Brominated oxime nucleophiles are efficiently reactivating cholinesterases inhibited by nerve agents.

Six novel brominated bis-pyridinium oximes were designed and synthesized to increase their nucleophilicity and reactivation ability of phosphorylated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Their pKa was valuably found lower to parent non-halogenated oximes. Stability tests showed that novel brominated oximes were stable in water, but the stability of di-brominated oximes was decreased in buffer solution and their degradation products were prepared and characterized. The reactivation screening of brominated oximes was tested on AChE and BChE inhibited by organophosphorus surrogates. Two mono-brominated oximes reactivated AChE comparably to non-halogenated analogues, which was further confirmed by reactivation kinetics. The acute toxicity of two selected brominated oximes was similar to commercially available oxime reactivators and the most promising brominated oxime was tested in vivo on sarin- and VX-poisoned rats. This brominated oxime showed interesting CNS distribution and significant reactivation effectiveness in blood. The same oxime resulted with the best protective index for VX-poisoned rats.

Direct Chiral Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Monoacylglycerol and Diacylglycerol Isomers for the Study of Lipase-Catalyzed Hydrolysis of Triacylglycerols.

The fast and selective separation method of intact monoacylglycerol (MG) and diacylglycerol (DG) isomers using chiral supercritical fluid chromatography-mass spectrometry (SFC-MS) was developed and employed to study lipase selectivity in the hydrolysis of triacylglycerols (TGs). The synthesis of 28 enantiomerically pure MG and DG isomers was performed in the first stage using the most commonly occurring fatty acids in biological samples such as palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and docosahexaenoic acids. To develop the SFC separation method, different chromatographic conditions such as column chemistry, mobile phase composition and gradient, flow rate, backpressure, and temperature were carefully assessed. Our SFC-MS method used a chiral column based on a tris(3,5-dimethylphenylcarbamate) derivative of amylose and neat methanol as a mobile phase modifier, which provides baseline separation of all the tested enantiomers in 5 min. This method was used to evaluate hydrolysis selectivity of lipases from porcine pancreas (PPL) and Pseudomonas fluorescens (PFL) using nine TGs differing in acyl chain length (14-22 carbon atoms) and number of double bonds (0-6) and three DG regioisomer/enantiomers as hydrolysis intermediate products. PFL exhibited preference of the fatty acyl hydrolysis from the sn-1 position of TG more pronounced for the substrates with long polyunsaturated acyls, while PPL did not show considerable stereoselectivity to TGs. Conversely, PPL preferred hydrolysis from the sn-1 position of prochiral sn-1,3-DG regioisomer, whereas PFL exhibited no preference. Both lipases showed selectivity for the hydrolysis of outer positions of DG enantiomers. The results show complex reaction kinetics of lipase-catalyzed hydrolysis given by different stereoselectivities for substrates.

Structure-Guided Design of N-Methylpropargylamino-Quinazoline Derivatives as Multipotent Agents for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a complex disease with an unknown etiology. Available treatments, limited to cholinesterase inhibitors and N-methyl-d-aspartate receptor (NMDAR) antagonists, provide symptomatic relief only. As single-target therapies have not proven effective, rational specific-targeted combination into a single molecule represents a more promising approach for treating AD, and is expected to yield greater benefits in alleviating symptoms and slowing disease progression. In the present study, we designed, synthesized, and biologically evaluated 24 novel N-methylpropargylamino-quinazoline derivatives. Initially, compounds were thoroughly inspected by in silico techniques determining their oral and CNS availabilities. We tested, in vitro, the compounds' effects on cholinesterases and monoamine oxidase A/B (MAO-A/B), as well as their impacts on NMDAR antagonism, dehydrogenase activity, and glutathione levels. In addition, we inspected selected compounds for their cytotoxicity on undifferentiated and differentiated neuroblastoma SH-SY5Y cells. We collectively highlighted II-6h as the best candidate endowed with a selective MAO-B inhibition profile, NMDAR antagonism, an acceptable cytotoxicity profile, and the potential to permeate through BBB. The structure-guided drug design strategy applied in this study imposed a novel concept for rational drug discovery and enhances our understanding on the development of novel therapeutic agents for treating AD.

A systematic evaluation of the cucurbit[7]uril pharmacokinetics and toxicity after a single dose and short-term repeated administration in mice.

