Are cannabidiol and Δ⁹-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review

Cannabidiol (CBD) and Δ⁹-tetrahydrocannabivarin (THCV)

At last count, 108 phytocannabinoids have been characterized in various chemovars of the plant (Hanuš, 2008). The other phytocannabinoids of greatest clinical interest are CBD and THCV. THC and CBD are ‘sister’ molecules, biosynthesized by nearly identical enzymes in Cannabis – expressions of two alleles at a single gene locus (de Meijer et al., 2003). THC and CBD are C₂₁ terpenophenols with pentyl alkyl tails, whereas THCV is a C₁₉ propyl-tailed analogue of THC. Cannabis biosynthesizes these compounds as carboxylic acids, for example, THC-carboxylic acid (2-COOH-THC). When heated, dried or exposed to light, the parent compounds are decarboxylated.

Fundamentally, THC mimics AEA and 2-AG by acting as a partial agonist at CB₁ and CB₂ receptors (Mechoulam et al., 1998). But rather than simply substituting for AEA and 2-AG, cannabis and its many constituents work, in part, by ‘kick-starting’ the endocannabinoid system (McPartland and Guy, 2004). CBD, in particular, gained attention early in this regard. Several landmark studies published in the previous century have shown interactions between CBD and THC (see Box 1).

Meta-analysis

Many narrative reviews have been written about THC, CBD and THCV, and cannabis as a ‘synergistic shotgun’ (McPartland and Pruitt, 1999; McPartland and Russo, 2001; Pertwee, 2004; 2005; 2008; Russo and Guy, 2006; Mechoulam et al., 2007; Zuardi, 2008; Izzo et al., 2009; Russo, 2011). CBD by itself has many therapeutic properties: anxiolytic, antidepressant, antipsychotic, anticonvulsant, anti-nausea, antioxidant, anti-inflammatory, anti-arthritic and antineoplastic.

The ability of CBD to antagonize THC has been the focus of many narrative reviews, as well as the studies listed in Box 1. This has led to the assumption that CBD exerts a direct pharmacodynamic blockade of THC. The pharmacological community tends to view CBD and THCV as negative modulators of CB₁ receptor agonists. This view may be due to a superficial interpretation of the available pharmacological data. Hence, CBD and THCV would appear to mirror the mechanism of first-generation CB₁ receptor inverse agonists known as cannABinoid ANTagonists (‘abants’), such as rimonabant, taranabant, otenabant and ibipinabant. Rimonabant was developed as an anti-obesity agent and marketed as an adjuvant to diet and exercise for weight loss in obese individuals. It was subsequently withdrawn from the market due to adverse psychiatric side effects (Bermudez-Silva et al., 2010).

We hypothesize that CBD and THCV may, under certain conditions, ‘antagonize’ the effects of THC via mechanisms other than direct CB₁ receptor blockade. The purpose of this article is to review mechanistic studies (in vitro and ex vivo) of CBD and THCV. Rather than a narrative review, we will conduct a meta-analysis. The results of this synthesis of mechanistic studies will be applied to an emerging discussion: do CBD and THCV act as natural ‘abants’?

Search strategy and study selection

We briefly describe the analysis here. For details regarding inclusion criteria, heterogeneity tests, subgroup analysis, quality assessment and publication bias, see Supporting Information Appendix S1.

PubMed (www.ncbi.nlm.nih.gov/pubmed/) was searched from 1988 (beginning with Devane et al., 1988) through December 2013, using the following Boolean search string: (cannabidiol OR tetrahydrocannabivarin) AND (animal OR affinity OR efficacy) NOT (behavioral OR behavioural). References identified by the search strategy were scanned for inclusion and exclusion criteria by three independent reviewers who resolved disagreements by consensus.

Inclusion criteria included studies of CBD or THCV, their carboxylic acids (CBD-acid, THCV-acid), as well as CBD- or THCV-enriched plant extracts (‘botanical drug substances,’ CBD-BDS, THCV-BDS). Included studies reported in vitro or ex vivo mechanistic data (receptor affinity and efficacy assays), detailed in Supporting Information Appendix S1.

The rather broad search strategy retrieved many articles that were subsequently excluded as irrelevant. Excluded topics included (i) review articles or publications with duplicated data; (ii) animal studies or in vivo studies without mechanisms or an identified molecular target; (iii) studies of synthetic analogues, or metabolites of CBD or THCV; (iv) human clinical trials lacking mechanistic analysis; urinary metabolites of CBD and THCV and their use in drug testing; characterizations of cannabinoid drug delivery systems; and (v) other irrelevant topics (see Supporting Information Appendix S1 for elaboration).

