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1.
Arch Toxicol ; 96(2): 571-583, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34962578

RESUMO

The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M-1 min-1) towards VX and a preferential hydrolysis of the more toxic P(-) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg-1 i.v.: PTE-2 dosed at 1.3 mg kg-1 i.v. (PTE-2.1) and 2.6 mg kg-1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg-1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.


Assuntos
Substâncias para a Guerra Química/toxicidade , Compostos Organotiofosforados/toxicidade , Hidrolases de Triester Fosfórico/farmacologia , Animais , Caulobacteraceae/enzimologia , Inibidores da Colinesterase/toxicidade , Masculino , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Engenharia de Proteínas , Ratos , Ratos Wistar , Estereoisomerismo
2.
Arch Toxicol ; 95(8): 2815-2823, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34160649

RESUMO

Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M-1 min-1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC-MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(-) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.


Assuntos
Caulobacteraceae/enzimologia , Agentes Neurotóxicos/metabolismo , Hidrolases de Triester Fosfórico/metabolismo , Catálise , Cromatografia Líquida , Hidrólise , Mutação , Agentes Neurotóxicos/química , Agentes Neurotóxicos/toxicidade , Hidrolases de Triester Fosfórico/genética , Estereoisomerismo , Especificidade por Substrato , Espectrometria de Massas em Tandem
3.
Org Biomol Chem ; 18(27): 5218-5227, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32602497

RESUMO

The highly toxic nerve agent VX is a methylphosphonothioate that degrades via three pathways in aqueous solution, namely through the hydrolysis of the P-O or P-S bonds, or the cleavage of the C-S bond at the 2-aminoethyl residue. In the latter case, an aziridinium ion and a phosphonothioate is formed. Here it is shown that acyclic or cyclic cucurbiturils inhibit these reactions in phosphate buffer at physiological pH and thus stabilise the nerve agent. When using unbuffered basic solutions as the reaction medium, however, in which the P-S or P-O bonds are normally hydrolysed preferentially, cucurbiturils turned out to strongly shift VX degradation towards the cleavage of the C-S bond. Cucurbit[7]uril, in particular, has a so pronounced effect under suitable conditions that it almost completely suppresses the formation of products resulting from the other degradation pathways. Investigations involving VX analogues in combination with computational methods suggest that one reason for the reaction control exerted by the cucurbiturils is the preorganisation of VX for aziridinium ion formation. In addition, cucurbit[7]uril also lowers the transition state of the reaction by stabilising the positive charge developing on the way to the product. Cucurbiturils thus have a marked effect on the reactivity of a highly toxic nerve agent, which potentially allows using them for decontamination purposes.


Assuntos
Substâncias para a Guerra Química/química , Compostos Macrocíclicos/química , Compostos Organotiofosforados/química , Ciclização , Hidrólise , Cinética
4.
Molecules ; 25(13)2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32630769

RESUMO

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.


Assuntos
Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Compostos Organotiofosforados/química , Compostos Organotiofosforados/farmacologia , Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/sangue , Inibidores da Colinesterase/farmacocinética , Reativadores da Colinesterase/farmacologia , Estabilidade de Medicamentos , Humanos , Agentes Neurotóxicos/química , Agentes Neurotóxicos/farmacologia , Cloreto de Obidoxima/química , Cloreto de Obidoxima/farmacologia , Compostos Organotiofosforados/sangue , Compostos Organotiofosforados/farmacocinética , Relação Estrutura-Atividade
5.
Arch Toxicol ; 93(7): 1881-1891, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31069408

