Publications by authors named "Franz Worek"

181 Publications

Inhibition of an organophosphate-detoxifying bacterial phosphotriesterase by albumin and plasma thiol components.

Toxicol Lett 2021 Oct 22;350:194-201. Epub 2021 Jul 22.

Chair of Biological Chemistry, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany. Electronic address:

The phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE) is a naturally occurring enzyme that catalyzes the hydrolysis of organophosphate (OP) nerve agents as well as pesticides and offers a potential treatment of corresponding intoxications. While BdPTE mutants with improved catalytic efficiencies against several OPs have been described, unexpectedly, less efficient breakdown of an OP was observed upon application in an animal model compared with in vitro measurements. Here, we describe detailed inhibition studies with the high-activity BdPTE mutant 10-2C3(C59M/C227A) by human plasma components, indicating that this enzyme is inhibited by serum albumin. The inhibitory activity is mediated by depletion of crucial zinc ions from the BdPTE active site, either via the known high-affinity zinc binding site of albumin or via chemical complex formation with its free thiol side chain at position Cys34. Albumin pre-charged with zinc ions or carrying a chemically blocked Cys34 side chain showed significantly reduced inhibitory activity; in fact, the combination of both measures completely abolished BdPTE inhibition. Consequently, the available zinc ion concentration in blood plays an important role for BdPTE activity in vivo and should be taken into account for therapeutic development and application of a catalytic OP scavenger.
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http://dx.doi.org/10.1016/j.toxlet.2021.07.011DOI Listing
October 2021

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.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Aug 15;1179:122693. Epub 2021 Apr 15.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, D-80937 Munich, Germany. Electronic address:

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 = 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.
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http://dx.doi.org/10.1016/j.jchromb.2021.122693DOI Listing
August 2021

Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro.

Arch Toxicol 2021 08 23;95(8):2815-2823. Epub 2021 Jun 23.

Institut für Pharmakologie und Toxikologie der Bundeswehr, 80937, Munich, Germany.

Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured k/K values were as high as 6.5 and 1.5 × 10 M min with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC-MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(-) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.
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http://dx.doi.org/10.1007/s00204-021-03094-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298220PMC
August 2021

Development of versatile and potent monoquaternary reactivators of acetylcholinesterase.

Arch Toxicol 2021 03 31;95(3):985-1001. Epub 2021 Jan 31.

Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05, Hradec Kralove, Czech Republic.

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.
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http://dx.doi.org/10.1007/s00204-021-02981-wDOI Listing
March 2021

Translating the Concept of Bispecific Antibodies to Engineering Heterodimeric Phosphotriesterases with Broad Organophosphate Substrate Recognition.

Biochemistry 2020 11 4;59(45):4395-4406. Epub 2020 Nov 4.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.

We have adopted the concept of bispecific antibodies, which can simultaneously block or cross-link two different biomolecular targets, to create bispecific enzymes by exploiting the homodimeric quaternary structure of bacterial phosphotriesterases (PTEs). The PTEs from and , whose engineered variants can efficiently hydrolyze organophosphorus (OP) nerve agents and pesticides, respectively, have attracted considerable interest for the treatment of the corresponding intoxications. OP nerve agents and pesticides still pose a severe toxicological threat in military conflicts, including acts of terrorism, as well as in agriculture, leading to >100000 deaths per year. In principle, engineered conventional homodimeric PTEs may provoke hydrolytic inactivation of individual OPs , and their application as catalytic bioscavengers via administration into the bloodstream has been proposed. However, their narrow substrate specificity would necessitate therapeutic application of a set or mixture of different enzymes, which complicates biopharmaceutical development. We succeeded in combining subunits from both enzymes and to stabilize their heterodimerization by rationally designing electrostatic steering mutations, thus breaking the natural C2 symmetry. The resulting bispecific enzyme from two PTEs with different bacterial origin exhibits an ultrabroad OP substrate profile and allows the efficient detoxification of both nerve agents and pesticides. Our approach of combining two active sites with distinct substrate specificities within one artificial dimeric biocatalyst-retaining the size and general properties of the original enzyme without utilizing protein mixtures or much larger fusion proteins-not only should facilitate biological drug development but also may be applicable to oligomeric enzymes with other catalytic activities.
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http://dx.doi.org/10.1021/acs.biochem.0c00751DOI Listing
November 2020

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

Molecules 2020 Jul 2;25(13). Epub 2020 Jul 2.

