Author Report for: Zueva IV

One of the main problems in the treatment of poisoning with organophosphorus (OPs) inhibitors of acetylcholinesterase (AChE) is low ability of existing reactivators of AChE that are used as antidotes to cross the blood-brain barrier (BBB). In this work, modified cationic liposomes were developed that can penetrate through the BBB and deliver the reactivator of AChE pralidoxime chloride (2-PAM) into the brain. Liposomes were obtained on the basis of phosphatidylcholine and imidazolium surfactants. To obtain the composition optimized in terms of charge, stability, and toxicity, the molar ratio of surfactant/lipid was varied. For the systems, physicochemical parameters, release profiles of the substrates (rhodamine B, 2-PAM), hemolytic activity and ability to cause hemagglutination were evaluated. Screening of liposome penetration through the BBB, analysis of 2-PAM pharmacokinetics, and in vivo AChE reactivation showed that modified liposomes readily pass into the brain and reactivate brain AChE in rats poisoned with paraoxon (POX) by 25%. For the first time, an assessment was made of the ability of imidazolium liposomes loaded with 2-PAM to reduce the death of neurons in the brains of mice. It was shown that intravenous administration of liposomal 2-PAM can significantly reduce POX-induced neuronal death in the hippocampus.

A series of new compounds in which uracil and 3,6-dimethyluracil moieties are bridged with different spacers were prepared and evaluated in vitro for the acetyl- and butyrylcholinesterase (AChE and BChE) inhibitory activities. These bisuracils are shown to be very effective inhibitors of AChE, inhibiting the enzyme at nano- and lower molar concentrations with extremely high selectivity for AChE vs. BChE. Kinetic analysis showed that the lead compound 2h acts as a slow-binding inhibitor of AChE and possess a long drug-target residence time (tau = 1/k(off) = 18.6 +/- 7.5smin). Moreover, compound 2h ameliorated muscle weakness in myasthenia gravis rat model with a lower effective dose and longer lasting effect than pyridostigmine bromide. Besides, it was shown that compound 2h has an effect of increasing efficiency of antidotal therapy as a pretreatment for poisoning by organophosphates.

Novel derivatives based on 6-methyluracil and condensed uracil, 2,4-quinazoline-2,4-dione, were synthesized with terminal meta- and para-benzoate moieties in polymethylene chains at the N atoms of the pyrimidine ring. In the synthesized compounds, the polymethylene chains were varied from having tris- to hexamethylene chains and quaternary ammonium groups; varying substituents (ester, salt, acid) at benzene ring were introduced into the chains and benzoate moieties. In vivo biological experiments demonstrated the potency of these compounds in decreasing the number of beta-amyloid plaques and their suitability for the treatment of memory impairment in a transgenic model of Alzheimer's disease.

Chitosan-decorated liposomes were proposed for the first time for the intranasal delivery of acetylcholinesterase (AChE) reactivator pralidoxime chloride (2-PAM) to the brain as a therapy for organophosphorus compounds (OPs) poisoning. Firstly, the chitosome composition based on phospholipids, cholesterol, chitosans (Cs) of different molecular weights, and its arginine derivative was developed and optimized. The use of the polymer modification led to an increase in the encapsulation efficiency toward rhodamine B (RhB; ~85%) and 2-PAM (~60%) by 20% compared to conventional liposomes. The formation of monodispersed and stable nanosized particles with a hydrodynamic diameter of up to 130 nm was shown using dynamic light scattering. The addition of the polymers recharged the liposome surface (from -15 mV to +20 mV), which demonstrates the successful deposition of Cs on the vesicles. In vitro spectrophotometric analysis showed a slow release of substrates (RhB and 2-PAM) from the nanocontainers, while the concentration and Cs type did not significantly affect the chitosome permeability. Flow cytometry and fluorescence microscopy qualitatively and quantitatively demonstrated the penetration of the developed chitosomes into normal Chang liver and M-HeLa cervical cancer cells. At the final stage, the ability of the formulated 2-PAM to reactivate brain AChE was assessed in a model of paraoxon-induced poisoning in an in vivo test. Intranasal administration of 2-PAM-containing chitosomes allows it to reach the degree of enzyme reactivation up to 35 +/- 4%.