RESUMO

We present the forensic analyses of plasma samples of human victims exposed to sulfur mustard (SM) in a crisis region in the Middle East in 2015. A few hours after exposure, poisoned persons showed typical signs and symptoms of percutaneous SM exposure including erythema and later on blisters and hardly healing skin wounds. Blood samples were collected 15 days after poisoning to be analyzed for the presence of long-lived protein-adduct biomarkers to verify SM poisoning. We applied a novel bioanalytical toolbox targeting four human serum albumin-derived biomarkers that were made accessible after plasma proteolysis. These adducts contained the SM-specific hydroxyethylthioethyl moiety either bound to the thiol group of a cysteine residue (C34*) or to the side-chain carboxylic group of a glutamic acid residue (E230*). Peptide biomarkers were produced from plasma of the victims using proteinase K (C34*PF), pronase (C34*P) and pepsin (AE230*VSKL and LQQC34*PFEDHVKL) for enzymatic protein cleavage. Separation and detection were carried out by selective micro-liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS). In addition to this site-specific adduct detection, a general approach after alkaline hydrolysis of the plasma protein fraction was applied. Liberated thiodiglycol (TDG) was derivatized with heptafluorobutyric anhydride and detected by gas chromatography-electron ionization mass spectrometry (GC-EI MS). The different bioanalytical methods yielded congruent results confirming SM poisoning for all patients who showed clinical signs and symptoms. This is the first time that real cases of SM poisoning were confirmed and presented by such a broad compilation of protein-derived biomarkers.


Assuntos
Substâncias para a Guerra Química/intoxicação , Toxicologia Forense/métodos , Gás de Mostarda/intoxicação , Albumina Sérica Humana/química , Biomarcadores/sangue , Substâncias para a Guerra Química/química , Humanos , Gás de Mostarda/química , Intoxicação/sangue , Ligação Proteica , Proteólise , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
6.
Arch Toxicol ; 90(9): 2131-2145, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27349770

RESUMO

The implementation of the Chemical Weapon Convention (CWC), prohibiting the development, production, storage and use of chemical weapons by 192 nations and the ban of highly toxic OP pesticides, especially class I pesticides according to the WHO classification, by many countries constitutes a great success of the international community. However, the increased interest of terrorist groups in toxic chemicals and chemical warfare agents presents new challenges to our societies. Almost seven decades of research on organophosphorus compound (OP) toxicology was mainly focused on a small number of OP nerve agents despite the fact that a huge number of OP analogues, many of these agents having comparable toxicity to classical nerve agents, were synthesized and published. Only limited physicochemical, toxicological and medical information on nerve agent analogues is available in the open literature. This implies potential gaps of our capabilities to detect, to decontaminate and to treat patients if nerve agent analogues are disseminated and may result in inadequate effectiveness of newly developed countermeasures. In summary, our societies may face new, up to now disregarded, threats by toxic OP which calls for increased awareness and appropriate preparedness of military and civilian CBRN defense, a broader approach for new physical and medical countermeasures and an integrated system of effective detection, decontamination, physical protection and treatment.


Assuntos
Terrorismo Químico , Substâncias para a Guerra Química/toxicidade , Intoxicação por Organofosfatos/etiologia , Compostos Organofosforados/toxicidade , Praguicidas/toxicidade , Animais , Antídotos/uso terapêutico , Substâncias para a Guerra Química/química , Substâncias para a Guerra Química/farmacocinética , Descontaminação , Planejamento em Desastres , Humanos , Estrutura Molecular , Intoxicação por Organofosfatos/terapia , Compostos Organofosforados/química , Compostos Organofosforados/farmacocinética , Praguicidas/química , Praguicidas/farmacocinética , Medição de Risco , Relação Estrutura-Atividade , Testes de Toxicidade
7.
Angew Chem Int Ed Engl ; 55(41): 12668-72, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27627873

RESUMO

Sulfonatocalix[4]arenes with an appended hydroxamic acid residue can detoxify VX and related V-type neurotoxic organophosphonates with half-lives down to 3 min in aqueous buffer at 37 °C and pH 7.4. The detoxification activity is attributed to the millimolar affinity of the calixarene moiety for the positively charged organophosphonates in combination with the correct arrangement of the hydroxamic acid group. The reaction involves phosphonylation of the hydroxamic acid followed by a Lossen rearrangement, thus rendering the mode of action stoichiometric rather than catalytic. Nevertheless, these calixarenes are currently the most efficient low-molecular-weight compounds for detoxifying persistent V-type nerve agents under mild conditions. They thus represent lead structures for novel antidotes that allow treatment of poisonings by these highly toxic chemicals.