Bundeswehr Institute of Pharmacology and Toxicology, D-80937 Munich, Germany.

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 -alkyl, --alkyl and ,-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.
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http://dx.doi.org/10.3390/molecules25133029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412149PMC
July 2020

Influence of cyclic and acyclic cucurbiturils on the degradation pathways of the chemical warfare agent VX.

Org Biomol Chem 2020 07;18(27):5218-5227

Technische Universität Kaiserslautern, Fachbereich Chemie - Organische Chemie, Erwin-Schrödinger-Straße, 67663 Kaiserslautern, Germany.

The highly toxic nerve agent VX is a methylphosphonothioate that degrades via three pathways in aqueous solution, namely through the hydrolysis of the P-O or P-S bonds, or the cleavage of the C-S bond at the 2-aminoethyl residue. In the latter case, an aziridinium ion and a phosphonothioate is formed. Here it is shown that acyclic or cyclic cucurbiturils inhibit these reactions in phosphate buffer at physiological pH and thus stabilise the nerve agent. When using unbuffered basic solutions as the reaction medium, however, in which the P-S or P-O bonds are normally hydrolysed preferentially, cucurbiturils turned out to strongly shift VX degradation towards the cleavage of the C-S bond. Cucurbit[7]uril, in particular, has a so pronounced effect under suitable conditions that it almost completely suppresses the formation of products resulting from the other degradation pathways. Investigations involving VX analogues in combination with computational methods suggest that one reason for the reaction control exerted by the cucurbiturils is the preorganisation of VX for aziridinium ion formation. In addition, cucurbit[7]uril also lowers the transition state of the reaction by stabilising the positive charge developing on the way to the product. Cucurbiturils thus have a marked effect on the reactivity of a highly toxic nerve agent, which potentially allows using them for decontamination purposes.
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http://dx.doi.org/10.1039/d0ob01167cDOI Listing
July 2020

Organophosphorus compounds and oximes: a critical review.

Arch Toxicol 2020 07 6;94(7):2275-2292. Epub 2020 Jun 6.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany.

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.
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http://dx.doi.org/10.1007/s00204-020-02797-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367912PMC
July 2020

Synthesis and in vitro evaluation of novel non-oximes for the reactivation of nerve agent inhibited human acetylcholinesterase.

Chem Biol Interact 2020 Aug 23;326:109139. Epub 2020 May 23.

TNO, Lange Kleiweg 137, 2288, GJ Rijswijk, the Netherlands.

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.
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http://dx.doi.org/10.1016/j.cbi.2020.109139DOI Listing
August 2020

Diagnostics and treatment of nerve agent poisoning-current status and future developments.

Ann N Y Acad Sci 2020 11 21;1479(1):13-28. Epub 2020 Mar 21.

Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany.

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, bioscavengers 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.
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http://dx.doi.org/10.1111/nyas.14336DOI Listing
November 2020

Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity.

Int J Mol Sci 2020 Mar 1;21(5). Epub 2020 Mar 1.

Institute of Biochemistry and Cell Biology, CNR, 80131 Naples, Italy.

Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from . The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 10 M s against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.
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http://dx.doi.org/10.3390/ijms21051683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084321PMC
March 2020

Early diagnosis of nerve agent exposure with a mobile test kit and implications for medical countermeasures: a trigger to react.

BMJ Mil Health 2020 Apr 20;166(2):99-102. Epub 2020 Feb 20.

Military Medical Academy, National Poison Control Center, Belgrade, Serbia.

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.
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http://dx.doi.org/10.1136/jramc-2019-001310DOI Listing
April 2020

Impact of soman and acetylcholine on the effects of propofol in cultured cortical networks.

Toxicol Lett 2020 Apr 16;322:98-103. Epub 2020 Jan 16.

