Repurposing of 8-Hydroxyquinoline-Based Butyrylcholinesterase and Cathepsin B Ligands as Potent Nonpeptidic Deoxyribonuclease I Inhibitors.

A library of 31 butyrylcholinesterase (BChE) and cathepsin B (CatB) inhibitors was screened in vitro for inhibition of deoxyribonuclease I (DNase I). Compounds 22, 8 and 7 are among the most potent synthetic non-peptide DNase I inhibitors reported to date. Three 8-hydroxyquinoline analogues inhibited both DNase I and BChE with IC50 values below 35 µM and 50 nM, respectively, while two nitroxoline derivatives inhibited DNase I and Cat B endopeptidase activity with IC50 values below 60 and 20 µM. Selected derivatives were screened for various co-target binding affinities at dopamine D2 and D3 , histamine H3 and H4 receptors and inhibition of 5-lipoxygenase. Compound 8 bound to the H3 receptor and is highlighted as the most promising multifunctional ligand with a favorable pharmacokinetic profile and one of the most potent non-peptide DNase I inhibitors. The present study demonstrates that 8-hydroxyquinoline is a structural fragment critical for DNase I inhibition in the presented series of compounds.

Relative bioavailability enhancement of simvastatin via dry emulsion systems: Comparison of spray drying and fluid bed layering technology.

Comprehensive comparisons of similar lipid based drug delivery systems produced by different technologies are scarce. Spray drying and fluid bed layering technologies were compared with respect to the process and product characteristics of otherwise similar simvastatin loaded dry emulsion systems. Fluid bed layering provided higher process yield (83.3% vs 71.5%), encapsulation efficiency (80.0% vs 68.4%), relative one month product stability (93.8% vs 85.5%), larger and more circular particles (336 µm vs 56 µm) and lower median oil droplet size after product reconstitution in water (2.85 µm vs 4.27 µm), compared to spray drying. However, spray dried products exhibited higher drug content (22.2 mg/g vs 9.34 mg/g). An in-vivo pharmacokinetic study in rats was performed and a pharmacokinetic model was developed in order to compare the optimised simvastatin loaded dry emulsion systems, a simvastatin glyceride mimetic loaded in the dry emulsion and a simvastatin loaded SMEDDS with a reference physical mixture. Of the formulation tested, fluid bed layered pellets excelled and provided a 115% relative increase in bioavailability. Among the two technologies, fluid bed layering provided dry emulsion products with higher relative bioavailability and better product characteristics for further processing into final dosage forms.

Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside.

Galectin-8 has gained attention as a potential new pharmacological target for the treatment of various diseases, including cancer, inflammation, and disorders associated with bone mass reduction. To that end, new molecular probes are needed in order to better understand its role and its functions. Herein we aimed to improve the affinity and target selectivity of a recently published galectin-8 ligand, 3-O-[1-carboxyethyl]-ß-d-galactopyranoside, by introducing modifications at positions 1 and 3 of the galactose. Affinity data measured by fluorescence polarization show that the most potent compound reached a KD of 12 µM. Furthermore, reasonable selectivity versus other galectins was achieved, making the highlighted compound a promising lead for the development of new selective and potent ligands for galectin-8 as molecular probes to examine the protein's role in cell-based and in vivo studies.

Synthesis and Evaluation of Antioxidant Properties of 2-Substituted Quinazolin-4(3H)-ones.

Quinazolinones represent an important scaffold in medicinal chemistry with diverse biological activities. Here, two series of 2-substituted quinazolin-4(3H)-ones were synthesized and evaluated for their antioxidant properties using three different methods, namely DPPH, ABTS and TEACCUPRAC, to obtain key information about the structure-antioxidant activity relationships of a diverse set of substituents at position 2 of the main quinazolinone scaffold. Regarding the antioxidant activity, ABTS and TEACCUPRAC assays were more sensitive and gave more reliable results than the DPPH assay. To obtain antioxidant activity of 2-phenylquinazolin-4(3H)-one, the presence of at least one hydroxyl group in addition to the methoxy substituent or the second hydroxyl on the phenyl ring in the ortho or para positions is required. An additional ethylene linker between quinazolinone ring and phenolic substituent, present in the second series (compounds 25a and 25b), leads to increased antioxidant activity. Furthermore, in addition to antioxidant activity, the derivatives with two hydroxyl groups in the ortho position on the phenyl ring exhibited metal-chelating properties. Our study represents a successful use of three different antioxidant activity evaluation methods to define 2-(2,3-dihydroxyphenyl)quinazolin-4(3H)-one 21e as a potent antioxidant with promising metal-chelating properties.