m-(Tert-butyl) trifluoroacetophenone (TFK), a slow-binding inhibitor of acetylcholinesterase (AChE), a transition state analog of acetylcholine, was investigated as a potential neuroprotectant of central and peripheral AChE against organophosphate paraoxon (POX) toxicity. Acute toxicity and pharmacological effects of TFK were investigated on mice and rats. Intraperitoneal administered TFK has low acute toxicity in mice (LD(50) = 19 mg/kg). Effects on motor function as investigated by rotarod and open field tests showed that TFK up to 5 mg/kg did not alter motor coordination and stereotypical exploration behavior of mice. Passive avoidance test showed that 1 or 5 mg/kg TFK restored memory impairment in scopolamine-induced Alzheimer's disease-like dementia in rats. Pretreatment of mice with 5 mg/kg TFK, 2-3 hrs before challenge by 2xLD(50) POX provided a modest and short protection against POX toxicity. Futhermore, analysis of POX-induced neuronal degeneration by using fluoro-jade B staining showed that TFK pretreatment, at the dose 5mg/kg before POX challenge, significantly reduced the density of apoptotic cells in hippocampus and entorhinal cortex of mice. Thus, TFK is capable of reducing POX-induced neurotoxicity.

New lipid-based nanomaterials and multi-target directed ligands (MTDLs) based on sterically hindered phenol, containing a quaternary ammonium moiety (SHP-s-R, with s = 2,3) of varying hydrophobicity (R = CH2Ph and CnH2n+1, with n = 8, 10, 12, 16), have been prepared as potential drugs against Alzheimer's disease (AD). SHP-s-R are inhibitors of human cholinesterases with antioxidant properties. The inhibitory potency of SHP-s-R and selectivity ratio of cholinesterase inhibition were found to significantly depend on the length of the methylene spacer (s) and alkyl chain length. The compound SHP-2-16 showed the best IC50 for human AChE and the highest selectivity, being 30-fold more potent than for human BChE. Molecular modeling of SHP-2-16 binding to human AChE suggests that this compound is a dual binding site inhibitor that interacts with both the peripheral anionic site and catalytic active site. The relationship between self-assembly parameters (CMC, solubilization capacity, aggregation number), antioxidant activity and a toxicological parameter (hemolytic action on human red blood cells) was investigated. Two sterically hindered phenols (SHP-2-Bn and SHP-2-R) were loaded into L-alpha-phosphatidylcholine (PC) nanoparticles by varying the SHP alkyl chain length. For the brain AChE inhibition assay, PC/SHP-2-Bn/SHP-2-16 nanoparticles were administered to rats intranasally at a dose of 8 mg kg-1. The Morris water maze experiment showed that scopolamine-induced AD-like dementia in rats treated with PC/SHP-2-Bn/SHP-2-16 nanoparticles was significantly reduced. This is the first example of cationic SHP-phospholipid nanoparticles for inhibition of brain cholinesterases realized by the use of intranasal administration. This route has promising potential for the treatment of AD.

The nanotechnological approach is an innovative strategy of high potential to achieve reactivation of organophosphorus-inhibited acetylcholinesterase in central nervous system. It was previously shown that pralidoxime chloride-loaded solid lipid nanoparticles (2-PAM-SLNs) are able to protect the brain against pesticide (paraoxon) central toxicity. In the present work, we increased brain AChE reactivation efficacy by PEGylation of 2-PAM-SLNs using PEG-lipid N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt) (DSPE-PEG(2000)) as a surface-modifier of SLNs. To perform pharmacokinetic study, a simple, sensitive (LLOQ 1.0 ng/ml) high-performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization by multiple reaction monitoring mode (HPLC-APCI-MS) was developed. The method was compared to mass spectrometry with electrospray ionization. The method was validated for linearity, accuracy, precision, extraction recovery, matrix effect and stability. Acetophenone oxime was used as the internal standard for the quantification of 2-PAM in rat plasma and brain tissue after intravenous administration. 2-PAM-DSPE-PEG(2000)-SLNs of mean size about 80 nm (PDI = 0.26), zeta-potential of -55 mV and of high in vitro stability, prolonged the elimination phase of 2-PAM from the bloodstream more than 3 times compared to free 2-PAM. An increase in reactivation of POX-inhibited human brain acetylcholinesterase up to 36.08 +/- 4.3% after intravenous administration of 2-PAM-DSPE-PEG(2000)-SLNs (dose of 2-PAM is 5 mg/kg) was achieved. The result is one of the first examples where this level of brain acetylcholinesterase reactivation was achieved. Thus, the implementation of different approaches for targeting and modifying nanoparticles' surface gives hope for improving the antidotal treatment of organophosphorus poisoning by marketed reactivators.

