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.

Phenomic Microglia Diversity as a Druggable Target in the Hippocampus in Neurodegenerative Diseases.

Phenomics, the complexity of microglia phenotypes and their related functions compels the continuous study of microglia in disease animal models to find druggable targets for neurodegenerative disorders. Activation of microglia was long considered detrimental for neuron survival, but more recently it has become apparent that the real scenario of microglia morphofunctional diversity is far more complex. In this review, we discuss the recent literature on the alterations in microglia phenomics in the hippocampus of animal models of normal brain aging, acute neuroinflammation, ischemia, and neurodegenerative disorders, such as AD. Microglia undergo phenomic changes consisting of transcriptional, functional, and morphological changes that transform them into cells with different properties and functions. The classical subdivision of microglia into M1 and M2, two different, all-or-nothing states is too simplistic, and does not correspond to the variety of phenotypes recently discovered in the brain. We will discuss the phenomic modifications of microglia focusing not only on the differences in microglia reactivity in the diverse models of neurodegenerative disorders, but also among different areas of the brain. For instance, in contiguous and highly interconnected regions of the rat hippocampus, microglia show a differential, finely regulated, and region-specific reactivity, demonstrating that microglia responses are not uniform, but vary significantly from area to area in response to insults. It is of great interest to verify whether the differences in microglia reactivity may explain the differential susceptibility of different brain areas to insults, and particularly the higher sensitivity of CA1 pyramidal neurons to inflammatory stimuli. Understanding the spatiotemporal heterogeneity of microglia phenomics in health and disease is of paramount importance to find new druggable targets for the development of novel microglia-targeted therapies in different CNS disorders. This will allow interventions in three different ways: (i) by suppressing the pro-inflammatory properties of microglia to limit the deleterious effect of their activation; (ii) by modulating microglia phenotypic change to favor anti-inflammatory properties; (iii) by influencing microglia priming early in the disease process.

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.

The Protective Effect of CBD in a Model of In Vitro Ischemia May Be Mediated by Agonism on TRPV2 Channel and Microglia Activation.

Cannabinoids, used for centuries for recreational and medical purposes, have potential therapeutic value in stroke treatment. Cannabidiol (CBD), a non-psychoactive compound and partial agonist of TRPV2 channels, is efficacious in many neurological disorders. We investigated the effects of CBD or Δ9-tetrahydrocannabinol (THC) in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Neuronal TRPV2 expression decreased after OGD, but it increased in activated, phagocytic microglia. CBD increased TRPV2 expression, decreased microglia phagocytosis, and increased rod microglia after OGD. THC had effects contrary to those of CBD. Our results show that cannabinoids have different effects in ischemia. CBD showed neuroprotective effects, mediated, at least in part, by TRPV2 channels, since the TRPV2 antagonist tranilast blocked them, while THC worsened the neurodegeneration caused by ischemia. In conclusion, our results suggest that different cannabinoid molecules play different roles in the mechanisms of post-ischemic neuronal death. These different effects of cannabinoid observed in our experiments caution against the indiscriminate use of cannabis or cannabinoid preparations for recreational or therapeutic use. It was observed that the positive effects of CBD may be counteracted by the negative effects caused by high levels of THC.

Co-expression of the SARS-CoV-2 entry receptors ACE2 and TMPRSS2 in healthy human conjunctiva.

The purpose of this study was to evaluate the expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in an immortalized human conjunctival epithelial cell line and in healthy human conjunctiva excised during ocular surgery, using Western blot, confocal microscopy and immunohistochemistry. The Western blot showed that ACE2 and TMPRSS2 proteins were expressed in human immortalized conjunctival cells, and this was confirmed by confocal microscopy images, that demonstrated a marked cellular expression of the viral receptors and their co-localization on the cell membranes. Healthy conjunctival samples from 11 adult patients were excised during retinal detachment surgery. We found the expression of ACE2 and TMPRSS2 in all the conjunctival surgical specimens analyzed and their co-localization in the superficial conjunctival epithelium. The ACE2 Western blot levels and immunofluorescence staining for ACE2 were variable among specimens. These results suggest the susceptibility of the conjunctival epithelium to SARS-CoV-2 infection, even though with a possible interindividual variability.

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.

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.

Impact of BMI on HER2+ metastatic breast cancer patients treated with pertuzumab and/or trastuzumab emtansine. Real-world evidence.

