Compounds and extracts

CBC; CBD; CBG; CBN; cannabidiol acid (CBDA); cannabigerol acid (CBGA); cannabidivarin (CBDV); cannabigevarin (CBGV); cannabidivarin acid (CBDVA); Δ⁹-tetrahydrocannabinol (THC); Δ⁹-tetrahydrocannabinol acid (THCA); Δ⁹-tetrahydrocannabivarin (THCV); Δ⁹-tetrahydrocannabivarin acid (THCVA); and the corresponding BDS (extracts prepared from Cannabis sativa L. botanical raw material) were provided by GW Pharma Ltd (Salisbury, UK). The compounds were of at least 95% purity. The amount of each principal cannabinoid in the corresponding BDS varied between 40% and 70% (% w/w of extract) depending upon the BDS tested. The amount of each major cannabinoid in the BDS, expressed as a %, was used to calculate the amount of the BDS necessary to obtain the equimolar amount of the corresponding pure cannabinoid in the various experiments. Thus, for example, if the amount of a given cannabinoid in a given BDS was 70%, the amount of BDS to be administered to cells to yield a given final concentration of the cannabinoid was calculated from the molecular weight of the cannabinoid and the amount in milligrams of the BDS (as if the BDS only contained the given cannabinoid) divided by 0.7. The chemical profile of minor cannabinoids present in each BDS was unique to each BDS, as was that of non-cannabinoid components. Thus, each BDS has a unique chemical profile (‘chemical fingerprint’).

TRP calcium assays

HEK-293 cells stably over-expressing recombinant rat TRPA1 rat TRPM8, rat TRPV2 or human TRPV1 were selected by G-418 (Geneticin; 600 µg·mL⁻¹), grown on 100 mm diameter Petri dishes as monolayers in minimum essential medium supplemented with non-essential amino acids, 10% fetal bovine serum and 2 mM glutamine, and maintained under 5% CO₂ at 37°C. Stable expression of each channel was checked by quantitative-PCR. On the day of the experiment, the cells were loaded for 1 h at 25°C with the cytoplasmic calcium indicator Fluo-4AM (Invitrogen) 4 µM in dimethyl sulphoxide containing 0.02% Pluronic F-127 (Invitrogen, Carlsbad, CA, USA). After loading, cells were washed twice in Tyrode’s buffer (145 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl₂, 1.2 mM MgCl₂, 10 mM d-glucose and 10 mM HEPES, pH 7.4), resuspended in the same buffer, and transferred to the quartz cuvette of the spectrofluorimeter (ex λ = 488 nm; em λ = 516 nm) (Perkin-Elmer LS50B equipped with PTP-1 Fluorescence Peltier System; Perkin-Elmer Life and Analytical Sciences, Waltham, MA, USA) under continuous stirring. Experiments were carried by measuring cell fluorescence before and after the addition of various concentrations of phytocannabinoids. The values of the effect on [Ca²⁺]_i in wild-type (i.e. not transfected with any construct) HEK-293 cells were taken as baselines and subtracted from the values obtained from transfected cells. The potency of test compounds (EC₅₀ values) were determined as the concentration of test substances required to produce half-maximal increases in [Ca²⁺]_i. The efficacy of the agonists was determined by comparing their effect with the analogous effect observed with 4 µM ionomycin. The effects of the phytocannabinoids on TRPA1 are expressed as a percentage of the effect obtained with 100 µM allylisothiocyanate [mustard oil (MO)]. Antagonist/desensitizing behaviour was evaluated against capsaicin (0.1 µM) for TRPV1; icilin (0.25 µM) for TRPM8; MO (10 µM) for TRPA1; or lysophosphatidylcholine (LPC) (3 µM) for TRPV2 by adding the compounds in the quartz cuvette 5 min before stimulation of cells with agonists. Data are expressed as the concentration exerting a half-maximal inhibition of agonist effect (IC₅₀). The effect on [Ca²⁺]_iexerted by the agonist alone was taken as 100%. Some experiments were also carried out to antagonize the effects of CBDV and CBC on TRPA1-mediated elevation of [Ca²⁺]_i with AP18 (Petrus et al., 2007) or HC030031 (McNamara et al., 2007), which were added to cells 5 min before the agonists. All determinations were at least performed in triplicate. Dose-response curves were fitted by a sigmoidal regression with variable slope. Curve fitting and parameter estimation were performed with GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA).

