First in Class Dual Non-ATP-Competitive Glycogen Synthase Kinase 3ß/Histone Deacetylase Inhibitors as a Potential Therapeutic to Treat Alzheimer's Disease.

Despite recent FDA approvals, Alzheimer's disease (AD) still represents an unmet medical need. Among the different available therapeutic approaches, the development of multitarget molecules represents one of the most widely pursued. In this work, we present a second generation of dual ligands directed toward highly networked targets that are deeply involved in the development of the disease, namely, Histone Deacetylases (HDACs) and Glycogen Synthase Kinase 3ß (GSK-3ß). The synthesized compounds are highly potent GSK-3ß, HDAC2, and HDAC6 inhibitors with IC50 values in the nanomolar range of concentrations. Among them, compound 4 inhibits histone H3 and tubulin acetylation at 0.1 µM concentration, blocks hyperphosphorylation of tau protein, and shows interesting immunomodulatory and neuroprotective properties. These features, together with its ability to cross the blood-brain barrier and its favorable physical-chemical properties, make compound 4 a promising hit for the development of innovative disease-modifying agents.

Phenolic and Antioxidant Characterization of Fruit By-Products for Their Nutraceuticals and Dietary Supplements Valorization under a Circular Bio-Economy Approach.

Agri-food by-products, obtained as waste from the food industry, negatively impact the global economy and the environment. In order to valorize waste materials from fruit juices and tomato sauces as upcycled materials rich in health-promoting compounds, they were characterized in terms of polyphenolic and protein content. The results obtained were compared with those collected for their final products. The recovery of polyphenols was performed via ultrasound-assisted extraction (UAE). A high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed and validated to depict the quali-quantitative polyphenolic profile of both the by-products and the final products. The antioxidant capacity of the resulting extracts was characterized by UV-Vis spectrophotometric assays in terms of total phenolic content (TPC) and total antioxidant status (TAS). Moreover, the protein content was assessed with the Kjeldahl method too. The results highlighted a significant quantity of polyphenols remaining in peach, apricot, and apple by-products, which were able to exert an antioxidant activity (in the range of 4.95 ± 5.69 × 10-1 to 7.06 ± 7.96 × 10-1 mmol Trolox 100 g-1 of dry weight (DW) sample). Conversely, the tomato by-products were highly rich in proteins (11.0 ± 2.00 to 14.4 ± 2.60 g of proteins 100 g-1 DW). The results proved that all by-products may potentially be sustainable ingredients with nutritional and functional value in a circular bio-economy prospect.

Integrated analytical approaches for the characterization of Spirulina and Chlorella microalgae.

Microalgae are well-known for their content of bioactive molecules such as pigments, proteins, fatty acids, polysaccharides, vitamins and antioxidants, all of which are of great interest in the preparation of a wide range of products such as food, cosmetics and nutraceuticals. The purpose of this project was the analytical characterization of commercial dry microalgal biomass: four samples of Chlorella and five of Spirulina were analysed in order to highlight their content in terms of micro/macro nutrients. The research was oriented towards the development and validation of accurate, fast and reproducible methods for the nutritional assessment of algal biomasses, aiming to provide a guiding methodology. The lipid profiles of algal matrixes were analysed for the content of saturated, unsaturated and polyunsaturated fatty acids. The process was divided into two phases: firstly, the extraction and determination of the total lipids and pigment content; secondly, the trans-esterification of the extracted lipid-pigment portion in order to analyse fatty acid methyl esters (FAMEs) with a GC-MS method. A fingerprinting of MUFAs and PUFAs was obtained regarding microalgae species. The determination of total carotenoids and chlorophylls content in the lipid extracts was evaluated through a fast UV-Vis spectrophotometric analysis, which was validated by a new HPLC-DAD analysis. Furthermore, the total antioxidant activity of each lipid extract was determined along with the determination of the microalgae protein content. Then, with the aid of the principal component analysis (PCA) plots, the two microalgae were clustered in terms of their micro/macro nutrients, for differentiating their properties. Spirulina, resulting to have a greater antioxidant activity, supposedly due to a higher content in pigments and higher protein concentration, could be suggested for an appropriate diet for sporting people. Chlorella, instead, showed a more balanced profile of PUFAs and MUFAs and its use could be suggested for cosmetics and vegan diets. This paper puts forward an overall analytical approach, sustained by a multivariate analysis, for emphasising content differences and activity of two different microalgae strains, in order to underline specific claims for each class, addressed to defined final users.