Organophosphorus (OP) compounds represent a serious health hazard worldwide. The dominant mechanism of their action results from covalent inhibition of acetylcholinesterase (AChE). Standard therapy of acute OP poisoning is partially effective. However, prophylactic administration of reversible or pseudo-irreversible AChE inhibitors before OP exposure increases the efficiency of standard therapy. The purpose of the study was to test the duration of the protective effect of a slow-binding reversible AChE inhibitor (C547) in a mouse model against acute exposure to paraoxon (POX). It was shown that the rate of inhibition of AChE by POX in vitro after pre-inhibition with C547 was several times lower than without C547. Ex vivo pre-incubation of mouse diaphragm with C547 significantly prevented the POX-induced muscle weakness. Then it was shown that pre-treatment of mice with C547 at the dose of 0.01 mg/kg significantly increased survival after poisoning by 2xLD(50) POX. The duration of the pre-treatment was effective up to 96 h, whereas currently used drug for pre-exposure treatment, pyridostigmine at a dose of 0.15 mg/kg was effective less than 24 h. Thus, long-lasting slow-binding reversible AChE inhibitors can be considered as new potential drugs to increase the duration of pre-exposure treatment of OP poisoning.

Hydroxyethyl bearing gemini surfactants, alkanediyl-alpha,-bis(N-hexadecyl-N-2-hydroxyethyl-N-methylammonium bromide), 16-s-16(OH), were used to augment phosphatidylcholine based liposomes to achieve higher stability and enhanced cellular uptake and penetration. The developed liposomes were loaded with rhodamine B, doxorubicin hydrochloride, pralidoxime chloride to investigate release properties, cytotoxicity in vitro, as well as ability to cross the blood-brain barrier. At molar ratios of 35:1 (lipid:surfactant) the formulation was found to be of low toxicity, stable for two months, and able to deliver rhodamine B beyond the blood-brain barrier in rats. In vivo, pharmacokinetics of free and formulated 2-PAM in plasma and brain were evaluated, liposomal 2-PAM was found to reactivate 27% of brain acetylcholinesterase, which is, to our knowledge, the first example of such high degree of reactivation after intravenous administration of liposomal drug.

In this study, novel derivatives based on 6-methyluracil and condensed uracil were synthesized, namely, 2,4-quinazoline-2,4-dione with -(ortho-nitrilebenzylethylamino) alkyl chains at the N atoms of the pyrimidine ring. In this series of synthesized compounds, the polymethylene chains were varied from having tetra- to hexamethylene chains, and secondary NH, tertiary ethylamino, and quaternary ammonium groups were introduced into the chains. The molecular modeling of the compounds indicated that they could function as dual binding site acetylcholinesterase inhibitors, binding to both the peripheral anionic site and active site. The data from in vitro experiments show that the most active compounds exhibit affinity toward acetylcholinesterase within a nanomolar range, with selectivity for acetylcholinesterase over butyrylcholinesterase reaching four orders of magnitude. In vivo biological assays demonstrated the potency of these compounds in the treatment of memory impairment using an animal model of Alzheimer disease.

Enzyme-catalyzed hydrolysis of echothiophate, a P-S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with Km = 0.20 +/- 0.03 mM and kcat = 5.4 +/- 1.6 min(-1). The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 +/- 0.2 mM; kcat = 53400 min(-1)). With a kcat/Km = (2.6 +/- 1.6) x 10(7) M(-1)min(-1), GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P-S bonded OPs by thiol-free OP hydrolases.

Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with K(i) = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with K(i)* = 0.53 nM. Reversible binding of TFK provides a long residence time, = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the "isomerization" step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called "transition state analog", followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer's disease and protection of central AChE against organophosphorus compounds.

