José Martínez-Orgado

Cannabidiol for neurodegenerative disorders: important new clinical applications for this phytocannabinoid?

Cannabidiol (CBD) is a phytocannabinoid with therapeutic properties for numerous disorders exerted through molecular mechanisms that are yet to be completely identified. CBD acts in some experimental models as an anti‐inflammatory, anticonvulsant, anti‐oxidant, anti‐emetic, anxiolytic and antipsychotic agent, and is therefore a potential medicine for the treatment of neuroinflammation, epilepsy, oxidative injury, vomiting and nausea, anxiety and schizophrenia, respectively. The neuroprotective potential of CBD, based on the combination of its anti‐inflammatory and anti‐oxidant properties, is of particular interest and is presently under intense preclinical research in numerous neurodegenerative disorders. In fact, CBD combined with Δ9‐tetrahydrocannabinol is already under clinical evaluation in patients with Huntingtons disease to determine its potential as a disease‐modifying therapy. The neuroprotective properties of CBD do not appear to be exerted by the activation of key targets within the endocannabinoid system for plant‐derived cannabinoids like Δ9‐tetrahydrocannabinol, i.e. CB1 and CB2 receptors, as CBD has negligible activity at these cannabinoid receptors, although certain activity at the CB2 receptor has been documented in specific pathological conditions (i.e. damage of immature brain). Within the endocannabinoid system, CBD has been shown to have an inhibitory effect on the inactivation of endocannabinoids (i.e. inhibition of FAAH enzyme), thereby enhancing the action of these endogenous molecules on cannabinoid receptors, which is also noted in certain pathological conditions. CBD acts not only through the endocannabinoid system, but also causes direct or indirect activation of metabotropic receptors for serotonin or adenosine, and can target nuclear receptors of the PPAR family and also ion channels.

Neuroprotective Effects of the Nonpsychoactive Cannabinoid Cannabidiol in Hypoxic-Ischemic Newborn Piglets

To test the neuroprotective effects of the nonpsychoactive cannabinoid cannabidiol (CBD), piglets received i.v. CBD or vehicle after hypoxia-ischemia (HI: temporary occlusion of both carotid arteries plus hypoxia). Nonhypoxic-ischemic sham-operated piglets remained as controls. Brain damage was studied by near-infrared spectroscopy (NIRS) and amplitude-integrated electroencephalography (aEEG) and by histologic assessment (Nissl and FluoroJadeB staining). In HI+vehicle, HI led to severe cerebral hemodynamic and metabolic impairment, as reflected in NIRS by an increase in total Hb index (THI) and a decrease in the fractional tissue oxygenation extraction (FTOE); in HI+CBD the increase of THI was blunted and FTOE remained similar to SHAM. HI profoundly decreased EEG amplitude, which was not recovered in HI+vehicle, indicating cerebral hypofunction; seizures were observed in all HI+vehicle. In HI+CBD, however, EEG amplitude recovered to 46.4 ± 7.8% baseline and seizures appeared only in 4/8 piglets (both p < 0.05). The number of viable neurons decreased and that of degenerating neurons increased in HI+vehicle; CBD reduced both effects by more than 50%. CBD administration was free from side effects; moreover, CBD administration was associated with cardiac, hemodynamic, and ventilatory beneficial effects. In conclusion, administration of CBD after HI reduced short-term brain damage and was associated with extracerebral benefits.

