José A. Ramos

PHARMACOLOGICAL AND BIOCHEMICAL INTERACTIONS BETWEEN OPIOIDS AND CANNABINOIDS

Opioids and cannabinoids are among the most widely consumed drugs of abuse in humans. A number of studies have shown that both types of drugs share several pharmacological properties, including hypothermia, sedation, hypotension, inhibition of both intestinal motility and locomotor activity and, in particular, antinociception. Moreover, phenomena of cross-tolerance or mutual potentiation of some of these pharmacological effects have been reported. In recent years, these phenomena have supported the possible existence of functional links in the mechanisms of action of both types of drugs. The present review addresses the recent advances in the study of pharmacological interactions between opioids and cannabinoids, focusing on two aspects: antinociception and drug addiction. The potential biochemical mechanisms involved in these pharmacological interactions are also discussed together with possible therapeutic implications of opioid-cannabinoid interactions.

The endogenous cannabinoid system and brain development

Cannabinoid receptors and their endogenous ligands constitute a novel modulatory system that is involved in specific brain functions, such as nociception, control of movement, memory and neuroendocrine regulation. Recently, it has also been suggested that this system is involved in brain development. Studies have used a variety of techniques to elucidate the effects of cannabinoids during development, as well as to characterize the presence of elements of the endogenous cannabinoid system (receptors and ligands) in the developing brain. Collectively, they suggest that endocannabinoids participate in brain development through the activation of second-messenger-coupled cannabinoid receptors.

Changes in endocannabinoid contents in the brain of rats chronically exposed to nicotine, ethanol or cocaine

Despite recent data suggesting that the endocannabinoid transmission is a component of the brain reward system and plays a role in dependence/withdrawal to different habit-forming drugs, only a few studies have examined changes in endocannabinoid ligands and/or receptors in brain regions related to reinforcement processes after a chronic exposure to these drugs. Recently, we carried out a comparative analysis of the changes in cannabinoid CB(1) receptor density in several rat brain regions caused by chronic exposure to some of the most powerful habit-forming drugs. In the present study, we have extended this objective by examining changes in the brain contents of arachidonoylethanolamide (AEA) and 2-arachidonoyl-glycerol (2-AG), the endogenous ligands for cannabinoid receptors, in animals chronically exposed to cocaine, nicotine or ethanol. Results were as follows. Cocaine was the drug exhibiting the minor number of effects, with only a small, but significant, decrease in the content of 2-AG in the limbic forebrain. In contrast, chronic alcohol exposure caused a decrease in the contents of both AEA and 2-AG in the midbrain, while it increased AEA content in the limbic forebrain. This latter effect was also observed after chronic nicotine exposure together with an increase in AEA and 2-AG contents in the brainstem. In contrast, the hippocampus, the striatum and the cerebral cortex exhibited a decrease in AEA and/or 2-AG contents after chronic nicotine exposure. We also tested the effect of chronic nicotine on brain CB(1) receptors, which had not been investigated before, and found an almost complete lack of changes in mRNA levels or binding capacity for these receptors. In summary, our results, in concordance with previous data on CB(1) receptors, indicate that the three drugs tested here produce different changes in endocannabinoid transmission. Only in the case of alcohol and nicotine, we observed a common increase in AEA contents in the limbic forebrain. This observation is important considering that this region is a key area for the reinforcing properties of habit-forming drugs, which might support the involvement of endocannabinoid transmission in some specific events of the reward system activated by these drugs.

Cannabinoids provide neuroprotection against 6-hydroxydopamine toxicity in vivo and in vitro: Relevance to Parkinson's disease