DAGL-α assay

Human recombinant DAGL-α was originally over-expressed with high efficiency in COS-7 cells and therefore these cells were used for this assay instead of HEK-293 cells, which, moreover, also express the native enzyme (Bisogno et al., 2003). COS-7 cells, over-expressing DAGL-α, were homogenized in Tris–HCl buffer, pH 7. The homogenates were centrifuged at 4°C at 800×g (5 min) and then at 10 000×g (25 min). The 10 000×g membrane fraction was incubated in Tris–HCl 50 mM at pH 7.0 at 37°C for 20 min, with synthetic 1-[¹⁴C]-oleoyl-2-arachidonoyl-glycerol (1.0 mCi·mmol⁻¹, 25 µM) in the presence of vehicle or varying concentrations of test compounds. After the incubation, lipids were extracted with two volumes of CHCl₃/MeOH 2/1 (by volume). The extracts were lyophilized under vacuum and then purified by TLC on silica on polypropylene plates using CHCl₃/CH₃OH/NH₄OH (85/15/1 by volume) as the eluting system. Bands corresponding to [¹⁴C]-oleic acid were cut, and their radioactivity was counted with a β-counter.

MAGL assay

MAGL activity was measured using the cytosol derived from the 10 000×g fraction of homogenates from wild-type COS cells and synthetic 2 [³H]-arachidonoylglycerol; 100 µg of protein and 10 µM (10 000 cpm) [³H]-2-AG for each assay were incubated for 20 min a 37°C in the presence or absence of various concentrations of phytocannabinoids in Tris-HCl buffer, pH 7. [³H]-glycerol produced from the MAGL hydrolysis of [³H]-2-AG was measured by scintillation counting of the aqueous phase after extraction of the incubation mixture with two volumes of CHCl3/CH3OH (1:1 by volume) and used as a measure of enzyme activity.

FAAH assay

The effect of increasing concentrations of the compounds on the enzymatic hydrolysis of [¹⁴C]-AEA was studied using membranes prepared from rat brain. Membranes (70–100 µg) were incubated in the presence or absence of varying concentrations of the compounds and [¹⁴C]-AEA (10 000 cpm, 2.5 µM) in 50 mM Tris-HCl, pH 9, for 30 min at 37°C. [¹⁴C]-ethanolamine produced from [¹⁴C]-AEA hydrolysis was used to calculate FAAH activity and was measured by scintillation counting of the aqueous phase after extraction of the incubation mixture with two volumes of CHCl3/CH3OH (1:1 by volume). Data are expressed as the concentration exerting a half maximal inhibition (IC₅₀).

NAAA assays

HEK-293 cells in which the cDNA encoding the human enzyme was stably over-expressed (HEK-NAAA cells), were used for a specific enzymatic assay of NAAA as described previously (Saturnino et al., 2010). Briefly, the 12 000 membrane fraction of homogenized cells was suspended in phosphate-buffered saline (pH 7.4) and subjected to two cycles of freezing and thawing and used in the assay. The enzyme preparation (50 µg per sample) was incubated at 37°C for 30 min with 100 µM [¹⁴C]-N-palmitoylethanolamine (10 000 cpm per sample) in a solution of citrate/sodium phosphate 50 mM (pH 5.2) and 0.1% Triton X-100, containing varying concentrations of the phytocannabinoids. The reaction was terminated by the addition of chloroform/methanol (1:1 by volume) and quantification of [¹⁴C]-ethanolamine was carried out by using a β-counter (TRI-carb 2100TR; Perkin-Elmer) to quantify the [¹⁴C]-ethanolamine formed by the enzymatic activity.