Combining cross-coupling reaction and Knoevenagel condensation in the synthesis of glyco-BODIPY probes for DC-SIGN super-resolution bioimaging.

Lectins are involved in a wide range of carbohydrate mediated recognition processes. Therefore, the availability of highly performant fluorescent tools tailored for lectin targeting and able to efficiently track events related to such key targets is in high demand. We report here on the synthesis of the glyco-BODIPYs 1 and 2, based on the efficient combination of a Heck-like cross coupling and a Knoevenagel condensation, which revealed efficient in addressing lectins. In particular, glyco-BODIPY 1 has two glycosidase stable C-mannose residues, which act as DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) targeting modules. By using live-cell fluorescence microscopy, we proved that BODIPY-mannose 1 was efficiently taken up by immune cells expressing DC-SIGN receptors. Super-resolution stimulated emission depletion (STED) microscopy further revealed that the internalized 1 localized in membranes of endosomes, proving that 1 is a reliable tool also in STED applications. Of note, glyco-BODIPY 1 contains an aryl-azido group, which allows further functionalization of the glycoprobe with bioactive molecules, thus paving the way for the use of 1 for tracking lectin-mediated cell internalization in diverse biological settings.

Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors.

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. Selective inhibition of its catalytic activities is therefore a viable approach for the treatment of these diseases. However, the development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. We previously reported 7H-furo[3,2-g]chromen-7-one (psoralen)-based compounds with an oxathiazolone warhead as selective inhibitors of the chymotrypsin-like (ß5i) subunit of immunoproteasome. Here, we describe the influence of the electrophilic warhead variations at position 3 of the psoralen core on the inhibitory potencies. Despite mapping the chemical space with different warheads, all compounds showed decreased inhibition of the ß5i subunit of immunoproteasome in comparison to the parent oxathiazolone-based compound. Although suboptimal, these results provide crucial information about structure-activity relationships that will serve as guidance for the further design of (immuno)proteasome inhibitors.

Synthesis, In Vitro Antioxidant Properties and Distribution of a New Cyanothiophene-Based Phenolic Compound in Olive Oil-In-Water Emulsions.

We synthesized and determined the antioxidant activity and distribution of a new cyanothiophene-based compound, N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3,5-dihydroxybenzamide (SIM-53B), in intact stripped olive oil-in-water emulsion. The in vitro antioxidant properties of SIM-53B were evaluated and compared to those for Trolox and resveratrol. Addition of an emulsifier (Tween 20) creates a narrow region, the aqueous-oil interface, and the distribution of SIM-53B can be described by two partition constants: PWI (between aqueous/interfacial regions) and POI (between oil/interfacial regions). The effects of emulsifier concentration expressed in terms of the volume fraction, ΦI, and O/W ratio were also evaluated on its distribution. SIM-53B is predominantly distributed (>90%) in the interfacial region of 1:9 (O/W) olive oil-in-water emulsions at the lowest emulsifier volume fraction (ΦI = 0.005) and only a small fraction is located in the aqueous (<5%) and the oil (<5%) regions. Besides, the concentration of SIM-53B in the interfacial region of the emulsions is ~170-190-fold higher than the stoichiometric concentration, emphasizing the compartmentalization effects. Results suggest that the emulsifier volume fraction is a key parameter that may modulate significantly its concentration in the interface. Our study suggests that cyanothiophene-based compounds may be interesting additives for potential lipid protection in biomembranes or other lipid-based systems.

Degradation of polysorbates 20 and 80 catalysed by histidine chloride buffer.

Polysorbates are amphiphilic, non-ionic surfactants, and they represent one of the key components of biopharmaceuticals. They serve as stabilisers, and their degradation can cause particle formation, which has been an industry-wide issue over the past decade. To determine the influence of the buffers most frequently used in biopharmaceuticals on polysorbate degradation, an accelerated stability study was carried out using placebo formulations containing 0.02% polysorbates and 20 mM buffers (pH 5.5, 6.5). These included histidine chloride, sodium citrate, sodium succinate and sodium phosphate buffers. The rate of polysorbate degradation was highest in histidine chloride buffer, and therefore we further focused on the mechanism here. The predominant degradation pathway of polysorbates in this buffer was ester hydrolysis, catalysed by the imidazole moiety of the histidine. Interestingly, the presence of therapeutic proteins in the formulations slowed histidine-catalysed degradation of polysorbates in 50% of cases, with negligible degradation seen otherwise. This emphasises the complex nature of the interactions between the components of biopharmaceutical drug products. Nonetheless, there are disadvantages of using histidine chloride buffers in biopharmaceuticals that contain polysorbates. Careful consideration should be given to selection of excipients used in parenteral formulations, whereby compatibility between buffer and surfactant is of key importance.

