VX Penetration Following Percutaneous Poisoning: A Dermal Microdialysis Study in the Guinea Pig.

ABSTRACT VX, a potent organophosphorus compound, acts primarily by irreversibly inhibiting acetylcholinesterase resulting in an accumulation of acetylcholine, which produces the characteristic signs of nerve agent poisoning. VX is a low-volatility agent, and therefore the most likely route of absorption into the body is via the skin. This study demonstrates for the first time that it is possible to follow the time course of percutaneous VX penetration using the technique of dermal microdialysis and that VX is absorbed through the skin of the anesthetized guinea pig in a concentration-dependent manner. A linear microdialysis probe (5-kDa cut-off) was implanted in the dermis of the back of the guinea pig and perfused (5 muL/min) with physiological Ringer's solution. VX (296 or 592 mug/kg) was applied (33 muL/kg) over the site of the microdialysis probe and dialysate samples collected for up to 6 h. The VX dialysate concentration was measured by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Quantitation was performed over the range 0.1 to 100 ng/mL and the calibration was linear. VX was detected within 15 min, reaching a peak at 30 min following both VX doses. After this time the VX concentration decreased. There was a clear dose-dependent recovery of VX in the dialysate and the total amount recovered was statistically significant between the two doses. This study has clearly shown that microdialysis can be used to follow the time course of the percutaneous absorption of VX in the anesthetized guinea pig and will be used in future studies to develop improved medical countermeasures. Crown Copyright (c) 2007 Dstl.

A novel approach to assessing percutaneous VX poisoning in the conscious guinea-pig.

Nerve agents like VX (S-2-diisopropylaminoethyl-O-ethyl-methylphosphonothiolate) are potent irreversible acetylcholinesterase (AChE) inhibitors. Following percutaneous nerve agent exposure there is a slower rate of absorption, later onset and longer duration of signs of poisoning. Relatively little is known about the physiological effects of percutaneously applied nerve agent in unanaesthetised laboratory animals. Heart rate (ECG), brain electrical activity (EEG), body temperature, locomotor activity and clinical signs were monitored following percutaneous application of VX to conscious guinea-pigs.A fall in heart rate (bradycardia) preceded incapacitation following the highest VX dose, and occurred in the absence of incapacitation at the lower doses. Following the highest dose of VX (0.592 mg kg(-1)) three out of four animals died within 24 h. The lower two doses of VX (0.296 and 0.148 mg kg(-1)), produced extended periods of bradycardia in the absence of observable signs of poisoning. Bradycardia preceded, or occurred in the absence of, a temperature decrease; seizure-like EEG changes were not observed at any of the VX doses tested. Acetylcholinesterase activity was significantly inhibited in the blood and most brain areas at 48 h. There were significant dose-related decreases in body weight at 24 and 48 h following VX. This preliminary study suggests that decreased heart rate may be an early sign of the toxic effects of VX, whereas temperature and observable clinical signs are not good early indicators of percutaneous VX poisoning in this animal model. Future studies will use this model to assess the benefit of administering medical countermeasures in response to a defined decrease in heart rate.

Development of next generation medical countermeasures to nerve agent poisoning.

Medical countermeasures provide a key role in the UK integrated approach to chemical defence and are aimed at preventing or mitigating the effects of exposure to nerve agents. It is UK policy that medical countermeasures will be licensed products. Demonstration of efficacy relies on extrapolation of animal-derived data to man which means that species selection is extremely important. For the foreseeable future it is likely that a combination of pretreatment and therapy will be required to provide protection against nerve agent poisoning. There is a longer-term aspiration to develop a post poisoning-therapy which would reduce the reliance on pretreatment, prevent or mitigate the effects of exposure to all nerve agents and decrease the requirement for three autoinjectors. Immediate therapy comprising physostigmine (0.2mg/kg), hyoscine hydrobromide (4mg/kg) and HI-6 (93.6mg/kg) protected all animals against the lethal effects of a supralethal dose of GD, when given 1min after nerve agent poisoning in the absence of any pretreatment. In contrast when hyoscine hydrobromide was replaced with hyoscine methyl nitrate most of the animals died within 24h, whereas when an equal mixture of hyoscine hydrobromide and hyoscine methyl nitrate was used all the animals survived. None of these animals had an intussusception. It would not be possible to deliver these doses of HI-6 to a human from a single autoinjector device. Recent studies have shown that a lower dose of HI-6 (7mg/kg) which can be delivered via an autoinjector, in combination with physostigmine and hyoscine hydrobromide provides good protection against the lethal effects of a supralethal dose of GD. A number of animals died between 6 and 24h and had an intussusception. The surviving animals did not begin to regain weight until 48h after poisoning. In contrast when a mixture of hyoscine hydrobromide and hyoscine methyl nitrate was used, one animal died within 15min, the other animals all survived, regained weight from 24h and did not have an intussusception. These studies will now be extended to include other agents and will be taken forward to studies in non-human primates where the incidence of intussusception will be closely monitored.

