Reactive skin decontamination lotion (RSDL) safety with clinical antiseptics and hemostatic agents.

Reactive skin decontamination lotion (RSDL) is a Health Canada approved product used by the Canadian Armed Forces for removal and inactivation of toxic chemicals on skin. Although it is considered very safe when used as directed, questions have been raised regarding whether topical RSDL in the medical setting will react exothermically with antiseptic compounds on the casualty's epidermis that could result in thermal burns. Benchtop experiments were conducted to investigate reactivity of RSDL with various antiseptic compounds or hemostatic agents. Temperature changes were closely monitored in three different volume ratios, 1:10, 1:1, and 10:1 over a time course of 16 minutes. Chlorine based bleaches versus RSDL were included as a positive control and were the only combination that exhibited a significant exothermic reaction capable of causing minor thermal burns. RSDL was also evaluated with antiseptic solution applied to swine epidermal tissue without observation of visual irritation; then in lacerated skeletal muscle tissue which resulted in no measured temperature change. The conclusion of this study is that antiseptics and hemostatic agents can be used as required on a patient decontaminated with RSDL as no exothermic reaction will occur.

Cerebral blood flow and oxygenation in rat brain after soman exposure.

Nerve agent exposure can cause debilitating neurological damage even with treatment. Currently accepted treatments involve attenuating the cholinergic crisis and seizure onset but do not focus directly on neuroprotection. Hence, there is a need for improved treatments to reduce neurological deficits. It is important to understand the pathophysiology of nerve agent mediated injury in order to identify effective treatment targets. Nerve agent-induced seizures are believed to be the main contributor to the neuropathology. Recently seizures have been shown to cause vascular changes that may actually attenuate neurological damage. This study evaluated the effect of soman-induced convulsive seizures on the relationship between CNS oxygen consumption and supply. To simultaneously assess changes in oxygenation and perfusion, rats were implanted with permanently fixed fiber-optic tissue oxygen sensing probes in the motor cortex and imaged with continuous arterial spin labelling MRI to measure cerebral blood flow. Baseline tissue oxygen tension (ptO2) and cerebral blood flow (CBF) were measured in isoflurane anaesthetized rats at least one day prior to soman or saline exposure. Rats were pretreated with HI-6 dimethansulfonate and atropine methyl nitrate (125 mg/kg and 20 mg/kg; intraperitoneal) followed by a convulsive dose of soman (90 µg/kg; subcutaneous) or equal volume of saline. Three additional treatments of HI-6/AMN were administered to improve survival. At 1.5 -hs after exposure, ptO2 and cerebral blood flow measurements were conducted. There was a significant decrease in CBF 1.5 -hs following soman exposure but no change in ptO2 was found. When we correlated ptO2 and CBF, for a given ptO2, there was lower CBF following soman exposure. This may indicate metabolism is inhibited, possibly because of mitochondrial impairment, therefore reducing oxygen demand. These data show hypoperfusion in brain following soman exposure which would be expected to contribute to soman-related neuropathology.

Quantitative T2 MRI is predictive of neurodegeneration following organophosphate exposure in a rat model.

Organophosphorus compounds, such as chemical warfare nerve agents and pesticides, are known to cause neurological damage. This study measured nerve agent-related neuropathology and determined whether quantitative T2 MRI could be used as a biomarker of neurodegeneration. Quantitative T2 MRI was performed using a 9.4 T MRI on rats prior to and following soman exposure. T2 images were taken at least 24 h prior, 1 h and 18-24 h after soman exposure. Rats were pre- and post-treated with HI-6 dimethanesulfonate and atropine methyl nitrate. A multicomponent T2 acquisition and analysis was performed. Brains were stained with Fluoro-Jade C to assess neurodegeneration. Rats exposed to soman developed behavioral expression of electrographic seizures. At 18-24 h after soman exposure, significant increases in T2, a possible marker of edema, were found in multiple regions. The largest changes were in the piriform cortex (before: 47.7 ± 1.4 ms; 18-24 h: 82.3 ± 13.4 ms). Fluoro-Jade C staining showed significant neurodegeneration 18-24 h post exposure. The piriform cortex had the strongest correlation between the change in relaxation rate and percent neurodegeneration (r = 0.96, p < 0.001). These findings indicate there is regionally specific neurodegeneration 24 h after exposure to soman. The high correlation between T2 relaxivity and histopathology supports the use of T2 as a marker of injury.

