Verification of Exposure to Novichok Nerve Agents Utilizing a Semitargeted Human Butyrylcholinesterase Nonapeptide Assay.

Novichok (NV) nerve agents were recently added to the list of Schedule 1 chemicals of the Chemical Weapons Convention. There is a well-accepted method for assessment of nerve agent exposure based on mass spectrometric analysis of a nonapeptide with the serine-198 residue modified by the nerve agent, but this approach has not yet been reported for the class of NV agents and requires the availability of reference standards, which may be a limitation for NV agent exposure assessment. Thus, a goal of this study was to first verify the utility of the nonapeptide method for the characterization of human plasma samples exposed in vitro to the NV agents A-230, A-232, and A-234. A second aim was to evaluate the possibility of identifying unknown exposures by applying precursor ion scanning in combination with high resolution mass spectrometry (HRMS). Thus, precursor ion scanning, with a generic fragment ion (m/z 778) of the nonapeptide, was used to pinpoint any modified nonapeptide, while HRMS was used for structural elucidation of the adduct moiety. By this approach, use of HRMS enabled differentiation between adducts of agents with similar molecular masses. A new unique feature that could be exploited for NV nonapeptide analysis was that the modification was released from the peptide during fragmentation in the mass spectrometer and was detected in the low-mass region of the mass spectrum. This low-mass region was extremely informative and contributed to the assignment of the structure of the particular agent used, which is especially important in case no reference materials are available. The presented method is important for verification purposes by the Organisation for Prohibition of Chemical Weapons (OPCW), e.g., in case of investigations of alleged use of NV agents, and for regular forensic investigations.

Mass spectrometric analysis of adducts of sulfur mustard analogues to human plasma proteins: approach towards chemical provenancing in biomedical samples.

The primary aim of this study was to identify biomarkers of exposure to some so-called Schedule 1 sulfur mustard (HD) analogues, in order to facilitate and expedite their retrospective analysis in case of alleged use of such compounds. Since these HD analogues can be regarded as model compounds for possible impurities of HD formed during synthesis processes, the secondary aim was to explore to which extent these biomarkers can be used for chemical provenancing of HD in case biomedical samples are available. While the use of chemical attribution signatures (CAS) for neat chemicals or for environmental samples has been addressed quite frequently, the use of CAS for investigating impurities in biomedical samples has been addressed only scarcely. Human plasma was exposed to each of the five HD analogues. After pronase or proteinase K digestion of precipitated protein and sample work-up, the histidine (His) and tripeptide (CPF) adducts to proteins were analyzed, respectively. Adducts of the analogues could still be unambiguously identified next to the main HD adducts in processed plasma samples after exposure to HD mixed with each of the analogues, at a 1% level relative to HD. In conclusion, we have identified plasma protein adducts of a number of HD analogues, which can be used as biomarkers to assess an exposure to these Schedule 1 chemicals. We have shown that adducts of these analogues can still be analyzed after work-up of plasma samples which had been exposed to these analogues in a mixture with HD, supporting the hypothesis that biomedical sample analysis might be useful for chemical provenancing.

Biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach using LC-HRMS/MS and chemometrics.

