Worek F

General

Full name : Worek Franz

First name : Franz

Mail : Bundeswehr Institute of Pharmacology and Toxicology\; Neuherbergstrasse 11\; Munich\; 80937

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Country : Germany

Email : franz.worek@lrz.uni-muenchen.de

Phone : +498931682930

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References (222)

Title : Effects of organophosphates on precision-cut kidney slices - Hoeffner_2024_Toxicol.Mech.Methods__1
Author(s) : Hoeffner C , Worek F , Amend N
Ref : Toxicol Mech Methods , :1 , 2024
Abstract : Organophosphate (OP) poisoning, both accidental and with suicidal intent, is a global medical challenge. While the primary toxicity of these pesticides is based on the inhibition of acetylcholinesterase (AChE), case reports describe patients developing OP-mediated renal insufficiency. We set out to investigate possible pathomechanisms utilizing rat precision-cut kidney slices (PCKS). Depending on the method of investigation, PCKS were observed for a maximum of 10 days. PCKS exposed to OP compounds (malaoxon, malathion, paraoxon, parathion) showed a dose-dependent loss of viability and a reduction of total protein content over the course of 10 days. A concentration of 500 microM OP showed the most differences between OP compounds. After two days of incubation parathion showed a significantly lower level of viability than malathion. The respective effects of paraoxon and malaoxon were not significantly different from control. However, effects of OP were only observed in concentrations exceeding those that were needed to achieve significant AChE inhibition in rat kidney tissue. In addition, we observed histological changes, without inducing LDH leakage. Overall, results suggest that OP exert effects in kidney tissue, that exceed those expected from the sole inhibition of AChE and vary between compounds. Without signs of necrosis, findings call for studies that address other possible pathomechanisms including inflammatory response, oxidative stress or activation of apoptosis to further understand nephrotoxicity of OP compounds. Monitoring oxon concentration over time, we demonstrated reduced enzyme inhibiting properties in the presence of PCKS, suggesting interactions between OP compound and kidney tissue.
ESTHER : Hoeffner_2024_Toxicol.Mech.Methods__1
PubMedSearch : Hoeffner_2024_Toxicol.Mech.Methods__1
PubMedID: 38745427

Title : Restoration of nerve agent impaired neuromuscular transmission in rat diaphragm by bispyridinium non-oximes - Structure-activity relationships - Amend_2024_Toxicology__153741
Author(s) : Amend N , Timperley CM , Bird M , Green AC , Worek F , Seeger T
Ref : Toxicology , :153741 , 2024
Abstract : Organophosphate (OP) poisoning is currently treated with atropine, oximes and benzodiazepines. The nicotinic signs, i.e., respiratory impairment, can only be targeted indirectly via the use of oximes as reactivators of OP-inhibited acetylcholinesterase. Hence, compounds selectively targeting nicotinic acetylcholine receptors (nAChRs) might fundamentally improve current treatment options. The bispyridinium compound MB327 has previously shown some therapeutic effect against nerve agents in vitro and in vivo. Nevertheless, compound optimization was deemed necessary, due to limitations (e.g., toxicity and efficacy). The current study investigated a series of 4-tert-butyl bispyridinium compounds and of corresponding bispyridinium compounds without substituents in a rat diaphragm model using an indirect field stimulation technique. The length of the respective linker influenced the ability of the bispyridinium compounds to restore muscle function in rat hemidiaphragms. The current data show structure-activity relationships for a series of bispyridinium compounds and provide insight for future structure-based molecular modeling.
ESTHER : Amend_2024_Toxicology__153741
PubMedSearch : Amend_2024_Toxicology__153741
PubMedID: 38311098

Title : MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor - Nitsche_2024_Toxicol.Lett_392_94
Author(s) : Nitsche V , Hofner G , Kaiser J , Gertzen CGW , Seeger T , Niessen KV , Steinritz D , Worek F , Gohlke H , Paintner FF , Wanner KT
Ref : Toxicol Lett , 392 :94 , 2024
Abstract : Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR, as well. In this study, UNC0642 has been utilized as a reporter ligand to establish new Binding Assays for this target. These assays follow the concept of MS Binding Assays for which by assessing the amount of bound reporter ligand by mass spectrometry no radiolabeled material is required. According to the results of the performed MS Binding Assays comprising saturation and competition experiments it can be concluded, that UNC0642 used as a reporter ligand addresses the MB327 binding site of the Torpedo-nAChR. This is further supported by the outcome of ex vivo studies carried out with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of UNC0646, an analog of UNC0642 with the highest binding affinity, in the recently proposed binding site of MB327 (MB327-PAM-1). With UNC0642 addressing the MB327 binding site of the Torpedo-nAChR, this and related quinazoline derivatives represent a promising starting point for the development of novel ligands of the nAChR as antidotes for the treatment of intoxications with organophosphorus compounds. Further, the new MS Binding Assays are a potent alternative to established assays and of particular value, as they do not require the use of radiolabeled material and are based on a commercially available compound as reporter ligand, UNC0642, exhibiting one of the highest binding affinities for the MB327 binding site known so far.
ESTHER : Nitsche_2024_Toxicol.Lett_392_94
PubMedSearch : Nitsche_2024_Toxicol.Lett_392_94
PubMedID: 38216073

Title : Screening for new ligands of the MB327-PAM-1 binding site of the nicotinic acetylcholine receptor - Sichler_2024_Toxicol.Lett_394_23
Author(s) : Sichler S , Hofner G , Nitsche V , Niessen KV , Seeger T , Worek F , Paintner FF , Wanner KT
Ref : Toxicol Lett , 394 :23 , 2024
Abstract : Intoxications with organophosphorus compounds (OPCs) effect a severe impairment of cholinergic neurotransmission that, as a result of overstimulation may lead to desensitization of nicotinic acetylcholine receptors (nAChRs) and finally to death due to respiratory paralysis. So far, therapeutics, that are capable to address and revert desensitized neuromuscular nAChRs into their resting, i.e. functional state are still missing. Still, among a class of compounds termed bispyridinium salts, which are characterized by the presence of two pyridinium subunits, constituents have been identified, that can counteract organophosphate poisoning by resensitizing desensitized nAChRs. According to comprehensive modeling studies this effect is mediated by an allosteric binding site at the nAChR termed MB327-PAM-1 site. For MB327, the most prominent representative of the bispyridinium salts and all other analogues studied so far, the affinity for the aforementioned binding site and the intrinsic activity measured in ex vivo and in in vivo experiments are distinctly too low, to meet the criteria to be fulfilled for therapeutic use. Hence, in order to identify new compounds with higher affinities for the MB327-PAM-1 binding site, as a basic requirement for an enhanced potency, two compound libraries, the ChemDiv library with 60 constituents and the Tocriscreen Plus library with 1280 members have been screened for hit compounds addressing the MB327-PAM-1 binding site, utilizing the [(2)H(6)]MB327 MS Binding Assay recently developed by us. This led to the identification of a set of 10 chemically diverse compounds, all of which exhibit an IC(50) value of >= 10 microM (in the [(2)H(6)]MB327 MS Binding Assay), which had been defined as selection criteria. The three most affine ligands, which besides a quinazoline scaffold share similarities with regard to the substitution pattern and the nature of the substituents, are UNC0638, UNC0642 and UNC0646. With binding affinities expressed as pK(i) values of 6.01 +/- 0.10, 5.97 +/- 0.05 and 6.23 +/- 0.02, respectively, these compounds exceed the binding affinity of MB327 by more than one log unit. This renders them promising starting points for the development of drugs for the treatment of organophosphorus poisoning by addressing the MB327-PAM-1 binding site of the nAChR.
ESTHER : Sichler_2024_Toxicol.Lett_394_23
PubMedSearch : Sichler_2024_Toxicol.Lett_394_23
PubMedID: 38387764

Title : The use of bispyridinium non-oxime analogues for the restoration of nerve agent impaired neuromuscular transmission in rat hemidiaphragms - Structure optimization - Amend_2024_Toxicol.Lett__
Author(s) : Amend N , Timperley CM , Bird M , Green AC , Worek F , Seeger T
Ref : Toxicol Lett , : , 2024
Abstract : Organophosphate pesticide poisoning challenges health care systems worldwide. Furthermore, nerve agents remain a continuous threat. The treatment options for organophosphate poisoning have virtually been unchanged for decades, relying on symptomatic treatment and the use of oximes to indirectly restore neuromuscular function. Hence, compounds targeting directly nicotinic acetylcholine receptors (nAChRs) might substantially improve treatment options. The current study investigated a series of bispyridinium analogues with a trimethylene or 2,2'-diethyloxy linker in a rat hemidiaphragm model, using indirect field stimulation. Methyl- and ethyl-substituted bispyridinium analogues restored neuromuscular function up to 37 +/- 17% (MB419, a 3-methyl analogue) at a stimulation frequency of 20Hz. The bispyridinium analogues with a 2- or 3-methyl group, or a 2- or 3-ethyl group, tended towards a higher restoration of neuromuscular function than those with a 4-methyl or 4-ethyl group, respectively. The current data can be used for future studies to optimize structure-based molecular modeling of compounds targeting the nAChR.
ESTHER : Amend_2024_Toxicol.Lett__
PubMedSearch : Amend_2024_Toxicol.Lett__
PubMedID: 38723915

Title : Synthesis and Biological Evaluation of Novel MB327 Analogs as Resensitizers for Desensitized Nicotinic Acetylcholine Receptors after Intoxication with Nerve Agents - Bernauer_2024_Toxicol.Lett__
Author(s) : Bernauer T , Nitsche V , Kaiser J , Gertzen CGW , Hofner G , Niessen KV , Seeger T , Steinritz D , Worek F , Gohlke H , Wanner KT , Paintner FF
Ref : Toxicol Lett , : , 2024
Abstract : Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062 and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.
ESTHER : Bernauer_2024_Toxicol.Lett__
PubMedSearch : Bernauer_2024_Toxicol.Lett__
PubMedID: 38759939

Title : Suitability of human HepaRG cells and liver spheroids as in vitro model to investigate the bioactivation of the organothiophosphate pesticide parathion - Horn_2024_Toxicol.In.Vitro_97_105811
Author(s) : Horn G , Worek F
Ref : Toxicol In Vitro , 97 :105811 , 2024
Abstract : Organophosphorus compounds (OP) constitute a large group of chemicals including pesticides and nerve agents. Organothiophosphate pesticides require cytochrome P450-mediated oxidative desulphuration in the liver to form corresponding oxons, which are potent inhibitors of the enzyme acetylcholinesterase (AChE). Human HepaRG cells are a promising tool to study liver-specific functions and have been shown to maintain drug metabolizing enzymes. This research describes for the first time the in vitro metabolic activation of an organothiophosphate to its active oxon by two different HepaRG cell-based models. Monolayer cultures and liver spheroids were exposed to the model OP parathion and the quantification of the corresponding oxon was performed with an AChE inhibition assay. Our results showed a time- and dose-dependent cytochrome P450 catalyzed bioactivation and a superior metabolism capacity of the monolayer HepaRG model in comparison with the liver spheroids. Finally, HepaRG cells can be assessed as a metabolically competent cell model intermediate between cell-free preparations and intact animals and as suitable to study OP metabolism in the human liver.
ESTHER : Horn_2024_Toxicol.In.Vitro_97_105811
PubMedSearch : Horn_2024_Toxicol.In.Vitro_97_105811
PubMedID: 38521251

Title : Uncharged mono- and bisoximes: In search of a zwitterion to countermeasure organophosphorus intoxication - Gorecki_2024_Chem.Biol.Interact__110941
Author(s) : Gorecki L , Markova A , Hepnarova V , Zivna N , Junova L , Hrabinova M , Janousek J , Kobrlova T , Prchal L , Jun D , Soukup O , Horn G , Worek F , Marek J , Korabecny J
Ref : Chemico-Biological Interactions , :110941 , 2024
Abstract : The current study imposes a new class of organophosphorus (OP)-inhibited cholinesterase reactivators by conceptualizing a family of asymmetric bisoximes with various reactivating scaffolds. Several novel nucleophilic warheads were investigated, putting forward 29 novel reactivating options, by evaluating their nucleophilicity and ability to directly decompose OP compounds. Adopting the so-called zwitterionic strategy, 17 mono-oxime and nine bisoxime reactivators were discovered with major emphasis on the bifunctional-moiety approach. Compounds were compared with clinically used standards and other known experimentally highlighted reactivators. Our results clearly favor the concept of asymmetric bisoximes as leading reactivators in terms of efficacy and versatility. These top-ranked compounds were characterized in detail by reactivation kinetics parameters and evaluated for potential CNS availability. The highlighted molecules 55, 57, and 58 with various reactivating warheads, surpassed the reactivating potency of pralidoxime and several notable uncharged reactivators. The versatility of lead drug candidate 55 was also inspected on OP-inhibited butyrylcholinesterase, revealing a much higher rate compared to existing clinical antidotes.
ESTHER : Gorecki_2024_Chem.Biol.Interact__110941
PubMedSearch : Gorecki_2024_Chem.Biol.Interact__110941
PubMedID: 38493910

Title : Inhibition kinetics of acetylcholinesterase and butyrylcholinesterase from various species by 2-(2-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide (CBDP) - Horn_2024_Toxicol.Lett_396_28
Author(s) : Horn G , Rappengluck S , Worek F
Ref : Toxicol Lett , 396 :28 , 2024
Abstract : The aerotoxic syndrome has been associated with exposure to tricresyl phosphate (TCP), which is used as additive in hydraulic fluids and engine lubricants. The toxic metabolite 2-(2-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide (CBDP) is formed from the TCP isomer tri-ortho-cresyl phosphate (TOCP) in vivo and is known to react with the active site serine in acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) resulting in the inhibition of the enzymes. Previous in vitro studies showed pronounced species differences in the inhibition kinetics of cholinesterases by organophosphorus compounds (OP), which must be considered in the development of relevant animal models for the investigation of OP poisoning and the aerotoxic syndrome. The present study was designed to investigate the inhibition kinetics of human, Cynomolgus monkey, pig, mini pig, guinea pig, mouse, and rat AChE as well as BChE by CBDP under standardized conditions. There were similar rate constants for the inhibition (k(i)) of human, Cynomolgus monkey and mouse AChE by CBDP. In contrast, the k(i) values obtained for guinea pig, mini pig, pig, and rat AChE were 2.8- to 5.9-fold lower than that of human AChE. The results of the present study confirmed CBDP as one of the most potent inhibitors of human BChE, indicating a k(i) value of 3.24 +/- 0.33 x10(8)M(-1)min(-1), which was about 1,140-fold higher than that of human AChE. Accordingly, a markedly more pronounced inhibition rate of BChE from the species guinea pig, mini pig, pig, rat, Cynomolgus monkey, and mouse by CBDP was found as compared to those of AChE from the respective sources, indicating 2.0- to 89.6-fold higher k(i) values.
ESTHER : Horn_2024_Toxicol.Lett_396_28
PubMedSearch : Horn_2024_Toxicol.Lett_396_28
PubMedID: 38642675

Title : A novel binding site in the nicotinic acetylcholine receptor for MB327 can explain its allosteric modulation relevant for organophosphorus-poisoning treatment - Kaiser_2023_Toxicol.Lett_373_160
Author(s) : Kaiser J , Gertzen CGW , Bernauer T , Hofner G , Niessen KV , Seeger T , Paintner FF , Wanner KT , Worek F , Thiermann H , Gohlke H
Ref : Toxicol Lett , 373 :160 , 2023
Abstract : Organophosphorus compounds (OPCs) are highly toxic compounds that can block acetylcholine esterase (AChE) and thereby indirectly lead to an overstimulation of muscarinic and nicotinic acetylcholine receptors (nAChRs). The current treatment with atropine and AChE reactivators (oximes) is insufficient to prevent toxic effects, such as respiratory paralysis, after poisonings with various OPCs. Thus, alternative treatment options are required to increase treatment efficacy. Novel therapeutics, such as the bispyridinium non-oxime MB327, have been found to reestablish neuromuscular transmission by interacting directly with nAChR, probably via allosteric mechanisms. To rationally design new, more potent drugs addressing nAChR, knowledge of the binding mode of MB327 is fundamental. However, the binding pocket of MB327 has remained elusive. Here, we identify a new potential allosteric binding pocket (MB327-PAM-1) of MB327 located at the transition of the extracellular to the transmembrane region using blind docking experiments and molecular dynamics simulations. MB327 forms striking interactions with the receptor at this site. The interacting amino acids are highly conserved among different subunits and different species. Correspondingly, MB327 can interact with several nAChR subtypes from different species. We predict by rigidity analysis that MB327 exerts an allosteric effect on the orthosteric binding pocket and the transmembrane domain after binding to MB327-PAM-1. Furthermore, free ligand diffusion MD simulations reveal that MB327 also has an affinity to the orthosteric binding pocket, which agrees with recently published results that related bispyridinium compounds show inhibitory effects via the orthosteric binding site. The newly identified binding site allowed us to predict structural modifications of MB327, resulting in the more potent resensitizers PTM0062 and PTM0063.
ESTHER : Kaiser_2023_Toxicol.Lett_373_160
PubMedSearch : Kaiser_2023_Toxicol.Lett_373_160
PubMedID: 36503818

Title : AChE reactivation in Precision-cut lung slices following organophosphorus compound poisoning - Golitz_2023_Toxicol.Lett__
Author(s) : Golitz F , Herbert J , Worek F , Wille T
Ref : Toxicol Lett , : , 2023
Abstract : Precision-cut lung slices (PCLS) are a suitable model for analyzing the acetylcholinesterase (AChE) activity and subsequent effects after exposure to organophosphorus (OP) compounds. In this study, the AChE activity was determined in intact PCLS for the first time. Since the current standard therapy for OP poisoning (atropine + oxime + benzodiazepine) lacks efficiency, reliable models to study novel therapeutic substances are needed. Models should depict pathophysiological mechanisms and help to evaluate the beneficial effects of new therapeutics. Here PCLS were exposed to three organophosphorus nerve agents (OPNAs): sarin (GB), cyclosarin (GF), and VX. They were then treated with three reactivators: HI-6, obidoxime (OBI), and a non-oxime (NOX-6). The endpoints investigated in this study were the AChE activity and the airway area (AA) change. OPNA exposure led to very low residual AChE activities. Depending on the reactivator properties different AChE reactivation results were measured. GB-inhibited PCLS-AChE was reactivated best, followed by VX and GF. To substantiate these findings and to understand the connection between the molecular and the functional levels in a more profound way the results were correlated to the AA changes. These investigations underline the importance of reactivator use and point to the possibilities for future improvements in the treatment of OPNA-exposed victims.
ESTHER : Golitz_2023_Toxicol.Lett__
PubMedSearch : Golitz_2023_Toxicol.Lett__
PubMedID: 38160862

Title : Human HepaRG liver spheroids: cold storage protocol and study on pyridinium oxime-induced hepatotoxicity in vitro - Horn_2022_Chem.Biol.Interact__110285
Author(s) : Horn G , Kranawetvogl T , John H , Weigel C , Rauen U , Worek F , Wille T
Ref : Chemico-Biological Interactions , :110285 , 2022
Abstract : Oximes play an essential role in the therapy of organophosphorus compound (OP) poisoning by reactivating inhibited acetylcholinesterase. Impairment of liver function was observed in OP poisoning and associated with obidoxime treatment by some reports. In this study human three-dimensional HepaRG spheroids were used as complex in vitro model to investigate oxime-induced liver toxicity. In this context, cold storage of liver spheroids at 4 degreesC in standard culture medium and in optimized tissue preservation solutions of up to 72 h was assessed. Cold storage in standard culture medium resulted in a complete loss of viability whereas an optimized tissue preservation solution preserved viability. Separately from that liver spheroids were exposed to the four oximes pralidoxime, obidoxime, HI-6, MMB-4 and cytotoxicity (effective concentration, EC(50)) was determined with an ATP-based assay at several time points. The release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin secretion was measured in supernatants. The same parameters were assessed with diclofenac as positive hepatotoxic control and with the OP pesticides malathion and malaoxon alone or in the presence of obidoxime. All individual tested oximes and OP showed a low cytotoxicity with effective concentrations mostly >2,000 microM. In contrast, the exposure to malaoxon in the presence of 1,000 microM obidoxime resulted in a marked decrease of viability and an increased release of AST indicating risk of liver injury only if oxime antidotes are strongly overdosed.
ESTHER : Horn_2022_Chem.Biol.Interact__110285
PubMedSearch : Horn_2022_Chem.Biol.Interact__110285
PubMedID: 36442613

Title : Oximes should be used routinely in organophosphate poisoning - Thiermann_2022_Br.J.Clin.Pharmacol__
Author(s) : Thiermann H , Worek F
Ref : British Journal of Clinical Pharmacology , : , 2022
Abstract : In poisoning with organophosphorus compounds, patients can only profit from the regeneration of acetylcholinesterase, when the poison load has dropped below a toxic level. Every measure that allows an increase of synaptic AChE activity at the earliest is essential for timely termination of the cholinergic crisis. Only a drug induced reactivation allows to achieve fast restoration of the inhibited AChE. Obidoxime and pralidoxime have proved to be able to reactivate inhibited cholinesterase thereby saving life of poisoned animals. A plasma level of obidoxime or pralidoxime allowing reactivation in humans poisoned by OP can be adjusted. There is no doubt that obidoxime and pralidoxime are able to reactivate OP inhibited AChE activity in poisoned patients thereby increasing AChE activity and contributing substantially to terminate cholinergic crisis. Hence, a benefit may be expected when substantial reactivation is achieved. A test system allowing determination of red blood cell AChE activity, reactivatability, inhibitory equivalents and BChE activity is available for relatively low cost. If any reactivation is possible while inhibiting equivalents are present, oxime therapy should be maintained. In particular, when balancing the benefit risk assessment, obidoxime or palidoxime should be given as soon as possible and as long as a substantial reactivation may be expected.
ESTHER : Thiermann_2022_Br.J.Clin.Pharmacol__
PubMedSearch : Thiermann_2022_Br.J.Clin.Pharmacol__
PubMedID: 35023196

Title : A case report of severe pirimiphos-methyl intoxication: Clinical findings and cholinesterase status - Zellner_2022_Front.Pharmacol_13_1102160
Author(s) : Zellner T , Rabe C , von der Wellen-Pawlowski J , Hansen D , John H , Worek F , Eyer F
Ref : Front Pharmacol , 13 :1102160 , 2022
Abstract : A 63-year-old male was admitted to a district hospital after ingesting ethanol and pirimiphos-methyl (PM) with suicidal intentions. History included alcoholic cirrhosis with alcoholism, adiposity, diabetes with cerebral microangiopathy, chronic renal insufficiency, heparin-induced thrombocytopenia, and status post necrotizing fasciitis. Emergency medical service reported an alert patient without signs of cholinergic crisis; activated charcoal and atropine were administered. Upon hospital arrival, he received fluid resuscitation, activated charcoal, and atropine. He was transferred to a toxicology unit the next day. On admission, he had no cholinergic signs (dry mucous membranes, warm skin, and mydriatic pupils) requiring small atropine doses (0.5smg per hour). Four hours after admission, he developed bradycardia and respiratory distress, necessitating intubation. He received atropine by continuous infusion for 7sdays (248smg total) and obidoxime (bolus and continuous infusion). PM, pirimiphos-methyl-oxon (PMO), and phosphorylated tyrosine (Tyr) adducts derived from human serum albumin were analyzed in vivo. Cholinesterase status (acetylcholinesterase (AChE), butyrylcholinesterase (BChE), inhibitory activity of patient plasma and reactivatability, and phosphorylated BChE-derived nonapeptides) was measured in vivo. Obidoxime and atropine were monitored. PM and PMO were detectable, PM with maximum concentration -24sh post admission (p.a.) and PMO at -18sh p.a. Tyr adducts were detectable. AChE in vivo was suppressed on admission, increased continuously after starting obidoxime, and reached maximum activity after -30sh. AChE in vivo and reactivatability remained at the same level until the end of monitoring. BChE was already suppressed on admission; termination of the antidote treatment was possible after BChE had recovered to 1/5th of its normal value and extubation was possible after BChE had recovered to 2/5th. While a substantial part of BChE was already aged on admission, aging continued peaking at -24sh p.a. After initiating obidoxime treatment, plasma levels increased until obidoxime plasma levels reached a steady state. On admission, plasma atropine level was low; it increased with the start of the continuous infusion. Afterward, the level dropped to a steady state. The clinical course was characterized by bouts of pneumonia, necessitating re-intubation and prolonged ventilation, sepsis, delirium, and a peripheral neuropathy. After psychiatric evaluation, the patient was discharged to a neurological rehabilitation facility after 77 days of hospital care.
ESTHER : Zellner_2022_Front.Pharmacol_13_1102160
PubMedSearch : Zellner_2022_Front.Pharmacol_13_1102160
PubMedID: 36618943

Title : Broad-spectrum antidote discovery by untangling the reactivation mechanism of nerve agent inhibited acetylcholinesterase - Lindgren_2022_Chemistry_28_e202200678
Author(s) : Lindgren C , Forsgren N , Hoster N , Akfur C , Artursson E , Edvinsson L , Svensson R , Worek F , Ekstrom , Linusson A
Ref : Chemistry , 28 :e202200678 , 2022
Abstract : Reactivators are vital for the treatment of organophosphorus nerve agent (OPNA) intoxication but new alternatives are needed due to their limited clinical applicability. The toxicity of OPNAs stems from covalent inhibition of the essential enzyme acetylcholinesterase (AChE), which reactivators relieve via a chemical reaction with the inactivated enzyme. Here, we present new strategies and tools for developing reactivators. We discover suitable inhibitor scaffolds by using an activity-independent competition assay to study non-covalent interactions with OPNA-AChEs and transform these inhibitors into broad-spectrum reactivators. Moreover, we identify determinants of reactivation efficiency by analysing reactivation and prereactivation kinetics together with structural data. Our results show that new OPNA reactivators can be discovered rationally by exploiting detailed knowledge of the reactivation mechanism of OPNA-inhibited AChE.
ESTHER : Lindgren_2022_Chemistry_28_e202200678
PubMedSearch : Lindgren_2022_Chemistry_28_e202200678
PubMedID: 35420233
Gene_locus related to this paper: mouse-ACHE

Title : Development of versatile and potent monoquaternary reactivators of acetylcholinesterase - Gorecki_2021_Arch.Toxicol__
Author(s) : Gorecki L , Hepnarova V , Karasova JZ , Hrabinova M , Courageux C , Dias J , Kucera T , Kobrlova T , Muckova L , Prchal L , Malinak D , Jun D , Musilek K , Worek F , Nachon F , Soukup O , Korabecny J
Ref : Archives of Toxicology , : , 2021
Abstract : To date, the only treatments developed for poisoning by organophosphorus compounds, the most toxic chemical weapons of mass destruction, have exhibited limited efficacy and versatility. The available causal antidotes are based on reactivation of the enzyme acetylcholinesterase (AChE), which is rapidly and pseudo-irreversibly inhibited by these agents. In this study, we developed a novel series of monoquaternary reactivators combining permanently charged moieties tethered to position 6- of 3-hydroxypyridine-2-aldoxime reactivating subunit. Highlighted representatives (21, 24, and 27; also coded as K1371, K1374, and K1375, respectively) that contained 1-phenylisoquinolinium, 7-amino-1-phenylisoquinolinium and 4-carbamoylpyridinium moieties as peripheral anionic site ligands, respectively, showed efficacy superior or comparable to that of the clinically used standards. More importantly, these reactivators exhibited wide-spectrum efficacy and were minutely investigated via determination of their reactivation kinetics in parallel with molecular dynamics simulations to study their mechanisms of reactivation of the tabun-inhibited AChE conjugate. To further confirm the potential applicability of these candidates, a mouse in vivo assay was conducted. While K1375 had the lowest acute toxicity and the most suitable pharmacokinetic profile, the oxime K1374 with delayed elimination half-life was the most effective in ameliorating the signs of tabun toxicity. Moreover, both in vitro and in vivo, the versatility of the agents was substantially superior to that of clinically used standards. Their high efficacy and broad-spectrum capability make K1374 and K1375 promising candidates that should be further investigated for their potential as nerve agents and insecticide antidotes.
ESTHER : Gorecki_2021_Arch.Toxicol__
PubMedSearch : Gorecki_2021_Arch.Toxicol__
PubMedID: 33517499

Title : Organophosphorus pesticides exhibit compound specific effects in rat precision-cut lung slices (PCLS): mechanisms involved in airway response, cytotoxicity, inflammatory activation and antioxidative defense - Tigges_2021_Arch.Toxicol__
Author(s) : Tigges J , Worek F , Thiermann H , Wille T
Ref : Archives of Toxicology , : , 2021
Abstract : Organophosphorus compound pesticides (OP) are widely used in pest control and might be misused for terrorist attacks. Although acetylcholinesterase (AChE) inhibition is the predominant toxic mechanism, OP may induce pneumonia and formation of lung edema after poisoning and during clinical treatment as life-threatening complication. To investigate the underlying mechanisms, rat precision-cut lung slices (PCLS) were exposed to the OP parathion, malathion and their biotransformation products paraoxon and malaoxon (100-2000 micromol/L). Airway response, metabolic activity, release of LDH, cytokine expression and oxidative stress response were analyzed. A concentration-dependent inhibition of airway relaxation was observed after exposure with the oxon but not with the thion-OP. In contrast, cytotoxic effects were observed for both forms in higher concentrations. Increased cytokine expression was observed after exposure to parathion and paraoxon (IL-6, GM-CSF, MIP-1alpha) and IL-6 expression was dependent on NFkappaB activation. Intracellular GSH levels were significantly reduced by all four tested OP but an increase in GSSG and HO-1 expression was predominantly observed after malaoxon exposure. Pretreatment with the antioxidant N-acetylcysteine reduced malaoxon but not paraoxon-induced cytotoxicity. PCLS as a 3D lung model system revealed OP-induced effects depending on the particular OP. The experimental data of this study contribute to a better understanding of OP toxicity on cellular targets and may be a possible explanation for the variety of clinical outcomes induced by different OP.
ESTHER : Tigges_2021_Arch.Toxicol__
PubMedSearch : Tigges_2021_Arch.Toxicol__
PubMedID: 34778934

Title : Release of protein-bound nerve agents by excess fluoride from whole blood: GC-MS\/MS method development, validation, and application to a real-life denatured blood sample - Koller_2021_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1179_122693
Author(s) : Koller M , Thiermann H , Worek F , Wille T
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 1179 :122693 , 2021
Abstract : 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: R(2) = 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.
ESTHER : Koller_2021_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1179_122693
PubMedSearch : Koller_2021_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1179_122693
PubMedID: 34171608

Title : Post-VX exposure treatment of rats with engineered phosphotriesterases - Stigler_2021_Arch.Toxicol__
Author(s) : Stigler L , Kohler A , Koller M , Job L , Escher B , Potschka H , Thiermann H , Skerra A , Worek F , Wille T
Ref : Archives of Toxicology , : , 2021
Abstract : 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 (k(cat)/K(M) = 21 to 38 x 10(6) 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 (~ 2LD(50)) 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.
ESTHER : Stigler_2021_Arch.Toxicol__
PubMedSearch : Stigler_2021_Arch.Toxicol__
PubMedID: 34962578

Title : Diagnostics and treatment of nerve agent poisoning-current status and future developments - Amend_2020_Ann.N.Y.Acad.Sci__
Author(s) : Amend N , Niessen KV , Seeger T , Wille T , Worek F , Thiermann H
Ref : Annals of the New York Academy of Sciences , : , 2020
Abstract : Although 193 states have committed to the Chemical Weapons Convention and 98% of the declared chemical weapons stockpiles have been destroyed so far, nerve agent poisoning remains a lingering threat. The recent dissemination of sarin in Syria, the assassination of Kim Jong-Nam in Malaysia, and the assault on Sergei Skripal in the United Kingdom underline the need for effective treatment. The current therapeutic options of a muscarinic receptor antagonist, an oxime, and an anticonvulsant have been unchanged for decades. Therefore, new therapeutic strategies, for example, bio scavengers and receptor-active substances, are promising concepts that have to be examined for their benefits and limitations. In order to facilitate rapid diagnosis in challenging clinical situations, point-of-care diagnostics and detection are of importance. Therapeutic guidance concerning the duration and success of the current oxime therapy via determination of the cholinesterase status can contribute to an optimal use of resources. In summary, the challenges of current and future therapies for nerve agent poisoning and key diagnostic devices will be discussed.
ESTHER : Amend_2020_Ann.N.Y.Acad.Sci__
PubMedSearch : Amend_2020_Ann.N.Y.Acad.Sci__
PubMedID: 32198755

Title : Early diagnosis of nerve agent exposure with a mobile test kit and implications for medical countermeasures: a trigger to react - Wille_2020_BMJ.Mil.Health__
Author(s) : Wille T , Djordjevic S , Worek F , Thiermann H , Vucinic S
Ref : BMJ Mil Health , : , 2020
Abstract : Recent uses of nerve agents underline the need of early diagnosis as trigger to react (initiating medical countermeasures, avoiding cross-contamination). As organophosphorus (OP) pesticide poisoning exerts the same pathomechanism, that is, inhibition of the pivotal enzyme acetylcholinesterase (AChE), a portable cholinesterase (ChE) test kit was applied in an emergency room for rapid diagnosis of OP poisoning. OP nerve agents or pesticides result in the inhibition of AChE. As AChE is also expressed on erythrocytes, patient samples are easily available. However, in most clinics only determination of plasma butyrylcholinesterase (BChE) is established which lacks a pathophysiological correlate, shows higher variability in the population and behaves different regarding inhibition by OP and reactivation by oximes. The ChE test kit helped to diagnose atypical cases of OP poisoning, for example, missing of typical muscarinic symptoms, and resulted in administration of pralidoxime, the oxime used in Serbia. The ChE test kit also allows an initial assessment whether an oxime therapy is successful. In one case report, AChE activity increased after oxime administration indicating therapeutic success whereas BChE activity did not. With only BChE at hand, this therapeutic effect would have been missed. As inhibition of AChE or BChE activity is determined, the CE-certified device is a global diagnostic tool for all ChE inhibitors including carbamates which might also be misused as chemical weapon. The ChE test kit is a helpful point-of-care device for the diagnosis of ChE inhibitor poisoning. Its small size and easy menu-driven use advocate procurement where nerve agent and OP pesticide exposure are possible.
ESTHER : Wille_2020_BMJ.Mil.Health__
PubMedSearch : Wille_2020_BMJ.Mil.Health__
PubMedID: 32086265

Title : Organophosphorus compounds and oximes: a critical review - Worek_2020_Arch.Toxicol_94_2275
Author(s) : Worek F , Thiermann H , Wille T
Ref : Archives of Toxicology , 94 :2275 , 2020
Abstract : Organophosphorus (OP) pesticides and nerve agents still pose a threat to the population. Treatment of OP poisoning is an ongoing challenge and burden for medical services. Standard drug treatment consists of atropine and an oxime as reactivator of OP-inhibited acetylcholinesterase and is virtually unchanged since more than six decades. Established oximes, i.e. pralidoxime, obidoxime, TMB-4, HI-6 and MMB-4, are of insufficient effectiveness in some poisonings and often cover only a limited spectrum of the different nerve agents and pesticides. Moreover, the value of oximes in human OP pesticide poisoning is still disputed. Long-lasting research efforts resulted in the preparation of countless experimental oximes, and more recently non-oxime reactivators, intended to replace or supplement the established and licensed oximes. The progress of this development is slow and none of the novel compounds appears to be suitable for transfer into advanced development or into clinical use. This situation calls for a critical analysis of the value of oximes as mainstay of treatment as well as the potential and limitations of established and novel reactivators. Requirements for a straightforward identification of superior reactivators and their development to licensed drugs need to be addressed as well as options for interim solutions as a chance to improve the therapy of OP poisoning in a foreseeable time frame.
ESTHER : Worek_2020_Arch.Toxicol_94_2275
PubMedSearch : Worek_2020_Arch.Toxicol_94_2275
PubMedID: 32506210

Title : A case report of cholinesterase inhibitor poisoning: cholinesterase activities and analytical methods for diagnosis and clinical decision making - Amend_2020_Arch.Toxicol__
Author(s) : Amend N , Langgartner J , Siegert M , Kranawetvogl T , Koller M , John H , Pflugler C , Mogele-Schmid C , Worek F , Thiermann H , Wille T
Ref : Archives of Toxicology , : , 2020
Abstract : Suicidal ingestion of organophosphorus (OP) or carbamate (CM) compounds challenges health care systems worldwide, particularly in Southeast Asia. The diagnosis and treatment of OP or CM poisoning is traditionally based on the clinical appearance of the typical cholinergic toxidrome, e.g. miosis, salivation and bradycardia. Yet, clinical signs might be inconclusive or even misleading. A current case report highlights the importance of enzymatic assays to provide rapid information and support clinicians in diagnosis and rational clinical decision making. Furthermore, the differentiation between OP and CM poisoning seems important, as an oxime therapy will most probably not provide benefit in CM poisoning, but-as every pharmaceutical product-it might result in adverse effects. The early identification of the causing agent and the amount taken up in the body are helpful in planning of the therapeutic regimen including experimental strategies, e.g. the use of human blood products to facilitate scavenging of the toxic agent. Furthermore, the analysis of biotransformation products and antidote levels provides additional insights into the pathophysiology of OP or CM poisoning. In conclusion, cholinesterase activities and modern analytical methods help to provide a more effective treatment and a thorough understanding of individual cases of OP or CM poisoning.
ESTHER : Amend_2020_Arch.Toxicol__
PubMedSearch : Amend_2020_Arch.Toxicol__
PubMedID: 32303803

Title : In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase - Worek_2020_Molecules_25_
Author(s) : Worek F , Thiermann H , Koller M , Wille T
Ref : Molecules , 25 : , 2020
Abstract : 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.
ESTHER : Worek_2020_Molecules_25_
PubMedSearch : Worek_2020_Molecules_25_
PubMedID: 32630769

Title : Synthesis and in vitro evaluation of novel non-oximes for the reactivation of nerve agent inhibited human acetylcholinesterase - de Koning_2020_Chem.Biol.Interact__109139
Author(s) : De Koning MC , Horn G , Worek F , van Grol M
Ref : Chemico-Biological Interactions , :109139 , 2020
Abstract : Since several decades oximes have been used as part of treatment of nerve agent intoxication with the aim to restore the biological function of the enzyme acetylcholinesterase after its covalent inhibition by organophosphorus compounds such as pesticides and nerve agents. Recent findings have illustrated that, besides oximes, certain Mannich phenols can reactivate the inhibited enzyme very effectively, and may therefore represent an attractive complementary class of reactivators. In this paper we further probe the effect of structural variation on the in vitro efficacy of Mannich phenol based reactivators. Thus, we present the synthesis of 14 compounds that are close variants of the previously reported 4-amino-2-(1-pyrrolidinylmethyl)-phenol, a very effective non-oxime reactivator, and 3 dimeric Mannich phenols. All compounds were assessed for their ability to reactivate human acetylcholinesterase inhibited by the nerve agents VX, tabun, sarin, cyclosarin and paraoxon in vitro. It was confirmed that the potency of the compounds is highly sensitive to small structural changes, leading to diminished reactivation potency in many cases. However, the presence of 4-substituted alkylamine substituents (as exemplified with the 4-benzylamine-variant) was tolerated. More surprisingly, the dimeric compounds demonstrated non-typical behavior and displayed some reactivation potency as well. Both findings may open up new avenues for designing more effective non-oxime reactivators.
ESTHER : de Koning_2020_Chem.Biol.Interact__109139
PubMedSearch : de Koning_2020_Chem.Biol.Interact__109139
PubMedID: 32454005

Title : COPD and asthma therapeutics for supportive treatment in organophosphate poisoning - Herbert_2019_Clin.Toxicol.(Phila)__1
Author(s) : Herbert J , Thiermann H , Worek F , Wille T
Ref : Clinical Toxicology (Phila) , :1 , 2019
Abstract : CONTEXT: Nerve agents like sarin or VX have repeatedly been used in military conflicts or homicidal attacks, as seen in Syria or Malaysia 2017. Together with pesticides, nerve agents assort as organophosphorus compounds (OP), which inhibit the enzyme acetylcholinesterase. To counteract subsequent fatal symptoms due to acetylcholine (ACh) accumulation, oximes plus atropine are administered, a regimen that lacks efficacy in several cases of OP poisoning. New therapeutics are in development, but still need evaluation before clinical employment. Supportive treatment with already approved drugs presents an alternative, whereby compounds from COPD and asthma therapy are likely options. A recent pilot study by Chowdhury et al. included beta2-agonist salbutamol in the treatment of OP-pesticide poisoned patients, yielding ambiguous results concerning the addition. Here, we provide experimental data for further investigations regarding the value of these drugs in OP poisoning. METHODS: By video-microscopy, changes in airway area were analyzed in VX-poisoned rat precision cut lung slices (PCLS) after ACh-induced airway contraction and subsequent application of selected anticholinergics/beta2-agonists. RESULTS: Glycopyrrolate and ipratropium efficiently antagonized an ACh-induced airway contraction in VX-poisoned PCLS (EC50 glycopyrrolate 15.8 nmol/L, EC50 ipratropium 2.3 nmol/L). beta2-agonists formoterol and salbutamol had only negligible effects when solely applied in the same setting. However, combination of formoterol or salbutamol with low dosed glycopyrrolate or atropine led to an additive effect compared to the sole application [50.6 +/- 8.8% airway area increase after 10 nmol/L formoterol +1 nmol/L atropine versus 11.7 +/- 9.2% (10 nmol/L formoterol) or 8.6 +/- 5.9% (1 nmol/L atropine)]. DISCUSSION: We showed antagonizing effects of anticholinergics and beta2-agonists on ACh-induced airway contractions in VX-poisoned PCLS, thus providing experimental data to support a prospective comprehensive clinical study. CONCLUSIONS: Our results indicate that COPD and asthma therapeutics could be a valuable addition to the treatment of OP poisoning.
ESTHER : Herbert_2019_Clin.Toxicol.(Phila)__1
PubMedSearch : Herbert_2019_Clin.Toxicol.(Phila)__1
PubMedID: 30696282

Title : The in vitro protective effects of the three novel nanomolar reversible inhibitors of human cholinesterases against irreversible inhibition by organophosphorous chemical warfare agents - Vitorovic-Todorovic_2019_Chem.Biol.Interact_13ChEPon_309_108714
Author(s) : Vitorovic-Todorovic MD , Worek F , Perdih A , Bauk SD , Vujatovic TB , Cvijetic IN
Ref : Chemico-Biological Interactions , 309 :108714 , 2019
Abstract : Acetylcholinesterase (AChE) is an enzyme which terminates the cholinergic neurotransmission, by hydrolyzing acetylcholine at the nerve and nerve-muscle junctions. The reversible inhibition of AChE was suggested as the pre-treatment option of the intoxications caused by nerve agents. Based on our derived 3D-QSAR model for the reversible AChE inhibitors, we designed and synthesized three novel compounds 8-10, joining the tacrine and aroylacrylic acid phenylamide moieties, with a longer methylene chain to target two distinct, toplogically separated anionic areas on the AChE. The targeted compounds exerted low nanomolar to subnanomolar potency toward the E. eel and human AChE's as well as the human BChE and showed mixed inhibition type in kinetic studies. All compounds were able to slow down the irreversible inhibition of the human AChE by several nerve agents including tabun, soman and VX, with the estimated protective indices around 5, indicating a valuable level of protection. Putative noncovalent interactions of the selected ligand 10 with AChE active site gorge were finally explored by molecular dynamics simulation suggesting a formation of the salt bridge between the protonated linker amino group and the negatively charged Asp74 carboxylate side chain as a significant player for the successful molecular recognition in line with the design strategy. The designed compounds may represent a new class of promising leads for the development of more effective pre-treatment options.
ESTHER : Vitorovic-Todorovic_2019_Chem.Biol.Interact_13ChEPon_309_108714
PubMedSearch : Vitorovic-Todorovic_2019_Chem.Biol.Interact_13ChEPon_309_108714
PubMedID: 31228470

Title : The arrhythmogenic potential of nerve agents and a cardiac safety profile of antidotes - A proof-of-concept study using human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) - Amend_2019_Toxicol.Lett_308_1
Author(s) : Amend N , Thiermann H , Worek F , Wille T
Ref : Toxicol Lett , 308 :1 , 2019
Abstract : The global use of organophosphorus compounds (OP) for pest control and nerve agents being used in military conflicts and for assassinations renders intoxications by these agents a public health concern. OP-poisoned patients often suffer from dysrhythmias which may ultimately result in death. In this study, human-induced pluripotent stem cells derived cardiomyocytes were exposed to OP compounds in a microelectrode array system (MEA). The MEA system is widely accepted to assess the proarrhythmic properties of (candidate) drugs. The directly acting cholinergic compounds acetylcholine and carbachol and the irreversible acetylcholinesterase inhibitor cyclosarin - a highly toxic nerve agent - were assessed. All three compounds induced a dose-dependent (up to 600 nmol/L) corrected field potential duration (FPDc) prolongation of 9.7 +/- 0.6% for carbachol, for 9.7 +/- 1.2% acetylcholine and 9.4 +/- 0.5% for cyclosarin. Additionally, the electrophysiological alterations of the clinically approved oxime reactivators obidoxime, pralidoxime and the oximes in development HI-6 and MMB-4 were investigated in the absence of OP. Neither of these oximes (up to a concentration of 300 mumol/L) caused dysrhythmia nor beat arrest. The competitive muscarinic receptor antagonist atropine as a cornerstone in the treatment of OP poisoning was also analyzed. Interestingly, atropine caused a drop in the beat rate which might result from a non-receptor action of this substance in the absence of OP. Atropine in combination with the OP nerve agent cyclosarin and the direct cholinergics acetylcholine or carabachol completely reversed the induced FPDc prolongation. However, the oxime HI-6 as potent reactivator of cyclosarin-inhibited AChE was not able to prevent the FPDc prolongation in this model. In conclusion, the current model allows the assessment of FPDc prolongation by the nerve agent cyclosarin, the cholinergic compounds carbachol, acetylcholine and the block of this effect by atropine.
ESTHER : Amend_2019_Toxicol.Lett_308_1
PubMedSearch : Amend_2019_Toxicol.Lett_308_1
PubMedID: 30858091