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198.H438.E325) was replaced by S198.H438.N322E in silico. Molecular dynamics for 1.5 mus and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198.H438.N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

A new spectrofluorimetric method more sensitive than the Ellman method was developed for determination of both acetylcholinesterase and butyrylcholinesterase activity and for kinetic analysis of these enzymes and their mutants. Two selected mutants of human butyrylcholinesterase (E197Q and E197G) were included in this work. As for the Ellman's method, substrates are thiocholine esters, but the chromogenic reagent, DTNB (dithio-bisnitro benzoic acid) is replaced by a fluorogenic probe, "Calbiochem Probe IV", (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl)acrylic acid methylester). Compared to the classical Ellman's method, the sensitivity of this new spectrofluorimetric assay is 2 orders of magnitude higher. The method allows measurement of activity in media containing <10(-11)M of cholinesterase active sites at low substrate concentrations, either under first order conditions, [S]<

Novel ammonium and betaine derivatives of p-tert-butylthiacalix[4]arene in cone and 1,3-alternate conformation were synthesized with high yields for the first time. The obtained compounds form in water spherical nanoparticles. It was shown by molecular docking calculations and in vitro experiments that amino and betaine derivatives can inhibit acetylcholinesterase and butyrylcholinesterase on the level of pyridostigmine while the toxicity of the obtained compounds is much lower than that of pyridostigmine.

Multitarget ligands (MTL) based on sterically hindered phenol and containing a quaternary ammonium moiety (SHP-n-Q) were synthesized. These compounds are inhibitors of cholinesterases with antioxidant properties. The inhibitory selectivity is 10-fold potent for BChE than for AChE. IC50 of SHP-n-Q for BChE is 20muM. SHP-n-Q and their nanosystems exhibit more pronounced antioxidant properties than the synthetic antioxidant (hindered phenol, butylated hydroxytoluene). These compounds display a low hemolytic activity against human red blood cells. The nanotechnological approach was used to increase the bioavailability of SHP-n-Q derivatives. For water soluble SHP-n-Q derivative, the self-assembled structures have a size close to 100nmat critical association concentration (0.01M). Mixed cationic liposomes based on l-alpha-phosphatidylcholine and SHP-n-Q of 100nm diameter were prepared. The stability, encapsulation efficacy and release from liposomes of a model drug, Rhodamine B, depend on the structure of SHP-n-Q. Cationic liposomes based on l-alpha-phosphatidylcholine and SHP-3-Q show a good stability in time (1year) and a sustained release (>65h). They are promising templates for the development of anti-Alzheimer MT-drug delivery systems.

New uncharged conjugates of 6-methyluracil derivatives with imidazole-2-aldoxime and 1,2,4-triazole-3-hydroxamic acid units were synthesized and studied as reactivators of organophosphate-inhibited cholinesterase. Using paraoxon (POX) as a model organophosphate, it was shown that 6-methyluracil derivatives linked with hydroxamic acid are able to reactivate POX-inhibited human acetylcholinesterase (AChE) in vitro. The reactivating efficacy of one compound (5b) is lower than that of pyridinium-2-aldoxime (2-PAM). Meanwhile, unlike 2-PAM, in vivo study showed that the lead compound 5b is able: (1) to reactivate POX-inhibited AChE in the brain; (2) to decrease death of neurons and, (3) to prevent memory impairment in rat model of POX-induced neurodegeneration.

Profound synaptic dysfunction contributes to early loss of short-term memory in Alzheimer's disease. This study was set up to analyze possible neuroprotective effects of two dual binding site inhibitors of acetylcholinesterase (AChE), a new 6-methyluracil derivative, C-35, and the clinically used inhibitor donepezil. Crystal structure of the complex between human AChE and C-35 revealed tight contacts of ligand along the enzyme active site gorge. Molecular dynamics simulations indicated that the external flexible part of the ligand establishes multiple transient interactions with the enzyme peripheral anionic site. Thus, C-35 is a dual binding site inhibitor of AChE. In transgenic mice, expressing a chimeric mouse/human amyloid precursor protein and a human presenilin-1 mutant, C-35 (5mg/kg, i.p) and donepezil (0.75mg/kg, i.p) partially reversed synapse loss, decreased the number of amyloid plaques, and restored learning and memory. To separate temporal symptomatic therapeutic effects, associated with the increased lifetime of acetylcholine in the brain, from possible disease-modifying effect, an experimental protocol based on drug withdrawal from therapy was performed. When administration of C-35 and donepezil was terminated three weeks after the trial started, animals that were receiving C-35 showed a much better ability to learn than those who received vehicle or donepezil. Our results provide additional evidence that dual binding site inhibitors of AChE have Alzheimer's disease-modifying action.

