and that this property is more pronounced with (+)-enantiomers, or when the C-7 and C-5′ are derivatized with a hydroxyl- and a DMH group, respectively. The most potent inhibitor found was (+)-7-hydroxy-5′-DMH-CBD. However, this compound exhibited high affinity for CB1 and CB2 receptors. Also (+)-5′-DMH-CBD was more active as a CB1 and CB2 receptor ligand than as an AMT inhibitor. By contrast, (−)-7-hydroxy-5′-DMH-CBD and (−)-5′-DMH-CBD, which were almost as potent as AM404 against the AMT, but, unlike AM404, had low affinity for the two cannabinoid receptors subtypes and no activity at VR1, may represent metabolically stable and relatively selective pharmacological tools for the study of AEA inactivation in vitro. The (−)-5′DMH-CBD is obtained by a facile, high yield synthesis (Baek et al., 1985) and may find application as therapeutic agent for those disorders where AEA exerts an endogenous tone with beneficial effects. The novel AMT inhibitors developed here should be tested also on the cellular uptake of palmitoylethanolamide, a natural anti-inflammatory AEA congener (Lambert & Di Marzo, 1999), since a recent study showed that CBD inhibits the facilitated transport of this compound into RBL–2H3 cells (Jacobsson & Fowler, 2001).
We have mentioned above that the (+)-enantiomers of the CBD analogues tested here on AEA cellular uptake were more potent inhibitors than the (−)-enantiomers. A certain enantio-specificity for the interaction with the AMT has been noted previously also for AEA analogues (see Khanolkar & Makriyannis, 1999, for review). It was also noted that the same stereochemical preference existed for the interaction of these compounds with FAAH, whereas the interaction with CB1 receptors followed the opposite enantio-selectivity (Khanolkar & Makriyannis, 1999). We noted that CBD inhibits FAAH more potently than the (+)-enantiomer. By contrast, all but one of the (+)-CBD analogues tested exhibited much higher affinity for CB1 receptors than their (−)-enantiomers. Thus, for CBD analogues, the stereochemical requisites for the interaction with the AMT and CB1 receptors are the same, and they may be opposite to those necessary for the interaction with FAAH. The binding data were indeed unexpected. In the tetrahydrocannabinol series the (−) (3R,4R) enantiomers bind to CB1 and have pharmacological activity in various typical cannabinoid assays, while the (+) (3S,4S) enantiomers are essentially inactive (Mechoulamet al., 1988; Howlett et al., 1990; Little et al., 1989; Jarbe et al., 1989). In the CBD series of compounds we observed here the opposite situation. The reason for this dichotomy is unknown. Further studies investigating the potency and efficacy of the compounds in functional assays of CB1 receptor-mediated activity need to be performed in order to fully assess the cannabimimetic activity of the compounds in the (+)-CBD series.
In conclusion, the present study has provided novel insights into the possible mechanism(s) of action of the natural cannabinoid CBD by identifying in VR1 receptors a novel potential molecular target for this compound. Furthermore, we have shown that potent inhibitors of AEA cellular uptake can be developed from certain chemical modifications of CBD, and have confirmed that CBD can act in principle also by inhibiting AEA inactivation. Future studies will be needed to address the question of whether vanilloid receptors or endogenous cannabinoids contribute to the anti-inflammatory and neuroprotective actions of CBD.
We thank the U.S. National Institute on Drug Abuse (grant DA 9789, to R. Mechoulam), the Israel Science Foundation (to R. Mechoulan), the Yeshaya Horowitz Association (to R. Mechoulam) and the MURST (3933 to V. Di Marzo) for support.
- anandamide membrane transporter
- cytosolic calcium concentration
- fatty acid amide hydrolase
- human hembryonic kidney
- HEK cells transfected with human VR1 cDNA
- vanilloid receptor of type 1
- AGURELL S., HALLDIN M., LINDGREN J.E., OHLSSON A., WIDMAN M., GILLESPIE H., HOLLISTER L. Pharmacokinetics and metabolism of delta-1-tetrahydrocannabinol and other cannabinoids with emphasis on man. Pharmacol. Rev. 1986;38:21–43. [PubMed]
- BAEK S.H., SREBNIK M., MECHOULAM R. Borontrifluoride on alumina–a modified Lewis acid reagent. An improved synthesis of cannabidiol. Tetrahedron Lett. 1985;26:1083–1086.