The Structural Integrity of the Model Lipid Membrane during Induced Lipid Peroxidation: The Role of Flavonols in the Inhibition of Lipid Peroxidation.

The structural integrity, elasticity, and fluidity of lipid membranes are critical for cellular activities such as communication between cells, exocytosis, and endocytosis. Unsaturated lipids, the main components of biological membranes, are particularly susceptible to the oxidative attack of reactive oxygen species. The peroxidation of unsaturated lipids, in our case 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), induces the structural reorganization of the membrane. We have employed a multi-technique approach to analyze typical properties of lipid bilayers, i.e., roughness, thickness, elasticity, and fluidity. We compared the alteration of the membrane properties upon initiated lipid peroxidation and examined the ability of flavonols, namely quercetin (QUE), myricetin (MCE), and myricitrin (MCI) at different molar fractions, to inhibit this change. Using Mass Spectrometry (MS) and Fourier Transform Infrared Spectroscopy (FTIR), we identified various carbonyl products and examined the extent of the reaction. From Atomic Force Microscopy (AFM), Force Spectroscopy (FS), Small Angle X-Ray Scattering (SAXS), and Electron Paramagnetic Resonance (EPR) experiments, we concluded that the membranes with inserted flavonols exhibit resistance against the structural changes induced by the oxidative attack, which is a finding with multiple biological implications. Our approach reveals the interplay between the flavonol molecular structure and the crucial membrane properties under oxidative attack and provides insight into the pathophysiology of cellular oxidative injury.

Protein Formulations Containing Polysorbates: Are Metal Chelators Needed at All?

Proteins are prone to post-translational modifications at specific sites, which can affect their physicochemical properties, and consequently also their safety and efficacy. Sources of post-translational modifications include oxygen and reactive oxygen species. Additionally, catalytic amounts of Fe(II) or Cu(I) can promote increased activities of reactive oxygen species, and thus catalyse the production of particularly reactive hydroxyl radicals. When oxidative post-translational modifications are detected in the biopharmaceutical industry, it is common practice to add chelators to the formulation. However, the resultant complexes with metals can be even more damaging. Indeed, this is supported here using an ascorbate redox system assay and peptide mapping. Ethylenediaminetetraacetic acid (EDTA) addition strongly accelerated the formation of hydroxyl radicals in an iron-ascorbate system, while diethylenetriaminepentaacetic acid (DTPA) addition did not. When Fe(III) was substituted with Cu(II), EDTA addition almost stopped hydroxyl radical production, whereas DTPA addition showed continued production, but at a reduced rate. Further, EDTA accelerated metal-catalysed oxidation of proteins, and thus did not protect them from Fe-mediated oxidative damage. As every formulation is unique, justification for EDTA or DTPA addition should be based on experimental data and not common practice.

Rational design to biologics development: The polysorbates point of view.

Formulation development is an essential part of any biopharmaceuticals development programme, and this will affect quality, safety and efficacy of the final drug product. The vast majority of biopharmaceuticals on the market are therapeutic proteins; however, these are less stable compared to conventional pharmaceuticals. To counter aggregation, denaturation and surface adsorption of proteins in solution, surfactants are added to the formulations; however, the choice of the best formulation is a challenge that is faced during formulation development. Polysorbates are the most widely used surfactants in the pharmaceutical industry and are presented in >80% of commercial monoclonal antibody formulations. In this review, we provide a general overview of polysorbates and their issues, and the characteristics that have to be taken into account during formulation development. Degradation of polysorbates, namely by hydrolysis and/or oxidation, is one of the main concerns associated with their use. Furthermore, degradation of polysorbates is determined by formulation composition, pH and storage conditions, therefore underlining the importance and complexity of protein formulation development using polysorbates. A need-based approach should be used for correct selection of excipients in protein formulations that contain polysorbates.

Iron-Binding and Anti-Fenton Properties of Novel Amino Acid-Derived Cyclic Imide Dioximes.