New mixed cationic liposomes based on L-alpha-phosphatidylcholine and dihexadecylmethylhydroxyethylammonium bromide (DHDHAB) were designed to overcome the BBB crossing by using the intranasal route. Synthesis and self-assembly of DHDHAB were performed. A low critical association concentration (0.01 mM), good solubilization properties toward hydrophobic dye Orange OT and antimicrobial activity against gram-positive bacteria Staphylococcus aureus (MIC=7.8 mug mL(-1)) and Bacillus cereus (MIC=7.8 mug mL(-1)), low hemolytic activities against human red blood cells (less than 10%) were achieved. Conditions for preparation of cationic vesicles and mixed liposomes with excellent colloidal stability at room temperature were determined. The intranasal administration of rhodamine B-loaded cationic liposomes was shown to increase bioavailability into the brain in comparison to the intravenous injection. The cholinesterase reactivator, 2-PAM, was used as model drug for the loading in cationic liposomes. 2-PAM-loaded cationic liposomes displayed high encapsulation efficiency ( approximately 90%) and hydrodynamic diameter close to 100 nm. Intranasally administered 2-PAM-loaded cationic liposomes were effective against paraoxon-induced acetylcholinesterase inhibition in the brain. 2-PAM-loaded liposomes reactivated 12 +/- 1% of brain acetylcholinesterase. This promising result opens the possibility to use marketed positively charged oximes in medical countermeasures against organophosphorus poisoning for reactivation of central acetylcholinesterase by implementing a non-invasive approach, via the "nose-brain" pathway.

A novel approach for brain protection against poisoning by organophosphorus agents is developed based on the combination treatment of dual delivery of two oximes. Pralidoxime chloride (2-PAM) and a novel reactivator, 6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxy picolinaldehyde oxime (3-HPA), have been loaded in solid-lipid nanoparticles (SLNs) to offer distinct release profile and systemic half-life for both oximes. To increase the therapeutic time window of both oximes, SLNs with two different compartments were designed to load each respective drug. Oxime-loaded SLNs of hydrodynamic diameter between 100 and 160nm and negative zeta potential (-30 to -25mV) were stable for a period of 10months at 4 degrees C. SLNs displayed longer circulation time in the bloodstream compared to free 3-HPA and free 2-PAM. Oxime-loaded SLNs were suitable for intravenous (iv) administration. Paraoxon-poisoned rats (0.8xLD50) were treated with 3-HPA-loaded SLNs and 2-PAM+3-HPA-loaded SLNs at the dose of 3-HPA and 2-PAM of 5mg/kg. Brain AChE reactivation up to 30% was slowly achieved in 5h after administration of 3-HPA-SLNs. For combination therapy with two oximes, a time-dependent additivity and increased reactivation up to 35% were observed.

Non-selective inhibitors of cholinesterases (ChEs) are clinically used for treatment of myasthenia gravis (MG). While being generally safe, they cause numerous adverse effects including induction of hyperactivity of urinary bladder and intestines affecting quality of patients life. In this study we have compared two ChEs inhibitors, a newly synthesized compound C547 and clinically used pyridostigmine bromide, by their efficiency to reduce muscle weakness symptoms and ability to activate contractions of urinary bladder in a rat model of autoimmune MG. We found that at dose effectively reducing MG symptoms, C547 did not affect activity of rat urinary bladder. In contrast, at equipotent dose, pyridostigmine caused a significant increase in tonus and force of spontaneous contractions of bladder wall. We also found that this profile of ChEs inhibitors translates into the preparation of human urinary bladder. The difference in action observed for C547 and pyridostigmine we attribute to a high level of pharmacological selectivity of C547 in inhibiting acetylcholinesterase as compared to butyrylcholinesterase. These results raise reasonable hope that selective acetylcholinesterase inhibitors should show efficacy in treating MG in human patients with a significant reduction in adverse effects related to hyperactivation of smooth muscles.

C-547, a potent slow-binding inhibitor of acetylcholinesterase (AChE) was intravenously administered to rat (0.05mg/kg). Pharmacokinetic profiles were determined in blood and different organs: extensor digitorum longus muscle, heart, liver, lungs and kidneys as a function of time. Pharmacokinetics (PK) was studied using non-compartmental and compartmental analyses. A 3-compartment model describes PK in blood. Most of injected C-547 binds to albumin in the bloodstream. The steady-state volume of distribution (3800ml/kg) is 15 times larger than the distribution volume, indicating a good tissue distribution. C-547 is slowly eliminated (kel=0.17 h(-1); T1/2=4h) from the bloodstream. Effect of C-547 on animal model of myasthenia gravis persists for more than 72h, even though the drug is not analytically detectable in the blood. A PK/PD model was built to account for such a pharmacodynamical (PD) effect. Long-lasting effect results from micro-PD mechanisms: the slow-binding nature of inhibition, high affinity for AChE and long residence time on target at neuromuscular junction (NMJ). In addition, NMJ spatial constraints i.e. high concentration of AChE in a small volume, and slow diffusion rate of free C-547 out of NMJ, make possible effective rebinding of ligand. Thus, compared to other cholinesterase inhibitors used for palliative treatment of myasthenia gravis, C-547 is the most selective drug, displays a slow pharmacokinetics, and has the longest duration of action. This makes C-547 a promising drug leader for treatment of myasthenia gravis, and a template for development of other drugs against neurological diseases and for neuroprotection.

