IMPACT OF DIFFERENT FORMULATIONS OF PHARMACEUTICAL CANNABIS-BASED EXTRACTS ON THE NEUROPROTECTIVE EFFECT IN CEREBELLAR GRANULE CELL CULTURES

Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e. g., epilepsy); however, their neuroprotective potential has not been widely investigated. In addition, there is still controversy about the impact of other factors in the beneficial effect of these extracts (e.g., the entourage effect, and oil formulations). We evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, and trace levels of Δ9- tetrahydrocannabinol and the acid form of CBD. Using primary cultures of cerebellar granule cells, we determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). The results revealed that EPI induced a significant reduction in the rotenone-induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se. EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions (i.e., entourage effect) between individual substances present in EPI occurred. In contrast, CBD crystals did show a different profile to EPI and XAL since a neurotoxic effect per se was observed at the higher concentrations assayed. Medium-chain triglyceride oil used in EPI formulation could explain this difference. Our data support a neuroprotective effect of EPI which may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based productAgencia Nacional de Investigación e Innovació

Impact of different formulations of pharmaceutical cannabis-based extracts on the neuroprotective effect in cerebellar granule cell cultures

Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e. g., epilepsy); however, their neuroprotective potential has not been widely investigated. In addition, there is still controversy about the impact of other factors in the beneficial effect of these extracts (e.g., the entourage effect, and oil formulations). We evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, and trace levels of Δ9-tetrahydrocannabinol and the acid form of CBD. Using primary cultures of cerebellar granule cells, we determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). The results revealed that EPI induced a significant reduction in the rotenone-induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se. EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions (i.e., entourage effect) between individual substances present in EPI occurred. In contrast, CBD crystals did show a different profile to EPI and XAL since a neurotoxic effect per se was observed at the higher concentrations assayed. Medium-chain triglyceride oil used in EPI formulation could explain this difference. Our data support a neuroprotective effect of EPI which may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based productAgencia Nacional de Investigación e Innovació

Uso medicinal de cannabinoides: su acción como agente neuroprotector y su biodisponibilidad cerebral

A pesar de los avances en el conocimiento sobre los cambios fisiopatológicos y moleculares de las diferentes enfermedades neurodegenerativas no existe cura para ninguna de ellas. Las terapias existentes, tanto farmacológicas como quirúrgicas, están destinadas a tratar la sintomatología de estasenfermedades y, en general, poseen importantes efectos adversos. Esto hace imprescindible la necesidad de trabajar en el desarrollo de nuevos tratamientos eficaces. Dada la naturaleza multifactorial de las enfermedades neurodegenerativas, se cree que una combinación de moléculas capaces de actuar simultáneamente sobre varios blancos celulares (acción polifarmacológica) va a tener un mayor potencial neuroprotector en comparación con aquellos fármacos con un solo mecanismo de acción. El proyecto de investigación propone evaluar el potencial neuroprotector de combinaciones de cannabinoides, en diferentes proporciones, usando modelos celulares. Además, busca estudiar algunos mecanismos de acción (dentro y fuera del sistema endocannabinoide) que podrían subyacer a la neuroprotección. Estos estudios permitirán avanzar en la caracterización de los cannabinoides y sus combinaciones para su eventual aplicación terapéutica para enfermedades neurodegenerativasAgencia Nacional de Investigación e Innovació

Neuroprotective effect of a pharmaceutical extract of cannabis with high content on CBD against rotenone in primary cerebellar granule cell cultures and the relevance of formulations

Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e.g., epilepsy); however, their neuroprotective potential has not been widely investigated. Using primary cultures of cerebellar granule cells, we evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, and trace levels of Δ9-tetrahydrocannabinol and the acid form of CBD. We determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). The results revealed that EPI induced a significant reduction in the rotenone induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se. EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions between individual substances present in EPI occurred. In contrast, CBD did show a different profile to EPI and XAL since a neurotoxic effect per se was observed at the higher concentrations assayed. Medium-chain triglyceride (MCT) oil used in EPI formulation could explain this difference. Our data support a neuroprotective effect of EPI which may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based products, which could be critical to avoid neurotoxicity at very high doses.Agencia Nacional de Investigación e Innovació