8.
Toxicol Lett ; 398: 13-18, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38857853

RESUMO

Phosphotriesterases (PTE) are a new and promising approach for the treatment of organophosphate poisoning, since the current therapy of such intoxications shows some limitations. A previous rat in vivo study confirmed the therapeutic effect of PTE, which were specifically designed for VX breakdown, and demonstrated rapid degradation of VX in whole blood samples. The present study now focuses on the degradation of VX and its distribution in organ tissues of the animals used in the aforementioned study. In order to gain a broader overview, we have extended the investigations to the VX metabolites EA-2192 and EMPA by using methods developed for an LC-ESI-MS/MS system. Applying these methods, we were able to verify the effectiveness of the PTE treatment and gained an overview of VX tissue distribution in poisoned but untreated rats.


Assuntos
Compostos Organotiofosforados , Hidrolases de Triester Fosfórico , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Animais , Compostos Organotiofosforados/toxicidade , Masculino , Distribuição Tecidual , Hidrolases de Triester Fosfórico/metabolismo , Ratos Wistar , Cromatografia Líquida , Ratos
9.
Toxicol Lett ; 393: 78-83, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38311194

RESUMO

Organ-on-a-chip technology is considered a next-generation platform in pharmacology and toxicology. Nevertheless, this novel technology still faces several challenges concerning the respective materials which are used for these microfluidic devices. Currently available organ-chips are most often based on polydimethylsiloxane (PDMS). However, this material has strong limitations regarding compound binding. The current study investigated options to reduce compound absorption of the highly toxic nerve agent VX (1000 µmol/L) in a commercially available organ-chip. In addition, surface effects on degradation products of VX were investigated. The alternative polymer cyclic olefin copolymers (CoC) showed significantly less compound absorption compared to PDMS. Furthermore, a coating of PDMS- and CoC-based chips was investigated. The biocompatible polymer polyethyleneimine (PEI) successfully modified PDMS and CoC surfaces and further reduced compound absorption. A previously examined VX concentration after 72 h of 141 ± 10 µmol/L VX could be increased to 442 ± 54 µmol/L. Finally, the respective concentrations of VX and degradation products accounted for > 90% of the initial concentration of 1000 µmol/L VX. The currently described surface modification might be a first step towards the optimization of organ-on-a-chip surfaces, facilitating a better comparability of different studies and results.


Assuntos
Agentes Neurotóxicos , Compostos Organotiofosforados , Agentes Neurotóxicos/toxicidade , Sistemas Microfisiológicos , Toxicocinética , Polímeros
10.
Toxicol Lett ; 388: 24-29, 2023 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-37827339

RESUMO

Organ-on-a-chip platforms are an emerging technology in experimental and regulatory toxicology (species-specific differences, ethical considerations). They address gaps between in vivo and in vitro models. However, there are still certain limitations considering material, setup and applicability. The current study examined the suitability of a commercially available polydimethylsiloxane-based (PDMS) organ-chip for the toxicokinetic characterization of the highly toxic nerve agent VX and the organophosphate pesticide parathion. The respective concentrations of 1000 µmol/L and 100 µmol/L VX and parathion were chosen deliberately high in order to study concentrations even if high compound absorption by PDMS might occur. Neuronal and liver spheroids, totaling 2 × 106 cells were used to study concentration changes of VX and parathion. In addition, VX enantiomers were quantified. The current study suggests a significant absorption of VX, respectively parathion by PDMS. This might require future investigation of alternative materials or coatings to limit absorption for organophosphorus compounds in toxicokinetic studies.


Assuntos
Compostos Organotiofosforados , Paration , Compostos Organofosforados/toxicidade , Microfluídica , Toxicocinética , Compostos Organotiofosforados/toxicidade , Dimetilpolisiloxanos
11.
Artigo em Inglês | MEDLINE | ID: mdl-34171608

