The measured CSF/plasma donepezil concentration ratio but not individually measured CSF and plasma concentrations significantly increase over 24 h after donepezil treatment in patients with Alzheimer's disease.

BACKGROUND: The acetylcholinesterase inhibitor donepezil is administered as a treatment for Alzheimer's disease (AD). However, the appropriate donepezil dosage is still a matter of debate. METHODS: Forty AD patients receiving 10 mg/day of donepezil were randomly divided into four groups based on the time of plasma and cerebrospinal fluid (CSF) sampling: 6 h (n = 5), 12 h (n = 12), 18 h (n = 6) and 24 h (n = 17) after donepezil administration. High-performance liquid chromatography measured the donepezil concentration in plasma samples and CSF samples collected at 4-time points. RESULTS: Plasma and CSF levels among the groups were not significantly different. Conversely, the CSF/plasma donepezil concentration ratio considerably increased in the 24 h group compared to the 6 h (p < 0.005) and 12 h (p < 0.05) groups. CONCLUSION: The measurement of the CSF/plasma donepezil concentration ratio could be used to better evaluate the optimal dose of donepezil.

Discovery and validation of quality markers of Fructus Aurantii against acetylcholinesterase using metabolomics and bioactivity assays.

Fructus Aurantii is a traditional medicated diet in East Asia. To determine the underlying chemical markers responsible for the quality and efficacy of Fructus Aurantii, a sensitive metabolomic method was applied to distinguish Fructus Aurantii in Jiangxi Province from other two geographical locations (Hunan Province and Chongqing City) in China. In the present study, multivariate analyses were adopted to compare chemical compositions in 21 batches of Fructus Aurantii samples. Among three geographical origins, 23 differential compounds were structurally identified. Serum pharmacochemistry exhibited that 22 components could be detected in rat serum. Six differential and absorbed components were selected as six potential markers. Statistical analysis revealed that the content of six markers varied widely in three origins of Fructus Aurantii. Six differential and absorbed components were evaluated further by biological activity. Neohesperidin, naringin, and meranzin showed inhibitory effect on acetylcholinesterase that regulates gastrointestinal motility in vitro and in silico, suggesting that these three components may be determined as the active biomarkers of Fructus Aurantii. These findings demonstrate the potential of biomarkers for identification and quality control of Fructus Aurantii.

Clinical utility of validated gas chromatography-ion trap mass spectrometry in patients with anticholinesterase pesticides poisoning.

Intentional or unintentional intake of anticholinesterase pesticides became common due to their extensive use in agricultural and domestic purposes, resulting in numerous poisoning cases. A simple, accurate, and sensitive gas chromatography-ion trap mass spectrometry-based method for the quantification of 12 anticholinesterase pesticides (monocrotophos, dimethoate, dichlorvos, azinphos-methyl, carbofuran, chlorpyrifos, dialifos, diazinon, malathion, parathion, methidathion, and terbufos) in serum was developed, and its utility in patients with alleged pesticides poisoning was assessed. The quantification was performed using liquid-liquid extraction by toluene/chloroform (4:1,v/v) with 500 µL of serum. On column limit of detection and limit of quantification were less than 50.00 µg/L. The recovery ranged from 97.54 to 103.23%. The calibration curves were linear (R2 > 0.9937). Accuracy was found to be between - 7.1 and 7.2%. Intra-day and inter-day reproducibility was less than 17% for the spiked quality control serum samples. The level of pesticide in serum quantified by the validated method correlated with clinical signs and symptoms, pseudo-cholinesterase activity, total atropine dose, length of hospital stay, and clinical outcome in 15 patients with alleged pesticide poisoning. The validated method may be used for monitoring and prognosis in patients with pesticide poisoning and diagnosis of poisoning in forensic toxicology.

Therapeutic Drug Monitoring of Rivastigmine and Donepezil Under Consideration of CYP2D6 Genotype-Dependent Metabolism of Donepezil.