Title : Efficacy of an organophosphorus hydrolase enzyme (OpdA) in human serum and minipig models of organophosphorus insecticide poisoning - Eddleston_2019_Clin.Toxicol.(Phila)__1
Author(s) : Eddleston M , Clutton RE , Taylor M , Thompson A , Worek F , John H , Thiermann H , Scott C
Ref : Clinical Toxicology (Phila) , :1 , 2019
Abstract : Objectives: Current therapeutic options for organophosphorus (OP) insecticide self-poisoning including atropine and oximes are inadequate and case fatality may exceed 20%. An OP hydrolase enzyme, OpdA, has been used for environmental cleansing of OP insecticides and prevented death in rat and non-human primate models of OP insecticide poisoning if given very quickly after exposure. We here tested OpdA's ability to break down OP insecticides in human serum and in clinically relevant minipig models of OP insecticide poisoning. Methods: Human serum was spiked with seven diverse WHO Class II OP insecticides (chlorpyrifos, quinalphos, diazinon, dimethoate, fenthion, phenthoate, and profenofos) and the effect of OpdA on degradation measured. The pharmacodynamic and clinical effects of OpdA treatment were studied in Gottingen minipigs orally poisoned with agricultural formulations of dimethoate EC40 or methyl parathion EC60; pharmacodynamic effects were also assessed in profenofos EC50-poisoned pigs. Results: OpdA effectively hydrolysed OP insecticides in human serum, with rates varying from 856 (SD 44) down to 0.107 (SD 0.01) moles of substrate hydrolysed/mole of enzyme/sec (kcat) for quinalphos and phenthoate, respectively, although at rates 2-3 log orders less than found in vitro in buffered solution. It showed clinical benefit in minipig models, reducing the dose of noradrenaline required to sustain an adequate mean arterial pressure after dimethoate (mean 0.149 [SD 0.10] mug/kg/h vs. 1.07 [SD 0.77] mug/kg/h, p < .0001) and methyl parathion (mean 0.077 [SD 0.08] mug/kg/h vs. 0.707 [SD 0.49] mug/kg/h, p < .0001) poisoning. OpdA reduced blood OP insecticide concentration and acetylcholinesterase inhibition after poisoning by dimethoate, methyl parathion, and profenofos insecticides. Conclusions: In vitro incubation of OpdA in human serum showed hydrolysis of diverse OP insecticides, although at lower rates than found in buffer solutions. This activity results in clinical and pharmacodynamic efficacy in vivo against several OP insecticides. These results support the testing of OpdA in further animal models before considering human trials to determine whether it may become an urgently required novel therapeutic agent for OP insecticide self-poisoning.
ESTHER : Eddleston_2019_Clin.Toxicol.(Phila)__1
PubMedSearch : Eddleston_2019_Clin.Toxicol.(Phila)__1
PubMedID: 31452424

Title : New Resensitizers for the Nicotinic Acetylcholine Receptor by Ligand-Based Pharmacophore Modeling - Wein_2019_Curr.Comput.Aided.Drug.Des_15_104
Author(s) : Wein T , Wanner KT , Rappengluck S , Sichler S , Niessen KV , Seeger T , Worek F , Thiermann H
Ref : Curr Comput Aided Drug Des , 15 :104 , 2019
Abstract : INTRODUCTION: Irreversible inhibition of the acetylcholinesterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. A direct intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach to the treatment with atropine and oximes. METHODS: The bispyridinium compound MB327 has been found to recover functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. Recent solid-supported membrane-based electrophysiological experiments have identified symmetrically substituted bispyridinium compounds e.g. MB327, MB583, and PTM0001 that are able to resensitize nAChR of Torpedo californica. In addition, six compounds have been found not to show any resensitizing potential and were thus classified as inactive. This set of active and inactive bispyridinium compounds was taken to develop a pharmacophore model and in silico screening of a virtual database of bispyridinium compounds to identify new compounds that are able to restore the functional activity of desensitized nAChR. RESULTS: Screening of a virtual compound database of symmetrically substituted bispyridinium compounds with the derived pharmacophore yielded several promising compounds which satisfy the pharmacophore and ought to have the same or even better resensitizing effect on nAChR as the parent compound MB327.
ESTHER : Wein_2019_Curr.Comput.Aided.Drug.Des_15_104
PubMedSearch : Wein_2019_Curr.Comput.Aided.Drug.Des_15_104
PubMedID: 29968541

Title : Human small bowel as model for poisoning with organophosphorus compounds - Marquart_2019_Toxicol.In.Vitro_57_76
Author(s) : Marquart K , Prokopchuk O , Wilhelm D , Worek F , Thiermann H , Martignoni ME , Wille T
Ref : Toxicol In Vitro , 57 :76 , 2019
Abstract : In previous experiments, human and rat small bowel samples have been successfully used to study the spasmolytic effect of (potential) therapeutics in carbamate-constricted bowel specimens. Additionally, transferability from rat to human data was shown in the previous study. In the present study, the effects of atropine, scopolamine, MB327, HI-6 as well as obidoxime were examined in organophosphorus-poisoned human small bowel specimens. All substances were tested with at least seven concentrations in samples previously exposed to the nerve agent sarin. Furthermore, the cholinesterase reactivation potential of all substances was investigated. The test substances displayed a spasmolytic effect allowing the calculation of dose-response curves and EC50s. The parasympatholytic compound scopolamine had the strongest relaxing effect (EC50=0.05muM) followed by atropine (EC50=0.07muM). HI-6 and obidoxime were capable to reactivate the sarin-inhibited cholinesterase activity in small bowel samples. Both substances restored AChE activity in a dose-dependent way, with HI-6 being more potent (HI-6 EC50=3.8muM vs obidoxime EC50=197.8muM). Summarizing, our isolated human small bowel setup is a suitable tool to investigate the smooth muscle relaxing effect of (candidate) therapeutics for organophosphorus compound poisoning i.e. sarin exposure in a complex 3D tissue model.
ESTHER : Marquart_2019_Toxicol.In.Vitro_57_76
PubMedSearch : Marquart_2019_Toxicol.In.Vitro_57_76
PubMedID: 30763608

Title : Development of MS Binding Assays targeting the binding site of MB327 at the nicotinic acetylcholine receptor - Sichler_2018_Toxicol.Lett_293_172
Author(s) : Sichler S , Hofner G , Rappengluck S , Wein T , Niessen KV , Seeger T , Worek F , Thiermann H , Paintner FF , Wanner KT
Ref : Toxicol Lett , 293 :172 , 2018
Abstract : The bispyridinium compound MB327 has been shown previously to have a positive pharmacological effect against poisoning with organophosphorous compounds (OPCs). The mechanism by which it exerts its therapeutic effect seems to be directly mediated by the nicotinic acetylcholine receptor (nAChR). In the present study, the development of mass spectrometry based binding assays (MS Binding Assays) for characterization of the binding site of MB327 at the nAChR from Torpedo californica is described. MS Binding Assays follow the principle of radioligand binding assays, but do not, in contrast to the latter, require a radiolabeled reporter ligand, as the readout is in this case based on mass spectrometric detection. For [(2)H6]MB327, a deuterated MB327 analogue employed as reporter ligand in the MS Binding Assays, an LC-ESI-MS/MS method was established allowing for its fast and reliable quantification in samples resulting from binding experiments. Using centrifugation for separation of non-bound [(2)H6]MB327 from target-bound [(2)H6]MB327 in saturation and autocompetition experiments (employing native MB327 as competitor) enabled reliable determination of specific binding. In this way, the affinities for [(2)H6]MB327 (Kd=15.5+/-0.9mumolL(-1)) and for MB327 (Ki=18.3+/-2.6mumolL(-1)) towards the nAChR could be determined for the first time. The almost exactly matching affinities for MB327 and [(2)H6]MB327 obtained in the MS Binding Assays are in agreement with potencies previously found in functional studies. In summary, our results demonstrate that the established MS Binding Assays represent a promising tool for affinity determination of test compounds towards the binding site of MB327 at the nAChR.
ESTHER : Sichler_2018_Toxicol.Lett_293_172
PubMedSearch : Sichler_2018_Toxicol.Lett_293_172
PubMedID: 29146291

Title : Human small bowel as a useful tool to investigate smooth muscle effects of potential therapeutics in organophosphate poisoning - Marquart_2018_Toxicol.Lett_293_235
Author(s) : Marquart K , Prokopchuk O , Worek F , Thiermann H , Martignoni ME , Wille T
Ref : Toxicol Lett , 293 :235 , 2018
Abstract : Isolated organs proofed to be a robust tool to study effects of (potential) therapeutics in organophosphate poisoning. Small bowel samples have been successfully used to reveal smooth muscle relaxing effects. In the present study, the effects of obidoxime, TMB-4, HI-6 and MB 327 were investigated on human small bowel tissue and compared with rat data. Hereby, the substances were tested in at least seven different concentrations in the jejunum or ileum both pre-contracted with carbamoylcholine. Additionally, the cholinesterase activity of native tissue was determined. Human small intestine specimens showed classical dose response-curves, similar to rat tissue, with MB 327 exerting the most potent smooth muscle relaxant effect in both species (human EC50=0.7x10(-5)M and rat EC50=0.7x10(-5)M). The AChE activity for human and rat samples did not differ significantly (rat jejunum=1351+/-166 mU/mg wet weight; rat ileum=1078+/-123 mU/mg wet weight; human jejunum=1030+/-258 mU/mg wet weight; human ileum=1293+/-243 mU/mg wet weight). Summarizing, our isolated small bowel setup seems to be a solid tool to investigate the effects of (potential) therapeutics on pre-contracted smooth muscle, with data being transferable between rat and humans.
ESTHER : Marquart_2018_Toxicol.Lett_293_235
PubMedSearch : Marquart_2018_Toxicol.Lett_293_235
PubMedID: 29154801

Title : Searching for putative binding sites of the bispyridinium compound MB327 in the nicotinic acetylcholine receptor - Wein_2018_Toxicol.Lett_293_184
Author(s) : Wein T , Hofner G , Rappengluck S , Sichler S , Niessen KV , Seeger T , Worek F , Thiermann H , Wanner KT
Ref : Toxicol Lett , 293 :184 , 2018
Abstract : Irreversible inhibition of the acetylcholine esterase upon intoxication with organophosphorus compounds leads to an accumulation of acetylcholine in the synaptic cleft and a subsequent desensitization of nicotinic acetylcholine receptors which may ultimately result in respiratory failure. The bispyridinium compound MB327 has been found to restore functional activity of nAChR thus representing a promising starting point for the development of new drugs for the treatment of organophosphate poisoning. In order to optimize the resensitizing effect of MB327 on nAChR, it would be very helpful to know the MB327 specific binding site to apply structure based molecular modeling. The binding site for MB327 at the nAChR is not known and so far goal of speculations, but it has been shown that MB327 does not bind to the orthosteric acetylcholine binding site. We have used docking calculations to screen the surface of nAChR for possible binding sites of MB327. The results indicate that at least two potential binding sites for MB327 at nAChR are present inside the channel pore. In these binding sites, MB327 intercalates between the gamma-alpha and beta-delta subunits of nAChR, respectively. Both putative MB327 binding sites show an unsymmetrical distribution of surrounding hydrophilic and lipophilic amino acids. This suggests that substitution of MB327-related bispyridinium compounds on one of the two pyridinium rings with polar substituents should have a favorable effect on the pharmacological function.
ESTHER : Wein_2018_Toxicol.Lett_293_184
PubMedSearch : Wein_2018_Toxicol.Lett_293_184
PubMedID: 29097222

Title : Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents - Porzio_2018_Sci.Rep_8_13773
Author(s) : Porzio E , Bettazzi F , Mandrich L , Del Giudice I , Restaino OF , Laschi S , Febbraio F , De Luca V , Borzacchiello MG , Carusone TM , Worek F , Pisanti A , Porcaro P , Schiraldi C , De Rosa M , Palchetti I , Manco G
Ref : Sci Rep , 8 :13773 , 2018
Abstract : Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.
ESTHER : Porzio_2018_Sci.Rep_8_13773
PubMedSearch : Porzio_2018_Sci.Rep_8_13773
PubMedID: 30214052

Title : Counteracting desensitization of human alpha7-nicotinic acetylcholine receptors with bispyridinium compounds as an approach against organophosphorus poisoning - Scheffel_2018_Toxicol.Lett_293_149
Author(s) : Scheffel C , Niessen KV , Rappengluck S , Wanner KT , Thiermann H , Worek F , Seeger T
Ref : Toxicol Lett , 293 :149 , 2018
Abstract : Irreversible inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and overstimulation of muscarinic and nicotinic receptors accounts for the acute toxicity of organophosphorus compounds (OP). Accordingly, the mainstay pharmacotherapy against poisoning by OP comprises the competitive muscarinic acetylcholine receptor antagonist atropine to treat muscarinic effects and, in addition, oximes to reactivate inhibited AChE. A therapeutic gap still remains in the treatment of desensitized nicotinic acetylcholine receptors following OP exposure. Hereby, nicotinic effects result in paralysis of the central and peripheral respiratory system if untreated. Thus, these receptors pose an essential target for therapeutic indication to address these life-threatening nicotinic symptoms of the cholinergic crisis. Identification of ligands regulating dynamic transitions between functional states by binding to modulatory sites appears to be a promising strategy for therapeutic intervention. In this patch clamp study, the ability of differently substituted bispyridinium non-oximes to "resensitize" i.e. to recover the activity of desensitized human homomeric alpha7-type nAChRs stably transfected in CHO cells was investigated and compared to the already described alpha7-specific positive allosteric modulator PNU-120596. The structures of these bispyridinium analogues were based on the lead structure of the tert-butyl-substituted bispyridinium propane MB327, which has been shown to have a positive therapeutic effect due to a non-competitive antagonistic action at muscle-type nAChRs in vivo and has been found to have a positive allosteric activity at neuronal receptors in vitro. Prior to test compounds, desensitization of halpha7-nAChRs was verified by applying an excess of nicotine revealing activation at low, and desensitization at high concentrations. Thereby, desensitization could be reduced by modulation with PNU-120596. Desensitization was further verified by dose-response profiles of agonists, carbamoylcholine and epibatidine in the absence and presence of PNU-120596. Although less pronounced than PNU-120596 and the lead structure MB327, bispyridinium compounds, particularly those substituted at position 3 and 4, resensitized the nicotine desensitized halpha7-nAChRs in a concentration-dependent manner and prolonged the mean channel open time. In summary, identification of more potent compounds able to restore nAChR function in OP intoxication is needed for development of a putative efficient antidote.
ESTHER : Scheffel_2018_Toxicol.Lett_293_149
PubMedSearch : Scheffel_2018_Toxicol.Lett_293_149
PubMedID: 29248576

Title : Midazolam is effective to reduce cortical network activity in organotypic cultures during severe cholinergic overstimulation with soman - Drexler_2018_Toxicol.Lett_297_19
Author(s) : Drexler B , Seeger T , Worek F , Thiermann H , Antkowiak B , Grasshoff C
Ref : Toxicol Lett , 297 :19 , 2018
Abstract : Intoxication with organophosphorus compounds can result in life-threatening organ dysfunction and refractory seizures. Sedation or hypnosis is essential to facilitate mechanical ventilation and control seizure activity. The range of indications for midazolam includes both hypnosis and seizure control. Since benzodiazepines cause sedation and hypnosis by dampening neuronal activity of the cerebral cortex, we investigated the drug's effect on action potential firing of cortical neurons in brain slices. Extensive cholinergic overstimulation was induced by increasing acetylcholine levels and simultaneously treating the slices with soman to block acetylcholinesterase activity. At control conditions midazolam reduced discharge rates (median/95% confidence interval) from 8.8 (7.0-10.5) Hz (in the absence of midazolam) to 2.2 (1.4-2.9) Hz (10 muM midazolam) and 1.6 (0.9-2.2) Hz (20 muM midazolam). Without midazolam, cholinergic overstimulation significantly enhanced neuronal activity to 13.1 (11.0-15.2) Hz. Midazolam attenuated firing rates during cholinergic overstimulation to 6.5 (4.8-8.2) Hz (10 muM midazolam) and 4.1 (3.3-6.0) Hz (20 muM midazolam), respectively. Thus, high cholinergic tone attenuated the drug's efficacy only moderately. These results suggest that midazolam is worth being tested as a promising drug to induce sedation and hypnosis in patients suffering from severe organophosphorous intoxication.
ESTHER : Drexler_2018_Toxicol.Lett_297_19
PubMedSearch : Drexler_2018_Toxicol.Lett_297_19
PubMedID: 30165091

Title : The oximes HI-6 and MMB-4 fail to reactivate soman-inhibited human and guinea pig AChE: A kinetic in vitro study - Worek_2018_Toxicol.Lett_293_216
Author(s) : Worek F , Thiermann H , Wille T
Ref : Toxicol Lett , 293 :216 , 2018
Abstract : Acetylcholinesterase (AChE) inhibited by the organophosphorus nerve (OP) agent soman underlies a spontaneous and extremely rapid dealkylation ("aging") reaction which prevents reactivation by oximes. However, in vivo studies in various, soman poisoned animal species showed a therapeutic effect of oximes, with the exact mechanism of this effect remaining still unclear. In order to get more insight and a basis for the extrapolation of animal data to humans, we applied a dynamic in vitro model with continuous online determination of AChE activity. This model allows to simulate the in vivo toxico- and pharmacokinetics between human and guinea pig AChE with soman and the oximes HI-6 and MMB-4 in order to unravel the species dependent kinetic interactions. It turned out that only HI-6 was able to slow down the ongoing inhibition of human AChE by soman without preventing final complete inhibition of the enzyme. Continuous perfusion of AChE with soman and simultaneous or delayed (8, 15 or 40min) oxime perfusion did not result in a relevant reactivation of AChE (less than 2%). In conclusion, the results of the present study indicate a negligible reactivation of soman-inhibited AChE by oximes at conditions simulating the in vivo poisoning by soman. The observed therapeutic effect of oximes in soman poisoned animals in vivo must be attributed to alternative mechanisms which may not be relevant in humans.
ESTHER : Worek_2018_Toxicol.Lett_293_216
PubMedSearch : Worek_2018_Toxicol.Lett_293_216
PubMedID: 28993240

Title : Electrophysiological investigation of the effect of structurally different bispyridinium non-oxime compounds on human alpha7-nicotinic acetylcholine receptor activity-An in vitro structure-activity analysis - Scheffel_2018_Toxicol.Lett_293_157
Author(s) : Scheffel C , Niessen KV , Rappengluck S , Wanner KT , Thiermann H , Worek F , Seeger T
Ref : Toxicol Lett , 293 :157 , 2018
Abstract : Organophosphorus compounds, including nerve agents and pesticides, exert their toxicity through irreversible inhibition of acetylcholinesterase (AChE) resulting in an accumulation of acetylcholine and functional impairment of muscarinic and nicotinic acetylcholine receptors. Current therapy comprises oximes to reactivate AChE and atropine to antagonize effects induced by muscarinic acetylcholine receptors. Nicotinic malfunction leading to depression of the central and peripheral respiratory system is not directly treated calling for alternative therapeutic interventions. In the present study, we investigated the electrophysiological properties of the human nAChR subtype alpha7 (halpha7-nAChR) and the functional effect of the 4-tert-butyl bispyridinium (BP) compound MB327 and of a series of novel substituted bispyridinium compounds on the receptors by an automated patch clamp technique. Activation of halpha7-nAChRs was induced by nicotine and acetylcholine demonstrating rapid cationic influx up to 100muM. Agonist-induced currents decayed within a few milliseconds revealing fast desensitization of the receptors. Application of higher agonist concentrations led to a decline of current amplitudes which seemed to be due to increasing receptor desensitization. When 100muM of agonist was coapplied with low concentrations of the well characterized alpha7-specific positive allosteric modulator PNU-120596 (1muM-10muM), the maximum response and duration of nAChR activation were markedly augmented indicating an elongated mean open-time of receptors and prevention of receptor desensitization. However, co-application of increasing PNU-120596 concentrations (>10muM) with agonist induced a decline of potentiated current responses. Although less pronounced than PNU-120596, six of the twenty tested substituted BP compounds, in particular those with a substituent at 3-position and 4-position at the pyridinium moieties, were found to potentiate current responses of halpha7-nAChRs, most pronounced MB327.This effect was clearly depended on the presence of the agonist indicating a positive allosteric mechanism of these compounds. Besides potentiation at low concentrations, these compounds seem to interact at different binding sites on halpha7-nAChRs since enhancement decreased at high concentrations. The residual fourteen BP compounds, possessing either an isopropyl-group or more than one group at the pyridinium moiety, antagonized nicotinic currents exhibiting IC50 of low up to high micromolar concentrations ( approximately 1muM-300muM).
ESTHER : Scheffel_2018_Toxicol.Lett_293_157
PubMedSearch : Scheffel_2018_Toxicol.Lett_293_157
PubMedID: 29191791

Title : Evaluation of the accuracy of ChE check mobile in measurement of acetylcholinesterase in pesticide poisoning - Shihana_2018_Clin.Toxicol.(Phila)__1
Author(s) : Shihana F , Worek F , Dassanayake GA , Rathgamage SH , Dhanarisi J , Buckley NA
Ref : Clinical Toxicology (Phila) , :1 , 2018
Abstract : BACKGROUND: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are used in clinical management to confirm the diagnosis and indicate the severity of organophosphorus and carbamate poisoning. ChE check mobile is a new portable cholinesterase testing system developed in Germany. The study aims to evaluate the accuracy of ChE check mobile compared to the standard reference method and Test-mate ChE system. METHODS: Patients with organophosphorus and carbamate poisoning were recruited from two general hospitals in Sri Lanka between September 2013 and November 2014. The AChE was measured using the three methods. RESULTS: Blood samples were collected from 185 self-poisoned patients (170 organophosphorus and 15 carbamate) and 20 normal individuals. ChE check mobile correlated well with spectrophotometer readings (Pearson's correlation coefficient 0.87) but gave higher values (Mean bias for AChE: +6.55 (95% CI: -11 to 24) U/g Hb). A similar positive bias from Test-mate results was also observed. Applying a correction factor derived from the volunteer samples (dividing by 1.353) greatly improved agreement in pesticide poisoned patients. CONCLUSIONS: ChE check mobile system allowed for rapid determination of AChE activity but gave somewhat higher AChE compared to other methods. Applying a correction factor of 1.353 provide a good agreement to both reference and Test-mate ChE machine in this setting.
ESTHER : Shihana_2018_Clin.Toxicol.(Phila)__1
PubMedSearch : Shihana_2018_Clin.Toxicol.(Phila)__1
PubMedID: 30451024

Title : Interactions between acetylcholinesterase, toxic organophosphorus compounds and a short series of structurally related non-oxime reactivators: Analysis of reactivation and inhibition kinetics in vitro - Horn_2018_Toxicol.Lett_299_218
Author(s) : Horn G , De Koning MC , van Grol M , Thiermann H , Worek F
Ref : Toxicol Lett , 299 :218 , 2018
Abstract : Poisoning by organophosphorus compounds (OP) is characterized by inhibition of the key enzyme acetylcholinesterase (AChE) and potentially fatal outcomes in humans. Insufficient efficacy of the standard therapy with atropine and AChE reactivators (oximes) against certain OP initiated synthesis of novel non-oxime reactivators basing on the common structure 4-amino-2-((diethylamino)methyl)phenol (ADOC). Recently, we reported of a pyrrolidine-bearing ADOC analogue (3 l) with a remarkable ability to reactivate OP-inhibited AChE. This in vitro study was undertaken to determine reactivity, affinity and overall reactivation constants of 3 l, the reference compound ADOC and two structural analogues with human AChE inhibited by paraoxon, sarin, cyclosarin and VX. The data showed a 10 to 34-fold reactivating potency of 3 l compared to ADOC mainly due to improved affinity. Additionally, various interactions between non-oximes, human or guinea pig (GP) AChE and structurally different OP were investigated: OP-inhibited guinea pig AChE was less amenable to reactivation by ADOC and 3 l than human AChE. Compound 3 l was considered as potential pretreatment to prevent AChE from irreversible inhibition by OP: In the presence of 10 muM 3 l inhibition of native human AChE was attenuated resulting in protective indices (PI) ranging from about 2.7 to 6.0. A combination of 3 l and the bispyridinium oxime HI-6 was tested to reactivate OP-inhibited AChE: The superior reactivator of the respective OP-AChE combination dominated the reactivation process and a synergistic effect could not be observed. In conclusion, novel non-oxime reactivators like 3 l may be considered as promising templates for the design of more potent therapeutics against poisoning by highly toxic OP.
ESTHER : Horn_2018_Toxicol.Lett_299_218
PubMedSearch : Horn_2018_Toxicol.Lett_299_218
PubMedID: 30312685

Title : Synthesis of a Series of Non-Symmetric Bispyridinium and Related Compounds and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor - Rappengluck_2018_ChemMedChem_13_2653
Author(s) : Rappengluck S , Sichler S , Hofner G , Wein T , Niessen KV , Seeger T , Paintner FF , Worek F , Thiermann H , Wanner KT
Ref : ChemMedChem , 13 :2653 , 2018
Abstract : The current standard therapy to counteract organophosphate intoxication is not effective in equal measure against all types of organophosphorus compounds (OPCs), as the outcome of oxime-induced reactivation of inactivated acetylcholinesterase (AChE) strongly depends on the particular OPC. In case the reactivation is insufficient, acetylcholine concentrations that rise to pathophysiological levels force the nicotinic acetylcholine receptor (nAChR) into a desensitized state and hence a functionally inactive state. As a consequence, neurotransmission is irreversibly disrupted at the neuromuscular junction. Previous electrophysiological studies identified the symmetric bispyridinium compound 1,1'-(propane-1,3-diyl)bis[4-(tert-butyl)pyridin-1-ium] diiodide (MB327) as a re-sensitizer of the desensitized nAChR. MB327 is thereby capable of restoring the functional activity. Very recently, in silico modeling studies suggested non-symmetric derivatives of MB327 as potential re-sensitizers with enhanced binding affinity and thus possible enhanced efficacy. In this study, 26 novel non-symmetric bispyridinium compounds and related derivatives were synthesized. For the synthesis of the highly polar target compounds in sufficient quantities, newly developed and highly efficient two-step procedures were used. Compounds were characterized in terms of their binding affinity toward the MB327 binding site at the nAChR using recently developed mass spectrometry (MS) Binding Assays. Regarding structure-affinity relationships at the MB327 binding site, the presence of two quaternary aromatic nitrogen centers as well as pyridinium systems with a tert-butyl group at the 4-position or a NMe2 group at the 3- or 4-positions appeared to be beneficial for high binding affinities.
ESTHER : Rappengluck_2018_ChemMedChem_13_2653
PubMedSearch : Rappengluck_2018_ChemMedChem_13_2653
PubMedID: 30362667

Title : Synthesis of a Series of Structurally Diverse MB327 Derivatives and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor - Rappengluck_2018_ChemMedChem_13_1806
Author(s) : Rappengluck S , Sichler S , Hofner G , Wein T , Niessen KV , Seeger T , Paintner FF , Worek F , Thiermann H , Wanner KT
Ref : ChemMedChem , 13 :1806 , 2018
Abstract : A novel series of 30 symmetric bispyridinium and related N-heteroaromatic bisquaternary salts with a propane-1,3-diyl linker was synthesized and characterized for their binding affinity at the MB327 binding site of nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Compounds targeting this binding site are of particular interest for research into new antidotes against organophosphate poisoning, as therapeutically active 4-tert-butyl-substituted bispyridinium salt MB327 was previously identified as a nAChR re-sensitizer. Efficient access to the target compounds was provided by newly developed methods enabling N-alkylation of sterically hindered or electronically deactivated heterocycles exhibiting a wide variety of functional groups. Determination of binding affinities toward the MB327 binding site at the nAChR, using a recently developed mass spectrometry (MS)-based Binding Assay, revealed that several compounds reached affinities similar to that of MB327 (pKi =4.73+/-0.03). Notably, the newly prepared lipophilic 4-tert-butyl-3-phenyl-substituted bispyridinium salt PTM0022 (3 h) was found to have significantly higher binding affinity, with a pKi value of 5.16+/-0.07, thus representing considerable progress toward the development of more potent nAChR re-sensitizers.
ESTHER : Rappengluck_2018_ChemMedChem_13_1806
PubMedSearch : Rappengluck_2018_ChemMedChem_13_1806
PubMedID: 29974635

Title : Fatal sarin poisoning in Syria 2013: forensic verification within an international laboratory network - John_2018_Forensic.Toxicol_36_61
Author(s) : John H , van der Schans MJ , Koller M , Spruit HET , Worek F , Thiermann H , Noort D
Ref : Forensic Toxicol , 36 :61 , 2018
Abstract : 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.
ESTHER : John_2018_Forensic.Toxicol_36_61
PubMedSearch : John_2018_Forensic.Toxicol_36_61
PubMedID: 29367863

Title : Discovery of a potent non-oxime reactivator of nerve agent inhibited human acetylcholinesterase - de Koning_2018_Eur.J.Med.Chem_157_151
Author(s) : De Koning MC , Horn G , Worek F , van Grol M
Ref : Eur Journal of Medicinal Chemistry , 157 :151 , 2018
Abstract : Organophosphorous (OP) compounds (such as nerve agents) inhibit the enzyme acetylcholinesterase (AChE) by covalent phosphylation of a key serine residue in the active site of the enzyme resulting in severe symptoms and ultimately death. OP intoxications are currently treated by administration of certain oxime compounds. The presently fielded oximes reactivate OP-inhibited AChE by liberating the phosphylated serine. Recent research towards new reactivators was predominantly devoted to design, synthesis and evaluation of new oxime-based compounds dedicated to overcoming some of the major limitations such as their intrinsic toxicity, their permanent charge which thwarts penetration of brain tissues and their inability to effectively reactivate all types of nerve agent inhibited AChEs. However, in over six decades of research only limited success has been achieved, indicating that there is a need for alternative classes of compounds that could reactivate OP-inhibited AChE. Recently, a number of non-oxime compounds was discovered in which the 4-amino-2-((diethylamino)methyl)phenol (ADOC) motif proved to be able to reactivate OP-inhibited AChE to some extent. In this paper several structural derivatives of ADOC were synthesized and screened for their ability to reactivate human AChE (hAChE) inhibited by the nerve agents VX, sarin, tabun, cyclosarin and paraoxon. We here disclose that one of those compounds showed a remarkable ability to reactivate OP-inhibited hAChE in vitro and that it is the most potent non-oxime reported to date.
ESTHER : de Koning_2018_Eur.J.Med.Chem_157_151
PubMedSearch : de Koning_2018_Eur.J.Med.Chem_157_151
PubMedID: 30096649

Title : Effect of cholinergic crisis on the potency of different emergency anaesthesia protocols in soman-poisoned rats - Marquart_2018_Clin.Toxicol.(Phila)__1
Author(s) : Marquart K , Herbert J , Amend N , Thiermann H , Worek F , Wille T
Ref : Clinical Toxicology (Phila) , :1 , 2018
Abstract : BACKGROUND: In a military or terrorist scenario, combination of organophosphorus compounds (OP) poisoning with physical trauma requiring surgical treatment and thus general anaesthesia are possible. Previous in vitro studies showed an altered potency of relevant anaesthetics during cholinergic crisis. Hence, it is not clear, which anaesthetics are suitable to achieve the necessary stage of surgical anaesthesia in OP poisoning. METHODS: In the present study, different anaesthetic regimens (ketamine-midazolam, propofol-fentanyl, thiopental-fentanyl), relevant in military emergency medicine, were examined in soman-poisoned rats. Clinical signs and cardiovascular variables were recorded continuously. Blood samples for acetylcholinesterase (AChE) activity were drawn. After euthanasia or death of the animals, brain and diaphragm were collected for cholinesterase assays. RESULTS: Propofol-fentanyl and thiopental-fentanyl resulted in surgical anaesthesia throughout the experiments. With ketamine-midazolam, surgical anaesthesia without respiratory impairment could not be achieved in pilot experiments (no soman challenge) and was therefore not included in the study. Soman-poisoned and control animals required a comparable amount of propofol-fentanyl or thiopental-fentanyl. In combination with atropine, significantly less propofol was needed. Survival rate was higher with thiopental compared to propofol. Atropine improved survival in both groups. Blood and tissue AChE activities were strongly inhibited after soman administration with and without atropine treatment. DISCUSSION: The current in vivo study did not confirm concerns of altered potency of existing anaesthetic protocols for the application of propofol or thiopental with fentanyl due to soman poisoning. Despite severe cholinergic crisis, sufficient anaesthetic depth could be achieved in all animals. CONCLUSION: Further experiments in in vivo models closer to human pharmaco- and toxicokinetics (e.g., swine) are required for confirmation of the initial findings and for improving extrapolation to humans.
ESTHER : Marquart_2018_Clin.Toxicol.(Phila)__1
PubMedSearch : Marquart_2018_Clin.Toxicol.(Phila)__1
PubMedID: 30307341

Title : In vitro pharmacological characterization of the bispyridinium non-oxime compound MB327 and its 2- and 3-regioisomers - Niessen_2018_Toxicol.Lett_293_190
Author(s) : Niessen KV , Seeger T , Rappengluck S , Wein T , Hofner G , Wanner KT , Thiermann H , Worek F
Ref : Toxicol Lett , 293 :190 , 2018
Abstract : The primary toxic mechanism of organophosphorus compounds, i.e. nerve agents or pesticides, is based on the irreversible inhibition of acetylcholinesterase. In consequence of the impaired hydrolysis, the neurotransmitter acetylcholine accumulates in cholinergic synapses and disturbs functional activity of nicotinic and muscarinic acetylcholine receptors by overstimulation and subsequent desensitization. The resulting cholinergic syndrome will become acute life-threatening, if not treated adequately. The current standard treatment, consisting of administration of a competitive mAChR antagonist (e.g. atropine) and an oxime (e.g. obidoxime, pralidoxime), is not sufficient in the case of soman or tabun intoxications. Consequently, alternative therapeutic options are necessary. An innovative approach comprises the use of compounds selectively targeting nAChRs, especially positive allosteric modulators, which increase the population of the conducting receptor state. MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)) is able to restore soman-blocked muscle-force in preparations of various species including human and was recently identified as "resensitizer". In contrast to the well-studied MB327, the pharmacological efficacy of the 2- and 3-tert-butylpyridinium propane regioisomers is unknown. As a first step, MB327 and its 3-regioisomer (PTM0001) and 2-regioisomer (PTM0002) were pharmacologically characterized using [(3)H]epibatidine binding assays, functional studies by solid supported membranes based electrophysiology, and in vitro muscle-force investigations of soman-poisoned rat hemidiaphragm preparations by indirect field stimulation technique. The results obtained from targets of different complexity (receptor, muscle tissue) showed that the pharmacological profiles of the 2- and 3-regioisomers were relatively similar to those of MB327. Furthermore, high concentrations showed inhibitory effects, which might critically influence the application as an antidote. Thus, more effective drugs have to be developed. Nevertheless, the combination of the methods presented is an effective tool for clarifying structure-activity relationships.
ESTHER : Niessen_2018_Toxicol.Lett_293_190
PubMedSearch : Niessen_2018_Toxicol.Lett_293_190
PubMedID: 29024789

Title : Pseudocatalytic scavenging of the nerve agent VX with human blood components and the oximes obidoxime and HI-6 - Wille_2017_Arch.Toxicol_91_1309
Author(s) : Wille T , von der Wellen J , Thiermann H , Worek F
Ref : Archives of Toxicology , 91 :1309 , 2017
Abstract : Despite six decades of extensive research in medical countermeasures against nerve agent poisoning, a broad spectrum acetylcholinesterase (AChE) reactivator is not yet available. One current approach is directed toward synthesizing oximes with high affinity and reactivatability toward butyrylcholinesterase (BChE) in plasma to generate an effective pseudocatalytic scavenger. An interim solution could be the administration of external AChE or BChE from blood products to augment pseudocatalytic scavenging with slower but clinically approved oximes to decrease nerve agent concentrations in the body. We here semiquantitatively investigate the ability of obidoxime and HI-6 to decrease the inhibitory activity of VX with human AChE and BChE from whole blood, erythrocyte membranes, erythrocytes, plasma, clinically available fresh frozen plasma and packed red blood cells. The main findings are that whole blood showed a VX concentration-dependent decrease in inhibitory activity with HI-6 being more potent than obidoxime. Using erythrocytes and erythrocyte membranes again, HI-6 was more potent compared to obidoxime. With freshly prepared plasma, obidoxime and HI-6 showed comparable results for the decrease in VX. The use of the clinically available blood products revealed that packed red blood cells showed similar kinetics as fresh erythrocytes. Fresh frozen plasma resulted in a slower and incomplete decrease in inhibitory plasma compared to freshly prepared plasma. In conclusion, the administration of blood products in combination with available oximes augments pseudocatalytic scavenging and might be useful to decrease the body load of persistent, highly toxic nerve agents.
ESTHER : Wille_2017_Arch.Toxicol_91_1309
PubMedSearch : Wille_2017_Arch.Toxicol_91_1309
PubMedID: 27358236

Title : An Unusual Dimeric Inhibitor of Acetylcholinesterase: Cooperative Binding of Crystal Violet - Allgardsson_2017_Molecules_22_
Author(s) : Allgardsson A , David Andersson C , Akfur C , Worek F , Linusson A , Ekstrom F
Ref : Molecules , 22 : , 2017
Abstract : Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by a rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for treatment of various cholinergic deficiencies, including Alzheimer's disease and myasthenia gravis. In a previous high throughput screening campaign, we identified the dye crystal violet (CV) as an inhibitor of AChE. Herein, we show that CV displays a significant cooperativity for binding to AChE, and the molecular basis for this observation has been investigated by X-ray crystallography. Two monomers of CV bind to residues at the entrance of the active site gorge of the enzyme. Notably, the two CV molecules have extensive intermolecular contacts with each other and with AChE. Computational analyses show that the observed CV dimer is not stable in solution, suggesting the sequential binding of two monomers. Guided by the structural analysis, we designed a set of single site substitutions, and investigated their effect on the binding of CV. Only moderate effects on the binding and the cooperativity were observed, suggesting a robustness in the interaction between CV and AChE. Taken together, we propose that the dimeric cooperative binding is due to a rare combination of chemical and structural properties of both CV and the AChE molecule itself.
ESTHER : Allgardsson_2017_Molecules_22_
PubMedSearch : Allgardsson_2017_Molecules_22_
PubMedID: 28867801
Gene_locus related to this paper: mouse-ACHE

Title : Application of the Ugi Multicomponent Reaction in the Synthesis of Reactivators of Nerve Agent Inhibited Acetylcholinesterase - de Koning_2017_J.Med.Chem_60_9376
Author(s) : De Koning MC , Joosen MJ , Worek F , Nachon F , van Grol M , Klaassen SD , Alkema DPW , Wille T , de Bruijn HM
Ref : Journal of Medicinal Chemistry , 60 :9376 , 2017
Abstract : Recently, a new class of reactivators of chemical warfare agent inhibited acetylcholinesterase (AChE) with promising in vitro potential was developed by the covalent linkage of an oxime nucleophile and a peripheral site ligand. However, the complexity of these molecular structures thwarts their accessibility. We report the compatibility of various oxime-based compounds with the use of the Ugi multicomponent reaction in which four readily accessible building blocks are mixed together to form a product that links a reactivating unit and a potential peripheral site ligand. A small library of imidazole and imidazolium reactivators was successfully synthesized using this method. Some of these compounds showed a promising ability to reactivate AChE inhibited by various types of CWA in vitro. Molecular modeling was used to understand differences in reactivation potential between these compounds. Four compounds were evaluated in vivo using sarin-exposed rats. One of the reactivators showed improved in vivo efficacy compared to the current antidote pralidoxime (2-PAM).
ESTHER : de Koning_2017_J.Med.Chem_60_9376
PubMedSearch : de Koning_2017_J.Med.Chem_60_9376
PubMedID: 29091431

Title : Oximes in organophosphate poisoning: 60 years of hope and despair - Worek_2016_Chem.Biol.Interact_259_93
Author(s) : Worek F , Thiermann H , Wille T
Ref : Chemico-Biological Interactions , 259 :93 , 2016
Abstract : The high number of annual fatalities following suicidal poisoning by organophosphorus (OP) pesticides and the recent homicidal use of the chemical warfare nerve agent sarin against civilian population in Syria underlines the continuous threat by these highly toxic agents. The need for an effective treatment of OP poisoning resulted in the implementation of a combination therapy with the muscarinic receptor antagonist atropine and an oxime for the reactivation of OP-inhibited acetylcholinesterase (AChE). Since the invention of the first clinically used oxime pralidoxime (2-PAM) in the 1950s ongoing research attempted to identify more effective oximes. In fact, several thousand oximes were synthesized in the past six decades. These include charged and non-charged compounds, mono- and bispyridinium oximes, asymmetric oximes, oximes with different substitutes and more recently non-oxime reactivators. Multiple in vitro and in vivo studies investigated the potential of oximes to reactivate OP-inhibited AChE and to reverse OP-induced cholinergic signs. Depending on the experimental model, the investigated species and the tested OP largely variable results were obtained by different laboratories. These findings and the inconsistent effectiveness of oximes in the treatment of OP-pesticide poisoned patients led to a continuous discussion on the value of oximes. In order to provide a forward-looking evaluation of the significance of oximes in OP poisoning multiple aspects, including intrinsic toxicity, in vitro reactivation potency, efficacy and pharmacokinetics, as well as the impact of the causative OP have to be considered. The different influencing factors in order to define the benefit and limitations of oximes in OP poisoning will be discussed.
ESTHER : Worek_2016_Chem.Biol.Interact_259_93
PubMedSearch : Worek_2016_Chem.Biol.Interact_259_93
PubMedID: 27125761

Title : Blaptica dubia as sentinels for exposure to chemical warfare agents - a pilot study - Worek_2016_Toxicol.Lett_262_12
Author(s) : Worek F , Seeger T , Neumaier K , Wille T , Thiermann H
Ref : Toxicol Lett , 262 :12 , 2016
Abstract : The increased interest of terrorist groups in toxic chemicals and chemical warfare agents presents a continuing threat to our societies. Early warning and detection is a key component for effective countermeasures against such deadly agents. Presently available and near term solutions have a number of major drawbacks, e.g. lack of automated, remote warning and detection of primarily low volatile chemical warfare agents. An alternative approach is the use of animals as sentinels for exposure to toxic chemicals. To overcome disadvantages of vertebrates the present pilot study was initiated to investigate the suitability of South American cockroaches (Blaptica dubia) as warning system for exposure to chemical warfare nerve and blister agents. Initial in vitro experiments with nerve agents showed an increasing inhibitory potency in the order tabun - cyclosarin - sarin - soman - VX of cockroach cholinesterase. Exposure of cockroaches to chemical warfare agents resulted in clearly visible and reproducible reactions, the onset being dependent on the agent and dose. With nerve agents the onset was related to the volatility of the agents. The blister agent lewisite induced signs largely comparable to those of nerve agents while sulfur mustard exposed animals exhibited a different sequence of events. In conclusion, this first pilot study indicates that Blaptica dubia could serve as a warning system to exposure of chemical warfare agents. A cockroach-based system will not detect or identify a particular chemical warfare agent but could trigger further actions, e.g. specific detection and increased protective status. By designing appropriate boxes with (IR) motion sensors and remote control (IR) camera automated off-site warning systems could be realized.
ESTHER : Worek_2016_Toxicol.Lett_262_12
PubMedSearch : Worek_2016_Toxicol.Lett_262_12
PubMedID: 27639501

Title : Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology - Niessen_2016_Toxicol.Lett_247_1
Author(s) : Niessen KV , Muschik S , Langguth F , Rappengluck S , Seeger T , Thiermann H , Worek F
Ref : Toxicol Lett , 247 :1 , 2016
Abstract : Organophosphorus compounds (OPC), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). Inhibited AChE results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nAChR) in the postsynaptic membrane is provoked. Direct targeting of nAChR to reduce receptor desensitisation might be an alternative therapeutic approach. For drug discovery, functional properties of potent therapeutic candidates need to be investigated in addition to affinity properties. Solid supported membrane (SSM)-based electrophysiology is useful for functional characterisation of ligand-gated ion channels like nAChRs, as charge translocations via capacitive coupling of the supporting membrane can be measured. By varying the agonist (carbamoylcholine) concentration, different functional states of the nAChR were initiated. Using plasma membrane preparations obtained from Torpedo californica electric organ, functional properties of selected nAChR ligands and non-oxime bispyridinium compounds were investigated. Depending on overall-size, the bispyridinium compounds enhanced or inhibited cholinergic signals induced by 100muM carbamoylcholine. Applying excessive concentrations of the agonist carbamoylcholine provoked desensitisation of the nAChRs, whereas addition of bispyridinium compounds bearing short alkyl linkers exhibited functional recovery of previously desensitised nAChRs. The results suggest that these non-oxime bispyridinium compounds possibly interacted with nAChR subtypes in a manner of a positive allosteric modulator (PAM). The described newly developed functional assay is a valuable tool for the assessment of functional properties of potential compounds such as nAChR modulating ligands, which might be a promising approach in the therapeutically treatment of OPC-poisonings.
ESTHER : Niessen_2016_Toxicol.Lett_247_1
PubMedSearch : Niessen_2016_Toxicol.Lett_247_1
PubMedID: 26851639

Title : Single treatment of VX poisoned guinea pigs with the phosphotriesterase mutant C23AL: Intraosseous versus intravenous injection - Wille_2016_Toxicol.Lett_258_198
Author(s) : Wille T , Neumaier K , Koller M , Ehinger C , Aggarwal N , Ashani Y , Goldsmith M , Sussman JL , Tawfik DS , Thiermann H , Worek F
Ref : Toxicol Lett , 258 :198 , 2016
Abstract : 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 ( approximately 2LD50). C23AL showed a Cmax of 0.63mumolL(-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 0.37mumolL(-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.
ESTHER : Wille_2016_Toxicol.Lett_258_198
PubMedSearch : Wille_2016_Toxicol.Lett_258_198
PubMedID: 27397758

