Toxicokinetic analysis of the highly toxic nerve agent VX in commercially available multi-organ-chips - Ways to overcome compound absorption.

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

The suitability of a polydimethylsiloxane-based (PDMS) microfluidic two compartment system for the toxicokinetic analysis of organophosphorus compounds.

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

Post-VX exposure treatment of rats with engineered phosphotriesterases.

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

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

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

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.

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

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

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

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

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.

Screening of chiral shift reagents suitable to generically separate the enantiomers of V-agents by 31P-NMR spectroscopy.

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

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

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

Fatal sarin poisoning in Syria 2013: forensic verification within an international laboratory network.

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

Detoxification of VX and Other V-Type Nerve Agents in Water at 37 °C and pH†7.4 by Substituted Sulfonatocalix[4]arenes.

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

Single treatment of VX poisoned guinea pigs with the phosphotriesterase mutant C23AL: Intraosseous versus intravenous injection.

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

Toxicology of organophosphorus compounds in view of an increasing terrorist threat.

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

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.

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.

Medical documentation, bioanalytical evidence of an accidental human exposure to sulfur mustard and general therapy recommendations.

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

Application of a dynamic in vitro model with real-time determination of acetylcholinesterase activity for the investigation of tabun analogues and oximes.

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.

Detoxification of organophosphorus pesticides and nerve agents through RSDL: efficacy evaluation by (31)P NMR spectroscopy.

Intoxication by organophosphorus compounds, especially by pesticides, poses a considerable risk to the affected individual. Countermeasures involve both medical intervention by means of antidotes as well as external decontamination to reduce the risk of dermal absorption. One of the few decontamination options available is Reactive Skin Decontamination Lotion (RSDL), which was originally developed for military use. Here, we present a (31)P NMR spectroscopy based methodology to evaluate the detoxification efficacy of RSDL with respect to a series of organophosphorus pesticides and nerve agents. Kinetic analysis of the obtained NMR data provided degradation half-lives proving that RSDL is also reasonably effective against organophosphorus pesticides. Unexpected observations of different RSDL degradation patterns are presented in view of its reported oximate-catalyzed mechanism of action.

Reactions of methylphosphonic difluoride with human acetylcholinesterase and oximes--Possible therapeutic implications.

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

In vitro toxicokinetic studies of cyclosarin: molecular mechanisms of elimination.

The toxicokinetics of in vitro elimination of highly toxic cyclosarin (GF) in biological systems revealed striking stereoselective differences in the range of 0.01µM to 1mM GF. While weak concentration dependency was detected for elimination of the toxic (-)-enantiomer indicating catalytic processes, elimination of less toxic (+)-GF followed unusual kinetics with relatively high concentration dependency. Fast initial GF binding in human heparinised plasma increased only at lower initial GF concentrations while (+)-GF binding strongly increased with decreasing GF concentration. In displacement experiments it was shown for the first time that GF binding on plasma components in rats and mice plasma was reversible. Investigations with human plasma require further methodical improvement. GF elimination by diisopropylfluorophosphatase (DFPase) wildtype as phosphotriesterase (PTE) model showed some similarities compared to human heparinised plasma. Impact of human serum albumin is negligible. When comparing kinetics of GF elimination with metabolite formation (fluoride and cyclohexyl methyl phosphonic acid, CHMPA), marked differences were detected. From the results a model was postulated illustrating possible steps of molecular mechanisms of GF interaction with plasma proteins including high affine fast initial binding followed by formation of metastable phosphonylated plasma proteins with subsequent hydrolysis and release of metabolites.

Tabun scavengers based on hydroxamic acid containing cyclodextrins.

Arrangement of several hydroxamic acid-derived substituents along the cavity of a cyclodextrin ring leads to compounds that detoxify tabun in TRIS-HCl buffer at physiological pH and 37.0 °C with half-times as low as 3 min.