Experimental Anesthesiology Section, Department of Anesthesiology and Intensive Care Medicine, Eberhard-Karls-University, Waldhoernlestrasse 22, 72072, Tuebingen, Germany. Electronic address:

Patients intoxicated with organophosphorous compounds may need general anaesthesia to enable mechanical ventilation or for control of epileptiform seizures. It is well known that cholinergic overstimulation attenuates the efficacy of general anaesthetics to reduce spontaneous network activity in the cortex. However, it is not clear how propofol, the most frequently used intravenous anaesthetic today, is affected. Here, we investigated the effects of cholinergic overstimulation induced by soman and acetylcholine on the ability of propofol to depress spontaneous action potential activity in organotypic cortical slices measured by extracellular voltage recordings. Cholinergic overstimulation by co-application of soman and acetylcholine (10 μM each) did not reduce the relative inhibition of propofol (1.0 μM; mean normalized action potential firing rate 0.49 ± 0.06 of control condition, p < 0.001, Wilcoxon signed rank test) but clearly reduced its efficacy. Co-application of atropine (10 nM) did not improve the efficacy. Propofol preserved its relative inhibitory potential but did not produce a degree of neuronal depression which can be expected to assure hypnosis in humans. Since a combination with atropine did not improve its efficacy, an increase in dosage will probably be necessary when propofol is used in victims suffering from organophosphorous intoxication.
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http://dx.doi.org/10.1016/j.toxlet.2020.01.012DOI Listing
April 2020

A catalytic bioscavenger with improved stability and reduced susceptibility to oxidation for treatment of acute poisoning with neurotoxic organophosphorus compounds.

Toxicol Lett 2020 Mar 28;321:138-145. Epub 2019 Dec 28.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany. Electronic address:

Organophosphorus (OP) nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE), engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.
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http://dx.doi.org/10.1016/j.toxlet.2019.12.030DOI Listing
March 2020

Screening of chiral shift reagents suitable to generically separate the enantiomers of V-agents by P-NMR spectroscopy.

Toxicol Lett 2020 Mar 2;320:28-36. Epub 2019 Dec 2.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany. Electronic address:

Fourteen amino acids protected at the N-terminal and at their side chains were screened for resolving the enantiomers of V-agents by NMR. While none of the shift reagents tested showed really effective separation in proton NMR, two of them (BOC-Gln(Xan)-OH, 16, and Z-Arg(Z)-OH), 21, with 16 superior to 21) were found suitable to separate the enantiomers of all V-agent homologues involved in the test by P-NMR. Molar ratios investigated were 1:0.5, 1:1, 1:1.5, 1:2, and 1:3 with the V-agent set to 1 throughout the experiments. All these ratios were more or less effective, but 1:3 was found to separate the V-agents the most reliable way. It is postulated that three chiral solvating molecules are then coordinated around the organophosphate: ion pair formation with the amino nitrogen of the V agent side chain, hydrogen bonding provided by the PO unit, and extension of coordination at the phosphorus atom itself. After chiral separation of VX by semi-preparative LC-MS the enantiomers were examined with both configurations of 16 releasing four different P NMR peaks which correspond to four different complexes: R-S, R-R, S-R, and S-S. Comparing these results with literature data it is assumed that (+)-VX corresponds to the R configuration and (-)-VX to the S-configuration.
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http://dx.doi.org/10.1016/j.toxlet.2019.12.004DOI Listing
March 2020

[Chemical warfare agent poisoning].

Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2019 Nov;62(11):1370-1377

Institut für Pharmakologie und Toxikologie der Bundeswehr, Neuherbergstr. 11, 80937, München, Deutschland.

Despite long-lasting international efforts to ban and disarm chemical warfare agents (CWAs), they pose an ongoing threat to the population. The reasons for this are existing remainders, inappropriately disposed of chemical munitions and availability of instructions for synthesis in open literature. Dissemination of CWAs during war, warlike conflicts and terrorist incidents has recently resulted in thousands of deaths. In this manuscript CWAs and comparable substances are presented and the signs and symptoms of poisoning with these substances are described. Aside from clear recommendations for the treatment of poisoning by the single groups of CWAs, parallels to well-known related poisonings including pathophysiological similarities are demonstrated. Moreover, aspects of detection, diagnosis and general management, such as decontamination, verification and antidote stockpiling, are described.According to the respective pathophysiological target, CWAs are classified as lung, skin, nerve and incapacitating agents. They are generally liquids at ambient room temperature and are more or less able to vaporise. In recent years, pharmaceutical-based agents (PBAs) came on board although they are not listed in the chemical warfare convention and therefore not listed as CWAs. Due to their high toxicity, however, they are mentioned here. PBAs comprise, for example, synthetic opioids which can act after inhalative respiration.During the rescue of affected victims, early detection of CWAs, restriction of access to the contaminated area and use of protective clothes and masks by first responders are necessary. Exposure should be terminated as soon as possible by removal of the victim from the hot zone and decontamination. The latter is also important to avoid secondary contamination of other persons or facilities located outside of the contaminated zone. According to the type of poisoning, therapy should be started as soon as possible.
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http://dx.doi.org/10.1007/s00103-019-03035-4DOI Listing
November 2019

Efficacy of an organophosphorus hydrolase enzyme (OpdA) in human serum and minipig models of organophosphorus insecticide poisoning.