The role of water in oxime-mediated reactivation of phosphylated cholinesterases (ChEs) has been asked with recurrence. To investigate oximate water structure changes in this reaction, reactivation of paraoxon-inhibited human acetylcholinesterase (AChE) was performed by the oxime asoxime (HI-6) at different pH in the presence and absence of lyotropic salts: a neutral salt (NaCl), a strong chaotropic salt (LiSCN) and strong kosmotropic salts (ammonium sulphate and phosphate HPO(4)(2-)). At the same time, molecular dynamic (MD) simulations of enzyme reactivation under the same conditions were performed over 100 ns. Reactivation kinetics showed that the low concentration of chaotropic salt up to 75 mM increased the percentage of reactivation of diethylphosphorylated AChE whereas kosmotropic salts lead only to a small decrease in reactivation. This indicates that water-breaker salt induces destructuration of water molecules that are electrostricted around oximate ions. Desolvation of oximate favors nucleophilic attack on the phosphorus atom. Effects observed at high salt concentrations (>100 mM) result either from salting-out of the enzyme by kosmotropic salts (phosphate and ammonium sulphate) or denaturing action of chaotropic LiSCN. MDs simulations of diethylphosphorylated hAChE complex with HI-6 over 100 ns were performed in the presence of 100 mM (NH(4))(2)SO(4) and 50 mM LiSCN. In the presence of LiSCN, it was found that protein and water have a higher mobility, i.e. water is less organized, compared with the ammonium sulphate system. LiSCN favors protein solvation (hydrophobic hydration) and breakage of elelectrostricted water molecules around of oximate ion. As a result, more free water molecules participated to reaction steps accompanying oxime-mediated dephosphorylation.

Solid lipid nanoparticles (SLNs) are among the most promising nanocarriers to target the blood-brain barrier (BBB) for drug delivery to the central nervous system (CNS). Encapsulation of the acetylcholinesterase reactivator, pralidoxime chloride (2-PAM), in SLNs appears to be a suitable strategy for protection against poisoning by organophosphorus agents (OPs) and postexposure treatment. 2-PAM-loaded SLNs were developed for brain targeting and delivery via intravenous (iv) administration. 2-PAM-SLNs displayed a high 2-PAM encapsulation efficiency ( approximately 90%) and loading capacity (maximum 30.8 +/- 1%). Drug-loaded particles had a mean hydrodynamic diameter close to 100 nm and high negative zeta potential (-54 to -15 mV). These properties contribute to improve long-term stability of 2-PAM-SLNs when stored both at room temperature (22 degrees C) and at 4 degrees C, as well as to longer circulation time in the bloodstream compared to free 2-PAM. Paraoxon-poisoned rats (2 x LD50) were treated with 2-PAM-loaded SLNs at a dose of 2-PAM of 5 mg/kg. 2-PAM-SLNs reactivated 15% of brain AChE activity. Our results confirm the potential use of SLNs loaded with positively charged oximes as a medical countermeasure both for protection against OPs poisoning and for postexposure treatment.