Body mass index (BMI) is a main indicator of obesity and its association with breast cancer is well established. However, little is known in the metastatic setting, especially in HER2-positive patients. We assessed the influence of BMI on clinical outcomes of patients treated with pertuzumab and/or trastuzumab emtansine (T-DM1) for HER2+ metastatic breast cancer (mBC). BMI was addressed as a categorical variable, being classified on the basis of the following ranges, that is, 18.5-24.9, 25-29.9, and 30.0-34.9, namely, normal weight, overweight, and Class I obesity. The outcomes chosen were progression-free survival to first-line chemotherapy (PFS1) and overall survival (OS). Overall (N = 709), no impact of BMI was observed on PFS1 (p = .15), while BMI ≥ 30 was associated with worse OS (p = .003). In subjects who progressed to first line (N = 575), analyzing data across PFS1 quartiles and strata of disease burden, BMI predicted lower PFS1 in patients within the I PFS1 quartile and with the lowest disease burden (p = .001). Univariate analysis showed a detrimental effect of BMI ≥ 30 on OS for women within the I PFS1 quartile (p = .03). Results were confirmed in multivariate analysis. According to PFS1 quartiles a higher percentage of patients with high BMI and low disease burden progressed within 6 months of therapy. The effect of BMI on prognosis was also confirmed in multivariate analysis of OS for overall population. In our cohort, a BMI ≥ 30 correlated with worse OS in patients with HER2+ mBC who received pertuzumab and/or T-DM1 but had no impact on PFS to first line. BMI predicted worse I PFS1 quartile.

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.

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.

iPSC-derived neurons profiling reveals GABAergic circuit disruption and acetylated α-tubulin defect which improves after iHDAC6 treatment in Rett syndrome.

Mutations in MECP2 gene have been identified in more than 95% of patients with classic Rett syndrome, one of the most common neurodevelopmental disorders in females. Taking advantage of the breakthrough technology of genetic reprogramming, we investigated transcriptome changes in neurons differentiated from induced Pluripotent Stem Cells (iPSCs) derived from patients with different mutations. Profiling by RNA-seq in terminally differentiated neurons revealed a prominent GABAergic circuit disruption along with a perturbation of cytoskeleton dynamics. In particular, in mutated neurons we identified a significant decrease of acetylated α-tubulin which can be reverted by treatment with selective inhibitors of HDAC6, the main α-tubulin deacetylase. These findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the treatment of Rett-associated epileptic behavior as well as for the definition of new therapeutic strategies for Rett syndrome.

Enhanced Neuroprotective Effects of Panax ginseng G115® and Ginkgo biloba GK501® Combinations In Vitro Models of Excitotoxicity.

Neurological-related disorders are seen as an increasingly important aspect of welfare. While conventional medicine is still the mainstay for the treatment of these diseases, it is becoming apparent that patients are also seeking more natural and preventative interventions. Panax ginseng G115® and Ginkgo biloba GK501® extracts alone or in combination were used in two in vitro experimental models of primary cultures exposed to excitotoxicity: rat organotypic hippocampal slices exposed to either 5 µM kainic acid or 10 µM N-Methyl-d-aspartate for 24 hours, and mixed cortical cells exposed to 300 µM NMDA for 10 min. Cell death in the Cornu Ammonis areas CA3 or CA1 subregions of slices was quantified by measuring propidium iodide fluorescence, whereas in cortical cells, it was assessed by measuring the amount of lactate dehydrogenase. In slices, treatment with extracts alone or in combination significantly attenuated CA3 and CA1 damage induced by exposure to kainic acid or NMDA, respectively. A similar neuroprotective effect was observed in cortical cells exposed to NMDA. Analysis of cell signaling pathways found that the two extracts induced an increase of the phosphorylation and they reversed the decrease of phosphorylation of ERK1/2 and Akt induced by kainic acid and NMDA in organotypic hippocampal slices. These results suggest that P. ginseng G115® and G. biloba GK501® extracts may mediate their effects by activating phosphorylation of ERK1/2 and Akt signaling pathways, protecting against excitotoxicity-induced damage in in vitro models.

Formulation of Nanomicelles to Improve the Solubility and the Oral Absorption of Silymarin.

Two novel nanomicellar formulations were developed to improve the poor aqueous solubility and the oral absorption of silymarin. Polymeric nanomicelles made of Soluplus and mixed nanomicelles combining Soluplus with d-α-tocopherol polyethylene glycol 1000 succinate (vitamin E TPGS) were prepared using the thin film method. Physicochemical parameters were investigated, in particular the average diameter, the homogeneity (expressed as polydispersity index), the zeta potential, the morphology, the encapsulation efficiency, the drug loading, the critical micellar concentration and the cloud point. The sizes of ~60 nm, the narrow size distribution (polydispersity index ≤0.1) and the encapsulation efficiency >92% indicated the high affinity between silymarin and the core of the nanomicelles. Solubility studies demonstrated that the solubility of silymarin increased by ~6-fold when loaded into nanomicelles. Furthermore, the physical and chemical parameters of SLM-loaded formulations stored at room temperature and in refrigerated conditions (4 °C) were monitored over three months. In vitro stability and release studies in media miming the physiological conditions were also performed. In addition, both formulations did not alter the antioxidant properties of silymarin as evidenced by the 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) assay. The potential of the nanomicelles to increase the intestinal absorption of silymarin was firstly investigated by the parallel artificial membrane permeability assay. Subsequently, transport studies employing Caco-2 cell line demonstrated that mixed nanomicelles statistically enhanced the permeability of silymarin compared to polymeric nanomicelles and unformulated extract. Finally, the uptake studies indicated that both nanomicellar formulations entered into Caco-2 cells via energy-dependent mechanisms.

Urine-derived podocytes-lineage cells: A promising tool for precision medicine in Alport Syndrome.

