Biological characterization of PM226, a chromenoisoxazole, as a selective CB2 receptor agonist with neuroprotective profile.

Cannabinoids have emerged as promising neuroprotective agents due to their capability to activate specific targets, which are involved in the control of neuronal homeostasis and survival. Specifically, those ligands that selectively target and activate the CB2 receptor may be useful for their anti-inflammatory and neuroprotective properties in various neurological disorders, with the advantage of being devoid of psychotropic effects associated with the activation of CB1 receptors. The aim of this work has been to investigate the neuroprotective properties of 7-(1,1-dimethylheptyl)-4,4-dimethyl-9-methoxychromeno[3,4-d]isoxazole (PM226), a compound derived from a series of chromeno-isoxazoles and -pyrazoles, which seems to have a promising profile related to the CB2 receptor. The compound binds selectively to this receptor with an affinity in the nanomolar range (Ki=12.8±2.4nM). It has negligible affinity for the CB1 receptor (Ki>40000nM) and no activity at the GPR55. PM226 was also evaluated in GTPγS binding assays specific to the CB2 receptor showing agonist activity (EC50=38.67±6.70nM). In silico analysis of PM226 indicated that it has a good pharmacokinetic profile and a predicted ability to cross the blood-brain barrier. Next, PM226 was investigated in an in vitro model to explore its anti-inflammatory/neuroprotective properties. Conditioned media were collected from LPS-stimulated cultures of BV2 microglial cell line in the absence or presence of different doses of PM226, and then media were added to cultured M213-2O neuronal cells to record their influence on cell viability evaluated using MTT assays. As expected, cell viability was significantly reduced by the exposure to these conditioned media, while the addition of PM226 attenuated this reduction in a dose-dependent manner. This effect was reversed by co-incubating with the CB2 antagonist SR144528, thus confirming the involvement of CB2 receptors, whereas the addition of PM226 to neuronal cultures instead cultured BV2 cells was not effective. PM226 has also been studied in an in vivo model of mitochondrial damage generated by intrastriatal application of malonate in rats. MRI analysis showed that PM226 administration decreased the volume of the striatal lesion caused by malonate, effect that was confirmed after the histopathological evaluation (Nissl staining, Iba-1 immunostaining and TUNEL assay) of striatal sections derived from malonate-lesioned rats in the absence or presence of PM226. Again, the beneficial effects of PM226 were dependent on the activation of CB2 receptors as they were reversed by blocking these receptors with AM630. Overall, PM226 has shown to have a promising neuroprotective profile derived from its ability to selectively activate CB2 receptor, so that it could be a useful disease-modifying agent in those neurodegenerative pathologies in which the activation of these receptors may have therapeutic value.

The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis, rather than enhancing striatal damage, protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG.

The cannabinoid CB2 receptor, which is activated by the endocannabinoid 2-arachidonoyl-glycerol (2-AG), protects striatal neurons from apoptotic death caused by the local administration of malonate, a rat model of Huntington's disease (HD). In the present study, we investigated whether endocannabinoids provide tonic neuroprotection in this HD model, by examining the effect of O-3841, an inhibitor of diacylglycerol lipases, the enzymes that catalyse 2-AG biosynthesis, and JZL184 or OMDM169, two inhibitors of 2-AG inactivation by monoacylglycerol lipase (MAGL). The inhibitors were injected in rats with the striatum lesioned with malonate, and several biochemical and morphological parameters were measured in this brain area. Similar experiments were also conducted in vitro in cultured M-213 cells, which have the phenotypic characteristics of striatal neurons. O-3841 produced a significant reduction in the striatal levels of 2-AG in animals lesioned with malonate. However, surprisingly, the inhibitor attenuated malonate-induced GABA and BDNF deficiencies and the reduction in Nissl staining, as well as the increase in GFAP immunostaining. In contrast, JZL184 exacerbated malonate-induced striatal damage. Cyclooxygenase-2 (COX-2) was induced in the striatum 24 h after the lesion simultaneously with other pro-inflammatory responses. The COX-2-derived 2-AG metabolite, prostaglandin E2 glyceryl ester (PGE2-G), exacerbated neurotoxicity, and this effect was antagonized by the blockade of PGE2-G action with AGN220675. In M-213 cells exposed to malonate, in which COX-2 was also upregulated, JZL184 worsened neurotoxicity, and this effect was attenuated by the COX-2 inhibitor celecoxib or AGN220675. OMDM169 also worsened neurotoxicity and produced measurable levels of PGE2-G. In conclusion, the inhibition of 2-AG biosynthesis is neuroprotective in rats lesioned with malonate, possibly through the counteraction of the formation of pro-neuroinflammatory PGE2-G, formed from COX-2-mediated oxygenation of 2-AG. Accordingly, MAGL inhibition or the administration of PGE2-G aggravates the malonate toxicity.