Pathophysiology of subarachnoid hemorrhage in the rat

Subarachnoid hemorrhage (SAH) causes brain damage, but the underlying mechanisms are poorly understood. An obstacle in SAH research is the lack of an adequate animal model. In this thesis, we developed a new approach to simulate SAH that involves the injection of blood into the prechiasmatic cistern of the rat. This model had several advantages when it was compared to the two commonest SAH rat models in the literature: unlike the endovascular perforation of the internal carotid artery model, it was reproducible, easy to perform and the mortality rate was acceptable, while when compared to the injection of blood into the cisterna magna model, the blood distribution and histological, hemodynamic and metabolic changes more closely resembled that found in patients with SAH. We studied various mechanisms that can cause brain damage after SAH. Among the many factors occurring during the first minutes-hours after SAH, the global reduction in cerebral blood flow (CBF), but not the changes in intracranial and perfusion pressure, seemed to be a main determinant of brain injury: the degree of its reduction was correlated with the amount of subarachnoid blood, the acute changes in the expression of the N-methyl-D-aspartate (NMDA) receptor subunits, the severity of subsequent inflammation, and most importantly, with delayed cell death and the mortality rate. Signs of acute metabolic derangements after severe SAH were detected by a reduction in cerebral oxygen extraction and altered levels of extracellular glucose, lactate and pyruvate. These changes in metabolism could not always be explained by ischemic episodes. Dying cells, mainly neurons, were seen in various areas of the brain in a large percentage of the surviving animals at 2 and 7 days after SAH. The involvement of apoptotic pathways in the brain damage after SAH was evidenced by the morphological (chromatin condensation and/or apoptotic bodies) and molecular features (upregulation of Bax and active caspase 3) of the damaged cells. Activation of an inflammatory cascade, comprising both parenchymal and vascular tissue, was also detected in the brain by the induction of intercellular adhesion molecule 1, OX6, ED1, tumor necrosis factor á, inducible nitric oxide synthase and nestin. In accordance with the view that inflammation caused brain damage, we found a marked overlapping between areas with dying cells and those with inflammation. Using in situ hybridization, a CBF-dependent early downregulation of the hippocampal NR2A, NR2B and NR3B subunit mRNA levels after SAH was observed. Although these changes may have played a pathogenic role following SAH, their causal relationship to subsequent cell death could not be established. In conclusion, this thesis describes a new and suitable SAH model in the rat. We showed that acute ischemic episodes, early metabolic derangements, a secondary inflammatory reaction and apoptosis are probable mechanisms which contribute to brain damage after SAH. The fact that cells were still dying at least 7 days later indicates that there is a temporal window during which adequate treatment may improve the final outcome after SAH

A Focus on Astrocyte Contribution to Parkinson’s Disease Etiology

Parkinson’s disease (PD) is an incurable neurodegenerative disease of high prevalence, characterized by the prominent death of dopaminergic neurons in the substantia nigra pars compacta, which produces dopamine deficiency, leading to classic motor symptoms. Although PD has traditionally been considered as a neuronal cell autonomous pathology, in which the damage of vulnerable neurons is responsible for the disease, growing evidence strongly suggests that astrocytes might have an active role in the neurodegeneration observed. In the present review, we discuss several studies evidencing astrocyte implications in PD, highlighting the consequences of both the loss of normal homeostatic functions and the gain in toxic functions for the wellbeing of dopaminergic neurons. The revised information provides significant evidence that allows astrocytes to be positioned as crucial players in PD etiology, a factor that needs to be taken into account when considering therapeutic targets for the treatment of the disease

Evaluación de la capacidad antioxidante de las variedades de Cannabis Alfa y Beta, comercializadas en farmacias del Uruguay

La ley de regulación del mercado de Cannabis en el Uruguay permitió el acceso a la población de dos variantes de Cannabis, alfa y beta, la primera con predominancia de variedad “índica” y la segunda de “sativa”. Escasos estudios se han realizado hasta ahora sobre los efectos producidos por dichas variedades. Reportes de la literatura muestran que varias de las acciones beneficiosas de extractos de Cannabis se deben a su capacidad antioxidante (CA), la cual podría estar relacionada con el contenido en fenoles totales, que varía según la variedad y el método de extracción. En este trabajo evaluamos la CA de las variedades alfa y beta luego de dos métodos de extracción: 1) EtOH 95% y 3 min de macerado y, 2) EtOH 70% y 10 min de sonicado. Además, evaluamos la CA luego de la descarboxilación por calor de estos extractos, y evaluamos el contenido de fenoles totales, CBD y CBDA. Resultados primarios muestran que el segundo método de extracción fue más eficaz en la extracción de fenoles totales, CBD y CBDA en ambas variedades y, la CA de la variedad beta fue mayor que la alfa. La descarboxilación de los extractos redujo el contenido de fenoles, disminuyó CBDA y aumentó de CBD, y no afectó la CA. En su conjunto, los resultados sugieren que la CA de las variedades no depende únicamente del contenido de fenoles totales, sino también de la forma química en que los compuestos se encuentren, principalmente los cannabinoides (por ejemplo, CBD).Agencia Nacional de Investigación e Innovació

Evaluación de la capacidad antioxidante de las variedades de Cannabis Alfa y Beta, comercializadas en farmacias del Uruguay

La ley de regulación del mercado de Cannabis en el Uruguay permitió el acceso a la población de dos variantes de Cannabis, alfa y beta, la primera con predominancia de variedad “índica” y la segunda de “sativa”. Escasos estudios se han realizado hasta ahora sobre los efectos producidos por dichas variedades. Reportes de la literatura muestran que varias de las acciones beneficiosas de extractos de Cannabis se deben a su capacidad antioxidante (CA), la cual podría estar relacionada con el contenido en fenoles totales, que varía según la variedad y el método de extracción. En este trabajo evaluamos la CA de las variedades alfa y beta luego de dos métodos de extracción: 1) EtOH 95% y 3 min de macerado y, 2) EtOH 70% y 10 min de sonicado. Además, evaluamos la CA luego de la descarboxilación por calor de estos extractos, y evaluamos el contenido de fenoles totales, CBD y CBDA. Resultados primarios muestran que el segundo método de extracción fue más eficaz en la extracción de fenoles totales, CBD y CBDA en ambas variedades y, la CA de la variedad beta fue mayor que la alfa. La descarboxilación de los extractos redujo el contenido de fenoles, disminuyó CBDA y aumentó de CBD, y no afectó la CA. En su conjunto, los resultados sugieren que la CA de las variedades no depende únicamente del contenido de fenoles totales, sino también de la forma química en que los compuestos se encuentren, principalmente los cannabinoides (por ejemplo, CBD).Agencia Nacional de Investigación e Innovació