Articles meeting inclusion and exclusion criteria were screened for supporting citations, and antecedent sources were retrieved. The search also included unpublished data communicated at research conferences, upon approval by the authors of the data. Lastly, we contacted world experts and asked them to contribute unpublished data (see Acknowledgements section).

Data extraction and synthesis

Extracted data included ligand (CBD or THCV), assay type, animal species, reported means, sample variance, sample size and methodological factors. Methodological factors (covariates) were extracted for use in subgroup analyses to test whether they exerted heterogeneous effects upon pooled means. Methodological factors were pre-specified, chosen in advance by a priori hypotheses based upon recognized methodological diversity among studies, and not undertaken after the results of the studies had been compiled (post hoc analyses).

Data were synthesized qualitatively (e.g. categorical data) or quantitatively [e.g. the continuous variables for affinity (K_i) and efficacy (E_MAX)]. A quantitatively synthesized result is reported as a pooled mean ± SEM. Optimal quantitative synthesis would employ a weighted pooled mean, which adjusts each study's mean divided by its SEM, because larger studies with less variance should carry more ‘weight’ in a meta-analysis. Unfortunately, many studies omitted variance data or sample size.

To determine whether pooling was statistically appropriate, the coefficient of variation (CV) was determined for each pooled mean. The CV measures data dispersion of a probability distribution, defined simply as the ratio of the SD to the mean (Reed et al., 2002). We applied the Cochrane ‘skew test’ to the CV (Higgins and Green, 2005), where CV ≥ 1 (i.e. SD ≥ mean) identifies a skewed mean with excessive heterogeneity. Skewed mean was submitted to Grubb's test (www.graphpad.com/quickcalcs/). Data identified as significant outliers (P < 0.05) were reported in Supporting Information Appendix S2 and S3 and withdrawn from synthesis. The CV-skew test could not be used for sample sizes of n ≤ 3. In those cases, we used simple pooled means.

CBD at CB₁ receptors: direct and indirect interactions

The pooled mean affinity of CBD at CB₁ receptors is K_i = 3245 ± 803 nM. The pooled mean was calculated from 1 human, 3 mice and 11 rat studies (Supporting Information Appendix S2). Species differences were not statistically different, including the human study (K_i = 1510 ± 100 nM). Several studies omitted quality measures such as statistical data (variance and/or sample size). Subgroup analysis of the five methodological factors produced surprising results. Only one factor – the use of crude brain homogenates – proved to be statistically relevant (Supporting Information Appendix S2). None of the other factors gave rise to data heterogeneity – species differences, class of tritiated ligand (agonist vs. antagonist) or even the use of non-specific probes (e.g. [³H]TMA-THC). One methodological factor could not be assessed – no studies used PMSF.

Eight studies tested CBD efficacy at CB₁ receptors, assayed with forskolin-stimulated adenylate cyclase (FsAC) or [³⁵S]GTPγS (Supporting Information Appendix S2). Six of these studies reported no measurable response or inconsistent dose–response curves hovering near zero. One study reported slight agonism, and one study reported slightly inverse agonism, both at high concentrations (≥10 μM).

Surprisingly, in some mechanistic studies, the effects of CBD could be reversed by CB₁ receptor inverse agonists, or were absent in CB₁ receptor knockout mice (n = 10 studies; Supporting Information Appendix S2). This suggests that CBD may exert ‘indirect agonism’ at CB₁ receptors – either augmenting CB1 constitutional activity or augmenting endocannabinoid tone. Regarding the first mechanism, Howlett et al. (1989) presented thermodynamic data that suggest that CBD may alter CB₁ receptor activity by increasing membrane fluidity. Sagredo et al. (2011) showed that Sativex^® (a mix of THC and CBD; GW Pharmaceuticals, Salisbury, UK) can up-regulate CB₁ receptor gene expression in a rat model of Huntington's disease. The second mechanism – augmenting endocannabinoid tone – is supported by studies showing that CBD or CBD-BDS inhibits AEA hydrolysis by FAAH (n = 5 studies; Supporting Information Appendix S2), with a pooled mean IC₅₀ = 19.8 ± 4.77 μM. Four rodent studies show that CBD inhibits the putative AEA transporter, with a pooled mean IC₅₀ = 10.2 ± 3.03 μM. Two studies reported CBD increasing 2-AG levels, 33 or 260% (Supporting Information Appendix S2).

