Biological Evaluation and In Vitro Characterization of ADME Profile of In-House Pyrazolo[3,4-d]pyrimidines as Dual Tyrosine Kinase Inhibitors Active against Glioblastoma Multiforme.

The therapeutic use of tyrosine kinase inhibitors (TKIs) represents one of the successful strategies for the treatment of glioblastoma (GBM). Pyrazolo[3,4-d]pyrimidines have already been reported as promising small molecules active as c-Src/Abl dual inhibitors. Herein, we present a series of pyrazolo[3,4-d]pyrimidine derivatives, selected from our in-house library, to identify a promising candidate active against GBM. The inhibitory activity against c-Src and Abl was investigated, and the antiproliferative profile against four GBM cell lines was studied. For the most active compounds endowed with antiproliferative efficacy in the low-micromolar range, the effects toward nontumoral, healthy cell lines (fibroblasts FIBRO 2-93 and keratinocytes HaCaT) was investigated. Lastly, the in silico and in vitro ADME properties of all compounds were also assessed. Among the tested compounds, the promising inhibitory activity against c-Src and Abl (Ki 3.14 µM and 0.44 µM, respectively), the irreversible, apoptotic-mediated death toward U-87, LN18, LN229, and DBTRG GBM cell lines (IC50 6.8 µM, 10.8 µM, 6.9 µM, and 8.5 µM, respectively), the significant reduction in GBM cell migration, the safe profile toward FIBRO 2-93 and HaCaT healthy cell lines (CC50 91.7 µM and 126.5 µM, respectively), the high metabolic stability, and the excellent passive permeability across gastrointestinal and blood-brain barriers led us to select compound 5 for further in vivo assays.

Olea europea L. Leaves and Hibiscus sabdariffa L. Petals Extracts: Herbal Mix from Cardiovascular Network Target to Gut Motility Dysfunction Application.

It is well known that diet and nutrition play a critical role in the etiopathogenesis of many disorders. On the other hand, nutrients or bioactive compounds can specifically target and control various aspects of the mechanism underlying the pathology itself, and, in this context, diseases related to intestinal motility disorders stand out. The Herbal Mix (HM) consisting of Olea europea L. leaf (OEE) and Hibiscus sabdariffa L. (HSE) extracts (13:2) has been proven to be a promising nutraceutical option for many diseases, but its potential in inflammatory-driven gastrointestinal disorders is still unexplored. In this study, HM effects on guinea-pig ileum and colon contractility (induced or spontaneous) and on human iNOS activity, as well as on human colorectal adenocarcinoma Caco-2 cells, were studied. Results showed that the HM can control the ileum and colon contractility without blocking the progression of the food bolus, can selectively inhibit iNOS and possesses a strong pro-apoptotic activity towards Caco-2 cells. In conclusion, the present results suggest that, in some diseases, such as those related to motility disorders, an appropriate nutritional approach can be accompanied by a correct use of nutraceuticals that could help not only in ameliorating the symptoms but also in preventing more severe, cancer-related conditions.

Sdox, a H2S releasing anthracycline, with a safer profile than doxorubicin toward vasculature.

Sdox is a synthetic H2S-releasing doxorubicin (Dox) less cardiotoxic and more effective than Dox in pre-clinical, Dox-resistant tumour models. The well-known anthracycline vascular toxicity, however, might limit Sdox clinical use. This study aimed at evaluating Sdox vascular toxicity in vitro, using Dox as reference compound. Both vascular smooth muscle A7r5 and endothelial EA.hy926 cells were more sensitive to Dox than Sdox, although both drugs equally increased intracellular free radical levels. Sdox released H2S in both cell lines. The H2S scavenger hydroxocobalamin partially reverted Sdox-induced cytotoxicity in A7r5, but not in EA.hy926 cells, suggesting a role for H2S in smooth muscle cell death. Markers of Sdox-induced apoptosis were significantly lower than, in A7r5 cells, and comparable to those of Dox in EA.hy926 cells. In A7r5 cells, Dox increased the activity of caspase 3, 8, and 9, Sdox affecting only that of caspase 3. Moreover, both drugs induced comparable DNA damage in A7r5 cells, while Sdox was less toxic than Dox in Ea.hy926 cells. In fresh aorta rings, only Dox weakly increased phenylephrine-induced contraction when endothelium was present. In rings cultured with both drugs for 7 days, Sdox blunted phenylephrine- and high K+-induced contractions though at a concentration 10-fold higher than that of Dox. In conclusion, Sdox may represent the prototype of an innovative anthracycline, effective against Dox-resistant tumours, displaying a more favourable vascular toxicity profile compared to the parent compound.