Fentanyl pharmacokinetics in blood of cancer patients by Gas Chromatography - Mass Spectrometry.

In order to find a correlation between Fentanyl action on pain and inter-individual variability in different cancer patients, the pharmacokinetic characterization of the drug becomes essential. Therefore, a gas chromatographic-mass spectrometric (GC-MS) in SIM mode analytical procedure has been developed and validated for the determination of Fentanyl in human blood. The sample preparation consisted of a liquid-liquid extraction (LLE) from whole blood. The analysis were carried out with Agilent 7820 A series gas chromatograph equipped with a 5977E series mass selective single quadrupole detector (MSD) with an electron impact (EI) source (70 eV), under a temperature gradient elution. The limit of detection (LoD) and the limit of quantification (LoQ) values were found to be 5.60E-02 ± 3.50E-02 ng mL-1 and 1.86E-01 ± 1.18E-01 ng mL-1 respectively. The developed method was found selective and sensitive and therefore suitable for a fast determination of Fentanyl in human blood and for its pharmacokinetic characterization. Blood samples from 31 cancer patients treated with transdermal Fentanyl (doses in the range of 12-100 µg h-1) were collected at fixed intervals during an overall exposure time of 72 h. The analysis of data and the pharmacokinetic parameters revealed dissimilar pharmacokinetic profiles in the patients examined. Patients were therefore grouped in three categories representing the different trends observed: high, medium and slow responders. These preliminary data provided significant outcomes for a correlation to clinical response.

Aß1-42 peptide toxicity on neuronal cells: A lipidomic study.

Currently Alzheimer's Disease (AD) pathological pathways, which lead to cell death and dementia, are not completely well-defined; in particular, the lipid changes in brain tissues that begin years before AD symptoms. Due to the central role of the amyloid aggregation process in the early phase of AD pathogenesis, we aimed at developing a lipidomic approach to evaluate the amyloid toxic effects on differentiated human neuroblastoma derived SH-SY5Y cells. First of all, this work was performed to highlight qualitative and relative quantitative lipid variations in connection with amyloid toxicity. Then, with an open outcome, the study was focused to find out some new lipid-based biomarkers that could result from the interaction of amyloid peptide with cell membrane and could justify neuroblastoma cells neurotoxicity. Hence, cells were treated with increasing concentration of Aß1-42 at different times, then the lipid extraction was carried out by protein precipitation protocol with 2-propanol-water (90:10 v/v). The LC-MS analysis of samples was performed by a RP-UHPLC system coupled with a quadrupole-time-of-flight mass spectrometer in comprehensive data - independent SWATH acquisition mode. Data processing was achieved by MS-DIAL. Each lipid class profile in SH-SY5Y cells treated with Aß1-42 was compared to the one obtained for the untreated cells to identify (and relatively quantify) some altered species in various lipid classes. This approach was found suitable to underline some peculiar lipid alterations that might be correlated to different Aß1-42 aggregation species and to explore the cellular response mechanisms to the toxic stimuli. The in vitro model presented has provided results that coincide with the ones in literature obtained by lipidomic analysis on cerebrospinal fluid and plasma of AD patients. Therefore, after being validated, this method could represent a way for the preliminary identification of potential biomarkers that could be researched in biological samples of AD patients.

Natural products as novel scaffolds for the design of glycogen synthase kinase 3ß inhibitors.

INTRODUCTION: The different and relevant roles of GSK-3 are of critical importance since they deal with development, metabolic homeostasis, cell polarity and fate, neuronal growth and differentiation as well as modulation of apoptotic potential. Given their involvement with different diseases, many investigations have been undertaken with the aim of discovering new and promising inhibitors for this target. In this context, atural products represent an invaluable source of active molecules. AREAS COVERED: In order to overcome issues such as poor pharmacokinetic properties or efficacy, frequently associated with natural compounds, different GSK-3ß inhibitors belonging to alkaloid or flavonoid classes have been subjected to structural modifications in order to obtain more potent and safer compounds. Herein, the authors report the results obtained from studies where natural compounds have been used as hits with the aim of providing new kinase inhibitors endowed with a better inhibitory profile. EXPERT OPINION: Structurally modification of natural scaffolds is a proven approach taking advantage of their pharmacological characteristics. Indeed, whatever the strategy adopted is and, despite the limitations associated with the structural complexity of natural products, the authors recommend the use of natural scaffolds as a promising strategy for the discovery of novel and potent GSK-3ß inhibitors.