In vivo efficacy of the Reactive Skin Decontamination Lotion (RSDL®) kit against organophosphate and carbamate pesticides.

In this study, we assessed the efficacy of the Reactive Skin Decontamination Lotion (RSDL®) Kit against parathion and aldicarb pesticide dermal exposure in a guinea pig model. The pesticides inhibit acetylcholinesterase (AChE) leading to signs and symptoms of hyperactivity of organs due to accumulation of acetylcholine. The RSDL Kit has been shown to physically remove and chemically degrade chemical warfare agents. Degradation occurs from a nucleophilic substitution reaction between an active ingredient in the RSDL lotion, potassium 2,3-butanedione monoximate (KBDO), with susceptible sites in these compounds. In the present study, guinea pigs dermally exposed to parathion and aldicarb were decontaminated with RSDL to mitigate the toxic effects of the pesticides. It is observed that animals exposed to 749 mg/kg of parathion (n = 3) died within 24 h without RSDL decontamination; however, RSDL-treated animals (n = 3) showed only mild signs of neurotoxicity. The RSDL-treated animals had an AChE inhibition of 0-58% while the untreated animals had up to 86% inhibition. Similarly, RSDL has been demostrated to prevent aldicarb neurotoxicity effects. The percent inhibition of AChE activity during the 24 h post challenge of 9 mg aldicarb/kg of animal weight ranged from 25% to 61% with severe signs of intoxication while only up to 5% with mild or no signs of intoxication in the case of RSDL-decontaminated animals. Generally, it has been shown that the toxic effects of the organophosphate and carbamate pesticides can be prevented via decontamination using the RSDL Kit.

Pharmacokinetics of Three Oximes in a Guinea Pig Model and Efficacy of Combined Oxime Therapy.

Organophosphorus nerve agents (NA) inhibit acetylcholinesterase (AChE) which results in the over-stimulation of both the central and peripheral nervous systems, creating a toxic syndrome that can be lethal if left untreated (Cannard, 2006). It is standard practice to treat Sarin (GB) intoxication with an oxime, an antimuscarinic such as atropine and an anticonvulsant. Three common oximes are available: HI-6, Pralidoxime (2-PAM) and Obidoxime (Obi), all possess a nucleophile that can break the NA-AChE covalent bond. However, each oxime's efficacy profile against various agents is different (Thiermann and Worek, 2018). In an effort to broaden therapeutic efficacy against a range of possible NA's, consideration should be given to the use of two oximes in combination. Using a guinea pig model, the first arm of this study was to determine the pharmacokinetics (PK) of HI-6 DMS, 2-PAM chloride and Obi chloride (at autoinjector equivalent doses) following intramuscular (i.m.) co-administration along with atropine to replicate either a single isometrically scaled dose (referred to in this study as a single autoinjector equivalent) of 2-PAM (and equimolar doses of Obi and HI-6) or double doses (referred to in this study as two autoinjector equivalents). The second arm of the study evaluated the efficacy of Obi and 2-PAM individually at a single or double autoinjector dose and also in combination against GB exposure. Pharmacokinetic profiles of each oxime were evaluated for both arms of the study and no significant change in parameters were reported. Improved cholinesterase reactivation was observed in a dose dependent manner with combined therapy showing similar reactivation to individual oximes alone at a two autoinjector equivalent dose. Seizure activity was reduced when combined oxime therapy was administered. This improvement was also reflected in the Racine seizure index score assigned at the end of the experimental period. To the best of our knowledge, this study is the first to evaluate and compare the pharmacokinetics of three oximes and the combination of two oximes (2-PAM and Obi) administered in naïve animals or those exposed to GB. Combined oxime therapy (Obi and 2-PAM) resulted in improved seizure control, increased cholinesterase reactivation peripherally and centrally and improved behavioral signs (Racine score). This study provides evidence that combination of oximes is effective, does not result in adverse events and that the pharmacokinetics of each oxime are not affected when administered in combination.

A newly developed oxime K203 is the most effective reactivator of tabun-inhibited acetylcholinesterase.