Hibiscus Flower and Olive Leaf Extracts Activate Apoptosis in SH-SY5Y Cells.

Compounds of natural origin may constitute an interesting tool for the treatment of neuroblastoma, the most prevalent extracranial solid tumor in children. PRES is a commercially available food supplement, composed of a 13:2 (w/w) extracts mix of Olea europaea L. leaves (OE) and Hibiscus sabdariffa L. flowers (HS). Its potential towards neuroblastoma is still unexplored and was thus investigated in human neuroblastoma SH-SY5Y cells. PRES decreased the viability of cells in a concentration-dependent fashion (24 h IC50 247.2 ± 31.8 µg/mL). Cytotoxicity was accompanied by an increase in early and late apoptotic cells (AV-PI assay) and sub G0/G1 cells (cell cycle analysis), ROS formation, reduction in mitochondrial membrane potential, and caspases activities. The ROS scavenger N-acetyl-L-cysteine reverted the cytotoxic effects of PRES, suggesting a key role played by ROS in PRES-mediated SH-SY5Y cell death. Finally, the effects of OE and HS extracts were singularly tested and compared to those of the corresponding mixture. OE- or HS-mediated cytotoxicity was always significantly lower than that caused by PRES, suggesting a synergic effect. In conclusion, the present findings highlight the potential of PRES for the treatment of neuroblastoma and offers the basis for a further characterization of the mechanisms underlying its effects.

Acacia catechu Willd. Extract Protects Neuronal Cells from Oxidative Stress-Induced Damage.

Oxidative stress (OS) and the resulting reactive oxygen species (ROS) generation and inflammation play a pivotal role in the neuronal loss occurring during the onset of neurodegenerative diseases. Therefore, promising future drugs that would prevent or slow down the progression of neurodegeneration should possess potent radical-scavenging activity. Acacia catechu Willd. heartwood extract (AC), already characterized for its high catechin content, is endowed with antioxidant properties. The aim of the present study was to assess AC neuroprotection in both human neuroblastoma SH-SY5Y cells and rat brain slices treated with hydrogen peroxide. In SH-SY5Y cells, AC prevented a decrease in viability, as well as an increase in sub-diploid-, DAPI positive cells, reduced ROS formation, and recovered the mitochondrial potential and caspase-3 activation. AC related neuroprotective effects also occurred in rat brain slices as a reversal prevention in the expression of the main proteins involved in apoptosis and signalling pathways related to calcium homeostasis following OS-mediated injury. Additionally, unbiased quantitative mass spectrometry allowed for assessing that AC partially prevented the hydrogen peroxide-induced altered proteome, including proteins belonging to the synaptic vesicle fusion apparatus. In conclusion, the present results suggest the possibility of AC as a nutraceutical useful in preventing neurodegenerative diseases.

In vitro toxicity evaluation of lomefloxacin-loaded MCM-41 mesoporous silica nanoparticles.