New Coumarin Derivatives as Cholinergic and Cannabinoid System Modulators.

In the last years, the connection between the endocannabinoid system (eCS) and neuroprotection has been discovered, and evidence indicates that eCS signaling is involved in the regulation of cognitive processes and in the pathophysiology of Alzheimer's disease (AD). Accordingly, pharmacotherapy targeting eCS could represent a valuable contribution in fighting a multifaceted disease such as AD, opening a new perspective for the development of active agents with multitarget potential. In this paper, a series of coumarin-based carbamic and amide derivatives were designed and synthesized as multipotent compounds acting on cholinergic system and eCS-related targets. Indeed, they were tested with appropriate enzymatic assays on acetyl and butyryl-cholinesterases and on fatty acid amide hydrolase (FAAH), and also evaluated as cannabinoid receptor (CB1 and CB2) ligands. Moreover, their ability to reduce the self-aggregation of beta amyloid protein (Aß42) was assessed. Compounds 2 and 3, bearing a carbamate function, emerged as promising inhibitors of hAChE, hBuChE, FAAH and Aß42 self-aggregation, albeit with moderate potencies, while the amide 6 also appears a promising CB1/CB2 receptors ligand. These data prove for the new compounds an encouraging multitarget profile, deserving further evaluation.

Targeting topoisomerase II with trypthantrin derivatives: Discovery of 7-((2-(dimethylamino)ethyl)amino)indolo[2,1-b]quinazoline-6,12-dione as an antiproliferative agent and to treat cancer.

Drugs targeting human topoisomerase II (topoII) are used in clinical practice since decades. Nevertheless, there is an urgent need for new and safer topoII inhibitors due to the emergence of secondary malignancies and the appearance of resistance mechanisms upon treatment with topoII-targeted anticancer drugs. In the present investigation, we report the discovery of a new topoII inhibitor, whose design was based on the structure of the natural product trypthantrin, a natural alkaloid containing a basic indoloquinazoline moiety. This new topoII inhibitor, here numbered compound 5, is found to inhibit topoII with an IC50 of 26.6 ± 4.7 µM. Notably, compound 5 is more potent than the template compound trypthantrin, and even than the widely used topoII-targeted clinical drug etoposide. In addition, compound 5 also exhibits high water solubility, and a promising antiproliferative activity on different tumor cell lines such as acute leukemia, colon, and breast cancer. In light of these results, compound 5 represents a promising lead for developing new topoII inhibitors as anti-cancer therapeutic agents.

Correction to "Discovery of the First-in-Class GSK-3ß/HDAC Dual Inhibitor as Disease-Modifying Agent To Combat Alzheimer's Disease".

[This corrects the article DOI: 10.1021/acsmedchemlett.8b00507.].

Discovery of novel benzofuran-based compounds with neuroprotective and immunomodulatory properties for Alzheimer's disease treatment.

To address the multifactorial nature of Alzheimer's Disease (AD), a multi-target-directed ligand approach was herein developed. As a follow-up of our previous studies, a small library of newly designed 2-arylbenzofuran derivatives was evaluated towards cholinesterases and cannabinoid receptors. The two most promising compounds, 8 and 10, were then assessed for their neuroprotective activity and for their ability to modulate the microglial phenotype. Compound 8 emerged as able to fight AD from several directions: it restored the cholinergic system by inhibiting butyrylcholinesterase, showed neuroprotective activity against Aß1-42 oligomers, was a potent and selective CB2 ligand and had immunomodulatory effects, switching microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype. Derivative 10 was a potent CB2 inverse agonist with promising immunomodulatory properties and could be considered as a tool for investigating the role of CB2 receptors and for developing potential immunomodulating drugs addressing the endocannabinoid system.

Discovery of the First-in-Class GSK-3ß/HDAC Dual Inhibitor as Disease-Modifying Agent To Combat Alzheimer's Disease.

