Wille T


Full name : Wille Timo

First name : Timo

Mail : Bundeswehr Institute of Pharmacology and Toxicology, Munich

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Country : Germany

Email : TimoWille@bundeswehr.org

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References (56)

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

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

Title : Aminoalkoxy-substituted coumarins: Synthesis and evaluation for reactivation of inhibited human acetylcholinesterase - Elsinghorst_2022_Arch.Pharm.(Weinheim)__e2200208
Author(s) : Elsinghorst PW , Wille T , Baric D , Mertens MD , Baumann M , Kuppers J , Gutschow M
Ref : Arch Pharm (Weinheim) , :e2200208 , 2022
Abstract : Reactivation of inhibited acetylcholinesterase remains an important therapeutic strategy for the treatment of poisoning by organophosphorus compounds, such as nerve agents or pesticides. Although drugs like obidoxime or pralidoxime have been used with considerable success, there is a need for new substances capable of reactivating acetylcholinesterase with a broader scope and increased efficacy. Possible screening candidates must fulfill two fundamental requirements: They must (i) show an affinity to acetylcholinesterase well balanced between sufficient binding and competitive inhibition and (ii) facilitate the nucleophilic cleavage of the phosphorylated catalytic serine residue. We attached a variety of nonaromatic primary and secondary amines to a coumarin core through selected alkoxy side linkers attached at coumarin positions 6 or 7 to obtain a small set of possible reactivators. Evaluation of their inhibition and reactivation potential in vitro showed some activity with respect to acetylcholinesterase inhibited by cyclosarin.
ESTHER : Elsinghorst_2022_Arch.Pharm.(Weinheim)__e2200208
PubMedSearch : Elsinghorst_2022_Arch.Pharm.(Weinheim)__e2200208
PubMedID: 35876340

Title : The Chemotype of Chromanones as a Privileged Scaffold for Multineurotarget Anti-Alzheimer Agents - Keuler_2022_ACS.Pharmacol.Transl.Sci_5_1097
Author(s) : Keuler T , Lemke C , Elsinghorst PW , Iriepa I , Chioua M , Martinez-Grau MA , Beadle CD , Vetman T , Lopez-Munoz F , Wille T , Bartz U , Deuther-Conrad W , Marco-Contelles J , Gutschow M
Ref : ACS Pharmacol Transl Sci , 5 :1097 , 2022
Abstract : The multifactorial nature of Alzheimer's disease necessitates the development of agents able to interfere with different relevant targets. A series of 22 tailored chromanones was conceptualized, synthesized, and subjected to biological evaluation. We identified one representative bearing a linker-connected azepane moiety (compound 19) with balanced pharmacological properties. Compound 19 exhibited inhibitory activities against human acetyl-, butyrylcholinesterase and monoamine oxidase-B, as well as high affinity to both the (1) and (2) receptors. Our study provides a framework for the development of further chromanone-based multineurotarget agents.
ESTHER : Keuler_2022_ACS.Pharmacol.Transl.Sci_5_1097
PubMedSearch : Keuler_2022_ACS.Pharmacol.Transl.Sci_5_1097
PubMedID: 36407962

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

Title : Release of protein-bound nerve agents by excess fluoride from whole blood: GC-MS\/MS method development, validation, and application to a real-life denatured blood sample - Koller_2021_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1179_122693
Author(s) : Koller M , Thiermann H , Worek F , Wille T
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 1179 :122693 , 2021
Abstract : In analogy to the fluoride-induced regeneration of butyrylcholinesterase (BChE) inhibited by nerve agents a method was developed and optimized for whole blood samples. Compared to the plasma method, regeneration grade was found to be higher for cyclosarin (GF), i-butylsarin from VR, and n-butylsarin from CVX, but lower for sarin (GB), fluorotabun from tabun (GA), and ethylsarin from VX. Regeneration grade of soman (GD) is the same for both matrices because it is released from serum albumin and not from cholinesterases. The method was fully validated for GB and GF to prove selectivity, linearity (n = 6), limit of determination (LOD1), reproducibility (within day (n = 8) and from day to day (n = 8)), effectiveness of extraction, matrix effect, and sample stability (after sample preparation and during three freeze/thaw cycles). The other agents were tested for selectivity, linearity (n = 2), limit of determination, and stability after sample preparation. The method showed high selectivity, good linearity up to the protein's saturation concentration (GB: R(2) = 0.9995, GF: 0.9968), and high reproducibility (GB: C.V. 5.9-13.7%, GF: 4.9-10.3%). The limits of determination (calculated from the spiked amount of the original agent) were found with 0.3 ng/mL VX, 0.5 ng/mL GB, 1 ng/mL VR, 0.5 ng/mL GA, 1 ng/mL CVX, and 8 ng/mL GD. In the case of GF, it was found with 4 ng/mL using Isolute ENV + SPE cartridges as for the other analytes and with 2.5 ng/mL using Isolute C8 EC SPE cartridges instead. This method was then applied to a denatured whole blood sample obtained from an individual exposed to GB. While previously only the GB metabolite isopropyl methylphosphonic acid (IMPA) could be detected in this blood sample it was now possible to successfully release GB from the blood proteins by excess fluoride.
ESTHER : Koller_2021_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1179_122693
PubMedSearch : Koller_2021_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1179_122693
PubMedID: 34171608