Cannabidiol administration after hypoxia-ischemia to newborn rats reduces long-term brain injury and restores neurobehavioral function.

Cannabidiol (CBD) demonstrated short-term neuroprotective effects in the immature brain following hypoxia-ischemia (HI). We examined whether CBD neuroprotection is sustained over a prolonged period. Newborn Wistar rats underwent HI injury (10% oxygen for 120 min after left carotid artery electrocoagulation) and then received vehicle (HV, n = 22) or 1 mg/kg CBD (HC, n = 23). Sham animals were similarly treated (SV, n = 16 and SC, n = 16). The extent of brain damage was determined by magnetic resonance imaging, histological evaluation (neuropathological score, 0-5), magnetic resonance spectroscopy and Western blotting. Several neurobehavioral tests (RotaRod, cylinder rear test[CRT],and novel object recognition[NOR]) were carried out 30 days after HI (P37). CBD modulated brain excitotoxicity, oxidative stress and inflammation seven days after HI. We observed that HI led to long-lasting functional impairment, as observed in all neurobehavioral tests at P37, whereas the results of HC animals were similar to those of sham animals (all p < 0.05 vs. HV). CBD reduced brain infarct volume by 17% (p < 0.05) and lessened the extent of histological damage. No differences were observed between the SV and SC groups in any of the experiments. In conclusion, CBD administration after HI injury to newborn rats led to long-lasting neuroprotection, with the overall effect of promoting greater functional rather than histological recovery. These effects of CBD were not associated with any side effects. These results emphasize the interest in CBD as a neuroprotective agent for neonatal HI.

The endocannabinoid system in Huntington's disease.

The hypokinetic profile of certain cannabinoid agonists becomes these compounds as promising medicines to attenuate the hyperkinesia that characterizes the first grades of Huntington's disease (HD) and that represents the major neurological abnormality in this disease. The fact that CB(1) receptors, the receptor type involved in motor effects of cannabinoid agonists, are significantly reduced in the basal ganglia during the progression of HD represents a convincing explanation for the hyperkinesia typical of this disorder and supports the usefulness of enhancing CB(1) receptor signaling in HD. However, further studies revealed that the key property that enables certain cannabinoid agonists to reduce hyperkinesia is their capability to directly activate vanilloid TRPV(1) receptors. Cannabinoids may also serve to delay/arrest the progression of HD by protecting striatal projection neurons from death. Several cannabinoid agonists have been tested for this purpose in various animal models of HD, and these studies revealed that the major characteristics that enable cannabinoids to provide neuroprotection are three: (i) a reduction in inflammatory events exerted through activating CB(2) receptors located in glial cells; (ii) a normalization of glutamate homeostasis, then limiting excitotoxicity, an effect that would be exerted through CB(1) receptors; and (iii) an antioxidant effect exerted by cannabinoid receptor-independent mechanisms. The changes experienced by the endocannabinoid signaling system during the striatal degeneration support this neuroprotective effect, particularly the up-regulatory responses proved by CB(2) receptors in glial cells recruited at lesioned sites. The present article will review the neurochemical and pharmacological bases that sustain the importance of the endocannabinoid system in the pathophysiology of HD, trying to collect the present information and the future lines for research on the therapeutic potential of this system in this disorder.

Colocalization of CB1 receptors with L1 and GAP-43 in forebrain white matter regions during fetal rat brain development: evidence for a role of these receptors in axonal growth and guidance.