A novel approach for medical countermeasures to nerve agent poisoning in the guinea-pig.

This study forms part of a larger programme of work aimed at developing improved medical countermeasures for nerve agent poisoning with less reliance on pretreatment. Therapy with N(6)-cyclopentyladenosine (CPA), physostigmine, hyoscine and HI-6 protected guinea-pigs against the incapacitating and lethal effects of a supralethal challenge of soman (135 microg/kg) when given 1 min after poisoning. CPA, however has well-recognised side effects that are likely to preclude it being licensed for use in humans so further refinements were made to the doses of the other therapy components to improve efficacy in the absence of CPA. An immediate therapy comprising physostigmine (0.2 mg/kg), hyoscine (4 mg/kg) and HI-6 (93.6 mg/kg), when given 1 min after nerve agent, provided good protection against the lethal effects of GA, GB, GD, GF and VX poisoning and reduced the duration of the signs of incapacitation and hypothermia. In the case of GA and GB poisoning some animals exhibited a short period of substantial incapacitation. Most animals continued to gain weight over the following 6 days without the need for further medical intervention. In the case of GA poisoning further medical intervention would be needed to ensure the longer term survival of all animals and it is likely that in the battlefield situation further medical treatment would be available within 2-4 h. The drug combination described in this paper protects against supralethal doses of a range of nerve agents, with minimal incapacitation in the absence of any pretreatment. Further modification and refinement of this therapy is required for human use and it may provide a way forward for development of medical countermeasures for the treatment of organophosphate poisoning in the wider community should there be a need.

Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents.

Enzymes that efficiently hydrolyze highly toxic organophosphorus nerve agents could potentially be used as medical countermeasures. As sufficiently active enzymes are currently unknown, we synthesized twelve fluorogenic analogues of organophosphorus nerve agents with the 3-chloro-7-oxy-4-methylcoumarin leaving group as probes for high-throughput enzyme screening. This set included analogues of the pesticides paraoxon, parathion, and dimefox, and the nerve agents DFP, tabun, sarin, cyclosarin, soman, VX, and Russian-VX. Data from inhibition of acetylcholinesterase, in vivo toxicity tests of a representative analogue (cyclosarin), and kinetic studies with phosphotriesterase (PTE) from Pseudomonas diminuta, and a mammalian serum paraoxonase (PON1), confirmed that the analogues mimic the parent nerve agents effectively. They are suitable tools for high-throughput screens for the directed evolution of efficient nerve agent organophosphatases.

Physostigmine and hyoscine improves protection against the lethal and incapacitating effects of nerve agent poisoning in the guinea-pig.

This study is drawn from a work programme aimed at developing improved medical counter measures for nerve agent poisoning. Guinea-pigs were administered pyridostigmine (5.1 microg/h) or physostigmine (4.7 microg/h) and hyoscine (1.94 microg/h) for 6 days via a subcutaneously implanted mini osmotic pump. Pyridostigmine inhibited red cell acetylcholinesterase (AChE) by 44.2 +/- 2.7% and plasma cholinesterase (ChE) by 29.9 +/- 1.8%. Physostigmine and hyoscine inhibited red cell AChE by 18.7 +/- 3.7% and plasma ChE by 44.1 +/- 3.1%. On day 6, animals were challenged with a lethal dose of tabun (GA; 125 microg/kg), sarin (GB; 51.2 microg/kg), soman (GD; 31.2 microg/kg), GF (50 microg/kg) or VX (11.25 microg/kg) administered by the subcutaneous route. Animals were closely observed for signs of poisoning. The time to the onset of signs of poisoning was similar for all the agents except for VX, which showed a delay compared to the other agents. Following pretreatment with either pyridostigmine or physostigmine and hyoscine most animals survived for 2-3 h following nerve agent administration. In contrast, only physostigmine and hyoscine prevented or reduced the duration of the signs of incapacitation and the temperature drop produced by all the agents. Pyridostigmine-pretreated animals showed little or no recovery from incapacitation prior to death. Physostigmine and hyoscine pretreatment provided statistically (P < 0.05) better protection against GB, GD and VX lethality (24 h) than pyridostigmine pretreatment and better protection against GA and GF lethality.