Eva de Lago

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.

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.

Cannabinoids and Neuroprotection in Basal Ganglia Disorders

Cannabinoids have been proposed as clinically promising neuroprotective molecules, as they are capable to reduce excitotoxicity, calcium influx, and oxidative injury. They are also able to decrease inflammation by acting on glial processes that regulate neuronal survival and to restore blood supply to injured area by reducing the vasoconstriction produced by several endothelium-derived factors. Through one or more of these processes, cannabinoids may provide neuroprotection in different neurodegenerative disorders including Parkinson’s disease and Huntington’s chorea, two chronic diseases that are originated as a consequence of the degeneration of specific nuclei of basal ganglia, resulting in a deterioration of the control of movement. Both diseases have been still scarcely explored at the clinical level for a possible application of cannabinoids to delay the progressive degeneration of the basal ganglia. However, the preclinical evidence seems to be solid and promising. There are two key mechanisms involved in the neuroprotection by cannabinoids in experimental models of these two disorders: first, a cannabinoid receptor-independent mechanism aimed at producing a decrease in the oxidative injury and second, an induction/upregulation of cannabinoid CB2 receptors, mainly in reactive microglia, that is capable to regulate the influence of these glial cells on neuronal homeostasis. Considering the relevance of these preclinical data and the lack of efficient neuroprotective strategies in both disorders, we urge the development of further studies that allow that the promising expectatives generated for these molecules progress from the present preclinical evidence till a real clinical application.

UCM707, an inhibitor of the anandamide uptake, behaves as a symptom control agent in models of Huntington's disease and multiple sclerosis, but fails to delay/arrest the progression of different motor-related disorders

To date, UCM707, (5Z,8Z,11Z,14Z)-N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide, has the highest potency and selectivity in vitro and in vivo as inhibitor of the endocannabinoid uptake. This may enable this compound to potentiate endocannabinoid transmission, with minimal side effects, in the treatment of several neurological disorders. In the present study, we examined whether the treatment with UCM707 produced beneficial effects, as other cannabinoid-related compounds have already shown, to alleviate motor deterioration or to delay/arrest neurodegeneration, in several models of neurological diseases such as Huntingtons disease (HD), Parkinsons disease (PD) and multiple sclerosis (MS). UCM707 exhibited a notable anti-hyperkinetic activity in a rat model of HD generated by bilateral intrastriatal application of 3-nitropropionic acid. This effect was possibly associated with an amelioration of GABA and glutamate deficits induced by the toxin in the globus pallidus and the substantia nigra, respectively. However, UCM707 did not protect against the death of GABAergic neurons that occurs in rats with striatal atrophy generated by unilateral application of malonate, another animal model of HD, which is more useful to test neuroprotective strategies. In addition, UCM707 did not provide neuroprotection in rats with unilateral lesions of the nigrostriatal dopaminergic neurons caused by 6-hydroxydopamine, a rat model of PD. This was possibly due to the fact that UCM707 is devoid of anti-oxidant properties since another uptake inhibitor, AM404, that has these properties acted as a protective agent. Lastly, UCM707 was also unable to inhibit the development of the neurological impairment of rats with experimental autoimmune encephalomyelitis (EAE), an acute model of MS. However, UCM707, like other endocannabinoid uptake inhibitors reported previously, significantly reduced spasticity of the hindlimbs in a chronic relapsing EAE mice, a chronic model of MS. In summary, UCM707 might be a promising compound in HD to alleviate motor symptoms, which represents an important goal considering the current lack of efficient pharmacological treatments in this basal ganglia disorder. However, the compound was unable to delay neurodegeneration in this disorder and also in PD. In addition, UCM707 did not produce any neurological recovery from inflammatory attack in an EAE rat model of MS, although it retained the classic anti-spastic action shown by other uptake inhibitors in the EAE mouse model of this disease.

UCM707, a potent and selective inhibitor of endocannabinoid uptake, potentiates hypokinetic and antinociceptive effects of anandamide.

To date, UCM707, N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide, has the highest potency and selectivity in vitro as inhibitor of the endocannabinoid transporter, which might make this compound useful in potentiating endocannabinoid transmission, with minimal side-effects, in the treatment of several disorders. However, there is no information about how UCM707 behaves in vivo as regards certain classic effects of endocannabinoids, such as hypomotility and antinociception. In the present work, we tested in rats the dose-response effects of UCM707 in the open-field and hot-plate tests, and, in particular, we analyzed whether this compound enhanced the hypokinetic and/or the antinociceptive actions of anandamide at a subeffective dose, using these two in vivo assays. UCM707, administered alone, had no effect on ambulatory, exploratory and stereotypic activities, time spent in inactivity and sensitivity to noxious heat, with only some small responses at the highest dose used. UCM707, administered at a dose that did not produce any effects by itself or these were very small, was, however, able to significantly potentiate the action of a dose of anandamide that did not produce any effects when it was administered alone. So, the combination of both compounds produced greater decreases in exploratory activity and, particularly in ambulation, increased the time spent in inactivity and the latency to respond to a painful stimulus. In summary, UCM707, as suggested by its in vitro properties, seems also to behave in vivo as a selective and potent inhibitor of the endocannabinoid transporter, showing negligible direct effects on the receptors for endocannabinoids but potentiating the action of these endogenous compounds. This compound is, thus, a promising tool, used alone or in combination with endocannabinoids, for the treatment of a variety of disorders.

