A-series agent A-234 belongs to a new generation of nerve agents. The poisoning of a former Russian spy Sergei Skripal and his daughter in Salisbury, England, in March 2018 led to the inclusion of A-234 and other A-series agents into the Chemical Weapons Convention. Even though five years have already passed, there is still very little information on its chemical properties, biological activities, and treatment options with established antidotes. In this article, we first assessed A-234 stability in neutral pH for subsequent experiments. Then, we determined its inhibitory potential towards human recombinant acetylcholinesterase (HssAChE; EC 3.1.1.7) and butyrylcholinesterase (HssBChE; EC 3.1.1.8), the ability of HI-6, obidoxime, pralidoxime, methoxime, and trimedoxime to reactivate inhibited cholinesterases (ChEs), its toxicity in rats and therapeutic effects of different antidotal approaches. Finally, we utilized molecular dynamics to explain our findings. The results of spontaneous A-234 hydrolysis showed a slow process with a reaction rate displaying a triphasic course during the first 72 h (the residual concentration 86.2%). A-234 was found to be a potent inhibitor of both human ChEs (HssAChE IC50 = 0.101 ± 0.003 μM and HssBChE IC50 = 0.036 ± 0.002 μM), whereas the five marketed oximes have negligible reactivation ability toward A-234-inhibited HssAChE and HssBChE. The acute toxicity of A-234 is comparable to that of VX and in the context of therapy, atropine and diazepam effectively mitigate A-234 lethality. Even though oxime administration may induce minor improvements, selected oximes (HI-6 and methoxime) do not reactivate ChEs in vivo. Molecular dynamics implies that all marketed oximes are weak nucleophiles, which may explain the failure to reactivate the A-234 phosphorus-serine oxygen bond characterized by low partial charge, in particular, HI-6 and trimedoxime oxime oxygen may not be able to effectively approach the A-234 phosphorus, while pralidoxime displayed low interaction energy. This study is the first to provide essential experimental preclinical data on the A-234 compound.
- MeSH
- acetylcholinesterasa MeSH
- antidota farmakologie MeSH
- butyrylcholinesterasa MeSH
- cholinesterasové inhibitory toxicita MeSH
- fosfor MeSH
- krysa rodu rattus MeSH
- kyslík MeSH
- lidé MeSH
- oximy farmakologie MeSH
- pralidoximové sloučeniny * MeSH
- pyridinové sloučeniny farmakologie MeSH
- reaktivátory cholinesterázy * farmakologie MeSH
- taurin analogy a deriváty MeSH
- trimedoxim farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- tisková chyba MeSH
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.
- MeSH
- acetylcholinesterasa MeSH
- Alzheimerova nemoc * krev mozkomíšní mok farmakoterapie MeSH
- cholinesterasové inhibitory * krev mozkomíšní mok terapeutické užití MeSH
- donepezil * krev mozkomíšní mok terapeutické užití MeSH
- indany terapeutické užití farmakologie MeSH
- lidé MeSH
- piperidiny farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- randomizované kontrolované studie MeSH
3-Quinuclidinyl benzilate (BZ) ranks among incapacitating military warfare agents. It acts as a competitive inhibitor on muscarinic receptors leading to non-lethal mental impairment. The present study aimed to investigate toxicokinetics of BZ in rats. Moreover, BZ can be exploited to produce a pharmacological model of Alzheimer's disease; thus, this paper focuses mainly on the BZ distribution to the brain. Wistar rats were administered i.p. with BZ (2 and 10 mg/kg). The BZ concentration was determined using LC-MS/MS in plasma, urine, bile, brain, kidney and liver. The sample preparation was based on a solid phase extraction (liquids) or protein precipitation (organ homogenates). The plasma concentration peaked at 3 min (204.5 ± 55.4 and 2185.5 ± 465.4 ng/ml). The maximal concentration in the brain was reached several minutes later. Plasma elimination half-life was 67.9 ± 3.4 in the 2 mg/kg group and 96.6 ± 27.9 in the 10 mg/kg group. BZ concentrations remained steady in the brain, with slow elimination (t1/2 506.9 ± 359.5 min). Agent BZ is excreted mainly via the urine. Steady BZ concentration in the brain could explain the previously published duration of the significant impairment in passive avoidance tasks in rats after an injection of BZ.
