The use of bispyridinium non-oxime analogues for the restoration of nerve agent impaired neuromuscular transmission in rat hemidiaphragms - Structure optimization.

Organophosphate pesticide poisoning challenges health care systems worldwide. Furthermore, nerve agents remain a continuous threat. The treatment options for organophosphate poisoning have virtually been unchanged for decades, relying on symptomatic treatment and the use of oximes to indirectly restore neuromuscular function. Hence, compounds targeting directly nicotinic acetylcholine receptors (nAChRs) might substantially improve treatment options. The current study investigated a series of bispyridinium analogues with a trimethylene or 2,2'-diethyloxy linker in a rat hemidiaphragm model, using indirect field stimulation. Methyl- and ethyl-substituted bispyridinium analogues restored neuromuscular function up to 37 ± 17% (MB419, a 3-methyl analogue) at a stimulation frequency of 20 Hz. The bispyridinium analogues with a 2- or 3-methyl group, or a 2- or 3-ethyl group, tended towards a higher restoration of neuromuscular function than those with a 4-methyl or 4-ethyl group, respectively. The current data can be used for future studies to optimize structure-based molecular modeling of compounds targeting the nAChR.

Protein networking: nicotinic acetylcholine receptors and their protein-protein-associations.

Nicotinic acetylcholine receptors (nAChR) are complex transmembrane proteins involved in neurotransmission in the nervous system and at the neuromuscular junction. nAChR disorders may lead to severe, potentially fatal pathophysiological states. To date, the receptor has been the focus of basic and applied research to provide novel therapeutic interventions. Since most studies have investigated only the nAChR itself, it is necessary to consider the receptor as part of its protein network to understand or elucidate-specific pathways. On its way through the secretory pathway, the receptor interacts with several chaperones and proteins. This review takes a closer look at these molecular interactions and focuses especially on endoplasmic reticulum biogenesis, secretory pathway sorting, Golgi maturation, plasma membrane presentation, retrograde internalization, and recycling. Additional knowledge regarding the nAChR protein network may lead to a more detailed comprehension of the fundamental pathomechanisms of diseases or may lead to the discovery of novel therapeutic drug targets.

Disulfide-adducts with cysteine residues in human serum albumin prove exposure to malodorous mercaptans in vitro.

Malodorants are mixtures containing mercaptans, which trigger the flight instinct upon exposure and might thus be deployed in military and civilian defense scenarios. Exposure to mercaptans might lead to unconsciousness, thus representing a possible threat for health. Therefore, we developed and validated a bioanalytical procedure for the simultaneous detection and identification of corresponding biomarkers for the verification of exposure to mercaptans. Disulfide-adducts of ethyl mercaptan (SEt), n-butyl mercaptan (SnBu), tert-butyl mercaptan (StBu) and iso-amyl mercaptan (SiAm) with cysteine (Cys) residues in human serum albumin (HSA) were formed by in vitro incubation of human plasma. After pronase-catalyzed proteolysis, reaction products were identified as adducts of the single amino acid Cys and the dipeptide cysteine-proline (Cys34Pro) detected by a sensitive µLC-ESI MS/MS method working in the scheduled multiple reaction monitoring (sMRM) mode. Dose-response studies showed linearity for the yield of Cys34Pro-adducts in the range from 6 nM to 300 µM of mercaptans in plasma and limits of identification (LOI) were in the range from 60 nM to 6 µM. Cys34-adducts showed stability for at least 6 days in plasma (37 °C). The presented disulfide-biomarkers expand the spectrum for bioanalytical verification procedures and might be helpful to prove exposure to malodorants.

Synthesis and Biological Evaluation of Novel MB327 Analogs as Resensitizers for Desensitized Nicotinic Acetylcholine Receptors after Intoxication with Nerve Agents.

Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062 and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.

Identification of ligands binding to MB327-PAM-1, a binding pocket relevant for resensitization of nAChRs.