BACKGROUND: Based on in vitro and in vivo rat experiments, the newly developed acetylcholinesterase (AChE) reactivator, K203, appears to be much more effective in the treatment of tabun poisonings than currently fielded oximes. METHODS: To determine if this reactivating efficacy would extend to humans, studies were conducted in vitro using human brain homogenate as the source of AChE. The efficacy of K203 was compared with commercially available oximes; pralidoxime, obidoxime and asoxime (HI-6). RESULTS: Reactivation studies showed that K203 was the most effective reactivator with a second order kinetic constant (kr) of 2142 min- 1. M- 1, which was 51 times higher than that obtained for obidoxime (kr = 42 min- 1. M- 1). Both pralidoxime and asoxime (HI-6) failed to significantly reactivate tabun-inhibited human AChE. DISCUSSION: According to these results and previous studies, using K203, it appears that oxime K203 is the most effective reactivator of tabun-inhibited cholinesterase in several species including humans and should be considered as a possible medical countermeasure to tabun exposure.

Assessment of brain oxygenation imbalance following soman exposure in rats.

Nerve agents (NAs) are potent organophosphorus (OP) compounds with applications in chemical warfare. OP compounds act by inhibiting acetylcholinesterase (AChE). Soman (O-pinacolyl methylphosphonofluoridate) is one of the most potent NAs. It is well known that small doses of NAs can be lethal, and that even non-lethal exposure leads to long-term mental debilitation/neurological damage. However, the neuropathology following exposure to sub-lethal nerve agents is not well understood. In this study, we examined changes in tissue oxygenation (pO2) in the cortex and hippocampus after a sub-lethal dose of soman [80-90 µg/kg; subcutaneous]. pO2 changes can provide information regarding oxygen delivery and utilization and may be indicative of a disruption in cerebral blood flow and/or metabolism. Changes in oxygenation were measured with chronically implanted oxygen sensors in awake and freely moving rats. Measurements were taken before, during, and after soman-induced convulsive seizures. Soman exposure resulted in an immediate increase in pO2 in the cortex, followed by an even greater increase that precedes the onset of soman-induced convulsive seizures. The rise in hippocampus pO2 was delayed relative to the cortex, although the general pattern of brain oxygenation between these two regions was similar. After convulsive seizures began, pO2 levels declined but usually remained hyperoxygenated. Following the decline in pO2, low frequency cycles of large amplitude changes were observed in both the cortex and hippocampus. This pattern is consistent with recurring seizures. Measuring real-time changes in brain pO2 provides new information on the physiological status of the brain following soman exposure. These results highlight that the measurement of brain oxygenation could provide a sensitive marker of nerve agent exposure and serve as a biomarker for treatment studies.

Comparison of tranexamic acid plasma concentrations when administered via intraosseous and intravenous routes.

INTRODUCTION: There is a lack of information regarding intraosseous (IO) administration of tranexamic acid (TXA). Our hypothesis was that a single bolus IO injection of TXA will have a similar pharmacokinetic profile to TXA administered at the same dose IV. METHODS: Sixteen male Landrace cross swine (mean body weight 27.6±2.6kg) were divided into an IV group (n=8) and an IO group (n=8). Each animal received 30mg/kg TXA via an IV or IO catheter, respectively. Jugular blood samples were collected for pharmacokinetic analysis over a 3h period. The maximum TXA plasma concentration (Cmax) and corresponding time as well as distribution half-life, elimination half-life, area under the curve, plasma clearance and volume of distribution were calculated. One- and two-way analysis of variance for repeated measures (time, group) with Tukey's and Bonferonni post hoc tests were used to compare TXA plasma concentrations within and between groups, respectively. RESULTS: Plasma concentrations of TXA were significantly higher (p<0.0001) in the IV group during the TXA infusion. Cmax occurred at 4min after initiation of the bolus in the IV group (9.36±3.20ng/µl) and at 5min after initiation of the bolus in the IO group (4.46±0.49ng/µl). Plasma concentrations were very similar from the completion of injection onwards. There were no significant differences between the two administration routes for any other pharmacokinetic variables measured. CONCLUSION: The results of this study support pharmacokinetic bioequivalence of IO and IV administration of TXA.

Reversed-phase ion-pair chromatography-diode array detection of the bispyridinium compound MB327: plasma analysis of a potential novel antidote for the treatment of organophosphorus poisoning.