BACKGROUND: The efficacy of acetylcholinesterase inhibitors (AChE-I) might depend on blood concentration. While rivastigmine metabolism is independent of the cytochrome P450 system, its isoenzymes, especially CYP2D6, metabolize donepezil. CYP2D6 polymorphisms can cause altered enzyme activity resulting in lower or higher than expected drug concentrations of donepezil. OBJECTIVE: We investigated correlations between clinical efficacy and serum concentrations of rivastigmine and donepezil under special consideration of CYP 2D6 genotype or gene dose-dependent metabolism of donepezil. METHODS: Serum concentrations of donepezil and rivastigmine were measured by liquid chromatography - tandem mass spectrometry (LC-MS/MS). Real-time quantitative polymerase chain reaction (PCR) and allele-specific PCR were performed to assess CYP2D6 genotype and gene dose. RESULTS: Patients treated with rivastigmine (n=28) or donepezil (n=48) were included in the study. Both gene dose and metabolism type significantly predicted the level of donepezil serum concentration (p=0.019 and p=0.013, respectively). In the rivastigmine group, changes of the word list delayed recall subtest before treatment and under stable medication were significantly associated with rivastigmine serum levels (ß=0.465; p=0.018). Drug serum concentrations were outside the recommended range in a substantial percentage of participants, which might have contributed to poor correlations between changes in cognitive measures and drug concentrations. Donepezil serum concentrations significantly depended on CYP2D6 gene dose. CONCLUSION: Testing AChE-I serum concentration should be considered in patients without clinical response to treatment or those with severe side effects. Patients with donepezil drug levels outside the recommended range might additionally profit from CYP2D6 genotyping or treatment with an AChE-I independent of CYP metabolism.

In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase.

The implementation of the Chemical Weapons Convention (CWC) in 1997 was a milestone in the prohibition of chemical warfare agents (CWA). Yet, the repeated use of CWA underlines the ongoing threat to the population. Organophosphorus (OP) nerve agents still represent the most toxic CWA subgroup. Defensive research on nerve agents is mainly focused on the "classical five", namely tabun, sarin, soman, cyclosarin and VX, although Schedule 1 of the CWC covers an unforeseeable number of homologues. Likewise, an uncounted number of OP pesticides have been produced in previous decades. Our aim was to determine the in vitro inhibition kinetics of selected organophosphono- and organophosphorothioates with human AChE, as well as hydrolysis of the agents in human plasma and reactivation of inhibited AChE, in order to derive potential structure-activity relationships. The investigation of the interactions of selected OP compounds belonging to schedule 1 (V-agents) and schedule 2 (amiton) of the CWC with human AChE revealed distinct structural effects of the P-alkyl, P-O-alkyl and N,N-dialkyl residues on the inhibitory potency of the agents. Irrespective of structural modifications, all tested V-agents presented as highly potent AChE inhibitors. The high stability of the tested agents in human plasma will most likely result in long-lasting poisoning in vivo, having relevant consequences for the treatment regimen. In conclusion, the results of this study emphasize the need to investigate the biological effects of nerve agent analogues in order to assess the efficacy of available medical countermeasures.

Bioactivities of 7'­ethoxy­trans­feruloyltyramine from Portulaca oleracea L. and its metabolism in rats using ultra­high­performance liquid chromatography electrospray coupled with quadrupole time­of­flight mass spectrometry.

This research aims to study the antioxidation and anticholinesterase activities of 7'-ethoxy-trans-feruloyltyramine (ETFT), which was an alkaloid isolated from Portulaca oleracea for the first time. Furthermore, its main metabolites and metabolic pathways in rats were also explored. The antioxidation and anticholinesterase effects of ETFT were, respectively, examined using 1,1-diphenyl-2-picrylhydrazyl assay and modified Ellman's method. The results showed that ETFT exhibited both the good antioxidant and anticholinesterase effects. Its main metabolites in rats were implemented, and nine metabolites were finally found in the rat's plasma and urine, including the oxidation, reduction, hydrolysis, glucuronidation, sulfation, and glutathionylation process.