Cannabinoids have been reported to provide neuroprotection in acute and chronic neurodegeneration. In this study, we examined whether they are also effective against the toxicity caused by 6-hydroxydopamine, both in vivo and in vitro, which may be relevant to Parkinsons disease (PD). First, we evaluated whether the administration of cannabinoids in vivo reduces the neurodegeneration produced by a unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. As expected, 2 weeks after the application of this toxin, a significant depletion of dopamine contents and a reduction of tyrosine hydroxylase activity in the lesioned striatum were noted, and were accompanied by a reduction in tyrosine hydroxylase-mRNA levels in the substantia nigra. None of these events occurred in the contralateral structures. Daily administration of delta9-tetrahydrocannabinol (delta9-THC) during these 2 weeks produced a significant waning in the magnitude of these reductions, whereas it failed to affect dopaminergic parameters in the contralateral structures. This effect of delta9-THC appeared to be irreversible since interruption of the daily administration of this cannabinoid after the 2-week period did not lead to the re-initiation of the 6-hydroxydopamine-induced neurodegeneration. In addition, the fact that the same neuroprotective effect was also produced by cannabidiol (CBD), another plant-derived cannabinoid with negligible affinity for cannabinoid CB1 receptors, suggests that the antioxidant properties of both compounds, which are cannabinoid receptor-independent, might be involved in these in vivo effects, although an alternative might be that the neuroprotection exerted by both compounds might be due to their anti-inflammatory potential. As a second objective, we examined whether cannabinoids also provide neuroprotection against the in vitro toxicity of 6-hydroxydopamine. We found that the non-selective cannabinoid agonist HU-210 increased cell survival in cultures of mouse cerebellar granule cells exposed to this toxin. However, this effect was significantly lesser when the cannabinoid was directly added to neuronal cultures than when these cultures were exposed to conditioned medium obtained from mixed glial cell cultures treated with HU-210, suggesting that the cannabinoid exerted its major protective effect by regulating glial influence to neurons. In summary, our results support the view of a potential neuroprotective action of cannabinoids against the in vivo and in vitro toxicity of 6-hydroxydopamine, which might be relevant for PD. Our data indicated that these neuroprotective effects might be due, among others, to the antioxidant properties of certain plant-derived cannabinoids, or exerted through the capability of cannabinoid agonists to modulate glial function, or produced by a combination of both mechanisms.

Presence of cannabinoid binding sites in the brain from early postnatal ages.

The present study demonstrates the presence of cannabinoid receptors in the brain from early postnatal ages. Specific and saturable binding was observed in the forebrain and remaining brain from early postnatal ages (2 and 5 days after birth). Female neonate forebrain exhibited a higher receptor density at 2 days after birth than males, but this trend was inverted at 5 days. From postnatal day 10, the receptors could be measured in more defined brain areas, i.e. the striatum, limbic forebrain and ventral mesencephalon. The ontogeny of the receptors in these three areas was relatively similar, exhibiting a progressive increase which maximised on days 30 or 40 and then subsequently decreased to adult values. Subtle sexual dimorphism was found in the striatum and ventral mesencephalon but not the limbic forebrain.

Increased cannabinoid CB1 receptor binding and activation of GTP‐binding proteins in the basal ganglia of patients with Parkinson's syndrome and of MPTP‐treated marmosets

Recent evidence obtained in rat models of Parkinsons disease showed that the density of cannabinoid CB1 receptors and their endogenous ligands increase in basal ganglia. However, no data exists from post‐mortem brain of humans affected by Parkinsons disease or from primate models of the disorder. In the present study, we examined CB1 receptor binding and the magnitude of the stimulation by WIN55,212‐2, a specific CB1 receptor agonist, of [35S]GTPγS binding to membrane fractions from the basal ganglia of patients affected by Parkinsons disease. In Parkinsons disease, WIN55,212‐2‐stimulated [35S]GTPγS binding in the caudate nucleus, putamen, lateral globus pallidus and substantia nigra was increased, thus indicating a more effective activation of GTP‐binding protein‐coupled signalling mechanisms via CB1 receptors. This was accompanied by an increase in CB1 receptor binding in the caudate nucleus and the putamen, although no changes were observed in the lateral globus pallidus and the substantia nigra. Because Parkinsons disease patients had been chronically treated with l‐DOPA, brains were studied from normal common marmosets and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated animals with and without chronic L‐DOPA treatment. MPTP‐lesioned marmosets had increased CB1 receptor binding in the caudate nucleus and the putamen compared to control marmosets, as well as increased stimulation of [35S]GTPγS binding by WIN55,212‐2. However, following l‐DOPA treatment these parameters returned towards control values. The results indicate that a nigro‐striatal lesion is associated with an increase in CB1 receptors in the basal ganglia in humans and nonhuman primates and that this increase could be reversed by chronic l‐DOPA therapy. The data suggest that CB1 receptor blockade might be useful as an adjuvant for the treatment of parkinsonian motor symptoms.