Title : Post-VX exposure treatment of rats with engineered phosphotriesterases - Stigler_2021_Arch.Toxicol__
Author(s) : Stigler L , Kohler A , Koller M , Job L , Escher B , Potschka H , Thiermann H , Skerra A , Worek F , Wille T
Ref : Archives of Toxicology , : , 2021
Abstract : The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (k(cat)/K(M) = 21 to 38 x 10(6) M(-1) min(-1)) towards VX and a preferential hydrolysis of the more toxic P(-) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD(50)) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg(-1) i.v.: PTE-2 dosed at 1.3 mg kg(-1) i.v. (PTE-2.1) and 2.6 mg kg(-1) i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg(-1) i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.
ESTHER : Stigler_2021_Arch.Toxicol__
PubMedSearch : Stigler_2021_Arch.Toxicol__
PubMedID: 34962578

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

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

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

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

Title : In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase - Worek_2020_Molecules_25_
Author(s) : Worek F , Thiermann H , Koller M , Wille T
Ref : Molecules , 25 : , 2020
Abstract : The implementation of the Chemical Weapons Convention (CWC) in 1997 was a milestone in the prohibition of chemical warfare agents (CWA). Yet, the repeated use of CWA underlines the ongoing threat to the population. Organophosphorus (OP) nerve agents still represent the most toxic CWA subgroup. Defensive research on nerve agents is mainly focused on the "classical five", namely tabun, sarin, soman, cyclosarin and VX, although Schedule 1 of the CWC covers an unforeseeable number of homologues. Likewise, an uncounted number of OP pesticides have been produced in previous decades. Our aim was to determine the in vitro inhibition kinetics of selected organophosphono- and organophosphorothioates with human AChE, as well as hydrolysis of the agents in human plasma and reactivation of inhibited AChE, in order to derive potential structure-activity relationships. The investigation of the interactions of selected OP compounds belonging to schedule 1 (V-agents) and schedule 2 (amiton) of the CWC with human AChE revealed distinct structural effects of the P-alkyl, P-O-alkyl and N,N-dialkyl residues on the inhibitory potency of the agents. Irrespective of structural modifications, all tested V-agents presented as highly potent AChE inhibitors. The high stability of the tested agents in human plasma will most likely result in long-lasting poisoning in vivo, having relevant consequences for the treatment regimen. In conclusion, the results of this study emphasize the need to investigate the biological effects of nerve agent analogues in order to assess the efficacy of available medical countermeasures.
ESTHER : Worek_2020_Molecules_25_
PubMedSearch : Worek_2020_Molecules_25_
PubMedID: 32630769

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

Title : Chromenones as Multineurotargeting Inhibitors of Human Enzymes - Lemke_2019_ACS.Omega_4_22161
Author(s) : Lemke C , Christmann J , Yin J , Alonso JM , Serrano E , Chioua M , Ismaili L , Martinez-Grau MA , Beadle CD , Vetman T , Dato FM , Bartz U , Elsinghorst PW , Pietsch M , Muller CE , Iriepa I , Wille T , Marco-Contelles J , Gutschow M
Ref : ACS Omega , 4 :22161 , 2019
Abstract : The complex nature of multifactorial diseases, such as Morbus Alzheimer, has produced a strong need to design multitarget-directed ligands to address the involved complementary pathways. We performed a purposive structural modification of a tetratarget small-molecule, that is contilisant, and generated a combinatorial library of 28 substituted chromen-4-ones. The compounds comprise a basic moiety which is linker-connected to the 6-position of the heterocyclic chromenone core. The syntheses were accomplished by Mitsunobu- or Williamson-type ether formations. The resulting library members were evaluated at a panel of seven human enzymes, all of which being involved in the pathophysiology of neurodegeneration. A concomitant inhibition of human acetylcholinesterase and human monoamine oxidase B, with IC50 values of 5.58 and 7.20 muM, respectively, was achieved with the dual-target 6-(4-(piperidin-1-yl)butoxy)-4H-chromen-4-one (7).
ESTHER : Lemke_2019_ACS.Omega_4_22161
PubMedSearch : Lemke_2019_ACS.Omega_4_22161
PubMedID: 31891098

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

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

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

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

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

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

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

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

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

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

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

Title : A novel fluorogenic probe for the investigation of free thiols: Application to kinetic measurements of acetylcholinesterase activity - Mertens_2016_Toxicol.Lett_244_161
Author(s) : Mertens MD , Bierwisch A , Li T , Gutschow M , Thiermann H , Wille T , Elsinghorst PW
Ref : Toxicol Lett , 244 :161 , 2016
Abstract : A novel coumarin-derived thiol probe, based on the thiol-promoted cleavage of a quenching 2,4-dinitrobenzenesulfonyl group is described. The probe shows a sensitive fluorescence turn-on and sufficient solubility in aqueous environments. As a proof of concept, a new assay for AChE activity was developed as a useful addition to the established Ellman method. The observed reaction kinetics followed an asymmetric sigmoidal pattern and were successfully evaluated applying a three parameter Gompertz equation. Providing a linear relationship between the detected fluorescence formation curves and corresponding enzyme activities, this probe appears as a valuable tool for AChE activity measurements.
ESTHER : Mertens_2016_Toxicol.Lett_244_161
PubMedSearch : Mertens_2016_Toxicol.Lett_244_161
PubMedID: 26494253

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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