In the case of poisoning by organophosphorus nerve agents or pesticides, there is still a lack of pharmacological treatment of the cholinergic crisis selectively targeting the nicotinic acetylcholine receptor. Recently, the compound MB327 was identified as a potential novel lead structure to close this gap, thus demanding a quantitative assay for initial pharmacokinetic (PK) studies. MB327 is a salt consisting of the dicationic bispyridinium compound (BPC) 1,1´-(propane-1,3-diyl)bis(4-tert-butylpyridinium) and two iodide counter ions. Due to the permanent positive charge of the BPC, an isocratic reversed-phase ion-pair chromatographic separation (RPIPC) was developed using heptanesulfonic acid as ion-pairing reagent and 45% v/v methanol as organic modifier (1 mL/min). Selective UV-detection (230 nm) was done by a diode array detector (DAD) for reliable, rugged, precise (RSD < 7%) and accurate (96-104%) quantitative analysis of 50 µL swine plasma (linear range 1-1000 µg BPC/mL plasma, lower limit of quantification 2 µg/mL). During method validation, diverse parameters essential for the chromatographic process were investigated to generate van´t Hoff, van Deemter and width plots allowing calculation of thermodynamic data like the distribution constant K (5.7 ± 0.3), change in enthalpy, ΔH(0) : -23.66 kJ/mol, and entropy, ΔS(0) : -65 J/(mol*K). In addition, RPIPC-DAD analysis enabled calculation of molar absorptivities of the BPC, ε230 : 17 400 ± 1100 L/(mol*cm), and iodide, ε230 : 9900 ± 400 L/(mol*cm), which determination was hampered by interference with each other in conventional cuvette UV-spectrophotometric measurements. Finally, the RPIPC-DAD procedure was applied to samples from an in vivo study of swine.

In vitro and in vivo toxicological studies of V nerve agents: molecular and stereoselective aspects.

In vitro inhibition data of cholinesterases (ChEs) and reactivation with HI 6 are presented for separated VX and VR enantiomers with high purity (enantiomer excess >99.999%). Inhibition rate constants for (-)-VR were fourfold higher than for (-)-VX. Marked higher stereoselectivity of ChEs inhibition was observed for VR compared with VX enantiomers. Low/no reactivation was determined for respective (+)-enantiomers. Results were related to orientation of (-)- and (+)-enantiomers in ChEs active sites. In vivo in swine, absorption rate constants were practically identical for VX and VR enantiomers after percutaneous application of 3xLD50 underlining relevance of amine group and postulated equilibria shifts between charged, uncharged, open and cyclic form (skin depot). In vivo toxicokinetics of VX and VR enantiomers differed markedly after 4h. Elimination of VX was much slower compared with VR. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition in vivo differed for VX and VR. In vivo spontaneous reactivation was not observed for VX-inhibited AChE while VR-inhibited AChE was much faster spontaneously reactivated than expected and AChE inhibition by VR was slower than expected. Progredient BChE inhibition was detected after VX application while VR inhibited BChE weakly. Possible explanation may be impact of the agents on hemodynamics and different metabolisms. Thus, due to increase of the V agents' blood concentration after atropine administration (depot release) the present standard therapy should be thoroughly reconsidered.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of titanium oxide-enriched peptides for detection of aged organophosphorus adducts on human butyrylcholinesterase.

Exposure to nerve agents or organophosphorus (OP) pesticides can have life-threatening effects. Human plasma butyrylcholinesterase (BChE) inactivates these poisons by binding them to Ser198. After hours or days, these OP adducts acquire a negative charge by dealkylation in a process called aging. Our goal was to develop a method for enriching the aged adduct to facilitate detection of exposure. Human BChE inhibited by OP toxicants was incubated for 4 days to 6 years. Peptides produced by digestion with pepsin were enriched by binding to titanium oxide (TiO2) and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. It was found that with two exceptions, all aged OP adducts in peptide FGES198AGAAS were enriched by binding to Titansphere tips. Cresyl saligenin phosphate yielded two types of aged adduct, cresylphosphate and phosphate, but only the phosphate adduct bound to Titansphere. The nerve agent VR yielded no aged adduct, supporting crystal structure findings that the VR adduct on BChE does not age. The irreversible nature of aged OP adducts was demonstrated by the finding that after 6 years at room temperature in sterile pH 7.0 buffer, the adducts were still detectable. It was concluded that TiO2 microcolumns can be used to enrich aged OP-modified BChE peptide.