Lomefloxacin (LF) is interesting as a model molecule from a safety point of view because of its high potential for serious adverse drug effects (i.e. phototoxic reactions). In this study, MCM-41 mesoporous silica nanoparticles (MCM-41) were loaded with lomefloxacin, aiming to overcome the drug's intrinsic cytotoxicity. The good biocompatibility of the empty drug carrier (0.1-1.0 mg/ml) was established by the absence of red blood cell lysis (hemolysis assay). The cytotoxicity of empty MCM-41 and lomefloxacin-loaded MCM-41 (LF-MCM-41) was evaluated by using a battery of in vitro cytotoxicity assays: Alamar blue, lactate dehydrogenase release and reactive oxygen species formation by dichlorofluorescein assay. Three cell cultures models: hepatoma HepG2, fibroblasts L929 and endothelial EA.hy926 cells were used to compare the cytotoxicity and reactive oxygen species formation by free drug, empty MCM-41, and LF-MCM-41. The findings from the study indicated that empty MCM-41 (0.1-1.0 mg/ml) showed a low cytotoxic potential in HepG2, followed by L929 and EA.hy926 cells. Lomefloxacin loading in MCM-41 mesoporous silica nanocarrier reduced the cytotoxicity of the free lomefloxacin, especially in the high concentration (1.0 mg/ml MCM-41, containing 120 µg/ml LF). L929 and EA.hy926 cells were more sensitive to the protective effects of LF-MCM-41, compared to HepG2 cells. The results indicate that an improvement in lomefloxacin safety might be expected after incorporation in an appropriate drug delivery system.

Nitric Oxide/Cyclic GMP-Dependent Calcium Signalling Mediates IL-6- and TNF-α-Induced Expression of Glial Fibrillary Acid Protein.

Astrocyte activation is characterized by hypertrophy with increased glial fibrillary acidic protein (GFAP), whose expression may involve pro-inflammatory cytokines. In this study, the effects of pro-inflammatory IL-6 and TNF-α and anti-inflammatory cytokines IL-4 and IL-10 on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling, intracellular calcium concentration ([Ca2+]i) and GFAP expression were investigated. In human glioblastoma astrocytoma U-373 MG cells, IL-6 and TNF-α, but not IL-4 or IL-10, increased iNOS, cGMP, [Ca2+]i and GFAP expression. The inhibitors of iNOS (1400 W), soluble guanylyl cyclase (ODQ) and IP3 receptors (ryanodine and 2-APB) reversed the increase in cGMP or [Ca2+]i, respectively, and prevented GFAP expression. In rat striatal slices, IL-6 and TNF-α, at variance with IL-4 and IL-10, promoted a concentration-dependent increase in Ca2+ efflux, an effect prevented by 1400 W, ODQ and RY/2APB. These data were confirmed by in vivo studies, where IL-6, TNF-α or the NO donor DETA/NO injected in the striatum of anaesthetised rats increased cGMP levels and increased GFAP expression. The present findings point to NO/cGMP-dependent calcium signalling as part of the mechanism mediating IL-6- and TNF-α-induced GFAP expression. As this process plays a fundamental role in driving neurotoxicity, targeting NO/cGMP-dependent calcium signalling may constitute a new approach for therapeutic interventions in neurological disorders.

Olive Leaves and Hibiscus Flowers Extracts-Based Preparation Protect Brain from Oxidative Stress-Induced Injury.

Oxidative stress (OS) arising from tissue redox imbalance, critically contributes to the development of neurodegenerative disorders. Thus, natural compounds, owing to their antioxidant properties, have promising therapeutic potential. Pres phytum (PRES) is a nutraceutical product composed of leaves- and flowers-extracts of Olea europaea L. and Hibiscus sabdariffa L., respectively, the composition of which has been characterized by HPLC coupled to a UV-Vis and QqQ-Ms detector. As PRES possess antioxidant, antiapoptotic and anti-inflammatory properties, the aim of this study was to assess its neuroprotective effects in human neuroblastoma SH-SY5Y cells and in rat brain slices subjected to OS. PRES (1-50 µg/mL) reverted the decrease in viability as well as the increase in sub-diploid-, DAPI-and annexin V-positive-cells, reduced ROS formation, recovered the mitochondrial potential and caspase-3 and 9 activity changes caused by OS. PRES (50-100 µg/mL) neuroprotective effects occurred also in rat brain slices subjected to H2O2 challenge. Finally, as the neuroprotective potential of PRES is strictly related to its penetration into the brain and a relatively good pharmacokinetic profile, an in-silico prediction of its components drug-like properties was carried out. The present results suggest the possibility of PRES as a nutraceutical, which could help in preventing neurodegenerative diseases.