Characterization of the Neuroprotective Effect of the Cannabinoid Agonist WIN-55212 in an In Vitro Model of Hypoxic-Ischemic Brain Damage in Newborn Rats

Brain slices from 7-d-old Wistar rats were exposed to oxygen-glucose deprivation (OGD) for 30 min. OGD slices were incubated with vehicle or with the CB1/CB2 cannabinoid agonist WIN55212 (50 μM), the CB1 agonist arachidonyl-2-chloroethylamide (ACEA) (50 μM), or the CB2 agonist JW133 (50 μM), alone or combined with the CB1 and CB2 receptor antagonist SR 141716 (50 μM) or SR 144528 (50 μM), respectively. Neuronal damage was assessed by histologic analysis and spectrophotometric determination of lactate dehydrogenase (LDH) efflux into the incubation medium. Additionally, medium glutamate levels were determined by high-performance liquid chromatography (HPLC) and those of tumor necrosis factor α (TNF-α) by enzyme-linked immunosorbent assay. Finally, inducible nitric oxide synthase (iNOS) and CB1/CB2 receptor expression were determined in slices homogenate by Western blot. Both CB1 and CB2 receptors were expressed in slices. OGD increased CB1 expression, cellular damage, LDH efflux, glutamate and TNF-α release, and inducible nitric oxide synthase (iNOS) expression; WIN55212 inhibited all these actions. SR141716 and SR144528 inhibited the effect of R(+)-WIN-55212-2 (WIN), as well as the reduction of LDH efflux by ACEA and JW133, respectively. In conclusion, WIN55212 afforded robust neuroprotection in the forebrain slices exposed to OGD, by acting on glutamatergic excitotoxicity, TNF-α release, and iNOS expression; this neuroprotective effect seemed to be mediated by CB1 and CB2 receptors.

Mechanisms of cannabidiol neuroprotection in hypoxic–ischemic newborn pigs: Role of 5HT1A and CB2 receptors

The mechanisms underlying the neuroprotective effects of cannabidiol (CBD) were studied in vivo using a hypoxic-ischemic (HI) brain injury model in newborn pigs. One- to two-day-old piglets were exposed to HI for 30 min by interrupting carotid blood flow and reducing the fraction of inspired oxygen to 10%. Thirty minutes after HI, the piglets were treated with vehicle (HV) or 1 mg/kg CBD, alone (HC) or in combination with 1 mg/kg of a CB₂ receptor antagonist (AM630) or a serotonin 5HT(1A) receptor antagonist (WAY100635). HI decreased the number of viable neurons and affected the amplitude-integrated EEG background activity as well as different prognostic proton-magnetic-resonance-spectroscopy (H(±)-MRS)-detectable biomarkers (lactate/N-acetylaspartate and N-acetylaspartate/choline ratios). HI brain damage was also associated with increases in excitotoxicity (increased glutamate/N-acetylaspartate ratio), oxidative stress (decreased glutathione/creatine ratio and increased protein carbonylation) and inflammation (increased brain IL-1 levels). CBD administration after HI prevented all these alterations, although this CBD-mediated neuroprotection was reversed by co-administration of either WAY100635 or AM630, suggesting the involvement of CB₂ and 5HT(1A) receptors. The involvement of CB₂ receptors was not dependent on a CBD-mediated increase in endocannabinoids. Finally, bioluminescence resonance energy transfer studies indicated that CB₂ and 5HT(1A) receptors may form heteromers in living HEK-293T cells. In conclusion, our findings demonstrate that CBD exerts robust neuroprotective effects in vivo in HI piglets, modulating excitotoxicity, oxidative stress and inflammation, and that both CB₂ and 5HT(1A) receptors are implicated in these effects.

The activation of cannabinoid CB2 receptors stimulates in situ and in vitro beta-amyloid removal by human macrophages.