ACU assay

The effect of compounds on ACU was analysed on rat basophilic leukaemia cells by using 2.5 µM (10 000 cpm) [¹⁴C]-AEA. Cells were incubated with [¹⁴C]-AEA for 90 s at 37°C, in the presence or absence of varying concentrations of the compounds. Residual [¹⁴C]-AEA in the incubation medium after extraction with CHCl₃/CH₃OH 2:1 (by volume) was determined by scintillation counting of the lyophilized organic phase and was used as a measure of the AEA taken up by cells. Specific AEA uptake was determined by subtracting uptake at 4°C from uptake at 37°C. Data are expressed as the concentration exerting 50% inhibition of AEA uptake (IC₅₀) calculated by GraphPad software (Graph-Pad Software Inc., San Diego, CA, USA).

Activity at TRPA1 channels

In Table 1, the efficacies and potencies of cannabinoids at eliciting a TRPA1-mediated increase in intracellular Ca²⁺ in HEK-293 cells over-expressing the channel are reported. HEK-293 cells were stably transfected with the plasmid pcDNA3 containing the rat recombinant TRPA1 cDNA, thus generating cells (denoted hereafter as ‘TRPA1-HEK-293’) that express high levels of TRPA1 transcript (De Petrocellis et al., 2008). The compounds tested induced TRPA1-mediated Ca²⁺ elevation with efficacy comparable with that of allylisothiocyanate, the most potent being CBC (EC₅₀ = 90 nM) and CBD (EC₅₀ = 110 nM), in good agreement with the EC₅₀ values of 60 nM and 96 nM, respectively, previously published by our research group (De Petrocellis et al., 2008). The rank order of potency of the pure compounds was as follows: CBC > CBD > CBN > CBDV > CBG > THCV > CBGV > THCA > CBDA > CBGA > THCVA. Therefore, none of the pure compounds was more potent than CBC, CBD and CBN as TRPA1 agonists. These compounds as well as CBDV and CBG were more potent than allylisothiocyanate, whereas the acid cannabinoids were the least active in this test. Except for THCA and CBN, all the pure cannabinoids also exhibited higher efficacy than allylisothiocyanate. Interestingly, THCV-BDS was the most potent (EC₅₀ = 70 nM), and other BDS (i.e. those of THCV, THCA, CBGV, CBDA, THCVA) were more potent (EC₅₀ = 0.07–5.8 µM) than the corresponding pure compounds in this assay (Table 1). At high concentrations (>10 µM), some compounds also caused an effect on [Ca²⁺]_i in cells that had not been transfected with any TRP construct (wild-type HEK-293 cells), and the values obtained from the latter cells were subtracted from those obtained in transfected cells. Importantly, all the compounds found here to activate TRPA1 also desensitized this channel to subsequent (5 min after addition of the cannabinoid) stimulation by MO allyisothiocyanates (Table 1). Finally, to provide conclusive evidence for the specificity of the effects, some compounds (i.e. CBDV and CBC) were also given to transfected cells that had been pre-incubated for 5 min with the TRPA1 antagonists AP18 (Petrus et al., 2007) or HC030031(McNamara et al., 2007). The effect of CBDV (10 µM) was reduced by AP18 (50 µM) by 63.6 ± 7.1% and by HC030031 (50 µM) by 42.0 ± 3.4%. These antagonists reduced the effect of MO isothiocyanate (10 µM) by 51.0 ± 8.2% and 51.5 ± 5.2%,respectively. The effect of CBC (10 µM) was reduced by AP18 (50 µM) by 55.8 ± 6.0%. AP18 (10 µM) reduced the effects of CBC and MO isothiocyanate by 43.2 ± 3.8 and 48.6 ± 4.1% respectively (data are means ± SD of n = 3 determinations).

Table 1

Efficacy and potency of pure cannabinoids and of most of their corresponding ‘botanical drug substance’ (BDS) as functional agonists at TRPA1 channels, and the potency of pure cannabinoids as desensitizers of TRPA1 to the effect of allyl …