Chemical Characterisation and Antihypertensive Effects of Locular Gel and Serum of Lycopersicum esculentum L. var. "Camone" Tomato in Spontaneously Hypertensive Rats.

Blood pressure control in hypertensive subjects calls for changes in lifestyle, especially diet. Tomato is widely consumed and rich in healthy components (i.e., carotenoids, vitamins and polyphenols). The aim of this study was to evaluate the chemical composition and antihypertensive effects of locular gel reconstituted in serum of green tomatoes of "Camone" variety. Tomato serum and locular gel were chemically characterised. The antihypertensive effects of the locular gel in serum, pure tomatine, and captopril, administered by oral gavage, were investigated for 4 weeks in male spontaneously hypertensive and normotensive rats. Systolic blood pressure and heart rate were monitored using the tail cuff method. Body and heart weight, serum glucose, triglycerides and inflammatory cytokines, aorta thickness and liver metabolising activity were also assessed. Locular gel and serum showed good tomatine and polyphenols content. Significant reductions in blood pressure and heart rate, as well as in inflammatory blood cytokines and aorta thickness, were observed in spontaneously hypertensive rats treated both with locular gel in serum and captopril. No significant effects were observed in normotensive rats. Green tomatoes locular gel and serum, usually discarded during tomato industrial processing, are rich in bioactive compounds (i.e., chlorogenic acid, caffeic acid and rutin, as well as the glycoalkaloids, α-tomatine and dehydrotomatine) that can lower in vivo blood pressure towards healthier values, as observed in spontaneously hypertensive rats.

Thymus vulgaris L. Essential Oil Solid Formulation: Chemical Profile and Spasmolytic and Antimicrobial Effects.

A new Thymus vulgaris L. solid essential oil (SEO) formulation composed of liquid EO linked to solid excipients has been chemically analysed and evaluated for its intestinal spasmolytic and antispastic effects in ex vivo ileum and colon of guinea pig and compared with liquid EO and excipients. Liquid EO and solid linked EO were analysed by original capillary electrochromatography coupled to diode array detection (CEC-DAD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodologies. The main bioactive constituents are thymol and carvacrol, with minor constituents for a total of 12 selected analysed compounds. Liquid EO was the most effective in decreasing basal contractility in ileum and colon; excipients addiction permitted normal contractility pattern in solid linked EO SEO. In ileum and colon, the Thymus vulgaris L. solid formulation exerted the relaxant activity on K+-depolarized intestinal smooth muscle as well as liquid EO. The solid essential oil exhibits antimicrobial activity against different strains (Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli, Salmonella Thyphimurium, Candida albicans) similarly to liquid oil, with activity against pathogen, but not commensal strains (Bifidobacterium Breve, Lactobacillus Fermentum) in intestinal homeostasis. Therefore, Thymus vulgaris L. solid essential oil formulation can be proposed as a possible spasmolytic and antispastic tool in medicine.

ß-Naphthoflavone and Ethanol Reverse Mitochondrial Dysfunction in A Parkinsonian Model of Neurodegeneration.

The 1-methyl-4-phenylpyridinium (MPP+) is a parkinsonian-inducing toxin that promotes neurodegeneration of dopaminergic cells by directly targeting complex I of mitochondria. Recently, it was reported that some Cytochrome P450 (CYP) isoforms, such as CYP 2D6 or 2E1, may be involved in the development of this neurodegenerative disease. In order to study a possible role for CYP induction in neurorepair, we designed an in vitro model where undifferentiated neuroblastoma SH-SY5Y cells were treated with the CYP inducers ß-naphthoflavone (ßNF) and ethanol (EtOH) before and during exposure to the parkinsonian neurotoxin, MPP+. The toxic effect of MPP+ in cell viability was rescued with both ßNF and EtOH treatments. We also report that this was due to a decrease in reactive oxygen species (ROS) production, restoration of mitochondrial fusion kinetics, and mitochondrial membrane potential. These treatments also protected complex I activity against the inhibitory effects caused by MPP+, suggesting a possible neuroprotective role for CYP inducers. These results bring new insights into the possible role of CYP isoenzymes in xenobiotic clearance and central nervous system homeostasis.