Clin Toxicol (Phila) 2020 05 27;58(5):397-405. Epub 2019 Aug 27.

CSIRO Biocatalysis & Synthetic Biology Team, Black Mountain Science and Innovation Park, Canberra, Australia.

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. 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. 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 (k) for quinalphos and phenthoate, respectively, although at rates 2-3 log orders less than found 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] μg/kg/h vs. 1.07 [SD 0.77] μg/kg/h,  < .0001) and methyl parathion (mean 0.077 [SD 0.08] μg/kg/h vs. 0.707 [SD 0.49] μg/kg/h,  < .0001) poisoning. OpdA reduced blood OP insecticide concentration and acetylcholinesterase inhibition after poisoning by dimethoate, methyl parathion, and profenofos insecticides. 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 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.
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http://dx.doi.org/10.1080/15563650.2019.1655149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114914PMC
May 2020

The in vitro protective effects of the three novel nanomolar reversible inhibitors of human cholinesterases against irreversible inhibition by organophosphorous chemical warfare agents.

Chem Biol Interact 2019 Aug 19;309:108714. Epub 2019 Jun 19.

National Institute of Chemistry, Hajdrihova 19, Ljubljana, Slovenia; Innovation Center of The Faculty of Chemistry, University of Belgrade, Studentski Trg 12-16, Serbia.

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.
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http://dx.doi.org/10.1016/j.cbi.2019.06.027DOI Listing
August 2019

Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts.

Arch Toxicol 2019 07 8;93(7):1881-1891. Epub 2019 May 8.

Bundeswehr Institute of Pharmacology and Toxicology (InstPharmToxBw), Neuherbergstr. 11, 80937, Munich, Germany.

We present the forensic analyses of plasma samples of human victims exposed to sulfur mustard (SM) in a crisis region in the Middle East in 2015. A few hours after exposure, poisoned persons showed typical signs and symptoms of percutaneous SM exposure including erythema and later on blisters and hardly healing skin wounds. Blood samples were collected 15 days after poisoning to be analyzed for the presence of long-lived protein-adduct biomarkers to verify SM poisoning. We applied a novel bioanalytical toolbox targeting four human serum albumin-derived biomarkers that were made accessible after plasma proteolysis. These adducts contained the SM-specific hydroxyethylthioethyl moiety either bound to the thiol group of a cysteine residue (C*) or to the side-chain carboxylic group of a glutamic acid residue (E*). Peptide biomarkers were produced from plasma of the victims using proteinase K (C*PF), pronase (C*P) and pepsin (AE*VSKL and LQQC*PFEDHVKL) for enzymatic protein cleavage. Separation and detection were carried out by selective micro-liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS). In addition to this site-specific adduct detection, a general approach after alkaline hydrolysis of the plasma protein fraction was applied. Liberated thiodiglycol (TDG) was derivatized with heptafluorobutyric anhydride and detected by gas chromatography-electron ionization mass spectrometry (GC-EI MS). The different bioanalytical methods yielded congruent results confirming SM poisoning for all patients who showed clinical signs and symptoms. This is the first time that real cases of SM poisoning were confirmed and presented by such a broad compilation of protein-derived biomarkers.
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http://dx.doi.org/10.1007/s00204-019-02461-2DOI Listing
July 2019

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).

Toxicol Lett 2019 Jun 8;308:1-6. Epub 2019 Mar 8.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany. Electronic address:

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 μmol/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.
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http://dx.doi.org/10.1016/j.toxlet.2019.03.003DOI Listing
June 2019

Human small bowel as model for poisoning with organophosphorus compounds.

Toxicol In Vitro 2019 Jun 11;57:76-80. Epub 2019 Feb 11.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany. Electronic address:

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 ECs. The parasympatholytic compound scopolamine had the strongest relaxing effect (EC = 0.05 μM) followed by atropine (EC = 0.07 μM). 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 EC = 3.8 μM vs obidoxime EC = 197.8 μM). 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.
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http://dx.doi.org/10.1016/j.tiv.2019.02.010DOI Listing
June 2019

COPD and asthma therapeutics for supportive treatment in organophosphate poisoning.