CBD may affect the pharmacokinetics of THC when the two are co-administered (n = 17 studies; Supporting Information Appendix S2). One of these pharmacokinetic studies predates the isolation of pure THC – Loewe and Modell (1941) stated that CBD ‘synergized’ hypnotic action in mice induced by isomeric tetrahydrocannabinols. CBD may impair THC hydrolysis by CYP450 enzymes. The results of CYP studies vary due to species differences, timing (CBD pre-administration vs. co-administration) and specific CYP isoenzymes. Recent human studies show no pharmacokinetic interaction between THC and CBD at clinically relevant dosing (Supporting Information Appendix S2).

The effect of CBD on CYPs is important because these enzymes metabolize THC into 11-OH-THC. This metabolite may be up to four times more psychoactive than THC, according to a rat discriminative study (Browne and Weissman, 1981). The affinity and efficacy of 11-OH-THC at CB₁ receptors has not been measured, although the affinity of 11-OH-Δ⁸-THC was K_i = 25.8 nM, displacing [³H]HU-243 from rCB₁ COS cells (Rhee et al., 1997). In the same assay, Δ⁹-THC exhibited K_i = 80.3 nM (Rhee et al., 1997) or K_i = 39.5 nM (Bayewitch et al., 1996). The significance of CBD modulating the metabolism of THC into 11-OH-THC is an important variable that remains to be explored.

CBD may antagonize cannabinoid-induced effects. Six in vitro studies demonstrate that CBD can antagonize CP55,940- or WIN55212-2-induced efficacy (Supporting Information Appendix S2), with a pooled mean K_B = 88.5 ± 18.46 nM. This unexpected K_B is 37-fold more potent than the K_i of CBD in binding assays. This suggests an indirect mechanism, that is, mediated by (an)other target(s). The K_B of CBD is 147-fold higher (less potent) than the K_B of rimonabant, when this inverse agonist was run in parallel (n = 3 studies; Supporting Information Appendix S2). Note that this antagonism can be visualized by juxtaposing two log concentration–response curves; for example, the curve of CP55,940 for stimulation of [³⁵S]GTPγS binding to CB₁ receptors, compared to the curve of CP55,940 for such stimulation in the presence of CBD. A competitive antagonist would produce a parallel rightward shift in the curve of CP55,940. However, Petitet et al. (1998) found CBD to produce a parallel shift in the curve of CP55,940 that was downward rather than rightward in its direction, an effect that a competitive antagonist is not expected to produce.

CBD at targets in the ‘expanded endocannabinoid system’

The ability of CBD to antagonize a cannabinoid agonist in an efficacy assay may be confounded by the impact of CBD on other receptors or enzymes present in the assay. In Table 1, we summarize studies regarding CBD affinity and efficacy at other metabotropic receptors and ion channels in the ‘expanded endocannabinoid system.’

CBD shows low affinity at CB₂ receptors (Table 1). Its efficacy at CB₂ receptors suggests weak inverse agonism at concentrations that may not be pharmacologically relevant. However, CBD antagonizes CP55,940 signalling at CB₂ receptors with a K_B potency not commensurate with its K_i. This suggests again an indirect mechanism of action. At GPR18, two studies suggest that CBD acts as a partial agonist and antagonizes THC (Supporting Information Appendix S2). The direct affinity and efficacy of CBD at GPR55 has not been measured, but it can antagonize GPR55 agonists (Supporting Information Appendix S2).

Table 1 highlights CBD signalling at transient receptor potential (TRP) channels, which have been characterized as ‘ionotropic cannabinoid receptors’ (Di Marzo et al., 2002). We find a species difference at TRPV1 channels and the pooled mean E_MAX at human TRPV1 is 53.4% ± 5.03 (Table 1), whereas a single study of rat TRPV1 channels reports an E_MAX of 21% (Qin et al., 2008). At TRPA1 channels, the pooled mean E_MAX of CBD (Table 1) is considerably less than that of CBD-BDS (163.6 ± 11.9%; De Petrocellis et al., 2011). This suggests that terpenoids and other plant substances in the extract may enhance CBD activity at TRPA1 channels, either pharmacokinetically or pharmacodynamically. The same study showed that CBD-BDS also showed slightly greater efficacy than pure CBD at TRPV1 channels (Supporting Information Appendix S2). CBD acts as an antagonist at TRPM8, unlike its agonist activity at other TRP channels (Table 1).