Evaluation of the neuroprotective effect of cannabinoids in a rat model of Parkinson's disease : Importance of antioxidant and cannabinoid receptor-independent properties

We have recently demonstrated that two plant-derived cannabinoids, Delta9-tetrahydrocannabinol and cannabidiol (CBD), are neuroprotective in an animal model of Parkinsons disease (PD), presumably because of their antioxidant properties. To further explore this issue, we examined the neuroprotective effects of a series of cannabinoid-based compounds, with more selectivity for different elements of the cannabinoid signalling system, in rats with unilateral lesions of nigrostriatal dopaminergic neurons caused by local application of 6-hydroxydopamine. We used the CB1 receptor agonist arachidonyl-2-chloroethylamide (ACEA), the CB2 receptor agonist HU-308, the non-selective agonist WIN55,212-2, and the inhibitors of the endocannabinoid inactivation AM404 and UCM707, all of them administered i.p. Daily administration of ACEA or WIN55,212-2 did not reverse 6-hydroxydopamine-induced dopamine (DA) depletion in the lesioned side, whereas HU-308 produced a small recovery that supports a possible involvement of CB2 but not CB1 receptors. AM404 produced a marked recovery of 6-hydroxydopamine-induced DA depletion and tyrosine hydroxylase deficit in the lesioned side. Possibly, this is caused by the antioxidant properties of AM404, which are derived from the presence of a phenolic group in its structure, rather than by the capability of AM404 to block the endocannabinoid transporter, because UCM707, another transporter inhibitor devoid of antioxidant properties, did not produce the same effect. None of these effects were observed in non-lesioned contralateral structures. We also examined the timing for the effect of CBD to provide neuroprotection in this rat model of PD. We found that CBD, as expected, was able to recover 6-hydroxydopamine-induced DA depletion when it was administered immediately after the lesion, but it failed to do that when the treatment started 1 week later. In addition, the effect of CBD implied an upregulation of mRNA levels for Cu,Zn-superoxide dismutase, a key enzyme in endogenous defenses against oxidative stress. In summary, our results indicate that those cannabinoids having antioxidant cannabinoid receptor-independent properties provide neuroprotection against the progressive degeneration of nigrostriatal dopaminergic neurons occurring in PD. In addition, the activation of CB2 (but not CB1) receptors, or other additional mechanisms, might also contribute to some extent to the potential of cannabinoids in this disease.

The endogenous cannabinoid system and the basal ganglia: biochemical, pharmacological, and therapeutic aspects

New data strengthen the idea of a prominent role for endocannabinoids in the modulation of a wide variety of neurobiological functions. Among these, one of the most important is the control of movement. This finding is supported by 3 lines of evidence: (1) the demonstration of a powerful action, mostly inhibitory in nature, of synthetic and plant-derived cannabinoids and, more recently, of endocannabinoids on motor activity; (2) the presence of the cannabinoid CB(1) receptor subtype and the recent description of endocannabinoids in the basal ganglia and the cerebellum, the areas that control movement; and (3) the fact that CB(1) receptor binding was altered in the basal ganglia of humans affected by several neurological diseases and also of rodents with experimentally induced motor disorders. Based on this evidence, it has been suggested that new synthetic compounds that act at key steps of endocannabinoid activity (i.e., more-stable analogs of endocannabinoids, inhibitors of endocannabinoid reuptake or metabolism, antagonists of CB(1) receptors) might be of interest for their potential use as therapeutic agents in a variety of pathologies affecting extrapyramidal structures, such as Parkinsons and Huntingtons diseases. Currently, only a few data exist in the literature studying such relationships in humans, but an increasing number of journal articles are revealing the importance of this new neuromodulatory system and arguing in favour of the funding of more extensive research in this field. The present article will review the current knowledge of this neuromodulatory system, trying to establish the future lines for research on the therapeutic potential of the endocannabinoid system in motor disorders.