Inhibition of human AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) by an alkylammonium derivative of 6-methyluracil, C-547, a potential drug for the treatment of MG (myasthenia gravis) was studied. Kinetic analysis of AChE inhibition showed that C-547 is a slow-binding inhibitor of type B, i.e. after formation of the initial enzyme.inhibitor complex (Ki=140 pM), an induced-fit step allows establishment of the final complex (Ki*=22 pM). The estimated koff is low, 0.05 min(-1) On the other hand, reversible inhibition of human BChE is a fast-binding process of mixed-type (Ki=1.77 muM; Ki'=3.17 muM). The crystal structure of mouse AChE complexed with C-547 was solved at 3.13 A resolution. The complex is stabilized by cation-pi, stacking and hydrogen-bonding interactions. Molecular dynamics simulations of the binding/dissociation processes of C-547 and C-35 (a non-charged analogue) to mouse and human AChEs were performed. Molecular modelling on mouse and human AChE showed that the slow step results from an enzyme conformational change that allows C-547 to cross the bottleneck in the active-site gorge, followed by formation of tight complex, as observed in the crystal structure. In contrast, the related non-charged compound C-35 is not a slow-binding inhibitor. It does not cross the bottleneck because it is not sensitive to the electrostatic driving force to reach the bottom of the gorge. Thus C-547 is one of the most potent and selective reversible inhibitors of AChE with a long residence time, tau=20 min, longer than for other reversible inhibitors used in the treatment of MG. This makes C-547 a promising drug for the treatment of this disease.

We report a novel class of carbamate-type ChE inhibitors, structural analogs of pyridostigmine. A small library of congeneric pyridoxine-based compounds was designed, synthesized and evaluated for AChE and BChE enzymes inhibition in vitro. The most active compounds have potent enzyme inhibiting activity with IC50 values in the range of 0.46-2.1muM (for AChE) and 0.59-8.1muM (for BChE), with moderate selectivity for AChE comparable with that of pyridostigmine and neostigmine. Acute toxicity studies using mice models demonstrated excellent safety profile of the obtained compounds with LD50 in the range of 22-326mg/kg, while pyridostigmine and neostigmine are much more toxic (LD50 3.3 and 0.51mg/kg, respectively). The obtained results pave the way to design of novel potent and safe cholinesterase inhibitors for symptomatic treatment of neuromuscular disorders.

This work highlights the H-function of Tb(III)-doped silica nanoparticles in aqueous solutions of acetic acid as a route to sense acetylcholinesterase-catalyzed hydrolysis of acetylcholine (ACh). The H-function results from H+-induced quenching of Tb(III)-centered luminescence due to protonation of Tb(III) complexes located close to silica/water interface. The H-function can be turned on/switched off by the concentration of complexes within core or nanoparticle shell zones, by the silica surface decoration and adsorption of both organic and inorganic cations on silica surface. Results indicate the optimal synthetic procedure for making nanoparticles capable of sensing acetic acid produced by enzymatic hydrolysis of acetylcholine. The H-function of nanoparticles was determined at various concentrations of ACh and AChE. The measurements show experimental conditions for fitting the H-function to Michaelis-Menten kinetics. Results confirm that reliable fluorescent monitoring AChE-catalyzed hydrolysis of ACh is possible through the H-function properties of Tb(III)-doped silica nanoparticles.

Novel 6-methyluracil derivatives with omega-(substituted benzylethylamino)alkyl chains at the nitrogen atoms of the pyrimidine ring were designed and synthesized. The numbers of methylene groups in the alkyl chains were varied along with the electron-withdrawing substituents on the benzyl rings. The compounds are mixed-type reversible inhibitors of cholinesterases, and some of them show remarkable selectivity for human acetylcholinesterase (hAChE), with inhibitory potency in the nanomolar range, more than 10 000-fold higher than that for human butyrylcholinesterase (hBuChE). Molecular modeling studies indicate that these compounds are bifunctional AChE inhibitors, spanning the enzyme active site gorge and binding to its peripheral anionic site (PAS). In vivo experiments show that the 6-methyluracil derivatives are able to penetrate the blood-brain barrier (BBB), inhibiting brain-tissue AChE. The most potent AChE inhibitor, 3 d (1,3-bis[5-(o-nitrobenzylethylamino)pentyl]-6-methyluracil), was found to improve working memory in scopolamine and transgenic APP/PS1 murine models of Alzheimer's disease, and to significantly decrease the number and area of beta-amyloid peptide plaques in the brain.