Alport Syndrome (ATS) is a rare genetic disorder caused by collagen IV genes mutations, leading to glomerular basement membrane damage up to end-stage renal disease. Podocytes, the main component of the glomerular structure, are the only cells able to produce all the three collagens IV alpha chains associated with ATS and thus, they are key players in ATS pathogenesis. However, podocytes-targeted therapeutic strategies have been hampered by the difficulty of non-invasively isolating them and transcripts-based diagnostic approaches are complicated by the inaccessibility of other COL4 chains-expressing cells. We firstly isolated podocyte-lineage cells from ATS patients' urine samples, in a non-invasive way. RT-PCR analysis revealed COL4A3, COL4A4, and COL4A5 expression. Transcripts analysis on RNA extracted from patient's urine derived podocyte-lineage cells allowed defining the pathogenic role of intronic variants, namely one mutation in COL4A3 (c.3882+5G>A), three mutations in COL4A4 (c.1623+2T>A, c.3699_3706+1del, c.2545+143T>A), and one mutation in COL4A5 (c.3454+2T>C). Therefore, our cellular model represents a novel tool, essential to unequivocally prove the effect of spliceogenic intronic variants on transcripts expressed exclusively at a glomerular level. This process is a key step for providing the patient with a definite molecular diagnosis and with a proper recurrence risk. The established system also opens up the possibility of testing personalized therapeutic approaches on disease-relevant cells.

Ethanol Toxicity During Brain Development: Alterations of Excitatory Synaptic Transmission in Immature Organotypic Hippocampal Slice Cultures.

BACKGROUND: The developing brain is particularly vulnerable to alcohol: Drinking during pregnancy can lead to a number of physical, learning, and behavioral disorders in the newborn. It has been demonstrated that immature and mature brain tissues display a differential sensitivity to ethanol (EtOH) toxicity and that cerebral structure and function are diversely impaired according to the stage of synaptic maturation. METHODS: Rat organotypic hippocampal slice cultures were exposed for 7 days to EtOH (100 to 300 mM) after 2 days (immature) or 10 days (mature) of culture in vitro; EtOH was then removed from the medium, and 24 hours later, slices were analyzed by fluorescence microscopy, Western blotting, electrophysiology, and electron microscopy to explore the molecular mechanisms of EtOH toxicity in the developing hippocampus. RESULTS: EtOH withdrawal elicited a selective CA1 pyramidal cell injury in mature slices, but not in immature slices. A significant increase in the expression of pre- and postsynaptic proteins in mature slices revealed that slice maturation is presumably associated with the development of new synapses. Incubation with chronic EtOH for 7 days and its removal from the medium induced a significant decrease in GluA1 and GluA2 expression levels; a significant reduction in the expression of synaptophysin and GluN2A was observed only after EtOH withdrawal. Whole-cell patch-clamp recordings showed that incubation with EtOH for 7 days induced a significant decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency in CA1 pyramidal cells of immature slices and a trend toward a decrease in sEPSC amplitude. Electron microscopy revealed a disorganization of neurotubuli in immature slices after chronic exposure to EtOH. CONCLUSIONS: These results indicate that prolonged incubation with EtOH and its subsequent withdrawal from the medium induce an impairment of excitatory synaptic transmission and possibly an incorrect formation of neuronal circuits in developing hippocampus in vitro, which is suggestive of mechanisms that may lead to mental retardation in fetal alcohol spectrum disorders.

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.

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-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..

Mild activation of poly(ADP-ribose) polymerase (PARP) is neuroprotective in rat hippocampal slice models of ischemic tolerance.

Ischemic tolerance is a phenomenon in which exposure to a mild preconditioning stress results in resistance to a subsequent lethal ischemic insult. Here we investigated the role of poly(ADP-ribose) polymerase (PARP) in the development of ischemic tolerance by using organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later. We developed models of pharmacological preconditioning by exposing slices to subtoxic concentrations of either N-methyl-D-aspartate (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) and then, 24 h later, to 30 min OGD. Under these conditions, we observed a significant reduction in OGD-induced CA1 damage. Exposure of slices to the PARP-1 and -2 inhibitors TIQ-A, PJ-34 and UPF 1069 during preconditioning prevented the development of OGD tolerance in a concentration-dependent manner. NMDA and DHPG preconditioning increased the activity of PARP, as detected by immunoblots using antibodies against the poly(ADP-ribose) polymer product, but was not associated with consumption of cellular NAD(+) or ATP. Neuroprotection induced by preconditioning was also prevented by the caspase inhibitor Z-VAD-FMK. The modest but significant increase in caspase-3/7 induced by preconditioning, however, was not associated with PARP-1 cleavage, as occurred with staurosporine. Finally, TIQ-A prevented the activation of ERK1/2 and Akt induced by NMDA preconditioning, suggesting that the protective mechanism evoked by PARP requires activation of these prosurvival mediators. Our results suggest that preconditioning with appropriate pharmacological stimuli may promote neuroprotective mechanisms triggered by the sublethal activation of two otherwise deleterious executioners such as PARP and caspase-3/7.

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.