Non-cholinergic intervention of sarin nerve agent poisoning.

The protective effects of selected anesthetic regimens on sarin (GB) were investigated in domestic swine. At 30% oxygen, the toxicity of this agent in isoflurane anesthetized animals (LD(50)=10.1µg/kg) was similar to literature sited values in awake swine (LD(50)=11.8µg/kg) and slightly higher than that of both ketamine (LD(50)=15.6µg/kg) and propofol (LD(50)=15.3µg/kg) anesthetized swine. Use of 100% oxygen in ketamine anesthetized animals resulted in three-fold protective effects compared to 30% oxygen. Use of 100% oxygen in both isoflurane and propofol anesthetized animals, compared to 30% resulted in profound protection against GB poisoning (>33×). There were no differences in the severity of the poisoning or recovery time in animals treated over dose ranges of 10-350µg/kg (isoflurane) or 15-500µg/kg GB (propofol). Survivors of high GB challenges that were revived from propofol anesthetic exhibited no signs of cognitive impairment seven days later. Protective treatments did not attenuate cholinesterase (ChE) inhibition; survivors of otherwise supralethal GB concentrations exhibited very low blood ChE activities. This work indicates that propofol has protective effects against GB, and that oxygen tension may have an important role in treating nerve agent casualties. More importantly, it demonstrates that non-cholinergic protective mechanisms exist that may be exploited in the future development of medical countermeasures against organophosphorous nerve agents.

Application of an enantioselective LC-ESI MS/MS procedure to determine R- and S-hyoscyamine following intravenous atropine administration in swine.

S-hyoscyamine (S-hyo) is a natural plant tropane alkaloid acting as a muscarinic receptor (MR) antagonist. Its racemic mixture (atropine) is clinically used in pre-anaesthesia, ophthalmology and for the antidotal treatment of organophosphorus (OP) poisoning with nerve agents or pesticides even though R-hyo exhibits no effects on MR. Further investigative research is required to optimize treatment of OP poisoning. Swine are often the animal model utilized due to similarities in physiology and antidote response to humans. However, no studies have been reported that elucidated differences in the kinetics of R- and S-hyo. Therefore, the concentration-time profiles of total hyo as well as both enantiomers were analyzed in plasma after intravenous administration of atropine sulfate (Atr(2) SO(4) , 100 µg/kg) to anaesthetized swine. For quantification plasma samples were incubated separately with human serum (procedure A) and rabbit serum (procedure B). The rabbit serum used contained atropinesterase, which is suitable for stereoselective hydrolysis of S-hyo, while human serum does not hydrolyze either enantiomer. After incubation samples were precipitated and the supernatant was analyzed by RP-HPLC-ESI MS/MS. Procedure A allowed determination of total hyo and procedure B remaining R-hyo concentrations. S-hyo was calculated as the difference of the two procedures. Concentration data were regressed by a two-phase decay according to a two-compartment open model revealing similar kinetics for both enantiomers thus indicating distribution, metabolism and elimination without obvious stereoselective preference in tested swine.

Simultaneous quantification of VX and its toxic metabolite in blood and plasma samples and its application for in vivo and in vitro toxicological studies.

The present study was initiated to develop a sensitive and highly selective method for the simultaneous quantification of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) and its toxic metabolite (EA-2192) in blood and plasma samples in vivo and in vitro. For the quantitative detection of VX and EA-2192 the resolution was realized on a HYPERCARB HPLC phase. A specific procedure was developed to isolate both toxic analytes from blood and plasma samples. The limit of detection was 0.1 pg/ml and the absolute recovery of the overall sample preparation procedure was 74% for VX and 69% for EA-2192. After intravenous and percutaneous administration of a supralethal doses of VX in anaesthetised swine both VX and EA-2192 could be quantified over 540 min following exposure. This study is the first to verify the in vivo formation of the toxic metabolite EA-2192 after poisoning with the nerve agent VX. Further toxicokinetic and therapeutic studies are required in order to determine the impact of EA-2192 on the treatment of acute VX poisoning.

