Koller M

References (20)

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 : 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 : 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 : 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 : 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 : 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 : 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 : 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 : Purity of antidotal oxime HI-6 DMS as an active pharmaceutical ingredient for auto-injectors and infusions - Bogan_2012_Drug.Test.Anal_4_199
Author(s) : Bogan R , Koller M , Klaubert B
Ref : Drug Test Anal , 4 :199 , 2012
Abstract : As reactivators of inhibited acetylcholinesterase, oximes are essential antidotes in poisoning by organophosphorus compounds. Due to its superior efficacy in cases of soman, cyclosarin, and sarin poisoning, the oxime HI-6 represents a promising option for an active pharmaceutical ingredient (API) in the further development of antidote therapy for nerve agent poisoning. Developmental lots of HI-6 DMS (dimethanesulfonate) provided by different manufacturers were examined with respect to their content and purity with a view to their future use as an API. There are distinct differences in the HI-6 content from three manufacturers. With respect to purity, gradual differences arise with the known synthetic by-products as well as with unknown accompanying compounds. It became apparent that in the case of a modified synthesis using protective groups, the proportion of some synthesis by-products decreases considerably. With one exception, they are thus below the reporting threshold for API in accordance with pertinent regulatory guidelines. In HI-6, an unknown impurity always occurs, whose percentage necessitates identification due to regulations. This unknown impurity, which has not been described so far, could be identified as an isomer. These findings supply data required for the description of pharmaceutical quality in accordance with module 3 of a Common Technical Document (CTD). They thus contribute to the marketing authorization of this substance as an API for auto-injectors and infusions.
ESTHER : Bogan_2012_Drug.Test.Anal_4_199
PubMedSearch : Bogan_2012_Drug.Test.Anal_4_199
PubMedID: 22362586

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 : 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 : 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 : 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 : 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 : Reversed enantioselectivity of diisopropyl fluorophosphatase against organophosphorus nerve agents by rational design - Melzer_2009_J.Am.Chem.Soc_131_17226
Author(s) : Melzer M , Chen JC , Heidenreich A , Gab J , Koller M , Kehe K , Blum MM
Ref : J Am Chem Soc , 131 :17226 , 2009
Abstract : Diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is an efficient and robust biocatalyst for the hydrolysis of a range of highly toxic organophosphorus compounds including the nerve agents sarin, soman, and cyclosarin. In contrast to the substrate diisopropyl fluorophosphate (DFP) the nerve agents possess an asymmetric phosphorus atom, which leads to pairs of enantiomers that display markedly different toxicities. Wild-type DFPase prefers the less toxic stereoisomers of the substrates which leads to slower detoxification despite rapid hydrolysis. Enzyme engineering efforts based on rational design yielded two quadruple enzyme mutants with reversed enantioselectivity and overall enhanced activity against tested nerve agents. The reversed stereochemical preference is explained through modeling studies and the crystal structures of the two mutants. Using the engineered mutants in combination with wild-type DFPase leads to significantly enhanced activity and detoxification, which is especially important for personal decontamination. Our findings may also be of relevance for the structurally related enzyme human paraoxonase (PON), which is of considerable interest as a potential catalytic in vivo scavenger in case of organophosphorus poisoning.
ESTHER : Melzer_2009_J.Am.Chem.Soc_131_17226
PubMedSearch : Melzer_2009_J.Am.Chem.Soc_131_17226
PubMedID: 19894712

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 : 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