Pharmacokinetic Evaluation by Modeling and Simulation Analysis of a Donepezil Patch Formulation in Healthy Male Volunteers.

INTRODUCTION: This study characterized the pharmacokinetics (PKs) of a donepezil patch formulation currently under development, using mixed effect modeling analysis, and explored optimal patch dosing regimens in comparison with the donepezil oral formulation. METHODS: PK data used in this analysis were from 60 healthy Korean male subjects participating in two Phase I studies, where subjects received single or multiple doses of donepezil of 43.75, 87.5, and 175 mg via patches, and 12 of them received a single oral dose of 10 mg of donepezil, followed by a single dose of donepezil via a patch. Donepezil PKs were analyzed by nonlinear mixed effect modeling using NONMEM software. RESULTS: A well-stirred model with two-compartment distribution and delayed absorption was chosen as the best model for the oral formulation. The PKs of donepezil after the patch applications were best described by a two-compartment linear model with zero-order absorption (D2) and absorption delay. The relative bioavailability (BA) of donepezil after the patch application compared with oral dosing was described to be affected by the duration of patch application. CONCLUSION: PK simulations based on the chosen PK models suggested that, overall, donepezil exposure in plasma is similar whether with 10 mg of oral donepezil every 24 h or a 175 mg patch every 72 h, and likewise with 5 mg of oral donepezil every 24 h or an 87.5 mg patch every 72 h.

Understanding the bioconjugation reaction of phenthoate with human serum albumin: New insights from experimental and computational approaches.

Organophosphates are chemical pollutants that are existed widely in the environment, but the reactions of these agents with blood proteins are still not fully clarified. The current story was to analyze the static and dynamic interactions between human serum albumin (HSA) and phenthoate and then uncover the impact of the conjugations on the acetylcholinesterase (AChE) activity at the microscopic scale. Experimental results revealed clearly that the bioconjugate of the HSA-phenthoate was yielded and the conformation of HSA can produce autoregulation during the reaction. Dynamic reaction processes suggested that the conformational flexibility of the specific protein domain was changed significantly in equilibrium, and the electrostatic interaction energy played a major role in total energy of the biosystems, which matches the results of wet experiment and molecular docking. We also found that the modes of homologous proteins-phenthoate have obvious distinctions, and this point is related closely to the local dynamic flexibility of biomolecular structures. Additionally, the degree of bioconjugation of the HSA-phenthoate is positively associated with the enzymatic activity of target AChE, which may be attributed to the competitive reactions between HSA and AChE. Evidently, this scenario could provide useful molecular information for the systematic exploration of the toxicokinetics of organophosphorus compounds.

Development of HPLC method coupled with fluorescence detection for simultaneous determination of donepezil HCl and trazodone HCl in spiked human plasma and tablets dosage forms.

OBJECTIVES: Elderly people with dementia are commonly suffered from sleep disorders. So, the use of Donepezil hydrochloride as anti-Alzheimer drug and Trazodone hydrochloride as antidepressants with hypnotic action is very important in these cases. This study reports about novel and sensitive RP-HPLC method with fluorescence detection for simultaneous bioanalytical determination of Donepezil hydrochloride (DON) and co-administered, Trazodone hydrochloride (TRA) in their pure forms, spiked human plasma and tablets. MATERIALS AND METHODS: Elution of both drugs was achieved with excellent resolution using a RP-C18 Hypersil Gold column and an isocratic mobile phase consisting of phosphate buffer (50mm, pH 4.6): methanol: acetonitrile (60:35:5) with a flow rate of 1.5mL/min and 20µL as injection volume. A Fluorescence detector at 300nm for excitation and 400nm for emission was used. RESULTS: Retention times were 4.3 and 6.3min for Donepezil hydrochloride and Trazodone hydrochloride, respectively. Linearity ranges of the assay were 25-1000 and 50-5000ng/mL and the limits of detection (LOD) and quantitation (LOQ) were 8.52, 15.47 and 25.81, 46.89ng/mL for Donepezil hydrochloride and Trazodone hydrochloride, respectively. CONCLUSION: The high sensitivity of the proposed method enabled the successful determination of the cited drugs in spiked human plasma with mean percentage of recoveries of 91.58±3.34 and 100.30±5.11 for Donepezil hydrochloride and Trazodone hydrochloride, respectively.

