In vitro and in vivo pharmacology of synthetic olivetol- or resorcinol-derived cannabinoid receptor ligands

Introduction

Over the past few years, two receptors have gained growing importance as targets for the discovery of new potential therapeutic, and particularly analgesic and anti-inflammatory, drugs, namely the cannabinoid receptors subtype 1 and 2 (CB₁ and CB₂). These receptors were identified during studies on the molecular mode of action of a plant natural product, the psychotropic principle of Cannabis, Δ⁹-tetrahydrocannabinol (THC). Agonists of both cannabinoid receptor subtypes (see for review Iversen and Chapman, 2002) produce strong anti-nociceptive effects in animal models of chronic, neuropathic and inflammatory pain (see for review Di Marzo et al., 2002). Also ‘indirect’ agonists of cannabinoid receptors, that is, inhibitors of endogenous cannabinoid (endocannabinoid) inactivation, have been suggested as possible leads for the development of analgesic drugs (Lichtman et al., 2004a, 2004b). Finally, although activation of CB₂ causes pain relief (Costa et al., 2004; Ibrahim et al., 2006), recent evidence also points to cannabinoid CB₂ receptor antagonists/inverse agonists as promising anti-inflammatory agents (Iwamura et al., 2001; Ueda et al., 2005; Lunn et al., 2006; Oka et al., 2006). This finding is seen by some as paradoxical, but it reflects a typical property of endocannabinoids, which can behave as mediators often playing opposing roles under pathological (and especially neurodegenerative and inflammatory) states, depending on the phase of the disorder, the cellular source of the endocannabinoids and the cellular localization and biological effect of their molecular target. For example, both the CB₁ receptor inverse agonist SR141716A and the inhibitor of endocannabinoid inactivation VDM-11 are protective against β-amyloid-induced amnesia in mice (Mazzola et al., 2003; van der Stelt et al., 2006) depending on the phase of the disorder at which the two substances are administered. It has been recently shown that also SR141716A can induce paradoxical analgesic and anti-inflammatory actions, thus behaving as an agonist (Croci et al., 2003; Costa et al., 2005), an effect that might be due to targeting of non-CB₁ receptors by this compound or to the fact that, with CB₁ receptors blocked, endocannabinoids might tonically activate other targets involved in pain and inflammation. Indeed, an agonist-like neuroprotective effect of SR141716A against ischaemia in vivo was recently suggested to be due to indirect activation of vanilloid transient receptor potential vanilloid type 1 (TRPV1) receptors (Pegorini et al., 2006).

Some contradictory results obtained with cannabinoid receptor ligands on the pharmacological control of pain and inflammation are probably also due to the fact that some of these compounds might behave as partial or full agonists or as neutral antagonists or inverse agonists also depending on the activation state of cannabinoid receptors. Thus, if the pathological condition is such to cause a strong elevation of endocannabinoid levels, a potent but inefficacious agonist might behave as an antagonist. This was recently demonstrated with THC, which, compared to the endocannabinoid 2-arachidonoylglycerol (2-AG), is a potent but partial agonist at cannabinoid receptors, and, accordingly, abolishes the cannabinoid CB₂ receptor-mediated migration of human natural killer cells (Kishimoto et al., 2005) and occludes the CB₁-mediated depolarization-induced inhibition of excitatory neurotransmission in the hippocampus (Straiker and Mackie, 2005), two effects mediated by 2-AG. By contrast, with little or no endogenous activation of cannabinoid receptors, a partial agonist will still behave as such, and it will be possible to distinguish between a neutral antagonist (which should be inactive in this case) and an inverse agonists (which should still be active and behave in an opposite way as compared to agonists and partial agonists). Species differences can also play a role in determining the overall activity of a certain pharmacological tool, as in the case of the CB₂ inverse agonist SR144528, which exacerbates formalin-induced pain in mice (Calignano et al., 1998), and inhibits it in rats (Beaulieu et al., 2000). These observations underscore the importance of always measuring the amounts of endocannabinoids during physiological or pathological conditions, and of developing new tools that act as neutral antagonists. To date, only neutral CB₁ receptor antagonists have been reported (see for review Pertwee, 2005), but no neutral CB₂ antagonists.

