Human plasma-derived BuChE as a stoichiometric bioscavenger for treatment of nerve agent poisoning.

Potent organophosphorous (OP) agents, such as VX, are hazardous by absorption through the skin and are resistant to conventional pharmacological antidotal treatments. The residence time of a stoichiometric bioscavenger, human butyrylcholinesterase (huBuChE), in the plasma more closely matches that of VX than do the residence times of conventional therapy drugs (oxime, anti-muscarinic, anticonvulsant). Intramuscular (i.m.) huBuChE afforded almost complete protection when administered prior to the onset of observable cholinergic signs of VX poisoning, but once signs of poisoning became evident the efficacy of i.m. huBuChE decreased. A combination of nerve agent therapy drugs (oxime, anti-muscarinic, anticonvulsant) with huBuChE (i.m.) protected 100% (8/8) of guinea-pigs from a lethal dose of VX (0.74 mg/kg) to 48 h, even when administered on signs of poisoning. Survival was presumed to be due to immediate alleviation of the cholinergic crisis by the conventional pharmacological treatment drugs, in conjunction with bioscavenger that prevented further absorbed agent reaching the AChE targets. Evidence to support this proposed mechanism of action was obtained from PKPD experiments in which multiple blood samples and microdialysate samples were collected from individual conscious ambulatory animals. Plasma concentrations of intramuscularly-administered atropine, diazepam and HI-6 reached a peak within 15 min and were eliminated rapidly within 4h. Plasma concentrations of huBuChE administered by the i.m. route took approximately 24h to reach a peak, but were well-maintained over the subsequent 7days. Thus, the pharmacological therapy rapidly treated the initial signs of poisoning, whilst the bioscavenger provided prolonged protection by neutralising further nerve agent entering the bloodstream and preventing it from reaching the target organs.

Post-exposure therapy with human butyrylcholinesterase following percutaneous VX challenge in guinea pigs.

CONTEXT: Human butyrylcholinesterase (huBuChE) has potential utility as a post-exposure therapy following percutaneous nerve agent poisoning as there is a slower absorption of agent by this route and hence a later onset of poisoning. METHODS. We used surgically implanted radiotelemetry devices to monitor heart rate, EEG, body temperature and locomotor activity in guinea pigs challenged with VX via the percutaneous route. RESULTS. Treatment with huBuChE (24.2 mg/kg, i.m.) at 30 or 120 min following percutaneous VX (~2.5 × LD(50)) protected 9 out of 10 animals from lethality. When i.m. huBuChE administration was delayed until the onset of observable signs of systemic cholinergic poisoning, only one out of six animals survived to 7 days. Survival increased to 50% when the same dose of huBuChE was given intravenously at the onset of signs of poisoning. This dose represents approximately 1/10th the stoichiometric equivalent of the dose of VX administered (0.74 mg/kg). Intramuscular administration of huBuChE (24.2 mg/kg) alone did not produce any changes in heart rate, brain electrical activity, temperature or locomotion compared to saline control. Survival following VX and huBuChE treatment was associated with minimal incapacitation and observable signs of poisoning, and the mitigation or prevention of detrimental physiological changes (e.g. seizure, bradycardia and hypothermia) observed in VX + saline-treated animals. At 7 days, cholinesterase activity in the erythrocytes and most brain areas of guinea pigs that received huBuChE at either 18 h prior to or 30 min following VX was not significantly different from that of naïve, weight-matched control animals. CONCLUSION. Percutaneous VX poisoning was successfully treated using post-exposure therapy with huBuChE bioscavenger. The opportunity for post-exposure treatment may have particular relevance in civilian settings, and this is a promising indication for the use of huBuChE.

Post-exposure therapy with recombinant human BuChE following percutaneous VX challenge in guinea-pigs.

Poisoning by nerve agents via the percutaneous (p.c.) route is an issue because the slow absorption of agent could result in poisoning which outlasts the protection provided by conventional pharmacological therapy. The bioscavenger approach is based on the concept of binding nerve agent in the bloodstream, thus preventing nerve agent from reaching the target tissues and inhibiting acetylcholinesterase activity. One bioscavenger that has been extensively studied is human butyrylcholinesterase (huBuChE). Protexia® is a pegylated form of recombinant huBuChE. We used a guinea-pig model of p.c. nerve agent poisoning, using an implanted telemetry system to collect physiological data. Guinea-pigs were poisoned with the nerve agent VX (0.74 mg/kg) (∼2.5 × LD50). Two hours following VX exposure, Protexia (72 mg/kg) or saline control was administered intramuscularly. All guinea-pigs treated with Protexia (n=8) survived, compared to no survivors in a saline-treated control group (n=8). Survival following VX and Protexia treatment was associated with minimal incapacitation and observable signs of poisoning, and the mitigation or prevention of the detrimental physiological changes (e.g. seizure, bradycardia and hypothermia) observed in control animals. The opportunity for post-exposure treatment may have utility in both civilian and military scenarios, and this is a promising indication for the use of a bioscavenger.

Efficacy and physiological effects of human butyrylcholinesterase as a post-exposure therapy against percutaneous poisoning by VX in the guinea-pig.

The physiological effects of human plasma-derived butyrylcholinesterase (huBuChE) administration and its modulation of the effects of percutaneous VX challenge are poorly understood. Percutaneously administered nerve agents are more slowly absorbed than inhaled agents; consequently, signs of poisoning occur later, with a longer duration. Telemetry was used to monitor heart rate, EEG, temperature and activity in guinea-pigs. Treatment with huBuChE at 30 or 120 min following percutaneous VX challenge ( approximately 2.5 x LD(50)) provided 100% protection from lethality. When huBuChE administration was delayed until the onset of observable signs of poisoning only 1 out of 6 animals survived to the end of the experiment at 7 days. This study adds to the body of evidence demonstrating the efficacy of huBuChE in animals by describing the successful therapeutic use of a protein bioscavenger as a post-exposure treatment against dermal exposure to VX up to 2h post-exposure. This study simultaneously used telemetric methods to show that the efficacy of huBuChE is linked to the prevention of detrimental physiological changes observed in control VX-treated animals. Post-exposure therapy is a promising additional indication for the concept of use of this material, and one that has particular relevance in a civilian exposure scenario.

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.