CBD at other molecular targets

CBD is a promiscuous compound with activity at multiple targets. Promiscuity is governed by ligand structure, and cannabinoids exhibit the characteristics of promiscuous ligands: molecular mass >400 g·mol⁻¹, and partition coefficient (CLogP) scores between 2 and 7 (Hopkins, 2008).

CBD inhibits adenosine uptake (pooled IC₅₀ = 122 nM minus one outlier, n = 3 studies; Supporting Information Appendix S2) and this mechanism is likely to exert indirect agonism at adenosine receptors. Consistent with this, the beneficial effects of CBD in animal models of inflammation are blocked by adenosine A_2A receptor antagonists (n = 8 studies). CBD may also regulate 5-HT levels, although six studies report disparate results. CBD has slight affinity for the 5-HT_1A receptor (K_i ∼ 16 μM; Supporting Information Appendix S2), although its efficacy at 5-HT_1A receptors is inconsistent. Several beneficial effects of CBD in vivo are blocked by 5-HT_1A receptor antagonists (Supporting Information Appendix S2).

CBD exerts a positive allosteric modulation of α3 glycine receptors (pooled EC₅₀ = 11.0 μM, n = 3 studies; Supporting Information Appendix S2). NMR analysis revealed a direct interaction between CBD and S296 in the third transmembrane domain of α3 glycine receptors. The cannabinoid-induced analgesic effect was absent in mice lacking the α3 glycine receptors (Xiong et al., 2011). CBD can go nuclear, as it affects PPARγ receptors (IC₅₀ = 5 μM; Supporting Information Appendix S2), and its beneficial effects are blocked by PPARγ antagonists (n = 5 studies). A dozen studies indicate that CBD allosterically modulates other receptors: α₁-adrenoceptors, dopamine D₂, GABA_A, μ- and δ-opioid receptors – some positively, some negatively, none with great efficacy (Supporting Information Appendix S2). CBD modulates intracellular calcium levels, via T-type and L-type voltage-regulated Ca²⁺ channels and mitochondrial Na⁺/Ca²⁺ exchange (n = 7 studies).

The effects on the AA cascade are complex. Three studies indicate that CBD mobilizes AA by stimulating PLA₂ activity. The direction that AA goes from there is uncertain. CBD inhibits the metabolism of AA to LTB₄ by 5-lipoxygenase – pooled IC₅₀ = 3.1 ± 0.75 μM, n = 4 studies, although a fifth study reported no effect up to 80 μM (Supporting Information Appendix S2). Contradictory studies report CBD blocking the metabolism of AA to PGE₁ or TxB₂ (via COX-1) or PGE₂ (via COX-2). Early studies did not identify which COX isoenzyme they tested. Recent studies indicate that CBD attenuates serum PGE₂ in animal models of chronic pain, but this may or may not correlate with COX-2 protein expression (Supporting Information Appendix S2).

CBD clearly dampens NO production in animal models of acute and chronic inflammation – as measured by a reduction in nitrite levels or inducible NOS (iNOS) protein expression (n = 15 studies). A dozen studies show that CBD inhibits the expression of inflammatory cytokines and transcription factors (IL-1β, IL-2, IL-6, IL-8, TNF-α, IFN-γ, CCL3, CCL4, NF-κB).

CBD is a potent antioxidant; it reduces reactive oxygen species (ROS) induced by a variety of toxins and tissue insults (Supporting Information Appendix S2). However, one study suggests the opposite – that CBD hydroxyquinone, formed during hepatic microsomal metabolism of CBD, is capable of generating ROS and inducing cytotoxicity. Indeed, one mechanism by which CBD induces tumour cell apoptosis appears to be ROS generation (Supporting Information Appendix S2). In general, it appears that CBD can induce ROS in cancer cells and reduce ROS in healthy cells stimulated by agents that induce ROS formation. In fact, even in cancer cells, CBD inhibits ROS formation induced by H₂O₂ (Ligresti et al., 2006).