We have previously reported the development of several synthetic compounds derived from olivetol (the biosynthetic precursor of THC in Cannabis) or substituted resorcinol and fatty acids as ligands of cannabinoid CB₁ and CB₂ receptors (Brizzi et al., 2005) (Figure 1). We describe here the functional activity of two of these compounds, CB-25 and CB-52, and provide data indicating that, although they might behave as partial agonists at CB₁ receptors, they always act as neutral CB₂ antagonists in vitro. We also investigated the analgesic activity of the two compounds, and show that they appear to behave as CB₁ antagonists or partial agonists depending on the type of noxious stimulus used.

Materials and methods

Test of anti-nociceptive activity in normal rats

Animals

Male Wistar rats (250–300 g) and Swiss-Webster mice (40–45 g) were housed three per cage under controlled illumination (12 h light/12 h dark cycle; light on 0600 hours) and standard environmental conditions (ambient temperature 20–22°C, humidity 55–60%) for at least 1 week before the commencement of experiments. Rat chow and tap water were available ad libitum. All surgery and experimental procedures were performed during the light cycle and were approved by the Animal Ethics Committee of The Second University of Naples. Animal care was in compliance with European regulations on the protection of laboratory animals (OJ of EC L358/1 18/12/86). In agreement with the Ethical Guidelines of the IASP, all efforts were made to reduce both animal numbers and suffering during the experiments.

Surgical preparation

For intra-periaqueductal grey (PAG) microinjections, rats were anaesthetized with pentobarbital (60 mg kg⁻¹, intraperitoneally (i.p.)) and a 23-gauge, 12-mm-long stainless-steel guide cannula was stereotaxically lowered until its tip was 1.4 mm above the ventrolateral PAG by applying coordinates from the Atlas of Paxinos and Watson (1986) (A: −7.8 mm from bregma, L: 0.5 mm, V: 4.3 mm below the dura). The cannula was anchored with dental cement to a stainless-steel screw in the skull. We used a David Kopf stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) with the animal positioned on a homeothermic temperature control blanket (Harvard Apparatus Limited, Edenbridge, Kent, UK). Ventrolateral PAG was considered in this study because previous studies have shown in that area the presence of excitatory output neurons projecting monosynaptically to OFF neurons in the rostral ventromedial medulla (RVM) (Tortorici and Morgan, 2002). Direct intra-PAG administration of drugs or their vehicle (10% dimethyl sulphoxide (DMSO) in artificial cerebrospinal fluid (ACSF) of composition (in mM) KCl 2.5, NaCl 125, MgCl₂ 1.18 and CaCl₂ 1.26) was achieved with a stainless-steel cannula connected by a polyethylene tube to a Hamilton 1-microlitre syringe, inserted through the guide cannula and extended 1.4 mm beyond the tip of the guide cannula to reach the ventrolateral PAG. Volumes of 200 nl drug solutions, or vehicle, were injected into the ventrolateral PAG over a period of 60 s and the injection cannula gently removed 5 min later. At the end of the experiment, a volume of 200 nl of neutral red (0.1%) was also injected in the ventrolateral PAG 40–50 min before killing the rat. Rats were then perfused intracardially with 20 ml phosphate-buffered solution (PBS) followed by 20 ml of 10% formalin solution in PBS. The brains were removed and immersed in a saturated formalin solution for 2 days. The injection sites were ascertained by using two consecutive sections (40 μm), one stained with cresyl violet to identify nuclei and the other one unstained to determine dye spreading. Only those rats whose microinjection site and diffusion were located within ventrolateral PAG were included in the results.

Thermoceptive responses

Changes in thermoceptive responses were evaluated according to Hargreaves et al. (1988) using a Plantar Test Apparatus (Ugo Basile, Varese, Italy). On the day of the experiment, rats were placed in a plastic cage (22 cm × 17 cm × 14 cm; length × width × height) with a glass floor. After 1 h habituation period, the plantar surface of the hind paw was exposed to a beam of radiant heat through the glass floor. The radiant heat source consisted of an infrared bulb (Osram halogen-bellaphot bulb; 8 V, 50 W). A photoelectric cell detected light reflected from the paw and turned off the lamp when paw movement interrupted the reflected light. The paw withdrawal latency was automatically displayed to the nearest 0.1 s; the cutoff time was 25 s in order to prevent tissue damage. The latency of nociceptive reaction was measured in seconds under basal condition and after intra-PAG treatment with drugs. Each rat served as its own control, the latency to nociceptive response being measured both before and after treatments. At time 0, five baseline responses were already obtained for each animal at 15 min intervals and averaged. Rats received CB-25 (1 or 2.5 mg kg⁻¹, i.p.), CB-52 (1 and 5 mg kg⁻¹, i.p.) or WIN55,212-2 (2.5 mg kg⁻¹, i.p.). CB-25 and CB-52 (1 mg kg⁻¹, i.p.) were also given in combination with WIN55,212-2 (2.5 mg kg⁻¹, i.p.). We chose these doses of CB-25 and CB-52 on the basis of previous in vitro and in vivo studies of their potencies and receptor affinities relative to the reference compound CP55,940 (Brizzi et al., 2005; Vigano et al., 2005).