RESUMO

In analogy to the fluoride-induced regeneration of butyrylcholinesterase (BChE) inhibited by nerve agents a method was developed and optimized for whole blood samples. Compared to the plasma method, regeneration grade was found to be higher for cyclosarin (GF), i-butylsarin from VR, and n-butylsarin from CVX, but lower for sarin (GB), fluorotabun from tabun (GA), and ethylsarin from VX. Regeneration grade of soman (GD) is the same for both matrices because it is released from serum albumin and not from cholinesterases. The method was fully validated for GB and GF to prove selectivity, linearity (n = 6), limit of determination (LOD1), reproducibility (within day (n = 8) and from day to day (n = 8)), effectiveness of extraction, matrix effect, and sample stability (after sample preparation and during three freeze/thaw cycles). The other agents were tested for selectivity, linearity (n = 2), limit of determination, and stability after sample preparation. The method showed high selectivity, good linearity up to the protein's saturation concentration (GB: R2 = 0.9995, GF: 0.9968), and high reproducibility (GB: C.V. 5.9-13.7%, GF: 4.9-10.3%). The limits of determination (calculated from the spiked amount of the original agent) were found with 0.3 ng/mL VX, 0.5 ng/mL GB, 1 ng/mL VR, 0.5 ng/mL GA, 1 ng/mL CVX, and 8 ng/mL GD. In the case of GF, it was found with 4 ng/mL using Isolute ENV + SPE cartridges as for the other analytes and with 2.5 ng/mL using Isolute C8 EC SPE cartridges instead. This method was then applied to a denatured whole blood sample obtained from an individual exposed to GB. While previously only the GB metabolite isopropyl methylphosphonic acid (IMPA) could be detected in this blood sample it was now possible to successfully release GB from the blood proteins by excess fluoride.


Assuntos
Fluoretos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Agentes Neurotóxicos/análise , Organofosfatos/sangue , Espectrometria de Massas em Tandem/métodos , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Humanos , Limite de Detecção , Modelos Lineares , Agentes Neurotóxicos/química , Agentes Neurotóxicos/metabolismo , Organofosfatos/química , Organofosfatos/metabolismo , Reprodutibilidade dos Testes
12.
Toxicol Lett ; 320: 28-36, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31805340

RESUMO

Fourteen amino acids protected at the N-terminal and at their side chains were screened for resolving the enantiomers of V-agents by NMR. While none of the shift reagents tested showed really effective separation in proton NMR, two of them (BOC-Gln(Xan)-OH, 16, and Z-Arg(Z)2-OH), 21, with 16 superior to 21) were found suitable to separate the enantiomers of all V-agent homologues involved in the test by 31P-NMR. Molar ratios investigated were 1:0.5, 1:1, 1:1.5, 1:2, and 1:3 with the V-agent set to 1 throughout the experiments. All these ratios were more or less effective, but 1:3 was found to separate the V-agents the most reliable way. It is postulated that three chiral solvating molecules are then coordinated around the organophosphate: ion pair formation with the amino nitrogen of the V agent side chain, hydrogen bonding provided by the PO unit, and extension of coordination at the phosphorus atom itself. After chiral separation of VX by semi-preparative LC-MS the enantiomers were examined with both configurations of 16 releasing four different 31P NMR peaks which correspond to four different complexes: R-S3, R-R3, S-R3, and S-S3. Comparing these results with literature data it is assumed that (+)-VX corresponds to the RP configuration and (-)-VX to the SP-configuration.


Assuntos
Aminoácidos/química , Espectroscopia de Ressonância Magnética/métodos , Compostos Organofosforados/química , Fósforo/química , Solventes/química , Cromatografia Líquida , Ligação de Hidrogênio , Estrutura Molecular , Compostos Organofosforados/isolamento & purificação , Espectroscopia de Prótons por Ressonância Magnética , Estereoisomerismo , Espectrometria de Massas em Tandem
13.
J Am Chem Soc ; 131(47): 17226-32, 2009 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-19894712

RESUMO

Diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range of highly toxic organophosphorus compounds including the nerve agents sarin, soman, and cyclosarin. In contrast to the substrate diisopropyl fluorophosphate (DFP) the nerve agents possess an asymmetric phosphorus atom, which leads to pairs of enantiomers that display markedly different toxicities. Wild-type DFPase prefers the less toxic stereoisomers of the substrates which leads to slower detoxification despite rapid hydrolysis. Enzyme engineering efforts based on rational design yielded two quadruple enzyme mutants with reversed enantioselectivity and overall enhanced activity against tested nerve agents. The reversed stereochemical preference is explained through modeling studies and the crystal structures of the two mutants. Using the engineered mutants in combination with wild-type DFPase leads to significantly enhanced activity and detoxification, which is especially important for personal decontamination. Our findings may also be of relevance for the structurally related enzyme human paraoxonase (PON), which is of considerable interest as a potential catalytic in vivo scavenger in case of organophosphorus poisoning.