THCV at CB₁ and CB₂

Affinity studies show that THCV binds to CB₁ receptors with significant potency. There may be species differences – at human CB₁ receptors transfected into CHO cells, mean K_i = 5.47 ± 4.02 nM (n = 2 studies); at mouse brain membranes, mean K_i = 61.0 ± 14.40 nM (n = 2); and at rat brain membranes (n = 1), mean K_i = 286 ± 43 nM. The rat study used [³H]SR141716A, whereas others used [³H]CP55,940 (Supporting Information Appendix S3).

Four studies tested the efficacy of THCV at CB₁ receptors. THCV did not inhibit or stimulate [³⁵S]GTPγS binding to mouse whole brain membranes (n = 3 studies) or to rat membranes (cortical, cerebellar or piriform cortical membranes) at concentrations up to 10 μM. This suggests that THCV targets the CB₁ receptor as a neutral antagonist rather than as an antagonist/inverse agonist. The single study that tested FsAC in human CB₁ receptors in CHO cells produced signs of inverse agonism at concentrations of 10, 100 and 1000 nM (Supporting Information Appendix S3).

In the presence of other cannabinoids, THCV binds to CB₁, in vitro, in a manner that gives rise to competitive antagonism rather than to agonism. More specifically, THCV produces significant parallel rightward shifts in the log concentration–response curves of established CB₁ receptor agonists such as CP55,940 or WIN55212-2, in [³⁵S]GTPγS binding to mouse whole brain membranes, and in the inhibition of electrically evoked contractions of mouse isolated vasa deferentia. Pooling five studies (Supporting Information Appendix S3) results in a K_B = 64.2 ± 14.14 nM. Thus, the K_B of THCV is in the same range of concentrations as its K_i.

The five studies hint at ‘probe dependence’. That is, THCV has greater potency when antagonizing WIN55212-2 than when antagonizing CP55,940, although the difference falls short of statistical significance (Supporting Information Appendix S3). Two studies of THCV as an antagonist in the vas deferens assay also hint at probe dependence – the potency of THCV is not the same for all CB₁ receptor agonists. In rank order: AEA (K_B = 1.2 nM), methAEA (K_B = 2.6 nM), WIN55212-2 (K_B = 3.15 nM), CP55,940 (K_B = 10.3 nM), THC (K_B = 96.7 nM) (Supporting Information Appendix S3). THCV also reverses WIN55212-2-induced decreases of miniature inhibitory postsynaptic current frequency at mouse cerebellar interneuron-Purkinje cell synapses, albeit with much less potency than the CB₁ receptor inverse agonist AM251, which was tested in parallel (Supporting Information Appendix S3).

At human CB₂ receptors transfected into CHO cells, THCV has significant affinity, with pooled mean K_i = 124.7 ± 64.55 nM (n = 3 studies; Supporting Information Appendix S3). THCV acts as a partial agonist at these receptors as measured by [³⁵S]GTPγS binding or FsAC assays, pooled E_MAX = 56.7 from basal at 1–10 μM, EC₅₀ = 74.2 ± 34.4 nM (n = 3 studies; Supporting Information Appendix S3).

THCV may exert ‘indirect agonism’ at CB₁ and CB₂ receptors by augmenting endocannabinoid tone. It does this by inhibiting the putative AEA transporter, inhibiting FAAH activity and inhibiting MAGL activity, albeit at 25–100 μM (n = 3 studies; Supporting Information Appendix S3). Whether or not THCV exerts ‘indirect agonism’ to an extent that can overcome its surmountable CB₁ receptor antagonism requires further experiments.

THCV at other targets

Unlike CBD, much less evidence exists for non-cannabinoid receptor-mediated effects of THCV, and this evidence has to do mostly with the capability of THCV to interact with ‘thermo-TRP’ channels. One study demonstrates that THCV acts as an agonist at rat TRPA1, human TRPV1 and rat TRPV2-4 channels and a potent antagonist at rat TRPM8 channels (Supporting Information Appendix S3). Data regarding its effects at GPR55 are controversial, and the potentiation of 5-HT_1A receptors was observed only at the concentration of 100 nM (Supporting Information Appendix S3).

CBD at CB₁ receptors: direct and indirect interactions

Let us return to our original question: does CBD act as a natural ‘abant'? Data clearly show that CBD does not act directly at CB₁ receptors. The affinity of CBD at CB₁ receptors (K_i = 3245 nM) is at least three orders of magnitude less than that of rimonabant (K_d = 1.0 nM in rat, 2.9 nM in human; McPartland et al., 2007). Several efficacy studies made direct comparisons of [³⁵S]GTPγS binding: CBD showed no measurable activity, whereas rimonabant elicited strong inverse agonism with a mean I_MAX = −35.5% (Supporting Information Appendix S2).