Title : Reactivation of nerve agent-inhibited human acetylcholinesterase by obidoxime, HI-6 and obidoxime+HI-6: Kinetic in vitro study with simulated nerve agent toxicokinetics and oxime pharmacokinetics - Worek_2016_Toxicology_350-352_25
Author(s) : Worek F , Koller M , Thiermann H , Wille T
Ref : Toxicology , 350-352 :25 , 2016
Abstract : 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.
ESTHER : Worek_2016_Toxicology_350-352_25
PubMedSearch : Worek_2016_Toxicology_350-352_25
PubMedID: 27153754

Title : In vitro evaluation of the catalytic activity of paraoxonases and phosphotriesterases predicts the enzyme circulatory levels required for in vivo protection against organophosphate intoxications - Ashani_2016_Chem.Biol.Interact_259_252
Author(s) : Ashani Y , Leader H , Aggarwal N , Silman I , Worek F , Sussman JL , Goldsmith M
Ref : Chemico-Biological Interactions , 259 :252 , 2016
Abstract : Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme]pl), established prior to the OP challenge, with the severity of symptoms and survival rates of intoxicated animals. The major objective was to apply a theoretical approach to estimate a lower limit for (kcat/KM)[Enzyme]pl that will be adequate for establishing the desired kcat/KM value and plasma concentration of efficacious catalytic bioscavengers. Published data sets by our group and others, from in vivo protection experiments executed in the absence of any supportive medicine, were analyzed. The kcat/KM values of eight OP hydrolyzing enzymes and their plasma concentrations in four species exposed to OPs via s.c., i.m. and oral gavage, were analyzed. Our results show that regardless of the OP type and the animal species employed, sign-free animals were observed following bioscavenger treatment provided the theoretically estimated time period required to detoxify 96% of the OP (t96%) in-vivo was </=10 s. This, for example, can be achieved by an enzyme with kcat/KM = 5 x 107 M-1 min-1 and a plasma concentration of 0.4 muM ((kcat/KM)[Enzyme]pl = 20 min-1). Experiments in which animals were intoxicated by i.v. OP injections did not always conform to this rule, and in some cases resulted in high mortality rates. We suggest that in vivo evaluation of catalytic scavengers should avoid the unrealistic bolus i.v. route of OP exposure.
ESTHER : Ashani_2016_Chem.Biol.Interact_259_252
PubMedSearch : Ashani_2016_Chem.Biol.Interact_259_252
PubMedID: 27163850

Title : Repetitive obidoxime treatment induced increase of red blood cell acetylcholinesterase activity even in a late phase of a severe methamidophos poisoning: A case report - Steinritz_2016_Toxicol.Lett_244_121
Author(s) : Steinritz D , Eyer F , Worek F , Thiermann H , John H
Ref : Toxicol Lett , 244 :121 , 2016
Abstract : Accidental self-poisoning or deliberate use in suicidal intent of organophosphorus pesticides (OPP), which are widely used in agriculture, represent a health problem worldwide. Symptoms of poisoning are characterized by acute cholinergic crisis caused by inhibition of acetylcholinesterase. A 75-year-old male patient ingested 20ml of an OPP solution containing 10% methamidophos in suicidal intent. In the course of poisoning typical clinical symptoms of cholinergic crisis (miosis, bradycardia, hypotension, hypersalivation and impairment of neurologic status) were evident. Butyryl (plasma) cholinesterase (BChE) and red blood cell acetylcholinesterase (RBC-AChE) revealed decreased activities, thus specific treatment with the enzyme reactivator obidoxime was started. Inhibitory activity of the patient's plasma indicated significant amounts of persisting methamidophos in the circulation and was still found on day 4 after ingestion. Due to missing spontaneous breathing on day 6, obidoxime was administered again. Afterwards a significant increase of RBC-AChE activity was found. The patient was extubated on day 10 and a restitution ad integrum was achieved. In conclusion, obidoxime is a potent reactivator of OPP-inhibited AChE. A repetitive and prolonged administration of obidoxime should be considered in cases of severe methamidophos poisoning and should be tailored with an advanced analytical biomonitoring.
ESTHER : Steinritz_2016_Toxicol.Lett_244_121
PubMedSearch : Steinritz_2016_Toxicol.Lett_244_121
PubMedID: 26200596

Title : Kinetics of pesticide degradation by human fresh frozen plasma (FFP) in vitro - von der Wellen_2016_Toxicol.Lett_244_124
Author(s) : von der Wellen J , Bierwisch A , Worek F , Thiermann H , Wille T
Ref : Toxicol Lett , 244 :124 , 2016
Abstract : There is an ongoing debate about the benefit of fresh frozen plasma (FFP) infusion in organophosphorus (OP) pesticide-poisoned patients. This prompted us to investigate the kinetics of OP pesticide degradation by FFP with an enzymatic assay in vitro. Degradation was rapid with shortest half-lives of 19.5s for chlorpyrifos-oxon, 6.3min for paraoxon-ethyl and 17.9min for dichlorvos. Heptenophos (78.0min), mevinphos (101.8min), profenofos (162.3min) and malaoxon (179.7min) showed half-lives of up to 3h. Substantial longer degradation half-lives of 69.7-80.8h were determined with chlorfenvinphos and bromfenvinphos. Methamidophos and omethoate showed no degradation by FFP indicated by half-lives similar to spontaneous hydrolysis. In conclusion, degradation by FFP depends on the particular OP pesticide and the used FFP batch.
ESTHER : von der Wellen_2016_Toxicol.Lett_244_124
PubMedSearch : von der Wellen_2016_Toxicol.Lett_244_124
PubMedID: 26220518

Title : Investigation of the reactivation kinetics of a large series of bispyridinium oximes with organophosphate-inhibited human acetylcholinesterase - Winter_2016_Toxicol.Lett_244_136
Author(s) : Winter M , Wille T , Musilek K , Kuca K , Thiermann H , Worek F
Ref : Toxicol Lett , 244 :136 , 2016
Abstract : The limited effectiveness of the established oximes obidoxime and pralidoxime resulted in ongoing research on novel oximes for the reactivation of acetylcholinesterase (AChE) inhibited by organophosphorus compounds (OP). In order to get more insight into the ability of bispyridinium oximes to reactivate human AChE inhibited by structurally different OP the reactivation kinetics of 31 compounds was determined with tabun-, cyclosarin- and paraoxon-inhibited AChE under identical experimental conditions. The determined affinity (KD), reactivity (kr) and hybrid reactivation rate constants (kr2) enabled theoretical calculations and gave insight into distinct structural features which are important for the reactivation of AChE inhibited by different OP. Several oximes with superior reactivating potency towards selective OP-AChE conjugates were identified but none of the tested oximes can be considered as a broad spectrum reactivator. In the end, the data of this and previous studies gives rise to the question whether further modifications of the bispyridinium structure could ever result in a universal reactivator or whether future research should be directed to different templates.
ESTHER : Winter_2016_Toxicol.Lett_244_136
PubMedSearch : Winter_2016_Toxicol.Lett_244_136
PubMedID: 26210933

Title : An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination - Del Giudice_2016_Biotechnol.Bioeng_113_724
Author(s) : Del Giudice I , Coppolecchia R , Merone L , Porzio E , Carusone TM , Mandrich L , Worek F , Manco G
Ref : Biotechnol Bioeng , 113 :724 , 2016
Abstract : In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1) . The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces. Biotechnol. Bioeng. 2016;113: 724-734. (c) 2015 Wiley Periodicals, Inc.
ESTHER : Del Giudice_2016_Biotechnol.Bioeng_113_724
PubMedSearch : Del Giudice_2016_Biotechnol.Bioeng_113_724
PubMedID: 26416557

Title : Catalytic efficiencies of directly evolved phosphotriesterase variants with structurally different organophosphorus compounds in vitro - Goldsmith_2016_Arch.Toxicol_90_2711
Author(s) : Goldsmith M , Eckstein S , Ashani Y , Greisen P, Jr. , Leader H , Sussman JL , Aggarwal N , Ovchinnikov S , Tawfik DS , Baker D , Thiermann H , Worek F
Ref : Archives of Toxicology , 90 :2711 , 2016
Abstract : The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k cat/K M) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k cat/K M values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k cat/K M values >107 M-1 min-1 with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.
ESTHER : Goldsmith_2016_Arch.Toxicol_90_2711
PubMedSearch : Goldsmith_2016_Arch.Toxicol_90_2711
PubMedID: 26612364

Title : On-site analysis of acetylcholinesterase and butyrylcholinesterase activity with the ChE check mobile test kit-Determination of reference values and their relevance for diagnosis of exposure to organophosphorus compounds - Worek_2016_Toxicol.Lett_249_22
Author(s) : Worek F , Schilha M , Neumaier K , Aurbek N , Wille T , Thiermann H , Kehe K
Ref : Toxicol Lett , 249 :22 , 2016
Abstract : Poisoning by organophosphorus compounds (OP) still poses a major medical challenge. Diagnosis of clinical signs of OP poisoning is still the most important parameter for the initiation of specific treatment. However, in case of unspecific signs and of delayed onset of cholinergic crisis a rapid, reliable and on-site analysis of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity would be of great value. Recently the ChE check mobile, a CE-certified ready to use kit for the determination of whole blood AChE and BChE activities, was developed. Here, we evaluated whole blood AChE and BChE reference values with samples taken from 181 male and 61 female volunteers and analyzed them on-site with the ChE check mobile test kit. The analysis of the data revealed a large inter-individual variability (BChE>AChE), only a small sex difference for AChE but a significant difference for BChE activities. The now available normal range values enable an evaluation of determined AChE and BChE activities in case of suspected exposure to OP nerve agents and pesticides. However, the large inter-individual variability of AChE and BChE activities calls for the determination of pre-exposure values in specific subpopulations in order to enable the diagnosis of low-level OP exposure.
ESTHER : Worek_2016_Toxicol.Lett_249_22
PubMedSearch : Worek_2016_Toxicol.Lett_249_22
PubMedID: 27033775

Title : Catalytic bioscavengers in nerve agent poisoning: A promising approach? - Worek_2016_Toxicol.Lett_244_143
Author(s) : Worek F , Thiermann H , Wille T
Ref : Toxicol Lett , 244 :143 , 2016
Abstract : The repeated use of the nerve agent sarin against civilians in Syria in 2013 emphasizes the continuing threat by chemical warfare agents. Multiple studies demonstrated a limited efficacy of standard atropine-oxime treatment in nerve agent poisoning and called for the development of alternative and more effective treatment strategies. A novel approach is the use of stoichiometric or catalytic bioscavengers for detoxification of nerve agents in the systemic circulation prior to distribution into target tissues. Recent progress in the design of enzyme mutants with reversed stereo selectivity resulting in improved catalytic activity and their use in in vivo studies supports the concept of catalytic bioscavengers. Yet, further research is necessary to improve the catalytic activity, substrate spectrum and in vivo biological stability of enzyme mutants. The pros and cons of catalytic bioscavengers will be discussed in detail and future requirements for the development of catalytic bioscavengers will be proposed.
ESTHER : Worek_2016_Toxicol.Lett_244_143
PubMedSearch : Worek_2016_Toxicol.Lett_244_143
PubMedID: 26200600

Title : Kinetic analysis of interactions of amodiaquine with human cholinesterases and organophosphorus compounds - Bierwisch_2016_Toxicol.Lett_246_49
Author(s) : Bierwisch A , Wille T , Thiermann H , Worek F
Ref : Toxicol Lett , 246 :49 , 2016
Abstract : Standard therapy of poisoning by organophosphorus compounds (OP) is a combined administration of an anti-muscarinic drug (e.g. atropine) and an oxime as reactivator of inhibited acetylcholinesterase (AChE). Limited efficacy of clinically used oximes against a variety of OPs was shown in numerous studies, calling for research on novel reactivators of OP-inhibited AChE. Recently, reactivation of OP-inhibited AChE by the antimalarial drug amodiaquine was reported. In the present study, amodiaquine and its interactions with human cholinesterases in presence or absence of OP nerve agents was investigated in vitro. Thereby, reversible inhibition of human cholinesterases by amodiaquine (AChE>>BChE) was observed. Additionally, a mixed competitive-non-competitive inhibition type of amodiaquine with human AChE was determined. Slow and partial reactivation of sarin-, cyclosarin- and VX-inhibited cholinesterases by amodiaquine was recorded, amodiaquine failed to reactivate tabun-inhibited human cholinesterases. Amodiaquine, being a potent, reversible AChE inhibitor, was tested for its potential benefit as a pretreatment to prevent complete irreversible AChE inhibition by the nerve agent soman. Hereby, amodiaquine failed to prevent phosphonylation and resulted only in a slight increase of AChE activity after removal of amodiaquine and soman. At present the molecular mechanism of amodiaquine-induced reactivation of OP-inhibited AChE is not known, nevertheless amodiaquine could be considered as a template for the design of more potent non-oxime reactivators.
ESTHER : Bierwisch_2016_Toxicol.Lett_246_49
PubMedSearch : Bierwisch_2016_Toxicol.Lett_246_49
PubMedID: 26851641

Title : Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6 - Allgardsson_2016_Proc.Natl.Acad.Sci.U.S.A_113_5514
Author(s) : Allgardsson A , Berg L , Akfur C , Hornberg A , Worek F , Linusson A , Ekstrom F
Ref : Proc Natl Acad Sci U S A , 113 :5514 , 2016
Abstract : Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.
ESTHER : Allgardsson_2016_Proc.Natl.Acad.Sci.U.S.A_113_5514
PubMedSearch : Allgardsson_2016_Proc.Natl.Acad.Sci.U.S.A_113_5514
PubMedID: 27140636
Gene_locus related to this paper: human-ACHE

Title : Reactivation kinetics of 31 structurally different bispyridinium oximes with organophosphate-inhibited human butyrylcholinesterase - Horn_2015_Arch.Toxicol_89_405
Author(s) : Horn G , Wille T , Musilek K , Kuca K , Thiermann H , Worek F
Ref : Archives of Toxicology , 89 :405 , 2015
Abstract : Organophosphorus compounds (OP) are bound to human butyrylcholinesterase (BChE) and endogenous or exogenous BChE may act as a stoichiometric scavenger. Adequate amounts of BChE are required to minimize toxic OP effects. Simultaneous administration of BChE and oximes may transfer the enzyme into a pseudo-catalytic scavenger. The present study was initiated to determine the reactivation kinetics of 31 structurally different bispyridinium oximes with paraoxon-, tabun- and cyclosarin-inhibited human BChE. Human plasma was incubated with OP and the reactivation of inhibited BChE was tested with multiple oxime concentrations followed by nonlinear regression analysis for the determination of reactivity, affinity and overall reactivation constants. The generated data indicate that the tested oximes have a low-to-negligible reactivating potency with paraoxon- and tabun-inhibited human BChE. Several oximes showed a moderate-to-high potency with cyclosarin-inhibited BChE. Thus, the present study indicates that bispyridinium oximes are obviously not suitable to serve as reactivators of human BChE inhibited by different OP and it is doubtful whether further modifications of the bispyridinium template will lead to more potent reactivators. In the end, novel structures of oxime and non-oxime reactivators are urgently needed for the development of human BChE into an effective pseudo-catalytic scavenger.
ESTHER : Horn_2015_Arch.Toxicol_89_405
PubMedSearch : Horn_2015_Arch.Toxicol_89_405
PubMedID: 24912784

Title : Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines - Ring_2015_PLoS.One_10_e0135811
Author(s) : Ring A , Strom BO , Turner SR , Timperley CM , Bird M , Green AC , Chad JE , Worek F , Tattersall JE
Ref : PLoS ONE , 10 :e0135811 , 2015
Abstract : Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.
ESTHER : Ring_2015_PLoS.One_10_e0135811
PubMedSearch : Ring_2015_PLoS.One_10_e0135811
PubMedID: 26274808

Title : Application of a dynamic in vitro model with real-time determination of acetylcholinesterase activity for the investigation of tabun analogues and oximes - Worek_2015_Toxicol.In.Vitro_30_514
Author(s) : Worek F , Herkert NM , Koller M , Thiermann H , Wille T
Ref : Toxicol In Vitro , 30 :514 , 2015
Abstract : Tabun-inhibited acetylcholinesterase (AChE) is rather resistant towards reactivation by oximes in vitro while in vivo experiments showed some protection of animals poisoned by this chemical warfare nerve agent after treatment with an oxime and atropine. In addition, AChE inhibited by close tabun analogues, N,N-diethyltabun and N,N-di-n-propyltabun was completely resistant towards reactivation by oximes. In order to get more insight into potential mechanisms of this oxime resistance experiments with these toxic agents and the oximes obidoxime, 2-PAM, MMB-4 and HI-6 were performed utilizing a dynamic model with real-time determination of AChE activity. This experimental setup allowed the investigation of reactivation with minimized side reactions. The determined reactivation constants with tabun-inhibited human AChE were in good agreement with previously reported constants determined with a static model. N,N-diethyl- and N,N-di-n-propyltabun-inhibited human AChE could not be reactivated by oximes which indicates that the inadequate oxime effect was not due to re-inhibition by phosphonyloximes. Additional experiments with tabun-inhibited human and Rhesus monkey AChE revealed that no reactivation occurred with HI-6. These data give further support to the assumption that an interaction of tabun with residues in the active site gorge of AChE prevents effective reactivation by oximes, a mechanism which may also be the reason for the total oxime resistance of N,N-diethyl- and N,N-di-n-propyltabun-inhibited human AChE.
ESTHER : Worek_2015_Toxicol.In.Vitro_30_514
PubMedSearch : Worek_2015_Toxicol.In.Vitro_30_514
PubMedID: 26368669

Title : In vitro and in vivo toxicological studies of V nerve agents: molecular and stereoselective aspects - Reiter_2015_Toxicol.Lett_232_438
Author(s) : Reiter G , Muller S , Hill I , Weatherby K , Thiermann H , Worek F , Mikler J
Ref : Toxicol Lett , 232 :438 , 2015
Abstract : In vitro inhibition data of cholinesterases (ChEs) and reactivation with HI 6 are presented for separated VX and VR enantiomers with high purity (enantiomer excess >99.999%). Inhibition rate constants for (-)-VR were fourfold higher than for (-)-VX. Marked higher stereoselectivity of ChEs inhibition was observed for VR compared with VX enantiomers. Low/no reactivation was determined for respective (+)-enantiomers. Results were related to orientation of (-)- and (+)-enantiomers in ChEs active sites. In vivo in swine, absorption rate constants were practically identical for VX and VR enantiomers after percutaneous application of 3xLD(5)(0) underlining relevance of amine group and postulated equilibria shifts between charged, uncharged, open and cyclic form (skin depot). In vivo toxicokinetics of VX and VR enantiomers differed markedly after 4h. Elimination of VX was much slower compared with VR. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition in vivo differed for VX and VR. In vivo spontaneous reactivation was not observed for VX-inhibited AChE while VR-inhibited AChE was much faster spontaneously reactivated than expected and AChE inhibition by VR was slower than expected. Progredient BChE inhibition was detected after VX application while VR inhibited BChE weakly. Possible explanation may be impact of the agents on hemodynamics and different metabolisms. Thus, due to increase of the V agents' blood concentration after atropine administration (depot release) the present standard therapy should be thoroughly reconsidered.
ESTHER : Reiter_2015_Toxicol.Lett_232_438
PubMedSearch : Reiter_2015_Toxicol.Lett_232_438
PubMedID: 25448275

Title : Effect of reversible ligands on oxime-induced reactivation of sarin- and cyclosarin-inhibited human acetylcholinesterase - Scheffel_2015_Toxicol.Lett_232_557
Author(s) : Scheffel C , Thiermann H , Worek F
Ref : Toxicol Lett , 232 :557 , 2015
Abstract : Poisoning by organophosphorus compounds (OP) used as pesticides and nerve agents is due to irreversible inhibition of the enzyme acetylcholinesterase (AChE). Oximes have been widely recognized for their potency to reactivate the inhibited enzyme. The limited efficacy of currently available oximes against a broad spectrum of OP-compounds initiated novel research efforts to improve oxime-based treatment. Hereby, oxime-induced reactivation of OP-inhibited non-human AChE was reported to be accelerated by different AChE-ligands. To investigate this concept with AChE from human source, the inhibitory potency, binding properties and the potential enhancement of oxime-induced reactivation of OP-inhibited AChE by structurally different AChE-ligands was assessed. Several ligands competed with the oxime for the AChE binding-site impairing reactivation of OP-inhibited AChE whereas a markedly accelerated reactivation of sarin-inhibited enzyme by obidoxime was recorded in the presence of edrophonium, galanthamine and donepezil. Enhancement of oxime-induced reactivation with ligands was presumably subject to prevention of re-inhibition by the reaction product phosphonyloxime (POX). In the end, the results of the present study did not confirm that AChE-ligands directly accelerate the reactivation of OP-inhibited AChE by oximes, but indirectly by prevention of re-inhibition by the reaction product POX. This may be due to different experimental conditions and species differences between human and non-human AChE of previous experiments with non-human AChE.
ESTHER : Scheffel_2015_Toxicol.Lett_232_557
PubMedSearch : Scheffel_2015_Toxicol.Lett_232_557
PubMedID: 25522658

Title : Small-scale purification of butyrylcholinesterase from human plasma and implementation of a muLC-UV\/ESI MS\/MS method to detect its organophosphorus adducts - John_2015_Drug.Test.Anal_7_947
Author(s) : John H , Breyer F , Schmidt C , Mizaikoff B , Worek F , Thiermann H
Ref : Drug Test Anal , 7 :947 , 2015
Abstract : Human butyrylcholinesterase (hBChE) is a serine hydrolase (EC 3.1.1.8) present in all mammalian tissues and the bloodstream. Similar to acetylcholinesterase, the enzyme reacts with organophosphorus compounds (OP) like nerve agents or pesticides that cause enzyme inhibition (BChE adducts). These adducts represent valuable biomarkers for analytical verification of OP exposure. For establishment of these mass spectrometry based methods sufficient amounts of hBChE in high purity are required. Unfortunately, commercial lots are of inappropriate purity thus favouring in-house isolation. Therefore, we developed a small scale procedure to isolate hBChE from citrate plasma. After precipitation by polyethylene glycol (8% w/v and 20% w/v PEG 6000) hBChE was purified from plasma by four consecutive chromatographic steps including anion exchange, affinity extraction and size exclusion. Protein elution was monitored on-line by UV-absorbance (280 nm) followed by continuous fractionation for off-line analysis of (1) hBChE enzyme activity by Ellman assay, (2) protein purity by gel electrophoresis, and (3) protein identity by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Numerous major impurities separated from hBChE were identified. The purified material was used for in vitro incubation with diverse OP to establish a mu-liquid chromatography-ultra violet detection/electrospray ionization tandem-mass spectrometric method (muLC-UV/ESI MS/MS) for detection of hBChE adducts suitable for verification analysis. Analytical data for diverse OP pesticides including deuterated analogues as well as G- and V-type nerve agents and their precursor are summarized. This method was successfully applied to plasma samples provided by the Organisation for the Prohibition of Chemical Weapons (OPCW) for the 4th Biomedical Exercise. Copyright (c) 2015 John Wiley & Sons, Ltd.
ESTHER : John_2015_Drug.Test.Anal_7_947
PubMedSearch : John_2015_Drug.Test.Anal_7_947
PubMedID: 25828536

Title : Reactions of methylphosphonic difluoride with human acetylcholinesterase and oximes - Possible therapeutic implications - Worek_2014_Toxicol.Lett_231_92
Author(s) : Worek F , Elsinghorst P , Koller M , Thiermann H
Ref : Toxicol Lett , 231 :92 , 2014
Abstract : Highly toxic organophosphorus (OP) nerve agents are well characterized regarding chemical, biological and toxicological properties and the effectiveness of standard atropine and oxime therapy. Open literature data on the key nerve agent precursor methylphosphonic difluoride (DF) are scarce. To fill this gap the reactions of DF and its main degradation product methylphosphonofluoridic acid (MF) with human acetylcholinesterase (AChE) and the oximes obidoxime, HI-6 and 2-PAM were investigated in vitro. DF and MF were found to be weak inhibitors of human AChE being at least five orders less potent compared to the nerve agent sarin. Incubation of human AChE with millimolar DF and MF and subsequent addition of obidoxime and HI-6 resulted in a concentration-dependent decrease of AChE activity. This effect was not observed when incubating highly diluted AChE with oximes. The most likely explanation for this phenomenon is an inhibitory effect of phosphonyloximes formed by direct reaction of DF or MF with obidoxime and HI-6. These data indicate that high DF doses, resulting in millimolar blood and tissue DF/MF concentrations, are necessary to induce cholinergic signs and that under these conditions treatment with obidoxime and HI-6 may even worsen the poisoning.
ESTHER : Worek_2014_Toxicol.Lett_231_92
PubMedSearch : Worek_2014_Toxicol.Lett_231_92
PubMedID: 25240274

Title : Adaptation of a dynamic in vitro model with real-time determination of butyrylcholinesterase activity in the presence of cyclosarin and an oxime - Worek_2014_Toxicol.In.Vitro_29_162
Author(s) : Worek F , Horn G , Wille T , Thiermann H
Ref : Toxicol In Vitro , 29 :162 , 2014
Abstract : The well-established dynamic in vitro model for the real-time determination of acetylcholinesterase activity was modified for use of human butyrylcholinesterase (BChE) activity. Human plasma as BChE source was layered on a syringe filter and the enzyme reactor was continuously perfused with phosphate buffer, butyrylthiocholine and Ellman's reagent at pH 7.4 and 37 degrees C which resulted in a stable BChE activity for up to 240min. Then, the model was applied for investigating the suitability of human BChE in combination with an oxime (HLo 7) to serve as a 'pseudo-catalytic' scavenger of the organophosphorus nerve agent cyclosarin. The application of different perfusion protocols demonstrated the ability of BChE-oxime combinations to prevent BChE from irreversible inhibition by cyclosarin even at toxicologically relevant concentrations. In the end, this model seems to be suitable for the investigation of human plasma BChE as an endogenous, 'pseudo-catalytic' scavenger of a variety of nerve agents.
ESTHER : Worek_2014_Toxicol.In.Vitro_29_162
PubMedSearch : Worek_2014_Toxicol.In.Vitro_29_162
PubMedID: 25450746

Title : V-type nerve agents phosphonylate ubiquitin at biologically relevant lysine residues and induce intramolecular cyclization by an isopeptide bond - Schmidt_2014_Anal.Bioanal.Chem_406_5171
Author(s) : Schmidt C , Breyer F , Blum MM , Thiermann H , Worek F , John H
Ref : Anal Bioanal Chem , 406 :5171 , 2014
Abstract : Toxic organophosphorus compounds (e.g., pesticides and nerve agents) are known to react with nucleophilic side chains of different amino acids (phosphylation), thus forming adducts with endogenous proteins. Most often binding to serine, tyrosine, or threonine residues is described as being of relevance for toxicological effects (e.g., acetylcholinesterase and neuropathy target esterase) or as biomarkers for post-exposure analysis (verification, e.g., albumin and butyrylcholinesterase). Accordingly, identification of novel protein targets might be beneficial for a better understanding of the toxicology of these compounds, revealing new bioanalytical verification tools, and improving knowledge on chemical reactivity. In the present study, we investigated the reaction of ubiquitin (Ub) with the V-type nerve agents Chinese VX, Russian VX, and VX in vitro. Ub is a ubiquitous protein with a mass of 8564.8 Da present in the extra- and intracellular space that plays an important physiological role in several essential processes (e.g., proteasomal degradation, DNA repair, protein turnover, and endocytosis). Reaction products were analyzed by matrix-assisted laser desorption/ionization-time-of-flight- mass spectrometry (MALDI-TOF MS) and mu-high-performance liquid chromatography online coupled to UV-detection and electrospray ionization MS (muHPLC-UV/ESI MS). Our results originally document that a complex mixture of at least mono-, di, and triphosphonylated Ub adducts was produced. Surprisingly, peptide mass fingerprint analysis in combination with MALDI and ESI MS/MS revealed that phosphonylation occurred with high selectivity in at least 6 of 7 surface-exposed lysine residues that are essential for the biological function of Ub. These reaction products were found not to age. In addition, we herein report for the first time that phosphonylation induced intramolecular cyclization by formation of an isopeptide bond between the epsilon-amino group of a formerly phosphonylated lysine and the side chain of an adjacent acidic glutamic acid residue.
ESTHER : Schmidt_2014_Anal.Bioanal.Chem_406_5171
PubMedSearch : Schmidt_2014_Anal.Bioanal.Chem_406_5171
PubMedID: 24652148

Title : Efficacy of the rePON1 mutant IIG1 to prevent cyclosarin toxicity in vivo and to detoxify structurally different nerve agents in vitro - Worek_2014_Arch.Toxicol_88_1257
Author(s) : Worek F , Seeger T , Goldsmith M , Ashani Y , Leader H , Sussman JL , Tawfik DS , Thiermann H , Wille T
Ref : Archives of Toxicology , 88 :1257 , 2014
Abstract : The potent human toxicity of organophosphorus (OP) nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy. An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases (PON1). Recently, a chimeric PON1 mutant, IIG1, was engineered toward the hydrolysis of the toxic isomers of soman and cyclosarin with high in vitro catalytic efficiency. In order to investigate the suitability of IIG1 as a catalytic bioscavenger, an in vivo guinea pig model was established to determine the protective effect of IIG1 against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of IIG1 (1 mg/kg) prevented systemic toxicity in cyclosarin (~2LD50)-poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity, and protected erythrocyte AChE activity partially. A lower IIG1 dose (0.2 mg/kg) already prevented mortality and reduced systemic toxicity. IIG1 exhibited a high catalytic efficiency with a homologous series of alkylmethylfluorophosphonates but had low efficiency with the phosphoramidate tabun and was virtually ineffective with the nerve agent VX. This quantitative analysis validated the model for predicting in vivo protection by catalytic bioscavengers based on their catalytic efficiency, the level of circulating enzyme, and the dose of the intoxicating nerve agent. The in vitro and in vivo results indicate that IIG1 may be considered as a promising candidate bioscavenger to protect against the toxic effects of a range of highly toxic nerve agents.
ESTHER : Worek_2014_Arch.Toxicol_88_1257
PubMedSearch : Worek_2014_Arch.Toxicol_88_1257
PubMedID: 24477626

Title : Freeze-drying of HI-6-loaded recombinant human serum albumin nanoparticles for improved storage stability - Dadparvar_2014_Eur.J.Pharm.Biopharm_88_510
Author(s) : Dadparvar M , Wagner S , Wien S , Worek F , von Briesen H , Kreuter J
Ref : Eur J Pharm Biopharm , 88 :510 , 2014
Abstract : Severe intoxications with organophosphates require the immediate administration of atropine in combination with acetyl cholinesterase (AChE) reactivators such as HI-6. Although this therapy regimen enables the treatment of peripheral symptoms, the blood-brain barrier (BBB) restricts the access of the hydrophilic antidotes to the central nervous system which could lead to a fatal respiratory arrest. Therefore, HI-6-loaded albumin nanoparticles were previously developed to enhance the transport across this barrier and were able to reactivate organophosphate-(OP)-inhibited AChE in an in vitro BBB model. Since HI-6 is known to be moisture-sensitive, the feasibility of freeze-drying of the HI-6-loaded nanoparticles was investigated in the present study using different cryo- and lyoprotectants at different concentrations. Trehalose and sucrose (3%, w/v)-containing formulations were superior to mannitol concerning the physicochemical parameters of the nanoparticles whereas trehalose-containing samples were subject of a prolonged storage stability study at temperatures between -20 degrees C and +40 degrees C for predetermined time intervals. Shelf-life computations of the freeze-dried HI-6 nanoparticle formulations revealed a shelf-life time of 18months when stored at -20 degrees C. The formulations' efficacy was proven in vitro by reactivation of OP-inhibited AChE after transport over a porcine brain capillary endothelial cell layer model.
ESTHER : Dadparvar_2014_Eur.J.Pharm.Biopharm_88_510
PubMedSearch : Dadparvar_2014_Eur.J.Pharm.Biopharm_88_510
PubMedID: 24995841

Title : Post-exposure treatment of VX poisoned guinea pigs with the engineered phosphotriesterase mutant C23: A proof-of-concept study - Worek_2014_Toxicol.Lett_231_45
Author(s) : Worek F , Seeger T , Reiter G , Goldsmith M , Ashani Y , Leader H , Sussman JL , Aggarwal N , Thiermann H , Tawfik DS
Ref : Toxicol Lett , 231 :45 , 2014
Abstract : The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(-) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (kcat/KM=5x106M-1min-1). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5mgkg-1 i.v.) 5min after s.c. challenge with VX ( approximately 2LD50) prevented systemic toxicity. A lower C23 dose (2mgkg-1) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15min post VX) with 5mgkg-1 C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(-)-VX enantiomer that correlates with the C23 blood levels and its kcat/KM value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single post-exposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication.
ESTHER : Worek_2014_Toxicol.Lett_231_45
PubMedSearch : Worek_2014_Toxicol.Lett_231_45
PubMedID: 25195526

Title : In vitro kinetics of nerve agent degradation by fresh frozen plasma (FFP) - Wille_2014_Arch.Toxicol_88_301
Author(s) : Wille T , Thiermann H , Worek F
Ref : Archives of Toxicology , 88 :301 , 2014
Abstract : Great efforts have been undertaken in the last decades to develop new oximes to reactivate acetylcholinesterase inhibited by organophosphorus compounds (OP). So far, a broad-spectrum oxime effective against structurally diverse OP is still missing, and alternative approaches, e.g. stoichiometric and catalytic scavengers, are under investigation. Fresh frozen plasma (FFP) has been used in human OP pesticide poisoning which prompted us to investigate the in vitro kinetics of OP nerve agent degradation by FFP. Degradation was rapid and calcium-dependent with the G-type nerve agents tabun, sarin, soman and cyclosarin with half-lives from 5 to 28 min. Substantially longer and calcium-independent degradation half-lives of 23-33 h were determined with the V-type nerve agents CVX, VR and VX. However, at all the tested conditions, the degradation of V-type nerve agents was several-fold faster than spontaneous hydrolysis. Albumin did not accelerate the degradation of nerve agents. In conclusion, the fast degradation of G-type nerve agents by FFP might be a promising tool, but would require transfusion shortly after poisoning. FFP does not seem to be suitable for detoxifying relevant agent concentrations in case of human poisoning by V-type nerve agents.
ESTHER : Wille_2014_Arch.Toxicol_88_301
PubMedSearch : Wille_2014_Arch.Toxicol_88_301
PubMedID: 24057572

Title : Effectiveness of a substituted beta-cyclodextrin to prevent cyclosarin toxicity in vivo - Worek_2014_Toxicol.Lett_226_222
Author(s) : Worek F , Seeger T , Zengerle M , Kubik S , Thiermann H , Wille T
Ref : Toxicol Lett , 226 :222 , 2014
Abstract : Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and an oxime has a limited efficacy. An alternative approach is the development of stoichiometric or catalytic (bio-)scavengers which should be able to prevent systemic toxicity. Recently, a beta-cyclodextrin derivative, 6-OxP-CD, bearing a pyridinium oximate in 6-position of one glucose unit was synthetized and shown to possess a promising detoxification potential against a variety of alkyl methylfluorophosphonates in vitro. In order to investigate the suitability of 6-OxP-CD as a small molecule scavenger an in vivo guinea pig model was established to determine the protective effect of 6-OxP-CD against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of 6-OxP-CD (100mg/kg) prevented systemic toxicity in cyclosarin ( approximately 2LD50) poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity but did not protect erythrocyte AChE activity. A lower 6-OxP-CD dose (50mg/kg) reduced systemic toxicity and prevented mortality in all animals. Thus, the results of this proof of concept study indicate that 6-OxP-CD may be considered as a potential small molecule scavenger to protect against the toxic effects of a range of highly toxic OP nerve agents.
ESTHER : Worek_2014_Toxicol.Lett_226_222
PubMedSearch : Worek_2014_Toxicol.Lett_226_222
PubMedID: 24561299

Title : Investigations of kinetic interactions between lipid emulsions, hydroxyethyl starch or dextran and organophosphorus compounds - Von Der Wellen_2013_Clin.Toxicol.(Phila)_51_918
Author(s) : von der Wellen J , Worek F , Thiermann H , Wille T
Ref : Clinical Toxicology (Phila) , 51 :918 , 2013
Abstract : Abstract Context Numerous studies demonstrated a limited efficacy of clinically used oximes in case of poisoning by various organophosphorus compounds. A broad spectrum oxime antidote covering all organophosphorus nerve agents and pesticides is still missing and effective (bio-)scavengers have not yet been marketed. Objective. The interactions of the available and clinically approved hydroxyethyl starch, dextran and lipid emulsions with organophosphorus nerve agents and pesticides were investigated in order to provide an in vitro base for the evaluation of these compounds in human organophosphorus poisoning. Materials and methods. The degradation kinetics of organophosphorus compounds by the glucose derivatives and lipid emulsions were investigated with an acetylcholinesterase inhibition assay. Results. The incubation of organophosphorus compounds with TRIS-Ca(2+) buffer resulted in a time-dependent degradation of the nerve agents with half-lives of 42 min for cyclosarin, 49 min for sarin, 99 min for tabun, 107 min for soman 19 h for malaoxon and 54 h for VX. In contrast, incubation with all tested compounds resulted in a stabilisation of the organophosphorus compounds. Discussion. Our results suggest that binding of lipophilic organophosphorus compounds could result in a reduced spontaneous and enzyme-induced degradation of the toxic compounds. Conclusion. High dose lipid emulsions and glucose derivatives stabilised organophosphorus compounds in vitro.
ESTHER : Von Der Wellen_2013_Clin.Toxicol.(Phila)_51_918
PubMedSearch : Von Der Wellen_2013_Clin.Toxicol.(Phila)_51_918
PubMedID: 24199642

Title : Structural requirements for effective oximes - Evaluation of kinetic in vitro data with phosphylated human AChE and structurally different oximes - Worek_2013_Chem.Biol.Interact_203_125
Author(s) : Worek F , Wille T , Koller M , Thiermann H
Ref : Chemico-Biological Interactions , 203 :125 , 2013
Abstract : Treatment of poisoning by various organophosphorus (OP) nerve agents with established acetylcholinesterase (AChE) reactivators (oximes) is insufficient. In consequence, extensive research programs have been undertaken in various countries in the past decades to identify more effective oximes. The efficacy of new compounds has been investigated with different in vitro and in vivo models which hamper the comparison of results from different laboratories. The crucial mechanism of action of oximes is the reactivation of phosphylated AChE. The kinetic properties of these compounds can be quantified in vitro with isolated AChE from different origin. It was tempting to evaluate the reactivation kinetics of a series of oximes with various OP inhibitors performed under identical experimental conditions in order to get insight into structural requirements for adequate affinity and reactivity towards inhibited AChE. The determination of reactivation rate constants with bispyridinium oximes having different linkers, bearing oxime group(s) at different positions and having in part additional substituents revealed that (a) the reactivating potency was dependent on the position of the oxime groups and of additional substituents, (b) small modifications of the oxime structure had an in part marked effect on the kinetic properties and (c) no single oxime had an adequate reactivating potency with AChE inhibited by structurally different OP. These and previous studies underline the necessity to investigate in detail the kinetic properties of novel oximes and that the identification of a single oxime being effective against a broad range of structurally different OP will remain a major challenge.
ESTHER : Worek_2013_Chem.Biol.Interact_203_125
PubMedSearch : Worek_2013_Chem.Biol.Interact_203_125
PubMedID: 22827894

Title : Limitations and challenges in treatment of acute chemical warfare agent poisoning - Thiermann_2013_Chem.Biol.Interact_206_435
Author(s) : Thiermann H , Worek F , Kehe K
Ref : Chemico-Biological Interactions , 206 :435 , 2013
Abstract : Recent news from Syria on a possible use of chemical warfare agents made the headlines. Furthermore, the motivation of terrorists to cause maximal harm shifts these agents into the public focus. For incidents with mass casualties appropriate medical countermeasures must be available. At present, the most important threats arise from nerve agents and sulfur mustard. At first, self-protection and protection of medical units from contamination is of utmost importance. Volatile nerve agent exposure, e.g. sarin, results in fast development of cholinergic crisis. Immediate clinical diagnosis can be confirmed on-site by assessment of acetylcholinesterase activity. Treatment with autoinjectors that are filled with 2mg atropine and an oxime (at present obidoxime, pralidoxime, TMB-4(Trimedoxime) or HI-6) are not effective against all nerve agents. A more aggressive atropinisation has to be considered and more effective oximes (if possible with a broad spectrum or a combination of different oximes) as well as alternative strategies to cope with high acetylcholine levels at synaptic sites should be developed. A further gap exists for the treatment of patients with sustained cholinergic crisis that has to be expected after exposure to persistent nerve agents, e.g. VX. The requirement for long-lasting artificial ventilation can be reduced with an oxime therapy that is optimized by using the cholinesterase status for guidance or by measures (e.g. scavengers) that are able to reduce the poison load substantially in the patients. For sulfur mustard poisoning no specific antidote is available until now. Symptomatic measures as used for treatment of burns are recommended together with surgical or laser debridement. Thus, huge amounts of resources are expected to be consumed as wound healing is impaired. Possible depots of sulfur mustard in tissues may aggravate the situation. More basic knowledge is necessary to improve substantially therapeutic options. The use of stem cells may provide a new and promising option.
ESTHER : Thiermann_2013_Chem.Biol.Interact_206_435
PubMedSearch : Thiermann_2013_Chem.Biol.Interact_206_435
PubMedID: 24091052

Title : Investigation of kinetic interactions between approved oximes and human acetylcholinesterase inhibited by pesticide carbamates - Wille_2013_Chem.Biol.Interact_206_569
Author(s) : Wille T , Kaltenbach L , Thiermann H , Worek F
Ref : Chemico-Biological Interactions , 206 :569 , 2013
Abstract : Carbamates are widely used for pest control and act primarily by inhibition of insect and mammalian acetylcholinesterase (AChE). Accidental or intentional uptake of carbamates may result in typical signs and symptoms of cholinergic overstimulation which cannot be discriminated from those of organophosphorus pesticide poisoning. There is an ongoing debate whether standard treatment with atropine and oximes should be recommended for human carbamate poisoning as well, since in vitro and in vivo animal data indicate a deleterious effect of oximes when used in combination with the N-methyl carbamate carbaryl. Therefore, we performed an in vitro kinetic study to investigate the effect of clinically used oximes on carbamoylation and decarbamoylation of human AChE. It became evident that pralidoxime and obidoxime in therapeutic concentrations aggravate the inhibition of AChE by carbaryl and propoxur, with obidoxime being substantially more potent compared to 2-PAM. However, obidoxime had no impact on the decarbamoylation kinetics. Hence, the administration of 2-PAM and especially of obidoxime to severely propoxur and carbaryl poisoned humans cannot be recommended.
ESTHER : Wille_2013_Chem.Biol.Interact_206_569
PubMedSearch : Wille_2013_Chem.Biol.Interact_206_569
PubMedID: 23962483

Title : The value of novel oximes for treatment of poisoning by organophosphorus compounds - Worek_2013_Pharmacol.Ther_139_249
Author(s) : Worek F , Thiermann H
Ref : Pharmacol Ther , 139 :249 , 2013
Abstract : Poisoning by organophosphorus compounds (OP) still is a major therapeutic problem. Intentional OP pesticide poisoning results in up to 300.000 deaths each year and highly toxic OP nerve agents pose a permanent threat for the civilian population and military forces. The therapeutic value of clinically used oximes, pralidoxime, obidoxime and TMB-4, in human OP pesticide poisoning is under debate. Moreover, these oximes lack efficacy in poisoning by various nerve agents. An innumerable number of novel oximes have been synthesized in the past five decades to provide more effective oximes and compounds with improved blood-brain-barrier penetration. Novel compounds were tested with largely different experimental protocols in vitro and in animals in vivo. The lack of comparable experimental conditions and the absence of human in vivo studies hamper a well-founded evaluation of the available data. At present, it appears that only a small number of (bispyridinium) oximes show superior potency and efficacy against individual OP. However, until now, no oxime with sufficient broad-spectrum activity against structurally different OP pesticides and nerve agents is available. An interim solution may be the combination of two oximes with overlapping reactivation spectrum. In conclusion, the unsatisfying situation calls for studies with standardized and comparable experimental conditions in order to allow a sound assessment of available and novel oximes.
ESTHER : Worek_2013_Pharmacol.Ther_139_249
PubMedSearch : Worek_2013_Pharmacol.Ther_139_249
PubMedID: 23603539

Title : Affinities of bispyridinium non-oxime compounds to [(3)H]epibatidine binding sites of Torpedo californica nicotinic acetylcholine receptors depend on linker length - Niessen_2013_Chem.Biol.Interact_206_545
Author(s) : Niessen KV , Seeger T , Tattersall JE , Timperley CM , Bird M , Green C , Thiermann H , Worek F
Ref : Chemico-Biological Interactions , 206 :545 , 2013
Abstract : The toxicity of organophosphorus nerve agents or pesticides arises from accumulation of acetylcholine and overstimulation of both muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) due to inhibition of acetylcholinesterase (AChE). Standard treatment by administration of atropine and oximes, e.g., obidoxime or pralidoxime, focuses on antagonism of mAChRs and reactivation of AChE, whereas nicotinic malfunction is not directly treated. An alternative approach would be to use nAChR active substances to counteract the effects of accumulated acetylcholine. Promising in vitro and in vivo results were obtained with the bispyridinium compounds SAD-128 (1,1'-oxydimethylene bis(4-tert-butylpyridinium) dichloride) and MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)), which were partly attributed to their interaction with nAChRs. In this study, a homologous series of unsubstituted and 4-tert-butyl-substituted bispyridinium compounds with different alkane linker lengths was investigated in competition binding experiments using [(3)H]epibatidine as a reporter ligand. Additionally, the effect of the well-characterised MB327 on the [(3)H]epibatidine equilibrium dissociation (KD) constant in different buffers was determined. This study demonstrated that divalent cations increased the affinity of [(3)H]epibatidine. Since quaternary ammonium molecules are known to inhibit AChE, the obtained affinity constants of the tested bispyridinium compounds were compared with the inhibition of human AChE. In competition experiments, bispyridinium derivatives of longer linker length displaced [(3)H]epibatidine and inhibited AChE strongly. Bispyridinium compounds with short linkers, at most, have an allosteric interaction with the [(3)H]epibatidine binding sites and barely inhibited AChE. In dependence on alkane linker length, the bispyridinium compounds seemed to interact at different binding sites. However, the exact binding sites of the bispyridinium compounds responsible for the positive pharmacological effects have still not been identified, making predictive drug design difficult.
ESTHER : Niessen_2013_Chem.Biol.Interact_206_545
PubMedSearch : Niessen_2013_Chem.Biol.Interact_206_545
PubMedID: 24157926