In vivo and in vitro metabolism and pharmacokinetics of cholinesterase inhibitor deoxyvasicine from aerial parts of Peganum harmala Linn in rats via UPLC-ESI-QTOF-MS and UPLC-ESI-MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Peganum harmala Linn are a Uighur traditional medicinal herb in China used to treat amnesia, bronchial asthma, and cough. Deoxyvasicine (DVAS), a potent cholinesterase inhibitor exhibiting anti-senile dementia activity, is one of the chief active ingredients in aerial parts of P. harmala and plays a key role in mediating the pharmacological effects of P. harmala. However, the metabolic profiling and in vivo pharmacokinetic characteristics of DVAS still remain unknown. AIM OF THE STUDY: The aim of this present study was to investigate the metabolism and pharmacokinetic properties of DVAS in rats by using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method. MATERIALS AND METHODS: The metabolic profiling of DVAS was evaluated in vitro and in vivo by rat liver microsomes (RLMs) incubation and by rat bio-specimens, such as urine, feces, plasma, and bile, after the oral administration of 45 mg/kg DVAS. An efficient and sensitive UPLC-ESI-MS/MS method was developed and validated to simultaneously determine DVAS and its major four metabolites, namely, vasicine, deoxyvasicinone, vasicinone, and 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-3-ß-D-glucuronide in rat plasma. For pharmacokinetic studies, 32 Sprague-Dawley rats were randomly divided into four groups, namely, intravenous dosage group (2 mg/kg DVAS) and three oral dosage groups (5, 15, and 45 mg/kg DVAS). In addition, the activity of the components in plasma after intravenous administration of DVAS was evaluated by in vitro anti-butyrylcholinesterase (BChE) assays. RESULTS: A total of 23 metabolites were found in RLMs, plasma, urine, feces, and bile by UPLC-ESI-QTOF-MS. The metabolic pathway of DVAS in vivo and in vitro mainly involved hydroxylation, dehydrogenation, acetylation, methylation, glucuronidation, and O-sulphate conjugation, and the C-3 and C-9 sites were the main metabolic soft spots. All 23 metabolites were detected in the urine sample, and 13, 8, 22, and 6 metabolites were identified from rat feces, plasma, bile, and RLMs, respectively. The standard curves of DVAS and four metabolites in rat plasma showed good linearity in the concentration range of 0.82-524.00 ng/mL with acceptable selectivity, precision, accuracy, recovery, and stability. DVAS exhibited linear dose-proportional pharmacokinetics at doses of 5, 15, and 45 mg/kg after oral administration, and the average oral absolute bioavailability of DVAS was 47.46%. The in vitro anti-BChE assays implied that the inhibitive activities were mainly due to the different concentrations of prototype DVAS. CONCLUSIONS: DVAS can be rapidly absorbed and excreted by blood, and it is also extensively metabolized in vivo, and the anti-BChE activity in blood is mainly attributed to DVAS. These findings can lay a foundation for new drug development for DVAS.

Skinfold thickness for rivastigmine patch application in Alzheimer's disease.

