Anti-Inflammatory Properties of Cannabidiol and Beta-Caryophyllene Alone or Combined in an In Vitro Inflammation Model.

Cannabis contains over 500 different compounds, including cannabinoids, terpenoids, and flavonoids. Cannabidiol (CBD) is a non-psychoactive constituent, whereas beta-caryophyllene (BCP) is one of most the well-known terpenoids of Cannabis sativa. In recent years, there has been an emerging idea that the beneficial activities of these compounds are greater when they are combined. The aim of this study was to evaluate the anti-inflammatory effect of CBD and BCP using the in vitro model of lipopolysaccharide (LPS)-stimulated human keratinocytes (HaCaT) cells. The vitality of the cells was quantified using LDH and MTT assays. The levels of the following pro-inflammatory proteins and genes were quantified: IL-1ß, COX-2, and phospho-NF-κB p65 (p-p65) through Western blotting (WB) and interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) through quantitative real-time polymerase chain reaction (RT-qPCR). When present in the incubation medium, CBD and BCP reduced the increased levels of pro-inflammatory proteins (IL-1ß, COX-2, and p-NF-kB) induced by LPS. The anti-inflammatory effects of CBD were blocked by a PPARγ antagonist, whereas a CB2 antagonist was able to revert the effects of BCP. Selected concentrations of CBD and BCP were able to revert the increases in the expression of pro-inflammatory genes (IL-1ß, IL-6, and TNFα), and these effects were significant when the drugs were used in combination. Our results suggest that CBD and BCP work in concert to produce a major anti-inflammatory effect with good safety profiles.

Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

In the central nervous system, some specific phosphodiesterase (PDE) isoforms modulate pathways involved in neuronal plasticity. Accumulating evidence suggests that PDE9 may be a promising therapeutic target for neurodegenerative diseases. In the current study, computational techniques were used to identify a nature-inspired PDE9 inhibitor bearing the scaffold of an isoflavone, starting from a database of synthetic small molecules using a ligand-based approach. Furthermore, docking studies supported by molecular dynamics investigations allowed us to evaluate the features of the ligand-target complex. In vitro assays confirmed the computational results, showing that the selected compound inhibits the enzyme in the nanomolar range. Additionally, we evaluated the expression of gene and protein levels of PDE9 in organotypic hippocampal slices, observing an increase following exposure to kainate (KA). Importantly, the PDE9 inhibitor reduced CA3 damage induced by KA in a dose-dependent manner in organotypic hippocampal slices. Taken together, these observations strongly support the potential of the identified nature-inspired PDE9 inhibitor and suggest that such a molecule could represent a promising lead compound to develop novel therapeutic tools against neurological diseases..

Evaluation of Conventional and Hyaluronic Acid-Coated Thymoquinone Liposomes in an In Vitro Model of Dry Eye.

Dry eye disease (DED) is a common ocular disorder characterized by an inadequate lubrication of the eye by tears leading to inflammation and the alteration of the ocular surface. Current treatments are often limited due to their side effects and ineffectiveness. Thymoquinone (TQ) is a natural compound present in the essential oil of Nigella sativa L., with anti-inflammatory and antioxidant activities. In this study, conventional and hyaluronic acid-coated liposomes were developed to improve TQ activity at ocular level. In the present study, the cytoprotective effects of TQ or TQ liposomes were assessed against oxidative and inflammatory processes in human corneal epithelial cells (HCE-2). Hyperosmolarity conditions (450 mOsm) were used as a model of DED. Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6) and tumor necrosis factor (TNFα) were quantified by quantitative real-time polymerase chain reaction (RT-qPCR); COX-2 and Phospho-NF-κB p65 (p-p65) by Western blotting (WB). Moreover, the mitochondrial reactive oxygen species (mtROS) levels were measured by MitoSOX assay. The hyperosmotic treatment induced a significant increase of the proinflammatory genes and proteins expression that were significantly decreased in the liposomes-treated cells. The coincubation with hyaluronic acid-coated liposomes significantly reverted the increase of mtROS production, evidently stimulated by the hyperosmotic stress. Our data suggest that TQ-loaded liposomes have potential as a therapeutic agent in dry eye disease, improving the TQ efficacy.

Virtual screening and in vitro experiments highlight cannabidiol as a drug-like phosphodiesterase 9 inhibitor.