The inactivity of CBD in vitro is confirmed by in vivo studies. CBD does not elicit the classic CB₁-mediated tetrad of hypolocomotion, analgesia, catalepsy and hypothermia (Long et al., 2010). In drug discrimination studies, CBD failed to generalize with THC (Järbe et al., 1977; Zuardi et al., 1981) and did not alter the discriminative stimulus effects of THC (Vann et al., 2008). Its lack of cannabimimetic effects is well known in humans (Hollister, 1973).

CBD exerts CB₁ receptor agonist-like activity in some in vitro functional assays at high concentrations (>10 μM), which can be reversed by CB₁ receptor inverse agonists, and is absent in CB₁ receptor knockouts. This probably occurs by CBD augmenting endocannabinoid tone. Pooled rodent studies show moderate inhibition of FAAH and the putative AEA transporter. CBD may also augment endocannabinoid tone by activating PLA₂, thus mobilizing AA – the feedstock for AEA and 2-AG synthesis.

On the contrary, CBD inhibits adenosine uptake, thereby acting as an indirect agonist at A_2A receptors. Agonism of these receptors in post-synaptic cells prevents glutamate mGlu₅ receptor-mediated release of AEA and 2-AG through A_2A/mGlu₅ heteromers (Lerner et al., 2010). These opposing effects – CBD augmenting endocannabinoid tone and augmenting adenosine tone – often strike a balance in favour of the former: Robust rodent studies indicate that CBD augments endocannabinoid tone (e.g. Campos et al., 2013,2013). One clinical study showed that AEA levels increased in schizophrenic patients given CBD 800 mg·day⁻¹ (Leweke et al., 2012). Future in vitro studies of CBD affinity and efficacy in the presence or absence of PMSF or MAFP would be instructive (these are FAAH inhibitors that prevent the synthesis of AEA). Knocking down tonic adenosine A_2A receptor signalling may also shed light on this complex situation (Savinainen et al., 2003).

Our second paradoxical finding is the in vitro capacity of CBD to ‘functionally antagonize’ cannabinoid-induced activity at CB₁ receptors. However, the K_B of CBD (88.5 ± 18.46 nM) is far higher (implying less potency) than that of rimonabant (mean K_B of 0.6 ± 0.41 nM when tested in parallel in the same studies; Supporting Information Appendix S2). Indeed, CBD in vivo mostly behaved in a different way from that of rimonabant. For example, CBD produced no effect on food intake, energy expenditure or insulin sensitivity in obese mice (Cawthorne et al., 2008). Its anxiolytic, antidepressant and anti-nausea effects are all opposite to those reported for rimonabant (Pertwee, 2008) (Box 2).

Non-CB₁ receptor mechanisms of CBD antagonism of THC

Earlier we proposed that the functional antagonism of THC by CBD may be mediated by a non-CB₁ receptor mechanism of action. Pharmacodynamic antagonism arises when one drug diminishes the effect of another drug by targeting different receptors or enzymes. For example, dry mouth caused by a sympathomimetic drug is antagonized by a cholinergic drug.

Non-CB₁ receptor mechanisms of CBD potentiation of THC

CBD potentiates some effects of THC in an additive or synergistic fashion. Williamson (2001) has reviewed the mathematical definitions of synergy. Wilkinson et al. (2005) provided examples of synergy within polypharmaceutical cannabis extracts. CBD may potentiate the behavioural effects of THC via pharmacokinetic mechanisms, for example, increasing the area under the curve of THC in blood and brain (Klein et al., 2011). Pharmacodynamic interactions arise when CBD and THC act at separate but interrelated receptor sites. Landmark studies demonstrating the potentiation of THC by CBD or by CBD-rich cannabis extracts are summarized in Box 1 and Box 3.

THCV at CB₁ receptors

As discussed previously, meta-analysis indicates that THCV acts as a neutral CB₁ and CB₂ receptor antagonist, at least in most in vitro studies. Indeed, evidence has also emerged that THCV can block CB₁ receptors in vivo, as indicated by its ability to oppose (i) anti-nociception induced by THC in the mouse tail-flick test and by CP55,940 in the rat hot plate test; (ii) hypothermia induced in mice by THC; and (iii) CP55,940-induced inhibition of rat locomotor activity (Pertwee et al., 2007; García et al., 2011).