The endocannabinoid system is a promising therapeutic target in a wide variety of diseases. However, the non-desirable psychotropic effects of natural and synthetic cannabinoids have largely counteracted their clinical usefulness. These effects are mostly mediated by cannabinoid receptors of the CB(1) type, that exhibit a wide distribution in neuronal elements of the CNS. Thus, the presence of other elements of this system in the CNS, such as CB(2) receptors, may open new possibilities for the development of cannabinoid-based therapies. These receptors are almost absent from the CNS in normal conditions but are up-regulated in glial cells under chronic neuroinflammatory stimuli, as has been described in Alzheimers disease. To understand the functional role of these receptors, we tested their role in the process of beta-amyloid removal, that is currently considered as one of the most promising experimental approaches for the treatment of this disease. Our results show that a CB(2) agonist (JWH-015) is capable of inducing the removal of native beta-amyloid removal from human frozen tissue sections as well as of synthetic pathogenic peptide by a human macrophage cell line (THP-1). Remarkably, this effect was achieved at low doses (maximum effect at 10 nM) and was specific for this type of cells, as U373MG astrocytoma cells did not respond to the treatment. The effect was CB(2)-mediated, at least partially, as the selective CB(2) antagonist SR144528 prevented the JWH-015-induced plaque removal in situ. These data corroborate the possible therapeutic interest of CB(2) cannabinoid specific chemicals in the treatment of Alzheimers disease.

The Cannabinoid Agonist Win55212 Reduces Brain Damage in an In Vivo Model of Hypoxic-Ischemic Encephalopathy in Newborn Rats

Neonatal hypoxic-ischemic encephalopathy (NHIE) is a devastating condition for which effective therapeutic treatments are still unavailable. Cannabinoids emerge as neuroprotective substances in adult animal studies; therefore, we aimed herein to test whether cannabinoids might reduce brain damage induced by hypoxia-ischemia (HI) in newborn rats. Thus, 7-d-old Wistar rats (P7) were exposed to 8% O2 for 120 min after left carotid artery ligature, then received s.c. vehicle (VEH) (HI+VEH), the cannabinoid agonist WIN55212 (WIN) (0.1 mg/kg), or WIN with the CB1 or CB2 receptor antagonist SR141617 (SR1) (3 mg/kg) or SR141588 (SR2) (2 mg/kg). Brain damage was assessed by magnetic resonance imaging (MRI) at 1, 3, and 7 d after the insult. At the end of the experiment, MRI findings were corroborated by histology (Nissl staining). HI+VEH showed an area of cytotoxic and vasogenic edema at 24 h after the insult, then evolving to necrosis. HI+WIN showed a similar damaged area at 24 h after the insult, but the final necrotic area was reduced by 66%. Coadministration of either SR1 or SR2 reversed the effects of WIN. In conclusion, likely by activating CB1 and CB2 receptors, WIN afforded robust neuroprotection in newborn rats after HI.

Neuroprotection by the cannabinoid agonist WIN-55212 in an in vivo newborn rat model of acute severe asphyxia.

This study was designed to evaluate the neuroprotective effect of the cannabinoid agonist WIN-55212 after inducing acute severe asphyxia in newborn rats. The left common carotid artery was ligated in anaesthetised 7-day-old Wistar rats, which were then asphyxiated by inhaling 100% nitrogen for 10 min. Pups recovering from asphyxia were s.c. administered vehicle (n=23), WIN-55212 (0.1 mg/kg, n=18), or WIN-55212 plus the CB1 receptor antagonist SR141716 (3 mg/kg, n=10). Pups undergoing a sham operation served as controls (n=12). Coronal sections of the brain were obtained on the 14th day after surgery and observed under light microscope after Nissl or Fluoro-Jade B (FJB) staining, to respectively quantify surviving or degenerating neurones in the CA1 area of the hippocampus and parietal cortex. Acute asphyxia led to early neurone loss amounting to 19% in the hippocampus and 29% in the cortex (both ANOVA P<0.05 vs. control). Delayed neurone loss occurred in the proportions 13% in the hippocampus and 20% in the cortex (both ANOVA P<0.05 vs. control). Neuronal loss was fully prevented by WIN-55212 administration. Co-administration of SR141716 failed to modify the protective effect of WIN-55212 on early neuronal death, but abolished the WIN-55212-induced prevention of delayed neuronal death. We conclude that when administered after acute severe asphyxia in newborn rats, WIN-55212 shows a neuroprotective effect, reducing both early and delayed neurone loss. This effect is achieved through two parallel CB1-dependent and -independent mechanisms.