Groups of 10 animals per treatment were used, with each animal used for one treatment only. The results are expressed as a percentage of the maximum possible effect (%MPE), using the following formula:

Results

Functional activity of CB-25 and CB-52 at the mouse CB₁ receptor

Adenylate cyclase assays

As reported previously (Brizzi et al., 2005), CB-25 behaved as an inverse agonist by enhancing forskolin-induced cAMP formation by mouse neuroblastoma N18TG2 cells (EC₅₀=20±3 nM, max. stimulation=48±5%, means±s.e., N=4), which constitutively express CB₁, but not CB₂, receptors (see also Brizzi et al., 2005). By contrast, CB-52 behaved as a partial agonist in this assay by inhibiting forskolin-induced cAMP formation with lower efficacy as compared to WIN55,212-2 (IC₅₀=450±35 and 20±2 nM, max. inhibition 40±3 and 63±5%, respectively, means±s.e., N=4; see also Brizzi et al., 2005). The effect of CB-25 (100 nM) was blocked by WIN55,212-2 (5 nM, a concentration inactive by itself) (8.2±4.5% stimulation, P>0.05), whereas the effect of CB-52 (500 nM) was blocked by the CB₁ antagonist SR141716A (10 nM) (3.1±1.0% inhibition, P>0.05).

GTP-γ-S binding assays

Both CB-25 and CB-52 stimulated [³²S]GTP-γ-S binding to membranes from mouse brain (EC₅₀=100 and 11 nM, maximum Stimulation was 48 and 16%, respectively), thus behaving as rather potent agonists, albeit inefficacious as compared to the potent full CB₁ receptor agonist CP55,940 (Figure 2).

Functional activity of CB-25 and CB-52 at the human CB₁ receptor

Both CB-25 and CB-52 inhibited forskolin-induced cAMP formation by hCB₁-CHO cells (EC₅₀=1600 and 2600 nM, max. inhibition=68 and 62%, respectively), thus behaving as partial agonists in this assay as compared to the potent CB₁ receptor ligand CP55,940 (Figure 3a and b). No effect on forskolin-induced cAMP formation was observed in non-transfected CHO cells.

Functional activity of CB-25 and CB-52 at the human CB₂ receptor

Unlike CP55,940, neither CB-25 nor CB-52 significantly inhibited forskolin-induced cAMP formation by hCB₂-CHO cells at concentrations up to 10 μM. Furthermore, neither compound stimulated [³²S]GTP-γ-S binding to membranes from hCB₂-CHO cells over the same range of concentrations (Figure 4a). However, CB-25 (100 nM) and CB-52 (1 μM) antagonized the effect of CP55,940 on [³²S]GTP-γ-S binding to these membranes (K_b 5.4 and 9.8 nM, respectively), thus behaving as neutral antagonists at the human CB₂ receptor (Figure 4b and c).

Effect of CB-25 and CB-52 on nociception in normal rats

The systemic administration of CB-25 (1 and 2.5 mg kg⁻¹, i.p.) and CB-52 (1 and 5 mg kg⁻¹, i.p.) caused a long-lasting and dose-dependent decrease of the pain threshold to thermal stimulation as assessed in the plantar test in rats at the highest doses used (Figure 5a and b). By contrast, the potent cannabinoid receptor agonist WIN55,212-2 (2.5 mg kg⁻¹, i.p.) caused a strong anti-nociceptive effect, which was blocked by pretreatment with low doses (1 mg kg⁻¹, i.p.) of CB-25 and CB-52, which were inactive when given alone (Figure 5a and b).