Results from other in vivo experiments have shown that THCV can suppress food consumption and body weight in non-fasted mice, like AM251 (Riedel et al., 2009), and like rimonabant, THCV can reduce signs of motor inhibition in rats caused by 6-hydroxydopamine (García et al., 2011). It remains to be established whether THCV produced these effects through CB₁ receptor inverse agonism or because it was competitively antagonizing CB₁ receptor-mediated effects of one or more endogenously released endocannabinoids.

However, it is noteworthy that, like the established neutral CB₁ receptor antagonists, THCV does not share the ability (i) of SR141716A or AM251 to produce signs of nausea in rats (Rock et al., 2013) or (ii) of SR141716A to produce an anxiogenic-like reaction in the rat light–dark immersion model of anxiety-like behaviour, or a suppression of saccharin hedonic reactions of rats in the taste reactivity test of palatability processing (O'Brien et al., 2013). Furthermore, in some in vivo settings where neutral antagonists produce effects, THCV does not. For example, THCV did not reduce food intake and body weight in obese mice, even though it did improve insulin resistance in these animals (Wargent et al., 2013). Furthermore, THCV did not reduce food deprivation-induced food intake in mice (Izzo et al., 2013).

THCV has anti-epileptiform actions in the rat pentylenetetrazole seizure model (Hill et al., 2010). This is in contrast to rimonabant safety data, where seizures were occasionally observed (Bermudez-Silva et al., 2010). Rimonabant significantly increased seizure duration and seizure frequency in the rat pilocarpine model of epilepsy (Wallace et al., 2003). Pro-convulsant effects were also observed with AM251 in a model of generalized seizures (Shafaroodi et al., 2004).

Also, when given in vivo at doses well above those at which it can block CB₁ receptors, THCV produces signs of CB₁ receptor activation. For example, in experiments performed with mice, THCV has been shown to induce (i) immobility in the Pertwee ring test, albeit with a potency 4.8 times less than that of THC, and (ii) anti-nociception in the tail-flick test (Gill et al., 1970; Pertwee et al., 2007). Importantly, although SR141716A was found to antagonize the antinociceptive effect of THCV, it did not give rise to any significant antagonism of THCV-induced hypothermia or ring immobility (Pertwee et al., 2007). THCV also decreased pain behaviour in the formalin test; this effect appeared to be CB₁ and CB₂ receptor-mediated (Bolognini et al., 2010). It remains to be established how THCV produces apparent CB₁ receptor activation in vivo but not in vitro at doses above those at which it can block CB₁ receptors both in vivo and in vitro. One possibility is the ability of THCV to inhibit endocannabinoid re-uptake (see previous discussion), with indirect activation of cannabinoid receptors. Models of intestinal transit are often used as assays of CB₁ receptor activation and THCV inhibits upper intestinal transit in a dose-dependent manner (Izzo et al., 2013). However, this effect was not antagonized by a dose of rimonabant inactive per se. This study, together with the hypothermia and immobility assay, indicates that THCV can have cannabimimetic effects, which appear to be independent of the CB₁ receptor.

The ‘abants’ were withdrawn from the market because of their worsening of anxiety and depression in obese patients. In contrast, THCV has very little effect in animal models of anxiety and depression. For example, the dose of THCV required to impart anxiogenic effects in the elevated plus maze is 30-fold higher than that of rimonabant (Izzo et al., 2013).

The observed similarities or differences between THCV and rimonabant (Box 4) may depend upon several factors. These include (i) the initial physiopathological status of the cell/tissue/organ, and the degree of expression therein of functional CB₁ receptors, which might affect the activity of different ligands of the same receptor in different manners; (ii) the presence or absence in the cell/tissue/organ of non-CB₁, non-CB₂ receptor targets for THCV – in particular, TRP channels and CB₂ receptors, particularly when THCV is administered at doses higher than those required for CB₁ receptor antagonism, might counteract some of the CB₁ receptor antagonism-mediated effects of THCV, but not others; (iii) the fact that at higher doses, THCV might start behaving as a CB₁ receptor agonist, thus clearly counteracting some of the effects due to CB₁ receptor neutral antagonism; (iv) different pharmacokinetic properties of the two compounds, particularly, but not limited to, under pathological conditions that may alter their tissue distribution and catabolism.