RATIONALE: Rivastigmine patches are used for patients with Alzheimer's disease (AD), but little is known about the serum concentration of rivastigmine and its metabolite or clinical adherence in relation to skinfold thickness after rivastigmine patch application. OBJECTIVES: The aim of this study was to examine the association between rivastigmine and NAP 226-90 serum concentration and skinfold thickness and to determine the appropriate skinfold thickness for the use of rivastigmine patch in patients with AD. METHODS: Patients with AD who continuously used rivastigmine patches (4.6 mg/24 h, 5 cm2) for more than 6 months were recruited. The serum concentrations of rivastigmine and NAP 226-90 were measured. Skinfold thickness was measured using a Lange Skinfold Caliper. RESULTS: In total, 91 patients with AD (40 men and 51 women) participated in this study on skinfold thickness measurement. Among them, 27 patients were examined for rivastigmine and NAP 226-90 serum concentrations, with mean concentrations of 1.0 ± 0.6 ng/mL and 3.6 ± 3.6 ng/mL, respectively. The skinfold thickness in the subscapular area was significantly negatively correlated with the NAP 226-90 serum concentration (Spearman's rank correlation coefficient = - 0.47, P = .01). In addition, patients with AD and a subscapular skinfold thickness of ≥25 mm exhibited a significantly high risk of decreased Mini-Mental Status Examination score and nonadherence to a rivastigmine patch (odds ratio 3.00; 95% confidence interval = 1.076-8.366, P = .03). CONCLUSIONS: Subscapular skinfold thickness was significantly negatively correlated with the NAP 226-90 serum concentration and may be considered an appropriate predictor of response and adherence to clinical application of a rivastigmine patch.

Análise do impacto do uso de organofosforados e carbamatos em trabalhadores rurais de um município da região noroeste do estado do Rio Grande do Sul / Impact analysis of the use of organophosphates and carbamates rural workers of the municipality in a northwest region of Rio Grande do Sul

Anualmente milhões de agricultores são intoxicados no mundo, e destes, mais de 20 mil morrem em consequência da exposição a agrotóxicos. Intoxicações por organofosforados (OF) e carbamatos (CAR) representam as maiores ameaças à saúde dos trabalhadores rurais. Os OF e CAR atuam na inibição da enzima colinesterase, sendo assim a inibição desta mostra-se um excelente indicador da severidade da intoxicação. O objetivo deste estudo foi analisar o impacto do uso de OF e CAR em trabalhadores rurais na cidade de Mato Queimado/RS. Foi realizado um estudo transversal, prospectivo e experimental. Investigaramse 27 trabalhadores rurais expostos. Foram realizadas coletas sanguíneas e dados epidemiográficos nos meses de fevereiro e março de 2014. A atividade da colinesterase foi determinada através do método bioquímico cinético colorimétrico. A faixa etária média dos participantes foi 34,6 anos (± 8,5). A forma de contato mais prevalente foi a aplicação do produto (88,9%). O tempo médio de exposição foi de 10,7 anos. 70,4% relataram usar equipamentos de proteção individual (EPI), sendo mais frequente o uso de máscara (55,5%). A média dos valores de colinesterase para foi de 3244,45 U/I (± 345,8), níveis estes abaixo dos de referência. Através dos resultados obtidos nesta pesquisa torna-se imprescindível a utilização de meios de monitoramento biológico dos trabalhadores rurais na finalidade de prevenção e promoção da saúde.

Annually millions of rural workers are intoxicated in the world, and of these, more than 20,000 die as a result of exposure to pesticides. Intoxication by insecticides organophosphate (OF) and carbamates (CAR) represent the greatest threats to the health of rural workers. OF CAR and act on the inhibition of cholinesterase enzyme, thus inhibition of this proves to be an excellent indicator of the severity of the intoxication. The objective of this study was to analyze the impact of using OF CAR and in rural workers in the city of Mato Queimado/RS. A cross-sectional, prospective and experimental study was conducted. Twenty-three rural workers exposed were investigated. Sample collection and data demographic were conducted in February and March 2014. The cholinesterase activity was determined by biochemical kinetic colorimetric method. The average age of participants was 34.6 years (± 8.5). The most prevalent form of contact is via the application of the product (88.9%). The mean duration of exposure was 10.7 years. Still, 70.4% reported using personal protective equipment (PPE), more frequent use of mask (55.5%). The average values for cholinesterase was 3244.45 U/l (± 345.8) levels below those of the reference. The results obtained in this study are essential to use biological monitoring means of rural workers in purpose of prevention and health promotion.

Serum ß-glucuronidase activity as a biomarker for acute cholinesterase inhibitor pesticide poisoning.