Decreased endocannabinoid levels in the brain and beneficial effects of agents activating cannabinoid and/or vanilloid receptors in a rat model of multiple sclerosis

Recent studies have addressed the changes in endocannabinoid ligands and receptors that occur in multiple sclerosis, as a way to explain the efficacy of cannabinoid compounds to alleviate spasticity, pain, tremor, and other signs of this autoimmune disease. Using Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, we recently found a decrease in cannabinoid CB1 receptors mainly circumscribed to the basal ganglia, which could be related to the motor disturbances characteristic of these rats. In the present study, using the same model, we explored the potential changes in several neurotransmitters in the basal ganglia that might be associated with the motor disturbances described in these rats, but we only found a small increase in glutamate contents in the globus pallidus. We also examined whether the motor disturbances and the changes of CB1 receptors found in the basal ganglia of EAE rats disappear after the treatment with rolipram, an inhibitor of type IV phosphodiesterase able to supress EAE in different species. Rolipram attenuated clinical decline, reduced motor inhibition, and normalized CB1 receptor gene expression in the basal ganglia. As a third objective, we examined whether EAE rats also exhibited changes in endocannabinoid levels as shown for CB1 receptors. Anandamide and 2-arachidonoylglycerol levels decreased in motor related regions (striatum, midbrain) but also in other brain regions, although the pattern of changes for each endocannabinoid was different. Finally, we hypothesized that the elevation of the endocannabinoid activity, following inhibition of endocannabinoid uptake, might be beneficial in EAE rats. AM404, arvanil, and OMDM2 were effective to reduce the magnitude of the neurological impairment in EAE rats, whereas VDM11 did not produce any effect. The beneficial effects of AM404 were reversed by blocking TRPV1 receptors with capsazepine, but not by blocking CB1 receptors with SR141716, thus indicating the involvement of endovanilloid mechanisms in these effects. However, a role for CB1 receptors is supported by additional data showing that CP55,940 delayed EAE progression. In summary, our data suggest that reduction of endocannabinoid signaling is associated with the development of EAE in rats. We have also proved that the reduction of CB1 receptors observed in these rats is corrected following treatment with a compound used in EAE such as rolipram. In addition, the direct or indirect activation of vanilloid or cannabinoid receptors may reduce the neurological impairment experienced by EAE rats, although the efficacy of the different compounds examined seems to be determined by their particular pharmacodynamic and pharmacokinetic characteristics.

Chronic exposure to morphine, cocaine or ethanol in rats produced different effects in brain cannabinoid CB1 receptor binding and mRNA levels

Recent evidence suggest that the endocannabinoid system might be a component of the brain reward system and, then, play a role, not only in cannabinoid tolerance/dependence, but also in dependence/withdrawal to other drugs of abuse. However, there are not many studies that compare the changes in endocannabinoid ligands and/or receptors in brain regions (particularly in those areas related to reinforcement processes) during dependence to opiates, cocaine or alcohol. The present study addressed this objective, by examining the changes in CB(1) receptor binding (measured by [3H]-CP55,940 autoradiography) and its mRNA levels (measured by in situ hybridization) in different brain regions of animals chronically exposed to morphine, cocaine or ethanol. The results showed that these three drugs produced different changes in CB(1) receptor binding and mRNA levels, a finding that precludes the existence of a common alteration of the endocannabinoid system during dependence states to these habit-forming drugs. Thus, chronic ethanol exposure was usually uneffective in altering both CB(1) receptor binding and mRNA levels in all regions examined. In contrast, chronic cocaine exposure produced significant changes only at the level of CB(1) receptor mRNA, with decreases of the transcript levels in the ventromedial hypothalamic nucleus and the superficial and deep layers of the cerebral cortex, but no changes in the hippocampal, motor and limbic structures. Finally, chronic morphine exposure increased the density of CB(1) receptors in the medial caudate-putamen, but decreased their mRNA levels in this region and also in the lateral caudate-putamen and the cerebellum. In limbic structures, chronic morphine exposure increased both binding and mRNA levels for CB(1) receptors in the septum nuclei. Binding was also increased in the nucleus accumbens, but reduced in the basolateral amygdala. In hippocampal structures, chronic morphine exposure reduced CB(1) receptor binding in the dentate gyrus, although mRNA levels were unaffected in this region, but increased in the CA2 subfield of the Ammons horn. The results indicate that mechanisms of dependence for alcohol, cocaine and morphine are different in terms of their impact on the endocannabinoid system. Alcohol did not produce any effects on CB(1) receptor binding and mRNA levels, whereas cocaine only affected transcript levels in selected regions and morphine produced divergent and region-dependent effects.