Assuntos
Substâncias para a Guerra Química/metabolismo , Compostos Organofosforados/metabolismo , Hidrolases de Triester Fosfórico/metabolismo , Substâncias para a Guerra Química/química , Substâncias para a Guerra Química/farmacologia , Cristalização , Cinética , Modelos Moleculares , Sistema Nervoso/efeitos dos fármacos , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Hidrolases de Triester Fosfórico/química , Estereoisomerismo
14.
Forensic Toxicol ; 36(1): 61-71, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29367863

RESUMO

During the United Nations fact-finding mission to investigate the alleged use of chemical warfare agents in the Syrian Arab Republic in 2013, numerous tissues from a deceased female victim, who had displayed symptoms of cholinergic crisis, were collected. The Organisation for the Prohibition of Chemical Weapons (OPCW) authorized two specialized laboratories in the Netherlands and Germany for forensic analysis of these samples. Diverse modern mass spectrometry (MS)-based procedures in combination with either liquid chromatography (LC) or gas chromatography (GC) separation were applied. A variety of biotransformation products of the nerve agent sarin was detected, including the hydrolysis product O-isopropyl methylphosphonic acid (IMPA) as well as covalent protein adducts with e.g., albumin and human butyrylcholinesterase (hBChE). IMPA was extracted after sample acidification by solid-phase extraction and directly analyzed by LC-tandem-MS with negative electrospray ionization (ESI). Protein adducts were found, either by fluoride-induced reactivation applying GC-MS techniques or by LC-MS-based detection after positive ESI for proteolyzed proteins yielding phosphonylated tyrosine residues or a specific phosphonylated hBChE-derived nonapeptide. These experimental results provided unambiguous evidence for a systemic intoxication and were the first proving the use of sarin in the ongoing bellicose conflict. This scenario underlines the requirement for qualified and specialized analytical laboratories to face repeated violation of the Chemical Weapons Convention.

15.
Biochem Pharmacol ; 73(11): 1807-17, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17382909

RESUMO

The high number of fatalities due to poisoning by organophosphorus compound-based (OP) pesticides and the availability of highly toxic OP-type chemical warfare agents (nerve agents) emphasize the necessity for an effective medical treatment. Acute OP toxicity is mainly caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment. However, human AChE inhibited by certain OP, e.g. the phosphoramidates tabun and fenamiphos, is rather resistant towards reactivation by oximes while AChE inhibited by others, e.g. the phosphoramidate methamidophos is easily reactivated by oximes. To get more insight into a potential structure-activity relationship human AChE was inhibited by 16 different tabun analogues and the time-dependent reactivation by 1mM obidoxime, TMB-4, MMB-4, HI 6 or HLö 7, the reactivation kinetics of obidoxime and the kinetics of aging and spontaneous reactivation were investigated. A clear structure-activity relationship of aging, spontaneous and oxime-induced reactivation kinetics could be determined with AChE inhibited by N-monoalkyl tabun analogues depending on the chain length of the N-alkyl residue. N,N-dialkyl analogues bearing ethyl and n-propyl residues were completely resistant towards reactivation while N,N-di-i-propyl tabun was highly susceptible towards reactivation by oximes. AChE inhibited by phosphonoamidate analogues of tabun, bearing a N,N-dimethyl and N,N-diethyl group, could be reactivated and had comparable reactivation kinetics with obidoxime. These results in conjunction with previous data with organophosphates and organophosphonates emphasizes the necessity for kinetic studies as basis for future work on structural analysis with human AChE and for the development of effective broad-spectrum oximes.