Effect of acute soman exposure on GABA(A) receptors in rat hippocampal slices and cultured hippocampal neurons.

Exposure of the central nervous system to organophosphorus (OP) nerve agents causes seizures and neuronal cell death. Benzodiazepines are commonly used to treat seizures induced by OPs. However, it is known that soman-induced seizures are particularly resistant to benzodiazepine treatment, as compared with other OPs. This study investigated the effect of soman on γ-aminobutyric acid (GABA) neurotransmission in acute rat hippocampal slices and the surface expression of GABA(A) receptors in cultured rat hippocampal neurons. Results showed that GABA-mediated inhibitory post synaptic currents (IPSCs) are significantly reduced by soman in a concentration-dependent manner in acute rat hippocampal slices. Furthermore, confocal microscopic and cell-based ELISA assays revealed that soman caused rapid internalization of GABA(A) receptors in cultured rat hippocampal neurons. The effect of soman on GABA(A)R endocytosis was not due to inhibition of acetylcholinesterase (AChE) because (1) the acetylcholine muscarinic receptor antagonist atropine did not block soman-induced GABA(A)R endocytosis; and (2) physostigmine, at concentrations that completely inhibit AChE activity, did not cause GABA(A)R endocytosis. Moreover, blocking of the N-methyl-D-aspartate (NMDA) receptors by 2-amino-5-phosphonovalerate (APV) had no effect on soman-induced GABA(A)R endocytosis, suggesting that the soman effect was not secondary to glutamate receptor over activation. Regardless of the exact mechanism, the observation that soman induces rapid GABA(A)R endocytosis may have significant implications in the development of effective countermeasures against soman-induced seizures.

Comparative protective effects of HI-6 and MMB-4 against organophosphorous nerve agent poisoning.

The oximes pralidoxime (2-PAM), its dimethanesulphonate salt derivative P2S, and obidoxime (toxogonin) are currently licensed and fielded for the treatment of chemical warfare (CW) organophosphorous (OP) nerve agent poisoning. While they are effective against several of the identified threat CW OP agents, they have little efficacy against others such as soman (GD) and cyclosarin (CF). In addition, they are also significantly less effective than other investigational oximes against the nerve agent known as Russian VX (RVX). Among the oximes currently being investigated, two in particular, HI-6 (asoxime) and MMB-4 (ICD-039, methoxime) have been proposed as replacement therapies for the currently licensed oximes. HI-6 has been safely used in individuals to treat OP insecticide poisoning, as well as in human volunteers, although its efficacy against OP nerve agent poisoning in humans cannot be demonstrated due to ethical considerations. It is currently available for use in defined military settings in Canada, Sweden and the Czech Republic, and is also under development in a number of other countries. The oxime MMB-4 has not yet been studied clinically, but is fielded by the Czech Republic, and is being developed by the United States armed services as a replacement for the currently fielded 2-PAM. This review compares the effectiveness of HI-6 and MMB-4 against nerve agent threats where comparisons can be made. HI-6 has been demonstrated to be generally a superior reactivator of nerve agent inhibited enzyme, particularly with human and non-human primate derived enzyme, and has also shown better protective effects against the lethality of most OP agents in a variety of species. Both compounds appear to be clearly superior to the available oximes, obidoxime and 2-PAM.

Development and validation of a sensitive HPLC method for the quantification of HI-6 in guinea pig plasma and evaluated in domestic swine.

A rapid and small volume assay to quantify HI-6 in plasma was developed to further the development and licensing of an intravenous formulation of HI-6. The objective of this method was to develop a sensitive and rapid assay that clearly resolved HI-6 and an internal standard in saline and plasma matrices. A fully validated method using ion-pair HPLC and 2-PAM as the internal standard fulfilled these requirements. Small plasma samples of 35 microL were extracted using acidification, filtration and neutralization. Linearity was shown for over 4 microg/mL to 1mg/mL with accuracy and precision within 6% relative error at the lower limit of detection. This method was utilized in the pharmacokinetic analysis HI-6 dichloride (2Cl) and HI-6 dimethane sulfonate (DMS) in anaesthetized guinea pigs and domestic swine following an intravenous bolus administration. From the resultant pharmacokinetic parameters a target plasma concentration of 100 microM was established and maintained in guinea pigs receiving an intravenous infusion. This validated method allows for the analysis of low volume samples, increased sample numbers and is applicable to the determination of pharmacokinetic profiles and parameters.