The growing interest on the therapeutic potential against neurodegeneration of Cannabis sativa extracts, and of phytocannabinoids in particular, is paralleled by a limited understanding of the undergoing biochemical pathways in which these natural compounds may be involved. Computational tools are nowadays commonly enrolled in the drug discovery workflow and can guide the investigation of macromolecular targets for such molecules. In this contribution, in silico techniques have been applied to the study of C. sativa constituents at various extents, and a total of seven phytocannabinoids and four terpenes were considered. On the side of ligand-based virtual screening, physico-chemical descriptors were computed and evaluated, highlighting the phytocannabinoids possessing suitable drug-like properties to potentially target the central nervous system. Our previous findings and literature data prompted us to investigate the interaction of these molecules with phosphodiesterases (PDEs), a family of enzymes being studied for the development of therapeutic agents against neurodegeneration. Among the compounds, structure-based techniques such as docking and molecular dynamics (MD), highlighted cannabidiol (CBD) as a potential and selective PDE9 ligand, since a promising calculated binding energy value (-9.1 kcal/mol) and a stable interaction in the MD simulation timeframe were predicted. Additionally, PDE9 inhibition assay confirmed the computational results, and showed that CBD inhibits the enzyme in the nanomolar range in vitro, paving the way for further development of this phytocannabinoid as a therapeutic option against neurodegeneration.

Antidepressant-induced increase in GluA2 expression does not translate in changes of AMPA receptor-mediated synaptic transmission at CA3/CA1 synapses in rats.

Chronic treatment with serotonin selective reuptake inhibitors or tryciclic antidepressant drugs in rodents has been shown to increase the expression of GluA1 and/or GluA2 AMPA receptor (AMPAR) subunits in several brain areas, including the hippocampus. These changes in AMPAR composition have been suggested to result in increased glutamatergic neurotransmission and possibly underlie enhanced hippocampal synaptic plasticity through the increased availability of calcium-permeable AMPARs, specifically at CA3/CA1 synapses. However, the possibility that chronic treatment with antidepressants actually results in strengthened glutamatergic neurotransmission in CA1 has poorly been investigated. Here, we studied whether chronic treatment with the multimodal antidepressant drug trazodone mimicked the effect of paroxetine on the expression of AMPAR subunits in male wistar rat hippocampus and whether these drugs produced a parallel facilitation of field excitatory postsynaptic potentials (fEPSP) responses evoked by activation of CA3/CA1 synapses in dorsal hippocampal slices. In addition, we investigated whether the quality of glutamatergic AMPARs involved in basal neurotransmission was changed by altered subunit expression, e.g. leading to appearance of calcium-permeable AMPARs. We found a significant increase in GluA2 subunit expression following treatment with trazodone or paroxetine for twenty-one days, but not after seven-days treatment. In contrast, we did not find any significant changes in fEPSP responses supporting either a facilitation of glutamatergic neurotransmission in basal conditions or the appearance of functional calcium-permeable AMPARs at CA3/CA1 pyramidal neuron synapses. Thus, neurochemically-detected increases in the expression of AMPAR subunits cannot directly be extrapolated in increased number of functioning receptors and/or facilitated basal neurotransmission.

The Neuroprotective Effects of mGlu1 Receptor Antagonists Are Mediated by an Enhancement of GABAergic Synaptic Transmission via a Presynaptic CB1 Receptor Mechanism.

In this study, we investigated the cross-talk between mGlu1 and CB1 receptors in modulating GABA hippocampal output in whole-cell voltage clamp recordings in rat hippocampal acute slices, in organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD) and in gerbils subjected to global ischemia. CB1 receptor expression was studied using immunohistochemistry and the CA1 contents of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by LC-MS/MS. Our results show that mGlu1 receptor antagonists enhance sIPSCs in CA1 pyramidal cells and the basal and ischemic hippocampal release of GABA in vivo in a manner that is mediated by CB1 receptor activation. In hippocampal slices exposed to OGD and in ischemic gerbils, mGlu1 receptor antagonists protected CA1 pyramidal cells against post-ischemic injury and this effect was reduced by CB1 receptor activation. OGD induced a transient increase in the hippocampal content of AEA and this effect is prevented by mGlu1 receptor antagonist. Finally, OGD induced a late disruption of CB1 receptors in the CA1 region and the effect was prevented when CA1 pyramidal cells were protected by mGlu1 antagonists. Altogether, these results suggest a cooperative interaction between mGlu1 receptors and the endocannabinoid system in the mechanisms that lead to post-ischemic neuronal death.