Since its first employment in World War I, chlorine gas has often been used as chemical warfare agent. Unfortunately, after suspected release, it is difficult to prove the use of chlorine as a chemical weapon and unambiguous verification is still challenging. Furthermore, similar evidence can be found for exposure to chlorine gas and other, less harmful chlorinating agents. Therefore, the current study aims to use untargeted high resolution mass spectrometric analysis of chlorinated biomarkers together with machine learning techniques to be able to differentiate between exposure of plants to various chlorinating agents. Green spire (Euonymus japonicus), stinging nettle (Urtica dioica), and feathergrass (Stipa tenuifolia) were exposed to 1000 and 7500 ppm chlorine gas and household bleach, pool bleach, and concentrated sodium hypochlorite. After sample preparation and digestion, the samples were analyzed by liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). More than 150 chlorinated compounds including plant fatty acids, proteins, and DNA adducts were tentatively identified. Principal component analysis (PCA) and linear discriminant analysis (LDA) showed clear discrimination between chlorine gas and bleach exposure and grouping of the samples according to chlorine concentration and type of bleach. The identity of a set of novel biomarkers was confirmed using commercially available or synthetic reference standards. Chlorodopamine, dichlorodopamine, and trichlorodopamine were identified as specific markers for chlorine gas exposure. Fenclonine (Cl-Phe), 3-chlorotyrosine (Cl-Tyr), 3,5-dichlorotyrosine (di-Cl-Tyr), and 5-chlorocytosine (Cl-Cyt) were more abundantly present in plants after chlorine contact. In contrast, the DNA adduct 2-amino-6-chloropurine (Cl-Ade) was identified in both types of samples at a similar level. None of these chlorinated biomarkers were observed in untreated samples. The DNA adducts Cl-Cyt and Cl-Ade could clearly be identified even three months after the actual exposure. This study demonstrates the feasibility of forensic biomarker profiling in plants to distinguish between exposure to chlorine gas and bleach.

Chlorinated organic compounds in concrete as specific markers for chlorine gas exposure.

The formation of chlorinated organic compounds in concrete debris exposed to reactive chlorine was studied to search for markers specific to chlorine gas exposure. Concrete materials of different origins were exposed to a range of species of reactive chlorine including bleach, humid and dry chlorine gas at different concentrations. Chlorinated organic compounds in concrete extracts were analysed by targeted gas and liquid chromatography-tandem mass spectrometry (GC-MS/MS and LC-MS/MS) and by non-targeted screening using the corresponding high-resolution techniques (GC-HRMS and LC-HRMS). Overall, different levels and species of chlorinated organic compounds namely chlorophenols, chlorobenzenes, chloromethoxyphenols, chloromethylbenzenes and chloral hydrate were identified in these chlorinated concrete extracts; two examples of diagnostic markers for neat chlorine exposure were trichloromethylbenzene and tetrachlorophenol. The old concrete samples from the 1930s and 1950s had the most chlorinated organic compounds after exposure to neat chlorine gas. Lignin or lignin degradation products were identified as probable candidates for phenolic precursor molecules in the concrete samples. Multivariate data analysis (OPLS-DA) shows distinct patterns for bleach and chlorine exposure. The chlorinated chemicals and specific markers for chlorine gas discovered in our research assist other laboratories in forensic investigations of chlorine gas attacks.

Verification of exposure to chemical warfare agents through analysis of persistent biomarkers in plants.

The continuing threats of military conflicts and terrorism may involve the misuse of chemical weapons. The present study aims to use environmental samples to find evidence of the release of such agents at an incident scene. A novel approach was developed for identifying protein adducts in plants. Basil (Ocimum basilicum), bay laurel leaf (Laurus nobilis) and stinging nettle (Urtica dioica) were exposed to 2.5 to 150 mg m-3 sulfur mustard, 2.5 to 250 mg m-3 sarin, and 0.5 to 25 g m-3 chlorine gas. The vapors of the selected chemicals were generated under controlled conditions in a dedicated set-up. After sample preparation and digestion, the samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS), respectively. In the case of chlorine exposure, it was found that 3-chloro- and 3,5-dichlorotyrosine adducts were formed. As a result of sarin exposure, the o-isopropyl methylphosphonic acid adduct to tyrosine could be analyzed, and after sulfur mustard exposure the N1- and N3-HETE-histidine adducts were identified. The lowest vapor exposure levels for which these plant adducts could be detected, were 2.5 mg m-3 for sarin, 50 mg m-3 for chlorine and 12.5 mg m-3 for sulfur mustard. Additionally, protein adducts following a liquid exposure of only 2 nmol Novichock A-234, 0.4 nmol sarin and 0.2 nmol sulfur mustard could still be observed. For both vapor and liquid exposure, the amount of adduct formed increased with the level of exposure. In all cases synthetic reference standards were used for unambiguous identification. The window of opportunity for investigation of agent exposure through the analysis of plant material was found to be remarkably long. Even three months after the actual exposure, the biomarkers could still be detected in the living plants, as well as in dried leaves. An important benefit of the current method is that a relatively simple and generic sample work-up procedure can be applied for all agents studied. In conclusion, the presented work clearly demonstrates the possibility of analyzing chemical warfare agent biomarkers in plants, which is useful for forensic reconstructions, including the investigation into alleged use in conflict areas.