Assuntos
Acetilcolinesterase/metabolismo , Envelhecimento/metabolismo , Amidas/farmacologia , Ácidos Fosfóricos/farmacologia , Acetilcolinesterase/efeitos dos fármacos , Reativadores da Colinesterase , Humanos , Cinética , Cloreto de Obidoxima/química , Cloreto de Obidoxima/farmacologia , Organofosfatos/química , Organofosfatos/farmacologia , Oximas/química , Oximas/farmacologia , Relação Estrutura-Atividade
16.
Artigo em Inglês | MEDLINE | ID: mdl-17888747

RESUMO

The present study was initiated to develop a sensitive method for the analysis of cyclosarin (O-cyclohexyl methylphosphonofluoridate, GF) enantiomers in biological samples utilizing classical configurations of GC-MS and automated solid phase extraction. To achieve this goal, a specific procedure had to be developed to extract cyclosarin from swine blood samples thereby stabilising and minimising the racemisation/deracemisation of its enantiomers. The chiral stationary phase was GAMMA DEX (gamma cyclodextrin), on which GF and deuterated GF enantiomers were baseline-resolved. The limit of detection was 1 pg for (-)-GF with GC-EI-MS and 5 pg for (+)-GF with GC-NCI-MS. The absolute recovery of the overall procedure for sample preparation was 85%. After an intravenous infusion of a supralethal dose of GF in anaesthetised swine only (-)-GF could be quantified, (+)-GF was not detected.


Assuntos
Substâncias para a Guerra Química/análise , Substâncias para a Guerra Química/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Compostos Organofosforados/sangue , Compostos Organofosforados/química , Animais , Hemólise , Compostos Organofosforados/administração & dosagem , Sensibilidade e Especificidade , Estereoisomerismo , Suínos
17.
Toxicology ; 350-352: 25-30, 2016 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-27153754

RESUMO

Despite extensive research for decades no effective broad-spectrum oxime for the treatment of poisoning by a broad range of nerve agents is available. Previous in vitro and in vivo data indicate that the combination of in service oximes could be beneficial. To investigate the ability of obidoxime, HI-6 and the combination of both oximes to reactivate inhibited human AChE in the presence of sarin, cyclosarin or tabun we adopted a dynamic in vitro model with real-time and continuous determination of AChE activity to simulate inhalation nerve agent exposure and intramuscular oxime administration. The major findings of this kinetic study are that the extent and velocity of reactivation is dependent on the nerve agent and the oxime-specific reactivating potency. The oxime-induced reactivation of inhibited human AChE in the presence of nerve agents is markedly impaired and the combination of obidoxime and HI-6 had no additive effect but could broaden the spectrum. In conclusion, these data indicate that a combination of obidoxime and HI-6 would be beneficial for the treatment of poisoning by a broad spectrum of nerve agents and could present an interim solution until more effective and broad-spectrum reactivators are available.


Assuntos
Reativadores da Colinesterase/farmacologia , Modelos Biológicos , Agentes Neurotóxicos/toxicidade , Cloreto de Obidoxima/farmacologia , Oximas/farmacologia , Compostos de Piridínio/farmacologia , Acetilcolinesterase/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Reativadores da Colinesterase/administração & dosagem , Reativadores da Colinesterase/farmacocinética , Quimioterapia Combinada , Humanos , Exposição por Inalação , Injeções Intramusculares , Cloreto de Obidoxima/administração & dosagem , Cloreto de Obidoxima/farmacocinética , Organofosfatos/toxicidade , Compostos Organofosforados/toxicidade , Oximas/administração & dosagem , Oximas/farmacocinética , Compostos de Piridínio/administração & dosagem , Compostos de Piridínio/farmacocinética , Sarina/toxicidade , Toxicocinética
18.
Toxicol Lett ; 258: 198-206, 2016 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-27397758

RESUMO

The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge (∼2LD50). C23AL showed a Cmax of 0.63µmolL(-1) after i.o. and i.v. administration of 2mgkg(-1) providing a stable plasma profile up to 180min experimental duration with 0.41 and 0.37µmolL(-1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning.