Cannabidiol inhibits microglia activation and mitigates neuronal damage induced by kainate in an in-vitro seizure model.

BACKGROUND: Epilepsy is one of the most common brain disorder and, despite the possible use of several therapeutic options, many patients continue to have seizures for their entire lifespan and they need new therapeutic approaches. In the last years the interest on the non-psychoactive compounds present in Cannabis sativa has massively increased, and cannabidiol (CBD) has been shown to be effective in the treatment of different types of neurological disorders and neurodegenerative diseases such as epilepsy, ischemia, multiple sclerosis and Alzheimer's Disease. METHODS: We investigated the effects of the selected cannabinoids, Δ9-tetrahydrocannabinol (THC), CBD and cannabigerol (CBG) in rat organotypic hippocampal slices exposed to kainate, an in vitro seizure model. Cell death in the cornu Ammonis 3 (CA3) hippocampal subregion was quantified by propidium iodide fluorescence. Morphological analysis and tissue organization were examined by immunohistochemistry and confocal microscopy and microglia activation and polarization was evaluated using flow cytometry and morphology analysis. RESULTS: When present in the incubation medium, cannabidiol reduced dose-dependent CA3 injury induced by kainate. Conversely, incubation with THC exacerbated hippocampal damage. The neuroprotective effects of cannabidiol were blocked by TRPV1, TRPV2, 5-HT1A, and PPARγ antagonists. Confocal microscopy confirmed that CBD but not THC had a significant protective effect against neuronal damage and tissue disorganization caused by kainate. Cannabidiol incubation significantly block the microglia activation from the M0 to M1 phenotype observed in the kainate in-vitro seizure model, pushing toward a transition from M0 to M2. CONCLUSIONS: Our results suggest that CBD mitigated neuronal damage induced by kainate and blocked the transition from the M0 to the M1 phenotype.

Experimental Models for Testing the Efficacy of Pharmacological Treatments for Neonatal Hypoxic-Ischemic Encephalopathy.

Representing an important cause of long-term disability, term neonatal hypoxic-ischemic encephalopathy (HIE) urgently needs further research aimed at repurposing existing drug as well as developing new therapeutics. Since various experimental in vitro and in vivo models of HIE have been developed with distinct characteristics, it becomes important to select the appropriate preclinical screening cascade for testing the efficacy of novel pharmacological treatments. As therapeutic hypothermia is already a routine therapy for neonatal encephalopathy, it is essential that hypothermia be administered to the experimental model selected to allow translational testing of novel or repurposed drugs on top of the standard of care. Moreover, a translational approach requires that therapeutic interventions must be initiated after the induction of the insult, and the time window for intervention should be evaluated to translate to real world clinical practice. Hippocampal organotypic slice cultures, in particular, are an invaluable intermediate between simpler cell lines and in vivo models, as they largely maintain structural complexity of the original tissue and can be subjected to transient oxygen-glucose deprivation (OGD) and subsequent reoxygenation to simulate ischemic neuronal injury and reperfusion. Progressing to in vivo models, generally, rodent (mouse and rat) models could offer more flexibility and be more cost-effective for testing the efficacy of pharmacological agents with a dose-response approach. Large animal models, including piglets, sheep, and non-human primates, may be utilized as a third step for more focused and accurate translational studies, including also pharmacokinetic and safety pharmacology assessments. Thus, a preclinical proof of concept of efficacy of an emerging pharmacological treatment should be obtained firstly in vitro, including organotypic models, and, subsequently, in at least two different animal models, also in combination with hypothermia, before initiating clinical trials.

Neuronal and Astrocytic Morphological Alterations Driven by Prolonged Exposure with Δ9-Tetrahydrocannabinol but Not Cannabidiol.