There is recent evidence supporting the notion that the cannabinoid signaling system plays a modulatory role in the regulation of cell proliferation and migration, survival of neural progenitors, neuritic elongation and guidance, and synaptogenesis. This assumption is based on the fact that cannabinoid 1-type receptors (CB(1) receptors) and their ligands emerge early in brain development and are abundantly expressed in certain brain regions that play key roles in these processes. We have recently presented in vivo evidence showing that this modulatory action might be exerted through regulating the synthesis of the cell adhesion molecule L1 that is also a key element for those processes. To further explore this issue, we conducted here immunohistochemical studies aimed at determining the cellular substrates of CB(1) receptor-L1 interactions in the rat brain during late fetal development. In this period, we previously found that the activation of CB(1) receptors increased L1 synthesis in several forebrain white matter regions but not in gray matter areas. Using double labeling studies, we observed here colocalization of both proteins in fiber tracts including the corpus callosum, the adjacent subcortical white matter, the internal capsule and the anterior commissure. Experiments conducted with cultures of fetal rat cortical nerve cells revealed that L1 is present mainly in neurons but not in glial cells. This fact, together with the results obtained in the double labeling studies, would indicate that L1 and CB(1) receptors should possibly be present in axons elongating through these white matter tracts, or, alternatively, in migrating neurons. Further experiments confirmed the presence of CB(1) receptors in elongating axons, since these receptors colocalized with growth-associated protein 43 (GAP-43), a marker of growth cones, but not with synaptophysin, a marker of active synaptic terminals, in the same forebrain white matter regions. Lastly, using cultured fetal rat cortical neurons, we also observed that the activation of cannabinoid receptors increased the levels of the full-length L1 and altered those of some active proteolytic fragments of this protein whose generation has been associated with specific steps in the process of neuritic elongation in cultured neurons. In summary, we have demonstrated that the effects caused by cannabinoid agonists on L1 are facilitated by the colocalization of this cell adhesion molecule with CB(1) receptors in several forebrain white matter regions during fetal brain development. We have provided strong evidence that this phenomenon occurs in axons elongating through these white matter tracts, and we have explored in vitro how cannabinoid receptors influence L1 levels. Considering the role played by L1 in different events related to neural development, our observations support the occurrence of a physiological mechanism by which the cannabinoid system might regulate the process of axonal growth and guidance through regulating the synthesis and function of L1.

Cannabinoid CB2 receptors in human brain inflammation.

The presence of functional cannabinoid CB2 receptors in the CNS has provoked considerable controversy over the past few years. Formerly considered as an exclusively peripheral receptor, it is now accepted that it is also present in limited amounts and distinct locations in the brain of several animal species, including humans. Furthermore, the inducible nature of these receptors under neuroinflammatory conditions, in contrast to CB1, makes them attractive targets for the development of novel therapeutic approaches. In fact, the undesired psychoactive effects caused by CB1 activation have largely limited the clinical use of cannabinoid-related compounds that act on these receptors. In this review some recent findings on the antiinflammatory properties of CB2 receptors are presented, as well as new perspectives that have been obtained based on studies of human postmortem brain samples. In addition, various working hypotheses are also proposed and discussed.

Role of the endocannabinoid system in Alzheimer's disease: new perspectives.

The role of the endocannabinoid system in several diseases is currently under intense study. Among these, Alzheimer's disease may be a new promising area of research. We have recently reported the existence of profound changes in the location and density of several elements of this system in Alzheimer's disease tissue samples, indicating that a non-neuronal endocannabinoid system is up-regulated in activated glia. Additional data from other groups suggest that glial cells may be important elements in the regulation of endocannabinoid system activity, both in health as in disease. Some of these aspects are briefly discussed in the present review.

[Paraquat poisoning: clinical and anatomopathologic aspects in 3 cases]. / Intoxicación por paraquat: aspectos clínicos y anatomopatológicos en tres casos.

3 cases of suicide due to ingestion of 150, 200 and 4 gr. of paraquat, respectively, are presented. Two of them were brothers and agricultural industrialists, the other one was a farmer. The first two cases died 16 and 10 hours after intake. The farmer died 21 days after ingestion. All of them had progressive hypoxemia and renal failure the two brothers had heart conduction system glycemia (23 mg/dl) a few hours after ingestion. The postmortem study showed edema, hemorrhage and congestion of the lungs, alveolitis, fibrosis and lobar atelectasia; renal tubular necrosis adrenal necrosis; colestasia; hepatic steatosis focal miocarditis. The clinical evolution, particularly short in the first two patients, is commented on, as well as the post-mortem findings, comparing them with those described in the literature. Finally, due to the high toxicity of this herbicide, we insist in prompt transport to herbicide, we insist in prompt transport to the hospital in order to apply early treatment.

Acute thallium poisoning: an evaluation of different forms of treatment.

Hemodialysis, forced potassium diuresis, chelating agents per os, and Dithiocarb given intravenously during short periods of time were used for the treatment of acute thallium poisoning (ingestion of 750 mg of thallium sulfate), and the effectiveness of these different therapeutic procedures was analyzed. Chelating agents per os (Prussian blue, Dithiocarb, and Dithiozone) were ineffective in our patient, since fecal excretion of thallium was very low and unmodified by them. Forced potassium diuresis and hemodialysis were very useful therapeutic measures, especially in the first 12 days following ingestion. Dithiocarb perfusion seems to be the most effective method for enhancing urinary thallium excretion. This method might be most useful in the treatment of thallium poisoning if its deleterious effects could be eliminated.