We present a novel route for the preparation of amino acid-derived cyclic imide dioxime derivatives. Readily accessible amino acids were conveniently converted to their corresponding cyclic imide dioximes in simple synthetic steps. The aim of this work was to describe and compare the iron-chelating and antioxidant properties of synthesized compounds in relation to their molecular structure, and in particular, which of those features are essential for iron(II)-chelating ability. The glutarimide dioxime moiety has been established as an iron(II)-binding motif and imparts potent anti-Fenton properties to the compounds. Compound 3 was shown to strongly suppress hydroxyl radical formation by preventing iron cycling via Fe-complexation. These findings provide insights into the structural requirements for achieving anti-Fenton activity and highlight the potential use of glutarimide dioximes as antioxidants.

Comparison of the NMR and the Acid Value Determination Methods for Quality Control of Input Polysorbates.

Polysorbates (PS) are the most common non-ionic surfactants used in protein formulations. Their degradation has been studied intensively in recent years. Ester bond hydrolysis is one of many pathways of PS degradation that can lead to accumulation of free fatty acids (FFAs) and particle formation. The distribution and quantity of FFAs in PSs impacts directly on product quality. Characterization of input PS is highly relevant, because the initial content of FFAs differs greatly between manufacturers. The purpose of this study was to set up a quick and simple analytical method for the quantitative evaluation of FFAs in PS. The content of FFAs was measured for selected PS 20 and 80, using two methods, 1H nuclear magnetic resonance spectroscopy (1H NMR) and the European pharmacopoeia method for determining acid value (IA). These methods have been evaluated using the method of standard addition and, based on the results, they are interchangeable. It was concluded that 1H NMR is a useful tool for quality control of input PS and a rapid method for indicating the rate of PS degradation by hydrolysis and oxidation. Further, a newly discovered impurity in PS raw material, the long chain ketone 12-tricosanone, can be identified using 1H NMR.

A focused structure-activity relationship study of psoralen-based immunoproteasome inhibitors.

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. The development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. Here, we describe a focused series of psoralen-based inhibitors of the ß5i subunit of the immunoproteasome with different substituents placed at position 4'. The most promising compound was further evaluated through changes at position 3 of the psoralen ring. Despite a small decrease in the inhibitory potency in comparison with the parent compound, we were able to improve the selectivity against other subunits of both the immunoproteasome and the constitutive proteasome. The most potent compounds discriminated between both proteasome types in cell lysates and also showed a decrease in cytokine secretion in peripheral blood mononuclear cells.

Multi-target-directed ligands for treating Alzheimer's disease: Butyrylcholinesterase inhibitors displaying antioxidant and neuroprotective activities.

The limited clinical efficacy of current symptomatic treatment and minute effect on progression of Alzheimer's disease has shifted the research focus from single targets towards multi-target-directed ligands. Here, a potent selective inhibitor of human butyrylcholinesterase was used as the starting point to develop a new series of multifunctional ligands. A focused library of derivatives was designed and synthesised that showed both butyrylcholinesterase inhibition and good antioxidant activity as determined by the DPPH assay. The crystal structure of compound 11 in complex with butyrylcholinesterase revealed the molecular basis for its low nanomolar inhibition of butyrylcholinesterase (Ki = 1.09 ±â€¯0.12 nM). In addition, compounds 8 and 11 show metal-chelating properties, and reduce the redox activity of chelated Cu2+ ions in a Cu-ascorbate redox system. Compounds 8 and 11 decrease intracellular levels of reactive oxygen species, and are not substrates of the active efflux transport system, as determined in Caco2 cells. Compound 11 also protects neuroblastoma SH-SY5Y cells from toxic Aß1-42 species. These data indicate that compounds 8 and 11 are promising multifunctional lead ligands for treatment of Alzheimer's disease.

Microstructure evaluation of dermally applicable liquid crystals as a function of water content and temperature: Can electron paramagnetic resonance provide complementary data?

Insight into the microstructure of lyotropic liquid crystals (LCs) is of crucial importance for development of novel dermal delivery systems. Our aim was to evaluate the phase behaviour of dermally applicable LCs composed of isopropyl myristate/Tween 80/lecithin/water, along the dilution line, where phase transitions are predominantly driven by increased water content. Additionally, identification of LC temperature dependence is of great importance for skin application. Selected LCs were evaluated using electron paramagnetic resonance (EPR) plus conventionally used methods of polarization microscopy, small-angle X-ray scattering, differential scanning calorimetry, and rheological measurements. Depending on water content, LCs formed diverse microstructures, from (pseudo)hexagonal (LC1) and lamellar (LC2-LC7) liquid crystalline phases that possibly co-exist with rod-like micelles (LC4-LC7), to a transitional micellar phase (LC8). Furthermore, the LCs microstructure remained unaltered within the tested temperature range. EPR was shown to detect microstructural transitions of LCs and to provide complementary data to other techniques. These data thus confirm the applicability of EPR as a complementary technique for better understanding of LC microstructural transitions that are expected to contribute greatly to studies oriented towards the drug release characteristics from such systems.