Cannabis derivatives are largely used in the general population for recreational and medical purposes, with the highest prevalence among adolescents, but chronic use and abuse has raised medical concerns. We investigated the prolonged effects of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in organotypic hippocampal slices from P7 rats cultured for 2 weeks. Cell death in the CA1 subregion of slices was quantified by propidium iodide (PI) fluorescence, pre-synaptic and post-synaptic marker proteins were analysed by Western blotting and neurodegeneration and astrocytic alterations by NeuN and GFAP by immunofluorescence and confocal laser microscopy. The statistical significance of differences was analysed using ANOVA with a post hoc Dunnett w-test (PI fluorescence intensities and Western blots) or Newman-Keuls (immunohistochemistry data) for multiple comparisons. A probability value (P) of < 0.05 was considered significant. Prolonged (72 h) THC or CBD incubation did not induce cell death but caused modifications in the expression of synaptic proteins and morphological alterations in neurons and astrocytes. In particular, the expression of PSD95 was reduced following incubation for 72 h with THC and was increased following incubation with CBD. THC for 72 h caused disorganisation of CA1 stratum pyramidalis (SP) and complex morphological modifications in a significant number of pyramidal neurons and in astrocytes. Our results suggest that THC or CBD prolonged exposure induce different effects in the hippocampus. In particular, 72 h of THC exposure induced neuronal and glia alterations that must draw our attention to the effects that relatively prolonged use might cause, especially in adolescents.

Neuroprotective Effects of Cannabidiol but Not Δ9-Tetrahydrocannabinol in Rat Hippocampal Slices Exposed to Oxygen-Glucose Deprivation: Studies with Cannabis Extracts and Selected Cannabinoids.

(1) Background: Over the past 10 years, a number of scientific studies have demonstrated the therapeutic potential of cannabinoid compounds present in the Cannabis Sativa and Indica plants. However, their role in mechanisms leading to neurodegeneration following cerebral ischemia is yet unclear. (2) Methods: We investigated the effects of Cannabis extracts (Bedrocan, FM2) or selected cannabinoids (Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol) in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD), an in vitro model of forebrain global ischemia. Cell death in the CA1 subregion of slices was quantified by propidium iodide fluorescence, and morphological analysis and tissue organization were examined by immunohistochemistry and confocal microscopy. (3) Results: Incubation with the Bedrocan extract or THC exacerbated, whereas incubation with the FM2 extract or cannabidiol attenuated CA1 injury induced by OGD. Δ9-THC toxicity was prevented by CB1 receptor antagonists, the neuroprotective effect of cannabidiol was blocked by TRPV2, 5-HT1A, and PPARγ antagonists. Confocal microscopy confirmed that CBD, but not THC, had a significant protective effect toward neuronal damage and tissue disorganization caused by OGD in organotypic hippocampal slices. (4) Conclusions: Our results suggest that cannabinoids play different roles in the mechanisms of post-ischemic neuronal death. In particular, appropriate concentrations of CBD or CBD/THC ratios may represent a valid therapeutic intervention in the treatment of post-ischemic neuronal death.

Changes in physical activity levels, eating habits and psychological well-being during the Italian COVID-19 pandemic lockdown: Impact of socio-demographic factors on the Florentine academic population.

The confinement and lockdown imposed by the COVID-19 pandemic have produced restrictions in the lifestyle of Italian citizens with variations in their psychological well-being. The aim of the study was to identify changes and relationship with socio-demographic parameters. An online survey was administered to 1383 subjects (1007 females and 307 males) working in the University of Florence, Italy. Three validated questionnaires were used for the survey: the Global Physical Activity Questionnaire, the Med Diet Score and the Psychological General Well-Being Index-A. All the subjects were asked to complete the questionnaires twice, in order to attain a picture of the habits before and a later time point during confinement. Our results show that work-related physical activity was decreased, along with an increase in sedentary behaviour (from 07:22±03:20 to 08:49±03:41 h:min; p<0.001, ES = 0.38), whereas recreational physical activity was increased (vigorous exercise varied from 568.5 ± 838.6 to 833.7 ± 1263.0 METs; p<0.002, ES = 0.25). Eating habits changed according to the place where meals were eaten, with an increased habit for breakfast and snacks and a slight increase in alcohol consumption. Psychological well-being decreased (Index from 21.4±3.9 to 18.0±5.3; p<0.001, ES = 0.723), especially in terms of vitality and positive thinking. The socio-demographic variables affecting these variations were mostly represented by age, gender and working conditions: young age and self-employment conditions can be considered factors for the changes in daily habits induced by confinement that may affect psychological well-being.

Neuroprotective Effects of Thymoquinone by the Modulation of ER Stress and Apoptotic Pathway in In Vitro Model of Excitotoxicity.

