Colorimetric detection of acetylcholinesterase and its inhibitor based on thiol-regulated oxidase-like activity of 2D palladium square nanoplates on reduced graphene oxide.

A convenient and sensitive colorimetric assay for acetylcholinesterase (AChE) and its inhibitor has been designed based on the oxidase-like activity of {100}-faceted Pd square nanoplates which are grown in situ on reduced graphene oxide (PdSP@rGO). PdSP@rGO can effectively catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) without the assistance of H2O2 to generate blue oxidized TMB (oxTMB) with a characteristic absorption peak at 652 nm. In the presence of AChE, acetylthiocholine (ATCh), a typical AChE substrate, is hydrolyzed to thiocholine (TCh). The generated TCh can effectively inhibit the PdSP@rGO-triggered chromogenic reaction of TMB via cheating with Pd, resulting in color fading and decrease in absorbance. Thus, a sensitive probe for AChE activity is constructed with a working range of 0.25-5 mU mL-1 and  a limit of detection (LOD) of 0.0625 mU mL-1. Furthermore, because of the inhibition effect of tacrine on AChE, tacrine is also detected through the colorimetric AChE assay system within the concentrations range 0.025-0.4 µM with a LOD of 0.00229 µM. Hence, a rapid and facile colorimetric procedure to sensitively detect AChE and its inhibitor can be anticipated through modulating the oxidase-like activity of PdSP@rGO. Colorimetric method for detection of AChE and its inhibitor is established by modulating the oxidase mimetic activity of {100}-faceted Pd square nanoplates on reduced graphene oxide (PdSP@rGO).

Physicochemical evaluation of new tetrahydroacridine and iodobenzoic acid hybrids as the next step in the design of potential drugs for treating Alzheimer's disease.

Tacrine derivatives containing iodobenzoic acid were developed as a novel multitarget-directed ligand and find potential application in the treatment of Alzheimer's disease. The aim of this study is to perform a physicochemical profile of this series. Experimental log P and pKa values were determined and compared with those already calculated. The results indicated better values of the tested compounds than the values predicted using computer software. The stability report was obtained using the developed HPLC method. The stability assay in different environment conditions provided information about the photosensitivity of these compounds and a proper method for the storage of this series of compounds.

Colorimetric assay of acetylcholinesterase inhibitor tacrine based on MoO2 nanoparticles as peroxidase mimetics.

Molybdenum dichalcogenides MoX2 (X=S, Se) have been found to possess intrinsic peroxidase-like activity. However, molybdenum oxides (MoO2) as peroxidase mimetics have not been exploited yet. Herein, MoO2 nanoparticles were synthesized by a simple hydrothermal method and found to possess the peroxidase-like activity for the first time. MoO2 nanoparticles could catalyze the oxidation of 3,3',5,5'-tetrametylbenzidine (TMB) by H2O2 to produce a blue-color product (oxTMB). The catalytic property and mechanism were investigated by stead-state kinetics experiment and free radicals scavenging experiment, respectively. Acetylcholinesterase (AChE) could catalyze the hydrolysis of acetylthiocholine chloride (ATCh) into thiocholine (TCh), which could reduce oxTMB to decrease the absorbance in solution. In the presence of AChE inhibitor tacrine, the generation of TCh was inhibited and the absorbance was preserved. Based on these properties, a colorimetric assay method was developed for AChE inhibitor tacrine. This work not only broadens the application of the peroxidase mimetics, but also overcome the disadvantages of traditional methods such as expensive, complex and vulnerable to background interference for colorimetric assay of AChE inhibitor.

Molecular imaging identifies age-related attenuation of acetylcholine in retrosplenial cortex in response to acetylcholinesterase inhibition.

The neurotransmitter of the cholinergic system, acetylcholine plays a major role in the brain's cognitive function and is involved in neurodegenerative disorders. Here, we present age-related alterations of acetylcholine levels after administration of the acetylcholinesterase inhibitor drug tacrine in normal mice. Using a quantitative, robust and molecular-specific mass spectrometry imaging method we found that tacrine administration significantly raised acetylcholine levels in most areas of sectioned mice brains, inter alia the striatum, hippocampus and cortical areas. However, acetylcholine levels in retrosplenial cortex were significantly lower in 14-month-old than in 12-week-old animals following its administration, indicating that normal aging affects the cholinergic system's responsivity. This small brain region is interconnected with an array of brain networks and is involved in numerous cognitive tasks. Simultaneous visualization of distributions of tacrine and its hydroxylated metabolites in the brain revealed a significant decrease in levels of the metabolites in the 14-month-old mice. The results highlight strengths of the imaging technique to simultaneously investigate multiple molecular species and the drug-target effects in specific regions of the brain. The proposed approach has high potential in studies of neuropathological conditions and responses to neuroactive treatments.