Title : Functionalized cyclodextrins bearing an alpha nucleophile - A promising way to degrade nerve agents - Estour_2013_Chem.Biol.Interact_203_202
Author(s) : Estour F , Letort S , Muller S , Kalakuntla RK , Le Provost R , Wille T , Reiter G , Worek F , Lafont O , Gouhier G
Ref : Chemico-Biological Interactions , 203 :202 , 2013
Abstract : Organophosphorus nerve agents are irreversible inhibitors of acetylcholinesterase. Current treatment of nerve agent poisoning has limited efficacy and more efficient medical countermeasures need to be developed. A promising approach is to design chemical scavengers more stable during storage and less immunogenic than bioscavengers. Furthermore, they could be produced at lowest production costs. Cyclodextrins are attractive cyclic oligosaccharides that can be used to develop chemical scavengers of organophosphorus nerve agents. Their abilities to form inclusion and non-inclusion complexes with organic substrates are useful to trap chemical warfare agents. Selective introduction of an alpha-nucleophile residue on the secondary face of beta-cyclodextrin allowed to obtain supramolecular derivatives active against organophosphorus compounds. The degradation activity of these monosubstituted cyclodextrins was determined against paraoxon and chemical warfare agents. These tests showed that the structure of the scavengers mainly influences the interaction between the organophosphorus substrate, or its reaction products, and the cyclodextrin moiety. All the tested G-type agents were efficiently degraded. According to the binding modes of cyclosarin, some oligosaccharidic scavengers led to an enantioselective degradation of this nerve agent. These promising derivatives open the way to further investigations of new structural modifications to reach more sophisticated and efficient scavengers for prophylactic and curative medical applications.
ESTHER : Estour_2013_Chem.Biol.Interact_203_202
PubMedSearch : Estour_2013_Chem.Biol.Interact_203_202
PubMedID: 23123247

Title : Reactivation of Plasma Butyrylcholinesterase by Pralidoxime Chloride in Patients Poisoned by WHO Class II Toxicity Organophosphorus Insecticides - Konickx_2013_Toxicol.Sci_136_274
Author(s) : Konickx LA , Worek F , Jayamanne S , Thiermann H , Buckley NA , Eddleston M
Ref : Toxicol Sci , 136 :274 , 2013
Abstract : Some clinicians assess the efficacy of pralidoxime in organophosphorus (OP) poisoned patients by measuring reactivation of butyrylcholinesterase (BCHE). However, the degree of BCHE inhibition varies by OP insecticide, and it is unclear how well oximes reactivate BCHE in vivo. We aimed to assess the usefulness of BCHE activity to monitor pralidoxime treatment by studying its reactivation after pralidoxime administration to patients with laboratory-proven World Health Organization (WHO) class II OP insecticide poisoning. Patient data were derived from 2 studies, a cohort study (using a bolus treatment of 1g pralidoxime chloride) and a randomized controlled trial (RCT) (comparing 2g pralidoxime over 20min, followed by an infusion of 0.5g/h, with placebo). Two grams of pralidoxime variably reactivated BCHE in patients poisoned by 2 diethyl OP insecticides, chlorpyrifos and quinalphos; however, unlike acetylcholinesterase reactivation, this reactivation was not sustained. It did not reactivate BCHE inhibited by the dimethyl OPs dimethoate or fenthion. The 1-g dose produced no reactivation. Pralidoxime produced variable reactivation of BCHE in WHO class II OP-poisoned patients according to the pralidoxime dose administered, OP ingested, and individual patient. The use of BCHE assays for monitoring the effect of pralidoxime treatment is unlikely to be clinically useful.
ESTHER : Konickx_2013_Toxicol.Sci_136_274
PubMedSearch : Konickx_2013_Toxicol.Sci_136_274
PubMedID: 24052565

Title : New modified beta-cyclodextrin derivatives as detoxifying agents of chemical warfare agents (I). Synthesis and preliminary screening: Evaluation of the detoxification using a half-quantitative enzymatic assay - Kalakuntla_2013_Toxicol.Lett_216_200
Author(s) : Kalakuntla RK , Wille T , Le Provost R , Letort S , Reiter G , Muller S , Thiermann H , Worek F , Gouhier G , Lafont O , Estour F
Ref : Toxicol Lett , 216 :200 , 2013
Abstract : Current treatments of organophosphorus nerve agents poisoning are imperfect, and more efficient medical countermeasures need to be developed. Chemical scavengers based on beta-cyclodextrin displayed promising results, but further investigations have to be performed to evaluate the possibility of application of substituted cyclodextrins as potential detoxification agents. Herein, five new cyclodextrins scavengers were synthesized. New optimal conditions for regioselectively monosubstitution of beta-cyclodextrin at O-2 position were then studied to access to key intermediates. After these optimizations, a new series of three permethylated derivatives was developed, and two compounds bearing an alpha-nucleophilic group via a three carbon atoms linker were prepared. The ability of these five scavengers to detoxify nerve agents (cyclosarin, soman, tabun and VX) was evaluated by a semi-quantitative biological assay. All the modified cyclodextrins significantly decreased the inhibitory effect of chemical warfare G agents on acetylcholinesterase activity. For this purpose, we showed that the specific interactions between the organophosphorus compound and the oligosaccharidic moiety of the scavenger played a pivotal role in the detoxification process.
ESTHER : Kalakuntla_2013_Toxicol.Lett_216_200
PubMedSearch : Kalakuntla_2013_Toxicol.Lett_216_200
PubMedID: 23201439

Title : Fourteenth International Medical Chemical Defence Conference 2013 Translation of experimental research for improved treatment of chemical warfare agents -
Author(s) : Wille T , Worek F , Thiermann H
Ref : Chemico-Biological Interactions , 206 :433 , 2013
PubMedID: 23920049

Title : Restoration of soman-blocked neuromuscular transmission in human and rat muscle by the bispyridinium non-oxime MB327 in vitro - Seeger_2012_Toxicology_294_80
Author(s) : Seeger T , Eichhorn M , Lindner M , Niessen KV , Tattersall JE , Timperley CM , Bird M , Green AC , Thiermann H , Worek F
Ref : Toxicology , 294 :80 , 2012
Abstract : The standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes is not sufficiently effective against all types of nerve agents. Alternative therapeutic strategies are required and bispyridinium non-oximes, acting as nicotinic antagonists, were identified as promising compounds. A previous study showed that the di(methanesulfonate) salt of the bispyridinium compound MB327 could restore soman-impaired neuromuscular function in vitro and improve survival of sarin, soman and tabun poisoned guinea pigs in vivo. Here, by using the indirect field stimulation technique, the ability of MB327 to counteract soman-impaired neuromuscular transmission was investigated in human intercostal muscle and rat diaphragm preparations. MB327 restored muscle force in a concentration-dependent manner in both species without reactivating soman-inhibited acetylcholinesterase. The therapeutic effect of MB327 could be washed out, indicating a direct effect at the nicotinic receptor level. Also the ability of MB327 to restore respiratory muscle function could be demonstrated for the first time in rat and human tissue. In combination with previous in vitro and in vivo findings MB327 may be considered a promising compound for the treatment of nerve agent poisoning and further studies are needed to identify optimized drug combinations, concentrations and dosing intervals to provide an effective therapy for OP poisoning.
ESTHER : Seeger_2012_Toxicology_294_80
PubMedSearch : Seeger_2012_Toxicology_294_80
PubMedID: 22349640

Title : A role for solvents in the toxicity of agricultural organophosphorus pesticides - Eddleston_2012_Toxicology_294_94
Author(s) : Eddleston M , Street JM , Self I , Thompson A , King T , Williams N , Naredo G , Dissanayake K , Yu LM , Worek F , John H , Smith S , Thiermann H , Harris JB , Eddie Clutton R
Ref : Toxicology , 294 :94 , 2012
Abstract : Organophosphorus (OP) insecticide self-poisoning is responsible for about one-quarter of global suicides. Treatment focuses on the fact that OP compounds inhibit acetylcholinesterase (AChE); however, AChE-reactivating drugs do not benefit poisoned humans. We therefore studied the role of solvent coformulants in OP toxicity in a novel minipig model of agricultural OP poisoning. Gottingen minipigs were orally poisoned with clinically relevant doses of agricultural emulsifiable concentrate (EC) dimethoate, dimethoate active ingredient (AI) alone, or solvents. Cardiorespiratory physiology and neuromuscular (NMJ) function, blood AChE activity, and arterial lactate concentration were monitored for 12h to assess poisoning severity. Poisoning with agricultural dimethoate EC40, but not saline, caused respiratory arrest within 30 min, severe distributive shock and NMJ dysfunction, that was similar to human poisoning. Mean arterial lactate rose to 15.6 [SD 2.8] mM in poisoned pigs compared to 1.4 [0.4] in controls. Moderate toxicity resulted from poisoning with dimethoate AI alone, or the major solvent cyclohexanone. Combining dimethoate with cyclohexanone reproduced severe poisoning characteristic of agricultural dimethoate EC poisoning. A formulation without cyclohexanone showed less mammalian toxicity. These results indicate that solvents play a crucial role in dimethoate toxicity. Regulatory assessment of pesticide toxicity should include solvents as well as the AIs which currently dominate the assessment. Reformulation of OP insecticides to ensure that the agricultural product has lower mammalian toxicity could result in fewer deaths after suicidal ingestion and rapidly reduce global suicide rates.
ESTHER : Eddleston_2012_Toxicology_294_94
PubMedSearch : Eddleston_2012_Toxicology_294_94
PubMedID: 22365945

Title : Comparative kinetics of organophosphates and oximes with erythrocyte, muscle and brain acetylcholinesterase - Herkert_2012_Toxicol.Lett_209_173
Author(s) : Herkert NM , Freude G , Kunz U , Thiermann H , Worek F
Ref : Toxicol Lett , 209 :173 , 2012
Abstract : There is an ongoing debate whether oximes can effectively counteract the effects of organophosphorus compounds (OP) on brain acetylcholinesterase (AChE) activity and whether there are differences in the kinetic properties of brain and erythrocyte AChE. In order to investigate the kinetics of AChE from different tissues and species the well established dynamically working in vitro model with real-time determination of membrane-bound AChE activity was adapted for use with brain AChE. The enzyme reactor, that was loaded with brain, erythrocyte or muscle AChE, was continuously perfused with substrate and chromogen while AChE activity was on-line analyzed in a flow-through detector. It was possible to determine the Michaelis-Menten constants of human erythrocyte, muscle and brain AChE which were almost identical. In addition, the inhibition kinetics of sarin and paraoxon as well as the reactivation kinetics of obidoxime and HI 6 were determined with human, swine and guinea pig brain and erythrocyte AChE. It was found that the inhibition and reactivation kinetics of brain and erythrocyte AChE were highly comparable in all tested species. These data support the view that AChE from different tissue has similar kinetic properties and that brain AChE is comparably susceptible toward reactivation by oximes.
ESTHER : Herkert_2012_Toxicol.Lett_209_173
PubMedSearch : Herkert_2012_Toxicol.Lett_209_173
PubMedID: 22230262

Title : Reactivation kinetics of a series of related bispyridinium oximes with organophosphate-inhibited human acetylcholinesterase--Structure-activity relationships - Worek_2012_Biochem.Pharmacol_83_1700
Author(s) : Worek F , Wille T , Koller M , Thiermann H
Ref : Biochemical Pharmacology , 83 :1700 , 2012
Abstract : Despite extensive research in the last six decades, oximes are the only available drugs which enable a causal treatment of poisoning by organophosphorus compounds (OP). However, numerous in vitro and in vivo studies demonstrated a limited ability of these oximes to reactivate acetylcholinesterase (AChE) inhibited by different OP pesticides and nerve agents. New oximes were mostly tested for their therapeutic efficacy by using different animal models and for their reactivating potency with AChE from different species. Due to the use of different experimental protocols a comparison of data from the various studies is hardly possible. Now, we found it tempting to determine the reactivation kinetics of a series of bispyridinium oximes bearing one or two oxime groups at different positions and having an oxybismethylene or a trimethylene linker under identical conditions with human AChE inhibited by structurally different OP. The data indicate that the position of the oxime group(s) is decisive for the reactivating potency and that different positions of the oxime groups are important for different OP inhibitors while the nature of the linker, oxybismethylene or trimethylene, is obviously of minor importance. Hence, these and previous data emphasize the necessity for thorough kinetic investigations of OP-oxime-AChE interactions and underline the difficulty to develop a broad spectrum oxime reactivator which is efficient against structurally different OP inhibitors.
ESTHER : Worek_2012_Biochem.Pharmacol_83_1700
PubMedSearch : Worek_2012_Biochem.Pharmacol_83_1700
PubMedID: 22649796

Title : Competition radioligand binding assays for the investigation of bispyridinium compound affinities to the human muscarinic acetylcholine receptor subtype 5 (hM(5) ) - Niessen_2012_Drug.Test.Anal_4_292
Author(s) : Niessen KV , Tattersall JE , Timperley CM , Bird M , Green C , Thiermann H , Worek F
Ref : Drug Test Anal , 4 :292 , 2012
Abstract : Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with some nerve agents. Promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128 which was partly attributed to its interaction with nicotinic acetylcholine receptors. Previous studies indicate that bispyridinium compounds interact with muscarinic acetylcholine receptors as well. The muscarinic M(5) receptor is not well investigated compared to other subtypes, but could be important in the search for new drugs for treating nerve agent poisoning. A set of bispyridinium compounds structurally related to SAD-128 were tested in competition binding experiments with recombinant human M(5) muscarinic acetylcholine receptors. Five of the six investigated bispyridinium compounds interacted with the orthosteric binding site, with affinities in the low micromolar range. These data indicate that interaction of bispyridinium compounds with muscarinic receptors may contribute to their therapeutic efficacy.
ESTHER : Niessen_2012_Drug.Test.Anal_4_292
PubMedSearch : Niessen_2012_Drug.Test.Anal_4_292
PubMedID: 22362630

Title : Detoxification of tabun at physiological pH mediated by substituted beta-cyclodextrin and glucose derivatives containing oxime groups - Brandhuber_2012_Toxicology_302_163
Author(s) : Brandhuber F , Zengerle M , Porwol L , Tenberken O , Thiermann H , Worek F , Kubik S , Reiter G
Ref : Toxicology , 302 :163 , 2012
Abstract : The ability of 13 beta-cyclodextrin and 2 glucose derivatives containing substituents with oxime groups as nucleophilic components to accelerate the degradation of tabun at physiological pH has been evaluated. To this end, a qualitative and a quantitative enzymatic assay as well as a highly sensitive enantioselective GC-MS assay were used. In addition, an assay was developed that provided information about the mode of action of the investigated compounds. The results show that attachment of pyridinium-derived substituents with an aldoxime group in 3- or 4-position to a beta-cyclodextrin ring affords active compounds mediating tabun degradation. Activities differ depending on the structure, the number, and the position of the substituent on the ring. Highest activity was observed for a beta-cyclodextrin containing a 4-formylpyridinium oxime residue in 6-position of one glucose subunit, which detoxifies tabun with a half-time of 10.2 min. Comparison of the activity of this compound with that of an analog in which the cyclodextrin ring was replaced by a glucose residue demonstrated that the cyclodextrin is not necessary for activity but certainly beneficial. Finally, the results provide evidence that the mode of action of the cyclodextrin involves covalent modification of its oxime group rendering the scavenger inactive after reaction with the first tabun molecule.
ESTHER : Brandhuber_2012_Toxicology_302_163
PubMedSearch : Brandhuber_2012_Toxicology_302_163
PubMedID: 22982866

Title : Determination of acetylcholinesterase activity by the Ellman assay: a versatile tool for in vitro research on medical countermeasures against organophosphate poisoning - Worek_2012_Drug.Test.Anal_4_282
Author(s) : Worek F , Eyer P , Thiermann H
Ref : Drug Test Anal , 4 :282 , 2012
Abstract : Inhibition of acetylcholinesterase (AChE) is the main mechanism of action of organophosphorus compounds (OP), and AChE reactivators (oximes) are at present the only causal therapeutic approach. Being the key target of OP toxicity, AChE may serve as a valuable tool for diagnosis of OP exposure as well as for the investigation of the kinetics of interactions between OP and oximes. At present, the rapid, simple, and cheap spectrophotometric Ellman assay is widely used for diagnosis, therapeutic monitoring and in vitro kinetic investigations. Application of the assay for investigation of the interactions between AChE, inhibitors, and oximes requires the consideration of potential matrix effects (e.g. hemoglobin), side reactions (e.g. oximolysis of substrate) and other determinants (e.g. pH, temperature). By taking these factors into account, the Ellman assay allows the precise and reproducible determination of kinetic constants as a basis for the understanding of toxic OP effects and for the development of improved therapies against poisoning by OP. In addition, advanced applications of the Ellman assay, for example, in a dynamic in vitro model for the real-time activity determination of membrane-bound AChE, enables the proper investigation of relevant tissue, primarily respiratory muscle, and extends the applicability of this method.
ESTHER : Worek_2012_Drug.Test.Anal_4_282
PubMedSearch : Worek_2012_Drug.Test.Anal_4_282
PubMedID: 21998030

Title : Application of an enantioselective LC-ESI MS\/MS procedure to determine R- and S-hyoscyamine following intravenous atropine administration in swine - John_2012_Drug.Test.Anal_4_194
Author(s) : John H , Mikler J , Worek F , Thiermann H
Ref : Drug Test Anal , 4 :194 , 2012
Abstract : S-hyoscyamine (S-hyo) is a natural plant tropane alkaloid acting as a muscarinic receptor (MR) antagonist. Its racemic mixture (atropine) is clinically used in pre-anaesthesia, ophthalmology and for the antidotal treatment of organophosphorus (OP) poisoning with nerve agents or pesticides even though R-hyo exhibits no effects on MR. Further investigative research is required to optimize treatment of OP poisoning. Swine are often the animal model utilized due to similarities in physiology and antidote response to humans. However, no studies have been reported that elucidated differences in the kinetics of R- and S-hyo. Therefore, the concentration-time profiles of total hyo as well as both enantiomers were analyzed in plasma after intravenous administration of atropine sulfate (Atr(2) SO(4) , 100microg/kg) to anaesthetized swine. For quantification plasma samples were incubated separately with human serum (procedure A) and rabbit serum (procedure B). The rabbit serum used contained atropinesterase, which is suitable for stereoselective hydrolysis of S-hyo, while human serum does not hydrolyze either enantiomer. After incubation samples were precipitated and the supernatant was analyzed by RP-HPLC-ESI MS/MS. Procedure A allowed determination of total hyo and procedure B remaining R-hyo concentrations. S-hyo was calculated as the difference of the two procedures. Concentration data were regressed by a two-phase decay according to a two-compartment open model revealing similar kinetics for both enantiomers thus indicating distribution, metabolism and elimination without obvious stereoselective preference in tested swine.
ESTHER : John_2012_Drug.Test.Anal_4_194
PubMedSearch : John_2012_Drug.Test.Anal_4_194
PubMedID: 21964777

Title : Reactivation kinetics of a homologous series of bispyridinium bis-oximes with nerve agent-inhibited human acetylcholinesterase - Worek_2012_Arch.Toxicol_86_1379
Author(s) : Worek F , von der Wellen J , Musilek K , Kuca K , Thiermann H
Ref : Archives of Toxicology , 86 :1379 , 2012
Abstract : The reactivation of organophosphorus compound (OP)-inhibited acetylcholinesterase (AChE) by oximes is inadequate in case of different OP nerve agents. This fact led to the synthesis of numerous novel oximes by different research groups in order to identify more effective reactivators. In the present study, we investigated the reactivation kinetics of a homologous series of bispyridinium bis-oximes bearing a (E)-but-2-ene linker with tabun-, sarin-, and cyclosarin-inhibited human AChE. In part, marked differences in affinity and reactivity of the investigated oximes toward OP-inhibited human AChE were recorded. These properties depended on the position of the oxime groups and the inhibitor. None of the tested oximes was equally effective against all used OPs. In addition, the data indicate that a (E)-but-2-ene linker decreased in most cases the reactivating potency in comparison to oximes bearing an oxybismethylene linker, e.g., obidoxime and HI-6. The results of this study give further insight into structural requirements for oxime reactivators, underline the necessity to investigate the kinetic interactions of oximes and AChE with structurally different OP inhibitors, and point to the difficulty to develop an oxime reactivator which is efficient against a broad spectrum of OPs.
ESTHER : Worek_2012_Arch.Toxicol_86_1379
PubMedSearch : Worek_2012_Arch.Toxicol_86_1379
PubMedID: 22437842

Title : Photostability of antidotal oxime HI-6, impact on drug development - Bogan_2012_Drug.Test.Anal_4_208
Author(s) : Bogan R , Worek F , Koller M , Klaubert B
Ref : Drug Test Anal , 4 :208 , 2012
Abstract : HI-6 exhibits superior efficacy in the therapy of intoxication by different highly toxic organophosphorus nerve agents. Therefore HI-6 is a promising candidate for the development of new antidotes against nerve agents. For ethical and safety reasons antidotes containing HI-6 should get marketing authorization. Active pharmaceutical ingredients of medicinal products have to fulfil regulatory conditions in terms of purity and stability. Photostability is an essential parameter in this testing strategy. HI-6 was tested under conditions of ICH Q1B 'Photostability testing of new drug substances and products'. The data showed a marked degradation of HI-6 after exposure to daylight. The mechanism of degradation could be detected as photoisomerism. The light burden dependent rate of photoisomerism was followed quantitatively. Based on these quantitative results on the amount of light induced isomeric product a pharmacological qualification was made. A standardized in vitro test showed a decreased ability of light exposed HI-6 to reactivate sarin- and paraoxon-inhibited human acetylcholinesterase. These results have an impact on the further development of antidotes containing HI-6, as light protection will probably be necessary during handling, packaging, storage and application.
ESTHER : Bogan_2012_Drug.Test.Anal_4_208
PubMedSearch : Bogan_2012_Drug.Test.Anal_4_208
PubMedID: 22359386

Title : Quantification of pralidoxime (2-PAM) in urine by ion pair chromatography-diode array detection: application to in vivo samples from minipig - John_2012_Drug.Test.Anal_4_169
Author(s) : John H , Eddleston M , Eddie Clutton R , Worek F , Thiermann H
Ref : Drug Test Anal , 4 :169 , 2012
Abstract : Pralidoxime (2-PAM) is a monopyridinium oxime used as an antidote for the treatment of poisoning with organophosphorus (OP) compounds, for example, pesticides and nerve agents, reactivating OP-inhibited acetylcholinesterase. However, appropriate dosing and efficacy remains a matter of discussion requiring experimental data. Therefore, we developed and validated an ion pair chromatography-diode array detection (IPC-DAD) method suitable for quantitative analysis of 2-PAM in human and porcine urine. Before injection of 20 microl, urine was acidified with trichloroacetic acid, mixed with internal standard (pyridine-4-aldoxime, 4-PAO), and diluted with IPC solvent yielding a total dilution of 1:49.5 and a 100% recovery. Isocratic separation was carried out at 25 degrees C on a LiChrospher 60 RP-select B column (125 x 4.0 mm I.D.) using phosphate buffer (7.5 mM Na(2) HPO(4) , 7.5 mM KH(2) PO(4) , pH 2.6) mixed with octanesulfonate (2.5 mM) as ion pair reagent and acetonitrile (6% v/v) as organic modifier (1 ml/min). 2-PAM was detected at 293 nm and 4-PAO at 275 nm. The method is rugged, selective, and characterized by good intra-day and inter-day precision (RSD, 1.3-6.0%) and accuracy (88-100%) with a limit of detection at 4.9 microg/ml, a limit of quantification at 9.8 microg/ml, and a broad calibration range from 4.9-2500 microg/ml. The procedure was applied to urine samples obtained from dimethoate poisoned minipigs receiving 2-PAM therapy (intravenous bolus injection and infusion). Results indicate that 60-80% of infused 2-PAM is rapidly (within 1-2 h) excreted in the urine.
ESTHER : John_2012_Drug.Test.Anal_4_169
PubMedSearch : John_2012_Drug.Test.Anal_4_169
PubMedID: 22102522

Title : Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro - Herkert_2011_Toxicol.Appl.Pharmacol_253_7
Author(s) : Herkert NM , Schulz S , Wille T , Thiermann H , Hatz RA , Worek F
Ref : Toxicol Appl Pharmacol , 253 :7 , 2011
Abstract : Standard treatment of organophosphorus (OP) poisoning includes administration of an antimuscarinic (e.g., atropine) and of an oxime-based reactivator. However, successful oxime treatment in soman poisoning is limited due to rapid aging of phosphylated acetylcholinesterase (AChE). Hence, the inability of standard treatment procedures to counteract the effects of soman poisoning resulted in the search for alternative strategies. Recently, results of an in vivo guinea pig study indicated a therapeutic effect of physostigmine given after soman. The present study was performed to investigate a possible pre- and post-treatment effect of physostigmine on soman-inhibited human AChE given at different time intervals before or after perfusion with soman by using a well-established dynamically working in vitro model for real-time analysis of erythrocyte and muscle AChE. The major findings were that prophylactic physostigmine prevented complete inhibition of AChE by soman and resulted in partial spontaneous recovery of the enzyme by de-carbamylation. Physostigmine given as post-treatment resulted in a time-dependent reduction of the protection from soman inhibition and recovery of AChE. Hence, these date indicate that physostigmine given after soman does not protect AChE from irreversible inhibition by the OP and that the observed therapeutic effect of physostigmine in nerve agent poisoning in vivo is probably due to other factors.
ESTHER : Herkert_2011_Toxicol.Appl.Pharmacol_253_7
PubMedSearch : Herkert_2011_Toxicol.Appl.Pharmacol_253_7
PubMedID: 21402092

Title : Highly efficient cyclosarin degradation mediated by a beta-cyclodextrin derivative containing an oxime-derived substituent - Zengerle_2011_Beilstein.J.Org.Chem_7_1543
Author(s) : Zengerle M , Brandhuber F , Schneider C , Worek F , Reiter G , Kubik S
Ref : Beilstein J Org Chem , 7 :1543 , 2011
Abstract : The potential of appropriately substituted cyclodextrins to act as scavengers for neurotoxic organophosphonates under physiological conditions was evaluated. To this end, a series of derivatives containing substituents with an aldoxime or a ketoxime moiety along the narrow opening of the beta-cyclodextrin cavity was synthesized, and the ability of these compounds to reduce the inhibitory effect of the neurotoxic organophosphonate cyclosarin on its key target, acetylcholinesterase, was assessed in vitro. All compounds exhibited a larger effect than native beta-cyclodextrin, and characteristic differences were noted. These differences in activity were correlated with the structural and electronic parameters of the substituents. In addition, the relatively strong effect of the cyclodextrin derivatives on cyclosarin degradation and, in particular, the observed enantioselectivity are good indications that noncovalent interactions between the cyclodextrin ring and the substrate, presumably involving the inclusion of the cyclohexyl moiety of cyclosarin into the cyclodextrin cavity, contribute to the mode of action. Among the nine compounds investigated, one exhibited remarkable activity, completely preventing acetylcholinesterase inhibition by the (-)-enantiomer of cyclosarin within seconds under the conditions of the assay. Thus, these investigations demonstrate that decoration of cyclodextrins with appropriate substituents represents a promising approach for the development of scavengers able to detoxify highly toxic nerve agents.
ESTHER : Zengerle_2011_Beilstein.J.Org.Chem_7_1543
PubMedSearch : Zengerle_2011_Beilstein.J.Org.Chem_7_1543
PubMedID: 22238531

Title : In vitro kinetic interactions of DEET, pyridostigmine and organophosphorus pesticides with human cholinesterases - Wille_2011_Chem.Biol.Interact_190_79
Author(s) : Wille T , Thiermann H , Worek F
Ref : Chemico-Biological Interactions , 190 :79 , 2011
Abstract : The simultaneous use of the repellent DEET, pyridostigmine, and organophosphorus pesticides has been assumed as a potential cause for the Gulf War Illness and combinations have been tested in different animal models. However, human in vitro data on interactions of DEET with other compounds are scarce and provoked the present in vitro study scrutinizing the interactions of DEET, pyridostigmine and pesticides with human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE). DEET showed to be a weak and reversible inhibitor of hAChE and hBChE. The IC(50) of DEET was calculated to be 21.7mM DEET for hAChE and 3.2mM DEET for hBChE. The determination of the inhibition kinetics of pyridostigmine, malaoxon and chlorpyrifos oxon with hAChE in the presence of 5mM DEET resulted in a moderate reduction of the inhibition rate constant k(i). The decarbamoylation velocity of pyridostigmine-inhibited hAChE was not affected by DEET. In conclusion, the in vitro investigation of interactions between human cholinesterases, DEET, pyridostigmine, malaoxon and chlorpyrifos oxon showed a weak inhibition of hAChE and hBChE by DEET. The inhibitory potency of the tested cholinesterase inhibitors was not enhanced by DEET and it did not affect the regeneration velocity of pyridostigmine-inhibited AChE. Hence, this in vitro study does not give any evidence of a synergistic effect of the tested compounds on human cholinesterases.
ESTHER : Wille_2011_Chem.Biol.Interact_190_79
PubMedSearch : Wille_2011_Chem.Biol.Interact_190_79
PubMedID: 21354413

Title : In vitro kinetic interactions of DEET, pyridostigmine and organophosphorus pesticides with human cholinesterases - Response to the letter to the editor -
Author(s) : Wille T , Thiermann H , Worek F
Ref : Chemico-Biological Interactions , 193 :108 , 2011
PubMedID:

Title : Interaction of bispyridinium compounds with the orthosteric binding site of human alpha7 and Torpedo californica nicotinic acetylcholine receptors (nAChRs) - Niessen_2011_Toxicol.Lett_206_100
Author(s) : Niessen KV , Tattersall JE , Timperley CM , Bird M , Green C , Seeger T , Thiermann H , Worek F
Ref : Toxicol Lett , 206 :100 , 2011
Abstract : Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with different nerve agents. A direct pharmacologic intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach and promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128. In addition, a number of SAD-128 analogues improved neuromuscular transmission of soman-poisoned diaphragms in vitro. We investigated the interaction of six of these SAD-128 analogues with the orthosteric binding site of the human alpha7 nAChR and Torpedo californica nAChR with a high-throughput assay using radioactive ligands. The determined affinity constants indicate a weak interaction of three test compounds (K(i) in the micromolar range) with both receptors, but no interaction could be recorded with the other three test compounds. The six SAD-128 analogues showed a low intrinsic inhibitory potency with human acetylcholinesterase (IC(5)(0) > 400 muM). In conclusion, the results of the present study do not indicate a correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission and it is assumed that other mechanisms contribute to the therapeutic effect of the tested compounds.
ESTHER : Niessen_2011_Toxicol.Lett_206_100
PubMedSearch : Niessen_2011_Toxicol.Lett_206_100
PubMedID: 21703337

Title : Simultaneous quantification of VX and its toxic metabolite in blood and plasma samples and its application for in vivo and in vitro toxicological studies - Reiter_2011_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_879_2704
Author(s) : Reiter G , Mikler J , Hill I , Weatherby K , Thiermann H , Worek F
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 879 :2704 , 2011
Abstract : The present study was initiated to develop a sensitive and highly selective method for the simultaneous quantification of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) and its toxic metabolite (EA-2192) in blood and plasma samples in vivo and in vitro. For the quantitative detection of VX and EA-2192 the resolution was realized on a HYPERCARB HPLC phase. A specific procedure was developed to isolate both toxic analytes from blood and plasma samples. The limit of detection was 0.1 pg/ml and the absolute recovery of the overall sample preparation procedure was 74% for VX and 69% for EA-2192. After intravenous and percutaneous administration of a supralethal doses of VX in anaesthetised swine both VX and EA-2192 could be quantified over 540 min following exposure. This study is the first to verify the in vivo formation of the toxic metabolite EA-2192 after poisoning with the nerve agent VX. Further toxicokinetic and therapeutic studies are required in order to determine the impact of EA-2192 on the treatment of acute VX poisoning.
ESTHER : Reiter_2011_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_879_2704
PubMedSearch : Reiter_2011_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_879_2704
PubMedID: 21862421

Title : In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species - Herkert_2011_Toxicol.Lett_206_41
Author(s) : Herkert NM , Thiermann H , Worek F
Ref : Toxicol Lett , 206 :41 , 2011
Abstract : The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.
ESTHER : Herkert_2011_Toxicol.Lett_206_41
PubMedSearch : Herkert_2011_Toxicol.Lett_206_41
PubMedID: 21414391

Title : Evaluation of the Test-mate ChE (cholinesterase) field kit in acute organophosphorus poisoning - Rajapakse_2011_Ann.Emerg.Med_58_559
Author(s) : Rajapakse BN , Thiermann H , Eyer P , Worek F , Bowe SJ , Dawson AH , Buckley NA
Ref : Annals of Emergency Medicine , 58 :559 , 2011
Abstract : STUDY OBJECTIVE: Measurement of acetylcholinesterase (AChE) is recommended in the management of organophosphorus poisoning, which results in 200,000 deaths worldwide annually. The Test-mate ChE 400 is a portable field kit designed for detecting occupational organophosphorus exposure that measures RBC AChE and plasma cholinesterase (PChE) within 4 minutes. We evaluate Test-mate against a reference laboratory test in patients with acute organophosphorus self-poisoning. METHODS: This was a cross-sectional comparison study of 14 patients with acute organophosphorus poisoning between May 2007 and June 2008. RBC AChE and PChE were measured in 96 and 91 samples, respectively, with the Test-mate ChE field kit and compared with a reference laboratory, using the limits of agreement method (Bland and Altman), kappa statistics, and Spearman's correlation coefficients. RESULTS: There was good agreement between the Test-mate ChE and the reference laboratory for RBC AChE. The mean difference (Test-mate-reference) was -0.62 U/g hemoglobin, 95% limits of agreement -10.84 to 9.59 U/g hemoglobin. Good agreement was also observed between the categories of mild, moderate, and severe RBC AChE inhibition (weighted kappa 0.85; 95% confidence interval [CI] 0.83 to 0.87). Measurement of PChE also showed good agreement, with a mean difference (Test-mate-reference) of +0.06 U/mL blood, 95% limits of agreement -0.41 to 0.53 U/mL blood. Spearman's correlation coefficients were 0.87 (95% CI 0.81 to 0.91) for RBC AChE and 0.76 (95% CI 0.66 to 0.84) for PChE. Analysis for within-subject correlation of subjects did not change the limits of agreement. CONCLUSION: The Test-mate ChE field kit reliably provides rapid measurement of RBC AChE in acute organophosphorus poisoning.
ESTHER : Rajapakse_2011_Ann.Emerg.Med_58_559
PubMedSearch : Rajapakse_2011_Ann.Emerg.Med_58_559
PubMedID: 22098995

Title : Central respiratory effects on motor nerve activities after organophosphate exposure in a working heart brainstem preparation of the rat - Klein-Rodewald_2011_Toxicol.Lett_206_94
Author(s) : Klein-Rodewald T , Seeger T , Dutschmann M , Worek F , Morschel M
Ref : Toxicol Lett , 206 :94 , 2011
Abstract : The impact of organophosphorus compound (OP) intoxication on the activity of central respiratory circuitry, causing acetylcholinesterase (AChE) inhibition and accumulation of acetylcholine in the respiratory brainstem circuits, is not understood. We investigated the central effect of the OP Crotylsarin (CRS) on respiratory network activity using the working heart brainstem preparation, which specifically allows for the analysis of central drug effects without changes in brainstem oxygenation possibly caused by drug effects on peripheral cardio-respiratory activity. Respiratory network activity was determined from phrenic and hypoglossal or vagal nerve activities (PNA, HNA, VNA). To investigate combined central and peripheral CRS effects hypo-perfusion was used mimicking additional peripheral cardiovascular collapse. Systemic CRS application induced a brief central apnea and complete AChE-inhibition in the brainstem. Subsequently, respiration was characterised by highly significant reduced PNA minute activity, while HNA showed expiratory related extra bursting indicative for activation of un-specified oro-pharyngeal behaviour. During hypo-perfusion CRS induced significantly prolonged apnoea. In all experiments respiratory activity fully recovered after 1h. We conclude that CRS mediated AChE inhibition causes only transient central breathing disturbance. Apparently intrinsic brainstem mechanisms can compensate for cholinergic over activation. Nevertheless, combination of hypo-perfusion and CRS exposure evoke the characteristic breathing arrests associated with OP poisoning.
ESTHER : Klein-Rodewald_2011_Toxicol.Lett_206_94
PubMedSearch : Klein-Rodewald_2011_Toxicol.Lett_206_94
PubMedID: 21767620

Title : HI 6 human serum albumin nanoparticles--development and transport over an in vitro blood-brain barrier model - Dadparvar_2011_Toxicol.Lett_206_60
Author(s) : Dadparvar M , Wagner S , Wien S , Kufleitner J , Worek F , von Briesen H , Kreuter J
Ref : Toxicol Lett , 206 :60 , 2011
Abstract : The standard treatment of intoxication with organophosphorus (OP) compounds includes the administration of oximes acting as acetylcholinesterase (AChE) reactivating antidotes. However, the blood-brain barrier (BBB) restricts the rapid transport of these drugs from the blood into the brain in therapeutically relevant concentrations. Since human serum albumin (HSA) nanoparticles enable the delivery of a variety of drugs across the BBB into the brain, HI 6 dimethanesulfonate and HI 6 dichloride monohydrate were bound to these nanoparticles in the present study. The resulting sorption isotherms showed a better fit to Freundlich's empirical adsorption isotherm than to Langmuir's adsorption isotherm. At the pH of 8.3 maximum drug binding capacities of 344.8 mug and 322.6 mug per mg of nanoparticles were calculated for HI 6 dimethanesulfonate and HI 6 dichloride monohydrate, respectively. These calculated values are higher than the adsorption capacity of 93.5 mug/mg for obidoxime onto HSA nanoparticles determined in a previous study. In vitro testing of the nanoparticulate oxime formulations in primary porcine brain capillary endothelial cells (pBCEC) demonstrated an up to two times higher reactivation of OP-inhibited AChE than the free oximes. These findings show that nanoparticles made of HSA may enable a sufficient antidote OP-poisoning therapy with HI 6 derivatives even within the central nervous system (CNS).
ESTHER : Dadparvar_2011_Toxicol.Lett_206_60
PubMedSearch : Dadparvar_2011_Toxicol.Lett_206_60
PubMedID: 21726608

Title : Immobilization of Russian VX skin depots by localized cooling: implications for decontamination and medical countermeasures - Mikler_2011_Toxicol.Lett_206_47
Author(s) : Mikler J , Tenn C , Worek F , Reiter G , Thiermann H , Garrett M , Bohnert S , Sawyer TW
Ref : Toxicol Lett , 206 :47 , 2011
Abstract : The chemical weapon nerve agent known as Russian VX (VR) is a potent organophosphorus (OP) compound that is much less studied than its VX analogue with respect to toxicity, as well as to the effectiveness of several known countermeasures against it. An anaesthetized domestic swine model was utilized to assess several approaches in mitigating its toxicity, including the utility of cooling VR treated skin to increase the therapeutic window for treatment. The 6h LD(5)(0) for VR topically applied on the ear was 100 mug/kg. Treatment of VR exposed animals (5 x LD(5)(0)) with pralidoxime (2PAM) very poorly regenerated inhibited blood cholinesterase activity, but was partially effective in preventing signs of OP poisoning and increasing survival. In contrast, treatment with the Hagedorn oxime HI-6 reactivated cholinesterase, eliminated all signs of poisoning and prevented death. Decontamination with the Reactive Skin Decontaminant Lotion (RSDL) 15 min after VR exposure was completely effective in preventing death. Cooling of the VR exposure sites for 2 or 6h prevented signs of OP poisoning and death during the cooling period. However, these animals died very quickly after the cessation of cooling, unless they were treated with oxime or decontaminated with RSDL. Blood analyses showed that cooling of agent exposure sites delayed the entry of VR into the bloodstream. Medical treatment with HI-6 and to a lesser extent 2PAM, or decontamination with RSDL are effective in protecting against the toxic effects of cutaneous exposure to VR. Immobilizing this agent (and related compounds) within the dermal reservoir by cooling the exposure sites, dramatically increases the therapeutic window in which these medical countermeasures are effective.
ESTHER : Mikler_2011_Toxicol.Lett_206_47
PubMedSearch : Mikler_2011_Toxicol.Lett_206_47
PubMedID: 21704135

Title : Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase - Worek_2011_Toxicol.Lett_200_19
Author(s) : Worek F , Aurbek N , Wille T , Eyer P , Thiermann H
Ref : Toxicol Lett , 200 :19 , 2011
Abstract : Previous in vitro studies showed marked species differences in the reactivating efficiency of oximes between human and animal acetylcholinesterase (AChE) inhibited by organophosphorus (OP) nerve agents. These findings provoked the present in vitro study which was designed to determine the inhibition, aging, spontaneous and oxime-induced reactivation kinetics of the pesticide paraoxon, serving as a model compound for diethyl-OP, and the oximes obidoxime, pralidoxime, HI 6 and MMB-4 with human, Rhesus monkey, swine, rabbit, rat and guinea pig erythrocyte AChE. Comparable results were obtained with human and monkey AChE. Differences between human, swine, rabbit, rat and guinea pig AChE were determined for the inhibition and reactivation kinetics. A six-fold difference of the inhibitory potency of paraoxon with human and guinea pig AChE was recorded while only moderate differences of the reactivation constants between human and animal AChE were determined. Obidoxime was by far the most effective reactivator with all tested species. Only minor species differences were found for the aging and spontaneous reactivation kinetics. The results of the present study underline the necessity to determine the inhibition, aging and reactivation kinetics in vitro as a basis for the development of meaningful therapeutic animal models, for the proper assessment of in vivo animal data and for the extrapolation of animal data to humans.
ESTHER : Worek_2011_Toxicol.Lett_200_19
PubMedSearch : Worek_2011_Toxicol.Lett_200_19
PubMedID: 20971170

Title : Evaluation of 6,6'-dithionicotinic acid as alternative chromogen in a modified Ellman method--comparison in various species - Wille_2011_Toxicol.Mech.Methods_21_533
Author(s) : Wille T , Thiermann H , Worek F
Ref : Toxicol Mech Methods , 21 :533 , 2011
Abstract : For the diagnosis and therapy monitoring of intoxications with organophosphorus compounds, the determination of acetylcholinesterase (AChE) activity in whole blood is crucial. Usually, this testing is done with the colorimetric Ellman test using 412 nm wavelength and 5,5'-dithiobis-nitrobenzoic acid (DTNB) as chromogen. However, it has been described that the assay sensitivity is impaired by the Soret band of hemoglobin. In order to enable more sensitive determination of AChE activity in human, porcine, rat, and guinea pig whole blood dilutions, we tested the alternative chromogen 6,6'-dithionicotinic acid (DTNA) with an optimal absorption wavelength of 340 nm and compared it with a modified Ellman assay using a wavelength of 436 nm. DTNB resulted in a higher background absorption compared with DTNA in human and porcine blood samples, although the saturation of sulfhydryl groups was delayed with DTNA in all species. A slightly higher whole blood AChE activity was recorded in DTNB samples. In conclusion, the results of the present study with human, porcine, guinea pig, and rat whole blood do not provide evidence that using DTNA for the spectrophotometric determination of AChE activity in whole blood is superior to a modified Ellman assay using DTNB.
ESTHER : Wille_2011_Toxicol.Mech.Methods_21_533
PubMedSearch : Wille_2011_Toxicol.Mech.Methods_21_533
PubMedID: 21470076

Title : Kinetic prerequisites of oximes as effective reactivators of organophosphate-inhibited acetylcholinesterase: a theoretical approach - Worek_2011_J.Enzyme.Inhib.Med.Chem_26_303
Author(s) : Worek F , Aurbek N , Wille T , Eyer P , Thiermann H
Ref : J Enzyme Inhib Med Chem , 26 :303 , 2011
Abstract : The standard treatment of poisoning by organophosphorus compounds (OP) includes the reversible muscarine receptor antagonist atropine and oximes for the reactivation of OP-inhibited acetylcholinesterase (AChE). There is an ongoing discussion on the benefit of oxime therapy in OP pesticide poisoning, and experimental data indicate a limited efficacy of oximes against various nerve agents. Oxime effectiveness can be quantified in vitro by determination of the reactivity (k(r)) and affinity constants (1/K(D)). These constants can be used to calculate reactivation velocities and oxime concentrations necessary for the reactivation of a desired fraction of inhibited AChE. Model calculations indicate that a k(r) > 0.1 min(-1) and K(D) < 100 microM are minimal requirements for oxime effectiveness when reactivation is performed in the absence of free OP. In addition, the findings demonstrate that selective increase of either reactivity or affinity of an oxime would be insufficient. Hereby, it has to be taken into account that an increase of affinity to OP-inhibited AChE is generally accompanied by an increased affinity to native AChE and subsequent reduction in oxime tolerance. Hence, future developments of more effective oximes should consider kinetic demands by attempting to achieve a certain level of reactivity and affinity, preferentially towards OP-inhibited AChE.
ESTHER : Worek_2011_J.Enzyme.Inhib.Med.Chem_26_303
PubMedSearch : Worek_2011_J.Enzyme.Inhib.Med.Chem_26_303
PubMedID: 20807085