Desensitization of nicotinic acetylcholine receptors (nAChRs) can be induced by overstimulation with acetylcholine (ACh) caused by an insufficient degradation of ACh after poisoning with organophosphorus compounds (OPCs). Currently, there is no generally applicable treatment for OPC poisoning that directly targets the desensitized nAChR. The bispyridinium compound MB327, an allosteric modulator of nAChR, has been shown to act as a resensitizer of nAChRs, indicating that drugs binding directly to nAChRs can have beneficial effects after OPC poisoning. However, MB327 also acts as an inhibitor of nAChRs at higher concentrations and can thus not be used for OPC poisoning treatment. Consequently, novel, more potent resensitizers are required. To successfully design novel ligands, the knowledge of the binding site is of utmost importance. Recently, we performed in silico studies to identify a new potential binding site of MB327, MB327-PAM-1, for which a more affine ligand, UNC0646, has been described. In this work, we performed ligand-based screening approaches to identify novel analogs of UNC0646 to help further understand the structure-affinity relationship of this compound class. Furthermore, we used structure-based screenings and identified compounds representing four new chemotypes binding to MB327-PAM-1. One of these compounds, cycloguanil, is the active metabolite of the antimalaria drug proguanil and shows a higher affinity towards MB327-PAM-1 than MB327. Furthermore, cycloguanil can reestablish the muscle force in soman-inhibited rat muscles. These results can act as a starting point to develop more potent resensitizers of nAChR and to close the gap in the treatment after OPC poisoning.

Inhibition kinetics of acetylcholinesterase and butyrylcholinesterase from various species by 2-(2-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide (CBDP).

The aerotoxic syndrome has been associated with exposure to tricresyl phosphate (TCP), which is used as additive in hydraulic fluids and engine lubricants. The toxic metabolite 2-(2-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide (CBDP) is formed from the TCP isomer tri-ortho-cresyl phosphate (TOCP) in vivo and is known to react with the active site serine in acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) resulting in the inhibition of the enzymes. Previous in vitro studies showed pronounced species differences in the inhibition kinetics of cholinesterases by organophosphorus compounds (OP), which must be considered in the development of relevant animal models for the investigation of OP poisoning and the aerotoxic syndrome. The present study was designed to investigate the inhibition kinetics of human, Cynomolgus monkey, pig, mini pig, guinea pig, mouse, and rat AChE as well as BChE by CBDP under standardized conditions. There were similar rate constants for the inhibition (ki) of human, Cynomolgus monkey and mouse AChE by CBDP. In contrast, the ki values obtained for guinea pig, mini pig, pig, and rat AChE were 2.8- to 5.9-fold lower than that of human AChE. The results of the present study confirmed CBDP as one of the most potent inhibitors of human BChE, indicating a ki value of 3.24 ± 0.33 ×108M-1min-1, which was about 1,140-fold higher than that of human AChE. Accordingly, a markedly more pronounced inhibition rate of BChE from the species guinea pig, mini pig, pig, rat, Cynomolgus monkey, and mouse by CBDP was found as compared to those of AChE from the respective sources, indicating 2.0- to 89.6-fold higher ki values.

Effects of the nerve agent VX on hiPSC-derived motor neurons.

Poisoning with the organophosphorus nerve agent VX can be life-threatening due to limitations of the standard therapy with atropine and oximes. To date, the underlying pathomechanism of VX affecting the neuromuscular junction has not been fully elucidated structurally. Results of recent studies investigating the effects of VX were obtained from cells of animal origin or immortalized cell lines limiting their translation to humans. To overcome this limitation, motor neurons (MN) of this study were differentiated from in-house feeder- and integration-free-derived human-induced pluripotent stem cells (hiPSC) by application of standardized and antibiotic-free differentiation media with the aim to mimic human embryogenesis as closely as possible. For testing VX sensitivity, MN were initially exposed once to 400 µM, 600 µM, 800 µM, or 1000 µM VX and cultured for 5 days followed by analysis of changes in viability and neurite outgrowth as well as at the gene and protein level using µLC-ESI MS/HR MS, XTT, IncuCyte, qRT-PCR, and Western Blot. For the first time, VX was shown to trigger neuronal cell death and decline in neurite outgrowth in hiPSC-derived MN in a time- and concentration-dependent manner involving the activation of the intrinsic as well as the extrinsic pathway of apoptosis. Consistent with this, MN morphology and neurite network were altered time and concentration-dependently. Thus, MN represent a valuable tool for further investigation of the pathomechanism after VX exposure. These findings might set the course for the development of a promising human neuromuscular test model and patient-specific therapies in the future.