Structural study of the complex stereoselectivity of human butyrylcholinesterase for the neurotoxic V-agents.

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P(S) adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX(R)-(+) and VX(S)-(-) solutions lead to the formation of the P(S) and P(R) adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX(R)-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX(S)-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P(R) enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P(S) enantiomer and ages.

Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillus species.

We describe the development of a highly specific enzyme-based fluorescence resonance energy transfer (FRET) assay for easy and rapid detection both in vitro and in vivo of Bacillus spp., among which are the members of the B. cereus group. Synthetic substrates for B. anthracis proteases were designed and exposed to secreted enzymes of a broad spectrum of bacterial species. The rational design of the substrates was based on the fact that the presence of D-amino acids in the target is highly specific for bacterial proteases. The designed D-amino acids containing substrates appeared to be specific for B. anthracis but also for several other Bacillus spp. and for both vegetative cells and spores. With the use of mass spectrometry (MS), cleavage products of the substrates could be detected in sera of B. anthracis infected mice but not in healthy mice. Due to the presence of mirrored amino acids present in the substrate, the substrates showed high species specificity, and enzyme isolation and purification was redundant. The substrate wherein the D-amino acid was replaced by its L-isomer showed a loss of specificity. In conclusion, with the use of these substrates a rapid tool for detection of B. anthracis spores and diagnosis of anthrax infection is at hand. We are the first who present fluorogenic substrates for detection of bacterial proteolytic enzymes that can be directly applied in situ by the use of D-oriented amino acids.

Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum.

A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

WITHDRAWN: Characterization of botulinum progenitor neurotoxin A complexes by blue native gel electrophoresis and mass spectrometry.

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Toxicodynamic analysis of the combined cholinesterase inhibition by paraoxon and methamidophos in human whole blood.

Theoretical work has shown that the isobole method is not generally valid as a method for testing the absence or presence of interaction (in the biochemical sense) between chemicals. The present study illustrates how interaction can be tested by fitting a toxicodynamic model to the results of a mixture experiment. The inhibition of cholinesterases (ChE) in human whole blood by various dose combinations of paraoxon and methamidophos was measured in vitro. A toxicodynamic model describing the processes related to both OPs in inhibiting AChE activity was developed, and fit to the observed activities. This model, not containing any interaction between the two OPs, described the results from the mixture experiment well, and it was concluded that the OPs did not interact in the whole blood samples. While this approach of toxicodynamic modeling is the most appropriate method for predicting combined effects, it is not rapidly applicable. Therefore, we illustrate how toxicodynamic modeling can be used to explore under which conditions dose addition would give an acceptable approximation of the combined effects from various chemicals. In the specific case of paraoxon and methamidophos in whole blood samples, it was found that dose addition gave a reasonably accurate prediction of the combined effects, despite considerable difference in some of their rate constants, and mildly non-parallel dose-response curves. Other possibilities of validating dose-addition using toxicodynamic modeling are briefly discussed.

Toxicodynamic analysis of the inhibition of isolated human acetylcholinesterase by combinations of methamidophos and methomyl in vitro.

The applicability of dose addition to combinations of OP-esters and carbamates has been questioned based on theoretical considerations, but these have not been well supported by experimental findings. In the present study, the inhibition of AChE by combinations of methamidophos (an OP-ester) and methomyl (a carbamate) was examined in vitro. AChE inhibition was measured by the Ellman assay. We addressed the question of interaction between the OP-ester and carbamate by a toxicodynamic (TD) model reflecting the mechanism of action of the individual chemicals, without incorporating any interactions between them. The model was extended by including the experimental actions in the Ellman assay to correct for the difference in reactivation rates between phosphorylated and carbamylated AChE, which caused a bias in the observations from the assay. This zero-interactive TD model described the observations well, indicating that the OP-ester and carbamate did not interact. The applicability of dose addition was further explored by applying dose addition to the predicted inhibition by the TD model. Despite the differences in dynamics between methamidophos and methomyl, their dose-response curves were close to parallel, and dose addition gave a reasonably accurate prediction of the combined effects.