Development of a biosensing system for tacrine based on nitrogen-doped graphene quantum dots and acetylcholinesterase.

This work presents a novel fluorescent sensor for the determination of tacrine by combining the magnificent fluorescence properties of nitrogen-doped graphene quantum dots (N-GQDs) with the high potential of acetylcholinesterase (AChE) enzyme for screening its inhibitors. Tacrine was the first drug approved for Alzheimer's disease and it is currently being used in several therapeutic treatments given its activity as a reversible inhibitor of AChE. The principle of the developed biosensor relies on the fact that the native fluorescence of the synthesized N-GQDs is quenched by interaction with enzymatic reaction products, and the inclusion of tacrine in assay solution results in the gradual recovery of the original fluorescence in an inhibitor concentration-dependent manner. While N-GQD fluorescence was not directly affected by tacrine, the inclusion of an AChE based-enzymatic system allowed for its determination with a detection limit (S/N = 3) of 1.22 µM. This biosensor was demonstrated to be simple, rapid and reproducible (%RSD 4.87, n = 7) for analysis of tacrine in aqueous solutions.

Affinity binding-guided fluorescent nanobiosensor for acetylcholinesterase inhibitors via distance modulation between the fluorophore and metallic nanoparticle.

The magnitude of fluorescence enhancement was found to depend strongly on the distance between fluorophores and metal nanostructures in metal-enhanced fluorescence (MEF). However, the precise placement of the particle in front of the molecule with nanometer accuracy and distance control is a great challenge. We describe a method using acetylcholinesterase (AChE) to modulate the distance between a gold nanoparticle (AuNP) and the fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). We found that DDAO is a reversible mixed type-I AChE inhibitor. DDAO binds to the peripheral anionic site and penetrates into the active gorge site of AChE via inhibition kinetics test and molecular docking study. The affinity ligand DDAO bound to AChE which was immobilized onto AuNPs, and its fluorescence was sharply enhanced due to MEF. The fluorescence was reduced by distance variations between the AuNP and DDAO, which resulted from other inhibitors competitively binding with AChE and partly or completely displacing DDAO. Experimental results show that changes in fluorescence intensity are related to the concentration of inhibitors present in the solution. In addition, the nanobiosensor has high sensitivity, with detection limits as low as 0.4 µM for paraoxon and 10 nM for tacrine, and also exhibits different reduction efficiencies for the two types of inhibitor. Thus, instead of an inhibition test, a new type of affinity binding-guided fluorescent nanobiosensor was fabricated to detect AChE inhibitors, determine AChE inhibitor binding mode, and screen more potent AChE inhibitors. The proposed strategy may be applied to other proteins or protein domains via changes in the affinity ligand.

Rapid determination of tacrine and other drug metabolites in microsomal incubate by newly developed targeting algorithm on UHPLC/TOFMS.

A rapid simultaneous determination method for in vitro Cytochrome P450 (CYP) activity assay of 1,2,3,4-tetrahydroacridin-9-amine (tacrine) metabolites using ultra high performance liquid chromatography (UHPLC) coupled with computer-assisted in-source collision induced dissociation (CID) monitoring was investigated. In general, enzyme inhibition assays require quantitative analysis of incubates with drugs using various concentrations of substrates/inhibitors. The assay of CYP isozyme inhibition is an important informational step in the drug discovery process and, with the many substrates listed by the FDA, high-throughput qualitative and quantitative analyses are desired. Based on sub-2-micron packing material with reversed phase chromatography combined with a single liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS), a less than 1min analysis time is presented for two additional drugs. We successfully determined seven of the eight potential isomer metabolites for the drug tacrine in 2.5min using a 2mm internal diameterx100mm length column and applying in-source CID with our newly developed chromatographic peak deconvolution technique. Although two of the peaks were heavily fused at a peak width of less than 300ms, we could clearly identify these peaks by monitoring the chromatographic intensity difference of their fragment peaks on the mass spectrum.

New performance of biosensor technology for Alzheimer's disease drugs: in vitro comparison of tacrine and 7-methoxytacrine.

Two drugs were tested using electrochemical biosensor with immobilized acetylcholinesterase (AChE). The first was commercialized drug tacrine (known also as Cognex) used for treatment of cognitive manifestation of Alzheimer\'s disease (AD). The second one was its 7-methoxy derivate (7-MEOTA) that has not been marketed. We determined the IC50 (6.67+/-0.92)x10-7 M for tacrine and (1.66+/-1.43)x10-9 M for 7-MEOTA. In this in vitro study, 7-MEOTA acts as stronger inhibitor of AChE and in this way could be more favorable for treatment of cognitive manifestation of AD. Our study shows that biosensor technology could be used as a quick and cheap tool for testing of promising AChE inhibitors (AD drug candidates).