Title : The therapeutic use of localized cooling in the treatment of VX poisoning - Sawyer_2011_Toxicol.Lett_204_52
Author(s) : Sawyer TW , Mikler J , Worek F , Reiter G , Thiermann H , Tenn C , Weatherby K , Bohnert S
Ref : Toxicol Lett , 204 :52 , 2011
Abstract : The organophosphate (OP) nerve agent VX is a weaponized chemical warfare agent that has also been used by terrorists against civilians. This contact poison produces characteristic signs of OP poisoning, including miosis, salivation, mastication, dysrhythmias and respiratory distress prior to death. Although successful treatment of OP poisoning can be obtained through decontamination and/or oxime reactivation of agent-inhibited cholinesterase, medical countermeasures that increase the therapeutic window for these measures would be of benefit. An anaesthetized swine model was utilized to examine the effects of lethal VX exposure to the skin, followed by cooling the exposure site prior to decontamination or treatment. The cooling was simply accomplished by using crushed ice in grip-seal plastic bags applied to the exposure sites. Cooling of skin exposed to lethal doses of VX significantly increased the window of opportunity for successful decontamination using the Reactive Skin Decontaminant Lotion((R)) (RSDL((R))) or treatment with the oxime antidotes HI-6 and 2PAM. Analyses of blood VX levels showed that cooling acted to slow or prevent the entry of VX into the bloodstream from the skin. If the exposure site is known, the simple and non-invasive application of cooling provides a safe means with which to dramatically increase the therapeutic window in which decontamination and/or antidote treatment against VX are life-saving.
ESTHER : Sawyer_2011_Toxicol.Lett_204_52
PubMedSearch : Sawyer_2011_Toxicol.Lett_204_52
PubMedID: 21530621

Title : Reactivation of organophosphate-inhibited human acetylcholinesterase by isonitrosoacetone (MINA): a kinetic analysis - Worek_2011_Chem.Biol.Interact_194_91
Author(s) : Worek F , Thiermann H
Ref : Chemico-Biological Interactions , 194 :91 , 2011
Abstract : Treatment of poisoning by highly toxic organophosphorus compounds (OP) with atropine and an acetylcholinesterase (AChE) reactivator (oxime) is of limited effectiveness in case of different nerve agents and pesticides. One challenge is the reactivation of OP-inhibited brain AChE which shows inadequate success with charged pyridinium oximes. Recent studies with high doses of the tertiary oxime isonitrosoacetone (MINA) indicated a beneficial effect on central and peripheral AChE and on survival in nerve agent poisoned guinea pigs. Now, an in vitro study was performed to determine the reactivation kinetics of MINA with tabun-, sarin-, cyclosarin-, VX- and paraoxon-inhibited human AChE. MINA showed an exceptionally low affinity to inhibited AChE but, with the exception of tabun-inhibited AChE, a moderate to high reactivity. In comparison to the pyridinium oximes obidoxime, 2-PAM and HI-6 the affinity and reactivity of MINA was in most cases lower and in relation to the most effective reactivators, the second order reactivation constant of MINA was 500 to 3400-fold lower. Hence, high in vivo MINA concentrations would be necessary to achieve at least partial reactivation. This assumption corresponds to in vivo data showing a dose-dependent effect on reactivation and survival in animals. In view, of the toxic potential of MINA in animals human studies would be necessary to determine the tolerability and pharmacokinetics of MINA in order to enable a proper assessment of the value of this oxime as an antidote in OP poisoning.
ESTHER : Worek_2011_Chem.Biol.Interact_194_91
PubMedSearch : Worek_2011_Chem.Biol.Interact_194_91
PubMedID: 21930118

Title : Restoration of nerve agent inhibited muscle force production in human intercostal muscle strips with HI 6 - Seeger_2011_Toxicol.Lett_206_72
Author(s) : Seeger T , Niessen KV , Langer P , Gerhardus J , Worek F , Friess H , Bumm R , Mihaljevic AL , Thiermann H
Ref : Toxicol Lett , 206 :72 , 2011
Abstract : An important factor for successful therapy of poisoning with organophosphorus compounds (OP) is the rapid restoration of blocked respiratory muscle function. To achieve this goal, oximes are administered for reactivation of inhibited acetylcholinesterase (AChE). Unfortunately, clinically used oximes, e.g. obidoxime and pralidoxime, are of limited effectiveness in poisoning with different OP nerve agents requiring the search for alternative oximes, e.g. HI 6. In view of substantial species differences regarding reactivation properties of oximes, the effect of HI 6 was investigated with sarin, tabun and soman exposed human intercostal muscle. Muscle force production by indirect field stimulation and the activity of the human muscle AChE was assessed. 30 muM HI 6 resulted in an almost complete recovery of sarin blocked muscle force and in an increase of completely inhibited muscle AChE activity to approx. 30% of control. In soman or tabun exposed human intercostal muscle HI 6 (50 and 100 muM) had no effect on blocked muscle force or on inhibited human muscle AChE activity. In addition, HI 6 up to 1000 muM had no effect on soman blocked muscle force indicating that this oxime has no direct, pharmacological effect in human tissue. These results emphasize that sufficient reactivation of AChE is necessary for a beneficial therapeutic effect on nerve agent blocked neuromuscular transmission.
ESTHER : Seeger_2011_Toxicol.Lett_206_72
PubMedSearch : Seeger_2011_Toxicol.Lett_206_72
PubMedID: 21803135

Title : Optimized strategies to synthesize beta-cyclodextrin-oxime conjugates as a new generation of organophosphate scavengers - Le Provost_2011_Org.Biomol.Chem_9_3026
Author(s) : Le Provost R , Wille T , Louise L , Masurier N , Muller S , Reiter G , Renard PY , Lafont O , Worek F , Estour F
Ref : Org Biomol Chem , 9 :3026 , 2011
Abstract : A new generation of organophosphate (OP) scavengers was obtained by synthesis of beta-cyclodextrin-oxime derivatives 8-12. Selective monosubstitution of beta-cyclodextrin was the main difficulty in order to access these compounds, because reaction onto the oligosaccharide was closely related to the nature of the incoming group. For this purpose, non-conventional activation conditions were also evaluated. Intermediates 5 and 7 were then obtained with the better yields under ultrasounds irradiation. Finally, the desired compounds 8-10 were obtained from 5-7 in high purity by desilylation using potassium fluoride. Quaternarisation of compounds 8 and 9 was carried out. OP hydrolytic activity of compounds 8-12 was evaluated against cyclosarin (GF) and VX. None of the tested compounds was active against VX, but these five cyclodextrin derivatives detoxified GF, and the most active scavengers 10 and 11 allowed an almost complete hydrolysis of GF within 10 min. Even more fascinating is the fact that compounds 9 and 10 were able to hydrolyze enantioselectively GF.
ESTHER : Le Provost_2011_Org.Biomol.Chem_9_3026
PubMedSearch : Le Provost_2011_Org.Biomol.Chem_9_3026
PubMedID: 21373706

Title : Thirteenth International Medical Chemical Defence Conference 2011 New developments in the treatment of intoxications by chemical warfare agents with focus on neurotoxic agents -
Author(s) : Tenberken O , Worek F , Thiermann H
Ref : Toxicol Lett , 206 :3 , 2011
PubMedID: 21605643

Title : Atropine maintenance dosage in patients with severe organophosphate pesticide poisoning - Thiermann_2011_Toxicol.Lett_206_77
Author(s) : Thiermann H , Steinritz D , Worek F , Radtke M , Eyer P , Eyer F , Felgenhauer N , Zilker T
Ref : Toxicol Lett , 206 :77 , 2011
Abstract : Although the importance of atropine in therapy of organophosphate (OP) poisoning is generally recognized, its dosing is a matter of debate. A retrospective analysis of atropine dosing was undertaken in 34 patients who had been enrolled in a clinical study assessing obidoxime effectiveness in OP-poisoning. All patients were severely intoxicated (suicidal attempts) and required artificial ventilation. Atropine was administered routinely by intensive care physicians for life-threatening muscarinic symptoms, with the recommendation to favor low dosage. The pharmacological active enantiomere S-hyoscyamine was determined by a radioreceptor assay. When RBC-AChE activity ranged between 10% and 30%, S-hyoscyamine plasma concentrations of approx. 5 nmol L(-)(1) were sufficient. This concentration could be maintained with about 0.005 mg h(-)(1) kg(-)(1) atropine. Only when RBC-AChE was completely inhibited, therapy of cholinergic crisis required atropine doses up to 0.06 mg h(-)(1) kg(-)(1). Elimination half-life of S-hyoscyamine was 1.5 h, showing occasionally a second slow elimination phase with t((1/2))=12 h. Malignant arrhythmias were observed in some 10% of our cases, which occurred late and often in the absence of relevant glandular cholinergic signs, when the S-hyoscyamine concentration was below 2.5 nmol L(-)(1). Arrhythmias mostly resolved on reinstitution of atropine.
ESTHER : Thiermann_2011_Toxicol.Lett_206_77
PubMedSearch : Thiermann_2011_Toxicol.Lett_206_77
PubMedID: 21771644

Title : In vitro detoxification of cyclosarin (GF) by modified cyclodextrins - Muller_2011_Toxicol.Lett_200_53
Author(s) : Muller S , Koller M , Le Provost R , Lafont O , Estour F , Wille T , Thiermann H , Worek F , Reiter G
Ref : Toxicol Lett , 200 :53 , 2011
Abstract : Developing potent detoxification strategies for prophylaxis and therapy against organophosphate (OP) intoxication still represents a challenging task. Clinical application of numerous investigated substances including enzymes and low molecular scavengers like metal ions or nucleophiles could not yet be realised due to profound disadvantages. Presenting a promising attempt, cyclodextrins (CDs) efficiently enhance the degradation of some organophosphorus compounds. The present study examined the in vitro GF degradation mediated by three CDs and a nucleophilic precursor performed by mass spectrometric detection with ammonia chemical ionisation. All four compounds caused a notable enhancement of GF detoxification that was synergistically accelerated in the case of 2-O-(3-carboxy-4-iodosobenzyl)-beta-cyclodextrin (IBA-beta-CD) with the alpha-nucleophile 2-iodosobenzoic acid (IBA) grafted on the secondary face of beta-cyclodextrin (beta-CD). In vitro toxicokinetic investigations of CD derivatives are needed to evaluate the effect of slow terminal elimination phase of the more toxic (-)-GF shown for two CD-derivatives underlining the necessity of detecting the complete kinetic course of inactivation. The observed effect of fast high affinity binding (20-30%) represents an additional therapeutic option of an extremely rapid reduction of GF concentration in vivo. Distinctive differences in the course of reaction are detected depending on beta-CD-derivatives, allowing a first inference of possible mechanisms and relevance of attached substituents. However, further profound investigation needs to be done to evaluate the basis of a clinical application of substituted CDs as potential detoxification agents.
ESTHER : Muller_2011_Toxicol.Lett_200_53
PubMedSearch : Muller_2011_Toxicol.Lett_200_53
PubMedID: 21035528

Title : Chromatographic analysis of toxic phosphylated oximes (POX): a brief overview - Becker_2010_Drug.Test.Anal_2_460
Author(s) : Becker C , Worek F , John H
Ref : Drug Test Anal , 2 :460 , 2010
Abstract : Poisoning with organophosphorus compounds (OP), e.g. pesticides and nerve agents, causes inhibition of acetylcholinesterase (AChE) by phosphylation of the active site serine residue. Consequently, accumulation of stimulating acetylcholine in the synaptic cleft induces cholinergic crisis which ultimately may lead to death. For standard causal therapy, enzyme reactivators are administered representing oxime derivatives of quarternary pyridinium compounds, e.g. pralidoxime (2-PAM), obidoxime and HI 6. The mechanism of action includes removal of the phosphyl moiety by a nucleophilic attack of the oximate molecule substituting the enzyme and forming a phosphylated oxime (POX). POX is produced in stoichiometric amounts of reactivated enzyme and exhibits a significantly enhanced toxicity (inhibition rate constant) when compared to the parent OP. However, stability of POX under physiological conditions appears to be highly limited. Nevertheless, the presence of POX reveals a potential critical issue for both therapeutic efficacy in vivo and pharmacokinetic and pharmacodynamic (PK-PD) modelling based on cholinesterase activity data. Detailed characterization represents an important need for elaboration of the entire oxime pharmacology.Nevertheless, reports on POX toxicity and analysis are quite rare and may therefore be indicative of the challenge of POX analysis. This review provides a concise overview of chromatographic approaches applied to POX separation. Chromatography represents the key technology for POX purification and quantification in kinetic in vitro studies using buffers and biological fluids. Applications based on reversed-phase chromatography (RPC), ion pair chromatography (IPC) and an affinity approach as well as thin layer chromatography (TLC) are discussed and novel applications and data are presented.
ESTHER : Becker_2010_Drug.Test.Anal_2_460
PubMedSearch : Becker_2010_Drug.Test.Anal_2_460
PubMedID: 20882513

Title : GC-MS and LC-MS analysis of nerve agents in body fluids: intra-laboratory verification test using spiked plasma and urine samples - Koller_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1226
Author(s) : Koller M , Becker C , Thiermann H , Worek F
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 878 :1226 , 2010
Abstract : The purpose of this study was to check the applicability of different analytical methods for the identification of unknown nerve agents in human body fluids. Plasma and urine samples were spiked with nerve agents (plasma) or with their metabolites (urine) or were left blank. Seven random samples (35% of all samples) were selected for the verification test. Plasma was worked up for unchanged nerve agents and for regenerated nerve agents after fluoride-induced reactivation of nerve agent-inhibited butyrylcholinesterase. Both extracts were analysed by GC-MS. Metabolites were extracted from plasma and urine, respectively, and were analysed by LC-MS. The urinary metabolites and two blank samples could be identified without further measurements, plasma metabolites and blanks were identified in six of seven samples. The analysis of unchanged nerve agent provided five agents/blanks and the sixth agent after further investigation. The determination of the regenerated agents also provided only five clear findings during the first screening because of a rather noisy baseline. Therefore, the sample preparation was extended by a size exclusion step performed before addition of fluoride which visibly reduced baseline noise and thus improved identification of the two missing agents. The test clearly showed that verification should be performed by analysing more than one biomarker to ensure identification of the agent(s).
ESTHER : Koller_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1226
PubMedSearch : Koller_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1226
PubMedID: 20061191

Title : Paradox findings may challenge orthodox reasoning in acute organophosphate poisoning - Eyer_2010_Chem.Biol.Interact_187_270
Author(s) : Eyer P , Worek F , Thiermann H , Eddleston M
Ref : Chemico-Biological Interactions , 187 :270 , 2010
Abstract : It is generally accepted that inhibition of acetylcholinesterase (AChE) is the most important acute toxic action of organophosphorus compounds, leading to accumulation of acetylcholine followed by a dysfunction of cholinergic signaling. However, the degree of AChE inhibition is not uniformly correlated with cholinergic dysfunction, probably because the excess of essential AChE varies among tissues. Moreover, the cholinergic system shows remarkable plasticity, allowing modulations to compensate for dysfunctions of the canonical pathway. A prominent example is the living (-/-) AChE knockout mouse. Clinical experience indicates that precipitous inhibition of AChE leads to more severe poisoning than more protracted yet finally complete inhibition. The former situation is seen in parathion, the latter in oxydemeton methyl poisoning. At first glance, this dichotomy is surprising since parathion is a pro-poison and has to be activated to the oxon, while the latter is still the ultimate inhibitor. Also oxime therapy in organophosphorus poisoning apparently gives perplexing results: Oximes are usually able to reactivate diethylphosphorylated AChE, but the efficiency may be occasionally markedly smaller than expected from kinetic data. Dimethylphosphorylated AChE is in general less amenable to oxime therapy, which largely fails in some cases of dimethoate poisoning where aging was much faster than expected from a dimethylphosphorylated enzyme. Similarly, poisoning by profenofos, an O,S-dialkyl phosphate, leads to a rapidly aged enzyme. Most surprisingly, these patients were usually well on admission, yet their erythrocyte AChE was completely inhibited. Analysis of the kinetic constants of the most important reaction pathways, determination of the reactant concentrations in vivo and comparison with computer simulations may reveal unexpected toxic reactions. Pertinent examples will be presented and the potentially underlying phenomena discussed.
ESTHER : Eyer_2010_Chem.Biol.Interact_187_270
PubMedSearch : Eyer_2010_Chem.Biol.Interact_187_270
PubMedID: 19883634

Title : Aging mechanism of butyrylcholinesterase inhibited by an N-methyl analogue of tabun: implications of the trigonal-bipyramidal transition state rearrangement for the phosphylation or reactivation of cholinesterases - Nachon_2010_Chem.Biol.Interact_187_44
Author(s) : Nachon F , Carletti E , Worek F , Masson P
Ref : Chemico-Biological Interactions , 187 :44 , 2010
Abstract : Cholinesterases are the main target of organophosphorus nerve agents (OPs). Their inhibition results in cholinergic syndrome and death. The enzymes are inhibited by phosphylation of the catalytic serine enzyme, but can be reactivated by oximes to some extent. However, phosphylated cholinesterases undergo a side reaction that progressively prevents their reactivatability. This unimolecular reaction, termed "aging", has been investigated for decades. It was shown that most OP-ChE conjugates aged by O-dealkylation of an alkoxy substituent of the phosphorus atom, a mechanism involving the stabilization of a transient carbocation. In this paper we present structural data supporting a substitution-based mechanism for aging of the huBChE conjugate of an N-mono-methyl analogue of tabun. This mechanism involves an adjacent nucleophilic attack followed by Berry pseudorotation. A similar adjacent attack and subsequent rearrangement of the transition state have been recently proposed for tabun phosphylation of AChE. We suggest that a similar mechanism is also possible for oxime reactivation of phosphylated cholinesterases. This opens new perspectives in terms of reactivator design.
ESTHER : Nachon_2010_Chem.Biol.Interact_187_44
PubMedSearch : Nachon_2010_Chem.Biol.Interact_187_44
PubMedID: 20381476

Title : Comparative study of oxime-induced reactivation of erythrocyte and muscle AChE from different animal species following inhibition by sarin or paraoxon - Herkert_2010_Toxicol.Lett_194_94
Author(s) : Herkert NM , Aurbek N , Eyer P , Thiermann H , Worek F
Ref : Toxicol Lett , 194 :94 , 2010
Abstract : Standard treatment of acute poisoning by organophosphorus compounds (OP) includes administration of an antimuscarinic (e.g. atropine) and of an oxime-based reactivator of OP-inhibited acetylcholinesterase (AChE). A recently introduced dynamically working in vitro model with real-time determination of membrane-bound AChE activity was shown to be a very versatile and promising model to investigate oxime-induced reactivation kinetics of OP-inhibited enzyme. In this assay, human AChE from erythrocytes or muscle tissue was immobilized on a particle filter. This bioreactor was continuously perfused with substrate and chromogen and AChE activity was analyzed on-line in a flow-through detector. The model has been successfully adopted to Rhesus monkey, swine and guinea pig erythrocytes and intercostal muscle AChE. In addition, the basic kinetic constants of inhibition, aging, spontaneous- and oxime-induced-reactivation of erythrocyte AChE from these species were determined with a standard static model. The major findings were, in part substantial species differences in the inhibition (sarin, paraoxon) and reactivation kinetics (obidoxime, HI 6) of erythrocyte AChE, but comparable kinetics of inhibition and reactivation between erythrocyte and muscle AChE. Hence, these data provide further support of the assumption that erythrocyte AChE is an adequate surrogate of muscle (synaptic) AChE and admonish that major species differences have to be considered for the design and evaluation of therapeutic animal models.
ESTHER : Herkert_2010_Toxicol.Lett_194_94
PubMedSearch : Herkert_2010_Toxicol.Lett_194_94
PubMedID: 20156534

Title : Pharmacokinetics of obidoxime in patients poisoned with organophosphorus compounds - Thiermann_2010_Toxicol.Lett_197_236
Author(s) : Thiermann H , Eyer F , Felgenhauer N , Pfab R , Zilker T , Eyer P , Worek F
Ref : Toxicol Lett , 197 :236 , 2010
Abstract : OBJECTIVES: Reactivation of inhibited acetylcholinesterase (AChE) with oximes is a causal therapy of intoxication with organophosphorus compounds (OPs). Maximal oxime effects are expected when effective doses are administered as soon as possible and as long as reactivation can be anticipated. An obidoxime plasma level in the range of 10-20 microM was estimated as appropriate. The achievement of this target was assessed in 34 severely OP-poisoned patients. METHODS: After admission to the intensive care unit (ICU) the obidoxime regimen (250 mg i.v. as bolus, followed by 750 mg/24h) was started and maintained as long as reactivation was possible. Plasma concentrations of obidoxime were determined by HPLC. RESULTS: A total amount of 2269+/-1726 mg obidoxime was infused over 65 h+/-55 h resulting in a steady state plasma concentration of 14.5+/-7.3 microM. Obidoxime was eliminated with t(1/2(1)) 2.2 and t(1/2(2)) 14 h. The volumes of distribution amounted to 0.32+/-0.1L/kg (V((1))) and 0.28+/-0.12 (V((2)))L/kg. Postmortem examination of tissue in one patient showed obidoxime accumulation in cartilage, kidney and liver and pointed to brain concentrations similar to plasma concentration. CONCLUSIONS: Using the suggested obidoxime regimen, the targeted plasma concentration could be achieved. Obidoxime was eliminated biphasically and was well tolerated. This result allows the recommendation of using this definite regimen for adults also in case of mass casualties.
ESTHER : Thiermann_2010_Toxicol.Lett_197_236
PubMedSearch : Thiermann_2010_Toxicol.Lett_197_236
PubMedID: 20542100

Title : Assessment of neuromuscular dysfunction during poisoning by organophosphorus compounds - Thiermann_2010_Chem.Biol.Interact_187_265
Author(s) : Thiermann H , Seeger T , Gonder S , Herkert N , Antkowiak B , Zilker T , Eyer F , Worek F
Ref : Chemico-Biological Interactions , 187 :265 , 2010
Abstract : Dysfunction of respiratory muscles is a life-threatening complication in poisoning by organophosphorus compounds (OPs). It is both of central and peripheral origin due to impaired cholinergic signalling upon inhibition of acetylcholinesterase (AChE). The dysfunction at neuromuscular synapses is not amenable to anticholinergics and remains a therapeutic challenge. Thus, a clear understanding of the distinct mechanisms occurring at neuromuscular synapses is decisive for the development and improvement of therapeutic strategies, particularly with nerve agent poisoning, where clinical studies are prevented by ethical considerations. Using red blood cell AChE, the kinetics of OP induced inhibition, aging, and spontaneous and oxime-induced reactivation have been elucidated. In a dynamically working in vitro model with real-time determination of membrane-bound AChE, it was shown that the kinetic constants derived from erythrocyte AChE are comparable to muscle AChE in a given species. To assess, whether kinetic considerations of AChE activity are relevant for the neuromuscular function, organotypic spinal cord-skeletal muscle cocultures have been established. In this model neostigmine and VX affected neuromuscular transmission as anticipated from their known actions on AChE. Also oxime-induced restoration of the neuromuscular transmission was observed. These findings were confirmed by functional studies on diaphragm muscles of various species with determination of muscle force generation upon phrenic nerve or indirect electrical field stimulation techniques. Investigations with human intercostal muscles are in progress to assess the conditions in human tissue. The results obtained with paraoxon favourably correlate with data from clinical findings of parathion-poisoned patients where the correlation of neuromuscular transmission with the activity of erythrocyte AChE could be established. In conclusion, a variety of methods are available to follow the microscopic reactions occurring at the synaptic level. Due to the lack of clinical data with different OPs, e.g. nerve agents, well designed animal experiments, reflecting the human situation as close as possible, are indispensable for the development of new drugs against the deleterious OP effects.
ESTHER : Thiermann_2010_Chem.Biol.Interact_187_265
PubMedSearch : Thiermann_2010_Chem.Biol.Interact_187_265
PubMedID: 20036651

Title : Muscle force and acetylcholinesterase activity in mouse hemidiaphragms exposed to paraoxon and treated by oximes in vitro - Thiermann_2010_Toxicology_272_46
Author(s) : Thiermann H , Eyer P , Worek F
Ref : Toxicology , 272 :46 , 2010
Abstract : The therapy of organophosphorus compound (OP) poisoning is still a challenge to clinical toxicologists. To alleviate peripheral respiratory failure oximes, e.g. obidoxime and pralidoxime, are used to reactivate inhibited acetylcholinesterase (AChE) with the intention to restore the disturbed neuromuscular function. In severe human OP poisoning the persistence of poison may counteract effective reactivation by oximes. Therefore, the study was designed to investigate the effect of the clinically used oximes obidoxime, pralidoxime and the experimental compounds HI 6 and HLo 7 in the presence of different paraoxon concentrations. The mouse phrenic nerve-diaphragm preparation was used as a functional model. After washout of paraoxon remarkably low concentrations of obidoxime or HLo 7 were sufficient for restoration of paraoxon-impaired muscle force. In the presence of paraoxon, obidoxime was the most effective oxime and therapeutically used concentrations (10-20microM) were able to restore muscle function even in the presence of 1microM paraoxon. HLo 7 was less effective, but superior to HI 6 and pralidoxime. Generally, a reactivation of AChE to about 30-40% of normal was sufficient for restoration of muscle force. Thus, the data presented strongly support the administration of appropriately dosed oximes, preferably obidoxime, in paraoxon-poisoned patients to restore paraoxon-impaired muscle force.
ESTHER : Thiermann_2010_Toxicology_272_46
PubMedSearch : Thiermann_2010_Toxicology_272_46
PubMedID: 20385200

Title : Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC-ESI-MS\/MS and flow-injection-ESI-MS\/MS - John_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1234
Author(s) : John H , Eddleston M , Clutton RE , Worek F , Thiermann H
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 878 :1234 , 2010
Abstract : Dimethoate is an organophosphorus toxicant used in agri- and horticulture as a systemic broad-spectrum insecticide. It also exhibits toxic activity towards mammalian organism provoked by catalytic desulfuration in the liver producing its oxon-derivative omethoate thus inhibiting acetylcholinesterase, initiating cholinergic crisis and ultimately leading to death by respiratory paralysis and cardiovascular collapse. Pharmaco- and toxicokinetic studies in animal models help to broaden basic understanding of medical intervention by antidotes and supportive care. Therefore, we developed and validated a LC-ESI-MS/MS method suitable for the simultaneous, selective, precise (RSD(intra-day) 1-8%; RSD(inter-day) 5-14%), accurate (intra-day: 95-107%; inter-day: 90-115%), and robust quantification of both pesticides from porcine urine and plasma after deproteinization by precipitation and extensive dilution (1:11,250 for plasma and 1:40,000 for urine). Accordingly, lower limits of quantification (0.24-0.49 microg/ml plasma and 0.78-1.56 microg/ml urine) and lower limits of detection (0.12-0.24 microg/ml plasma and 0.39-0.78 microg/ml urine) were equivalent to quite low absolute on-column amounts (1.1-2.1 pg for plasma and 2.0-3.9 pg for urine). The calibration range (0.24-250 microg/ml plasma and 0.78-200 microg/ml urine) was subdivided into two linear ranges (r(2)>or=0.998) each covering nearly two orders of magnitude. The lack of any interfering peak in 6 individual blank specimens from plasma and urine demonstrated the high selectivity of the method. Furthermore, extensive sample dilution causing lowest concentration of potentially interfering matrix ingredients prompted us to develop and validate an additional flow-injection method (FI-ESI-MS/MS). Validation characteristics were as good as for the chromatographic method but sample throughput was enhanced by a factor of 6. Effects on ionization provoked by plasma and urine matrix from 6 individuals as well as in the presence of therapeutics (antidotes) administered in an animal study were investigated systematically underlying in the reliability of the presented methods. Both methods were applied to porcine samples derived from an in vivo animal study.
ESTHER : John_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1234
PubMedSearch : John_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1234
PubMedID: 20106727

Title : Evaluation of medical countermeasures against organophosphorus compounds: the value of experimental data and computer simulations - Worek_2010_Chem.Biol.Interact_187_259
Author(s) : Worek F , Aurbek N , Herkert NM , John H , Eddleston M , Eyer P , Thiermann H
Ref : Chemico-Biological Interactions , 187 :259 , 2010
Abstract : Despite extensive research for more than six decades on medical countermeasures against poisoning by organophosphorus compounds (OP) the treatment options are meagre. The presently established acetylcholinesterase (AChE) reactivators (oximes), e.g. obidoxime and pralidoxime, are insufficient against a number of nerve agents and there is ongoing debate on the benefit of oxime treatment in human OP pesticide poisoning. Up to now, the therapeutic efficacy of oximes was mostly evaluated in animal models but substantial species differences prevent direct extrapolation of animal data to humans. Hence, it was considered essential to establish relevant experimental in vitro models for the investigation of oximes as antidotes and to develop computer models for the simulation of oxime efficacy in different scenarios of OP poisoning. Kinetic studies on the various interactions between erythrocyte AChE from various species, structurally different OP and different oximes provided a basis for the initial assessment of the ability of oximes to reactivate inhibited AChE. In the present study, in vitro enzyme-kinetic and pharmacokinetic data from a minipig model of dimethoate poisoning and oxime treatment were used to calculate dynamic changes of AChE activities. It could be shown that there is a close agreement between calculated and in vivo AChE activities. Moreover, computer simulations provided insight into the potential and limitations of oxime treatment. In the end, such data may be a versatile tool for the ongoing discussion of the pros and cons of oxime treatment in human OP pesticide poisoning.
ESTHER : Worek_2010_Chem.Biol.Interact_187_259
PubMedSearch : Worek_2010_Chem.Biol.Interact_187_259
PubMedID: 19917271

Title : Toxicokinetics of tabun enantiomers in anaesthetized swine after intravenous tabun administration - Tenberken_2010_Toxicol.Lett_198_177
Author(s) : Tenberken O , Mikler J , Hill I , Weatherby K , Thiermann H , Worek F , Reiter G
Ref : Toxicol Lett , 198 :177 , 2010
Abstract : In the present study, we report the first in vivo toxicokinetic study of tabun (O-ethyl-N,N-dimethylphosphoramidocyanidate). The toxicokinetics of the enantiomers of tabun were investigated in anesthetized swine after intravenous administration of 3xLD(50) (161.4mug/kg) tabun. Blood samples were taken for gas chromatographic-mass spectrometric determination of the tabun enantiomers and for measurement of the activity of red blood cell acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE). The tabun enantiomers could be quantified in swine blood to a minimum concentration of 3.0pg/ml (18.5pM) and could be detected to a minimum concentration of 1.0pg/ml (6.2pM). The concentration-time profiles of both tabun enantiomers were best described by a bi-exponential equation. The elimination of (+)-tabun and (-)-tabun were comparable in the initial phase. In the terminal phase a remarkable difference was found, with terminal half lives of 11.5min for (+)-tabun and 23.1min for (-)-tabun. (+)-Tabun showed a markedly longer persistence in vivo than (+)-enantiomers of other G-type nerve agents and could be detected in all swine at least up to 30min post-injection, (-)-tabun at least up to 90min post-injection. These results demonstrate a rather rapid elimination of tabun enantiomers in vivo and may provide a toxicokinetic basis for the further development and optimization of medical countermeasures against this nerve agent.
ESTHER : Tenberken_2010_Toxicol.Lett_198_177
PubMedSearch : Tenberken_2010_Toxicol.Lett_198_177
PubMedID: 20599598

Title : Nanoparticulate transport of oximes over an in vitro blood-brain barrier model - Wagner_2010_PLoS.One_5_e14213
Author(s) : Wagner S , Kufleitner J , Zensi A , Dadparvar M , Wien S , Bungert J , Vogel T , Worek F , Kreuter J , von Briesen H
Ref : PLoS ONE , 5 :e14213 , 2010
Abstract : BACKGROUND: Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes. CONCLUSIONS/SIGNIFICANCE: With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulations are promising formulations for the treatment of the peripheral and the CNS after OP poisoning.
ESTHER : Wagner_2010_PLoS.One_5_e14213
PubMedSearch : Wagner_2010_PLoS.One_5_e14213
PubMedID: 21151975

Title : Kinetic analysis of interactions between alkylene-linked bis-pyridiniumaldoximes and human acetylcholinesterases inhibited by various organophosphorus compounds - Wille_2010_Biochem.Pharmacol_80_941
Author(s) : Wille T , Ekstrom F , Lee JC , Pang YP , Thiermann H , Worek F
Ref : Biochemical Pharmacology , 80 :941 , 2010
Abstract : The therapeutic approach of organophosphorus compound (OP) intoxications is to reactivate the inhibited enzyme acetylcholinesterase (AChE). Numerous studies demonstrated a limited efficacy of standard oxime-based reactivators against different nerve agents such as tabun and cyclosarin. This emphasizes research for more effective oximes. In the present study, reactivation kinetics of tabun-, sarin-, cyclosarin-, VX- or paraoxon-ethyl-inhibited human AChE (hAChE) with a homologous series of bis-ortho-pyridiniumaldoximes, Ortho-4 - Ortho-9, was investigated with a robot-assisted setting, allowing determination of second-order reactivation rate constants as well as model calculations. The reactivation constants of Ortho-4 - Ortho-9 resulted in marked differences of affinity and reactivity depending on the OP structure and the linker length of the oximes. In general, the K(D) values decreased with increasing linker length. Reactivity increased from Ortho-4 to Ortho-6 for PXE- and VX-inhibited hAChE and from Ortho-4 to Ortho-7 for GA-inhibited hAChE and decreased again with Ortho-8 and Ortho-9. In contrast, k(r) decreased with increasing linker length for sarin- and cyclosarin-inhibited hAChE. In view of the pronounced decrease of K(D) from Ortho-4 to Ortho-9, the k(r2) values increased with all tested OP. Hence, the ratios of K(I)/K(D) and of K(I)/k(r2) showed that in almost all cases the affinity of Ortho-N to the native hAChE was higher than to OP-inhibited enzyme. Model calculations indicated that Ortho-6 - Ortho-9 could be superior to obidoxime in reactivating tabun-inhibited hAChE. Finally, these data emphasize the need to develop oximes with a higher selective affinity towards OP-inhibited hAChE in order to minimize possible side effects.
ESTHER : Wille_2010_Biochem.Pharmacol_80_941
PubMedSearch : Wille_2010_Biochem.Pharmacol_80_941
PubMedID: 20510679

Title : Chromatographic preparation and kinetic analysis of interactions between tabun enantiomers and acetylcholinesterase - Tenberken_2010_Toxicol.Lett_195_142
Author(s) : Tenberken O , Thiermann H , Worek F , Reiter G
Ref : Toxicol Lett , 195 :142 , 2010
Abstract : The easy accessibility to highly toxic OP (organophosphorus)-type chemical warfare agents (nerve agents) underlines the necessity for an effective medical treatment. Acute OP toxicity is primarily caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment. However, the commercially available compounds, obidoxime and pralidoxime, are considered rather ineffective against various nerve agents, including tabun. OP-type chemical warfare agents include an asymmetrical P-atom and consist of at least two stereoisomers. Previous studies with the nerve agents sarin and soman showed marked differences between (-)- and (+)-P isomers regarding AChE inhibition and stability in biological matrices. Hence, stereoselectivity is a key parameter for the development of optimized treatment. In the present study, the tabun enantiomers were isolated by semi-preparative liquid-chromatography (LC) with offline analysis by GC-PCI-MS and final characterization of optical purity (99.98% (-)-tabun and 99.83% (+)-tabun) and specific optical rotation. The inhibition and reactivation kinetics of the tabun enantiomers were determined with human and swine AChE and the aging kinetics with human AChE. The results show a large difference in the inhibitory potency between (-)- and (+)-tabun. The determination of reactivation and aging kinetics indicates that both reactions are at least in part determined by the residual (-)-tabun contamination (0.17%) of the (+)-tabun preparation. These data provide further insight into the kinetic interactions between tabun enantiomers and AChE and may contribute to the development of more effective treatment options.
ESTHER : Tenberken_2010_Toxicol.Lett_195_142
PubMedSearch : Tenberken_2010_Toxicol.Lett_195_142
PubMedID: 20347021

Title : Kinetic analysis of interactions of different sarin and tabun analogues with human acetylcholinesterase and oximes: is there a structure-activity relationship? - Aurbek_2010_Chem.Biol.Interact_187_215
Author(s) : Aurbek N , Herkert NM , Koller M , Thiermann H , Worek F
Ref : Chemico-Biological Interactions , 187 :215 , 2010
Abstract : The repeated misuse of highly toxic organophosphorus compound (OP) based chemical warfare agents in military conflicts and terrorist attacks poses a continuous threat to the military and civilian sector. The toxic symptomatology of OP poisoning is mainly caused by inhibition of acetylcholinesterase (AChE, E.C. 3.1.1.7) resulting in generalized cholinergic crisis due to accumulation of the neurotransmitter acetylcholine (ACh) in synaptic clefts. Beside atropine as competitive antagonist of ACh at muscarinic ACh receptors oximes as reactivators of OP-inhibited AChE are a mainstay of standard antidotal treatment. However, human AChE inhibited by certain OP is rather resistant to oxime-induced reactivation. The development of more effective oxime-based reactivators may fill the gaps. To get more insight into a potential structure-activity relationship between human AChE, OPs and oximes in vitro studies were conducted to investigate interactions of different tabun and sarin analogues with human AChE and the oximes obidoxime and HI 6 by determination of various kinetic constants. Rate constants for the inhibition of human AChE by OPs, spontaneous dealkylation and reactivation as well as reactivation by obidoxime and HI 6 of OP-inhibited human AChE were determined. The recorded kinetic data did not allow a general statement concerning a structure-activity relationship between human AChE, OP and oximes.
ESTHER : Aurbek_2010_Chem.Biol.Interact_187_215
PubMedSearch : Aurbek_2010_Chem.Biol.Interact_187_215
PubMedID: 20105433

Title : Reactivation of organophosphate-inhibited human, Cynomolgus monkey, swine and guinea pig acetylcholinesterase by MMB-4: a modified kinetic approach - Worek_2010_Toxicol.Appl.Pharmacol_249_231
Author(s) : Worek F , Wille T , Aurbek N , Eyer P , Thiermann H
Ref : Toxicol Appl Pharmacol , 249 :231 , 2010
Abstract : Treatment of poisoning by highly toxic organophosphorus compounds (OP, nerve agents) is a continuous challenge. Standard treatment with atropine and a clinically used oxime, obidoxime or pralidoxime is inadequate against various nerve agents. For ethical reasons testing of oxime efficacy has to be performed in animals. Now, it was tempting to investigate the reactivation kinetics of MMB-4, a candidate oxime to replace pralidoxime, with nerve agent-inhibited acetylcholinesterase (AChE) from human and animal origin in order to provide a kinetic basis for the proper assessment of in vivo data. By applying a modified kinetic approach, allowing the use of necessary high MMB-4 concentrations, it was possible to determine the reactivation constants with sarin-, cyclosarin-, VX-, VR- and tabun-inhibited AChE. MMB-4 exhibited a high reactivity and low affinity towards OP-inhibited AChE, except of tabun-inhibited enzyme where MMB-4 had an extremely low reactivity. Species differences between human and animal AChE were low (Cynomolgus) to moderate (swine, guinea pig). Due to the high reactivity of MMB-4 a rapid reactivation of inhibited AChE can be anticipated at adequate oxime concentrations which are substantially higher compared to HI-6. Additional studies are necessary to determine the in vivo toxicity, tolerability and pharmacokinetics of MMB-4 in humans in order to enable a proper assessment of the value of this oxime as an antidote against nerve agent poisoning.
ESTHER : Worek_2010_Toxicol.Appl.Pharmacol_249_231
PubMedSearch : Worek_2010_Toxicol.Appl.Pharmacol_249_231
PubMedID: 20888357

Title : Development of a high-throughput screening for nerve agent detoxifying materials using a fully-automated robot-assisted biological assay - Wille_2010_Toxicol.In.Vitro_24_1026
Author(s) : Wille T , Thiermann H , Worek F
Ref : Toxicol In Vitro , 24 :1026 , 2010
Abstract : Developing improved medical countermeasures against chemical warfare agents (nerve agents) is urgently needed but time-consuming and costly. Here we introduce a robot-assisted liquid handling system with warming, cooling and incubating facilities to screen the detoxifying properties of biological and chemical materials against nerve agents. Two biological tests were established and plasma from various species, DFPase and three cyclodextrins were used as test materials. In test 1, plasma was mixed with sarin or VX and the inhibitory potency of the incubate was determined with human acetylcholinesterase (AChE) at 0, 30 and 60 min. In test 2, test materials and nerve agents were mixed and incubated. Between 0 and 40 min samples were taken and incubated for 3 min with AChE and the residual AChE inhibition was determined to enable the semi-quantitative evaluation of the detoxification kinetics. The automated assays proved to be highly reproducible. It was possible to pre-select detoxifying reagents with test 1 and to determine more detailed detoxifying kinetics with test 2. In conclusion, the automated assay may be considered as a versatile tool for the high-throughput screening of potential detoxifying materials against different nerve agents. With this two-step assay it is possible to screen effectively for detoxifying materials in a high-throughput system.
ESTHER : Wille_2010_Toxicol.In.Vitro_24_1026
PubMedSearch : Wille_2010_Toxicol.In.Vitro_24_1026
PubMedID: 19961920

Title : Development and validation of a sensitive gas chromatography-ammonia chemical ionization mass spectrometry method for the determination of tabun enantiomers in hemolysed blood and plasma of different species - Tenberken_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1290
Author(s) : Tenberken O , Worek F , Thiermann H , Reiter G
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 878 :1290 , 2010
Abstract : The aim of this study was to develop and validate a fast, sensitive and easily applicable GC-MS assay for the chiral quantification of the highly toxic organophosphorus compound tabun (O-ethyl-N,N-dimethylphosphoramidocyanidate, GA) in hemolysed swine blood for further use in toxicokinetic and toxicodynamic studies. These requirements were fulfilled best by a GC-MS assay with positive chemical ionization with ammonia (GC-PCI-MS). Separation was carried out on a beta-cyclodextrin capillary column (Supelco BetaDex 225) after reversed phase (C18) solid-phase extraction. The limit of detection was 1 pg/ml for each enantiomer (approximately 500 fg on column) and the limit of quantification 5 pg/ml. The GC-PCI-MS method was applied for the quantification of tabun enantiomers in spiked swine blood after hemolysis and in spiked plasma of different species including humans.
ESTHER : Tenberken_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1290
PubMedSearch : Tenberken_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1290
PubMedID: 19766064

Title : Improving the promiscuous nerve agent hydrolase activity of a thermostable archaeal lactonase - Merone_2010_Bioresour.Technol_101_9204
Author(s) : Merone L , Mandrich L , Porzio E , Rossi M , Muller S , Reiter G , Worek F , Manco G
Ref : Bioresour Technol , 101 :9204 , 2010
Abstract : The thermostable Phosphotriesterase-Like Lactonase from Sulfolobus solfataricus (SsoPox) hydrolyzes lactones and, at a lower rate, neurotoxic organophosphorus compounds. The persistent demand of detoxification tools in the field of agricultural wastes and restoring of conditions after terrorist acts prompted us to exploit SsoPox as a "starter" to evolve its ancillary nerve agents hydrolytic capability. A directed evolution strategy yielded, among several variants, the single mutant W263F with k(cat) and specificity constant against paraoxon 16- and 6-fold enhanced, respectively, compared to the wild type. Furthermore, a phenomenon of enzyme activation by SDS has been observed, which allowed to increase those values 150- and 28-fold, respectively. The activity of SsoPox against the deadly nerve gas Cyclosarin has been reported for the first time and proved to be substantially unaffected for variant W263F. Finally, outperforming efficiency of W263F was demonstrated, under severe stressing conditions, with respect to the best known phosphotriesterase PTE from Brevundimonas diminuta.
ESTHER : Merone_2010_Bioresour.Technol_101_9204
PubMedSearch : Merone_2010_Bioresour.Technol_101_9204
PubMedID: 20667718

Title : Comments on Efficacy of two new asymmetric bispyridinium oximes (K-27 and K-48) in rats exposed to diisopropylfluorophosphate: comparison with pralidoxime, obidoxime, trimedoxime, methoxime, and HI 6 -
Author(s) : Theirmann H , Worek F , Eyer P
Ref : Toxicol Mech Methods , 19 :334\; author reply 335 , 2009
PubMedID: 19778225

Title : Obidoxime in acute organophosphate poisoning: 1 - clinical effectiveness - Eyer_2009_Clin.Toxicol.(Phila)_47_798
Author(s) : Eyer F , Worek F , Eyer P , Felgenhauer N , Haberkorn M , Zilker T , Thiermann H
Ref : Clinical Toxicology (Phila) , 47 :798 , 2009
Abstract : OBJECTIVE: The effects of obidoxime in the treatment of organophosphate poisoning were assessed by comparing the clinical course with its effects on laboratory parameters relevant to poisoning. In this article we report clinical findings and activity of cholinesterase in plasma and acetylcholinesterase (AChE) in red blood cells. In a linked paper we describe changes in neuromuscular transmission and atropine concentrations in the same patient cohort. METHODS: We studied 34 atropinized patients with severe parathion, oxydemeton methyl, and dimethoate self-poisoning who were treated with obidoxime in a standard protocol. We measured the AChE activity in blood and related it to clinical features of organophosphate poisoning. RESULTS: Patients poisoned with parathion responded promptly to obidoxime (250 mg bolus followed by continuous infusion at 750 mg/day up to 1 week) with improvement of neuromuscular transmission and increased AChE activity. The effects were only transient in cases with the other poisons. Death (7/34) occurred late and was mostly due to complications rather than due to ongoing cholinergic crisis. CONCLUSIONS: Obidoxime appeared safe and reactivated AChE in parathion poisoning.
ESTHER : Eyer_2009_Clin.Toxicol.(Phila)_47_798
PubMedSearch : Eyer_2009_Clin.Toxicol.(Phila)_47_798
PubMedID: 19778163