- BAYEWITCH M., RHEE M.H., AVIDOR-REISS T., BREUER A., MECHOULAM R., VOGEL Z. (−)-Delta9-tetrahydrocannabinol antagonizes the peripheral cannabinoid receptor-mediated inhibition of adenylyl cyclase. J. Biol. Chem. 1996;271:9902–9905. [PubMed]
- BISOGNO T., MAURELLI S., MELCK D., De PETROCELLIS L., Di MARZO V. Biosynthesis, uptake, and degradation of anandamide and palmitoylethanolamide in leukocytes. J. Biol. Chem.1997;272:3315–3323. [PubMed]
- BORNHEIM L.M., CORREIA M.A. Effect of cannabidiol on cytochrome P-450 isozymes. Biochem. Pharmacol. 1989;38:2789–2794. [PubMed]
- BURSTEIN S., HUNTER S.A., RENZULLI L. Prostaglandins and cannabis XIV. Tolerance to the stimulatory actions of cannabinoids on arachidonate metabolism. J. Pharmacol. Exp. Ther.1985;235:87–91. [PubMed]
- CATERINA M.J., LEFFLER A., MALMBERG A.B., MARTIN W.J., TRAFTON J., PETERSEN-ZEITZ K.R., KOLTZENBURG M., BASBAUM A.I., JULIUS D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 2000;288:306–313. [PubMed]
- CONSROE P., MARTIN A., SINGH V. Antiepileptic potential of cannabidiol analogs. J. Clin. Pharmacol. 1981;21:428S–436S. [PubMed]
- CRAVATT B.F., GIANG D.K., MAYFIELD S.P., BOGER D.L., LERNER R.A., GILULA N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature.1996;384:83–87. [PubMed]
- DAVIS J.B., GRAY J., GUNTHORPE M.J., HATCHER J.P., DAVEY P.T., OVEREND P., HARRIES M.H., LATCHAM J., CLAPHAM C., ATKINSON K., HUGHES S.A., RANCE K., GRAU E., HARPER A.J., PUGH P.L., ROGERS D.C., BINGHAM S., RANDALL A., SHEARDOWN S.A. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature. 2000;405:183–187.[PubMed]
- De PETROCELLIS L., BISOGNO T., DAVIS J.B., PERTWEE R.G., Di MARZO V. Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity. FEBS Lett. 2000;483:52–56.[PubMed]
- DEVANE W.A., BREUER A., SHESKIN T., JARBE T.U.C., EISEN M., MECHOULAM R. A novel probe for the cannabinoid receptor. J. Med. Chem. 1992a;35:2065–2069. [PubMed]
- DEVANE W.A., HANUS L., BREUER A., PERTWEE R.G., STEVENSON L.A., GRIFFIN G., GIBSON D., MECHOULAM R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science. 1992b;258:1946–1949. [PubMed]
- DIB B., FALCHI M. Convulsions and death induced in rats by Tween 80 are prevented by capsaicin. Int. J. Tissue React. 1996;18:27–31. [PubMed]
- Di MARZO V., BISOGNO T., MELCK D., ROSS R., BROCKIE H., STEVENSON L., PERTWEE R., De PETROCELLIS L. Interactions between synthetic vanilloids and the endogenous cannabinoid system. FEBS Lett. 1998;436:449–454. [PubMed]
- Di MARZO V., BREIVOGEL C., BISOGNO T., MELCK D., PATRICK G., TAO Q., SZALLASI A., RAZDAN R.K., MARTIN B.R. Neurobehavioral activity in mice of N-vanillyl-arachidonyl-amide.Eur. J. Pharmacol. 2000b;406:363–374. [PubMed]
- Di MARZO V., FONTANA A., CADAS H., SCHINELLI S., CIMINO G., SCHWARTZ J.C., PIOMELLI D. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature. 1994;372:686–691. [PubMed]