Several evidence pointed out the role of epigenetics in Alzheimer's disease (AD) revealing strictly relationships between epigenetic and "classical" AD targets. Based on the reported connection among histone deacetylases (HDACs) and glycogen synthase kinase 3ß (GSK-3ß), herein we present the discovery and the biochemical characterization of the first-in-class hit compound able to exert promising anti-AD effects by modulating the targeted proteins in the low micromolar range of concentration. Compound 11 induces an increase in histone acetylation and a reduction of tau phosphorylation. It is nontoxic and protective against H2O2 and 6-OHDA stimuli in SH-SY5Y and in CGN cell lines, respectively. Moreover, it promotes neurogenesis and displays immunomodulatory effects. Compound 11 shows no lethality in a wt-zebrafish model (<100 µM) and high water solubility.

Dose administration maneuvers and patient care in tobramycin dry powder inhalation therapy.

The purpose of this work was to study a new dry powder inhaler (DPI) of tobramycin capable to simplify the dose administration maneuvers to maximize the cystic fibrosis (CF) patient care in antibiotic inhalation therapy. For the purpose, tobramycin/sodium stearate powder (TobraPS) having a high drug content, was produced by spray drying, characterized and the aerodynamic behavior was investigated in vitro using different RS01 DPI inhalers. The aerosols produced with 28, 56 or 112 mg of tobramycin in TobraPS powder using capsules size #3, #2 or #0 showed that there was quasi linear relationship between the amount loaded in the device and the FPD. An in vivo study in healthy human volunteers showed that 3-6 inhalation acts were requested by the volunteers to inhale 120 mg of TobraPS powder loaded in a size #0 capsule aerosolized with a prototype RS01 device, according to their capability to inhale. The amount of powder emitted at 4 kPa pressure drop at constant air flow well correlated with the in vivo emission at dynamic flow, when the same volume of air passed through the device. The novel approach for the administration of 112 mg of tobramycin in one capsule could improve the convenience and adherence of the CF patient to the antibiotic therapy.

Hydroxy-substituted trans-cinnamoyl derivatives as multifunctional tools in the context of Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. In recent years, a plethora of proteins and biochemical pathways has been proposed as possible targets to counteract neurotoxicity. Although the complex scenario is not completely elucidated, close relationships are emerging among some of these actors. In particular, increasing evidence has shown that aggregation of amyloid beta (Aß), glycogen synthase kinase 3ß (GSK-3ß) and oxidative stress are strictly interconnected and their concomitant modulation may have a positive and synergic effect in contrasting AD-related impairments. We designed compound 3 which demonstrated the ability to inhibit both GSK-3ß (IC50 = 24.36 ± 0.01 µM) and Aß42 self-aggregation (IC50 = 9.0 ± 1.4 µM), to chelate copper (II) and to act as exceptionally strong radical scavenger (kinh = 6.8 ± 0.5 · 105 M-1s-1) even in phosphate buffer at pH 7.4 (kinh = 3.2 ± 0.5 · 105 M-1s-1). Importantly, compound 3 showed high-predicted blood-brain barrier permeability, did not exert any significant cytotoxic effects in immature cortical neurons up to 50 µM and showed neuroprotective properties at micromolar concentration against toxic insult induced by glutamate.

Chalcone-based carbamates for Alzheimer's disease treatment.

AIM: Alzheimer's disease is a still untreatable multifaceted pathology, and drugs able to stop or reverse its progression are urgently needed. In this picture, the recent reformulation of the cholinergic hypothesis renewed the interest for acetylcholinesterase inhibitors. In this paper, a series of naturally inspired chalcone-based carbamates was designed to target cholinesterase enzymes and possibly generate fragments endowed with neuroprotective activity in situ. Results & methodology: All compounds presented in this study showed nanomolar potency for cholinesterase inhibition. Notably, fragment 11d also displayed an interesting neuroprotective profile. CONCLUSION: These new derivatives are able to simultaneously modulate different key targets involved in Alzheimer's disease, and could be regarded as promising starting points for the development of disease-modifying drug candidates. [Formula: see text].

Dry powders for the inhalation of ciprofloxacin or levofloxacin combined with a mucolytic agent for cystic fibrosis patients.