In vitro vascular toxicity assessment of NitDOX, a novel NO-releasing doxorubicin.

The conjugation of doxorubicin (DOX) with nitric oxide (NO)-releasing groups gave rise to novel anthracyclines, such as nitrooxy-DOX (NitDOX), capable to overcome multidrug resistance. The widely described anthracycline cardiovascular toxicity, however, might limit their clinical use. This study aimed to investigate NitDOX-induced effects, as potential hazard, on vascular smooth muscle A7r5 and endothelial EA.hy926 cell viability, on the mechanical activity of freshly and cultured rat aorta rings, as well as on Cav1.2 channels of A7r5 cells. DOX was used as a reference compound. Although an increase in intracellular radicals and a reduction in mitochondrial potential occurred upon treatment with both drugs, A7r5 and EA.hy926 cells proved to be more sensitive to DOX than to NitDOX. Both compounds promoted comparable effects in A7r5 cells, whereas NitDOX was less active than DOX in inducing DNA damage and in eliciting apoptotic-mediated cell death revealed as an increase in sub-diploid-, DAPI- and annexin V-positive- EA.hy926 cell percentage. Moreover, in EA.hy926 cells, NitDOX doubled basal NO content, while preincubation with the NO-scavenger PTIO increased NitDOX-induced cytotoxicity. DOX exhibited a negligible contracturing effect in endothelium-intact rings, while NitDOX induced a significant ODQ-sensible, vasodilation in endothelium-denuded rings. In arteries cultured with both drugs for 7 days, NitDOX prevented either phenylephrine- or KCl-induced contraction at a concentration 10-fold higher than that of DOX. These results demonstrate that NitDOX displays a more favourable vascular toxicity profile than DOX. Taking into account its greater efficacy against drug-resistant cells, NitDOX is worth of further investigations in preclinical and clinical settings.

3-Aryl-4-nitrobenzothiochromans S,S-dioxide: From Calcium-Channel Modulators Properties to Multidrug-Resistance Reverting Activity.

Our research groups have been involved for many years in studies aimed at identifying new active organic compounds endowed with pharmacological properties. In this work, we focused our attention on the evaluation of cardiovascular and molecular drug resistance (MDR) reverting activities of some nitrosubstituted sulphur-containing heterocycles. Firstly, we have examined the effects of 4-nitro-3-(4-methylphenyl)-3,6-dihydro-2H-thiopyran S,S-dioxide 5, and have observed no activity. Then we have extended our investigation to the 3-aryl-4-nitrobenzothiochromans S,S-dioxide 6 and 7, and have observed an interesting biological profile. Cardiovascular activities were assessed for all compounds using ex vivo studies, while the MDR reverting effect was evaluated only for selected compounds using tumor cell lines. All compounds were shown to affect cardiovascular parameters. Compound 7i exerted the most effect on negative inotropic activity, while 6d and 6f could be interesting molecules for the development of more active ABCB1 inhibitors. Both 6 and 7 represent structures of large possible biological interest, providing a scaffold for the identification of new ABCB1 inhibitors.

Apoptotic-Induced Effects of Acacia Catechu Willd. Extract in Human Colon Cancer Cells.