- Di MARZO V., LASTRES-BECKER I., BISOGNO T., DE PETROCELLIS L., MILONE A., DAVIS J.B., FERNANDEZ-RUIZ J.J. Hypolocomotor effects in rats of capsaicin and two long chain capsaicin homologues. Eur. J. Pharmacol. 2001;420:123–131. [PubMed]
- Di MARZO V., MELCK D., De PETROCELLIS L., BISOGNO T. Cannabimimetic fatty acid derivatives in cancer and inflammation. Prostaglandins Other Lipid Mediat. 2000a;61:43–61.[PubMed]
- GUIMARAES F.S., DE AQUIAR J.C., MECHOULAM R., BREUER A. Anxiolytic effect of cannabidiol derivatives in the elevated plus-maze. Gen. Pharmacol. 1994;25:161–194. [PubMed]
- HAMPSON A.J., GRIMALDI M., AXELROD J., WINK D. Cannabidiol and (−) Delta-9-tetrahydrocannabinol are neuroprotective. Proc. Natl. Acad. Sci. U.S.A. 1998;95:8268–8273.[PMC free article] [PubMed]
- HANSEN H.S., LAURITZEN L., MOESGAARD B., STRAND A.M., HANSEN H.H. Formation of N-acyl-phosphatidylethanolamines and N-acetylethanolamines: proposed role in neurotoxicity.Biochem. Pharmacol. 1998;55:719–725. [PubMed]
- HAYES P., MEADOWS H.J., GUNTHORPE M.J., HARRIES M.H., DUCKWORTH D.M., CAIRNS W., HARRISON D.C., CLARKE C.E., ELLINGTON K., PRINJHA R.K., BARTON A.J., MEDHURST A.D., SMITH G.D., TOPP S., MURDOCK P., SANGER G.J., TERRETT J., JENKINS O., BENHAM C.D., RANDALL A.D., GLOGER I.S., DAVIS J.B. Cloning and functional expression of a human orthologue of rat vanilloid receptor-1. Pain. 2000;88:205–215.[PubMed]
- HILLARD C.J., JARRAHIAN A. The movement of N-arachidonoylethanolamine (anandamide) across cellular membranes. Chem. Phys. Lipids. 2000;108:123–134. [PubMed]
- HOLZER P. Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons.Pharmacol. Rev. 1991;43:143–201. [PubMed]
- HOWLETT A.C., CHAMPION T.M., WILKEN G.H., MECHOULAM R. Stereochemical effects of 11-OH-delta-8-tetrahydrocannabinol-dimethylheptyl to inhibit adenylate cyclase and bind to the cannabinoid receptor. Neuropharmacology. 1990;29:161–165. [PubMed]
- JACOBSSON S.O., FOWLER C.J. Characterization of palmitoylethanolamide transport in mouse Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leukaemia cells: comparison with anandamide.Br. J. Pharmacol. 2001;132:1743–1754. [PMC free article] [PubMed]
- JARBE T.U.C., HILTUNEN A.J., MECHOULAM R. Stereospecificity of the discriminative stimulus functions of the dimethylheptyl homologs of 11-OH-delta-8-tetra-hydrocannabinol in rats and pigeons. J. Pharmacol. Exper. Ther. 1989;250:1000–1005. [PubMed]
- KHANOLKAR A.D., MAKRIYANNIS A. Structure-activity relationships of anandamide, an endogenous cannabinoid ligand. Life Sci. 1999;65:607–616. [PubMed]
- LAMBERT D.M., Di MARZO V. The palmitoylethanolamide and oleamide enigmas: are these two fatty acid amides cannabimimetic. Curr. Med. Chem. 1999;6:757–773. [PubMed]
- LEITE J.R., CARLINI E.A., LANDER N., MECHOULAM R. Anticonvulsant effect of (−) and (+) isomers of CBD and their dimethyl heptyl homologs. Pharmacol. 1982;124:141–146.