Cucurbit[n]urils are macrocyclic compounds capable of forming host-guest complexes with different molecules. In this study, we focused on cucurbit[7]uril (CB[7]) safety and pharmacokinetics. We investigated CB[7] cytotocixity in human renal cells ACHN using the xCELLigence system. We also determined maximum tolerated doses (MTD) and no observed adverse effect levels (NOAEL) after intramuscular (i.m.), intraperitoneal (i.p.), and intragastric (i.g.) administration in mice using clinical observation, blood biochemistry, and histopathology. At NOAELs, we studied its pharmacokinetics in plasma and kidneys. Finally, we performed a 7 day repeated-dose toxicity study at 50% of NOAEL after i.p. administration, assaying CB[7] concentration in plasma, brain, kidney, and liver; we also assessed the liver and kidney histopathology. In vitro, CB[7] did not show toxicity up to 0.94 mg/mL. MTDs in vivo were set at 300, 350, and 600 mg/kg, and NOAEL were established at 150, 100, and 300 mg/kg after i.m., i.p., and i.g. administration, respectively. Parenteral administration produced tissue damage mainly to the kidney, while i.g. administration caused only minor liver damage. Parenteral CB[7] administration led to fast elimination from blood, accompanied with kidney accumulation; absorption from the gastrointestinal tract was minimal. Short repeated i.p. administration was well tolerated. After initial CB[7] accumulation in blood and kidney, the concentrations stabilised and decreased during the experiment. Approximately 3.6% of animals showed signs of nephrotoxicity. Although CB[7] appears to be a promising molecule, nephrotoxicity may be the most critical drawback of its parenteral use, because the kidney represents the main organ of its elimination.

Charged pyridinium oximes with thiocarboxamide moiety are equally or less effective reactivators of organophosphate-inhibited cholinesterases compared to analogous carboxamides.

The organophosphorus antidotes, so-called oximes, are able to restore the enzymatic function of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) via cleavage of organophosphate from the active site of the phosphylated enzyme. In this work, the charged pyridinium oximes containing thiocarboxamide moiety were designed, prepared and tested. Their stability and pKa properties were found to be analogous to parent carboxamides (K027, K048 and K203). The inhibitory ability of thiocarboxamides was found in low µM levels for AChE and high µM levels for BChE. Their reactivation properties were screened on human recombinant AChE and BChE inhibited by nerve agent surrogates and paraoxon. One thiocarboxamide was able to effectively restore function of NEMP- and NEDPA-AChE, whereas two thiocarboxamides were able to reactivate BChE inhibited by all tested organophosphates. These results were confirmed by reactivation kinetics, where thiocarboxamides were proved to be effective, but less potent reactivators if compared to carboxamides.

Privileged multi-target directed propargyl-tacrines combining cholinesterase and monoamine oxidase inhibition activities.

Twenty-four novel compounds bearing tetrahydroacridine and N-propargyl moieties have been designed, synthesised, and evaluated in vitro for their anti-cholinesterase and anti-monoamine oxidase activities. Propargyltacrine 23 (IC50 = 21 nM) was the most potent acetylcholinesterase (AChE) inhibitor, compound 20 (IC50 = 78 nM) showed the best inhibitory human butyrylcholinesterase (hBChE) profile, and ligand 21 afforded equipotent and significant values on both ChEs (human AChE [hAChE]: IC50 = 0.095 ± 0.001 µM; hBChE: IC50 = 0.093 ± 0.003 µM). Regarding MAO inhibition, compounds 7, 15, and 25 demonstrated the highest inhibitory potential towards hMAO-B (IC50 = 163, 40, and 170 nM, respectively). In all, compounds 7, 15, 20, 21, 23, and 25 exhibiting the most balanced pharmacological profile, were submitted to permeability and cell viability tests. As a result, 7-phenoxy-N-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroacridin-9-amine hydrochloride (15) has been identified as a permeable agent that shows a balanced pharmacological profile [IC50 (hAChE) = 1.472 ± 0.024 µM; IC50 (hBChE) = 0.659 ± 0.077 µM; IC50 (hMAO-B) = 40.39 ± 5.98 nM], and consequently, as a new hit-ligand that deserves further investigation, in particular in vivo analyses, as the preliminary cell viability test results reported here suggest that this is a relatively safe therapeutic agent.

Allogeneic Bone Impregnated with Biodegradable Depot Delivery Systems for the Local Treatment of Joint Replacement Infections: An In Vitro Study.