The research for innovative treatments against colon adenocarcinomas is still a great challenge. Acacia catechu Willd. heartwood extract (AC) has health-promoting qualities, especially at the gastrointestinal level. This study characterized AC for its catechins content and investigated the apoptosis-enhancing effect in human colorectal adenocarcinoma HT-29 cells, along with its ability to spare healthy tissue. MTT assay was used to describe the time course, concentration dependence and reversibility of AC-mediated cytotoxicity. Cell cycle analysis and AV-PI and DAPI-staining were performed to evaluate apoptosis, together with ROS formation, mitochondrial membrane potential (MMP) changes and caspase activities. Rat ileum and colon rings were tested for their viability and functionality to explore AC effects on healthy tissue. Quantitative analysis highlighted that AC was rich in (±)-catechin (31.5 ± 0.82 mg/g) and (-)-epicatechin (12.5 ± 0.42 mg/g). AC irreversibly decreased cell viability in a concentration-dependent, but not time-dependent fashion. Cytotoxicity was accompanied by increases in apoptotic cells and ROS, a reduction in MMP and increases in caspase-9 and 3 activities. AC did not affect rat ileum and colon rings' viability and functionality, suggesting a safe profile toward healthy tissue. The present findings outline the potential of AC for colon cancer treatment.

4-Imidazo[2,1-b]thiazole-1,4-DHPs and neuroprotection: preliminary study in hits searching.

In the present work we describe the synthesis, characterization and evaluation of neuroprotective effects of a focused library of 4-imidazo[2,1-b]thiazole-1,4-dihydropyridines. Furthermore, the new dihydropyridines were subjected to functional in vitro assays in cardiac tissues and vascular smooth muscle to determine their possible selectivity in counteracting the effects of neurodegeneration. In particular the strategy adopted for designing the compounds involves the imidazo[2,1-b]thiazole nucleus. The observed properties show that substituents at C2 and C6 of the bicyclic scaffold are able to influence the cardiovascular parameters and the neuroprotective activity. In comparison to nifedipine, a set of derivatives such as compound 6, showed a neuroprotective profile of particular interest.

Inhibition of cell cycle progression by the hydroxytyrosol-cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells.

Hydroxytyrosol (HT), a polyphenol of olive oil, downregulates epidermal growth factor (EGFR) expression and inhibits cell proliferation in colon cancer (CC) cells, with mechanisms similar to that activated by the EGFR inhibitor, cetuximab. Here, we studied whether HT treatment would enhance the cetuximab inhibitory effects on cell growth in CC cells. HT-cetuximab combination showed greater efficacy in reducing cell growth in HT-29 and WiDr cells at concentrations 10 times lower than when used as single agents. This reduction was clearly linked to cell cycle blockade, occurring at G2/M phase. The cell cycle arrest in response to combination treatment is related to cyclins B, D1, and E, and cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 down-regulation, and to the concomitant over-expression of CDK inhibitors p21 and p27. HT and cetuximab stimulated a caspase-independent cell death cascade, promotedtranslocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus and activated the autophagy process. Notably, normal colon cells and keratinocytes were less susceptible to combo-induced cell death and EGFR downregulation. These results suggest a potential role of diet, containing olive oil, during cetuximab chemotherapy of colon tumor. HT may be a competent therapeutic agent in CC enhancing the effects of EGFR inhibitors.

Resveratrol: from diet to topical usage.

The stilbene derivative resveratrol (3,5,4'-trihydroxy-stilbene; RESV) has become the subject of interest of many researchers and the pharmaceutical industries due to its well-acclaimed beneficial biological activities. Although earlier research tended to focus on the effects of RESV on cardiovascular disorders, many other studies have described the beneficial effects of RESV in the areas of cancer chemoprevention and inflammation and interest of researchers on this compound is still increasing. It is now well accepted that the effect of RESV is not just due to its so called "antioxidant" activity but mainly (if not only) because of the ability of this compound to trigger cell signaling pathways and gene expression involved in cellular defense systems. Many "in vitro" studies on RESV did not take into account that although its oral absorption is about 75% it undergoes rapid metabolism and the concentration in the blood stream is almost undetectable. For this reason interest in the topical usage of RESV by cosmeceutical skin care brands has exponentially increased in the last decade reporting in general very promising results on its beneficial effect in protecting the skin from outdoor insults, but there is still some controversy on its topical usage mainly surrounding the concentration used. Therefore, more basic research on the topical application of RESV should be performed to better understand the way it prevents cutaneous damage and whether it could be recommended as a preventive skin aging agent for all skin insults.