Title : Reactivation of tabun-hAChE investigated by structurally analogous oximes and mutagenesis - Artursson_2009_Toxicology_265_108
Author(s) : Artursson E , Akfur C , Hornberg A , Worek F , Ekstrom F
Ref : Toxicology , 265 :108 , 2009
Abstract : The nerve agent tabun inhibits the essential enzyme acetylcholinesterase (AChE) by a rapid phosphoramidation of the catalytic serine residue. Oximes, such as K027 and HLo-7, can reactivate tabun-inhibited human AChE (tabun-hAChE) whereas the activity of their close structural analogue HI-6 is notably low. To investigate HI-6, K027 and HLo-7, residues lining the active-site gorge of hAChE were substituted and the effects on kinetic parameters for reactivation were determined. None of the mutants (Asp74Asn, Asp74Glu, Tyr124Phe, Tyr337Ala, Tyr337Phe, Phe338Val and Tyr341Ala) were able to facilitate HI-6-mediated reactivation of tabun-hAChE. In contrast, Tyr124Phe and Tyr337Phe induce a 2-2.5-fold enhancement of the bimolecular rate constant for K027 and HLo-7. The largest effects on the dissociation constant (3.5-fold increase) and rate constant (20-fold decrease) were observed for Tyr341Ala and Asp74Asn, respectively. These findings demonstrate the importance of residues located distant from the conjugate during the reactivation of tabun-hAChE.
ESTHER : Artursson_2009_Toxicology_265_108
PubMedSearch : Artursson_2009_Toxicology_265_108
PubMedID: 19761810

Title : Extreme variability in the formation of chlorpyrifos oxon (CPO) in patients poisoned by chlorpyrifos (CPF) - Eyer_2009_Biochem.Pharmacol_78_531
Author(s) : Eyer F , Roberts DM , Buckley NA , Eddleston M , Thiermann H , Worek F , Eyer P
Ref : Biochemical Pharmacology , 78 :531 , 2009
Abstract : Chlorpyrifos (CPF) is a pesticide that causes tens of thousands of deaths per year worldwide. Chlorpyrifos oxon (CPO) is the active metabolite of CPF that inhibits acetylcholinesterase. However, this presumed metabolite has escaped detection in human samples by conventional methods (HPLC, GC-MS, LC-MS) until now. A recently developed enzyme-based assay allowed the determination of CPO in the nanomolar range and was successfully employed to detect this metabolite. CPO and CPF were analysed in consecutive plasma samples of 74 patients with intentional CPF poisoning. A wide concentration range of CPO and CPF was observed and the ratio of CPO/CPF varied considerably between individuals and over time. The ratio increased during the course of poisoning from a mean of 0.005 in the first few hours after ingestion up to an apparent steady-state mean of 0.03 between 30 and 72h. There was a hundred-fold variation in the ratio between samples and the interquartile range (between individuals) indicated over half the samples had a 5-fold or greater variation from the mean. The ratio was independent of the CPF concentration and the pralidoxime regimen. CPO was present in sufficient quantities to explain any observed acetylcholinesterase inhibitory activity. The effectiveness of pralidoxime in reactivating the inhibited acetylcholinesterase is strongly dependent on the CPO concentration. Differences in clinical outcomes and the response to antidotes in patients with acute poisoning may occur due to inter-individual variability in metabolism.
ESTHER : Eyer_2009_Biochem.Pharmacol_78_531
PubMedSearch : Eyer_2009_Biochem.Pharmacol_78_531
PubMedID: 19433070

Title : Pralidoxime in acute organophosphorus insecticide poisoning--a randomised controlled trial - Eddleston_2009_PLoS.Med_6_e1000104
Author(s) : Eddleston M , Eyer P , Worek F , Juszczak E , Alder N , Mohamed F , Senarathna L , Hittarage A , Azher S , Jeganathan K , Jayamanne S , von Meyer L , Dawson AH , Sheriff MH , Buckley NA
Ref : PLoS Med , 6 :e1000104 , 2009
Abstract : BACKGROUND: Poisoning with organophosphorus (OP) insecticides is a major global public health problem, causing an estimated 200,000 deaths each year. Although the World Health Organization recommends use of pralidoxime, this antidote's effectiveness remains unclear. We aimed to determine whether the addition of pralidoxime chloride to atropine and supportive care offers benefit. METHODS AND FINDINGS: We performed a double-blind randomised placebo-controlled trial of pralidoxime chloride (2 g loading dose over 20 min, followed by a constant infusion of 0.5 g/h for up to 7 d) versus saline in patients with organophosphorus insecticide self-poisoning. Mortality was the primary outcome; secondary outcomes included intubation, duration of intubation, and time to death. We measured baseline markers of exposure and pharmacodynamic markers of response to aid interpretation of clinical outcomes. Two hundred thirty-five patients were randomised to receive pralidoxime (121) or saline placebo (114). Pralidoxime produced substantial and moderate red cell acetylcholinesterase reactivation in patients poisoned by diethyl and dimethyl compounds, respectively. Mortality was nonsignificantly higher in patients receiving pralidoxime: 30/121 (24.8%) receiving pralidoxime died, compared with 18/114 (15.8%) receiving placebo (adjusted hazard ratio [HR] 1.69, 95% confidence interval [CI] 0.88-3.26, p = 0.12). Incorporating the baseline amount of acetylcholinesterase already aged and plasma OP concentration into the analysis increased the HR for patients receiving pralidoxime compared to placebo, further decreasing the likelihood that pralidoxime is beneficial. The need for intubation was similar in both groups (pralidoxime 26/121 [21.5%], placebo 24/114 [21.1%], adjusted HR 1.27 [95% CI 0.71-2.29]). To reduce confounding due to ingestion of different insecticides, we further analysed patients with confirmed chlorpyrifos or dimethoate poisoning alone, finding no evidence of benefit.
CONCLUSIONS: Despite clear reactivation of red cell acetylcholinesterase in diethyl organophosphorus pesticide poisoned patients, we found no evidence that this regimen improves survival or reduces need for intubation in patients with organophosphorus insecticide poisoning. The reason for this failure to benefit patients was not apparent. Further studies of different dose regimens or different oximes are required.
ESTHER : Eddleston_2009_PLoS.Med_6_e1000104
PubMedSearch : Eddleston_2009_PLoS.Med_6_e1000104
PubMedID: 19564902

Title : Obidoxime in acute organophosphate poisoning: 2 - PK\/PD relationships - Thiermann_2009_Clin.Toxicol.(Phila)_47_807
Author(s) : Thiermann H , Worek F , Eyer P , Eyer F , Felgenhauer N , Zilker T
Ref : Clinical Toxicology (Phila) , 47 :807 , 2009
Abstract : OBJECTIVE: The effects of obidoxime in the treatment of organophosphate poisoning were assessed by biochemical and biological effect monitoring. In this article we report effects on neuromuscular function, oxime and atropine concentration, and relate them to acetylcholinesterase (AChE) activity. METHODS: We measured the activity of cholinesterase in plasma and AChE in red blood cells (RBC) and related these data with neuromuscular transmission analysis (ulnar nerve stimulation). Concomitantly, poison and oxon along with plasma obidoxime and atropine levels were measured at regular intervals. RESULTS: We found a close correlation between RBC-AChE activity and neuromuscular transmission and a reciprocal correlation between both the atropine maintenance dose and/or its plasma concentration. The steady state of RBC-AChE activity of reactivation and re-inhibition followed the course predicted by laboratory-determined reaction constants. CONCLUSIONS: Intense monitoring of organophosphate-poisoned patients allowed assessment of why a given obidoxime concentration was, or was not, able to counteract the re-inhibition of the RBC-AChE. RBC-AChE activity mirrors the function of n-receptor- and m-receptor-mediated cholinergic signaling as measured by neuromuscular transmission and atropine requirements.
ESTHER : Thiermann_2009_Clin.Toxicol.(Phila)_47_807
PubMedSearch : Thiermann_2009_Clin.Toxicol.(Phila)_47_807
PubMedID: 19778190

Title : Detoxification of nerve agents by a substituted beta-cyclodextrin: application of a modified biological assay - Wille_2009_Toxicology_265_96
Author(s) : Wille T , Tenberken O , Reiter G , Muller S , Le Provost R , Lafont O , Estour F , Thiermann H , Worek F
Ref : Toxicology , 265 :96 , 2009
Abstract : Chemical warfare agents (nerve agents) are still available and present a real threat to the population. Numerous in vitro and in vivo studies showed that various nerve agents, e.g. tabun and cyclosarin, are resistant towards standard therapy with atropine and oxime. Based on these facts we applied a modified biological assay for the easy, semi-quantitative testing of the detoxifying properties of the beta-cyclodextrin derivative CD-IBA. Cyclosarin, sarin, tabun and VX were incubated with CD-IBA for 1-50 min at 37 degrees C, then an aliquot was added to erythrocyte acetylcholinesterase (AChE) and the percentage of AChE inhibition was determined. The validity of the assay was confirmed by concomitant quantification of tabun by GC-MS. Different concentrations of cyclosarin were detoxified by CD-IBA in a concentration-dependent velocity. The ability to detoxify various nerve agents decreased in the order cyclosarin>sarin>tabun>>VX. Hereby, no detoxification of VX could be detected. Sarin was detoxified in a biphasic reaction with a fast reduction of inhibitory potential in the first phase and a slower detoxification in the second phase. CD-IBA detoxified tabun in a one phase decay and, compared to cyclosarin and sarin, a longer half-life was determined with tabun. The modified biological assay is appropriate for the initial semi-quantitative screening of candidate compounds for the detoxification of nerve agents. The beta-cyclodextrin derivative CD-IBA demonstrated its ability to detoxify different nerve agents.
ESTHER : Wille_2009_Toxicology_265_96
PubMedSearch : Wille_2009_Toxicology_265_96
PubMedID: 19800384

Title : The NADPH oxidase inhibitor diphenyleneiodonium is also a potent inhibitor of cholinesterases and the internal Ca(2+) pump - Tazzeo_2009_Br.J.Pharmacol_158_790
Author(s) : Tazzeo T , Worek F , Janssen L
Ref : British Journal of Pharmacology , 158 :790 , 2009
Abstract : BACKGROUND AND PURPOSE: Diphenyleneiodonium (DPI) is often used as an NADPH oxidase inhibitor, but is increasingly being found to have unrelated side effects. We investigated its effects on smooth muscle contractions and the related mechanisms. EXPERIMENTAL APPROACH: We studied isometric contractions in smooth muscle strips from bovine trachea. Cholinesterase activity was measured using a spectrophotometric assay; internal Ca(2+) pump activity was assessed by Ca(2+) uptake into smooth muscle microsomes. KEY RESULTS: Contractions to acetylcholine were markedly enhanced by DPI (10(-4) M), whereas those to carbachol (CCh) were not, suggesting a possible inhibition of cholinesterase. DPI markedly suppressed contractions evoked by CCh, KCl and 5-HT, and also unmasked phasic activity in otherwise sustained responses. Direct biochemical assays confirmed that DPI was a potent inhibitor of acetylcholinesterase and butyrylcholinesterase (IC(50) approximately 8 x 10(-6) M and 6 x 10(-7) M, respectively), following a readily reversible, mixed non-competitive type of inhibition. The inhibitory effects of DPI on CCh contractions were not mimicked by another NADPH oxidase inhibitor (apocynin), nor the Src inhibitors PP1 or PP2, ruling out an action through the NADPH oxidase signalling pathway. Several features of the DPI-mediated suppression of agonist-evoked responses (i.e. suppression of peak magnitudes and unmasking of phasic activity) are similar to those of cyclopiazonic acid, an inhibitor of the internal Ca(2+) pump. Direct measurement of microsomal Ca(2+) uptake revealed that DPI modestly inhibits the internal Ca(2+) pump. CONCLUSIONS AND IMPLICATIONS: DPI inhibits cholinesterase activity and the internal Ca(2+) pump in tracheal smooth muscle.
ESTHER : Tazzeo_2009_Br.J.Pharmacol_158_790
PubMedSearch : Tazzeo_2009_Br.J.Pharmacol_158_790
PubMedID: 19788497

Title : Monitoring of neuromuscular transmission in organophosphate pesticide-poisoned patients - Thiermann_2009_Toxicol.Lett_191_297
Author(s) : Thiermann H , Zilker T , Eyer F , Felgenhauer N , Eyer P , Worek F
Ref : Toxicol Lett , 191 :297 , 2009
Abstract : Thirty-four adult patients with severe organophosphorus compounds (OP) poisoning requiring artificial ventilation were enrolled in a clinical study and received atropine and obidoxime (250 mg i.v., followed by 750 mg/24 h) as antidotal treatment. Here, we re-analyzed the cholinesterase status (red blood cell acetylcholinesterase (RBC-AChE) activity, reactivatability of RBC-AChE, and plasma butyrylcholinesterase (Pl-BChE) activity) in relation to the neuromuscular transmission (NMT) data. When RBC-AChE activity ranged between 100% and 30% NMT was unimpaired after tetanic stimulation with frequencies up to 50 Hz. A further decrease in RBC-AChE activity was accompanied by a marked disturbance of NMT, being strongly impaired at AChE activities <5% of normal. Higher stimulation frequencies (>30 Hz) facilitated the discrimination of the types of impairment. The neuromuscular transmission was the best quantified by using the ratio of the ninth to the first amplitude, while the standard method was less discriminative. At RBC-AChE levels higher than 40% of normal weaning from the ventilator may be considered. Completely aged RBC-AChE as indicated by loss of reactivatability loses its guidance function. Then, steadily increasing Pl-BChE activity suggests lack of circulating poison. One-week later, neuromuscular transmission may be largely normal and patients could be weaned from the respirator if other complications are not withstanding.
ESTHER : Thiermann_2009_Toxicol.Lett_191_297
PubMedSearch : Thiermann_2009_Toxicol.Lett_191_297
PubMedID: 19793545

Title : Interaction of pentylsarin analogues with human acetylcholinesterase: a kinetic study - Worek_2009_Toxicol.Lett_187_119
Author(s) : Worek F , Herkert NM , Koller M , Aurbek N , Thiermann H
Ref : Toxicol Lett , 187 :119 , 2009
Abstract : Previous kinetic studies investigating the interactions between human acetylcholinesterase (AChE), structurally different organophosphorus compounds (OP) and oximes did not reveal a conclusive structure-activity relationship of the different reactions. The only exception was for a homologous series of methylphosphonofluoridates bearing C1-C4 O-n- or O-i-alkyl residues. Hence, it was tempting to investigate the kinetic interactions between different pentylsarin analogues, human AChE and two oximes, obidoxime and HI 6, in order to increase the understanding of structure-activity relationship between highly toxic OP and human AChE. The rate constants for the inhibition of human erythrocyte AChE by four pentylsarin compounds (k(i)), for the spontaneous dealkylation (aging, k(a)) and reactivation (k(s)) of inhibited AChE as well as for the oxime-induced reactivation of inhibited AChE by obidoxime and HI 6 reflected by the dissociation constant (K(D)) and the reactivity constant (k(r)) were determined. All pentylsarin analogues had a high inhibitory potency towards AChE. Inhibited AChE was subject to spontaneous reactivation which outweighed aging substantially. Pentylsarin-inhibited AChE could be reactivated by oximes, HI 6 being more potent than obidoxime. The determination of inhibition, reactivation and aging kinetics of pentylsarin analogues with human AChE extends the database on interactions between AChE and methylphosphonofluoridate homologues with C1-C4 n- and i-alkyl residues demonstrating a structure-activity relationship depending on the chain length with certain differences regarding inhibition and post-inhibitory reactions. Unfortunately, no structure-activity relationship could be observed for the oxime-induced reactivation of inhibited AChE. In view of previous results with numerous structurally different organophosphates, organophosphonates and phosphoramidates it has to be concluded that up to now kinetic studies did not provide decisive information for the development of more effective oxime-based reactivators.
ESTHER : Worek_2009_Toxicol.Lett_187_119
PubMedSearch : Worek_2009_Toxicol.Lett_187_119
PubMedID: 19429253

Title : Poisoning with the S-Alkyl organophosphorus insecticides profenofos and prothiofos - Eddleston_2009_QJM_102_785
Author(s) : Eddleston M , Worek F , Eyer P , Thiermann H , von Meyer L , Jeganathan K , Sheriff MH , Dawson AH , Buckley NA
Ref : Qjm , 102 :785 , 2009
Abstract : BACKGROUND: Many organophosphorus (OP) insecticides have either two O-methyl or two O-ethyl groups attached to the phosphorus atom. This chemical structure affects their responsiveness to oxime-induced acetylcholinesterase (AChE) reactivation after poisoning. However, several OP insecticides are atypical and do not have these structures. AIM: We aimed to describe the clinical course and responsiveness to therapy of people poisoned with two S-alkyl OP insecticides-profenofos and prothiofos. DESIGN: We set up a prospective cohort of patients with acute profenofos or prothiofos self-poisoning admitted to acute medical wards in two Sri Lankan district hospitals. Clinical observation was carried out throughout their inpatient stay; blood samples were taken in a subgroup for assay of cholinesterases and insecticide. RESULTS: Ninety-five patients poisoned with profenofos and 12 with prothiofos were recruited over 5 years. Median time to admission was 4 (IQR 3-7) h. Eleven patients poisoned with profenofos died (11/95; 11.6%, 95% CI 5.9-20); one prothiofos patient died (1/12; 8.3%, 95% CI 0.2-38). Thirteen patients poisoned with profenofos required intubation for respiratory failure (13/95; 13.7%, 95% CI 7.5-22); two prothiofos-poisoned patients required intubation. Both intubations and death occurred late compared with other OP insecticides. Prolonged ventilation was needed in those who survived-a median of 310 (IQR 154-349) h. Unexpectedly, red cell AChE activity on admission did not correlate with clinical severity-all patients had severe AChE inhibition (about 1% of normal) but most had only mild cholinergic features, were conscious, and did not require ventilatory support. CONCLUSION: Compared with other commonly used OP insecticides, profenofos and prothiofos are of moderately severe toxicity, causing relatively delayed respiratory failure and death. There was no apparent response to oxime therapy. The lack of correlation between red cell AChE activity and clinical features suggests that this parameter may not always be a useful marker of synaptic AChE activity and severity after OP pesticide poisoning.
ESTHER : Eddleston_2009_QJM_102_785
PubMedSearch : Eddleston_2009_QJM_102_785
PubMedID: 19737786

Title : Comparison of the oxime-induced reactivation of rhesus monkey, swine and guinea pig erythrocyte acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity - Herkert_2009_Toxicology_258_79
Author(s) : Herkert NM , Lallement G , Clarencon D , Thiermann H , Worek F
Ref : Toxicology , 258 :79 , 2009
Abstract : Recently, a dynamically working in vitro model with real-time determination of membrane-bound human acetylcholinesterase (AChE) activity was shown to be a versatile model to investigate oxime-induced reactivation kinetics of organophosphate- (OP) inhibited enzyme. In this assay, AChE was immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. Subsequently, AChE activity was continuously analyzed in a flow-through detector. Now, it was an intriguing question whether this model could be used with erythrocyte AChE from other species in order to investigate kinetic interactions in the absence of annoying side reactions. Rhesus monkey, swine and guinea pig erythrocytes were a stable and highly reproducible enzyme source. Then, the model was applied to the reactivation of sarin- and paraoxon-inhibited AChE by obidoxime or HI 6 and it could be shown that the derived reactivation rate constants were in good agreement to previous results obtained from experiments with a static model. Hence, this dynamic model offers the possibility to investigate highly reproducible interactions between AChE, OP and oximes with human and animal AChE.
ESTHER : Herkert_2009_Toxicology_258_79
PubMedSearch : Herkert_2009_Toxicology_258_79
PubMedID: 19428926

Title : Suitability of human butyrylcholinesterase as therapeutic marker and pseudo catalytic scavenger in organophosphate poisoning: a kinetic analysis - Aurbek_2009_Toxicology_259_133
Author(s) : Aurbek N , Thiermann H , Eyer F , Eyer P , Worek F
Ref : Toxicology , 259 :133 , 2009
Abstract : The widespread use of organophosphorus compounds (OPs) as pesticides and the frequent misuse of OP nerve agents in military conflicts or terrorist attacks emphasize the high clinical relevance of OP poisoning. The toxic symptomatology is caused by inhibition of acetylcholinesterase (AChE). A mainstay of standard antidotal treatment is atropine for antagonizing effects mediated by over stimulation of muscarinic ACh-receptors and oxime to reactivate OP-inhibited AChE. For therapeutic monitoring of oxime treatment in OP poisoning, measurement of erythrocyte AChE is suitable because erythrocyte AChE is an easily accessible surrogate for synaptic AChE. However, measurement of erythrocyte AChE is not standard practice. In contrast, determination of plasma butyrylcholinesterase (BChE) activity is in routine use for monitoring the benefit of oxime therapy. As oxime efficacy is limited with certain OPs (e.g. dimethoate, tabun, soman) alternative therapeutic approaches, e.g. the application of scavengers (BChE) which may sequester OPs before they reach their physiological target, are under investigation. To assess the eligibility of BChE as laboratory parameter and (pseudo catalytic or stoichiometric) scavenger in OP poisoning we initiated an in vitro study under standardized experimental conditions with the objective of determination of kinetic constants for inhibition, reactivation and aging of plasma BChE. It could be shown that, due to limited efficacy of obidoxime, pralidoxime, HI 6 and MMB4 with OP-inhibited BChE, plasma BChE activity is an inappropriate parameter for therapeutic monitoring of oxime treatment in OP poisoning. Furthermore, oxime-induced reactivation is too slow to accomplish a pseudo catalytic function, so that administered BChE may be merely effective as a stoichiometric scavenger.
ESTHER : Aurbek_2009_Toxicology_259_133
PubMedSearch : Aurbek_2009_Toxicology_259_133
PubMedID: 19428953

Title : Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun. - Carletti_2009_Biochem.J_421_97
Author(s) : Carletti E , Aurbek N , Gillon E , Loiodice M , Nicolet Y , Fontecilla-Camps JC , Masson P , Thiermann H , Nachon F , Worek F
Ref : Biochemical Journal , 421 :97 , 2009
Abstract : hBChE [human BChE (butyrylcholinesterase)] naturally scavenges OPs (organophosphates). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated ChEs (cholinesterases) can undergo a spontaneous time-dependent process called 'aging' during which the conjugate is dealkylated, leading to creation of an enzyme that cannot be reactivated. hBChE inhibited by phosphoramidates such as tabun displays a peculiar resistance to oxime-mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates by determining the kinetics of inhibition, reactivation (obidoxime {1,1'-(oxybis-methylene) bis[4-(hydroxyimino) methyl] pyridinium dichloride}, TMB-4(Trimedoxime) [1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide], HL 7 {1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate)}, HI-6 {1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate} and aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to a previous observation made on tabun-ChE conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that highlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides the structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment, or to design BChE-based catalytic bioscavengers.
ESTHER : Carletti_2009_Biochem.J_421_97
PubMedSearch : Carletti_2009_Biochem.J_421_97
PubMedID: 19368529
Gene_locus related to this paper: human-BCHE

Title : Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity - Eckert_2008_Biochem.Pharmacol_75_698
Author(s) : Eckert S , Eyer P , Herkert N , Bumm R , Weber G , Thiermann H , Worek F
Ref : Biochemical Pharmacology , 75 :698 , 2008
Abstract : The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex-GS, 0.22 microm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax and Potter-Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 microM) or HI 6 (30 microM) of sarin-inhibited human muscle acetylcholinesterase (0.142+/-0.004 min(-1) and 0.166+/-0.008 min(-1), respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker.
ESTHER : Eckert_2008_Biochem.Pharmacol_75_698
PubMedSearch : Eckert_2008_Biochem.Pharmacol_75_698
PubMedID: 17977518

Title : Effects of oximes on rate of decarbamylation of human red blood cell AChE measured with two different methods - Eckert_2008_Biochem.Pharmacol_75_1561
Author(s) : Eckert S , Eyer P , Melzer M , Thiermann H , Worek F
Ref : Biochemical Pharmacology , 75 :1561 , 2008
Abstract : Treatment regimen of poisonings by organophosphorus (OP) compounds usually includes oxime therapy. The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase (AChE), when the enzyme species is considered as irreversibly inhibited and resistant towards reactivation by oximes. Hence, oxime treatment probably comes too late in realistic scenarios. As an alternative, protecting part of the enzyme by reversible inhibition prior to soman exposure has been proposed. One means of protecting against soman poisoning is the prophylactic use of certain reversible inhibitors (carbamates) of AChE. The question whether there is a possibility of an interaction between pre-treating carbamates and oximes at AChE arises. Therefore we studied the effects of the oximes obidoxime, HI 6 and MMB-4 on the rate of decarbamylation for physostigmine- and pyridostigmine-inhibited human erythrocyte AChE both in a dynamically working in vitro model and a static cuvette system. Our results show that HI 6 increased the rate of decarbamylation for both physostigmine- and pyridostigmine-inhibited enzyme in both systems, the observed effect by HI 6 increasing with higher doses. Obidoxime had a slightly accelerating effect on the pyridostigmine-inhibited enzyme. MMB-4 applied to pyridostigmine-inhibited AChE in the static system only showed no difference to the experiments made in absence of oxime. No oxime showed a tendency to retard the rate of decarbamylation.
ESTHER : Eckert_2008_Biochem.Pharmacol_75_1561
PubMedSearch : Eckert_2008_Biochem.Pharmacol_75_1561
PubMedID: 18281016

Title : Predicting outcome using butyrylcholinesterase activity in organophosphorus pesticide self-poisoning - Eddleston_2008_QJM_101_467
Author(s) : Eddleston M , Eyer P , Worek F , Sheriff MH , Buckley NA
Ref : Qjm , 101 :467 , 2008
Abstract : BACKGROUND: The usefulness of a low butyrylcholinesterase (BuChE) activity on admission for predicting severity in acute organophosphorus (OP) insecticide poisoning has long been debated. Previous studies have been confounded by the inclusion of multiple insecticides with differing inhibitory kinetics. AIM: We aimed to assess the usefulness of admission BuChE activity, together with plasma OP concentration, for predicting death with two specific organophosphorus insecticides. DESIGN: A prospective cohort of self-poisoned patients. METHODS: We prospectively studied 91 and 208 patients with proven dimethoate or chlorpyrifos self-poisoning treated using a standard protocol. Plasma butyrylcholinesterase activity and OP concentration were measured on admission and clinical outcomes recorded. RESULTS: The usefulness of a plasma BuChE activity <600 mU/ml on admission varied markedly--while highly sensitive in chlorpyrifos poisoning (sensitivity 11/11 deaths; 100%, 95% CI 71.5-100), its specificity was only 17.7% (12.6-23.7). In contrast, while poorly sensitive for deaths in dimethoate poisoning [12/25 patients; 48%, (27.9-68.7)] it was reasonably specific [86.4% (75.7-93.6)]. A high OP concentration on admission was associated with worse outcome; however, a clear threshold concentration was only present for dimethoate poisoning. CONCLUSION: Plasma BuChE activity on admission can provide useful information; however, it must be interpreted carefully. It can only be used to predict death when the insecticide ingested is known and its sensitivity and specificity for that insecticide has been studied. Plasma concentration of some OP insecticides predicts outcome. The development of rapid bedside tests for OP detection may aid early assessment of severity.
ESTHER : Eddleston_2008_QJM_101_467
PubMedSearch : Eddleston_2008_QJM_101_467
PubMedID: 18375477

Title : Chromatographic resolution, characterisation and quantification of VX enantiomers in hemolysed swine blood samples - Reiter_2008_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_873_86
Author(s) : Reiter G , Mikler J , Hill I , Weatherby K , Thiermann H , Worek F
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 873 :86 , 2008
Abstract : The present study was initiated to develop a sensitive and highly selective method for the analysis of the enantiomers of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) in blood samples for toxicokinetic and therapeutic research. To achieve this goal, analytical and semi-preparative enantioseparation of VX were carried out with gas and liquid chromatography. The GC chiral stationary phase was HYDRODEX-beta-TBDAc (beta cyclodextrin), on which VX was baseline-resolved. On the chiral HPLC phase CHIRALCEL OD-H the enantiomers of VX were isolated with enantiomeric excess >99.99%. They were characterised by specific optical rotation (+/-25.8 deg ml dm(-1)g(-1) at 20 degrees C and 589 nm) and by determination of cholinesterase inhibition rate constants. For the quantitative chiral detection of VX the enantioresolution was realized on the HPLC chiral phase CHIRAL AGP. A specific procedure was developed to isolate VX from swine blood samples thereby stabilising its enantiomers. The limit of detection was 200 fg per enantiomer on column. The absolute recovery of the overall sample preparation procedure was 75%. After an intravenous and percutaneous administration of a supralethal dose of VX in anesthetised swine (+)-VX and (-)-VX could be quantified up to 720 min.
ESTHER : Reiter_2008_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_873_86
PubMedSearch : Reiter_2008_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_873_86
PubMedID: 18718824

Title : Are we using the right dose? - a tale of mole and gram -
Author(s) : Eyer P , Eddleston M , Thiermann H , Worek F , Buckley NA
Ref : British Journal of Clinical Pharmacology , 66 :451 , 2008
PubMedID: 18662291

Title : Reactions of isodimethoate with human red cell acetylcholinesterase - Eyer_2008_Biochem.Pharmacol_75_2045
Author(s) : Eyer P , Radtke M , Worek F
Ref : Biochemical Pharmacology , 75 :2045 , 2008
Abstract : Isodimethoate is a thermal decomposition product that is present in usual pesticide formulations of dimethoate. Owing to its PO structure the compound is a direct anticholinesterase agent whose properties, to the best of our knowledge, are presented here for the first time. Isodimethoate shows an inhibition rate constant towards human red blood cell acetylcholinesterase (AChE) of 2.3x10(3) M(-1) min(-1) (pH 7.4, 37 degrees C), indicating a somewhat higher potency than found with omethoate, the CYP450-mediated active metabolite of pure dimethoate. Isodimethoate-inhibited AChE shows fast spontaneous reactivation and aging kinetics (half-life 2.3 and 25 min, respectively). The inhibited, non-aged enzyme is readily reactivated by obidoxime (k(r)=9 min(-1); K(D)=0.1 mM) but hardly by pralidoxime at therapeutic concentrations. Interestingly, isodimethoate hydrolyzes readily in buffered solutions at pH 7.4 and 37 degrees C with liberation of methylmercaptan (half-life 16 min). Liberation of N-(methyl)mercaptoacetamide, the expected leaving group, was not observed. These properties make isodimethoate a hit-and-run agent that renders part of AChE non-reactivatable within a short period of time. The clinical consequences of exposure to or intentional ingestion of isodimethoate-containing dimethoate formulations are a partly untractable AChE shortly after incorporation. In fact, aging of AChE in dimethoate-poisoned patients on admission was much more advanced than expected from the reaction with omethoate. Manufacturers, researching scientists and clinical toxicologists should be aware of this problem.
ESTHER : Eyer_2008_Biochem.Pharmacol_75_2045
PubMedSearch : Eyer_2008_Biochem.Pharmacol_75_2045
PubMedID: 18384757

Title : Identical kinetics of human erythrocyte and muscle acetylcholinesterase with respect to carbamate pre-treatment, residual activity upon soman challenge and spontaneous reactivation after withdrawal of the inhibitors - Herkert_2008_Toxicology_246_188
Author(s) : Herkert NM , Eckert S , Eyer P , Bumm R , Weber G , Thiermann H , Worek F
Ref : Toxicology , 246 :188 , 2008
Abstract : The efficacy of oxime treatment in soman poisoning is limited due to rapid aging of inhibited acetylcholinesterase (AChE). Pre-treatment with carbamates was shown to improve antidotal treatment substantially. Recently, by using a dynamically working in vitro model with real-time determination of membrane-bound AChE activity, we were able to demonstrate that pre-inhibition of human erythrocyte AChE with pyridostigmine or physostigmine resulted in a markedly higher residual AChE activity after inhibition by soman or paraoxon than in the absence of reversible inhibitors. The purpose of the present study was to compare the effect of carbamate pre-treatment and soman challenge with human erythrocyte and muscle homogenate AChE. Both enzyme sources were immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. AChE activity was continuously analyzed in a flow-through detector. Pre-inhibition of AChE with pyridostigmine or physostigmine resulted in a concentration-dependent increase in carbamylation, residual activity after soman inhibition and fraction of decarbamylation AChE after discontinuation of the inhibitors without differences between human erythrocyte and muscle AChE. This data support the view that human erythrocyte AChE is an adequate surrogate marker for synaptic AChE in OP poisoning.
ESTHER : Herkert_2008_Toxicology_246_188
PubMedSearch : Herkert_2008_Toxicology_246_188
PubMedID: 18304715

Title : A structure-activity analysis of the variation in oxime efficacy against nerve agents - Maxwell_2008_Toxicol.Appl.Pharmacol_231_157
Author(s) : Maxwell DM , Koplovitz I , Worek F , Sweeney RE
Ref : Toxicol Appl Pharmacol , 231 :157 , 2008
Abstract : A structure-activity analysis was used to evaluate the variation in oxime efficacy of 2-PAM, obidoxime, HI-6 and ICD585 against nerve agents. In vivo oxime protection and in vitro oxime reactivation were used as indicators of oxime efficacy against VX, sarin, VR and cyclosarin. Analysis of in vivo oxime protection was conducted with oxime protective ratios (PR) from guinea pigs receiving oxime and atropine therapy after sc administration of nerve agent. Analysis of in vitro reactivation was conducted with second-order rate contants (k(r2)) for oxime reactivation of agent-inhibited acetylcholinesterase (AChE) from guinea pig erythrocytes. In vivo oxime PR and in vitro k(r2) decreased as the volume of the alkylmethylphosphonate moiety of nerve agents increased from VX to cyclosarin. This effect was greater with 2-PAM and obidoxime (>14-fold decrease in PR) than with HI-6 and ICD585 (<3.7-fold decrease in PR). The decrease in oxime PR and k(r2) as the volume of the agent moiety conjugated to AChE increased was consistent with a steric hindrance mechanism. Linear regression of log (PR-1) against log (k(r2)[oxime dose]) produced two offset parallel regression lines that delineated a significant difference between the coupling of oxime reactivation and oxime protection for HI-6 and ICD585 compared to 2-PAM and obidoxime. HI-6 and ICD585 appeared to be 6.8-fold more effective than 2-PAM and obidoxime at coupling oxime reactivation to oxime protection, which suggested that the isonicotinamide group that is common to both of these oximes, but absent from 2-PAM and obidoxime, is important for oxime efficacy.
ESTHER : Maxwell_2008_Toxicol.Appl.Pharmacol_231_157
PubMedSearch : Maxwell_2008_Toxicol.Appl.Pharmacol_231_157
PubMedID: 18508103

Title : Inhibition, reactivation and aging kinetics of highly toxic organophosphorus compounds: pig versus minipig acetylcholinesterase - Worek_2008_Toxicology_244_35
Author(s) : Worek F , Aurbek N , Wetherell J , Pearce P , Mann T , Thiermann H
Ref : Toxicology , 244 :35 , 2008
Abstract : Organophosphorus compound-based (OP) chemical warfare agents (nerve agents) represent a continuing threat to military forces and the civilian population. OPs act primarily by inhibiting acetylcholinesterase (AChE), the standard treatment for which includes AChE reactivators (oximes) in combination with antimuscarinic drugs. In the last decades, the efficacy of oximes has been investigated mostly in small animal models. In order to increase the predictive value of animal studies it is desirable to measure numerous physiological and biochemical parameters. This is difficult in small animals. Large animal models fulfil these requirements and swine are increasingly being used in toxicology studies. Swine breeds generally show considerable variability in different characteristics which may be minimised by the use of specially bred minipigs which have a known genetic background and health status. A comparative study was, therefore, initiated to investigate the kinetic properties of the White Landrace pig and Gottingen minipig AChE in respect of inhibition by the pesticide paraoxon; the nerve agents cyclosarin, VX and VR; the reactivation of inhibited AChE by oximes (obidoxime, pralidoxime and HI 6); and the aging and spontaneous reactivation of inhibited AChE. The determination of the respective kinetic constants found similarities between pig and minipig AChE which showed marked differences in comparison with human AChE values. This has to be considered in designing meaningful models for the investigation of oxime efficacy in pig or minipig experiments. The generated data indicate comparable kinetic properties of pig and minipig AChE and may provide a kinetic basis for extrapolation of data from pig studies to humans.
ESTHER : Worek_2008_Toxicology_244_35
PubMedSearch : Worek_2008_Toxicology_244_35
PubMedID: 18054823

Title : LC-MS-based procedures for monitoring of toxic organophosphorus compounds and verification of pesticide and nerve agent poisoning - John_2008_Anal.Bioanal.Chem_391_97
Author(s) : John H , Worek F , Thiermann H
Ref : Anal Bioanal Chem , 391 :97 , 2008
Abstract : Organophosphorus compounds (OPCs) are used worldwide as, e.g., flame retardants, plasticizers, and pesticides and remaining stockpiles of OPC nerve agents are present in military arsenals. These OPCs exhibit acute and potential chronic toxicity to man, the environment, and biota thus emphasizing the need for efficient analytical procedures to monitor potential risk to health. Therefore, this review discusses LC-MS-based procedures for OPC detection, addressing sample preparation, separation, ionization, and detection in comprehensive detail. For sample preparation conventional liquid-liquid extraction (LLE) and diverse solid-phase extraction (SPE) procedures are still used most frequently. Nevertheless, during the last three years a number of sophisticated novel methods have been introduced. Solid-phase microextraction (SPME), stir-bar-sorptive extraction (SBSE), membrane-assisted solvent extraction (MASE), and specifically designed molecularly imprinted polymers (MIP) exhibit high potential for frequent use in the future. Additional emphasis in this review is dedicated to the quite young history and current progress in ionization and MS detection of OPCs. The number of relevant published LC-MS reports has tripled in the last five years. This is especially due to the proliferating use of electrospray ionization (ESI), nowadays an indispensable and reliable tool for LC-MS coupling. LC-MS is becoming an appropriate complementary or replacement method for the more traditional GC-MS methods, and not only for non-volatile, hydrophilic, and ionic OPCs. The last section of this review covers recent approaches for verification of OPC poisoning. LC-MS-MS detection of phosphylated peptides generated from inhibited circulating serum butyrylcholinesterase (BChE) by valuable proteomics techniques enables proof of intoxication on the molecular level. Therefore, this review gives a comprehensive overview on the status quo of LC-MS-based OPC analysis in respect of both technical progress and relevant applications.
ESTHER : John_2008_Anal.Bioanal.Chem_391_97
PubMedSearch : John_2008_Anal.Bioanal.Chem_391_97
PubMedID: 18330546

Title : Inhibitory potency against human acetylcholinesterase and enzymatic hydrolysis of fluorogenic nerve agent mimics by human paraoxonase 1 and squid diisopropyl fluorophosphatase - Blum_2008_Biochemistry_47_5216
Author(s) : Blum MM , Timperley CM , Williams GR , Thiermann H , Worek F
Ref : Biochemistry , 47 :5216 , 2008
Abstract : A wide range of toxic organophosphorus pesticides and nerve agents is effectively hydrolyzed by the structurally related phosphotriesterase enzymes paraoxonase (PON1) from human plasma and diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris. Both enzymes have potential use as medical countermeasures and decontaminants. Enhanced enzymatic activity, stereochemical preference, and substrate variety are still the focus of ongoing research. Derivatives of pesticides and nerve agents bearing a fluorogenic leaving group were introduced for high-throughput screening of mutant libraries recently. We report the inhibitory potency of fluorogenic organophosphorus compounds with three different leaving groups [3-chloro-7-oxy-4-methylcoumarin, 7-oxy-4-methylcoumarin, 7-oxy-4-(trifluoromethyl)coumarin] toward human acetylcholinesterase (AChE) and report kinetic data for the enzymatic hydrolysis of these compounds by PON1 and DFPase. This is the first report of the hydrolysis of a substrate bearing a P-O bond to the leaving group by DFPase (its activity was believed to be restricted to cleavage of P-F and P-CN bonds). The reactivity of the enzymes toward the substrates is explained on the basis of structural reasoning and computational docking studies. We demonstrate that fluorogenic organophosphorus compounds can serve as valuable models for enzyme screening but also show that differences and limitations exist and have to be taken into account. The importance of using protein from human sources to obtain toxicological data for potential in vivo use is highlighted.
ESTHER : Blum_2008_Biochemistry_47_5216
PubMedSearch : Blum_2008_Biochemistry_47_5216
PubMedID: 18396898

Title : Reversible inhibition of acetylcholinesterase by carbamates or huperzine A increases residual activity of the enzyme upon soman challenge - Eckert_2007_Toxicology_233_180
Author(s) : Eckert S , Eyer P , Worek F
Ref : Toxicology , 233 :180 , 2007
Abstract : The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase, which makes the enzyme essentially intractable. Hence, oxime treatment probably comes too late in realistic scenarios. As an alternative, protecting part of the enzyme by reversible inhibition prior to soman exposure has been proposed. This strategy was successfully tested in animal experiments, but its efficacy still awaits complete understanding. In particular, it is unclear whether survival is improved by a higher residual activity of acetylcholinesterase during the acute phase, when the reversible and irreversible inhibitors are present together. In previous experiments with carbamate pre-treatment and paraoxon challenge we noticed an increased residual activity of erythrocyte acetylcholinesterase compared to non-pre-treatment. This result was encouraging to also test for comparable effects when using soman. Immobilized human erythrocytes were continuously perfused for real-time measurement of acetylcholinesterase activity by a modified Ellman method using 0.45mM acetylthiocholine. After having established the inhibition rate constant of soman, we tested the prophylactic potential of physostigmine, pyridostigmine and huperzine A. Pre-treatment with the reversible inhibitors inhibited the enzyme by 20-95%. Additional perfusion with 10nM soman for 30min resulted in a residual activity of 1-5%, at low and high pre-inhibition, respectively. The residual activity was markedly higher than in the absence of reversibly blocking agents (0.1%). After discontinuation of soman and the reversible inhibitors, enzyme activity recovered up to 30% following pre-inhibition by 50%. The experimental data agreed with computer simulations when feeding the kinetic-based model with the established rate constants. The results with soman essentially agreed with those obtained previously with paraoxon.
ESTHER : Eckert_2007_Toxicology_233_180
PubMedSearch : Eckert_2007_Toxicology_233_180
PubMedID: 17097792

Title : Direct reaction of oximes with crotylsarin, cyclosarin, or VX in vitro - Becker_2007_Arch.Toxicol_81_415
Author(s) : Becker G , Kawan A , Gutzeit D , Worek F , Szinicz L
Ref : Archives of Toxicology , 81 :415 , 2007
Abstract : The direct reaction of seven pyridinium oximes with the organophosphorus compounds (OPCs) crotylsarin, cyclosarin, and VX was studied by spectrophotometry. This method allows to quantify different parameters: (a) the half-life times (t (1/2)) of the oxime-OPC reactions on the basis of the changes in the absorption at the zwitterion (betaine) peak maximum, (b) the first- and second-order rate constants (k (1), k (2)), and (c) the maximum reaction velocities (v (max)). The results of the study show that the reaction velocity of the nerve agents with any of the oximes investigated decreased in the order crotylsarin > cyclosarin > VX. The comparison of the reaction rates of the three therapeutically used oximes (2-PAM, obidoxime, HI 6) with the respective OPC gave the highest rate for crotylsarin and cyclosarin with obidoxime and to a similar degree with HI 6, while in the case of VX the most reactive oxime was HI 6. The reaction velocity of the nerve agents with the monopyridinium oxime 2-PAM was lower as compared to the bispyridinium oximes (obidoxime, HI 6). The results obtained with the two sarin analogues indicate that the direct reaction with 2-PAM, obidoxime, or HI 6 could be used for non-corrosive decontamination purposes, especially, if sensitive biological surfaces like skin, mucous membranes, or wounds are considered. However, in view of the concentrations of nerve agents and oximes, which could be expected during OPC poisoning in man, the maximum reaction velocities would not be high enough to contribute markedly to the detoxication of nerve agents in vivo.
ESTHER : Becker_2007_Arch.Toxicol_81_415
PubMedSearch : Becker_2007_Arch.Toxicol_81_415
PubMedID: 17151865

Title : Testing of antidotes for organophosphorus compounds: experimental procedures and clinical reality - Eyer_2007_Toxicology_233_108
Author(s) : Eyer P , Szinicz L , Thiermann H , Worek F , Zilker T
Ref : Toxicology , 233 :108 , 2007
Abstract : According to current knowledge, inhibition of acetylcholinesterase (AChE) is a very important toxic action of organphosphorus compounds (OP). Hence, it is obvious to follow the AChE activity in order to quantify the degree of inhibition and to assess possible reactivation. Red blood cell (RBC)-AChE provides an easily accessible source to follow the AChE status also in humans. There are many reports underlining the appropriateness of RBC-AChE as a surrogate parameter that mirrors the synaptic enzyme. With this tool at hand, we can study the kinetic parameters of inhibition, spontaneous and oxime-induced reactivation, as well as aging with human RBCs under physiological conditions in vitro. Moreover, we can simulate the influence of inhibitor and reactivator on enzyme activity and can calculate what happens when both components change with time. Finally, we can correlate under controlled conditions the AChE-status in intoxicated patients with the clinical signs and symptoms and determine the time-dependent changes of the oxime and OP concentration. Data of a clinical trial performed in Munich to analyze the value of obidoxime has elucidated that obidoxime worked as expected from in vitro studies. Following a 250mg bolus, obidoxime was administered by continuous infusion at 750mg/24h aimed at maintaining a plasma concentration of 10-20microM obidoxime. This oxime concentration reactivated RBC-AChE>20% of normal in most cases of OP poisoning by diethylphosphoryl compounds within a few hours. The degree of reactivation fitted theoretical calculations very well when the obidoxime and paraoxon concentrations were fed into the model. Only in a few cases reactivation was much lower than expected. The reason for this effect is probably based on the polymorphism of paraoxonase (PON1) in that the (192)arginine phenotype does hardly hydrolyze the arising diethylphosphoryl obidoxime. While this variable may complicate a proper assessment even more, we are confident that the in vitro evaluation of all relevant kinetic data enables the prediction of probable effectiveness in humans. These studies also help to understand therapeutic failures and to define scenarios where oximes are virtually ineffective. These include poisonings with rapidly aging phosphylated AChE, late start with an effective oxime and too early discontinuation of oximes in poisonings with a persistent OP. The experience gathered with the experimental and therapeutic approaches to human poisoning by OP pesticides may be helpful when oximes have to be selected against nerve agents.
ESTHER : Eyer_2007_Toxicology_233_108
PubMedSearch : Eyer_2007_Toxicology_233_108
PubMedID: 17010492