Correction: Field, M. et al. AbobotulinumtoxinA (Dysport®), OnabotulinumtoxinA (Botox®), and IncobotulinumtoxinA (Xeomin®) Neurotoxin Content and Potential Implications for Duration of Response in Patients.

The authors wish to make the following corrections to their paper [...].

Development of an automated on-line pepsin digestion-liquid chromatography-tandem mass spectrometry configuration for the rapid analysis of protein adducts of chemical warfare agents.

Rapid monitoring and retrospective verification are key issues in protection against and non-proliferation of chemical warfare agents (CWA). Such monitoring and verification are adequately accomplished by the analysis of persistent protein adducts of these agents. Liquid chromatography-mass spectrometry (LC-MS) is the tool of choice in the analysis of such protein adducts, but the overall experimental procedure is quite elaborate. Therefore, an automated on-line pepsin digestion-LC-MS configuration has been developed for the rapid determination of CWA protein adducts. The utility of this configuration is demonstrated by the analysis of specific adducts of sarin and sulfur mustard to human butyryl cholinesterase and human serum albumin, respectively.

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.

AbobotulinumtoxinA (Dysport®), OnabotulinumtoxinA (Botox®), and IncobotulinumtoxinA (Xeomin®) Neurotoxin Content and Potential Implications for Duration of Response in Patients.

Botulinum neurotoxin type-A (BoNT-A) blocks the release of acetylcholine from peripheral cholinergic nerve terminals and is an important option for the treatment of disorders characterised by excessive cholinergic neuronal activity. Several BoNT-A products are currently marketed, each with unique manufacturing processes, excipients, formulation, and non-interchangeable potency units. Nevertheless, the effects of all the products are mediated by the 150 kDa BoNT-A neurotoxin. We assessed the quantity and light chain (LC) activity of BoNT-A in three commercial BoNT-A products (Dysport®; Botox®; Xeomin®). We quantified 150 kDa BoNT-A by sandwich ELISA and assessed LC activity by EndoPep assay. In both assays, we assessed the results for the commercial products against recombinant 150 kDa BoNT-A. The mean 150 kDa BoNT-A content per vial measured by ELISA was 2.69 ng/500 U vial Dysport®, 0.90 ng/100 U vial Botox®, and 0.40 ng/100 U vial Xeomin®. To present clinically relevant results, we calculated the 150 kDa BoNT-A/US Food and Drug Administration (FDA)-approved dose in adult upper limb spasticity: 5.38 ng Dysport® (1000 U; 2 × 500 U vials), 3.60 ng Botox® (400 U; 4 × 100 U vials), and 1.61 ng Xeomin® (400 U; 4 × 100 U vials). EndoPep assay showed similar LC activity among BoNT-A products. Thus, greater amounts of active neurotoxin are injected with Dysport®, at FDA-approved doses, than with other products. This fact might explain the long duration of action reported across multiple indications, which benefits patients, caregivers, clinicians, and healthcare systems.

The ultimate radiochemical nightmare: upon radio-iodination of Botulinum neurotoxin A, the introduced iodine atom itself seems to be fatal for the bioactivity of this macromolecule.