ß-glucuronidase (BG) activity is a promising biomarker for diagnosis and prognosis after exposure to organophosphorous (OP) pesticides. The aim of this study was to evaluate the changes in serum BG activity in patients with acute OP poisoning and to determine whether these changes correlate with the severity of poisoning. Thirty patients with anticholinesterase pesticide poisoning were included, besides 10 healthy volunteers as a control group. Serum activities of butyrylcholinesterase (BuChE) and BG were measured for each subject on admission, then after 12 and 24 h. Serum levels of BuChE and BG in poisoned patients were significantly different from the control subjects; these differences persisted in repeated measurements. Moreover, the serum levels showed significant differences within each group of the three time points. A significant negative correlation was found between the serum activities of BuChE and BG in all groups at the three time points. In conclusion, serum BG activity seems a reliable marker for OP poisoning even when measured at 24 h after poisoning.

Establishment of a quantitative bioanalytical method for an acetylcholinesterase inhibitor Ethyl 3-(2-(4-fluorophenyl) amino)-4-phenylthiazol-5-yl)-3-oxopropanoate including its physicochemical characterization and in vitro metabolite profiling using Liquid Chromatography-Mass Spectrometry.

Ethyl 3-(2-(4-fluorophenyl)amino)-4-phenylthiazo)-5-yl)-3-oxopropanoate is a novel molecule with potent acetylcholinesterase inhibition property. In this research, we have developed a rapid and selective RP-HPLC bioanalytical method for quantitative measurement of the molecule. The method has been validated following the USFDA bioanalytical method validation guideline. In addition, as a part of drug development, in vitro metabolite identification has also been performed. A Kromasil C18 column was used in eluting the molecule chromatographically. The optimized mobile phase was composed of a mixture of acetonitrile and 10 mM ammonium formate buffer (pH 4.6) in 70:30 ratio (v/v). The response of the molecule was found to be linear over a calibration range of 0.2 µg/mL to 12 µg/mL. The inter-day and intra-day accuracy of the method ranged from 89.95% to 101.90% and 99.84% to 104.08%, respectively. On the other hand, the precision (%CV) value for inter-day was in between 3.50% to 6.91% and for intra-day, it was 2.11% to 8.03%. The mean recovery of the molecule at three different quality control levels was more than 85%. The analyte was stable under different stability conditions including 12 h autosampler, 8 h bench top, 15 days long term at -20 °C and three freeze-thaw cycles. The method was applied to determine stability of the molecule in human plasma. The molecule was found to be stable with more than 90% remaining even after 120 min of incubation in plasma. Two metabolites each in rat liver microsome and human liver microsome has been identified.

Simultaneous Time-concentration Analysis of Soman and VX Adducts to Butyrylcholinesterase and Albumin by LC-MS-MS.

A sensitive method for the purification and determination of two protein adducts, organophosphorus (OP)-BChE and OP-albumin adducts, in a single sample using a simultaneous sample preparation method was developed and validated using liquid chromatography-tandem mass spectrometry. First, we isolated O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate (VX) and O-pinacolyl methylphosphonofluoridate (soman, GD)-BChE adducts using an immunomagnetic separation (IMS) method and the HiTrap™ Blue affinity column was subsequently used to isolate and purify VX and GD-albumin adducts from the plasma of rhesus monkeys exposed to nerve agents. Additionally, we examined the time-concentration profiles of two biomarkers, VX and GD-nonapeptides and VX and GD-tyrosines, derived from OP-BChE and OP-albumin adducts up to 8 weeks after exposure. Based on the results, we determined that VX and GD-tyrosine is more suitable than VX and GD-nonapeptide as a biomarker owing to its longevity. This integrated approach is expected to be applicable for the quantification of other OP-BChE and OP-albumin adducts in human plasma, thus serving as a potential generic assay for exposure to nerve agents.

Simple determination of some antidementia drugs in wastewater and human plasma samples by tandem dispersive liquid-liquid microextraction followed by high-performance liquid chromatography.