Suitability of human HepaRG cells and liver spheroids as in vitro model to investigate the bioactivation of the organothiophosphate pesticide parathion.

Organophosphorus compounds (OP) constitute a large group of chemicals including pesticides and nerve agents. Organothiophosphate pesticides require cytochrome P450-mediated oxidative desulphuration in the liver to form corresponding oxons, which are potent inhibitors of the enzyme acetylcholinesterase (AChE). Human HepaRG cells are a promising tool to study liver-specific functions and have been shown to maintain drug metabolizing enzymes. This research describes for the first time the in vitro metabolic activation of an organothiophosphate to its active oxon by two different HepaRG cell-based models. Monolayer cultures and liver spheroids were exposed to the model OP parathion and the quantification of the corresponding oxon was performed with an AChE inhibition assay. Our results showed a time- and dose-dependent cytochrome P450 catalyzed bioactivation and a superior metabolism capacity of the monolayer HepaRG model in comparison with the liver spheroids. Finally, HepaRG cells can be assessed as a metabolically competent cell model intermediate between cell-free preparations and intact animals and as suitable to study OP metabolism in the human liver.

Uncharged mono- and bisoximes: In search of a zwitterion to countermeasure organophosphorus intoxication.

The current study imposes a new class of organophosphorus (OP)-inhibited cholinesterase reactivators by conceptualizing a family of asymmetric bisoximes with various reactivating scaffolds. Several novel nucleophilic warheads were investigated, putting forward 29 novel reactivating options, by evaluating their nucleophilicity and ability to directly decompose OP compounds. Adopting the so-called zwitterionic strategy, 17 mono-oxime and nine bisoxime reactivators were discovered with major emphasis on the bifunctional-moiety approach. Compounds were compared with clinically used standards and other known experimentally highlighted reactivators. Our results clearly favor the concept of asymmetric bisoximes as leading reactivators in terms of efficacy and versatility. These top-ranked compounds were characterized in detail by reactivation kinetics parameters and evaluated for potential CNS availability. The highlighted molecules 55, 57, and 58 with various reactivating warheads, surpassed the reactivating potency of pralidoxime and several notable uncharged reactivators. The versatility of lead drug candidate 55 was also inspected on OP-inhibited butyrylcholinesterase, revealing a much higher rate compared to existing clinical antidotes.

Restoration of nerve agent impaired neuromuscular transmission in rat diaphragm by bispyridinium non-oximes - Structure-activity relationships.

Organophosphate (OP) poisoning is currently treated with atropine, oximes and benzodiazepines. The nicotinic signs, i.e., respiratory impairment, can only be targeted indirectly via the use of oximes as reactivators of OP-inhibited acetylcholinesterase. Hence, compounds selectively targeting nicotinic acetylcholine receptors (nAChRs) might fundamentally improve current treatment options. The bispyridinium compound MB327 has previously shown some therapeutic effect against nerve agents in vitro and in vivo. Nevertheless, compound optimization was deemed necessary, due to limitations (e.g., toxicity and efficacy). The current study investigated a series of 4-tert-butyl bispyridinium compounds and of corresponding bispyridinium compounds without substituents in a rat diaphragm model using an indirect field stimulation technique. The length of the respective linker influenced the ability of the bispyridinium compounds to restore muscle function in rat hemidiaphragms. The current data show structure-activity relationships for a series of bispyridinium compounds and provide insight for future structure-based molecular modeling.