Activity at TRPM8 channels

All the TRPM8 experiments were carried out at 22°C. None of the compounds tested activated TRPM8-mediated Ca²⁺ elevation in HEK-293 cells stably transfected with cDNA encoding the rat TRPM8 (denoted hereafter as ‘TRPM8-HEK-293’), and overexpressing high levels of TRPM8 transcript (De Petrocellis et al., 2007). Instead, when the compounds were given to cells 5 min before the known TRPM8 agonist, icilin (0.25 µM), they all, with the exception of CBC, antagonized the Ca²⁺ elevation response. As shown previously, icilin dose dependently elevated intracellular Ca²⁺ in TRPM8-HEK-293 cells, but not in non-transfected cells, with an EC₅₀ of 0.19 ± 0.03 µM (De Petrocellis et al., 2007) (Table 2). The rank order of potency of the pure compounds against icilin was: CBD > CBG > THCA > CBN > THCV > CBDV > CBGA > THCVA > CBGV > CBDA (IC₅₀ = 0.06–4.8 µM), but also some BDS (THCV, CBG, THCA) were very potent (IC₅₀ < 0.1 µM) at antagonizing TRPM8 (Table 2). A ‘CBG-free’ CBG-BDS was found to be inactive per se, but when added to pure CBG, the activity of CBG-BDS was restored and this compound was significantly more potent and efficacious at antagonizing TRPM8 than pure CBG (Figure 1), thus pointing to a synergistic effect between this cannabinoid and some of the components of its corresponding Cannabis extract.

Table 2

Potency of pure cannabinoids and of most of their corresponding ‘botanical drug substance’ (BDS) as functional antagonists at TRPM8 channels

Figure 1

Inhibition of TRPM8-mediated elevation of intracellular calcium by cannabigerol (CBG), a CBG-botanical drug substance rendered free of CBG [denoted as botanical extract (BDS)] and the reconstituted CBG-BDS (denoted as CBG-BDS reconstituted). Experiments …

Activity at TRPV1 channels

Given the previously reported effects of some cannabinoids on TRPV1 (Bisogno et al., 2001; Ligresti et al., 2006), it was interesting to assess whether those compounds belonging to this class that had not been tested before on TRPV1 can interact with this target. Apart from the previously reported stimulating effects of CBD and CBG on TRPV1 (Bisogno et al., 2001; Ligresti et al., 2006), we found here that THCV and CBGV, but much less so their acid analogues, also stimulated human TRPV1 (EC₅₀ = 1.0–2.0 µM), with the BDS being less efficacious than the corresponding cannabinoid, except for THCV-BDS (0.2 µM) (Table 3, Figure 2). Many compounds exhibited very weak efficacy at TRPV1 receptors, often hardly measurable. Therefore, the EC₅₀ values calculated for these compounds may have little pharmacological meaning. Nevertheless, several compounds also desensitized TRPV1 to its subsequent (5 min after addition of the cannabinoid) stimulation by capsaicin (Table 3).

Table 3

Efficacy and potency of pure cannabinoids and of most of their corresponding ‘botanical drug substance’ (BDS) as functional agonists at TRPV1 channels, and the potency of pure cannabinoids as desensitizers of TRPV1 to the effect of capsaicin …

Figure 2

(A) Dose-dependent effects of cannabigivarin (CBGV), Δ⁹-tetrahydrocannabiverin (THCV) and THCV-botanical drug substance (THCV-BDS) on TRPV1-mediated elevation of intracellular calcium in HEK-293 cells stably over-expressing the human recombinant …

Activity at TRPV2 channels

The pure compounds available were also tested on TRPV2, based on the previously reported agonistic effect of CBD on this channel (Qinet al., 2008). We stably transfected HEK-293 cells with the plasmid pcDNA3 containing the rat recombinant TRPV2 cDNA, thus generating cells (denoted hereafter as ‘TRPV2-HEK-293’) that, as assessed by real-time PCR, express high levels of TRPV2 transcript (data not shown). This transcript was absent in HEK-293 cells that had not been transfected with the pcDNA3 plasmid and were treated with only Lipofectamine (data not shown). Using our fluorometric test, we showed that rat TRPV2-HEK293 cells exhibit a sharp increase in intracellular [Ca²⁺]_i upon application of LPC or 2-aminoethoxydiphenyl borate, two well-established TRPV2 activators (Neeper et al., 2007; Monet et al., 2009). Using this test, we determined the concentration for half-maximal activation to be 3.4 ± 0.025 µM for LPC (Table 4, Figure 3). Analogous to the findings ofQin et al. (2008), THC and CBD, as well as several of the pure cannabinoids that were tested, increased [Ca²⁺]_i in TRPV2-HEK-293 cells. The EC₅₀ values are shown in Table 4, and the rank of potency was THC > CBD > CBGV > CBG > THCV > CBDV > CBN. Conversely, all the carboxylic acid cannabinoids and CBC were inactive or showed a very weak effect, as in the case of THCA, 18.4 ± 0.9 (3.0 ± 0.02) (Table 4). However, a 5 min preincubation of TRPV2-HEK-293 cells with CBGV, THC, CBG, CBD and THCV dose dependently abolished the elevation of [Ca²⁺]_i induced by LPC 3 µM (Table 4). Importantly, the order of potency for desensitization did not merely reflect the rank order of potency for channel activation.