Clin Toxicol (Phila) 2019 07 30;57(7):644-651. Epub 2019 Jan 30.

a Bundeswehr Institute of Pharmacology and Toxicology , Neuherbergstraße 11, Munich , Germany.

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 β2-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. 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/β2-agonists. Glycopyrrolate and ipratropium efficiently antagonized an ACh-induced airway contraction in VX-poisoned PCLS (EC glycopyrrolate 15.8 nmol/L, EC ipratropium 2.3 nmol/L). β2-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)]. We showed antagonizing effects of anticholinergics and β2-agonists on ACh-induced airway contractions in VX-poisoned PCLS, thus providing experimental data to support a prospective comprehensive clinical study. Our results indicate that COPD and asthma therapeutics could be a valuable addition to the treatment of OP poisoning.
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http://dx.doi.org/10.1080/15563650.2018.1540785DOI Listing
July 2019

Evaluation of the accuracy of "ChE check mobile" in measurement of acetylcholinesterase in pesticide poisoning.

Clin Toxicol (Phila) 2019 06 17;57(6):411-414. Epub 2018 Nov 17.

a South Asian Clinical Toxicology Research Collaboration, Faculty of Medicine , University of Peradeniya , Peradeniya , Sri Lanka.

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.
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http://dx.doi.org/10.1080/15563650.2018.1530778DOI Listing
June 2019

Synthesis of a Series of Non-Symmetric Bispyridinium and Related Compounds and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor.

ChemMedChem 2018 12 27;13(24):2653-2663. Epub 2018 Nov 27.

Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13, 81377, Munich, Germany.

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 NMe group at the 3- or 4-positions appeared to be beneficial for high binding affinities.
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http://dx.doi.org/10.1002/cmdc.201800539DOI Listing
December 2018

Interactions between acetylcholinesterase, toxic organophosphorus compounds and a short series of structurally related non-oxime reactivators: Analysis of reactivation and inhibition kinetics in vitro.

Toxicol Lett 2018 Dec 9;299:218-225. Epub 2018 Oct 9.

Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany. Electronic address:

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 μM 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.
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http://dx.doi.org/10.1016/j.toxlet.2018.10.004DOI Listing
December 2018

Effect of cholinergic crisis on the potency of different emergency anaesthesia protocols in soman-poisoned rats.

Clin Toxicol (Phila) 2019 05 11;57(5):343-349. Epub 2018 Oct 11.

a Bundeswehr Institute of Pharmacology and Toxicology , Munich , Germany.

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.
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http://dx.doi.org/10.1080/15563650.2018.1520241DOI Listing
May 2019

Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents.

Sci Rep 2018 09 13;8(1):13773. Epub 2018 Sep 13.

Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy.

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.
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http://dx.doi.org/10.1038/s41598-018-31751-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137069PMC
September 2018

Midazolam is effective to reduce cortical network activity in organotypic cultures during severe cholinergic overstimulation with soman.

Toxicol Lett 2018 Nov 27;297:19-23. Epub 2018 Aug 27.

Experimental Anesthesiology Section, Department of Anesthesiology and Intensive Care Medicine, Eberhard-Karls-University, Waldhoernlestrasse 22, 72072 Tuebingen, Germany. Electronic address:

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 μM midazolam) and 1.6 (0.9-2.2) Hz (20 μM 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 μM midazolam) and 4.1 (3.3-6.0) Hz (20 μM 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.
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http://dx.doi.org/10.1016/j.toxlet.2018.08.016DOI Listing
November 2018

Discovery of a potent non-oxime reactivator of nerve agent inhibited human acetylcholinesterase.

Eur J Med Chem 2018 Sep 6;157:151-160. Epub 2018 Aug 6.

TNO, Lange Kleiweg 137, 2288, GJ, Rijswijk, the Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ejmech.2018.08.016DOI Listing
September 2018

Synthesis of a Series of Structurally Diverse MB327 Derivatives and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor.

ChemMedChem 2018 09 1;13(17):1806-1816. Epub 2018 Aug 1.

Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.

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 (pK =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 pK value of 5.16±0.07, thus representing considerable progress toward the development of more potent nAChR re-sensitizers.
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http://dx.doi.org/10.1002/cmdc.201800325DOI Listing
September 2018
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