Title : Preparation and characterization of dialkylphosphoryl-obidoxime conjugates, potent anticholinesterase derivatives that are quickly hydrolyzed by human paraoxonase (PON1192Q) - Stenzel_2007_Biochem.Pharmacol_74_1390
Author(s) : Stenzel J , Worek F , Eyer P
Ref : Biochemical Pharmacology , 74 :1390 , 2007
Abstract : The potential of the most active pyridinium-4-aldoximes, such as obidoxime and trimedoxime, to reactivate phosphorylated acetylcholinesterase is not fully exploited because of inevitable formation of phosphoryloximes (POXs) with extremely high anticholinesterase activity. Hence, a topochemical equilibrium is expected at the active site, with the freshly reactivated enzyme being rapidly re-inhibited by POX produced during reactivation. In the present study, dimethylphosphoryl-, diethylphosphoryl-, and diisopropyl-obidoxime conjugates were generated and isolated in substance. Their inhibition rate of acetylcholinesterase from human red cell membranes was by a factor of 2250, 480 and 600 higher than that observed with paraoxon-methyl, paraoxon-ethyl, and diisopropyl phosphorofluoridate, respectively. All three POXs were hydrolyzed by human paraoxonase (PON1), with the alloenzyme PON1192Q being about 50-fold more active than PON1192R. The rate of hydrolysis, yielding obidoxime, was 1:6:0.03 for the three POXs, respectively. The rate of non-enzymic degradation, yielding obidoxime mononitrile, was similar with the three POXs and showed a high dependency on the reaction temperature (activation energy 83 kJ/mol), while enzymic hydrolysis required less energy (16 kJ/mol). To determine POX-hydrolase activity, we preferred a reaction temperature of 20 degrees C to reduce the noise of spontaneous degradation. A plot of POX-hydrolase versus salt-stimulated paraoxonase activity showed a highly discriminating power towards the PON1Q192R alloenzymes, which may be based on repulsive forces of the quaternary nitrogen atoms of the protonated arginine subtype and the bisquaternary POXs. It is concluded that the pharmacogenetic PON1Q192R polymorphism may be another contributor to the large variability of susceptible subjects seen in obidoxime-treated patients.
ESTHER : Stenzel_2007_Biochem.Pharmacol_74_1390
PubMedSearch : Stenzel_2007_Biochem.Pharmacol_74_1390
PubMedID: 17714697

Title : Reactivation of organophosphate-inhibited human AChE by combinations of obidoxime and HI 6 in vitro - Worek_2007_J.Appl.Toxicol_27_582
Author(s) : Worek F , Aurbek N , Thiermann H
Ref : J Appl Toxicol , 27 :582 , 2007
Abstract : Highly toxic organophosphorus-type (OP) chemical warfare agents (nerve agents) and OP pesticides may be used by terrorists and during military conflicts emphasizing the necessity for the development of effective medical countermeasures. The standard treatment with atropine and acetylcholinesterase (AChE) reactivators (oximes) is considered to be ineffective with certain nerve agents due to low oxime efficacy. Despite research over decades none of the oximes has turned out to be a broad spectrum reactivator to cover the whole range of potential threat agents. The prospective oxime HI 6 is a weak reactivator of tabun- and pesticide-inhibited AChE, while the established oxime obidoxime mainly lacks efficacy with cyclosarin-inhibited enzyme. In order to investigate the feasibility of combining obidoxime and HI 6, human AChE inhibited by sarin, cyclosarin, VX, tabun and paraoxon was reactivated by these oximes either alone or in combination. Two major findings of this study were that a combination of HI 6 and obidoxime did not impair reactivation, compared with HI 6 or obidoxime alone, but broadened the spectrum compared with the individual oximes. By using different oxime concentrations a combination of oxime doses may be suggested which could be an alternative to individual obidoxime or HI 6 autoinjectors.
ESTHER : Worek_2007_J.Appl.Toxicol_27_582
PubMedSearch : Worek_2007_J.Appl.Toxicol_27_582
PubMedID: 17370251

Title : Reevaluation of indirect field stimulation technique to demonstrate oxime effectiveness in OP-poisoning in muscles in vitro - Seeger_2007_Toxicology_233_209
Author(s) : Seeger T , Worek F , Szinicz L , Thiermann H
Ref : Toxicology , 233 :209 , 2007
Abstract : Organophosphorus (OP) pesticides or nerve agents cause severe intoxication by inhibition of acetylcholinesterase, finally resulting in death due to respiratory failure. The phrenic nerve diaphragm preparation is considered as the classic model to investigate the effect of OP intoxications and oxime treatment at the neuromuscular junction. However, this preparation is unsuitable for larger species or for muscle strips from biopsies where no nerve is available for stimulation. An alternative technique is the indirect field stimulation of muscles containing intramuscular nerve branches only. The proposed method by Wolthuis et al. [Wolthuis, O.L., Vanwersch, R.A.P., Van Der Wiel, H.J., 1981. The efficacy of some bis-pyridinium oximes as antidotes to soman in isolated muscles of several species including man. Eur. J. Pharmacol. 70, 355-369] was modified and experimentally reevaluated in isolated mouse diaphragms. To confirm that electrical field stimulation technique induced muscle contraction only via the neuromuscular endplate the nicotinic antagonists pancuronium or d-tubocurarine (1microM) were given. In the presence of a nicotinic antagonist hardly any contraction was blocked after indirect field stimulation technique with very short pulses (5micros, <0.6A), in contrast to direct muscle stimulation (broader pulse width, or higher amplitude >0.6A). During paraoxon circumfusion (20min, 1micromol/l) muscle force generation by indirect stimulation was almost completely blocked. Restoration of paralyzed muscle function to 80% of initial values could be achieved after paraoxon wash out (20min) and circumfusion with obidoxime (1micromol/l, 20min). This data correspond quite well to data shown earlier when using conventional nerve stimulation techniques.
ESTHER : Seeger_2007_Toxicology_233_209
PubMedSearch : Seeger_2007_Toxicology_233_209
PubMedID: 17250944

Title : Apoptosis in sulfur mustard treated A549 cell cultures - Steinritz_2007_Life.Sci_80_2199
Author(s) : Steinritz D , Emmler J , Hintz M , Worek F , Kreppel H , Szinicz L , Kehe K
Ref : Life Sciences , 80 :2199 , 2007
Abstract : The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.
ESTHER : Steinritz_2007_Life.Sci_80_2199
PubMedSearch : Steinritz_2007_Life.Sci_80_2199
PubMedID: 17229443

Title : Development of antidotes: problems and strategies - Szinicz_2007_Toxicology_233_23
Author(s) : Szinicz L , Worek F , Thiermann H , Kehe K , Eckert S , Eyer P
Ref : Toxicology , 233 :23 , 2007
Abstract : Antidotes against chemical warfare agents are "orphan drugs" given that these poisonings are rare. Therefore, they are of limited interest to the pharmaceutical industry. For this reason, and recognizing the increasing threat of terrorist or asymmetrical use of chemical warfare agents, the responsibility for research into medical countermeasures against these weapons is of primary interest to armies. Accordingly, the research activities of the Bundeswehr Institute of Pharmacology and Toxicology are dedicated to improving diagnosis, prophylaxis and therapy of individuals who are exposed to a chemical agent. Here, antidote development and testing are a high priority in the research program, particularly with respect to organophosphorus (OP) nerve agents and sulphur mustard. The Institute has been coordinating research activities undertaken in house and in collaboration with external researchers. The research program aims to develop primarily in vitro models to minimize the sacrifice of animals, using strategies, which involve human material early in antidote testing. An animal model using isolated mouse diaphragm demonstrated the correlation between AChE activity and neuromuscular function. A similar relationship was found between erythrocyte AChE and neuromuscular function in patients with acute OP pesticide poisoning. In vitro rate constants of the various reactions that are involved in enzyme inhibition and reactivation using human material were used for prediction of what would happen in vivo. This prediction could be confirmed in a patient with acute OP pesticide poisoning. Finally, computer models are being established to estimate the therapeutic effect of an antidote in various human poisoning scenarios. This approach is necessary to compensate for the lack of human clinical pharmacodynamic studies that are usually required for drug regulatory approval, given the obvious ethical issues preventing human volunteer studies with these agents.
ESTHER : Szinicz_2007_Toxicology_233_23
PubMedSearch : Szinicz_2007_Toxicology_233_23
PubMedID: 16949190

Title : Enzyme-kinetic investigation of different sarin analogues reacting with human acetylcholinesterase and butyrylcholinesterase - Bartling_2007_Toxicology_233_166
Author(s) : Bartling A , Worek F , Szinicz L , Thiermann H
Ref : Toxicology , 233 :166 , 2007
Abstract : The pertinent threat of using organophosphorus compound (OP)-type chemical warfare agents (nerve agents) during military conflicts and by non-state actors requires the continuous search for more effective medical countermeasures. OP inhibit acetylcholinesterase (AChE) and therefore standard treatment of respective poisoning includes AChE reactivators (oximes) in combination with antimuscarinic agents. Hereby, standard oximes, 2-PAM and obidoxime, are considered to be rather insufficient against various nerve agents. Numerous experimental oximes have been investigated in the last decades by in vitro and in vivo models. Recently, we studied the reactivating potency of several oximes with human AChE inhibited by structurally different OP and observed remarkable differences depending on the OP and oxime. In order to investigate structure-activity relationships we determined the various kinetic constants (inhibition, reactivation, aging) for a series of sarin analogues bearing a methyl, ethyl, n-propyl, n-butyl, i-propyl, i-butyl, cyclohexyl or pinacolyl group with human AChE and BChE. The rate constants for the inhibition of human erythrocyte AChE and plasma BChE by these OP (k(i)), for the spontaneous dealkylation (k(a)) and reactivation (k(s)) of OP-inhibited AChE and BChE as well as for the oxime-induced reactivation of OP-inhibited AChE and BChE by the oximes obidoxime, 2-PAM, HI 6, HLo 7 and MMB-4 were determined. With compounds bearing a n-alkyl group the inhibition rate constant increased with chain length. A relation between chain length and spontaneous reactivation velocity was also observed. In contrast, no structure-activity dependence could be observed for the oxime-induced reactivation of AChE and BChE inhibited by the compounds tested. In general, OP-inhibited AChE and BChE were susceptible towards reactivation by oximes. HLo 7 was the most potent reactivator followed by HI 6 and obidoxime while 2-PAM and MMB-4 were rather weak reactivators. These data indicate a potential structure-activity relationship concerning inhibition and spontaneous reactivation but not for oxime-induced reactivation.
ESTHER : Bartling_2007_Toxicology_233_166
PubMedSearch : Bartling_2007_Toxicology_233_166
PubMedID: 16904809

Title : Red blood cell acetylcholinesterase and plasma butyrylcholinesterase status: important indicators for the treatment of patients poisoned by organophosphorus compounds - Thiermann_2007_Arh.Hig.Rada.Toksikol_58_359
Author(s) : Thiermann H , Kehe K , Steinritz D , Mikler J , Hill I , Zilker T , Eyer P , Worek F
Ref : Arh Hig Rada Toksikol , 58 :359 , 2007
Abstract : Inhibition of acetylcholinesterase (AChE) is regarded as the primary toxic mechanism of organophosphorus compounds (OP). Therapeutic strategies are directed to antagonise overstimulation of muscarinic receptors with atropine and to reactivate inhibited AChE with oximes. Reactivation is crucial within the neuromuscular synapse, where atropine is ineffective, since peripheral neuromuscular block eventually leads to respiratory failure. Patients with OP intoxication have to be identified as early as possible. During an international NBC-defence exercise anesthetised pigs were poisoned with sarin, followed by treatment with atropine and oxime. Blood samples were drawn and red blood cell (RBC)-AChE activity determined with a fielded test system on-site. Within a few minutes the poisoning was verified. After administration of HI-6, RBC-AChE activity increased rapidly. Blood samples were reanalysed in our laboratory in Munich. Almost identical course of the AChE activities was recorded by both systems.The more comprehensive cholinesterase status was determined in Munich. Oxime administration can be stopped when AChE is aged completely, but has to be continued as long as poison is present in the body and reactivation is possible. To aid the on-site physician in optimising diagnosis and treatment, a fielded test system should be available to allow rapid determination of the complete cholinesterase status.
ESTHER : Thiermann_2007_Arh.Hig.Rada.Toksikol_58_359
PubMedSearch : Thiermann_2007_Arh.Hig.Rada.Toksikol_58_359
PubMedID: 17913691

Title : Lessons to be learnt from organophosphorus pesticide poisoning for the treatment of nerve agent poisoning - Thiermann_2007_Toxicology_233_145
Author(s) : Thiermann H , Szinicz L , Eyer P , Felgenhauer N , Zilker T , Worek F
Ref : Toxicology , 233 :145 , 2007
Abstract : The increasing threat of nerve agent use for terrorist purposes against civilian and military population calls for effective therapeutic preparedness. At present, administration of atropine and an oxime are recommended, although effectiveness of this treatment is not proved in clinical trials. Here, monitoring of intoxications with organophosphorus (OP) pesticides may be of help, as their actions are closely related to those of nerve agents and intoxication and therapy follow the same principles. To this end, the clinical course of poisoning and the effectiveness of antidotal therapy were investigated in patients requiring artificial ventilation being treated with atropine and obidoxime. However, poisoning with OP pesticides shows extremely heterogeneous pictures of cholinergic crisis frequently associated with clinical complications. To achieve valuable information for the therapy of nerve agent poisoning, cases resembling situations in nerve agent poisoning had to be extracted: (a) intoxication with OPs forming reactivatable OP-AChE-complexes with short persistence of the OP in the body resembling inhalational sarin intoxication; (b) intoxication with OPs resulting rapidly in an aged OP-AChE-complex resembling inhalational soman intoxication; (c) intoxications with OPs forming a reactivatable AChE-OP complex with prolonged persistence of the OP in the body resembling percutaneous VX intoxication. From these cases it was concluded that sufficient reactivation of nerve agent inhibited non-aged AChE should be possible, if the poison load was not too high and the effective oximes were administered early and with an appropriate duration. When RBC-AChE activity was higher than some 30%, neuromuscular transmission was relatively normal. Relatively low atropine doses (several milligrams) should be sufficient to cope with muscarinic symptoms during oxime therapy.
ESTHER : Thiermann_2007_Toxicology_233_145
PubMedSearch : Thiermann_2007_Toxicology_233_145
PubMedID: 17161895

Title : Recent advances in evaluation of oxime efficacy in nerve agent poisoning by in vitro analysis - Worek_2007_Toxicol.Appl.Pharmacol_219_226
Author(s) : Worek F , Eyer P , Aurbek N , Szinicz L , Thiermann H
Ref : Toxicol Appl Pharmacol , 219 :226 , 2007
Abstract : The availability of highly toxic organophosphorus (OP) warfare agents (nerve agents) underlines the necessity for an effective medical treatment. Acute OP toxicity is primarily caused by inhibition of acetylcholinesterase (AChE). Reactivators (oximes) of inhibited AChE are a mainstay of treatment, however, the commercially available compounds, obidoxime and pralidoxime, are considered to be rather ineffective against various nerve agents, e.g. soman and cyclosarin. This led to the synthesis and investigation of numerous oximes in the past decades. Reactivation of OP-inhibited AChE is considered to be the most important reaction of oximes. Clinical data from studies with pesticide-poisoned patients support the assumption that the various reactions between AChE, OP and oxime, i.e. inhibition, reactivation and aging, can be investigated in vitro with human AChE. In contrast to animal experiments such in vitro studies with human tissue enable the evaluation of oxime efficacy without being affected by species differences. In the past few years numerous in vitro studies were performed by different groups with a large number of oximes and methods were developed for extrapolating in vitro data to different scenarios of human nerve agent poisoning. The present status in the evaluation of new oximes as antidotes against nerve agent poisoning will be discussed.
ESTHER : Worek_2007_Toxicol.Appl.Pharmacol_219_226
PubMedSearch : Worek_2007_Toxicol.Appl.Pharmacol_219_226
PubMedID: 17112559

Title : Simulation of cholinesterase status at different scenarios of nerve agent exposure - Worek_2007_Toxicology_233_155
Author(s) : Worek F , Eyer P , Szinicz L , Thiermann H
Ref : Toxicology , 233 :155 , 2007
Abstract : The ongoing threat of homicidal use of organophosphorus-type chemical warfare agents ("nerve agents") during military conflicts and by terrorists underlines the necessity for effective medical countermeasures. Standard treatment with atropine and the established acetylcholinesterase (AChE) reactivators, obidoxime and pralidoxime, is considered to be ineffective with certain nerve agents due to low oxime effectiveness. From obvious ethical reasons only animal experiments can be used to evaluate new oximes as nerve agent antidotes. However, the extrapolation of data from animal to humans is hampered by marked species differences. Since reactivation of OP-inhibited AChE is considered to be the main mechanism of action of oximes, human erythrocyte AChE can be exploited to test the efficacy of new oximes. Recently, a dynamic computer model was developed which allows the calculation of AChE activities at different scenarios by combining enzyme kinetics (inhibition, reactivation, aging) with OP toxicokinetics and oxime pharmacokinetics. Now, this computer model was further extended by including the pharmaco- and enzyme kinetics of carbamate pretreatment. Simulations were performed for intravenous and percutaneous nerve agent exposure and intramuscular oxime treatment in the presence and absence of pyridostigmine pretreatment using published data. The model presented may serve as a tool for evaluating the impact of carbamate pretreatment on oxime-induced reactivation of inhibited AChE, for defining effective oxime concentrations and for optimizing oxime treatment. In addition, this model may be useful for the development of meaningful therapeutic strategies in animal experiments.
ESTHER : Worek_2007_Toxicology_233_155
PubMedSearch : Worek_2007_Toxicology_233_155
PubMedID: 16904807

Title : Kinetic analysis of reactivation and aging of human acetylcholinesterase inhibited by different phosphoramidates - Worek_2007_Biochem.Pharmacol_73_1807
Author(s) : Worek F , Aurbek N , Koller M , Becker C , Eyer P , Thiermann H
Ref : Biochemical Pharmacology , 73 :1807 , 2007
Abstract : 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 HLo 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.
ESTHER : Worek_2007_Biochem.Pharmacol_73_1807
PubMedSearch : Worek_2007_Biochem.Pharmacol_73_1807
PubMedID: 17382909

Title : Development of a dynamic model for real-time determination of membrane-bound acetylcholinesterase activity upon perfusion with inhibitors and reactivators - Eckert_2006_Biochem.Pharmacol_72_358
Author(s) : Eckert S , Eyer P , Muckter H , Worek F
Ref : Biochemical Pharmacology , 72 :358 , 2006
Abstract : Quantitative predictions of the course of acetylcholinesterase (AChE) activity, following interference of inhibitors and reactivators, are usually obscured by the time-dependent changes of all reaction partners. To mimic these dynamics we developed an in vitro model. Immobilized human erythrocyte ghosts in a bioreactor were continuously perfused while AChE activity was monitored by a modified Ellman method. The perfusion system consisted of two HPLC pumps with integrated quaternary low-pressure gradient formers that were programmed by a computer using commercial HPLC software. The combined eluates passed a particle filter (Millex-GS, 0.22 microm) containing a thin layer of erythrocytes that was immersed in a temperature-controlled water bath. The effluent passed a flow cell in a UV-vis detector, the signal of which was digitized, written to disc and calculated with curve fitting programs. AChE activity decreased by 3.4% within 2.5 h. The day-to-day variation of the freshly prepared bioreactor using the same enzyme source was +/-3.3%. Residual activity of 0.2% marked the limit of quantification. Following perfusion with paraoxon, pseudo first-order rate constants of inhibition were established that did not differ from results obtained in conventional assays. The same holds true for reactivation with obidoxime. The set-up presented allows freely programmable time-dependent changes of up to eight solvents to mimic pharmacokinetic profiles without accumulation of products. Due to some hysteresis in the system, reaction half-lives should be >3 min and concentration changes in critical compounds should exceed half-lives of 5 min. Otherwise, the system offers much flexibility and operates with high precision.
ESTHER : Eckert_2006_Biochem.Pharmacol_72_358
PubMedSearch : Eckert_2006_Biochem.Pharmacol_72_358
PubMedID: 16725113

Title : Kinetic analysis of the protection afforded by reversible inhibitors against irreversible inhibition of acetylcholinesterase by highly toxic organophosphorus compounds - Eckert_2006_Biochem.Pharmacol_72_344
Author(s) : Eckert S , Eyer P , Muckter H , Worek F
Ref : Biochemical Pharmacology , 72 :344 , 2006
Abstract : In organophosphate poisoning, the underlying mechanism of the therapeutic efficacy of carbamate prophylaxis, which was successfully tested in animal experiments, still awaits complete understanding. In particular, it is unclear whether survival is improved by increased acetylcholinesterase activity during the acute phase, when both carbamate and organophosphate are present. This question should be solved experimentally by means of a dynamically working in vitro model. Immobilized human erythrocytes were continuously perfused while acetylcholinesterase activity was monitored in real-time by a modified Ellman method. The concentrations of reversible inhibitors and of paraoxon were varied to assess the influence of both components on the enzyme activity under steady-state conditions. Physostigmine, pyridostigmine and huperzine A were tested for their prophylactic potential. Upon pretreatment with these reversible inhibitors the enzyme was inhibited by 20-90%. Additional perfusion with 1 microM paraoxon for 30 min resulted in a residual activity of 1-4%, at low and high pre-inhibition, respectively. The residual activity was significantly higher than in the absence of reversibly blocking agents (0.3%). After discontinuing paraoxon, the activity increased even in the presence of the reversible blockers. Stopping the reversibly blocking agents resulted in 10-35% recovery of the enzyme activity, depending on the degree of pre-inhibition. The experimental results agreed with computer simulations upon feeding with the essential reaction rate constants, showing that physostigmine was somewhat superior to pyridostigmine in enhancing residual activity in the presence of 1 microM paraoxon for 30 min. The model predicts that inhibitors with a faster dissociation rate, e.g. huperzine A, may be superior in case of a 'hit-and-run' poison such as soman.
ESTHER : Eckert_2006_Biochem.Pharmacol_72_344
PubMedSearch : Eckert_2006_Biochem.Pharmacol_72_344
PubMedID: 16780806

Title : Respiratory failure in acute organophosphorus pesticide self-poisoning - Eddleston_2006_Qjm_99_513
Author(s) : Eddleston M , Mohamed F , Davies JO , Eyer P , Worek F , Sheriff MH , Buckley NA
Ref : Qjm , 99 :513 , 2006
Abstract : BACKGROUND: Acute organophosphorus (OP) pesticide poisoning is a major clinical problem in the developing world. Textbooks ascribe most deaths to respiratory failure occurring in one of two distinct clinical syndromes: acute cholinergic respiratory failure or the intermediate syndrome. Delayed failure appears to be due to respiratory muscle weakness, but its pathophysiology is unclear. AIM: To describe the clinical patterns of OP-induced respiratory failure, and to determine whether the two syndromes are clinically distinct. DESIGN: Prospective study of 376 patients with confirmed OP poisoning.
METHODS: Patients were observed throughout their admission to three Sri Lankan hospitals. Exposure was confirmed by butyrylcholinesterase and blood OP assays.
RESULTS: Ninety of 376 patients (24%) required intubation: 52 (58%) within 2 h of admission while unconscious with cholinergic features. Twenty-nine (32%) were well on admission but then required intubation after 24 h while conscious and without cholinergic features. These two syndromes were not clinically distinct and had much overlap. In particular, some patients who required intubation on arrival subsequently recovered consciousness but could not be extubated, requiring ventilation for up to 6 days. DISCUSSION: Respiratory failure did not occur as two discrete clinical syndromes within distinct time frames. Instead, the pattern of failure was variable and overlapped in some patients. There seemed to be two underlying mechanisms (an early acute mixed central and peripheral respiratory failure, and a late peripheral respiratory failure) rather than two distinct clinical syndromes.
ESTHER : Eddleston_2006_Qjm_99_513
PubMedSearch : Eddleston_2006_Qjm_99_513
PubMedID: 16861715

Title : The liberation of thiocholine from acetylthiocholine (ASCh) by pralidoxime iodide (2=PAM) and other oximes (obidoxime and diacetylmonoxime) -
Author(s) : Worek F , Eyer P
Ref : Toxicol Lett , 167 :256 , 2006
PubMedID: 17113251

Title : Analysis of inhibition, reactivation and aging kinetics of highly toxic organophosphorus compounds with human and pig acetylcholinesterase - Aurbek_2006_Toxicology_224_91
Author(s) : Aurbek N , Thiermann H , Szinicz L , Eyer P , Worek F
Ref : Toxicology , 224 :91 , 2006
Abstract : Organophosphorus compounds (OP) are in wide spread use as pesticides and highly toxic OP may be used as chemical warfare agents (nerve agents). OP inhibit acetylcholinesterase (AChE), therefore, standard treatment includes AChE reactivators (oximes) in combination with antimuscarinic agents. In the last decades, the efficacy of oximes has been investigated in various animal models, mostly in rodents. However, extrapolating animal data to humans is problematical because of marked differences between rodents and humans concerning the toxicokinetics of nerve agents, the pharmacokinetics of antidotes and the AChE enzyme kinetics. In order to improve the understanding of species differences and to enable a more reliable extrapolation of animal data to humans a study was initiated to investigate the effect of highly toxic nerve agents, i.e. VX, Russian VX (VR) and Chinese VX (CVX), with human and pig erythrocyte AChE. Hereby, the rate constants for the inhibition of AChE by these OP (ki) and for the spontaneous dealkylation (ka) and reactivation (ks) of OP-inhibited AChE as well as for the oxime-induced reactivation of OP-inhibited AChE by the oximes obidoxime, 2-PAM, HI 6, HLo 7 and MMB-4 were determined. Compared to human AChE pig AChE showed a lower sensitivity towards the investigated OP. Furthermore, a slower spontaneous dealkylation and reactivation of pig AChE was recorded. The potency of the investigated oximes was remarkably lower with OP-inhibited pig AChE. These data may contribute to a better understanding of species differences and may provide a kinetic basis for extrapolation of data from pig experiments to humans.
ESTHER : Aurbek_2006_Toxicology_224_91
PubMedSearch : Aurbek_2006_Toxicology_224_91
PubMedID: 16720069

Title : Application of kinetic-based computer modelling to evaluate the efficacy of HI 6 in percutaneous VX poisoning - Aurbek_2006_Toxicology_224_74
Author(s) : Aurbek N , Thiermann H , Szinicz L , Eyer P , Worek F
Ref : Toxicology , 224 :74 , 2006
Abstract : The rife use of organophosphorus compounds (OP) as pesticides and the exertion of highly toxic OP-type chemical warfare agents (nerve agents) during military conflicts and terrorist attacks in the past emphasize the necessity of the development of effective therapeutic countermeasures. Presently, standard treatment of poisoning by OP includes administration of atropine as an antimuscarinic agent and of oximes, e.g. obidoxime or pralidoxime, as reactivators of OP-inhibited acetylcholinesterase (AChE), but is considered to be rather ineffective with certain nerve agents. The evaluation of new oximes as antidotes is only possible by implementation of animal experiments for ethical reasons and therefore complicated by a limited extrapolation of animal data to humans due to marked species differences. A computer simulation based on combination of AChE kinetic data (inhibition, reactivation, aging) with OP toxicokinetics and oxime pharmacokinetics allows the calculation of AChE activities at different scenarios and may facilitate to define effective oxime concentrations and to optimize oxime dosage in OP poisoning. On the base of species-specific kinetic data this model was used to calculate AChE activities in humans and pigs after percutaneous exposure to 5 x LD50 VX and treatment with HI 6. Due to marked species differences between human and pig AChE the HI 6 dose that is necessary to cause a comparable reactivation of VX-inhibited pig AChE is conspicuously higher. Hence, designing animal experiments with the aid of computer modeling may reduce the number of animal experiments and allow a more reliable extrapolation of animal data to humans.
ESTHER : Aurbek_2006_Toxicology_224_74
PubMedSearch : Aurbek_2006_Toxicology_224_74
PubMedID: 16740352

Title : Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study - Eddleston_2005_Lancet_366_1452
Author(s) : Eddleston M , Eyer P , Worek F , Mohamed F , Senarathna L , von Meyer L , Juszczak E , Hittarage A , Azhar S , Dissanayake W , Sheriff MH , Szinicz L , Dawson AH , Buckley NA
Ref : Lancet , 366 :1452 , 2005
Abstract : BACKGROUND: Although more than 100 organophosphorus insecticides exist, organophosphorus poisoning is usually regarded as a single entity, distinguished only by the compound's lethal dose in animals. We aimed to determine whether the three most common organophosphorus insecticides used for self-poisoning in Sri Lanka differ in the clinical features and severity of poisoning they cause.
METHODS: We prospectively studied 802 patients with chlorpyrifos, dimethoate, or fenthion self-poisoning admitted to three hospitals. Blood cholinesterase activity and insecticide concentration were measured to determine the compound and the patients' response to insecticide and therapy. We recorded clinical outcomes for each patient. FINDINGS: Compared with chlorpyrifos (35 of 439, 8.0%), the proportion dying was significantly higher with dimethoate (61 of 264, 23.1%, odds ratio [OR] 3.5, 95% CI 2.2-5.4) or fenthion (16 of 99, 16.2%, OR 2.2, 1.2-4.2), as was the proportion requiring endotracheal intubation (66 of 439 for chlorpyrifos, 15.0%; 93 of 264 for dimethoate, 35.2%, OR 3.1, 2.1-4.4; 31 of 99 for fenthion, 31.3%, 2.6, 1.6-4.2). Dimethoate-poisoned patients died sooner than those ingesting other pesticides and often from hypotensive shock. Fenthion poisoning initially caused few symptoms but many patients subsequently required intubation. Acetylcholinesterase inhibited by fenthion or dimethoate responded poorly to pralidoxime treatment compared with chlorpyrifos-inhibited acetylcholinesterase. INTERPRETATION: Organophosphorus insecticide poisoning is not a single entity, with substantial variability in clinical course, response to oximes, and outcome. Animal toxicity does not predict human toxicity since, although chlorpyrifos is generally the most toxic in rats, it is least toxic in people. Each organophosphorus insecticide should be considered as an individual poison and, consequently, patients might benefit from management protocols developed for particular organophosphorus insecticides.
ESTHER : Eddleston_2005_Lancet_366_1452
PubMedSearch : Eddleston_2005_Lancet_366_1452
PubMedID: 16243090

Title : Estimation of oxime efficacy in nerve agent poisoning: a kinetic approach - Worek_2005_Chem.Biol.Interact_157-158_349
Author(s) : Worek F , Szinicz L , Thiermann H
Ref : Chemico-Biological Interactions , 157-158 :349 , 2005
Abstract : Standard treatment of poisoning by organophosphorus compounds (OP) includes the administration of an anti-muscarinic, e.g. atropine, and of an acetylcholinesterase (AChE) reactivator (oxime). Two oximes, obidoxime and pralidoxime (2-PAM), are presently commercially available, yet, these compounds are considered to be of insufficient efficacy against certain nerve agents, e.g. soman and cyclosarin. In the past decades, numerous new oximes were synthesized and tested for their antidotal efficacy. The available data indicate that two Hagedorn oximes, HI 6 and HLo 7, are promising antidotes against various nerve agents. The efficacy of antidotes against nerve agent poisoning cannot be investigated in humans for ethical reasons. Therefore, it is necessary to use surrogate parameters for the evaluation of oxime efficacy. Reactivation of inhibited AChE is considered to be the main mechanism of action of oximes. Clinical data indicate that changes in erythrocyte AChE activity correlate to neuromuscular function indicating that interactions between AChE, inhibitor and oximes can be investigated in vitro with human erythrocyte AChE. Different theoretical models were used for the evaluation of reactivating efficacy of oximes with nerve agent-inhibited human AChE and for estimating effective oxime concentrations. The calculations demonstrate the marked differences between oximes in dependence of the inhibitor and provide a basis for the estimation of the required oxime dose as well as of dosing intervals.
ESTHER : Worek_2005_Chem.Biol.Interact_157-158_349
PubMedSearch : Worek_2005_Chem.Biol.Interact_157-158_349
PubMedID: 16266695

Title : Effect of metoclopramide and ranitidine on the inhibition of human AChE by VX in vitro - Bartling_2005_J.Appl.Toxicol_25_568
Author(s) : Bartling A , Thiermann H , Szinicz L , Worek F
Ref : J Appl Toxicol , 25 :568 , 2005
Abstract : The repeated misuse of highly toxic organophosphorus-type (OP) chemical warfare agents ('nerve agents') emphasizes the necessity for the development of effective medical countermeasures. The standard treatment with atropine and acetylcholinesterase (AChE) reactivators ('oximes') is considered to be ineffective with certain nerve agents due to low oxime efficacy. Therefore, pretreatment with carbamate-type compounds, e.g. pyridostigmine, was recommended to improve antidotal efficacy. Recently, the clinically used reversible AChE inhibitors metoclopramide (MCP) and ranitidine (RAN) were shown to exhibit some protective effect against the OP pesticide paraoxon in vitro and in vivo. The present study was undertaken to investigate a potential protective effect of MCP and RAN against inhibition of human AChE by the nerve agent VX (O-ethyl S-[2-(diisopropylamino)ethyl)methylphosphonothioate). Hemoglobin-free human erythrocyte membranes were incubated with various, human relevant MCP (0.5-2 microm) and RAN (0.5-5 microm) concentrations starting 1 min before addition of VX (1-40 nm). Both compounds failed to increase VX IC(50) values. In addition, human AChE was incubated with higher than human relevant therapeutic concentrations of MCP (1 microm-1 mm) and RAN (1 microm-2.0 mm) and inhibited by 40 nm VX. At concentrations higher than 100 microm MCP and RAN caused a concentration dependent increase of residual AChE activity 15 min after addition of VX. These data indicate that MCP and RAN may be ineffective in protecting human AChE against inhibition by the nerve agent VX at human relevant doses.
ESTHER : Bartling_2005_J.Appl.Toxicol_25_568
PubMedSearch : Bartling_2005_J.Appl.Toxicol_25_568
PubMedID: 16167316

Title : Formation and disposition of diethylphosphoryl-obidoxime, a potent anticholinesterase that is hydrolyzed by human paraoxonase (PON1) - Kiderlen_2005_Biochem.Pharmacol_69_1853
Author(s) : Kiderlen D , Eyer P , Worek F
Ref : Biochemical Pharmacology , 69 :1853 , 2005
Abstract : The potential of pyridinium-4-aldoximes, such as obidoxime, to reactivate diethylphosphorylated acetylcholinesterases is not fully exploited due to the inevitable formation of phosphoryloximes (POX) with high anticholinesterase activity. Mono(diethylphosphoryl) obidoxime (DEP-obidoxime) was isolated for the first time showing remarkable stability under physiological conditions (half-life 13.5min; pH 7.1; 37 degrees C). The half-life was considerably extended to 20h at 0 degrees C, which facilitated the preparation and allowed isolation by HPLC. The structure was confirmed by mass spectrometry and the degradation pattern. DEP-obidoxime decomposed by an elimination reaction forming the intermediate nitrile that hydrolyzed mainly into the pyridone and cyanide. The intermediates were prepared and confirmed by mass spectroscopy. DEP-Obidoxime was an extremely potent inhibitor of human acetylcholinesterase approaching a second-order rate constant of 10(9)M(-1)min(-1) (pH 7.4; 37 degrees C). The nitrile and the pyridone were still good reactivators. In the presence of human plasma DEP-obidoxime was hydrolyzed into parent obidoxime. Calcium-dependence and sensitivity towards chelators, substitution pattern by other divalent cations and protein-modifying agents all pointed to human paraoxonase (hPON1) as the responsible protein with POX-hydrolase activity. Subjects, probably belonging to the homozygous (192)arginine subtype, were virtually devoid of POX-hydrolase activity while a highly purified hPON1 of the homozygous (192)glutamine subtype exhibited particularly high POX-hydrolase activity. Two parathion-poisoned patients with high and low POX-hydrolase activity responded well and poorly, respectively, to obidoxime treatment although the former patient had higher plasma paraoxon levels than the poor responder. Hence, the POX-hydrolase associated PON1 subtype may be another contributor that modulates pyridinium-4-aldoxime effectiveness.
ESTHER : Kiderlen_2005_Biochem.Pharmacol_69_1853
PubMedSearch : Kiderlen_2005_Biochem.Pharmacol_69_1853
PubMedID: 15876422

Title : Effects of oximes on muscle force and acetylcholinesterase activity in isolated mouse hemidiaphragms exposed to paraoxon - Thiermann_2005_Toxicology_214_190
Author(s) : Thiermann H , Eyer P , Worek F , Szinicz L
Ref : Toxicology , 214 :190 , 2005
Abstract : Toxicity of organophosphates (OP) is caused by inhibition of acetylcholinesterase (AChE), resulting in accumulation of acetylcholine. While cholinolytics such as atropine are able to counteract muscarinic symptoms, they are unable to restore the impaired neuromuscular transmission (NMT). Here, oximes as potential reactivators of inhibited AChE may be effective. Until now, no unequivocal relation between oxime-induced increase in muscle force and reactivation has been demonstrated. To address this issue the isolated circumfused mouse hemidiaphragm was used as an experimental model. The muscle force generation upon tetanic stimuli was recorded during AChE inhibition by 1 microM paraoxon and after a wash-out period in the presence of obidoxime, pralidoxime and the experimental oximes HI 6, and HLo 7, 10 microM each. At the end of the experiments AChE activity was determined in the diaphragm homogenates by a radiometric assay. At 50-Hz stimulation, recovery was complete with obidoxime, nearly complete with HLo 7 but incomplete with HI 6 and pralidoxime. Only with obidoxime a significant increase in AChE activity was found. An increase of AChE to 10% of normal was sufficient to allow normal muscle force generation. When paraoxon was still present, obidoxime and HLo 7 were effective at 0.1 microM paraoxon, but failed so at paraoxon >1 microM. The data show different effectiveness of the oximes investigated in reactivation of muscle AChE and recovery of NMT after inhibition by paraoxon. Although an increase in muscle force by the oximes was accompanied by a measurable increase in AChE activity only in the case of obidoxime, the plot of muscle force against AChE activity as well as lacking evidence for a direct effect and adaptive processes indicate that reactivation of the enzyme is the main mechanism of NMT recovery. In agreement, in presence of AChE inhibitory concentrations of paraoxon during reactivation a reduced effectiveness of oximes was found.
ESTHER : Thiermann_2005_Toxicology_214_190
PubMedSearch : Thiermann_2005_Toxicology_214_190
PubMedID: 16040183

Title : Correlation between red blood cell acetylcholinesterase activity and neuromuscular transmission in organophosphate poisoning - Thiermann_2005_Chem.Biol.Interact_157-158_345
Author(s) : Thiermann H , Szinicz L , Eyer P , Zilker T , Worek F
Ref : Chemico-Biological Interactions , 157-158 :345 , 2005
Abstract : Assessment of effectiveness of oximes in severely organophosphate poisoned patients is hampered by sedation, artificial ventilation and other therapeutic measures as well as varying individual clinical courses due to, e.g. differences in type and amount of poison ingested or time elapsed before treatment starts. To evaluate oxime effects a suitable surrogate parameter would be helpful. Red blood cell acetylcholinesterase (RBC-AChE) is easily obtainable, shows a similar structure as synaptic enzyme and may be useful to reflect the AChE status at the synaptic site. Accordingly, it appeared rational to check whether RBC-AChE activity could be correlated with neuromuscular transmission (NMT), an easily accessible clinical parameter. The correlation was assessed in a clinical trial with severely OP-poisoned patients who were treated with obidoxime. The investigation revealed a good correlation between both parameters and showed, that a very low RBC-AChE activity (<10% of normal) was associated with a strongly impaired NMT marker, the so called decrement-phenomenon, RBC-AChE activity between 10 and 30% by impaired NMT with the decrement-increment-phenomenon and RBC activities above 30% generally by normal muscle function. Accordingly, RBC-AChE appears to be a suitable parameter for judgment of oxime effectiveness at the neuromuscular junction, one of the most important targets for therapy where atropine is ineffective in OP-poisoning.
ESTHER : Thiermann_2005_Chem.Biol.Interact_157-158_345
PubMedSearch : Thiermann_2005_Chem.Biol.Interact_157-158_345
PubMedID: 16263103

Title : Diagnostic aspects of organophosphate poisoning - Worek_2005_Toxicology_214_182
Author(s) : Worek F , Koller M , Thiermann H , Szinicz L
Ref : Toxicology , 214 :182 , 2005
Abstract : 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.
ESTHER : Worek_2005_Toxicology_214_182
PubMedSearch : Worek_2005_Toxicology_214_182
PubMedID: 16051411

Title : Evaluation of oxime efficacy in nerve agent poisoning: development of a kinetic-based dynamic model - Worek_2005_Toxicol.Appl.Pharmacol_209_193
Author(s) : Worek F , Szinicz L , Eyer P , Thiermann H
Ref : Toxicol Appl Pharmacol , 209 :193 , 2005
Abstract : The widespread use of organophosphorus compounds (OP) as pesticides and the repeated misuse of highly toxic OP as chemical warfare agents (nerve agents) emphasize the necessity for the development of effective medical countermeasures. Standard treatment with atropine and the established acetylcholinesterase (AChE) reactivators, obidoxime and pralidoxime, is considered to be ineffective with certain nerve agents due to low oxime effectiveness. From obvious ethical reasons only animal experiments can be used to evaluate new oximes as nerve agent antidotes. However, the extrapolation of data from animal to humans is hampered by marked species differences. Since reactivation of OP-inhibited AChE is considered to be the main mechanism of action of oximes, human erythrocyte AChE can be exploited to test the efficacy of new oximes. By combining enzyme kinetics (inhibition, reactivation, aging) with OP toxicokinetics and oxime pharmacokinetics a dynamic in vitro model was developed which allows the calculation of AChE activities at different scenarios. This model was validated with data from pesticide-poisoned patients and simulations were performed for intravenous and percutaneous nerve agent exposure and intramuscular oxime treatment using published data. The model presented may serve as a tool for defining effective oxime concentrations and for optimizing oxime treatment. In addition, this model can be useful for the development of meaningful therapeutic animal models.
ESTHER : Worek_2005_Toxicol.Appl.Pharmacol_209_193
PubMedSearch : Worek_2005_Toxicol.Appl.Pharmacol_209_193
PubMedID: 15904945

Title : Effect of organophosphorus hydrolysing enzymes on obidoxime-induced reactivation of organophosphate-inhibited human acetylcholinesterase - Herkenhoff_2004_Arch.Toxicol_78_338
Author(s) : Herkenhoff S , Szinicz L , Rastogi VK , Cheng TC , DeFrank JJ , Worek F
Ref : Archives of Toxicology , 78 :338 , 2004
Abstract : The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.
ESTHER : Herkenhoff_2004_Arch.Toxicol_78_338
PubMedSearch : Herkenhoff_2004_Arch.Toxicol_78_338
PubMedID: 14985944

Title : Reactivation and aging kinetics of human acetylcholinesterase inhibited by organophosphonylcholines - Worek_2004_Arch.Toxicol_78_212
Author(s) : Worek F , Thiermann H , Szinicz L
Ref : Archives of Toxicology , 78 :212 , 2004
Abstract : A great number of structurally different organophosphorus compounds (OPs) was synthesized in the past decades to be used as pesticides or chemical warfare agents. Methyl-fluorophosphonylcholines were found to be highly toxic OPs and the acetylcholinesterase (AChE) reactivator pralidoxime was shown to be unable to reactivate inhibited AChE. In the course of the development of more effective AChE reactivators, we have determined the reactivation rate constants of various oximes with human AChE inhibited by methylfluorophosphonylcholine (MFPCh), methylfluoro-beta-phosphonylcholine (MFP beta Ch) and methylfluorophosphonylhomocholine (MFPhCh). In addition, we investigated the potential influence of aging phenomena on the oxime efficacy. Human AChE inhibited by MFPCh, MFP beta Ch or MFPhCh was extremely resistant towards reactivation by oximes. Nevertheless, the newer compounds, HLo 7 and HI 6, were substantially more potent reactivators than obidoxime and pralidoxime. The low oxime efficacy was not due to rapid aging since no decrease in reactivatability was found over 96 h at 37 degrees C. Within this period a substantial spontaneous reactivation was observed, with MFPCh >MFP beta Ch >MFPhCh, which did not follow pseudo-first-order kinetics. In conclusion, the unexpected results, i.e., high resistance of inhibited AChE towards oxime reactivation and aging, and much lower resistance towards spontaneous reactivation, calls for further experiments at a molecular level for a better understanding of the interactions among AChE, its inhibitors and reactivators.
ESTHER : Worek_2004_Arch.Toxicol_78_212
PubMedSearch : Worek_2004_Arch.Toxicol_78_212
PubMedID: 14647978

Title : Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes - Worek_2004_Biochem.Pharmacol_68_2237
Author(s) : Worek F , Thiermann H , Szinicz L , Eyer P
Ref : Biochemical Pharmacology , 68 :2237 , 2004
Abstract : The wide-spread use of organophosphorus compounds (OP) as pesticides and the availability of highly toxic OP-type chemical warfare agents (nerve agents) underlines the necessity for an effective medical treatment. Acute OP toxicity is primarily caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment, however, the commercially available compounds, obidoxime and pralidoxime, are considered to be rather ineffective against various nerve agents. The antidotal efficacy of new oximes is primarily tested in animals for ethical reasons. However, the various interactions between AChE, OP and oximes can be investigated with human AChE which enables the direct assessment of oxime potency, thus excluding species differences. The kinetics of inhibition, reactivation and aging were investigated with human erythrocyte AChE, various structurally different OP (organophosphates, -phosphonates and phosphoramidates) and oximes (obidoxime, pralidoxime, HI 6, HLo 7). The inhibitory potency of OPs, reactivating potency of oximes and spontaneous reactivation and aging were strongly affected by the structural characteristics of the OPs and of the phosphyl-AChE-complex. The kinetic data emphasize the superior inhibitory potency of organophosphonates. AChE inhibited by various phosphoramidates was mostly resistant towards reactivation by oximes while phosphonylated AChE was easily reactivated. HLo 7 was most potent with phosphonylated AChE and obidoxime with AChE inhibited by organophosphates and phosphoramidates. With the exception of soman, OP-inhibited AChE aged rather slowly (t(1/2) 3-231 h) and reactivated spontaneously with some compounds. These results indicate that there is obviously no direct structure-activity relationship for the various interactions of human AChE, OPs and oximes.
ESTHER : Worek_2004_Biochem.Pharmacol_68_2237
PubMedSearch : Worek_2004_Biochem.Pharmacol_68_2237
PubMedID: 15498514