OBJECTIVE: This study aimed to design and characterize an inhalable dry powder of ciprofloxacin or levofloxacin combined with the mucolytics acetylcysteine and dornase alfa for the management of pulmonary infections in patients with cystic fibrosis. METHODS: Ball milling, homogenization in isopropyl alcohol and spray drying processes were used to prepare dry powders for inhalation. Physico-chemical characteristics of the dry powders were assessed via thermogravimetric analysis, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), X-ray diffractometry and scanning electron microscopy. The particle size distribution, dissolution rate and permeability across Calu-3 cell monolayers were analyzed. The aerodynamic parameters of dry powders were determined using the Andersen cascade impactor (ACI). RESULTS: After the micronization process, the particle sizes of the raw materials significantly decreased. X-ray and DSC results indicated that although ciprofloxacin showed no changes in its crystal structure, the structure of levofloxacin became amorphous after the micronization process. FT-IR spectra exhibited the characteristic peaks for ciprofloxacin and levofloxacin in all formulations. The dissolution rates of micro-homogenized and spray-dried ciprofloxacin were higher than that of untreated ciprofloxacin. ACI results showed that all formulations had a mass median aerodynamic diameter less than 5 µm; however, levofloxacin microparticles showed higher respirability than ciprofloxacin powders did. The permeability of levofloxacin was higher than those of the ciprofloxacin formulations. CONCLUSION: Together, our study showed that these methods could suitably characterize antibiotic and mucolytic-containing dry powder inhalers.

Fatty Acid Amide Hydrolase (FAAH), Acetylcholinesterase (AChE), and Butyrylcholinesterase (BuChE): Networked Targets for the Development of Carbamates as Potential Anti-Alzheimer's Disease Agents.

The modulation of the endocannabinoid system is emerging as a viable avenue for the treatment of neurodegeneration, being involved in neuroprotective and anti-inflammatory processes. In particular, indirectly enhancing endocannabinoid signaling to therapeutic levels through FAAH inhibition might be beneficial for neurodegenerative disorders such as Alzheimer's disease, effectively preventing or slowing the progression of the disease. Hence, in the search for a more effective treatment for Alzheimer's disease, in this paper, the multitarget-directed ligand paradigm was applied to the design of carbamates able to simultaneously target the recently proposed endocannabinoid system and the classic cholinesterase system, and achieve effective dual FAAH/cholinesterase inhibitors. Among the two series of synthesized compounds, while some derivatives proved to be extremely potent on a single target, compounds 9 and 19 were identified as effective dual FAAH/ChE inhibitors, with well-balanced nanomolar activities. Thus, 9 and 19 might be considered as new promising candidates for Alzheimer's disease treatment.

Multitarget Strategy to Address Alzheimer's Disease: Design, Synthesis, Biological Evaluation, and Computational Studies of Coumarin-Based Derivatives.

Alzheimer's disease (AD) is a major public health challenge that faces an aging global population. Current drug treatment has demonstrated only symptomatic efficacy, leaving an unmet medical need for a new generation of disease-modifying therapies. Following the multitarget-directed ligand approach, a small library of coumarin-based derivatives was designed and synthesized as a follow-up to our studies on AP2238, aimed at expanding its biological profile. The coumarin substitution pattern at the 6- or 7-position was modified by introducing alkyl chains of variable lengths and with different terminal amino functional groups. 3-(4-{[Benzyl(ethyl)amino]methyl}phenyl)-6-({5-[(7-methoxy-6H-indeno[2,1-b]quinolin-11-yl)amino]pentyl}oxy)-2H-chromen-2-one, bearing the bulkiest amine, emerged as a non-neurotoxic dual acetylcholinesterase (AChE)/butyrylcholinesterase (BuChE) inhibitor, potentially suitable for the treatment of the middle stage of AD. Furthermore, the introduction of a diethylamino spacer, as in 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-6-{[5-(diethylamino)pentyl]oxy}-2H-chromen-2-one and 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-7-[4-(diethylamino)butoxy]-2H-chromen-2-one, led to nanomolar human AChE inhibitors endowed with significant inhibitory activity toward Aß42 self-aggregation, whereas the reference compound was completely ineffective. Furthermore, 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-7-[4-(diethylamino)butoxy]-2H-chromen-2-one also showed promising neuroprotective behavior, which makes it a potential candidate for development into a disease-modifying agent.

Fluorinated benzophenone derivatives: balanced multipotent agents for Alzheimer's disease.

In an effort to develop multipotent agents against ß-secretase (BACE-1) and acetylcholinesterase (AChE), able to counteract intracellular ROS formation as well, the structure of the fluorinated benzophenone 3 served as starting point for the synthesis of a small library of 3-fluoro-4-hydroxy- analogues. Among the series, derivatives 5 and 12, carrying chemically different amino functions, showed a balanced micromolar potency against the selected targets. In particular, compound 12, completely devoid of toxic effects, seems to be a promising lead for obtaining effective anti-AD drug candidates.