Experimental evidence indicates that the activation of ionotropic glutamate receptors plays an important role in neurological disorders' models such as epilepsy, cerebral ischemia and trauma. The glutamate receptor agonist kainic acid (KA) induces seizures and excitotoxic cell death in the CA3 region of the hippocampus. Thymoquinone (TQ) is the most important component of the essential oil obtained from black cumin (Nigella sativa L.) seeds. It has many pharmacological actions including antioxidant, anti-inflammatory, and anti-apoptotic effects. TQ was used in an in vitro experimental model of primary cultures where excitotoxicity was induced. Briefly, rat organotypic hippocampal slices were exposed to 5 µM KA for 24 h. Cell death in the CA3 subregions of slices was quantified by measuring propidium iodide fluorescence. The cross-talk between TQ, ER stress and apoptotic pathways was investigated by Western blot. In untreated slices TQ (10 µM) induced a significant increase on the PSD95 levels and it decreased the excitotoxic injury induced by KA. Additionally, TQ was able to ameliorate the KA-induced increase in unfolded proteins GRP78 and GRP94 expression. Finally, TQ was able to partially rescue the reduction of the KA-induced apoptotic pathway activation. Our results suggest that TQ modulates the processes leading to post-kainate neuronal death in the CA3 hippocampal area.

Ethanol neurotoxicity is mediated by changes in expression, surface localization and functional properties of glutamate AMPA receptors.

Modifications in the subunit composition of AMPA receptors (AMPARs) have been linked to the transition from physiological to pathological conditions in a number of contexts, including EtOH-induced neurotoxicity. Previous work from our laboratory showed that EtOH withdrawal causes CA1 pyramidal cell death in organotypic hippocampal slices and changes in the expression of AMPARs. Here, we investigated whether changes in expression and function of AMPARs may be causal for EtOH-induced neurotoxicity. To this aim, we examined the subunit composition, localization and function of AMPARs in hippocampal slices exposed to EtOH by using western blotting, surface expression assay, confocal microscopy and electrophysiology. We found that EtOH withdrawal specifically increases GluA1 protein signal in total homogenates, but not in the post-synaptic density-enriched fraction. This is suggestive of overall increase and redistribution of AMPARs to the extrasynaptic compartment. At functional level, AMPA-induced calcium influx was unexpectedly reduced, whereas AMPA-induced current was enhanced in CA1 pyramidal neurons following EtOH withdrawal, suggesting that increased AMPAR expression may lead to cell death because of elevated excitability, and not for a direct contribution on calcium influx. Finally, the neurotoxicity caused by EtOH withdrawal was attenuated by the non-selective AMPAR antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt as well as by the selective antagonist of GluA2-lacking AMPARs 1-naphthyl acetyl spermine. We conclude that EtOH neurotoxicity involves changes in expression, surface localization and functional properties of AMPARs, and propose GluA2-lacking AMPARs as amenable specific targets for the development of neuroprotective drugs in EtOH-withdrawal syndrome.

A new ophthalmic formulation containing antiseptics and dexpanthenol: In vitro antimicrobial activity and effects on corneal and conjunctival epithelial cells.

Antibiotic resistance is increasing even in ocular pathogens, therefore the interest towards antiseptics in Ophthalmology is growing. The aim of this study was to analyze the in vitro antimicrobial efficacy and the in vitro effects of an ophthalmic formulation containing hexamidine diisethionate 0.05%, polyhexamethylene biguanide (PHMB) 0.0001% disodium edetate (EDTA) 0.01%, dexpanthenol 5% and polyvinyl alcohol 1.25% (Keratosept, Bruschettini, Genova, Italy) on cultured human corneal and conjunctival cells. The in vitro antimicrobial activity was tested on Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus mitis. For each microbial strain 10 µL of a 0.5 MacFarland standardized bacterial inoculum were incubated at 25 °C with 100 µL of ophthalmic solution for up to 6 h. After different periods of time, samples were inoculated on blood agar with 5% sheep blood. Moreover, a 0.5 MacFarland bacterial inoculum was seeded in triplicate on Mueller-Hinton Agar or on Mueller-Hinton Fastidious Agar; then a cellulose disc soaked with 50 µL of ophthalmic solution was applied on the surface of agar and plates were incubated for 18 h at 37 °C, in order to evaluate the inhibition of bacterial growth around the disc. Human corneal and conjunctival epithelial cells in vitro were incubated for 5, 10 and 15 min with Keratosept or its components. The cytotoxicity was assessed through the release of cytoplasmic enzyme lactate dehydrogenase (LDH) into the medium immediately after exposure to the drugs; the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the metabolic cell activity. Our results show that Keratosept ophthalmic solution gave an average logarithmic (log) reduction of bacterial load of 2.14 ± 0.35 within 6 h of exposure (p-value < 0.05 versus control saline solution). On agar plates, all microbial strains, excluding P. Aeruginosa, showed an inhibition zone of growth around the Keratosept-soaked discs. Keratosept and its components after 5 and 10 min did not show any cytotoxic effect on cultured corneal and conjunctival cells, and only after 15 min a significant reduction of cell viability and an increase of cytotoxicity compared to control (vehicle) was seen; dexpanthenol 5% and polyvinyl alcohol accelerated the wounding of corneal cells in vitro. In conclusion, Keratosept showed good antimicrobial activity on the tested strains; the ophthalmic solution and its components were safe and non-toxic for the corneal and conjunctival epithelial cells for 5 and 10 min at the concentrations analyzed, and dexpanthenol 5% and polyvinyl alcohol promoted the wounding of corneal cells.