Castanea sativa Mill. Bark Extract Protects U-373 MG Cells and Rat Brain Slices Against Ischemia and Reperfusion Injury.

Ischemic brain injury is one of the most important causes of death worldwide. The use of one-drug-multi-target agents based on natural compounds is a promising therapeutic option for cerebral ischemia due to their pleiotropic properties. This study assessed the neuroprotective properties of Castanea sativa Mill. bark extract (ENC) in human astrocytoma U-373 MG cells subjected to oxygen-glucose deprivation and reperfusion and rat cortical slices subjected to ischemia-like conditions or treated with glutamate or hydrogen peroxide. Neuroprotective effects were determined by assessing cells or slices viability (MTT assay), ROS formation (DCFH-DA assay), apoptosis (sub G0/G1 peak), nuclear fragmentation and chromatin condensation (DAPI staining) as well as changes in lysosomes and mitochondria morphology (Acridine Orange and Rhodamine123 staining, respectively). ENC treatment before injury on U-373 MG cells (5-50 µg/ml) and cortical slices (50-100 µg/ml) provided neuroprotection, while lower or higher concentrations (100 µg/ml U-373 MG cells, 200 µg/ml brain slices) were ineffective. ENC addition during reperfusion or after the injury was not found to be effective. The results suggest that ENC might hold potential as preventive neuroprotective agent, and indicate the importance of further studies exploring its mechanism of action. J. Cell. Biochem. 118: 839-850, 2017. © 2016 Wiley Periodicals, Inc.

Synthesis and biological evaluation of a new class of benzothiazines as neuroprotective agents.

Neurodegenerative diseases are disorders related to the degeneration of central neurons that gradually lead to various, severe alterations of cognitive and/or motor functions. Currently, for no such diseases does any pharmacological treatment exist able to arrest its progression. Riluzole (1) is a small molecule able to interfere with multiple cellular and molecular mechanisms of neurodegeneration, and is the only approved treatment of amyotrophic lateral sclerosis (ALS), the progression of which proved to significantly slow, thus increasing somewhat average survival. Here we report the synthesis of differently functionalized 4H-3,1-benzothiazine (5-6) and 2H-1,4-benzothiazine (7) series as superior homologues of 1. Biological evaluation demonstrated that amidine 4H-3,1-benzothiazine derivatives 5b-d can reduce glutamate and LDH release in the oxygen/glucose deprivation and reperfusion model (OGD/R) applied to brain slices with a higher potency than 1. Moreover the mentioned compounds significantly reduce glutamate- and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in neuroblastoma cells. In addition, the same compounds limit ROS formation in both neuronal preparations. Finally, 5c proved effective in inhibiting neuronal voltage-dependent Na+ and Ca2+-channels, showing a profile comparable with that of 1.

Human Tuberculosis I. Epidemiology, Diagnosis and Pathogenetic Mechanisms.

Mycobacterium tuberculosis (M. tuberculosis), an almost genetically monomorphic pathogen is a human parasite, transmitted mostly by humans and causes tuberculosis (TB). TB is firmly associated to poverty, although lack of proper nutrition and lowered immune status are contributing factors for disease development. TB remains second only to HIV/AIDS as the leading cause of mortality worldwide due to a single infectious agent and is responsible for nearly 1.5 million deaths annually. Some steps of the progress of our knowledge of M. tuberculosis physiology and its interactions with human beings, are reviewed here. This progress has provided fertile ground for improving diagnosis and cure of TB infection. For TB diagnostics laboratories in high-burden countries, primary isolation is the first step before performing drug susceptibility testing (DST) of M. tuberculosis. IGRA (interferon-γ release assay)-based tests for diagnosis of active TB are sufficiently fast, specific and sensitive to allow to contain infection and distinguish among latent TB infection and BCG vaccination individuals from those who have clinically resolved M. tuberculosis infection after anti-TB treatment.