Effect of CB-25 and CB-52 on basal activity of RVM cells in normal rats

We next studied the effects of CB-25 (2 and 4 nmol per rat), CB-52 (2 and 4 nmol per rat) and WIN55,212-2 (4 nmol per rat) locally injected into the ventrolateral PAG on the spontaneous firing activity of ‘ON’ and ‘OFF’ neurons of the RVM, which receive input innervation from the PAG. Moreover, CB-25 (2 nmol per rat) and CB-52 (2 nmol per rat) were also evaluated in combination with WIN55,212-2 (4 nmol per rat) in the same test. We found that, in agreement with their pro-nociceptive effects observed in the plantar test following i.p. administration, both compounds caused dose-dependent and prolonged inhibition of ‘OFF’ anti-nociceptive RVM neurons (Figure 6b and d) and stimulation of ‘ON’ pro-nociceptive RVM neurons (Figure 6a and c). Also in this case, WIN55,212-2 caused effects that were opposite to those induced by CB-25 and CB-52, and were attenuated by co-injection with the low, ineffective, doses of the two compounds.

Effects of CB-25 and CB-52 on the nocifensive behaviour induced by formalin in mice

Unlike their effects in normal rats, in mice treated with formalin, which respond with a typical biphasic nocifensive behaviour (paw flinching and licking) to the injection of the noxious agent, both CB-25 (2.5 or 5 mg kg⁻¹, i.p.) and CB-52 (1 or 5 mg kg⁻¹, i.p.) behaved like WIN55,212-2 (2.5 mg kg⁻¹, i.p.), that is, they dose-dependently inhibited both the first and second hyperalgesic phases (Figure 7a and b). CB-52 appeared to be more potent than CB-25. The effect of submaximal doses of CB-25 or CB-52 and WIN55,212-2 appeared to be additive (Figure 7c), thus suggesting a possible similar mechanism of action. Finally, the effect of both compounds was attenuated by pretreatment with selective antagonists of CB₁ and CB₂ receptors, SR141716A (0.5 mg kg⁻¹, i.p.) and AM630 (2 mg kg⁻¹, i.p.), respectively (Figure 7a and b), doses that were inactive by themselves.

Discussion

We have described here the results of a series of in vitro and in vivo pharmacological experiments aimed at (1) assessing for the first time the functional activity at mouse and human cannabinoid CB₁ and CB₂ receptors of two previously developed cannabinoid receptor ligands, CB-25 and CB-52 (Figure 1) (Brizzi et al., 2005), (2) establishing whether different functional activities can be observed for these compounds when using different in vitro bioassays carried out in different animal species and (3) investigating whether the same compound can exert agonist-like or antagonist-like analgesic activities in vivo depending on the type of experimental pain model used.

Our first set of data indicates that the compound with highest affinity for human CB₁ receptors, CB-25 (K_i=5.2 nM), behaves as a partial agonist at these receptors when using inhibition of forskolin-stimulated cAMP formation in intact hCB₁-CHO cells as a means to determine functional activity. Interestingly, in this assay, not only the efficacy of this compound was significantly lower than that of CP55,940, but also its potency (IC₅₀=1.6 μM) did not reflect its affinity for human CB₁ receptors. The same applies to CB-52, which also inefficiently inhibited forskolin-stimulated cAMP formation in intact hCB₁-CHO cells, although in this case the difference between affinity (K_i=210 nM) and potency (IC₅₀=1.4 μM) was less striking. Interestingly, when the functional activity of CB-25 and CB-52 was studied using mouse CB₁ receptors, that is, by examining their effect on either forskolin-stimulated cAMP formation in intact N18TG2 cells or GTP-γ-S binding to mouse brain membranes, the former compound behaved as an inverse agonist or as a partial agonist, respectively, whereas CB-52 always acted as a partial agonist. Also in the case of this latter compound, however, differences between the two assays were found, as its potency in the cAMP assay was significantly lower, and its efficacy higher, than in the GTP-γ-S binding assay. By contrast, the potency (EC₅₀=20–100 nM) and efficacy (48% stimulation) of CB-25 in the two assays were similar, although producing opposing effects on receptor functionality. In this case, we could not compare the potencies of the two compounds in the two assays with their affinity for mouse CB₁ receptors, as no binding data for CB-25 and CB-52 at the mouse CB₁ receptor are available. These findings support the concept that not only the potency but also the efficacy and the type (agonist, inverse agonist, partial agonist) of the functional activity of certain compounds at CB₁ receptors depend on the animal species as well as on the type of functional assay used. These differences may reflect the existence of CB₁ receptors that are pre-coupled or not to one or more types of G proteins, or of a higher or lower endogenous tone of endocannabinoids in the in vitro experimental model used. For example, one would expect to find higher pre-coupling of CB₁ receptors to G proteins in hCB₁-CHO cells (where the CB₁ receptor is overexpressed) than in N18TG2 cells (where the receptor is expressed constitutively), thus explaining why in the former cells one observes higher efficacy for both CB-52 and (although in opposing directions) CB-25. On the other hand, in intact cells (particularly N18TG2 cells, which produce high levels of the endocannabinoid 2-AG; Bisogno et al., 1997), one expects to find more endocannabinoids tonically activating the receptors than in cell membranes, and this might possibly explain why a potent but inefficacious agonist like CB-25 acts as inverse agonist in N18TG2 cells. Finally, differences between the ability to inhibit forskolin-stimulated cAMP in N18TG2 cells and to stimulate GTP-γ-S binding to mouse brain membranes might also be due to possibility that the two compounds, apart from CB₁, bind to and activate other G-protein-coupled receptors, which might be expressed (and assayed) to different extents in the two systems.