Title : Molar absorption coefficients for the reduced Ellman reagent: reassessment - Eyer_2003_Anal.Biochem_312_224
Author(s) : Eyer P , Worek F , Kiderlen D , Sinko G , Stuglin A , Simeon-Rudolf V , Reiner E
Ref : Analytical Biochemistry , 312 :224 , 2003
Abstract : The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.
ESTHER : Eyer_2003_Anal.Biochem_312_224
PubMedSearch : Eyer_2003_Anal.Biochem_312_224
PubMedID: 12531209

Title : Human parathion poisoning. A toxicokinetic analysis - Eyer_2003_Toxicol.Rev_22_143
Author(s) : Eyer F , Meischner V , Kiderlen D , Thiermann H , Worek F , Haberkorn M , Felgenhauer N , Zilker T , Eyer P
Ref : Toxicol Rev , 22 :143 , 2003
Abstract : The mortality rate of suicidal parathion poisoning is particularly high, the onset of fulminant cholinergic signs, and the patients frequently present to the emergency physician with life-threatening symptoms. Despite this uniformity, subsequent clinical course differs significantly among patients, mostly not as a result of different delays in treatment or insufficiency of primary care. Probably, the differences depend on the amount of poison absorbed and/or the disposition of the active poison, paraoxon. We followed the toxicokinetics of parathion and tried to quantify the actual poison load. To this end, we monitored parathion-intoxicated patients (patients requiring artificial ventilation) for plasma levels of parathion and paraoxon along with the activity of erythrocyte acetylcholinesterase and its reactivatability. Plasma obidoxime concentrations were followed as well as the cumulative urinary para-nitrophenol conjugate excretion as a measure of total poison load. All patients received a standard obidoxime scheme of a 250 mg bolus dose intravenously, followed by continuous infusion with 750 mg per 24 hours as long as reactivation could be expected (usually 1 week). All other treatment was instituted as judged by the physician. It was recommended to use atropine at low doses to achieve dry mucous membranes, no bronchoconstriction and no bradycardia. Usually 1-2 mg/h were sufficient. Seven selected cases are presented exemplifying toxicokinetic peculiarities. All patients were severely intoxicated, while the amount of parathion absorbed varied widely (between 0.12 and 4.4 g; lethal dose 0.02-0.1 g) and was generally much lower than anticipated from the reports of relatives. It remains open whether the discrepancies between reports and findings were due to exaggeration or to effective decontamination (including spontaneous vomiting, gastric lavage and activated charcoal). Absorption of parathion from the gastrointestinal tract was sometimes retarded, up to 5 days, resulting in fluctuating plasma profiles. The volume of distribution at steady-state (Vdss) of parathion was around 20 L/kg. Post-mortem analysis in one patient revealed a 66-fold higher parathion concentration in fat tissue compared with plasma, 16 days after ingestion. Biotransformation of parathion varied widely and was severely retarded in one patient receiving fluconazole during worsening of renal function, while phenobarbital (phenobarbitone) sedation (two cases) had apparently no effect. The proportion of plasma parathion to paraoxon varied from 0.3-30, pointing also to varying paraoxon elimination, as illustrated by one case with particularly low paraoxonase-1 activity. Obidoxime was effective at paraoxon concentrations below 0.5 microM, provided aging was not too advanced. This concentration correlated poorly with the paration concentration or the poison load. The data are discussed in light of the pertinent literature.
ESTHER : Eyer_2003_Toxicol.Rev_22_143
PubMedSearch : Eyer_2003_Toxicol.Rev_22_143
PubMedID: 15181664

Title : Equipotent cholinesterase reactivation in vitro by the nerve agent antidotes HI 6 dichloride and HI 6 dimethanesulfonate - Krummer_2002_Arch.Toxicol_76_589
Author(s) : Krummer S , Thiermann H , Worek F , Eyer P
Ref : Archives of Toxicology , 76 :589 , 2002
Abstract : The well-documented efficacy of HI 6 dichloride in reactivating acetylcholinesterase (AChE) inhibited by nerve agents is curtailed by its poor water-solubility at temperatures below 10 degrees C. This drawback can be circumvented by using HI 6 dimethanesulfonate, which has been developed in our laboratory. Since investigations on the efficacy of this new entity are lacking, it has been proposed that some bridging experiments be performed, aimed at demonstrating reactivator equivalence in vitro. The reactivating properties of the two salts were compared on human erythrocyte AChE inhibited with paraoxon, sarin, cyclosarin and agent VX. The comparison was extended to cynomolgus erythrocytes exposed in vitro for sarin and VX. Finally, mouse diaphragm preparations were circumfused with sarin, cyclosarin and VX and reactivation by each of the HI 6 salts was examined in muscle homogenates. AChE activity in erythrocyte suspensions was monitored by a modified Ellman procedure, muscle AChE by a radiometric assay. In all models tested no differences between the HI 6 salts could be detected ( P=0.05). From these data, equipotency in AChE reactivation by the two HI 6 salts can be anticipated.
ESTHER : Krummer_2002_Arch.Toxicol_76_589
PubMedSearch : Krummer_2002_Arch.Toxicol_76_589
PubMedID: 12373455

Title : Reactivation kinetics of acetylcholinesterase from different species inhibited by highly toxic organophosphates - Worek_2002_Arch.Toxicol_76_523
Author(s) : Worek F , Reiter G , Eyer P , Szinicz L
Ref : Archives of Toxicology , 76 :523 , 2002
Abstract : Standard treatment of poisoning by organophosphates (OP) includes the administration of an antimuscarinic agent, e.g. atropine, and of an acetylcholinesterase (AChE) reactivator (oxime). The presently available oximes, obidoxime and pralidoxime (2-PAM), are considered to be insufficient for highly toxic OPs, e.g. sarin. In the past decades numerous oximes were prepared and tested for their efficacy in OP poisoning, mostly in animal experiments. However, data indicate that the reactivating potency of oximes may be different in humans and animal species, which may hamper the extrapolation of animal data to humans and may pose a problem in the drug licensing of new compounds. In order to provide data for a better evaluation of the reactivating potency of oximes, experiments were undertaken to determine the reactivation rate constants of several oximes with human, rabbit, rat and guinea-pig AChE inhibited by the OPs sarin, cyclosarin and VX. The results show marked differences among the species, depending on the inhibitor and on the oxime, and indicate that the findings from animal experiments need careful evaluation before extrapolating these data to humans.
ESTHER : Worek_2002_Arch.Toxicol_76_523
PubMedSearch : Worek_2002_Arch.Toxicol_76_523
PubMedID: 12242610

Title : Effect of human plasma on the reactivation of sarin-inhibited human erythrocyte acetylcholinesterase - Worek_2000_Arch.Toxicol_74_21
Author(s) : Worek F , Eyer P , Kiderlen D , Thiermann H , Szinicz L
Ref : Archives of Toxicology , 74 :21 , 2000
Abstract : The reactivation of organophosphate-inhibited acetylcholinesterase (AChE) by oximes inevitably results in the formation of highly reactive phosphoryloximes (POX), which are able to re-inhibit the enzyme. In this study, the dependence of POX formation on AChE concentration was investigated with sarin-inhibited human erythrocyte AChE (EryAChE). A marked dependence was found with obidoxime but not with the experimental oxime HI 6, suggesting great differences in the decomposition rates of the respective POXs. At a physiological erythrocyte content the reactivation of EryAChE was markedly affected by POX with obidoxime and pralidoxime (2-PAM) but not with the newer oximes HI 6 and HLo 7. Addition of extensively dialysed, sarin-treated human plasma reduced the reactivation by obidoxime and 2-PAM even more. Obidoxime and 2-PAM were superior to HI 6 and HLo 7 in reactivating butyrylcholinesterase (BChE). This effect was pronounced in diluted plasma, but was obscured in concentrated plasma, probably because of re-inhibition by the generated POX. Addition of native erythrocytes to sarin-treated plasma resulted in marked inhibition of EryAChE in the presence of obidoxime, suggesting a higher affinity of the POX for EryAChE. The results indicate that obidoxime and 2-PAM may reactivate sarin-inhibited AChE insufficiently due to re-inhibition by the POX formed. In addition, the re-inhibition of Ery-AChE may be aggravated by the POX that is produced during BChE reactivation. These reactions must be regarded as therapeutically detrimental and disqualify those oximes which are capable of forming stable POX by reactivation of BChE.
ESTHER : Worek_2000_Arch.Toxicol_74_21
PubMedSearch : Worek_2000_Arch.Toxicol_74_21
PubMedID: 10817663

Title : The phosphoryl oxime-destroying activity of human plasma - Kiderlen_2000_Arch.Toxicol_74_27
Author(s) : Kiderlen D , Worek F , Klimmek R , Eyer P
Ref : Archives of Toxicology , 74 :27 , 2000
Abstract : The potential of obidoxime and other pyridinium-4-aldoximes to reactivate dimethyl- and diethylphosphorylated cholinesterases is markedly restricted by the inevitable formation of rather stable phosphoryl oximes (POXs) with high anticholinesterase activity. This effect is hardly seen with very dilute enzyme preparations, but becomes significant at physiological enzyme concentrations. Human plasma with the butyrylcholinesterase irreversibly blocked by soman was able to stimulate obidoxime-induced reactivation of concentrated erythrocyte acetylcholinesterase (Ery-AChE) to the same extent as was observed with a dilute preparation, suggesting phosphoryl oxime-destroying capacity. The inactivating factor, which was tentatively termed POX-hydrolase, had (1) a molecular weight of >100 kDa; (2) required Ca2+ , which could not be substituted by Zn2+ or Mg2+; and (3) lost its catalytic activity reversibly in the presence of ethylenediamine-tetraacetic acid (EDTA). The enzyme activity varied widely (20-fold) among different subjects and did not follow the activity pattern of human serum paraoxonase (PON1). Rabbit plasma with its particularly high paraoxonase content showed only weak POX-hydrolase activity. These data suggest POX-hydrolase to be a different entity. POX-hydrolase was most active with the putative phosphoryl-obidoxime from paraoxon-ethyl, less with the product from paraoxon-methyl and least with that from diisopropylfluorophosphate. The analogue TMB-4 reacted similarly to obidoxime. The putative phosphonyl oximes arising by the reaction of obidoxime with nerve agents were apparently not cleaved. The variation in POX-hydrolase activity may additionally contribute to the variable response to oxime therapy in patients with organophosphate insecticide poisoning.
ESTHER : Kiderlen_2000_Arch.Toxicol_74_27
PubMedSearch : Kiderlen_2000_Arch.Toxicol_74_27
PubMedID: 10817664

Title : Intoxication with huperzine A, a potent anticholinesterase found in the fir club moss - Felgenhauer_2000_J.Toxicol.Clin.Toxicol_38_803
Author(s) : Felgenhauer N , Zilker T , Worek F , Eyer P
Ref : J Toxicol Clinical Toxicology , 38 :803 , 2000
Abstract : BACKGROUND: Herbs from Lycopodium are generally reputed to be nontoxic and are occasionally used for preparing a salubrious tea. In Europe, the common Lycopodium clavatum can be easily confused with Lycopodium selago, the fir club moss. CASE REPORT: We report 2 patients who drank a tea, erroneously prepared from dried herbs of Lycopodium selago, which resulted in sweating, vomiting, diarrhea, dizziness, cramps, and slurred speech. These symptoms were suggestive of a cholinergic mechanism. To elucidate the active principle, aqueous extracts of Lycopodium selago were checked for their suspected anticholinesterase activity using human erythrocytes as an enzyme source in a modified Ellman assay. The extracts did exhibit significant anticholinesterase activity. The anticholinesterase(s) were most effectively extracted with dichloromethane and isolated by high-performance liquid chromatography. The major compound with anticholinesterase activity co-chromatographed with authentic huperzine A, but had a 2-3-fold higher inhibitory potency than the racemic standard. The amount of huperzine A found in the Lycopodium selago sample used for the tea preparation was calculated to be sufficient for a relevant acetylcholinesterase inhibition. CONCLUSION: The signs and symptoms of Lycopodium selago poisoning are consistent with the anticholinesterase activity of huperzine A and should favorably respond to atropine therapy. This report demonstrates once more that laymen should not be encouraged to gather their remedies from "Mother Nature" without advanced botanical knowledge.
ESTHER : Felgenhauer_2000_J.Toxicol.Clin.Toxicol_38_803
PubMedSearch : Felgenhauer_2000_J.Toxicol.Clin.Toxicol_38_803
PubMedID: 11192470

Title : Modern strategies in therapy of organophosphate poisoning - Thiermann_1999_Toxicol.Lett_107_233
Author(s) : Thiermann H , Szinicz L , Eyer F , Worek F , Eyer P , Felgenhauer N , Zilker T
Ref : Toxicol Lett , 107 :233 , 1999
Abstract : Considering the various microscopic reactions as well as toxicokinetic and pharmacokinetic principles in therapy of organophosphate poisoning, the administration of obidoxime by an initial bolus dose followed by continuous infusion appears rational. Using this protocol, six patients each with parathion or oxydemeton methyl poisoning were treated. In parathion poisoning, reactivation was possible up to 7 days. At paraoxon concentrations > 0.1 microM obidoxime only partially reactivated acetylcholinesterase (AChE) of erythrocytes in vivo although reactivation could be assessed in vitro, which roughly fitted theoretical calculations. AChE-inhibitory material was detected up to 5 days. Cholinergic signs soon subsided when AChE was above 20% of normal, and atropine plasma levels could be kept below 7 ng/ml. In one patient brain damage persisted. Oxydemeton methyl poisoning responded to obidoxime therapy only when the oxime was instituted shortly after poisoning. Out of six patients one died. No intermediate syndrome and no signs of permanent hepatic dysfunction were found in the 12 patients.
ESTHER : Thiermann_1999_Toxicol.Lett_107_233
PubMedSearch : Thiermann_1999_Toxicol.Lett_107_233
PubMedID: 10414801

Title : Dimethylphosphoryl-inhibited human cholinesterases: inhibition, reactivation, and aging kinetics - Worek_1999_Arch.Toxicol_73_7
Author(s) : Worek F , Diepold C , Eyer P
Ref : Archives of Toxicology , 73 :7 , 1999
Abstract : Human poisoning by organophosphates bearing two methoxy groups, e.g. by malathion, paraoxon-methyl, dimethoate and oxydemeton-methyl, is generally considered to be rather resistant to oxime therapy. Since the oxime effectiveness is influenced not only by its reactivating potential but also by inhibition, aging and spontaneous reactivation kinetics, experiments were performed with human acetyl- (AChE) and butyrylcholinesterase (BChE) to determine the respective kinetic constants. The efficacy of obidoxime in reactivating dimethylphosphoryl-AChE was 40, 9 and 3 times higher than of HI 6, pralidoxime and HL 7, respectively. Aging (t1/2 3.7 h) and spontaneous reactivation (t1/2 0.7 h) occurred concomitantly, with the portion of the aged enzyme being dependent on the presence of excess inhibitor. Calculation of steady-state AChE activity in the presence of inhibitor and oxime revealed that obidoxime was superior to pralidoxime. In addition, organophosphate concentrations up to 10(-6) M (paraoxon-methyl) and 10(-4) M (oxydemeton-methyl) could be counteracted at clinically relevant oxime concentrations (10 microM). These data indicate that oximes may effectively reactivate human dimethylphosphoryl-AChE. Failure of oximes may be attributed to megadose intoxications and to prolonged time intervals between poison uptake and oxime administration. The potency of the oximes to reactivate dimethylphosphoryl-BChE was much lower and the spontaneous reactivation slower (t1/2 9 h), while aging proceeded at a comparable rate. Thus, BChE activity determination for diagnosis and therapeutic monitoring may give no reliable information on AChE status.
ESTHER : Worek_1999_Arch.Toxicol_73_7
PubMedSearch : Worek_1999_Arch.Toxicol_73_7
PubMedID: 10207609

Title : Improved determination of acetylcholinesterase activity in human whole blood - Worek_1999_Clin.Chim.Acta_288_73
Author(s) : Worek F , Mast U , Kiderlen D , Diepold C , Eyer P
Ref : Clinica Chimica Acta , 288 :73 , 1999
Abstract : Determination of erythrocyte acetylcholinesterase (AChE) activity is the appropriate tool for the diagnosis of organophosphate exposure and intoxication. The original colorimetric Ellman procedure is disturbed by a high hemoglobin absorption at 412 nm. In our modified method the wavelength was changed to 436 nm. This reduced the indicator absorption to 80% and the hemoglobin absorption to 25%. The signal-to-noise ratio was further enhanced by reduction of pH and substrate concentration, thus making it possible to measure 3% residual activity. AChE activity was determined in whole blood samples in the presence of the selective butyrylcholinesterase inhibitor ethopropazine. Dilution of blood samples (1:100) stops secondary reactions in the presence of inhibitor (organophosphate) and reactivator (oxime). Normalization of the AChE activity to the hemoglobin content, determined as cyanmethemoglobin, prevented dilution errors. This modified approach provides a simple way for sensitive and precise determination of AChE activity in whole blood in the presence of organophosphates even with low-tech equipment.
ESTHER : Worek_1999_Clin.Chim.Acta_288_73
PubMedSearch : Worek_1999_Clin.Chim.Acta_288_73
PubMedID: 10529460

Title : Reactivating potency of obidoxime, pralidoxime, HI 6 and HLo 7 in human erythrocyte acetylcholinesterase inhibited by highly toxic organophosphorus compounds - Worek_1998_Arch.Toxicol_72_237
Author(s) : Worek F , Widmann R , Knopff O , Szinicz L
Ref : Archives of Toxicology , 72 :237 , 1998
Abstract : The treatment of poisoning by highly toxic organophosphorus compounds (nerve agents) is unsatisfactory. Until now, the efficacy of new potential antidotes has primarily been evaluated in animals. However, the extrapolation of these results to humans is hampered by species differences. Since oximes are believed to act primarily through reactivation of inhibited acetylcholinesterase (AChE) and erythrocyte AChE is regarded to be a good marker for the synaptic enzyme, the reactivating potency can be investigated with human erythrocyte AChE in vitro. The present study was undertaken to evaluate the ability of various oximes at concentrations therapeutically relevant in humans to reactivate human erythrocyte AChE inhibited by different nerve agents. Isolated human erythrocyte AChE was inhibited with soman, sarin, cyclosarin, tabun or VX for 30 min and reactivated in the absence of inhibitory activity over 5-60 min by obidoxime, pralidoxime, HI 6 or HL 7 (10 and 30 microM). The AChE activity was determined photometrically. The reactivation of human AChE by oximes was dependent on the organophosphate used. After soman, sarin, cyclosarin, or VX the reactivating potency decreased in the order HL 7 > HI 6 > obidoxime > pralidoxime. Obidoxime and pralidoxime were weak reactivators of cyclosarin-inhibited AChE. Only obidoxime and HL 7 reactivated tabun-inhibited AChE partially (20%), while pralidoxime and HI 6 were almost ineffective (5%). Therefore, HL 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans.
ESTHER : Worek_1998_Arch.Toxicol_72_237
PubMedSearch : Worek_1998_Arch.Toxicol_72_237
PubMedID: 9587020

Title : Inhibition, reactivation and aging kinetics of cyclohexylmethylphosphonofluoridate-inhibited human cholinesterases - Worek_1998_Arch.Toxicol_72_580
Author(s) : Worek F , Eyer P , Szinicz L
Ref : Archives of Toxicology , 72 :580 , 1998
Abstract : Cyclohexylmethylphosphonofluoridate (cyclosarin) is a highly toxic organophosphate, which was shown to be rather resistant to conventional oxime therapy. To give more insight into the inhibition, reactivation and aging kinetics, human acetyl-(AChE) and butyrylcholinesterase (BChE) were inhibited by cyclosarin (k2 of 7.4 and 3.8 x 10(8) M(-1) min(-1), respectively; pH 7.4, 37 degrees C) and reactivated with obidoxime, pralidoxime and three experimental oximes. The new oxime HL 7 (1-[[[4-aminocarbonyl)-pyridinio]-methoxy]-methyl]-2,4-bis-[ (hydroxyimino)methyl] pyridinium dimethanesulphonate) was shown to be superior to the other oximes. At oxime concentrations anticipated to be relevant in humans, obidoxime and pralidoxime were extremely weak reactivators of AChE. Aging velocity of BChE was almost fourfold higher compared to AChE (ka of 0.32 h(-1) and 0.08 h(-1), respectively). A substantial spontaneous reactivation was observed with AChE. These results support previous in vivo findings that obidoxime and pralidoxime are insufficient antidotes in cyclosarin poisoning. By contrast, HL 7 was shown to be an extremely potent reactivator of human AChE and BChE, which supports its position as a broad-spectrum oxime.
ESTHER : Worek_1998_Arch.Toxicol_72_580
PubMedSearch : Worek_1998_Arch.Toxicol_72_580
PubMedID: 9806430

Title : Acetylcholinesterase, a Versatile Protein -
Author(s) : Szinicz L , Thiermann H , Worek F , Eyer P
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :269 , 1998
PubMedID:

Title : Reappraisal of indications and limitations of oxime therapy in organophosphate poisoning - Worek_1997_Hum.Exp.Toxicol_16_466
Author(s) : Worek F , Backer M , Thiermann H , Szinicz L , Mast U , Klimmek R , Eyer P
Ref : Hum Exp Toxicol , 16 :466 , 1997
Abstract : 1 In vitro studies with human erythrocyte acetylcholinesterase (AChE) and the mouse diaphragm model were performed to unravel the various microscopic reaction parameters that contribute to the dynamic equilibrium of AChE inhibition, ageing and reactivation. These data may help to define more precisely the indications and limitations of oxime therapy in organophosphate (OP) poisoning. 2 Diethylphosphoryl-AChE resulting from intoxications with parathion, chlorpyrifos, chlorfenvinphos, diazinon and other OPs is characterized by slow spontaneous reactivation and low propensity for ageing. This kind of phosphorylated enzyme is particularly susceptible to reactivation by oximes. 3 None of the oximes tested (pralidoxime, obidoxime, HI 6 and HLo 7) can be regarded as a universally suitable reactivator. Obidoxime turned out to be the most potent and most efficacious oxime in reactivating AChE inhibited by various classes of OP insecticides and tabun. Obidoxime, however, was inferior to HI 6 against soman, sarin, cyclosarin and VX. Pralidoxime was generally less potent. 4 The kinetic data of reactivation established for diethylphosphoryl-AChE of human red cells indicate that the usually recommended dosage to attain a plasma concentration of 4 micrograms/ml does not permit exploitation of the full therapeutic potential of the oximes, in particular of pralidoxime. However, in suicidal mega-dose poisoning, oximes, even at optimal plasma concentrations, may be unable to cope with the fast re-inhibition of reactivated AChE in the first days following intoxication. 5 It is suggested that oximes be administered by continuous infusion following an initial bolus dose as long as reactivation can be expected and until permanent clinical improvement is achieved.
ESTHER : Worek_1997_Hum.Exp.Toxicol_16_466
PubMedSearch : Worek_1997_Hum.Exp.Toxicol_16_466
PubMedID: 9292287

Title : Factors influencing the efficacy of obidoxime in organophosphate pesticides poisoning [editorial] -
Author(s) : Zilker T , Felgenhauer N , Hibler A , Pfab R , Thiermann H , Worek F , Eyer P
Ref : Przegl Lek , 54 :662 , 1997
PubMedID: 9478082

Title : Reactivation by various oximes of human erythrocyte acetylcholinesterase inhibited by different organophosphorus compounds - Worek_1996_Arch.Toxicol_70_497
Author(s) : Worek F , Kirchner T , Backer M , Szinicz L
Ref : Archives of Toxicology , 70 :497 , 1996
Abstract : The new bispyridinium oximes HI 6 and HL 7 are promising antidotes against poisoning by highly toxic organophosphorus compounds, i.e. nerve agents. Until now, their ability to reactivate pesticide inhibited human acetylcholinesterase (AChE) has not been elucidated. For this purpose human erythrocyte AChE (EC 3.1.1.7) was inhibited (30 min) by chlorfenvinphos, dichlorvos, dicrotophos, heptenophos, mevinphos, monocrotophos, paraoxon, phosphamidon, trichlorfon, malaoxon, omethoate, oxydemeton-methyl or methamidophos by 85-98% of control. After removal of excess inhibitor, obidoxime, pralidoxime (2-PAM), HI 6 or HL 7 (10, 30 or 100 mumol/l) were added and the AChE activity was measured spectrophotometrically at various times thereafter (5-60 min). The oximes significantly, but not completely, reactivated organophosphate inhibited AChE. The velocity and extent of reactivation were dependent on the oxime and its concentration. In all cases obidoxime was superior to the three other oximes, followed by HL 7, 2-PAM and HI 6. In most cases obidoxime and HL 7 were most effective at 10 or 30 mumol/l while 2-PAM and HI 6 needed 100 mumol/l. These data suggest that 2-PAM HI 6 and HL 7 are less patent than obidoxime in reactivating human AChE inhibited by organophosphate pesticides.
ESTHER : Worek_1996_Arch.Toxicol_70_497
PubMedSearch : Worek_1996_Arch.Toxicol_70_497
PubMedID: 8783813

Title : Cardiorespiratory function in nerve agent poisoned and oxime + atropine treated guinea-pigs: effect of pyridostigmine pretreatment - Worek_1995_Arch.Toxicol_69_322
Author(s) : Worek F , Szinicz L
Ref : Archives of Toxicology , 69 :322 , 1995
Abstract : The effect of pyridostigmine pretreatment on the efficacy of oxime + atropine treatment in sarin and VX poisoning was investigated in a guinea-pig model with on-line monitoring of respiratory and circulatory parameters. The carotid artery, jugular vein and trachea were cannulated in female urethane-anesthetized Pirbright-white guinea-pigs. After baseline measurements the animals received pyridostigmine (PYR, 0.05 mumol/kg, i.v.), 30 min later sarin (100 or 200 micrograms/kg = 5 or 10 x LD50, i.v.) or VX (45 or 90 micrograms/kg = 10 or 20 x LD50, i.v.), followed 2 min later by atropine (10 mg/kg, i.v.) plus HI 6 or HL 7 (30 mumol/kg each, i.v.). Sixty-minute survival time and rate and respiratory and circulatory parameters were recorded. Diaphragm acetylcholinesterase (AChE) activity was determined spectrophotometrically. Identical groups without PYR were included for comparison. With regard to survival time and rate, PYR pretreatment slightly improved the efficacy of HI 6 plus atropine in sarin 5 x LD50 poisoned animals, reduced the efficacy of oxime + atropine treatment in the other sarin groups and had no effect in VX poisoning. Compared to non-pretreated oxime + atropine groups, PYR slightly improved respiratory function in sarin and in VX poisoning (only HI 6). PYR did not affect circulatory function in VX poisoning but reduced circulatory parameters in sarin poisoning to varying extent's. The oxime efficacy in reactivating diaphragm AChE decreased in the order sarin > VX without significant differences between pretreated and non-pretreated groups. The data suggest that pyridostigmine pretreatment does not enhance the efficacy of oxime + atropine in sarin or VX poisoning. poisoning
ESTHER : Worek_1995_Arch.Toxicol_69_322
PubMedSearch : Worek_1995_Arch.Toxicol_69_322
PubMedID: 7654137

Title : Effect of pyridostigmine pretreatment on cardiorespiratory function in tabun poisoning - Worek_1995_Human.Exp.Toxicol_14_634
Author(s) : Worek F , Kleine A , Szinicz L
Ref : Hum Exp Toxicol , 14 :634 , 1995
Abstract : 1. The effect of pyridostigmine on cardiorespiratory function after oxime + atropine injection was investigated in tabun poisoned guinea-pigs and without tabun poisoning. 2. The trachea, a carotid artery and jugular vein were cannulated in female urethane-anaesthetised Pirbright-white guinea-pigs. After baseline measurements the animals received pyridostigmine (0.05 mumol kg-1) and 30 min later atropine (29.5 mumol kg-1) plus obidoxime, HI 6 or HL 7 (30 or 100 mumol kg-1) or tabun (1.85 mumol kg-1 = 5 x LD50) followed by oxime + atropine treatment (all i.v.). Erythrocyte, brain and diaphragm acetylcholinesterase (AChE) activity were determined. Similar groups without pretreatment were included for comparison. 3. Pyridostigmine aggravated the oxime + atropine induced hypotension and prevented the increase in heart rate but not the respiratory stimulation. The pyridostigmine inhibited AChE recovered only in the 100 mumol kg-1 kg oxime groups at the end of the experiment. 4. In tabun poisoning, pyridostigmine reduced the oxime + atropine induced circulatory recovery and decreased the survival time and rate. It did not affect the therapeutic oxime + atropine effect on respiratory function. 5. These results suggest that pyridostigmine enhances oxime + atropine related circulatory depression which may be the reason for the reduced efficacy of oxime + atropine treatment in tabun poisoning. The possible mechanisms are discussed.
ESTHER : Worek_1995_Human.Exp.Toxicol_14_634
PubMedSearch : Worek_1995_Human.Exp.Toxicol_14_634
PubMedID: 7576830

Title : Effect of atropine and bispyridinium oximes on respiratory and circulatory function in guinea-pigs poisoned by sarin - Worek_1995_Toxicology_95_123
Author(s) : Worek F , Kirchner T , Szinicz L
Ref : Toxicology , 95 :123 , 1995
Abstract : During the past decade the oxime HI 6(1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2- [(hydroxyimino)methyl] pyridinium dichloride) was shown to improve survival in nerve agent poisoning (in combination with atropine). Recent studies indicate, that HL 7 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis [(hydroxyimino)methyl] pyridinium diiodide or dimethanesulfonate) is also an effective antidote in nerve agent poisoning but, with both oximes, data on restoration of respiration and circulation are scarce. The ability of HL 7 or HI 6 with atropine to improve the respiratory and circulatory function in sarin-poisoned guinea-pigs was therefore investigated. Female Dunkin-Hartley guinea-pigs were anaesthetised with urethane (1.8 g/kg) and the arteria carotis, vena jugularis and trachea were cannulated. After baseline measurements the animals received 100 or 200 micrograms/kg sarin, and 2 min later the antidotes (all i.v.): 10 mg/kg atropine sulfate or a combination of atropine and HL 7 or HI 6 (30 mumol/kg, each). Respiratory and circulatory parameters were recorded for the whole experimental period of 60 min or until the death of the animal. Brain and diaphragm acetylcholinesterase (AChE) activity was determined in each animal after the experiment. Poisoning by sarin resulted in a rapid respiratory arrest within 5 min. Atropine treatment was only partially effective in improving respiration after 100 micrograms/kg sarin but was ineffective after 200 micrograms/kg sarin. Therapy of sarin-poisoned animals with atropine plus oxime further improved respiration to various extents, restored circulation and increased survival time, HL 7 being more effective than HI 6. Diaphragm and brain AChE were reactivated by HL 7 and, to a minor extent, by HI 6. The results of this investigation suggest, that at equimolar doses (30 mumol/kg) the new bispyridinium dioxime HL 7 has a higher therapeutic efficacy in sarin-poisoned guinea-pigs when compared to HI 6 (both in combination with atropine).
ESTHER : Worek_1995_Toxicology_95_123
PubMedSearch : Worek_1995_Toxicology_95_123
PubMedID: 7825179

Title : Effects of atropine and soman on the pharmacokinetics of the oxime HLo 7 dimethanesulfonate in anesthetized guinea-pigs - Worek_1995_Drug.Chem.Toxicol_18_137
Author(s) : Worek F , Klimmek R , Szinicz L
Ref : Drug & Chemical Toxicology , 18 :137 , 1995
Abstract : The respiratory and circulatory effects and the pharmacokinetics of the bispyridinium dioxime HL 7 (30 mumol/kg) were investigated in guinea-pigs in combination with atropine (28.8 mumol/kg) or with atropine plus soman (0.44 mumol/kg = 5 x LD50). The pharmacokinetics and pharmacodynamics were studied in separate groups of animals. HL 7 had only minor effects on circulation and respiration. HL 7 plus atropine caused a transient, pronounced fall of blood pressure, tachycardia, and respiratory stimulation. The soman-induced bradycardia was completely restored by the antidotes, while respiration improved within a few minutes with the respiratory rate becoming stabilized at 50% of baseline. Plasma half-time (60-70 min), plasma clearance (6-7 ml.kg-1.min-1) and apparent volume of distribution (0.5-0.6 l/kg) of HL 7 did not differ between the groups, i.e. atropine and soman did not affect the kinetics of the oxime.
ESTHER : Worek_1995_Drug.Chem.Toxicol_18_137
PubMedSearch : Worek_1995_Drug.Chem.Toxicol_18_137
PubMedID: 7497908

Title : Arrhythmias in organophosphate poisoning: effect of atropine and bispyridinium oximes - Worek_1995_Arch.Int.Pharmacodyn.Ther_329_418
Author(s) : Worek F , Kleine A , Falke K , Szinicz L
Ref : Archives Internationales de Pharmacodynamie et de Therapie , 329 :418 , 1995
Abstract : The effect of atropine and of the bispyridinium oximes, HI6 and HL 7, on the electrocardiographic pattern was investigated in acutely nerve agent-poisoned guinea-pigs. The electrocardiographic, circulatory and respiratory parameters were recorded in female urethane-anaesthetized Pirbright-white guinea-pigs. After base line measurements, the animals received pyridostigmine (0.05 mumol/kg) and, 30 min later, tabun (5xLD50), sarin (5xLD50), soman (5xLD50 or 10xLD50) or VX (10xLD50 or 20xLD50), followed by saline or atropine (10 mg/kg) or atropine plus HI 6 or or HL 7 (30 mumol/kg) 2 minutes later. Nerve agent poisoning resulted in respiratory arrest within 2-3 minutes, followed by circulatory arrest a few minutes later in nontreated animals. Antidote treatment rapidly restored heart rate and mean arterial pressure and improved the respiratory function to various extent. The nerve agent injection caused a marked sinus bradycardia and a subsequent complete atrioventricular block within 1-2 minutes, followed by idioventricular rhythm. No ventricular tachyarrhythmias were observed in these groups just before death. Atropine and atropine plus oxime administration immediately restored sinus rhythm which persisted in animals with sufficient respiration > 50% of base line) throughout the observation period (60 minutes). In guinea-pigs with depressed respiratory function ( < 50%), intermittent ST-T wave alterations and second degree atrioventricular block were observed. In some cases, especially in tabun and soman (10xLD50) poisoning, sinus rhythm converted to deleterious ventricular tachycardia within 1 minute after treatment. These results suggest that atropine-containing antidote combinations may induce lethal arrhythmias in nerve agent poisoning, which may be of clinical importance during intravenous treatment of severe inhalative intoxications.
ESTHER : Worek_1995_Arch.Int.Pharmacodyn.Ther_329_418
PubMedSearch : Worek_1995_Arch.Int.Pharmacodyn.Ther_329_418
PubMedID: 8546540

Title : Treatment of tabun poisoned guinea-pigs with atropine, HLo 7 or HI 6: effect on respiratory and circulatory function - Worek_1994_Arch.Toxicol_68_231
Author(s) : Worek F , Kirchner T , Szinicz L
Ref : Archives of Toxicology , 68 :231 , 1994
Abstract : The oxime HI 6 (in combination with atropine) is considered to be an effective antidote in soman intoxication but was shown to be less effective in tabun poisoning. In contrast to HI 6, first in vitro studies with HL 7 demonstrated a reasonable reactivating potency at acetylcholinesterase (AChE) inhibited by soman and tabun. Therefore, the therapeutic efficacy of HL 7, HI 6 and obidoxime (with and without atropine) was compared in tabun poisoned guinea-pigs. In addition, the therapeutic effect of atropine in guinea-pigs poisoned by various doses of tabun was investigated. Female Pirbright-white guinea-pigs were anaesthetized with urethane (1.8 g/kg) and the carotid artery, jugular vein and trachea were cannulated. After baseline measurements the animals received tabun, 60, 180 or 300 micrograms/kg, and 2 min later the antidotes (all i.v.): obidoxine, HL 7, or HI 6 (30 or 100 mumol/kg, each) or atropine 10 mg/kg or a combination of atropine and one of the oximes. Respiratory and circulatory parameters were recorded for 60 min or until the death of the animal. Erythrocyte, brain and diaphragm AChE activity was determined in every animal after the experiment. Poisoning by tabun resulted in a rapid deterioration of respiratory function and respiratory arrest within 5 min. Atropine treatment was very effective in improving the respiratory function after tabun 60 micrograms/kg but was ineffective after tabun 300 micrograms/kg. However, circulatory parameters were restored almost completely in all atropine therapy groups. Therapy of tabun 300 microns/kg poisoned animals with atropine plus oxime (30 micromol/kg) improved respiration to a variable extent and restored circulation.
ESTHER : Worek_1994_Arch.Toxicol_68_231
PubMedSearch : Worek_1994_Arch.Toxicol_68_231
PubMedID: 8067895

Title : Effect of atropine, HLo 7 and HI 6 on respiratory and circulatory function in guinea-pigs poisoned by O-ethyl S-[2-(diisopropylamino) ethyl] methylphosponothioate (VX) - Worek_1994_Pharmacol.Toxicol_75_302
Author(s) : Worek F , Kirchner T , Szinicz L
Ref : Pharmacol Toxicol , 75 :302 , 1994
Abstract : In a guinea-pig model with on-line respiratory and circulatory monitoring the therapeutic efficacy of atropine, HL 7 and HI 6 in VX poisoning was compared. In female urethane-anaesthetized Pirbright-white guinea-pigs the a. carotis, v. jugularis and trachea were cannulated. After base line measurements the animals received VX (22.5, 45 or 90 micrograms/kg = 5, 10 or 20 x LD50) intravenously and 2 min. later the antidotes: HL 7 or HI 6 (30 mumol/kg, each) or atropine 10 mg/kg or a combination of atropine and one of the oximes (all intravenously). Respiratory and circulatory parameters were recorded for 60 min. or until death of the animal. Erythrocyte, brain and diaphragm acetylcholinesterase (AChE) activity was determined after the experiment. VX poisoning caused a rapid respiratory arrest within 4-5 min. Atropine treatment was effective in improving the respiratory function after VX, 22.5 micrograms/kg, but had only a small effect after the higher VX doses. The treatment of VX (10 or 20 x LD50) poisoned animals with oxime plus atropine improved respiration to various extents, restored circulation and prolonged the survival time, HL 7 being more effective than HI 6 after VX 90 micrograms/kg. Oximes alone were completely ineffective. Erythrocyte and diaphragm AChE was reactivated by HL 7 and, less effectively, by HI 6, while brain AChE remained almost completely inhibited in all groups. The results of this investigation demonstrate a reasonable efficacy of atropine after lower VX doses and of HL 7 and HI 6 (plus atropine) after high-dose VX poisoning, HL 7 being slightly more effective than HI 6.
ESTHER : Worek_1994_Pharmacol.Toxicol_75_302
PubMedSearch : Worek_1994_Pharmacol.Toxicol_75_302
PubMedID: 7870702

Title : Investigation of acute cardiovascular and respiratory toxicity of HLo 7 dimethanesulfonate and HI 6 dichloride in anaesthetized guinea-pigs - Worek_1993_Pharmacol.Toxicol_73_91
Author(s) : Worek F , Szinicz L
Ref : Pharmacol Toxicol , 73 :91 , 1993
Abstract : The bis-pyridinium dioxime HL 7 is considered to possess promising therapeutic properties in the treatment of organophosphate poisoning. Acute circulatory and respiratory effects of HL 7 and HI 6 were therefore compared in anaesthetized guinea-pigs. Female Pirbright white guinea-pigs were anaesthetized with urethane and the carotid artery, jugular vein and trachea were cannulated. Saline or atropine, 10 mg/kg, or HL 7 or HI 6 (30 or 100 mumol/kg, each) or atropine plus oxime were injected intravenously after base line measurements. Respiratory and circulatory parameters were recorded for 60 min., then blood was drawn for AChE measurement. Injection of HL 7 or HI 6 alone resulted in a temporary, dose-dependent hypotension, an almost unchanged heart rate and a slight respiratory stimulation. A more severe hypotension appeared after the administration of atropine plus HL 7 or HI 6. In these groups heart rate and respiration were markedly stimulated. Measurement of AChE activity in blood samples revealed no impairment by HL 7 or HI 6 with or without atropine. These results suggest that HL 7 has only transient effects on the cardiorespiratory system after intravenous administration and its safety regarding acute circulatory and respiratory toxicity is comparable to HI 6.
ESTHER : Worek_1993_Pharmacol.Toxicol_73_91
PubMedSearch : Worek_1993_Pharmacol.Toxicol_73_91
PubMedID: 8248012

Title : Atropine and oxime treatment in lethal soman poisoning of anaesthetized guinea-pigs: HLo 7 dimethanesulfonate versus HI 6 dichloride - Worek_1993_Pharmacol.Toxicol_72_13
Author(s) : Worek F , Szinicz L
Ref : Pharmacol Toxicol , 72 :13 , 1993
Abstract : The oxime HI 6 is considered to be effective in soman poisoning and less effective in tabun poisoning. Recently, HL 7 was shown to reactivate acetylcholinesterase (AChE) inhibited by soman and tabun. Therefore, the efficacy of HL 7 and HI 6 was compared in soman poisoned guinea-pigs. Female Pirbright-white guinea-pigs were anaesthetized with urethane (1.8 g/kg) and the a. carotis, v. jugularis and trachea were cannulated. After base line measurements soman 0.08 mg/kg (= 5 x LD50) or 0.16 mg/kg (= 10 x LD50) was injected intravenously, 2 min. later the antidotes were applied intravenously: HL 7 0.03 or 0.1 mmol/kg, HI 6 0.03 or 0.1 mmol/kg, atropine 10 mg/kg, or a combination of atropine and an oxime. Respiratory and circulatory parameters were recorded for 60 min. or until the death of the animal. The injection of 5 x LD50 soman resulted in a rapid respiratory arrest followed by circulatory failure in the soman and soman plus oxime groups (survival time about 7 min). Atropine restored the circulatory parameters to base line but was unable to provide a sufficient respiratory function (survival time 26 min.). The combination therapy with atropine plus HL 7 or HI 6 improved the respiration sufficiently, restored the circulation completely, and prolonged the survival time to about 50 min. Atropine treatment was insufficient in animals poisoned with 10 x LD50 soman. The combination of atropine and HL 7 or HI 6 improved respiration, circulation, and survival time to various extent. Despite of the striking therapeutic effect no reactivation of erythrocyte AChE by the antidotes was observed.
ESTHER : Worek_1993_Pharmacol.Toxicol_72_13
PubMedSearch : Worek_1993_Pharmacol.Toxicol_72_13
PubMedID: 8441735

Title : HLo 7 dimethanesulfonate, a potent bispyridinium-dioxime against anticholinesterases - Eyer_1992_Arch.Toxicol_66_603
Author(s) : Eyer P , Hagedorn I , Klimmek R , Lippstreu P , Loffler M , Oldiges H , Spohrer U , Steidl I , Szinicz L , Worek F
Ref : Archives of Toxicology , 66 :603 , 1992
Abstract : HL 7 dimethanesulfonate (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis [(hydroxyimino)methyl]pyridinium dimethanesulfonate) is a broad-spectrum reactivator against highly toxic organophosphorus compounds. The compound was synthesized by a new route with the carcinogenic bis(chloromethyl)ether being substituted by the non-mutagenic bis(methylsulfonoxymethyl)ether. The very soluble dimethanesulfonate of obidoxime was also prepared by this way. HL 7 dimethanesulfonate is the first water-soluble salt of HL 7 that should be suitable for the wet/dry autoinjector technology, because aqueous solutions of HL 7 are not very stable (calculated shelf-life 0.2 years when stored at 8 degrees C, 1 M solution, pH 2.5). The crystalline preparation contains 96% of the syn/syn-isomer, less than 2% of the syn/anti-isomer and some minor identified by-products. HL 7 was very efficient in reactivating acetylcholinesterase (AChE) blocked by organophosphates as long as ageing did not prevent dephosphylation. HL 7 was superior to HI 6 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2- [(hydroxyimino)methyl]pyridinium dichloride) in reactivating soman and sarin-inhibited AChE from erythrocytes, and literature data indicate that HL 7 exceeds HI 6 by far in reactivating tabun-inhibited AChE. In atropine-protected, soman-poisoned mice HL 7 was three times more potent than HI 6 (protective ratio 5 versus 2.5), and in sarin-poisoned mice HL 7 was 10 times more potent than HI 6 (protective ratio 8 for both oximes). In atropine-protected guinea-pigs HL 7 was less effective than HI 6 (protective ratio: 2.3 versus 5.2 for soman; 5.2 versus 6.8 for sarin; 4.3 versus 3.8 for tabun). The mean survival time of anaesthetized guinea-pigs exposed to 5 LD50 soman (6.3 min) was increased by atropine (27 min) and atropine + HL (57 min). HL 7 alone did not prolong the survival. The most impressive effect of HL 7 was on respiration: 3 min after i.v. injection of HL 7 and atropine, the depressed respiration increased rapidly to 60% of control and remained at that level during the observation period (60 min). With atropine alone, respiration recovered only slowly. Behavioural and physiologic parameters were determined in atropine-protected mice exposed to a sublethal soman dose. The running performance was significantly improved by HL 7. Even central symptoms, e.g. hypothermia and convulsions, were decreased markedly by HL 7 (evaluation 60 min after poisoning). The pharmacokinetic data for HL 7 in male beagle dogs are similar to those of HI 6.
ESTHER : Eyer_1992_Arch.Toxicol_66_603
PubMedSearch : Eyer_1992_Arch.Toxicol_66_603
PubMedID: 1482283