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.

Serial manipulator functional calibration for in vitro biomechanical testing.

Serial manipulators are often used in biomechanical testing of human joints because they are precise, repeatable instruments that can create interesting loading scenarios. Unfortunately, commercial serial manipulators often do not have acceptable global positional accuracy due to manufacturing tolerances, assembly errors, and other mechanical imperfections. Numerous calibration methods have been reported which calibrate geometric and non-geometric parameters to reduce static position errors under constant loading conditions. However, the manipulator's global accuracy during continuous motion with time-varying external loading conditions is often not addressed but is necessary for joint biomechanical testing. Using the Mitsubishi PA10-6CE as a case study, a novel functional calibration procedure was developed that performs both static and dynamic calibration. The calibration uses optimization techniques to populate a 34-parameter model that accounts for the robot's geometric and non-geometric parameters and significantly reduces the mean/peak static and dynamic position errors to 0.368/0.67 mm and 0.353/0.81 mm, respectively, while externally loaded.

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.

Pulmonary deposition of aerosolized Bacillus atrophaeus in a Swine model due to exposure from a simulated anthrax letter incident.

Dry anthrax spore powder is readily disseminated as an aerosol and it is possible that passive dispersion when opening a letter containing anthrax spores may result in lethal doses to humans. The specific aim of this study was to quantify the respirable aerosol hazard associated with opening an envelope/letter contaminated with a dry spore powder of the biological pathogen anthrax in a typical office environment. An envelope containing a letter contaminated with 1.0 g of dry Bacillus atrophaeus (BG) spores (pathogen simulant) was opened in the presence of an unrestrained swine model. Aerosolized spores were detected in the room in seconds and peak concentrations occurred by three minutes. The swine, located approximately 1.5 m from the source, was exposed to the aerosol for 28 min following the letter opening event and then moved to a clean room for 30 min. A necropsy was completed to determine the extent of in vivo spore deposition in the lungs. The median number of viable colony forming units (CFU) measured in the combined right and left lung was 21,200: the average mass of both lungs was 283 g. In excess of 100 CFU per gram of lung tissue was found at sites within the anterior, intermediate and posterior lobes. The results of this study confirmed that opening an envelope containing spores generated an aerosol spanning the respirable particle size range of 1-10 microm, and that normal respiration of swine led to spore deposition throughout the lungs. The observed deposition of spores in the lungs of the swine is within the LD(50) range of 2,500-55,000 estimated for humans for inhaled anthrax. Thus, there would appear to be a significant health risk to those individuals exposed to anthrax spores when opening a contaminated envelope.

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.

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.

Evaluation of a barrier cream against the chemical warfare agent VX using the domestic white pig.

The purpose of this study was to evaluate the efficacy of a novel barrier cream formulation at reducing the percutaneous toxicity of a 2xLD(50) liquid challenge of nerve agent (VX). The study was conducted in vitro and in vivo using the domestic pig. Pretreatment of the (inner ear skin) exposure site with barrier cream eliminated mortality, reduced cholinesterase inhibition and prevented any physiological or biochemical signs of intoxication. In contrast, untreated animals exposed to VX exhibited severe signs of intoxication, near total AChE inhibition and generally died within the (3 hr) exposure period (5/6 animals). Application of the barrier cream caused a significant decrease in the area of skin contaminated by VX. It was tentatively concluded that spreading was predominantly a surface phenomena (possibly mediated by capillary movement of the agent through the microrelief or between hair follicles) with little or no contribution from lateral diffusion within the stratum corneum. There was a disparity between the in vitro and in vivo skin absorption measurements that was ascribed to the absence of systemic clearance in vitro. However, both models indicated a substantial reservoir of VX within the skin, providing a potential strategy for future investigations into "catch-up therapies". In summary, the novel barrier cream formulation was effective against a 2xLD(50) (liquid, percutaneous) dose of VX applied for 3 hr. Further work should be conducted to investigate more pragmatic issues such as optimal reapplication frequency and environmental effects such as temperature and humidity.

The pharmacokinetics and pharmacodynamics of two HI-6 salts in swine and efficacy in the treatment of GF and soman poisoning.