- MeSH
- antagonisté muskarinových receptorů krev metabolismus toxicita moč MeSH
- chinuklidinylbenzilát krev metabolismus toxicita moč MeSH
- krysa rodu rattus MeSH
- metabolom MeSH
- moč MeSH
- mozek metabolismus MeSH
- potkani Wistar MeSH
- toxikokinetika MeSH
- žluč metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
S ohledem na události posledních let není riziko vojenského či teroristického zneužití vysoce toxických látek včetně zakázaných bojových otravných látek zanedbatelné. Katedra toxikologie a vojenské farmacie Fakulty vojenského zdravotnictví Univerzity obrany tudíž zavedla analytické metody a postupy úprav biologických vzorků (plasma, moč) pro stanovení identifikačních znaků (biomarkerů) vystavení nervově paralytickým látkám (sarinu, cyklosarinu, somanu, tabunu a V-látkám) a yperitu, čímž přináší do Armády České republiky schopnost potvrdit jejich zneužití vůči živé síle. Metody popsané v článku jsou založeny na průkazu metabolitů (O-alkyl methylfosfonových kyselin pro nervově paralytické látky a thiodiglykolu pro yperit) a produktů jejich reakcí s přítomnými biomolekulami (tyrosinových aduktů nervově paralytických látek a peptidových aduktů sirného yperitu) pomocí kapalinové a plynové chromatografie spojené s hmotnostní spektrometrií. Funkčnost těchto metod je ověřována a diskutována v rámci testu odborné způsobilosti pro biomedicínskou analýzu s mezinárodní účastí, který každoročně pořádá Organizace pro zákaz chemických zbraní. Katedra se těchto testů účastní od roku 2016, kdy se konal první ročník.
Taking into account the events of recent years, a risk of exposure to high toxic substances including chemical weapons is not negligible. Therefore the Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence introduced analytical and biological samples (plasma, urine) preparation methods for determination of biomarkers of exposure to nerve agent (sarin, cyclosarin, soman, tabun, VX and VR agent) and sulphur mustard. Evidence of chemical weapon use is based on detection of their metabolites (Alkyl methylphosphonic acids as metabolites of the organophosphorus nerve agents, thiodiglycol as a metabolite of sulphur mustard) and products of their reactions with biomolecules (tyrosine adducts of organophosphorus nerve agents, sulphur mustard-albumin adducts). Gas chromatography tandem mass spectrometry and liquid chromatography tandem mass spectrometry are used for determination of biomarkers. Functionality of described methods is validated and discussed participating in Biomedical proficiency test, which is organized by The Organisation for the Prohibition of Chemical Weapons every year.
3-Quinuclidinyl benzilate (QNB) is an anticholinergic compound that affects the nervous system. Its hallucinogenic action has led to its potential utility as an incapacitating warfare agent, and it is listed in Schedule 2 by the Organization for the Prohibition of Chemical Weapons. Although this compound has been known for a long time, limited information is available regarding its metabolism and mass spectrometric data of the metabolites, the information that could facilitate the identification of QNB in case of suspected intoxication. To the best of our knowledge, the analytical methods previously described in the literature are based on outdated procedures, which may result in a significantly lower number of observable metabolites. The aim of this work was to obtain deeper insight into QNB biotransformation using a combination of in vitro and in vivo approach. The development of a suitable method for the separation and detection of metabolites using mass spectrometry together with the identification of reliable diagnostic fragments for the unambiguous identification of QNB metabolites in the different biological matrices are also presented in this work. A screening of rat plasma, urine and tissue homogenates revealed 26 new metabolites related to the cytochrome P450 biotransformation pathway, which involves N-oxidation and hydroxylation(s) followed by O-methylation and O-glucuronosylation within phase II of the metabolism. A study showed that the brain is not metabolically active in the case of QNB and that the metabolites do not cross the blood-brain barrier; thus, the toxicodynamic effects are due to QNB itself. In addition, in vitro experiments performed using isolated human liver microsomes revealed N-oxidation as the principal metabolic pathway in human tissue. In light of current global events, the abuse of QNB by terrorists or para-military groups is a real possibility, and our findings may improve the detection systems used in laboratories involved in postexposure investigations.