Differential mechanisms of tolerance induced by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning.

We investigated the molecular events triggered by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning, that lead to neuroprotection against excitotoxic insults (AMPA or oxygen and glucose deprivation) in rat organotypic hippocampal slices, with particular attention on glutamate receptors and on cannabinoid system. We firstly evaluated the protein expression of NMDA and AMPA receptor subunits after preconditioning using western blot analysis performed in post-synaptic densities. We observed that following NMDA, but not DHPG preconditioning, the expression of GluA1 was significantly reduced and this reduction appeared to be associated with the internalization of AMPA receptors. Whole-cell voltage clamp recordings on CA1 pyramidal neurons of organotypic slices show that 24 hr after exposure to NMDA and DHPG preconditioning, AMPA-induced currents were significantly reduced. To clarify the mechanisms induced by DHPG preconditioning, we then investigated the involvement of the endocannabinoid system. Exposure of slices to the CB1 antagonist AM251 prevented the development of tolerance to AMPA toxicity induced by DHPG but not NMDA. Accordingly, the MAG-lipase inhibitor URB602, that increases arachidonoylglycerol (2-AG) content, but not the FAAH inhibitor URB597, that limits the degradation of anandamide, was also able to induce tolerance versus AMPA and OGD toxicity, suggesting that 2-AG is responsible for the DHPG-induced tolerance. In conclusion, preconditioning with NMDA or DHPG promotes differential neuroprotective mechanisms: NMDA by internalization of GluA1-AMPA receptors, DHPG by producing the endocannabinoid 2-AG.

Enhanced dissolution, permeation and oral bioavailability of aripiprazole mixed micelles: In vitro and in vivo evaluation.

Aripiprazole (ARP) is an antipsychotic drug approved for the treatment of schizophrenia. It is poorly water-soluble and undergoes extensive hepatic metabolism and P-gp efflux, which lead to poor bioavailability and increased dose-related side effects. This study focuses on the preparation of mixed micelles (MM) to enhance the aqueous solubility, oral bioavailability, and blood-brain barrier permeation of ARP. For this purpose, Soluplus and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) were selected for micelle preparation (ARP-MM). Micelles with borneol as penetration enhancer were also considered (ARP-B-MM). The optimized formulations have sizes of ca 50 nm, defined in distilled water, narrow size distribution (polydispersity index ≤0.1), and high encapsulation efficiency (greater than98%). Both formulations can be freeze-dried without losing their chemical-physical characteristics and are stable during storage for three months. The mixed micelles resulted stable in enzyme free-simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 6.8), and in serum. The in vitro ARP release was evaluated in the same biorelevant media, (SGF and SIF), and it disclosed that both micelles can give prolonged drug release. Furthermore, ARP solubility is greatly increased when loaded into mixed micelles. The absorption and efflux of ARP-loaded micelles were studied in vitro, employing two artificial membranes (Parallel Artificial Membrane Permeability Assay for the intestinal, PAMPA-GI, and the blood-brain barrier, PAMPA-BBB), to simulate the intestinal and brain epithelium, and the brain microvascular endothelial cell line hCMEC/D3. ARP-MM and ARP-B-MM increase the effective permeability of ARP by a factor of about three in the case of PAMPA-GI and about two for PAMPA-BBB. Furthermore, the P-gp mediated efflux was decreased by about six times in the case of ARP-MM and by about four times in the case of ARP-B-MM, compared to unformulated ARP. Finally, both ARP-loaded mixed micelles ameliorate the bioavailability of ARP, as demonstrated by the increase of the pharmacokinetic parameters, such as Cmax, AUC0-24h, and t1/2.