Rapid method for evaluation of cholinesterase inhibitors.

The fluorescence intensity of reversible inhibitor ethidium bromide fluorophore complex with equine blood butyryl cholinesterase decreases in the presence of inhibitor (tacrine) not fluorescing in the visible spectrum. An express method for tacrine evaluation is developed.

Determination of tacrine metabolites in microsomal incubate by high performance liquid chromatography-nuclear magnetic resonance/mass spectrometry with a column trapping system.

A column trapping system has been incorporated into high performance liquid chromatography-nuclear magnetic resonance-mass spectrometry (HPLC-NMR-MS) to reduce data acquisition time of NMR experiments. The system uses a trapping column to capture analytes after the HPLC column and back flush trapped analyte to the flow cell of the NMR probe for detection. A dilution solvent is mixed with eluent from HPLC column to reduce the influence of the organic content in the mobile phase before column trapping. The trapping column is also coupled with a mass spectrometer (MS) to get complementary MS data on the same peak. Studies on 1-hydroxylated 9-amino-1,2,3,4-tetrahydro-acridine (1-OH tacrine), indomethacin and testosterone with the column trapping system showed good recovery of analytes and over 3-fold mean increase in UV-VIS signal intensity. The time saving on NMR experiments with the column trapping system was demonstrated by the analysis of dog microsomal incubate with tacrine.

Electrochemical oxidation at carbon paste electrode of tacrine and 1-hydroxytacrine and differential pulse voltammetric determination of tacrine in pharmaceuticals and human urine.

The electrochemical oxidation of tacrine and its 1-OH-metabolite, has been studied by cyclic voltammetry and differential pulse voltammetry by using carbon paste electrodes. The peak current-concentration relationship was found to be linear up to 20 micrograms ml-1 with detection limits of 0.06 microgram ml-1 for tacrine and 0.18 microgram ml-1 for 1-OH-tacrine and quantitation limits of 0.20 microgram ml-1 for tacrine and 0.37 microgram ml-1 for 1-OH-tacrine. A method for determining tacrine by differential pulse voltammetry in pharmaceuticals and human urine, in the presence of 1-OH-tacrine, has been developed.

Spectrofluorimetric determination of tacrine in pharmaceuticals and spiked human serum.

A spectrofluorimetric method to determine tacrine is proposed and applied to the determination of tacrine in human serum and pharmaceuticals. The fluorimetric method allows the determination of 1-70 ng ml-1 of tacrine in aqueous solutions containing acetic acid-sodium acetate buffer (pH 5.6) with lambda exc = 242 nm and lambda em = 362 nm.

Capillary zone electrophoretic determination of some drugs against Alzheimer's disease.

A new capillary zone electrophoresis (CZE) method for the determination of tacrine (THA), 7-methoxytacrine (7-MTHA) and their basic metabolites (THAm, 7-MTHAm) in pharmaceutical and biological samples (urine and serum) was developed. Separation of all compounds by CZE was carried out using a 46.6 cm untreated fused-silica capillary applying 20 kV separation voltage using 50 mM phosphate buffer of pH 2.8 for THA and THAm and of pH 7.8 for 7-MTHA and 7-MTHAm as background electrolyte (BGE). Detection was carried out at 240 nm (THA and THAm) and 248 nm (7-MTHA and 7-MTHAm). THA and THAm were separated in less than 4 min while 7-MTHA and 7-MTHAm were separated in less than 7 min. The detection limits (SIN = 3) obtained were 3 ppb for THA and 4 ppb for 7-MTHA in aqueous solutions; 50 ppb for THA and 47 ppb for 7-MTHA for the determination in urine (diluted 1:10); 52 ppb for THA and 56 ppb for 7-MTHA, in deproteinized serum samples. The methods are suitable for therapeutic drug monitoring of the drugs.

Metabolic disposition of tacrine in primary suspensions of rat hepatocyte and in single-pass perfused liver: in vitro/in vivo comparisons.