Table 4

Efficacy and potency of pure cannabinoids as functional agonists at TRPV2 channels, and their potency as desensitizers of TRPV2 to the effect of lysophosphatidylcholine (LPC)

Figure 3

Dose-dependent effects of several pure cannabinoids on lysophosphatidylcholine (LPC, 3 µM)-induced, TRP channel of vanilloid type-2 (TRPV2)-mediated elevation of intracellular calcium in HEK-293 cells stably over-expressing the rat recombinant …

Activity at 2-AG biosynthesizing and degrading enzymes

Two enzymes involved in the biosynthesis of 2-AG from diacylglycerol precursors were cloned and named sn-1-specific DAGLα and β. DAGLα was found in the adult brain to be mostly localized in post-synaptic neurones (Bisogno et al., 2003). An MAGL inactive on AEA has been suggested to act as a major 2-AG degrading enzyme (Dinhet al., 2002). Both DAGL and MAGL are members of the large lipase-3 family of Ser hydrolases. Several cannabinoids were screened here as possible DAGLα and MAGL inhibitors. The inhibitory effect of compounds on DAGLα was tested in COS cells over-expressing human DAGLα, the most abundant of the two DAGLs in the adult brain (Bisogno et al., 2003); wild-type (i.e. non-transfected) COS cells also exhibited DAGL activity but this was significantly lower that the activity of cells transfected with human DAGLα cDNA and was subtracted from the latter when calculating the IC₅₀ values of the inhibitors (Bisogno et al., 2006). As reported in Table 5, CBDV (IC₅₀= 16.6 µM), and all the acids tested, exhibited inhibitory effects on DAGLα (IC₅₀ = 19.4–65.7 µM). Of all the BDS tested, DAGLα inhibitory activity was found only in the CBDA-BDS and THCA-BDS. Because, of the pure compounds, only CBDV and all the acids inhibited DAGLα, we reasoned that the acid of CBDV might be even more potent. Therefore, we prepared a few milligrams of CBDVA and tested it only in this assay. However, this compound was not more potent than CBDV and CBDA (Table 5).

Table 5

Efficacy and potency of pure cannabinoids and of most of their corresponding ‘botanical drug substance’ (BDS) as inhibitors of human recombinant DAGLα

The effect of the compounds on MAGL was studied in cytoplasmatic fractions of COS cells by using [³H]-2-AG as a substrate, as we had previously reported that the enzyme was abundantly expressed in this preparation (Bisogno et al., 2006). None of the pure compounds exhibited any significant inhibition up to 50 µM (Table 6), whereas some BDS exhibited significant MAGL inhibitory efficacy (IC₅₀ = 6.2–29.6 µM), CBG-BDS and THCA-BDS being the most potent (Table 6). Indeed, a ‘CBG-free’ CBG-BDS was found to be weakly active per se, but, when added to pure CBG, the activity of CBG-BDS was restored and this reconstituted extract was significantly more potent and efficacious at inhibiting MAGL than pure CBG (Figure 4).