Tolerance Induced by (S)-3,5-Dihydroxyphenylglycine Postconditioning is Mediated by the PI3K/Akt/GSK3ß Signalling Pathway in an In Vitro Model of Cerebral Ischemia.

Ischemic postconditioning (PostC) is an endogenous neuroprotective strategy for cerebral ischemia induced by low activation of glutamate receptors. We have previously shown that the application of the mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) 5 min after 30 min of oxygen and glucose deprivation (OGD) reduces CA1 damage in organotypic hippocampal slices by activating the PI3K-Akt signalling pathway. In order to extend these data, we analysed the production of reactive oxygen species (ROS) and the glycogen synthase kinase 3ß (GSK3ß) signalling pathway. Our results show that DHPG PostC was associated with a reduction in the formation of ROS that is massively increased 24 h after OGD exposure. This reduction was prevented by the PI3K inhibitor LY294002, indicating that there is a link between the PI3K/Akt pathway and the formation of ROS in the protective mechanisms of PostC. DHPG PostC also induces a transient increased in GSK3ß phosphorylation and inactivation that is followed by nuclear accumulation of ß-catenin, that probably lead to the up-regulation of neuroprotective genes. Our results propose GSK3ß as new target for neuroprotection, therefore, we verified that the two GSK3ß inhibitors N-(3-Chloro-4-methylphenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazol-2-amine (TC-G 24) and LiCl are neuroprotective agents in OGD and also can be used as PostC agents.

Neuroprotective effects of mGluR5 activation through the PI3K/Akt pathway and the molecular switch of AMPA receptors.

Previous studies have demonstrated that antagonists of mGluR1, but not mGluR5, are neuroprotective in models of cerebral ischemia. To investigate the individual roles of mGlu1 and mGlu5 receptors in in vitro model of cerebral ischemia we used low doses of the non-selective group I agonist DHPG and mGlu1 and mGlu5 selective positive allosteric modulators (PAMs). In hippocampal slices subjected to 30 min oxygen-glucose deprivation (OGD), DHPG (1 µM) and the mGluR5 PAM (VU0092273) significantly reduced OGD-induced CA1 injury monitored by propidium iodide staining of the slices and quantitative analysis of CA1 neurons. In contrast, the mGluR1 PAM (VU0483605) showed no neuroprotection. These protective effects of DHPG and VU0092273 were prevented by inhibition of PI3K/Akt pathway by LY294002. The mGluR5 PAM (VU0092273) also prevented GluA2 down-regulation triggered by ischemic injury, via PI3K/Akt pathway, revealing a further contribution to its neuroprotective effects by reducing the excitotoxic effects of increased Ca2+ influx through GluA2-lacking AMPA receptors. Furthermore, immunohistochemical assays confirmed the neuroprotective effect of VU0092273 and revealed activation of glia, indicating the involvement reactive astrogliosis in the mechanisms of neuroprotection. Our data suggest that selective activation/potentiation of mGluR5 signalling represents a promising strategy for the development of new interventions to reduce or prevent ischemia-induced neuronal death.

Niosomal Formulation of a Lipoyl-Carnosine Derivative Targeting TRPA1 Channels in Brain.

The transient receptor potential akyrin type-1 (TRPA1) is a non-selective cation channel playing a pivotal role in pain sensation and neurogenic inflammation. TRPA1 channels expressed in the central nervous system (CNS) have a critical role in the modulation of cortical spreading depression (CSD), which is a key pathophysiological basis of migraine pain. ADM_09 is a recently developed lipoic acid-based TRPA1 antagonist that is able to revert oxaliplatin-induced neuropathic pain and inflammatory trigeminal allodynia. In this context, aiming at developing drugs that are able to target TRPA1 channels in the CNS and promote an antioxidant effect, permeability across the blood-brain barrier (BBB) represents a central issue. Niosomes are nanovesicles that can be functionalized with specific ligands selectively recognized by transporters expressed on the BBB. In this work, the activity of ADM_09 on neocortex cultures was studied, and an efficient formulation to cross the BBB was developed with the aim of increasing the concentration of ADM_09 into the brain and selectively delivering it to the CNS rapidly after parenteral administration.