Cannabidiol reduces lipopolysaccharide-induced vascular changes and inflammation in the mouse brain: an intravital microscopy study.

BackgroundThe phytocannabinoid cannabidiol (CBD) exhibits antioxidant and antiinflammatory properties. The present study was designed to explore its effects in a mouse model of sepsis-related encephalitis by intravenous administration of lipopolysaccharide (LPS).MethodsVascular responses of pial vessels were analyzed by intravital microscopy and inflammatory parameters measured by qRT-PCR.ResultsCBD prevented LPS-induced arteriolar and venular vasodilation as well as leukocyte margination. In addition, CBD abolished LPS-induced increases in tumor necrosis factor-alpha and cyclooxygenase-2 expression as measured by quantitative real time PCR. The expression of the inducible-nitric oxide synthase was also reduced by CBD. Finally, preservation of Blood Brain Barrier integrity was also associated to the treatment with CBD.ConclusionsThese data highlight the antiinflammatory and vascular-stabilizing effects of CBD in endotoxic shock and suggest a possible beneficial effect of this natural cannabinoid.

Cannabidiol reduces brain damage and improves functional recovery after acute hypoxia-ischemia in newborn pigs.

Newborn piglets exposed to acute hypoxia-ischemia (HI) received i.v. cannabidiol (HI + CBD) or vehicle (HI + VEH). In HI + VEH, 72 h post-HI brain activity as assessed by amplitude-integrated EEG (aEEG) had only recovered to 42 ± 9% of baseline, near-infrared spectroscopy (NIRS) parameters remained lower than normal, and neurobehavioral performance was abnormal (27.8 ± 2.3 points, normal 36). In the brain, there were fewer normal and more pyknotic neurons, while astrocytes were less numerous and swollen. Cerebrospinal fluid concentration of neuronal-specific enolase (NSE) and S100β protein and brain tissue percentage of TNFα(+) cells were all higher. In contrast, in HI + CBD, aEEG had recovered to 86 ± 5%, NIRS parameters increased, and the neurobehavioral score normalized (34.3 ± 1.4 points). HI induced histological changes, and NSE and S100β concentration and TNFα(+) cell increases were suppressed by CBD. In conclusion, post-HI administration of CBD protects neurons and astrocytes, leading to histological, functional, biochemical, and neurobehavioral improvements.

Cannabinoids in Neurodegenerative Disorders and Stroke/Brain Trauma: From Preclinical Models to Clinical Applications

Cannabinoids form a singular family of plant-derived compounds (phytocannabinoids), endogenous signaling lipids (endocannabinoids), and synthetic derivatives with multiple biological effects and therapeutic applications in the central and peripheral nervous systems. One of these properties is the regulation of neuronal homeostasis and survival, which is the result of the combination of a myriad of effects addressed to preserve, rescue, repair, and/or replace neurons, and also glial cells against multiple insults that may potentially damage these cells. These effects are facilitated by the location of specific targets for the action of these compounds (e.g., cannabinoid type 1 and 2 receptors, endocannabinoid inactivating enzymes, and nonendocannabinoid targets) in key cellular substrates (e.g., neurons, glial cells, and neural progenitor cells). This potential is promising for acute and chronic neurodegenerative pathological conditions. In this review, we will collect all experimental evidence, mainly obtained at the preclinical level, supporting that different cannabinoid compounds may be neuroprotective in adult and neonatal ischemia, brain trauma, Alzheimer’s disease, Parkinson’s disease, Huntington’s chorea, and amyotrophic lateral sclerosis. This increasing experimental evidence demands a prompt clinical validation of cannabinoid-based medicines for the treatment of all these disorders, which, at present, lack efficacious treatments for delaying/arresting disease progression, despite the fact that the few clinical trials conducted so far with these medicines have failed to demonstrate beneficial effects.