Although progress is evident in the effective treatment of joint replacement-related infections, it still remains a serious issue in orthopedics. As an example, the local application of antibiotics-impregnated bone grafts supplies the high drug levels without systemic side effects. However, antibiotics in the powder or solution form could be a risk for local toxicity and do not allow sustained drug release. The present study evaluated the use of an antibiotic gel, a water-in-oil emulsion, and a PLGA microparticulate solid dispersion as depot delivery systems impregnating bone grafts for the treatment of joint replacement-related infections. The results of rheological and bioadhesive tests revealed the suitability of these formulations for the impregnation of bone grafts. Moreover, no negative effect on proliferation and viability of bone marrow mesenchymal stem cells was detected. An ex vivo dissolution test of vancomycin hydrochloride and gentamicin sulphate from the impregnated bone grafts showed a reduced burst and prolonged drug release. The PLGA-based formulation proved to be particularly promising, as one-day burst release drugs was only 15% followed with sustained antibiotics release with zero-order kinetics. The results of this study will be the basis for the development of a new product in the Tissue Section of the University Hospital for the treatment of bone defects and infections of joint replacements.

Effects of Charged Oxime Reactivators on the HK-2 Cell Line in Renal Toxicity Screening.

Oxime cholinesterase reactivators (oximes) are used to counteract organophosphate intoxication. Charged oximes are administered via intramuscular or intravenous injection when the majority of dose is unmetabolized and is excreted as urine. In this study, the effects of selected double charged oximes were determined in the HK-2 cell line as a model for renal toxicity screening. Some effects on dehydrogenase activity were found for obidoxime, asoxime (syn. HI-6), K027, and K203. The effects of K868 and K869 were found to be unreliable due to rapid degradation of both chlorinated oximes in the assay medium, resulting for K868 in an isoxazole-pyridinium product.

Effect of Oxime Encapsulation on Acetylcholinesterase Reactivation: Pharmacokinetic Study of the Asoxime-Cucurbit[7]uril Complex in Mice Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Oxime-based molecules are used for the treatment of patients to reactivate acetylcholinesterase (AChE) function after organophosphate intoxication. However, their efficacy is limited by low penetration through the blood-brain barrier and fast elimination. In this work, the cucurbit[7]uril (CB[7]) carrier was used for the encapsulation of the clinical agent asoxime to enhance brain bioavailability and the treatment window. We present a pharmacokinetic study of asoxime and the asoxime-CB[7] complex in an in vivo mouse model. Ultrahigh-performance liquid chromatography with electrospray ionization-mass spectrometry detection was developed to determine asoxime and CB[7] in biological fluids and tissues after thorough optimization of chromatographic conditions. The dihydroxypropane-silica stationary phase using hydrophilic interaction liquid chromatography conditions provided the best chromatographic performance. The final method was validated and applied for the pharmacokinetic study of mouse plasma, urine, bile, liver, kidney, and brain samples at different times after administration of asoxime and the asoxime-CB[7] complex. The results showed a greater than 3-fold increase in the area under the curve (AUC) in the brain for asoxime administered as a complex with CB[7] relative to that for the administration of asoxime alone. The effectiveness of the treatment strategy was evaluated using a reactivation study and a functional observatory battery. Protection of brain AChE activity is crucial for saving human lives or reducing the consequences of poisoning. The asoxime administered as a complex increased the brain activity by approximately 30% compared to that with atropine alone. CB[7] coadministration improved the AChE activity by 11%, which agrees with the higher asoxime AUC assessed in the pharmacokinetic study.

Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates.

The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.

Design, synthesis, and in vitro evaluation of BP-1-102 analogs with modified hydrophobic fragments for STAT3 inhibition.

Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed and synthesised with the aim to modify hydrophobic fragments of the molecules that are important for interaction with the STAT3 SH2 domain. The cytotoxic activity of the reference and novel compounds was evaluated using several human and two mouse cancer cell lines. BP-1-102 and its two analogs emerged as effective cytotoxic agents and were further tested in additional six human and two murine cancer cell lines, in all of which they manifested the cytotoxic effect in a micromolar range. Reference compound S3I-201.1066 was found ineffective in all tested cell lines, in contrast to formerly published data. The ability of selected BP-1-102 analogs to induce apoptosis and inhibition of STAT3 receptor-mediated phosphorylation was confirmed. The structure-activity relationship confirmed a demand for two hydrophobic substituents, i.e. the pentafluorophenyl moiety and another spatially bulky moiety, for effective cytotoxic activity and STAT3 inhibition.

Complex Analysis of Antioxidant Activity, Abscisic Acid Level, and Accumulation of Osmotica in Apple and Cherry In Vitro Cultures under Osmotic Stress.