The complexity of the functional activity at mouse CB₁ receptors of CB-25 and CB-52 is also illustrated by the surprising effects observed with the two compounds in two assays of anti-nociceptive activity, a typical property of cannabinoid receptor ligands, carried out in normal rats. The anti-nociceptive activity of cannabinoid receptor ligands in these assays was previously shown to be mediated only by CB₁ receptors, whereas CB₁, but not CB₂, receptor antagonists were found to induce hyperalgesia (Palazzo et al., 2001; de Novellis et al., 2005). When measuring either the threshold of thermal pain sensitivity in the plantar test or the spontaneous firing activity of pro- and anti-nociceptive neurons in the RVM, we observed that both CB-25 and CB-52, administered systemically or directly into the ventrolateral PAG, respectively, behaved as inverse agonists or as potent albeit partial agonists (see above), that is, as if they acted by counteracting a strong cannabinoid receptor-mediated, and possibly supraspinal, anti-nociceptive endogenous tone determined by the noxious stimulus (Walker et al., 1999). Indeed, the two compounds in both assays, and at doses inactive per se, also counteracted the anti-nociceptive effect of WIN55,212. Although it might fit with the in vitro functional profile observed here with CB-25 at mouse CB₁ receptors, or with CB-52 in the GTP-γ-S assay in mouse brain membranes (where the compound exhibited a very potent but inefficacious stimulation of binding), this interpretation of these in vivo pharmacological data, however, does not agree with some of the other data discussed above, that is, with (1) CB-52 activity in mouse N18TG2 cells (low potency and little efficacy at inhibiting cAMP formation) and (2) both CB-25 and CB-52 activity at human CB₁ receptors (inefficacy but also little potency at inhibiting cAMP formation). The two compounds investigated in the present study were previously shown (Brizzi et al., 2005) to be selective for cannabinoid receptors over enzymes that catalyse endocannabinoid inactivation (and hence potentially inactive as ‘indirect’ agonists of cannabinoid receptors) or over vanilloid TRPV1 receptors. However, we cannot rule out at this stage that they might induce their pro-nociceptive effects also via as yet uncharacterized non-CB₁, non-TRPV1 receptors (see for example Di Marzo et al., 2001).

The picture of the functional activity of CB-25 and CB-52 at human CB₂ receptors was far less complicated. Both compounds exhibited little or no efficacy when using either forskolin-stimulated cAMP formation in intact hCB₂-CHO cells or GTP-γ-S binding to membranes from similar cells, thus underscoring the importance of using similar cell systems, even when using different functional assays, to investigate the functional activity of cannabinoid receptor ligands in vitro. Furthermore, we found that the potency of CB-25 and CB-52 as antagonists of CP55,940-induced GTP-γ-S binding, either expressed as K_b values or as the minimal concentration required to exert a significant rightward shift of the dose–response curve of CP55,940, reflected their relative affinities for human recombinant CB₂ receptors.