- MeSH
- biotransformace MeSH
- chinuklidinylbenzilát MeSH
- hmotnostní spektrometrie MeSH
- krysa rodu rattus MeSH
- mozek * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Agent BZ (3-quinuclidinyl benzilate) is a centrally acting synthetic anticholinergic agent, considered as a potential military incapacitating chemical warfare agent. Despite its significance as a model compound in pharmacological research and its potential misuse in chemical attacks, few modern analytical methods for BZ determination in biological samples have been published. The goal of the present work is to develop and validate a sensitive and rapid LC-MS/MS method for the determination of agent BZ in rat plasma. The sample preparation was based on solid-phase extraction on C-18 cartridges. The reversed-phase HPLC coupled with the mass spectrometer with electrospray ionization in the positive ion-selective reaction monitoring mode was employed in the BZ analysis. Atropine was used as an internal standard. The presented method is selective, accurate, precise, and linear (r2 = 0.9947) in a concentration range from 0.5 ng/mL to 1 000 ng/mL and sensitive enough (limit of detection 0.2 ng/mL; limit of quantification 0.5 ng/mL) to determine the BZ plasma levels in rats exposed to 2 mg/kg and 10 mg/kg of BZ. The highest level of BZ in plasma was observed 5 minutes after intramuscular administration (154.6 ± 22.3 ng/mL in rats exposed to 2 mg/kg of BZ and 1024 ± 269 ng/mL in rats exposed to 10 mg/kg). After 48 h, no BZ was observed at detectable levels. This new method allows the detection and quantification of BZ in biological samples after exposure of an observed organism and it will be further optimized for other tissues to observe the distribution of BZ in organs.
- MeSH
- chinuklidinylbenzilát analýza krev MeSH
- cholinergní antagonisté analýza krev MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- krysa rodu rattus MeSH
- limita detekce MeSH
- potkani Wistar MeSH
- tandemová hmotnostní spektrometrie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Although reactive oxygen/nitrogen species (ROS/RNS) have a fundamental role in physiological processes, enhanced ROS/RNS production induced by exogenous sources, including drugs and other xenobiotics, may result in serious damage to biomolecules. Oxidative/nitrosative stress is being intensively investigated and might be responsible for a variety of health side effects. The present liquid chromatography-tandem mass spectrometry (LC-MS/MS) method provides reliable and accurate simultaneous measurement of malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) in cultured human hepatoma (HepG2) cells. Sample preparation process involving ultrasonic homogenization, alkaline hydrolysis of protein-bound MDA and 3-NT, deproteination, derivatization of MDA by 2,4-dinitrophenylhydrazine and solid-phase extraction was optimized, ensuring the isolation and purification of desired analytes. Additionally, nonprotein thiols and nonprotein disulfides were measured using HPLC-UV. The established lower limit of quantification (0.025 nmol/mL for MDA; 0.0125 nmol/mL for 3-NT) allowed their LC-MS/MS determination in HepG2 cells exposed to model oxidizing agent, tert-butyl hydroperoxide (t-BOOH). The results show significant changes in MDA and 3-NT concentrations and alterations in thiol redox-state in dependence on the t-BOOH concentration and duration of its incubation in HepG2 cells. Concurrent evaluation of oxidative/nitrosative stress biomarkers in the in vitro model may significantly facilitate assessment of toxicity of newly developed drugs in preclinical trials and thus improve their safety profile.
- MeSH
- buňky Hep G2 MeSH
- chromatografie kapalinová metody MeSH
- lidé MeSH
- limita detekce MeSH
- malondialdehyd analýza MeSH
- nádory jater metabolismus MeSH
- oxidační stres MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- tyrosin analogy a deriváty analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