Screening for new ligands of the MB327-PAM-1 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) effect a severe impairment of cholinergic neurotransmission that, as a result of overstimulation may lead to desensitization of nicotinic acetylcholine receptors (nAChRs) and finally to death due to respiratory paralysis. So far, therapeutics, that are capable to address and revert desensitized neuromuscular nAChRs into their resting, i.e. functional state are still missing. Still, among a class of compounds termed bispyridinium salts, which are characterized by the presence of two pyridinium subunits, constituents have been identified, that can counteract organophosphate poisoning by resensitizing desensitized nAChRs. According to comprehensive modeling studies this effect is mediated by an allosteric binding site at the nAChR termed MB327-PAM-1 site. For MB327, the most prominent representative of the bispyridinium salts and all other analogues studied so far, the affinity for the aforementioned binding site and the intrinsic activity measured in ex vivo and in in vivo experiments are distinctly too low, to meet the criteria to be fulfilled for therapeutic use. Hence, in order to identify new compounds with higher affinities for the MB327-PAM-1 binding site, as a basic requirement for an enhanced potency, two compound libraries, the ChemDiv library with 60 constituents and the Tocriscreen Plus library with 1280 members have been screened for hit compounds addressing the MB327-PAM-1 binding site, utilizing the [2H6]MB327 MS Binding Assay recently developed by us. This led to the identification of a set of 10 chemically diverse compounds, all of which exhibit an IC50 value of ≤ 10 µM (in the [2H6]MB327 MS Binding Assay), which had been defined as selection criteria. The three most affine ligands, which besides a quinazoline scaffold share similarities with regard to the substitution pattern and the nature of the substituents, are UNC0638, UNC0642 and UNC0646. With binding affinities expressed as pKi values of 6.01 ± 0.10, 5.97 ± 0.05 and 6.23 ± 0.02, respectively, these compounds exceed the binding affinity of MB327 by more than one log unit. This renders them promising starting points for the development of drugs for the treatment of organophosphorus poisoning by addressing the MB327-PAM-1 binding site of the nAChR.

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.

MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR, as well. In this study, UNC0642 has been utilized as a reporter ligand to establish new Binding Assays for this target. These assays follow the concept of MS Binding Assays for which by assessing the amount of bound reporter ligand by mass spectrometry no radiolabeled material is required. According to the results of the performed MS Binding Assays comprising saturation and competition experiments it can be concluded, that UNC0642 used as a reporter ligand addresses the MB327 binding site of the Torpedo-nAChR. This is further supported by the outcome of ex vivo studies carried out with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of UNC0646, an analog of UNC0642 with the highest binding affinity, in the recently proposed binding site of MB327 (MB327-PAM-1). With UNC0642 addressing the MB327 binding site of the Torpedo-nAChR, this and related quinazoline derivatives represent a promising starting point for the development of novel ligands of the nAChR as antidotes for the treatment of intoxications with organophosphorus compounds. Further, the new MS Binding Assays are a potent alternative to established assays and of particular value, as they do not require the use of radiolabeled material and are based on a commercially available compound as reporter ligand, UNC0642, exhibiting one of the highest binding affinities for the MB327 binding site known so far.

AChE reactivation in precision-cut lung slices following organophosphorus compound poisoning.

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.

Engineering of a phosphotriesterase with improved stability and enhanced activity for detoxification of the pesticide metabolite malaoxon.

Organophosphorus (OP) pesticides are still widely applied but pose a severe toxicological threat if misused. For in vivo detoxification, the application of hydrolytic enzymes potentially offers a promising treatment. A well-studied example is the phosphotriesterase of Brevundimonas diminuta (BdPTE). Whereas wild-type BdPTE can hydrolyse pesticides like paraoxon, chlorpyrifos-oxon and mevinphos with high catalytic efficiencies, kcat/KM >2 × 107 M-1 min-1, degradation of malaoxon is unsatisfactory (kcat/KM ≈ 1 × 104 M-1 min-1). Here, we report the rational engineering of BdPTE mutants with improved properties and their efficient production in Escherichia coli. As result, the mutant BdPTE(VRNVVLARY) exhibits 37-fold faster malaoxon hydrolysis (kcat/KM = 4.6 × 105 M-1 min-1), together with enhanced expression yield, improved thermal stability and reduced susceptibility to oxidation. Therefore, this BdPTE mutant constitutes a powerful candidate to develop a biocatalytic antidote for the detoxification of this common pesticide metabolite as well as related OP compounds.