BACKGROUND: Alzheimer's disease (AD) is the major age-related progressive neurodegenerative disorder. The brain of AD patients suffers from loss of cholinergic neurons and decreased number of synapses [1]. AD is caused by an imbalance between Abeta production and clearance, resulting in increased amount of Abeta in various forms [2]. Reduction of Abeta production and increasing clearance of Abeta pathogenic forms are key targets in the development of potential therapeutic agents for AD treatment. Unfortunately, only nosotropic approaches for treatment of AD are currently effective in humans. These approaches mainly focus on the inhibition of brain acetyl-cholinesterase (AChE) to increase lifetime of cerebral acetylcholine [3]. It is important to emphasize that AChE itself promotes the formation of Abeta fibrils in vitro and Abeta plaques in the cerebral cortex of transgenic mouse models of AD [4]. This property of AChE results from interaction between Abeta and the peripheral anionic site of the enzyme (PAS) [5]. Dual binding site inhibitors of both catalytic active site (CAS) and PAS can simultaneously improve cognition and slow down the rate of Abeta-induced neural degeneration. Unfortunately, the assortment of AChE PAS ligands is still extremely limited. OBJECTIVE: To study putative advantages of AChE non-charged PAS inhibitors based on 6-methyluracil derivatives for the treatment of Alzheimer's disease.
METHODS: In vitro studies. Concentration of drug producing 50% of AChE/BuChE activity inhibition (IC50) was measured using the method of Ellman et al. [6]. Toxicological experiments were performed using IP injection of the different compounds in mice. LD50, dose (in mg/kg) causing lethal effects in 50% of animals was taken as a criterion of toxicity [7]. The ability of compound to block in vitro AChE-induced Abeta1-40 aggregation was studied using a thioflavin T (ThT) fluorescent probe [8].In vivo biological assays. For in vivo blood-brain barrier permeation assay brains were removed 30 min after IP injection of LD50 dose of tested compound injection. The inhibitory potency was measured using the method of Ellman.Scopolamine and transgenic models of AD were used to evaluate the influence of compound 35 on spatial memory performance.Water solution of scopolamine was injected to mice (ip) 20 minutes before starting memory test during 14 days [9]. Mice were assigned to 7 groups, including 4 groups receiving injection (ip) of compound in different dosages, donepezil-treated mice (donepezil is conventionally used to treat Alzheimer's disease), positive and negative control groups. Double transgenic (APP/PS1) mice expressing a chimeric mouse/human amyloid precursor protein and a mutant of human presenilin-1 [10] were assigned to 4 groups, including transgenic animals injected (ip) with compound 35 or donepezil solution, positive (transgenes injected with water) and negative (wild-type mice) controls.To evaluate spatial memory performance, mice were trained on a reward alternation task using a conventional T-maze [11]. The criterion for a mouse having learned the rewarded alternation task was 3 consecutive days of at least 5 correct responses out of the 6 free trials.For beta-amyloid peptide load was evaluated quantitatively as a number and summary area of Thioflavine S fluorescent spots in cerebral cortex and hippocampal images using Image J program. Statistical analyses were performed using the Mann-Whitney test.
RESULTS: We evaluated the acute toxicity of the most active compounds. The most potent AChE inhibitor compound 35 (IC50 (AChE) = 5 +/- 0.5 nM) exhibited the lowest LD50 values (51 mg/kg) and inhibited brain AChE by more than 71 +/- 1%. Compound 35 at 10 nM, exhibited a significant (35 +/- 9%) inhibitory activity toward human AChE-induced Abeta aggregation.Scopolamine injection induced significant decrease in correct choice percentage in T-maze, as well as decrease in percentage of mice reaching criterion for learning the task by day 14. This memory deficit was relieved to some extent either by compound 35 (5 mg/kg) or donepezil (reference compound) treatment (0.75 mg/kg). Interestingly, higher doses of compound 35 (10 and 15 mg/kg) produced less therapeutic effect on spatial memory deficit.Group of APP/PS1 mice showed 3 times lower percentage of reaching behavioral criterion and lower percentage of correct choice in T-maze alternation task comparing to WT mice, whereas compound 35 (5 mg/kg) or Donepezil treatment effectively improved these parameters in APP/PS1 mice.Compound 35 treatment (5 mg/kg) during 14 days significantly reduced percentage of summary area and number of beta-amyloid peptide (betaAP) deposits visualized in sections of cerebral cortex, dentate gyrus, and hippocampal CA3 area in APP/PS1 mice. The most prominent reduction of betaAP load by compound 35 treatment was found in CA3 area and cerebral cortex. Meanwhile, Donepezil treatment (1 mg/kg) during 14 days significantly reduced betaAP load in cerebral cortex but not in dentate gyrus and CA3 area.
CONCLUSIONS: Experiments showed that the most potent AChE inhibitor compound 35 (6-methyluracil derivative) permeated the blood-brain barrier, improved working memory in the APP/PS1 transgenic mice and significantly reduced the number and area of Abeta plaques in the brain. Thus, compound 35 is a promising candidate as a bi-functional inhibitor of AChE for treatment of AD.