BACKGROUND: Botulinum neurotoxin A (BoNT-A) is a highly neurotoxic drug and frequently used in patients. Knowledge on the optimal way of administration of BoNT-A and its subsequent distribution is still rather limited. An accurate method for monitoring these processes might be the use of radiolabelled BoNT-A. In this paper, we report our feasibility study on labelling BoNT-A with high-dose iodine-125 ((125)I) via IODOGEN-coated BoNT-A method. METHODS: Using cetuximab as model substrate for BoNT-A, a miniaturization of the IODOGEN-coated mAb method was developed with special attention to the minimum required amount of the oxidant IODOGEN, while the amount of substrate, reaction volume and reaction time were downsized. Labelling efficiency and radiochemical purity were determined by TLC, integrity by SDS-PAGE and HPLC and immunoreactivity by cell-binding assay. BoNT-A (50 µg) was labelled with (125)I by coating with 2.5 µg IODOGEN, in a total reaction volume of 250 µL and a reaction time of 90 s. (125)I-BoNT-A was purified by size exclusion chromatography (PD10 column) using ascorbic acid solution (5 mg/ml, pH = 5) as eluent. Quality analysis of (125)I-BoNT-A was performed by an in vitro bladder strip model, an electrochemiluminescence assay and an Endopep assay. RESULTS: Cetuximab (50 µg) labelling with (125)I (15 to 150 MBq) resulted in a labelling efficiency of 70% to 80%, a radiochemical purity of >99%, an immunoreactivity of >95% and a retained integrity on SDS; HPLC analysis revealed partly affected integrity when 110 to 150 MBq (125)I was used, i.e. when the averaged I/mAb molar ratio exceeded 3. Addition of HEPES (20 mM) and lactose (1.25%) (lyophilized BoNT-A contains HEPES and lactose) decreased the labelling efficiency to 44% to 54%. BoNT-A (50 µg) labelling with (125)I (97.2 to 98.3 MBq) resulted in labelling efficiency of 51% to 52% with a radiochemical purity >98.5%, a specific activity of 150.5 to 152.9 MBq/nmol and an I/BoNT-A molar ratio of 1.86 to 1.90. The in vitro bladder strip model showed no bioactivity of (125)I-BoNT-A when compared to unlabelled BoNT-A. The electrochemiluminescence and Endopep assay demonstrated around 10% and 15% bioactivity of (125)I-BoNT-A compared to unlabelled BoNT-A, respectively. The remaining bioactivity correlates within the Poisson distribution with the amount of BoNT-A molecules that does not bear an iodine atom. CONCLUSIONS: BoNT-A was successfully radio-iodinated with an activity high enough to enable in vivo measurement of nanograms of BoNT-A, which could be used in studying optimization of administration techniques of BoNT-A. The bioactivity of a BoNT-A molecule is, however, lost upon the introduction of an iodine atom into the tyrosine moiety of this sensitive molecule.

Quantitation of ortho-cresyl phosphate adducts to butyrylcholinesterase in human serum by immunomagnetic-UHPLC-MS/MS.

Tri-ortho-cresyl phosphate (ToCP) is an anti-wear, flame retardant additive used in industrial lubricants, hydraulic fluids and gasoline. The neurotoxic effects of ToCP arise from the liver-activated metabolite 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (cresyl saligenin phosphate or CBDP), which inhibits esterase enzymes including butyrylcholinesterase (BChE). Following BChE adduction, CBDP undergoes hydrolysis to form the aged adduct ortho-cresyl phosphoserine (oCP-BChE), thus providing a biomarker of CBDP exposure. Previous studies have identified ToCP in aircraft cabin and cockpit air, but assessing human exposure has been hampered by the lack of a laboratory assay to confirm exposure. This work presents the development of an immunomagnetic-UHPLC-MS/MS method for the quantitation of unadducted BChE and the long-term CBDP biomarker, oCP-BChE, in human serum. The method has a reportable range from 2.0 ng/ml to 150 ng/ml, which is consistent with the sensitivity of methods used to detect organophosphorus nerve agent protein adducts. The assay demonstrated high intraday and interday accuracy (≥85%) and precision (RSD ≤ 15%) across the calibration range. The method was developed for future analyses of potential human exposure to CBDP. Analysis of human serum inhibited in vitro with CBDP demonstrated that the oCP-BChE adduct was stable for at least 72 h at 4, 22 and 37 °C. Compared to a previously reported assay, this method requires 75% less sample volume, reduces analysis time by a factor of 20 and demonstrates a threefold improvement in sensitivity. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Improvements of the fluoride reactivation method for the verification of nerve agent exposure.