With this apparently simpler scenario in mind, we tested CB-25 and CB-52 in a widely employed assay of peripheral pain, where not only CB₁ but also CB₂ receptor agonists and antagonists have been shown to produce effects, that is, the biphasic nocifensive behaviour observed in rodents following intra-paw injection of formalin (Calignano et al., 1998; Hanus et al., 1999; Beaulieu et al., 2000). Quite surprisingly, in consideration of the pro-nociceptive effects observed here in the plantar test in normal rats, both CB-25 and CB-52 behaved as analgesics against the two phases of formalin-induced behaviours in mice. As it has been often reported that cannabimimetic drugs can produce biphasic effects (see for example Sulcova et al., 1998), these seemingly paradoxical results might be simply due to the fact that we tested in vivo only two doses of the compounds, and hence might have missed observing either anti-nociceptive actions in the above-mentioned plantar test or pro-nociceptive effects in the formalin test. Alternatively, some of our data might suggest that the endogenous tone of endocannabinoids, hypothesized to be at the supraspinal level to explain the effects observed in the plantar test and on RVM neurons (see above), is absent in mice treated with formalin. In fact, although it was previously shown in formalin-treated mice that CB₁ and CB₂ receptor inverse agonists cause a pro-nociceptive effect, no evidence for an elevated endogenous tone of skin endocannabinoids has ever been reported following formalin injection in either mice or rats (Calignano et al., 1998; Beaulieu et al., 2000). Therefore, it is possible that in the absence of an endocannabinoid-induced CB₁/CB₂ tone in the paw skin of formalin-injected rats, CB-25 and CB-52, by being neutral CB₂ antagonists, act uniquely as CB₁ receptor partial agonists (see above) (a neutral antagonist will only exhibit pharmacological activity in the presence of an endogenous tone arising from endocannabinoid release, whereas an inverse agonist will also be active if the receptor is pre-coupled to G-proteins). This hypothesis is supported by our observations that (1) SR141716A (rimonabant), a selective CB₁ receptor inverse agonist, at a dose inactive per se, totally abolished CB-25 and CB-52 anti-nociceptive effects in formalin-treated rats, (2) these compounds affected significantly also the first phase of formalin-induced nociception, as one would expect from a partial CB₁ agonist, and (3) submaximal doses of CB-25 or CB-52 and WIN55,212-2 produced additive anti-nociceptive effects, thus suggesting a possible similar mechanism of action for the three compounds. However, the rank of potency/efficacy of CB-25 and CB-52 in this in vivo assay, where CB-52 was fully efficacious at doses lower than those necessary to CB-25 to exert a similar anti-hyperalgesic action, did not coincide with their rank of potency/efficacy at mouse CB₁ receptors observed here in vitro (see above), although this discrepancy might also be due to pharmacokinetic factors. More importantly, the effects of CB-25 and CB-52 were also abolished by the selective CB₂ receptor inverse agonist AM630, at a dose that is not expected to block CB₁ receptors and that was inactive per se. Therefore, additional mechanisms must be taken into account to explain the effects of the two compounds in the formalin test. For example, it is possible that CB-25 and CB-52, through some as yet unknown late compensatory mechanism, trigger the formation of anti-nociceptive endocannabinoids acting preferentially at CB₂ receptors at a stage and site when and where the two compounds cannot antagonize these receptors any longer. In other words, the two compounds might produce anti-nociception via as yet undefined CB₂-mediated mechanisms (that might indirectly result also in the activation of CB₁-mediated pathways). On the other hand, blockade by a CB₂ receptor antagonist is not always diagnostic of the direct participation of CB₂ receptors in the effect of a given compound, as shown by the finding that the analgesic actions of the cannabinoid receptor-inactive mediator, palmitoylethanolamide, are still reversed by the CB₂ antagonist/inverse agonist SR144528 (Calignano et al., 1998).

In conclusion, we have provided here the first functional data on CB-25 and CB-52, two ligands of cannabinoid CB₁ and CB₂ receptors derived from olivetol or resorcinol and fatty acids, previously developed by us and shown to be characterized by high metabolic stability with respect to both plant and endogenous cannabinoids (Brizzi et al., 2005). Our overall in vitro pharmacological data indicate that these compounds act in vitro mostly as partial agonists at CB₁ receptors and as neutral antagonists at CB₂ receptors. The in vivo data presented here, if interpreted in the light of the quantitatively and qualitatively different types of endogenous cannabinoid tone that are likely to occur in different types of pain, support in part these conclusions but need to be substantiated by further studies, while leaving open the possibility of additional mechanisms of action. Apart from describing the complex pharmacology of two potential new tools for the study of the endocannabinoid system, our data also highlight how different animal species and experimental models might lead to even opposing results in studies with cannabinoid drugs.

Conflict of interest

The authors state no conflict of interest.