Recombinant cellular model system for human muscle-type nicotinic acetylcholine receptor α12ß1δε.

The human muscle-type nicotinic acetylcholine receptor α12ß1δε (nAChR) is a complex transmembrane receptor needed for drug screening for disorders like congenital myasthenic syndromes and multiple pterygium syndrome. Until today, most models are still using the nAChR from Torpedo californica electric ray. A simple reproducible cellular system expressing functional human muscle-type nAChR is still missing. This study addressed this issue and further tested the hypothesis that different chaperones, both biological and chemical, and posttranslational modification supporting substances as well as hypothermic incubation are able to increase the nAChR yield. Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development. Graphical abstract created with http://biorender.com.

Trauma-toxicology: concepts, causes, complications.

Trauma and toxic substances are connected in several aspects. On the one hand, toxic substances can be the reason for traumatic injuries in the context of accidental or violent and criminal circumstances. Examples for the first scenario is the release of toxic gases, chemicals, and particles during house fires, and for the second scenario, the use of chemical or biological weapons in the context of terroristic activities. Toxic substances can cause or enhance severe, life-threatening trauma, as described in this review for various chemical warfare, by inducing a tissue trauma accompanied by break down of important barriers in the body, such as the blood-air or the blood-gut barriers. This in turn initiates a "vicious circle" as the contribution of inflammatory responses to the traumatic damage enhances the macro- and micro-barrier breakdown and often results in fatal outcome. The development of sophisticated methods for detection and identification of toxic substances as well as the special treatment of the intoxicated trauma patient is summarized in this review. Moreover, some highly toxic substances, such as the protein toxins from the pathogenic bacterium Clostridioides (C.) difficile, cause severe post-traumatic complications which significantly worsens the outcome of hospitalized patients, in particular in multiply injured trauma patients. Therefore, novel pharmacological options for the treatment of such patients are necessarily needed and one promising strategy might be the neutralization of the toxins that cause the disease. This review summarizes recent findings on the molecular and cellular mechanisms of toxic chemicals and bacterial toxins that contribute to barrier breakdown in the human body as wells pharmacological options for treatment, in particular in the context of intoxicated trauma patients. "trauma-toxicology" comprises concepts regrading basic research, development of novel pharmacological/therapeutic options and clinical aspects in the complex interplay and "vicious circle" of severe tissue trauma, barrier breakdown, pathogen and toxin exposure, tissue damage, and subsequent clinical complications.

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.

Evidence of nerve agent VX exposure in rat plasma by detection of albumin-adducts in vitro and in vivo.

VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys34(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys448(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 °C thus making the adducts highly suited for bioanalysis. Cys34(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 °C. For the first time, Cys34(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.

A pharmacologically pre-contracted smooth muscle bowel model for the study of highly-potent opioid receptor agonists and antagonists.

Isolated organ models are a versatile tool for pharmacological and toxicological research. Small bowel has been used to assess the inhibition of smooth muscle contraction by opioids. In the present study, we set out to establish a pharmacologically stimulated rat bowel model. The effects of carfentanil, remifentanil and the new synthetic opioid U-48800 and their respective antagonists naloxone, nalmefene and naltrexone were studied in a small bowel model in rats. The IC50 values of the tested opioids were as follows: carfentanil (IC50 = 0.02 µmol/L, CI 0.02-0.03 µmol/L) ≫ remifentanil (IC50 = 0.51 µmol/L, CI 0.40-0.66 µmol/L) ≫ U-48800 (IC50 = 1.36 µmol/L, CI 1.20-1.54 µmol/L). The administration of the opioid receptor antagonists naloxone, naltrexone and nalmefene led to progressive, parallel rightward shifts of the dose-response curves. Naltrexone was most potent in antagonizing the effects of U-48800, whereas naltrexone and nalmefene were most effective in antagonizing the effects of carfentanil. In summary, the current model seems to be a robust tool to study opioid effects in a small bowel model without the necessity of using electrical stimulation.