Plant response to osmotic stress is a complex issue and includes a wide range of physiological and biochemical processes. Extensive studies of known cultivars and their reaction to drought or salinity stress are very important for future breeding of new and tolerant cultivars. Our study focused on the antioxidant activity, accumulations of osmotica, and the content of abscisic acid in apple (cv. "Malinové holovouské", "Fragrance", "Rubinstep", "Idared", "Car Alexander") and cherry (cv. "Regina", "Napoleonova", "Kastánka", "Sunburst", "P-HL-C") cultivated in vitro on media containing different levels of polyethylene glycol PEG-6000. Our results indicated that the studied genotypes responded differently to osmotic stress manifested as reduction in the leaf relative water content (RWC) and increment in the activities of antioxidant enzymes, proline, sugars, and abscisic acid content. Overall, cherry cultivars showed a smaller decrease in percentage RWC and enzymatic activities, but enhanced proline content compared to the apple plants cultivars. Cultivars "Rubinstep", "Napoleonova", and "Kastánka" exhibited higher antioxidant capacity and accumulation of osmoprotectants like proline and sorbitol that can be associated with the drought-tolerance system.

Amaryllidaceae Alkaloids of Norbelladine-Type as Inspiration for Development of Highly Selective Butyrylcholinesterase Inhibitors: Synthesis, Biological Activity Evaluation, and Docking Studies.

Alzheimer's disease (AD) is a multifactorial neurodegenerative condition of the central nervous system (CNS) that is currently treated by cholinesterase inhibitors and the N-methyl-d-aspartate receptor antagonist, memantine. Emerging evidence strongly supports the relevance of targeting butyrylcholinesterase (BuChE) in the more advanced stages of AD. Within this study, we have generated a pilot series of compounds (1-20) structurally inspired from belladine-type Amaryllidaceae alkaloids, namely carltonine A and B, and evaluated their acetylcholinesterase (AChE) and BuChE inhibition properties. Some of the compounds exhibited intriguing inhibition activity for human BuChE (hBuChE), with a preference for BuChE over AChE. Seven compounds were found to possess a hBuChE inhibition profile, with IC50 values below 1 µM. The most potent one, compound 6, showed nanomolar range activity with an IC50 value of 72 nM and an excellent selectivity pattern over AChE, reaching a selectivity index of almost 1400. Compound 6 was further studied by enzyme kinetics, along with in-silico techniques, to reveal the mode of inhibition. The prediction of CNS availability estimates that all the compounds in this survey can pass through the blood-brain barrier (BBB), as disclosed by the BBB score.

1,2,4-Triazole-based anticonvulsant agents with additional ROS scavenging activity are effective in a model of pharmacoresistant epilepsy.

There are numerous studies supporting the contribution of oxidative stress to the pathogenesis of epilepsy. Prolonged oxidative stress is associated with the overexpression of ATP-binding cassette transporters, which results in antiepileptic drugs resistance. During our studies, three 1,2,4-triazole-3-thione derivatives were evaluated for the antioxidant activity and anticonvulsant effect in the 6 Hz model of pharmacoresistant epilepsy. The investigated compounds exhibited 2-3 times more potent anticonvulsant activity than valproic acid in 6 Hz test in mice, which is well-established preclinical model of pharmacoresistant epilepsy. The antioxidant/ROS scavenging activity was confirmed in both single-electron transfer-based methods (DPPH and CUPRAC) and during flow cytometric analysis of total ROS activity in U-87 MG cells. Based on the enzymatic studies on human carbonic anhydrases (CAs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), one can assume that the herein investigated drug candidates will not impair the cognitive processes mediated by CAs and will have minimal off-target cholinergic effects.

Synthesis, in vitro screening and molecular docking of isoquinolinium-5-carbaldoximes as acetylcholinesterase and butyrylcholinesterase reactivators.

The series of symmetrical and unsymmetrical isoquinolinium-5-carbaldoximes was designed and prepared for cholinesterase reactivation purposes. The novel compounds were evaluated for intrinsic acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) inhibition, when the majority of novel compounds resulted with high inhibition of both enzymes and only weak inhibitors were selected for reactivation experiments on human AChE or BChE inhibited by sarin, VX, or paraoxon. The AChE reactivation for all used organophosphates was found negligible if compared to the reactivation ability of obidoxime. Importantly, two compounds were found to reactivate BChE inhibited by sarin or VX better to obidoxime at human attainable concentration. One compound resulted as better reactivator of NEMP (VX surrogate)-inhibited BChE than obidoxime. The in vitro results were further rationalized by molecular docking studies showing future directions on designing potent BChE reactivators.

Acetylcholinesterase Inhibition of Diversely Functionalized Quinolinones for Alzheimer's Disease Therapy.