Internalization and Accumulation in Dendritic Cells of a Small pH-Activatable Glycomimetic Fluorescent Probe as Revealed by Spectral Detection.

DC-SIGN, an antigen-uptake receptor in dendritic cells (DCs), has a clear role in the immune response but, conversely, can also facilitate infection by providing entry of pathogens into DCs. The key action in both processes is internalization into acidic endosomes and lysosomes. Molecular probes that bind to DC-SIGN could thus provide a useful tool to study internalization and constitute potential antagonists against pathogens. So far, only large molecules have been used to directly observe DC-SIGN-mediated internalization into DCs by fluorescence visualization. We designed and synthesized an appropriate small glycomimetic probe. Two particular properties of the probe were exploited: activation in a low-pH environment and an aggregation-induced spectral shift. Our results indicate that small glycomimetic molecules could compete with antigen/pathogen for binding not only outside but also inside the DC, thus preventing the harmful action of pathogens that are able to intrude into DCs, for example, HIV-1.

In Silico Discovery of Novel Potent Antioxidants on the Basis of Pulvinic Acid and Coumarine Derivatives and Their Experimental Evaluation.

A pigment from the edible mushroom Xerocomus badius norbadione A, which is a natural derivative of pulvinic acid, was found to possess antioxidant properties. Since the pulvinic acid represents a novel antioxidant scaffold, several other derivatives were recently synthetized and evaluated experimentally, along with some structurally related coumarine derivatives. The obtained data formed the basis for the construction of several quantitative structure-activity and pharmacophore models, which were employed in the virtual screening experiments of compound libraries and for the prediction of their antioxidant activity, with the goal of discovering novel compounds possessing antioxidant properties. A final prioritization list of 21 novel compounds alongside 8 established antioxidant compounds was created for their experimental evaluation, consisting of the DPPH assay, 2-deoxyribose assay, ß-carotene bleaching assay and the cellular antioxidant activity assay. Ten novel compounds from the tetronic acid and barbituric acid chemical classes displayed promising antioxidant activity in at least one of the used assays, that is comparable to or even better than some standard antioxidants. Compounds 5, 7 and 9 displayed good activity in all the assays, and were furthermore effective preventers of oxidative stress in human peripheral blood mononuclear cells, which are promising features for the potential therapeutic use of such compounds.

Structure-based development of nitroxoline derivatives as potential multifunctional anti-Alzheimer agents.

Tremendous efforts have been dedicated to the development of effective therapeutics against Alzheimer's disease, which represents the most common debilitating neurodegenerative disease. Multifunctional agents are molecules designed to have simultaneous effects on different pathological processes. Such compounds represent an emerging strategy for the development of effective treatments against Alzheimer's disease. Here, we report on the synthesis and biological evaluation of a series of nitroxoline-based analogs that were designed by merging the scaffold of 8-hydroxyquinoline with that of a known selective butyrylcholinesterase inhibitor that has promising anti-Alzheimer properties. Most strikingly, compound 8g inhibits self-induced aggregation of the amyloid beta peptide (Aß1-42), inhibits with sub-micromolar potency butyrylcholinesterase (IC50=215 nM), and also selectively complexes Cu(2+). Our study thus designates this compound as a promising multifunctional agent for therapeutic treatment of Alzheimer's disease. The crystal structure of human butyrylcholinesterase in complex with compound 8g is also solved, which suggests ways to further optimize compounds featuring the 8-hydroxyquinoline scaffold.

How perifosine affects liposome-encapsulated drug delivery across a cell barrier.

BACKGROUND: The development of efficient drug delivery systems to transport therapeutics across barrier-forming cells remains a challenge. Recently it was shown that liposomes containing perifosine, a synthetic analog of lysophosphatidylcholine, efficiently deliver liposome encapsulated content across barrier-forming cells. METHODS: To elucidate the mechanism of the delivery, fluorescent and spin labeled analog of perifosine were synthesized and their transport from liposomes to the barrier-forming MDKC cells was measured. RESULTS & CONCLUSION: Perifosine analogs are rapidly transported from liposomes into cell membranes. The total amount of perifosine accumulated in plasma membranes seems to be the most important factor in efficient transepithelial transport of liposome-encapsulated substances. Lysolipid-containing liposomal formulations seem to be promising candidates as drug delivery systems in general.