The endocannabinoid system as a target for the treatment of neuronal damage

Importance of the field: Cannabinoids have been proposed as clinically promising neuroprotective molecules, based on their capability to normalize glutamate homeostasis, reducing excitotoxicity, to inhibit calcium influx, lowering intracellular levels and the subsequent activation of calcium-dependent destructive pathways, and to reduce the generation of reactive oxygen intermediates or to limit their toxicity, decreasing oxidative injury. Cannabinoids are also able to decrease local inflammatory events by acting on glial processes that regulate neuronal survival, and to restore blood supply by reducing vasocontriction produced by several endothelium-derived factors. Areas covered in this review: Current literature supporting these neuroprotective effects, particularly evidence generated during the last ten years, concentrating on targets within the cannabinoid signaling system that facilitate these effects. Acute or chronic neurodegenerative disorders where cannabinoids have shown neuroprotective effect. What the reader will gain: Most of the information reviewed here relates to preclinical studies. However, these molecules may progress from the present preclinical evidence to clinical applications. Take home message: Treatment of neurodegenerative disorders is a challenge for neuroscientists and neurologists. Unhappily, the efficacy of available medicines is still poor and there is an urgent need for novel neuroprotective agents. Cannabinoids can serve this purpose given their recognized antiexcitotoxic, antioxidant and anti-inflammatory properties.

Cannabinoids ameliorate disease progression in a model of multiple sclerosis in mice, acting preferentially through CB1 receptor-mediated anti-inflammatory effects

Multiple sclerosis (MS) is an autoimmune disease that affects the CNS and it is characterized by inflammation, demyelination, remyelination, gliosis and axonal damage that occur mainly in the spinal cord. Cannabinoids have been proposed as promising therapeutic agents in MS given their capability to alleviate specific MS symptoms (e.g., spasticity, pain). Although MS has been considered mainly an inflammatory disorder, recent evidence, however, revealed the importance of neurodegenerative events, opening the possibility that cannabinoid agonists, given their cytoprotective properties, may also serve to reduce oligodendrocyte death and axonal damage in MS. Thus, the treatment with WIN55,512-2, a potent CB(1) and CB(2) agonist, was reported to be effective to ameliorate tremor and spasticity in mice with chronic relapsing experimental autoimmune encephalomyelitis, a murine model of MS, but also to delay disease progression in this and other murine models of MS. The purpose of this investigation was to further explore the mechanism(s) underlying the amelioration in disease progression caused by WIN55,212-2. We have particularly focused on anti-glutamatergic and anti-inflammatory effects of this cannabinoid agonist. In this study, we used mice treated with myelin oligodendrocyte glycoprotein (MOG) that induces a progressive pattern of EAE and conducted the pharmacological experiments in early stages of the disease. As expected, the administration of WIN55,512-2 (5 mg/kg, i.p) had a positive effect in reducing neurological disability and improving motor coordination of EAE mice. Levels of glutamate and GABA in the spinal cord and also in the brainstem of EAE mice were similar to control animals, and, accordingly, they were not altered by the treatment with WIN55,212-2. However, EAE mice showed some subtle alterations in mRNA levels for the glutamate transporter GLT1 and, to a lesser extent, GLAST too, changes that were altered by the treatment with WIN55,212-2 in the spinal cord, but not in the brainstem. Regarding to inflammatory responses, EAE mice showed a marked up-regulation in mRNA levels for COX-2, inducible NOS and TNF-α in the spinal cord and the brainstem, these responses being attenuated after the treatment with WIN55,212-2. We also observed the presence of cell aggregates in the spinal cord of EAE mice that were significantly attenuated by the treatment with WIN55,212-2. Immunohistochemical analysis (with Iba-1 and Cd11b) of these aggregates indicated that they corresponded to microglia (resident macrophages) and peripheral macrophages. Lastly, experiments conducted with selective antagonists for the CB(1) (e.g. rimonabant) or CB(2) (e.g. AM-630) receptors revealed that WIN55,212-2 effects in EAE mice were mediated by the activation of CB(1) but not CB(2) receptors, as reflected the reversion of positive effects of this cannabinoid on neurological decline, TNF-α generation and accumulation of cell aggregates in the spinal cord with rimonabant, but not with AM-630. This was concordant with the lack of positive effects on neurological decline observed in EAE mice when they received HU-308, a selective CB(2) receptor agonist, instead WIN55,212-2. In summary, the treatment of EAE mice with the cannabinoid agonist WIN55,512-2 reduced their neurological disability and the progression of the disease. This effect was exerted through the activation of CB(1) receptors, which would exert a positive influence in the reduction of inflammatory events linked to the pathogenesis of this disease.