Assuntos
Antídotos/administração & dosagem , Mutação , Agentes Neurotóxicos/toxicidade , Intoxicação por Organofosfatos/tratamento farmacológico , Compostos Organotiofosforados/toxicidade , Fragmentos de Peptídeos/administração & dosagem , Hidrolases de Triester Fosfórico/administração & dosagem , Animais , Animais não Endogâmicos , Antídotos/metabolismo , Antídotos/farmacocinética , Antídotos/uso terapêutico , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacocinética , Proteínas de Bactérias/uso terapêutico , Medula Óssea , Cobaias , Inativação Metabólica , Injeções Intralesionais , Injeções Intramusculares , Injeções Intravenosas , Injeções Subcutâneas , Masculino , Agentes Neurotóxicos/análise , Agentes Neurotóxicos/metabolismo , Intoxicação por Organofosfatos/sangue , Intoxicação por Organofosfatos/etiologia , Intoxicação por Organofosfatos/metabolismo , Compostos Organotiofosforados/administração & dosagem , Compostos Organotiofosforados/antagonistas & inibidores , Compostos Organotiofosforados/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/uso terapêutico , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/farmacocinética , Hidrolases de Triester Fosfórico/uso terapêutico , Proteólise , Pseudomonas/enzimologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , Toxicocinética
19.
Toxicol Lett ; 244: 112-120, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26321678

RESUMO

Sulfur mustard (SM) is a chemical warfare agent (CWA) that was first used in World War I and in several military conflicts afterwards. The threat by SM is still present even today due to remaining stockpiles, old and abandoned remainders all over the world as well as to its ease of synthesis. CWA are banned by the Chemical Weapons Convention (CWC) interdicting their development, production, transport, stockpiling and use and are subjected to controlled destruction. The present case report describes an accidental exposure of three workers that occurred during the destruction of SM. All exposed workers presented a characteristic SM-related clinical picture that started about 4h after exposure with erythema and feeling of tension of the skin at the upper part of the body. Later on, superficial blister and a burning phenomenon of the affected skin areas developed. Similar symptoms occurred in all three patients differing severity. One patient presented sustained skin affections at the gluteal region while another patient came up with affections of the axilla and genital region. Fortunately, full recovery was observed on day 56 after exposure except some little pigmentation changes that were evident even on day 154 in two of the patients. SM-exposure was verified for all three patients using bioanalytical GC MS and LC MS/MS based methods applied to urine and plasma. Urinary biotransformation products of the ß-lyase pathway were detected until 5 days after poisoning whereas albumin-SM adducts could be found until day 29 underlining the beneficial role of adduct detection for post-exposure verification. In addition, we provide general recommendations for management and therapy in case of SM poisoning.


Assuntos
Acidentes , Vesícula/induzido quimicamente , Substâncias para a Guerra Química/intoxicação , Documentação , Eritema/induzido quimicamente , Irritantes/intoxicação , Gás de Mostarda/intoxicação , Pele/efeitos dos fármacos , Adulto , Vesícula/diagnóstico , Vesícula/terapia , Substâncias para a Guerra Química/metabolismo , Cromatografia Líquida , Eritema/diagnóstico , Eritema/terapia , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Masculino , Pessoa de Meia-Idade , Gás de Mostarda/metabolismo , Intoxicação/diagnóstico , Intoxicação/terapia , Ligação Proteica , Indução de Remissão , Albumina Sérica/metabolismo , Albumina Sérica Humana , Índice de Gravidade de Doença , Pele/patologia , Espectrometria de Massas em Tandem , Fatores de Tempo , Resultado do Tratamento
20.
Toxicology ; 214(3): 182-9, 2005 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-16051411

RESUMO

Organophosphate (OP)-type chemical warfare agents (nerve agents) present a constant threat to the population. Sensitive and specific methods for the detection and verification of exposure to nerve agents are required for diagnosis, therapeutic monitoring, health surveillance and forensic purposes. Determination of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in blood remains a mainstay for the fast initial screening but lacks sensitivity and specificity. Quantitative analysis of nerve agents and their degradation products in plasma and urine by mass spectrometric methods may prove exposure but is limited to hours or days after the incident due to the short residence time of the analytes. Investigation of protein adducts extends the time interval between exposure and sampling and may be suitable to detect low-level exposure. Definitive prove of exposure requires a spectrum of different methods, expensive and sophisticated equipment and will be limited to specialized laboratories.


Assuntos
Acetilcolinesterase/sangue , Butirilcolinesterase/sangue , Intoxicação por Organofosfatos , Humanos , Organofosfatos/sangue , Intoxicação/sangue , Intoxicação/diagnóstico
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