One of the most appropriate biomarkers for the verification of organophosphorus nerve agent exposure is the conjugate of the nerve agent to butyrylcholinesterase (BuChE). The phosphyl moiety of the nerve agent can be released from the BuChE enzyme by incubation with fluoride ions, after which the resulting organophosphonofluoridate can be analyzed with gas chromatography-mass spectrometry (GC-MS). This paper describes recent improvements of the fluoride-induced reactivation in human plasma or serum samples by enhancing the sample preparation with new solid-phase extraction cartridges and the MS analysis with large volume injections. Analysis is performed with thermal desorption GC with either mass selective detection with ammonia chemical ionization or high-resolution MS with electron impact ionization. The organophosphorus chemical warfare agents analyzed in this study are O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate, ethyl methylphosphonofluoridate, isopropyl methylphosphonofluoridate (sarin, GB), O-ethyl N,N-dimethylphosphoramidocyanidate, ethyl N,N-dimethylphosphoramidofluoridate, and cyclohexyl methylphosphonfluoridate. Detection limits of approximately 10 pg/mL plasma were achieved for all analytes, which corresponds to 0.09% inhibition with GB on a sample with normal BuChE levels.

Timing of decontamination and treatment in case of percutaneous VX poisoning: a mini review.

Low volatile organophosphorous nerve agents such as VX, will most likely enter the body via the skin. The pharmacokinetics of drugs such as oximes, atropine and diazepam, are not aligned with the variable and persistent toxicokinetics of the agent. Repeated administration of these drugs showed to improve treatment efficacy compared to a single injection treatment. Because of the effectiveness of continuous treatment, it was investigated to what extent a subchronic pretreatment with carbamate (pyridostigmine or physostigmine combined with either procyclidine or scopolamine) would protect against percutaneous VX exposure. Inclusion of scopolamine in the pretreatment prevented seizures in all animals, but none of the pretreatments affected survival time or the onset time of cholinergic signs. These results indicate that percutaneous poisoning with VX requires additional conventional treatment in addition to the current pretreatment regimen. Decontamination of VX-exposed skin is one of the most important countermeasures to mitigate the effects of the exposure. To evaluate the window of opportunity for decontamination, the fielded skin decontaminant Reactive Skin Decontaminant Lotion (RSDL) was tested at different times in hairless guinea pigs percutaneously challenged with 4× LD50 VX in IPA. The results showed that RSDL decontamination at 15 min after exposure could not prevent progressive blood cholinesterase inhibition and therefore would still require additional treatment. A similar decontamination regimen with RSDL at 90 min showed that it still might effectively increase the time window of opportunity for treatment. In conclusion, the delay in absorption presents a window of opportunity for decontamination and treatment. The continuous release of VX from the skin presents a significant challenge for efficacious therapy, which should ideally consist of thorough decontamination and continuous treatment.

Increasing oxime efficacy by blood-brain barrier modulation.

One of the shortcomings of current treatment of nerve agent poisoning is that oximes hardly penetrate the blood-brain barrier (BBB), whereas nerve agents easily do. Increasing the concentration of oximes in the brain, would therefore provide an attractive approach to improve medical countermeasures. An explanation for limited penetration might be that oximes are substrates for the active P-glycoprotein (Pgp) efflux transporter located in the BBB. Using quantitative brain microdialysis in rats, the effect of i.v. injected tariquidar, a non-competitive, specific Pgp-inhibitor, on HI-6 levels in blood and brain was investigated. It appeared that tariquidar enhanced HI-6 levels in the brain approximately 2-fold during the first hour after HI-6 administration, whereas plasma levels did not differ between the treatment groups. A subsequent proof-of-concept study in rats showed that soman-induced seizures and convulsions were prevented almost completely when they were, in addition to HI-6 and atropine, pretreated with tariquidar. Moreover, twice as much AChE activity was present in their brains as compared to control rats. These results in rats indicate that modulation of the BBB by a drug like tariquidar, which is non-toxic by itself, is of great value in enhancing the efficacy of oximes.