- LITTLE P.J., COMPTON D.R., MECHOULAM R., MARTIN B. Stereochemical effects of 11-OH-delta-8-THC-dimethylheptyl in mice and dogs. Pharmacol. Biochem. Behavior. 1989;32:661–666.[PubMed]
- LORTON D., LUBAHN C., ENGAN C., SCHALLER J., FELTEN D.L., BELLINGER D.L. Local application of capsaicin into the draining lymph nodes attenuates expression of adjuvant-induced arthritis. Neuroimmunomodulation. 2000;7:115–125. [PubMed]
- MAINGRET F., PATEL A.J., LAZDUNSKI M., HONORE E. The endocannabinoid anandamide is a direct and selective blocker of the background K(+) channel TASK-1. EMBO J. 2001;20:47–54.[PMC free article] [PubMed]
- MALFAIT A.M., GALLILY R., SUMARIWALLA P.F., MALIK A.S., ANDREAKOS E., MECHOULAM R., FELDMANN M. The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis. Proc. Natl. Acad. Sci. U.S.A.2000;97:9561–9566. [PMC free article] [PubMed]
- MARTIN A.R., CONSROE P., KANE V.V., SHAH V., SINGH V., LANDER N., MECHOULAM R., SREBNIK M. Structure-anticonvulsant activity relationships of cannabidiol analogs. NIDA Res. Monogr. 1987;79:48–58. [PubMed]
- MECHOULAM R. Recent advantages in cannabinoid research. Forsch. Komplementarmed. 1999;6:16–20. [PubMed]
- MECHOULAM R., GAONI Y. The absolute configuration of delta-1-tetrahydrocannabinol, the major active constituent of hashish. Tetrahedron Lett. 1967. pp. 1109–1111. [PubMed]
- MECHOULAM R., SHVO Y. The structure of cannabidiol. Tetrahedron. 1963;19:2073–2078. [PubMed]
- MECHOULAM R., FEIGENBAUM J.J., LANDER N., SEGAL M., JARBE T.U.C., HILTUNEN A.J., CONSROE P. Enantiomeric cannabinoids: stereospecificity of psychotropic activity. Experientia.1988;44:762–764. [PubMed]
- PERTWEE R.G. Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther. 1997;74:129–180. [PubMed]
- PERTWEE R.G. Pharmacology of cannabinoid receptor ligands. Curr. Med. Chem. 1999;6:635–664.[PubMed]
- RAKHSHAN F., DAY T.A., BLAKELY R.D., BARKER E.L. Carrier-mediated uptake of the endogenous cannabinoid anandamide in RBL-2H3 cells. J. Pharmacol. Exp. Ther. 2000;292:960–967. [PubMed]
- ROSS R.R., GIBSON T.M., BROCKIE H.C., LESLIE M., PASHMI G., CRAIB S.J., DI MARZO V., PERTWEE R.G. Structure-activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens. Br. J. Pharmacol.2001;132:631–640. [PMC free article] [PubMed]
- SMART D., GUNTHORPE M.J., JERMAN J.C., NASIR S., GRAY J., MUIR A.I., CHAMBERS J.K., RANDALL A.D., DAVIS J.B. The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1) Br. J. Pharmacol. 2000;129:227–230. [PMC free article] [PubMed]
- SRIVASTAVA M.D., SRIVASTAVA B.I., BROUHARD B. Delta9 tetrahydrocannabinol and cannabidiol alter cytokine production by human immune cells. Immunopharmacology. 1998;40:179–185.[PubMed]
- STRAUS S.E. Immunoactive cannabinoids: therapeutic prospects for marijuana constituents. Proc. Natl. Acad. Sci. U.S.A. 2000;97:9363–9364. [PMC free article] [PubMed]
- SZALLASI A., BLUMBERG P.M. Vanilloid (Capsaicin) receptors and mechanisms. Pharmacol. Rev.1999;51:159–212. [PubMed]
- SZALLASI A., Di MARZO V. New perspectives on enigmatic vanilloid receptors. Trends Neurosci.2000;23:491–497. [PubMed]
- TCHILIBON S., MECHOULAM R. Synthesis of a primary metabolite of cannabidiol. Org. Lett.2000;2:3301–3303. [PubMed]
- TOMINAGA M., CATERINA M.J., MALMBERG A.B., ROSEN T.A., GILBERT H., SKINNER K., RAUMANN B.E., BASBAUM A.I., JULIUS D. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron. 1998;21:531–543. [PubMed]
- UEDA N., PUFFENBARGER R.A., YAMAMOTO S., DEUTSCH D.G. The fatty acid amide hydrolase.Chem. Phys. Lipids. 2000;108:107–121. [PubMed]
- WATANABE K., KAYANO Y., MATSUNAGA T., YAMAMOTO I., YOSHIMURA H. Inhibition of anandamide amidase activity in mouse brain microsomes by cannabinoids. Biol. Pharm. Bull.1996;19:1109–1111. [PubMed]
- ZYGMUNT P.M., PETERSSON J., ANDERSSON D.A., CHUANG H., SORGARD M., Di MARZO V., JULIUS D., HOGESTATT E.D. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature. 1999;400:452–457. [PubMed]