Anesthetized pigs were injected i.m. with 500 mg HI-6 dichloride (HI-6 2Cl) (1-[[[4-(aminocarbonyl)-pyridinio]methoxy]methyl]-2[(hydroxyimino)methyl]pyridinium dichloride; CAS 34433-31-3)) or the molar equivalent of HI-6 dimethanesulphonate (HI-6 DMS) 633 mg. Plasma HI-6 concentrations were measured by HPLC (1, 3, 5, 10, 15, 30, 60 min and every 30 min until 4h or 6h following the i.v. or i.m. dose respectively) while a variety of physiological responses were continuously examined. HI-6 (500 mg 2Cl or 633 mg DMS) resulted in an identical pharmacokinetic profile unaffected by atropine co-administration. Neither HI-6 salt resulted in clinically significant changes in cardiovascular or respiratory function. HI-6 DMS (1899 mg i.v.) resulted in plasma HI-6 concentrations about 10 times higher than measured following i.m. 500 mg 2Cl or 633 mg DMS and resulted in small transitory effect on mean arterial pressure. Atropine plus HI-6 DMS (1-9 mg/kg or 127-172 mg/kg i.m.) protected up to 100% of guinea pigs exposed to 5 x LD50 of GF (cyclohexyl methyl phosphonoflouridate) or soman (pinacolyl methylphosphonofluoridate) (GD) respectively. The results suggest that the two HI-6 salts have a similar pharmacokinetic profile while HI-6 DMS appears extremely safe and effective against nerve agents and may be as suitable for human use.

Clinical aspects of percutaneous poisoning by the chemical warfare agent VX: effects of application site and decontamination.

O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate (VX) is an extremely toxic organophosphate nerve agent that has been weaponized and stockpiled in a number of different countries, and it has been used in recent terrorist events. It differs from other well-known organophosphate nerve agents in that its primary use is as a contact poison rather than as an inhalation hazard. For this reason, we examined the effects of application site and skin decontamination on VX toxicity in anesthetized domestic swine after topical application. VX applied to the surface of the ear rapidly resulted in signs of toxicity consistent with the development of cholinergic crisis, including apnea and death. VX on the epigastrium resulted in a marked delayed development of toxic signs, reduced toxicity, and reduction in the rate of cholinesterase depression compared with animals exposed on the ear. Skin decontamination (15 minutes post-VX on the ear) arrested the development of clinical signs and prevented further cholinesterase inhibition and death. These results confirm earlier work that demonstrates the importance of exposure site on the resultant toxicity of this agent and they also show that decontamination postexposure has the potential to be an integral and extremely important component of medical countermeasures against this agent.

Therapeutic effects of cooling swine skin exposed to sulfur mustard.

Recent world events have highlighted the need for effective medical therapies for chemical weapon injuries. Of the chemical weapon agents, perhaps one of the most widely used, both historically and most recently in the Iran-Iraq War, is sulfur mustard (HD). No effective antidotes exist for this vesicant agent and, to this day, HD casualties are treated entirely symptomatically. Previous work carried out in this laboratory has indicated that cooling HD-exposed tissue may ameliorate the resultant injury. To further examine this, an anesthetized domestic swine model was used to investigate whether alteration of skin temperature had any effect either visually or histopathologically on the development and progression of HD-induced skin lesions over 7 days. Exposure of swine skin to HD vapor resulted in lesions whose severity was exposure time related (4, 8, 12, and 16 minutes). Postdecontamination heating of skin above ambient temperature (approximately 39 degrees C) resulted in worsening of the lesion, whereas postdecontamination cooling (approximately 15 degrees C) for between 2 to 4 hours postexposure lessened the severity of HD-induced injury. The authors conclude that the early, noninvasive and simplistic act of cooling HD-exposed skin may have a salutary effect on the severity of HD-induced cutaneous lesions.

Site-specific percutaneous absorption of methyl salicylate and VX in domestic swine.

The site specificity of the percutaneous absorption of methyl salicylate (MeS) and the organophosphate nerve agent VX (O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate) was examined in anaesthetized domestic swine that were fully instrumented for physiological endpoints. Four different anatomical sites (ear, perineum, inguinal crease and epigastrium) were exposed to the MeS and the serum levels were measured over a 6-h time period. The dose absorbed at the ear region was 11 microg cm(-2) with an initial flux of 0.063 microg cm(-2)min(-1), whereas at the epigastrium region the dose absorbed was 3 microg cm(-2) with an initial flux of 0.025 microg cm(-2)min(-1). For this reason further studies were carried out with VX on the ear and the epigastrium only. In animals treated with agent on the epigastrium, blood cholinesterase (ChE) activity began to drop 90 min after application and continued to decline at a constant rate for the remainder of the experiment to ca. 25% of awake control activity. At this time there were negligible signs of poisoning and the medical prognosis was judged to be good. In contrast, the ChE activity in animals receiving VX on the ear decreased to 25% of awake control values within 45 min and levelled out at 5-6% by 120 min. Clinical signs of VX poisoning paralleled the ChE inhibition, progressing in severity over the duration of the exposure. It was judged that these animals would not survive. The dramatic site dependence of agent absorption leading to vastly different toxicological endpoints demonstrated in this model system has important ramifications for chemical protective suit development, threat assessment, medical countermeasures and contamination control protocols.