Table 6

Efficacy and potency of pure cannabinoids and of most of their corresponding ‘botanical drug substance’ (BDS) as inhibitors of COS cell MAGL

Figure 4

Inhibition of the effect of MAGL by cannabigerol (CBG), a CBG-botanical drug substance (CBG-BDS) as such or rendered free of CBG (denoted as BDS), and the reconstituted CBG-BDS (denoted as CBG-BDS reconstituted). Experiments were carried out in homogenates …

Activity at AEA and PEA degrading enzymes

AEA and PEA are inactivated by enzymatic hydrolysis catalyzed by amidases, the best characterized of which is the FAAH (Cravatt et al., 1996). All compounds were therefore evaluated in assays of FAAH using [¹⁴C]-AEA hydrolysis by rat brain membranes (which express FAAH as the only AEA hydrolyzing enzyme). The effects are shown inTable 7. It is noteworthy that CBD was the only compound that inhibited FAAH, this effect having been already reported previously (Watanabe et al., 1998).

Table 7

Efficacy and potency of pure cannabinoids and of most of their corresponding ‘botanical drug substance’ (BDS) as inhibitors of rat brain FAAH

PEA inactivation to palmitic acid and ethanolamine is catalyzed by FAAH and, more selectively, NAAA (Ueda et al., 2001). The screening of different cannabinoids allowed us to identify three BDS preparations that inhibited NAAA: CBC-BDS > CBG-BDS > CBGV-BDS (IC₅₀ = 14.2–24.4 µM). The only pure compound with appreciable inhibitory activity was CBDA (IC₅₀ = 23.0 µM) (Table 8). All these compounds were selective for NAAA, and were significantly much less active or inactive at FAAH, with IC₅₀ values always higher than 50 µM (Table 7).

Table 8

Efficacy and potency of pure cannabinoids and of most of their corresponding ‘botanical drug substance’ (BDS) as inhibitors of human recombinant NAAA

The extent and duration of AEA actions are determined by its extracellular concentration, controlled in turn by the rate of AEA synthesis and degradation. As the hydrolytic enzymes are intracellularly located, an efficient transport system allowing for the rapid trafficking of AEA through these topographically separated sites was proposed. Although this putative ‘carrier’ has yet to be cloned, some indirect biochemical and pharmacological evidence suggests its existence (Di Marzo et al., 1994; Ligresti et al., 2004;2010;). The 21 cannabinoid preparations were therefore evaluated for their activity on [¹⁴C]-AEA uptake by intact RBL-2H3 cells. Eleven were found to be active (Table 9), and interestingly none of the most potent preparations inhibited FAAH (Table 7). CBC = CBG > CBD inhibited AEA uptake most efficaciously and potently (IC₅₀ = 11.3–25.3 µM), but some BDS (i.e. those of THCVA, CBGV, CBDA, THCA) were even more potent (IC₅₀ = 5.8–12.5 µM) than the corresponding pure compounds in this assay (Table 9).

Table 9

Efficacy and potency of pure cannabinoids and of most of their corresponding ‘botanical drug substance’ (BDS) as inhibitors of anandamide cellular uptake by RBL-2H3 cells

This study was partly funded by GW Pharma, UK.

Abbreviations

2-AG

2-arachidonoylglycerol

5-HT

5-hydroxy-tryptamine

ACU

anandamide cellular uptake

AEA

anandamide

BDS

botanical drug substance

CB₁

cannabinoid receptor type-1

CB₂

cannabinoid receptor type-2

CBC

cannabichromene

CBD

cannabidiol

CBDA

cannabidiol acid

CBDV

propyl analogue of CBD or cannabidivarin

CBDVA

acid of the propyl analogue of CBD

CBG

cannabigerol

CBGA

cannabigerol acid

CBGV

propyl analogue of CBG or cannabigivarin

CBN

cannabinol

DAGLα

diacylglycerol lipase α

EC

endocannabinoid

FAAH

fatty acid amide hydrolase

MAGL

monoacylglycerol lipase

NAAA

N-acylethanolamine acid amide hydrolase

THC

Δ⁹-tetrahydrocannabinol

THCA

THC acid

THCV

propyl analogue of THC or tetrahydrocannabivarin

THCVA

tetrahydrocannabivarin acid

TRP

transient receptor potential

TRPA1

TRP channel of ankyrin type 1

TRPM8

TRP channel of melastatin type-8

TRPV1

TRP channel of vanilloid type-1

TRPV2

TRP channel of vanilloid type-2

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