1. Incubations of tacrine (1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate, THA) with a primary suspension of rat hepatocytes for 2 min resulted in formation of the 1-hydroxy derivative as the major metabolite with smaller amounts of the 2- and 4-hydroxy metabolites. 2. Apparent Vmax and Km for THA metabolism were 12.4 +/- 3.3 nmol/min/g liver and 0.98 +/- 0.34 microM respectively. 3. Incubations of THA for longer time-periods (> 10 min) resulted in irreversible binding of THA-derived radioactivity to hepatocellular protein. The apparent maximal rate of irreversible binding (Bmax) was 76.7 +/- 30.5 pmol equivalents bound/h/mg cell protein, whereas the apparent Kb for binding was 2.8 +/- 1.4 microM. 4. The kinetic parameters, Vmax and Km, were used to predict steady-state extraction ratios (ERSS) for various THA input concentrations (Cin) in single-pass perfused rat liver. At low input concentrations (0.72-0.85 microM; Cin < Km), ERSS of THA was approximately 1. For higher Cin (14.05, 20.72, 20.88 microM; Cin >> Km), the calculated ERSS was markedly decreased with 0.300, 0.296 and 0.261, respectively. 5. The intrinsic clearance of THA (Cli) estimated from in vitro hepatocyte data was 6.7 ml/min/g liver while the apparent oral THA clearance (Cloral) calculated from in vivo rat data was 6.6 ml/min/g liver.

Liquid chromatographic determination of tacrine and its metabolites in rat bile microdialysates.

A microbore liquid chromatographic method using a phenyl stationary phase was developed for the determination of 9-amino-1,2,3,4-tetrahydroacridine (tacrine, THA) and its metabolites in microdialysis samples of bile. Analysis of microdialysis samples requires analytical methods with low detection limits and small sample volume requirements. The method uses a 1-mm I.D. phenyl column and fluorescence detection. A detection limit of 0.25 ng/ml in a 5-microliters sample was achieved for THA. This method was then used to determine THA and THA-1-ol in the bile dialysate of a rat. Because of the small sample volume requirements, a 10-min temporal resolution was achieved for the microdialysis experiment. The low detection limit allowed the THA concentration in the bile to be monitored for more than 4 h following a 1.0 mg/kg i.v. dose of THA.

Measurements of tacrine and monoamines in brain by in vivo microdialysis argue against release of monoamines by tacrine at therapeutic doses.

1. The concentration of tacrine (tetrahydroaminoacridine or THA) in plasma, regions of brain and cerebral extracellular fluid has been studied in the rat at various times following injection of a dose of 5 mg kg-1, i.p. 2. The peak plasma THA concentration was 2.46 nmol ml-1, and occurred 30 min post injection and clearance was first order (t1/2 = 90 min). The concentration in the brain peaked between 30-60 min, and was around 30 times plasma concentration (striatum peak concentration = 65 +/- 3 nmol g-1). Extracellular cerebral concentration measured by in vivo microdialysis was similar to plasma concentration with the peak occurring 100 min post-injection. 3. No evidence was obtained by in vivo dialysis for THA inducing dopamine release from striatum or 5-hydroxytryptamine (5-HT) release from the frontal cortex. Enhanced release of dopamine did occur after (+)-amphetamine (5 mg kg-1, i.p.) injection, while KCl (100 mM) in the probe released both dopamine and 5-HT. 4. Since the minimum plasma THA concentration achieved in this study was at least twice that found in the plasma of patients given THA for the treatment of dementia, these results suggest that monoamine release in the brain does not occur during therapy.

Simultaneous determination of physostigmine and tetrahydroaminoacridine in a transdermal permeation study by high-performance liquid chromatography.

A selective and stability-indicating high-performance liquid chromatographic assay with diazepam as the internal standard was developed for simultaneously analyzing physostigmine and tetrahydroaminoacridine in skin samples, permeation diffusates and dosage form. Baseline resolution was achieved with an octadecyl column for physostigmine, its two degradation products and tetrahydroaminoacridine. Peak homogeneity of physostigmine and tetrahydroaminoacridine was confirmed. The calibration curves for both drugs in skin samples were established at 1-50 micrograms per 200 mg skin. Those for diffusate samples were at 10-50 ng per 50 microliters for physostigmine and 30-150 ng per 50 microliters for tetrahydroaminoacridine. The assay was reproducible with within-day and between-day variations of 5-6 and 4-10%, respectively. Application of the assay for in vitro transdermal permeation study was demonstrated.

Thin-layer chromatographic examination of the degradation of centbucridine in aqueous solutions.

Centbucridine (9-n-butylamino-1,2,3,4-tetrahydroacridine) is a new potent local anaesthetic. Its degradation in aqueous solutions has been investigated with the help of thin-layer chromatography. Apart from the degradation products 9-amino-1,2,3,4-tetrahydroacridine and 1,2,3,4-tetrahydroacridone, acridone was also found to be present. It is shown that the acridone is produced not through a dehydrogenation reaction but some other unknown pathway.