In this communication, we report the synthesis and cholinesterase (ChE)/monoamine oxidase (MAO) inhibition of 19 quinolinones (QN1-19) and 13 dihydroquinolinones (DQN1-13) designed as potential multitarget small molecules (MSM) for Alzheimer's disease therapy. Contrary to our expectations, none of them showed significant human recombinant MAO inhibition, but compounds QN8, QN9, and DQN7 displayed promising human recombinant acetylcholinesterase (hrAChE) and butyrylcholinesterase (hrBuChE) inhibition. In particular, molecule QN8 was found to be a potent and quite selective non-competitive inhibitor of hrAChE (IC50 = 0.29 µM), with Ki value in nanomolar range (79 nM). Pertinent docking analysis confirmed this result, suggesting that this ligand is an interesting hit for further investigation.

Donepezil + chromone + melatonin hybrids as promising agents for Alzheimer's disease therapy.

We describe herein the design, multicomponent synthesis and biological studies of new donepezil + chromone + melatonin hybrids as potential agents for Alzheimer's disease (AD) therapy. We have identified compound 14n as promising multitarget small molecule showing strong BuChE inhibition (IC50 = 11.90 ± 0.05 nM), moderate hAChE (IC50 = 1.73 ± 0.34 µM), hMAO A (IC50 = 2.78 ± 0.12 µM), and MAO B (IC50 = 21.29 ± 3.85 µM) inhibition, while keeping a strong antioxidant power (3.04 TE, ORAC test). Consequently, the results reported here support the development of new multitarget Donepezil + Chromone + Melatonin hybrids, such as compound 14n, as a potential drug for AD patients cure.

Improved detection of ß-N-methylamino-L-alanine using N-hydroxysuccinimide ester of N-butylnicotinic acid for the localization of BMAA in blue mussels (Mytilus edulis).

ß-N-Methylamino-L-alanine (BMAA) is an important non-protein amino acid linked to neurodegenerative diseases, specifically amyotrophic lateral sclerosis (ALS). Because it can be transferred and bioaccumulated higher up the food chain, it poses significant public health concerns; thus, improved detection methods are of prime importance for the identification and management of these toxins. Here, we report the successful use of N-hydroxysuccinimide ester of N-butylnicotinic acid (C4-NA-NHS) for the efficient separation of BMAA from its isomers and higher sensitivity in detecting BMAA compared to the current method of choice using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization. Implementation of this efficient method allowed localization of BMAA in the non-visceral tissues of blue mussels, suggesting that more efficient depuration may be required to remove this toxin prior to consumption. This is a crucial method in establishing the absence or presence of the neurotoxic amino acid BMAA in food, environmental or biomedical samples.

Formulation study of PLGA in situ films for topical delivery of salicylates.

A film-forming system (FFS) represents a convenient topical dosage form for drug delivery. In this study, a non-commercial poly(lactic-co-glycolic acid) (PLGA) was chosen to formulate an FFS containing salicylic acid (SA) and methyl salicylate (MS). This unique combination is advantageous from a therapeutic point of view, as it enabled modified salicylate release. It is beneficial from a technological perspective too, because it improved thermal, rheological, and adhesive properties of the in situ film. DSC revealed complete dissolution of SA and good miscibility of MS with the polymer. MS also ensures optimal viscoelastic and adhesive properties of the film, leading to prolonged and sustained drug release. The hydrolysis of MS to active SA was very slow at skin pH 5.5, but it apparently occurred at physiological pH 7.4. The film structure is homogeneous without cracks, unlike some commercial preparations. The dissolution study of salicylates revealed different courses in their release and the influence of MS concentration in the film. The formulated PLGA-based FFS containing 5 % SA and 10 % MS is promising for sustained and prolonged local delivery of salicylates, used mainly for keratolytic and anti-inflammatory actions and pain relief.

Towards cost-effective drug discovery: Reusable immobilized enzymes for neurological disease research.

Enzyme handling and utilization bears many challenges such as their limited stability, intolerance of organic solvents, high cost, or inability to reuse. Most of these limitations can be overcome by enzyme immobilization on the surface of solid support. In this work, the recombinant form of human cholinesterases and monoamine oxidases as important drug targets for neurological diseases were immobilized on the surface of magnetic non-porous microparticles by a non-covalent bond utilizing the interaction between a His-tag terminus on the recombinant enzymes and cobalt (Co2+) ions immobilized on the magnetic microparticles. This type of binding led to targeted enzyme orientation, which completely preserved the catalytic activity and allowed high reproducibility of immobilization. In comparison with free enzymes, the immobilized enzymes showed exceptional stability in time and the possibility of repeated use. Relevant Km, Vmax, and IC50 values using known inhibitors were obtained using particular immobilized enzymes. Such immobilized enzymes on magnetic particles could serve as an excellent tool for a sustainable approach in the early stage of drug discovery.