Design, synthesis and biological evaluation of new endocannabinoid transporter inhibitors

In the present work we describe the synthesis and the in vitro evaluation of a series of arachidonic acid derivatives of general structure I as endocannabinoid transporter inhibitors. In addition, we report the first in vivo studies of the most potent derivative (4, UCM707) within this series. The majority of compounds studied are highly potent (IC(50)=24-0.8 micro M) and selective endocannabinoid uptake inhibitors with very low affinities for either the enzyme fatty acid amide hydrolase (IC(50)=30-113 micro M) or for cannabinoid receptor subtype 1 (CB(1)), cannabinoid receptor subtype 2 (CB(2)) and vanilloid receptor subtype 1 (VR(1)) (K(i)=1000-10000 nM). Among them, (5Z,8Z,11Z,14Z)-N-(fur-3-ylmethyl)icosa-5,8,11,14-tetraenamide (UCM707) behaves as the most potent endocannabinoid transporter inhibitor described to date (IC(50)=0.8 micro M) and exhibits improved potency for the anandamide transporter, high selectivity for CB(1) and VR(1) receptors, and modest selectivity for CB(2). In vivo it enhances the analgesia and hypokinetic effects induced by a subeffective dose of anandamide.

Changes in Endocannabinoid Receptors and Enzymes in the Spinal Cord of SOD1G93A Transgenic Mice and Evaluation of a Sativex®-like Combination of Phytocannabinoids: Interest for Future Therapies in Amyotrophic Lateral Sclerosis

Cannabinoids afford neuroprotection in SOD1G93A mutant mice, an experimental model of amyotrophic lateral sclerosis (ALS). However, these mice have been poorly studied to identify alterations in those elements of the endocannabinoid system targeted by these treatments. Moreover, we studied the neuroprotective effect of the phytocannabinoid‐based medicine Sativex® in these mice.

Acyl-based anandamide uptake inhibitors cause rapid toxicity to C6 glioma cells at pharmacologically relevant concentrations.

Compounds blocking the uptake of the endogenous cannabinoid anandamide (AEA) have been used to explore the functions of the endogenous cannabinoid system in the CNS both in vivo and in vitro. In this study, the effects of four commonly used acyl‐based uptake inhibitors [N‐(4‐hydroxyphenyl)arachidonylamide (AM404), N‐(4‐hydroxy‐2‐methylphenyl) arachidonoyl amide (VDM11), (5Z,8Z,11Z,14Z)‐N‐(3‐furanylmethyl)‐5,8,11,14‐eicosatetraenamide (UCM707) and (9Z)‐N‐[1‐((R)‐4‐hydroxybenzyl)‐2‐hydroxyethyl]‐9‐octadecen‐amide (OMDM2)] and the related compound arvanil on C6 glioma cell viability were investigated. All five compounds reduced the ability of the cells to accumulate calcein, reduced the total nucleic acid content and increased the activity of lactate dehydrogenase recovered in the cell medium. AM404 (10 µm) and VDM11 (10 µm) acted rapidly, reducing cell viability after 3 h of exposure when cell densities of 5000 per well were used. In contrast, UCM707 (30 µm), OMDM2 (10 µm) and the related compound arvanil (10 µm) produced a more slowly developing effect on cell viability, although robust effects were seen after 6–9 h of exposure. At higher cell densities, the toxicities of AM404 and UCM707 were reduced. Comparison of the compounds with arachidonic acid, arachidonic acid methyl ester, AEA, arachidonoyl glycine and oleic acid suggested that the toxicity of the arachidonoyl‐based compounds was related primarily to the acyl side‐chain rather than the head group. A variety of pre‐treatments blocking possible metabolic pathways and receptor targets were tested, but the only consistent protective treatment against the effects of these compounds was the antioxidant N‐acetyl‐l‐cysteine. It is concluded that AM404, VDM11, UCM707 and OMDM2 produce a rapid loss of C6 glioma cell viability over the same concentration range as is required for the inhibition of AEA uptake in vitro, albeit with a longer latency. Such effects should be kept in mind when acyl‐derived compounds are used to probe the function of the endocannabinoid system in the CNS, particularly in chronic administration protocols.