Chromatographic resolution, characterisation and quantification of VX enantiomers in hemolysed swine blood samples.

The present study was initiated to develop a sensitive and highly selective method for the analysis of the enantiomers of the nerve agent VX (O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate) in blood samples for toxicokinetic and therapeutic research. To achieve this goal, analytical and semi-preparative enantioseparation of VX were carried out with gas and liquid chromatography. The GC chiral stationary phase was HYDRODEX-beta-TBDAc (beta cyclodextrin), on which VX was baseline-resolved. On the chiral HPLC phase CHIRALCEL OD-H the enantiomers of VX were isolated with enantiomeric excess >99.99%. They were characterised by specific optical rotation (+/-25.8 deg ml dm(-1)g(-1) at 20 degrees C and 589 nm) and by determination of cholinesterase inhibition rate constants. For the quantitative chiral detection of VX the enantioresolution was realized on the HPLC chiral phase CHIRAL AGP. A specific procedure was developed to isolate VX from swine blood samples thereby stabilising its enantiomers. The limit of detection was 200 fg per enantiomer on column. The absolute recovery of the overall sample preparation procedure was 75%. After an intravenous and percutaneous administration of a supralethal dose of VX in anesthetised swine (+)-VX and (-)-VX could be quantified up to 720 min.

Protective effects of N-methyl-D-aspartate receptor antagonism on VX-induced neuronal cell death in cultured rat cortical neurons.

Exposure of the central nervous system to organophosphorus (OP) nerve agents induces seizures and neuronal cell death. Here we report that the OP nerve agent, VX, induces apoptotic-like cell death in cultured rat cortical neurons. The VX effects on neurons were concentration-dependent, with an IC(50) of approximately 30 microM. Blockade of N-methyl-D-aspartate receptors (NMDAR) with 50 microM. D-2-amino-5-phosphonovalerate (APV) diminished 30 microM VX-induced total cell death, as assessed by alamarBlue assay and Hoechst staining. In contrast, neither antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) nor metabotropic glutamate receptors (mGluRs) had any effect on VX-induced neurotoxicity. VX-induced neuronal cell death could not be solely attributed to acetylcholinesterase (AChE) inhibition, since neither the reversible pharmacological cholinesterase inhibitor, physostigmine, nor the muscarinic receptor antagonist, atropine, affected VX-induced cell death. Importantly, APV was found to be therapeutically effective against VX-induced cell death up to 2 h post VX exposure. These results suggest that NMDARs, but not AMPARs or mGluRs, play important roles in VX-induced cell death in cultured rat cortical neurons. Based on their therapeutic effects, NMDAR antagonists may be beneficial in the treatment of VX-induced neurotoxicities.

Red blood cell acetylcholinesterase and plasma butyrylcholinesterase status: important indicators for the treatment of patients poisoned by organophosphorus compounds.

Inhibition of acetylcholinesterase (AChE) is regarded as the primary toxic mechanism of organophosphorus compounds (OP). Therapeutic strategies are directed to antagonise overstimulation of muscarinic receptors with atropine and to reactivate inhibited AChE with oximes. Reactivation is crucial within the neuromuscular synapse, where atropine is ineffective, since peripheral neuromuscular block eventually leads to respiratory failure. Patients with OP intoxication have to be identified as early as possible. During an international NBC-defence exercise anesthetised pigs were poisoned with sarin, followed by treatment with atropine and oxime. Blood samples were drawn and red blood cell (RBC)-AChE activity determined with a fielded test system on-site. Within a few minutes the poisoning was verified. After administration of HI-6, RBC-AChE activity increased rapidly. Blood samples were reanalysed in our laboratory in Munich. Almost identical course of the AChE activities was recorded by both systems.The more comprehensive cholinesterase status was determined in Munich. Oxime administration can be stopped when AChE is aged completely, but has to be continued as long as poison is present in the body and reactivation is possible. To aid the on-site physician in optimising diagnosis and treatment, a fielded test system should be available to allow rapid determination of the complete cholinesterase status.