In this work, a simple method, namely, tandem dispersive liquid-liquid microextraction, with a high sample clean-up is applied for the rapid determination of the antidementia drugs rivastigmine and donepezil in wastewater and human plasma samples. This method, which is based on two consecutive dispersive microextractions, is performed in 7 min. In the method, using a fast back-extraction step, the applicability of the dispersive microextraction methods in complicated matrixes is conveniently improved. This step can be performed in less than 2 min, and very simple tools are required for this purpose. To achieve the best extraction efficiency, optimization of the variables affecting the method was carried out. Under the optimized experimental conditions, the relative standard deviations for the method were in the range of 6.9-8.7%. The calibration curves were obtained in the range of 2-1100 ng/mL with good correlation coefficients, higher than 0.995, and the limits of detection ranged between 0.5 and 1.0 ng/mL.

Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma.

Organophosphorus nerve agent (OPNA) adducts formed with human butyrylcholinesterase (HuBuChE) can be used as biomarker of OPNA exposure. Indeed, intoxication by OPNAs can be confirmed by the LC/MS2 analysis of a specific HuBuChE nonapeptide on which OPNAs covalently bind. A fast, selective, and highly sensitive online method was developed to detect sarin and soman adducts in plasma, including immunoextraction by anti-HuBuChE antibodies, pepsin digestion on immobilized enzyme reactors (IMER), and microLC/MS2 analysis of the OPNA adducts. The potential of three different monoclonal antibodies, covalently grafted on sepharose, was compared for the extraction of HuBuChE. The online method developed with the most promising antibodies allowed the extraction of up to 100% of HuBuChE contained in plasma and the digestion of 45% of it in less than 40 min. Moreover, OPNA-HuBuChE adducts, aged OPNA adducts, and unadducted HuBuChE could be detected (with S/N > 2000), even in plasma spiked with a low concentration of OPNA (10 ng mL-1). Finally, the potential of this method was compared to approaches involving other affinity sorbents, already described for HuBuChE extraction. Graphical abstract Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of organophosphorous nerve agent adducts formed with human butyrylcholinesterase.

C-547, a 6-methyluracil derivative with long-lasting binding and rebinding on acetylcholinesterase: Pharmacokinetic and pharmacodynamic studies.

C-547, a potent slow-binding inhibitor of acetylcholinesterase (AChE) was intravenously administered to rat (0.05 mg/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 (3800 ml/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 = 4 h) from the bloodstream. Effect of C-547 on animal model of myasthenia gravis persists for more than 72 h, 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 percutaneous toxicokinetics of VX in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant.

This study used a damaged skin, porcine model to evaluate the in vivo efficacy of WoundStat™ for the decontamination of superficial, nerve agent-contaminated wounds. Anaesthetized animals were randomly assigned to either control (n = 7), no decontamination (n = 12) or WoundStat™ (n = 12) treatment groups. Pigs were exposed to a 5× LD50 dose of neat, radiolabelled S-[2-(diisopropylamino)ethyl]-O-ethyl methyl-phosphonothioate (VX; or equivalent volume of sterile saline for the control group) via an area of superficially damaged skin on the ear. WoundStat™ was applied at 30 seconds post-exposure to assigned animals. The VX contaminant (or saline) and decontaminant remained in place for the duration of the study (up to 6 hours). Physiological parameters and signs of intoxication were recorded during the exposure period. Skin and organ samples were taken post mortem for 14 C-VX distribution analyses. Blood samples were taken periodically for toxicokinetic and whole-blood acetylcholinesterase (AChE) activity analyses. VX exposure was accompanied by a rapid decrease in AChE activity in all animals, regardless of decontamination. However, decontamination significantly improved survival rate and time and reduced the severity of signs of intoxication. In addition, the distribution of 14 C-VX in key internal organs and post mortem blood samples was significantly lower in the WoundStat™ treatment group. This study demonstrates that WoundStat™ may be a suitable medical countermeasure for increasing both survival rate and time following VX exposure. The results also suggest that AChE activity is not a useful prognostic indicator.