Protexia (72 mg/kg; 0.699 ml/kg) administered 2 h following a supralethal dose of VX (0.740mg/kg) was effective at preventing nerve agent-induced lethality in 100% of cases. This is a significant result that expands previous findings (Tenn et al., 2008
; Lenz et al., 2010
), demonstrates the efficacy of Protexia in the guinea-pig and broadens the understanding of its physiological effects by using telemetric methods to monitor heart rate, temperature and activity in conscious animals over extended periods.
The adverse physiological changes seen following VX (bradycardia, hypothermia and seizure) were minimised or prevented in animals that received Protexia. Although some animals did have periods of reduced temperature and heart rate, these were less profound than those observed in saline-treated animals, and the Protexia group all recovered. Some animals that received Protexia did not show any observable signs of systemic cholinergic poisoning, (e.g. left) and their heart rates and body temperatures remained within the normal range.
For acute nerve agent poisoning, a stoichiometric relationship between scavenger and inhibitor was previously thought to be required for protection. The dose of Protexia used in this study was calculated to be stoichiometric to 296μg/kg of VX whereas the actual challenge dose of VX used was 740μg/kg. Although the Protexia dose used is lower than the stoichiometric dose, a large enough proportion of the applied VX must have been bound in the circulation to overcome the toxic effects, although in this study free agent levels in the blood were not measured. Following percutaneous poisoning in particular, only a small fraction of the applied VX enters the systemic circulation. Other workers have shown that 7 h following p.c. VX application the bioavailability of agent in blood was only 2.5% of the applied dose, albeit with a significant further increase expected after that time (van der Schans et al, 2003
). The bioavailability of pegylated rBuChE following i.m. administration in guinea-pigs has been determined to be approx. 46%, with a plasma half-life of approx. 44 h (Yue-Jin Huang et al., 2007
Intussusception was associated with the deaths of 4/8 animals in the saline-treated group. Intussusception is a consequence of nerve agent poisoning that has been previously reported following acute soman intoxication (Wetherell et al., 2007
) and has also been observed in previous studies following VX poisoning via the percutaneous route (Mumford et al., 2011
) and via microinstillation (Katos et al., 2007
). Full intussusception was observed in two saline-treated guinea-pigs, and a further two animals had changes in the gastrointestinal tract consistent with the initial stages of intussusception; one further animal had abnormal changes in the lower GI tract which were not judged to be the start of intussusception. Intussusception as a consequence of nerve agent poisoning generally develops over the course of several hours, typically between 6 and 24 h in guinea-pigs (Wetherell et al., 2007
). Some animals in the saline-treated group in the present study died from other effects of VX, before pathological signs consistent with intussusception, if present, would have developed.
At this dose of VX, not all guinea-pig would develop seizure, even in the absence of effective medical countermeasures. The seizure incidence in the saline control group in the present study (2/8) was comparable to that seen previously (Mumford et al, 2011
). Only one Protexia-treated animal developed seizure, but this was intermittent and did not appear to have been behaviourally detrimental. There could be long-term histopathological changes as a result of prolonged uncontrolled seizure activity (Taysse et al, 2006
), but that was not measured in this study.
Cholinesterase activity was measured using acetylthiolcholine as the substrate. This is therefore a non-selective measure of total cholinesterase activity. The brain area cholinesterase activities from the Protexia-treated animals were lower than those in the naïve weight-matched animals. It cannot be definitively stated that administration of Protexia protected brain cholinesterase: as the Protexia was administered 2h post VX challenge, inhibition of the cholinesterase by VX may have already been substantial at this point. In addition, the cholinesterase activity was measured following 7 days of a dynamic interaction between the endogenous cholinesterase inhibited by the VX, the exogenous huBuChE and de novo production of cholinesterase initiated at the time of poisoning. Interestingly, the erythrocyte cholinesterase levels in animals administered Protexia were elevated above controls after 7 days. This is in agreement with our previous findings (Mumford et al 2011
) and the explanation for this is not clear, but it may reflect compensatory up-regulation of endogenous enzyme in the intervening 6 days (Dorandeu et al, 2008
). We cannot rule out some residual BuChE activity in the red cells following the washing procedure, but the use of specific inhibitors is precluded due to the large excess of exogenously-administered BuChE present. The elevated plasma cholinesterase levels (over 7-fold higher than controls) measured after 7 days indicate a long retention time of the exogenously-administered recombinant huBuChE.
In the current study, therapy was administered at 2h post-poisoning. This was chosen as a time at which observable signs of systemic poisoning would not have commenced, and in all but one animal this was the case. In a more realistic scenario, therapy might not be administered until poisoning was evident. In that case, given the available pharmacokinetic data, Protexia given via the intramuscular route may not be completely effective in protecting against the nerve agent poisoning because the circulating levels of nerve agent would have already reached a toxicologically significant level in plasma and in the target tissues (in order to produce cholinergic signs of poisoning). It has been demonstrated(Lenz et al, 2010
) that following i.m. administration there is a substantial time lag before peak plasma concentration of the protein would be achieved, namely a Tmax of 4.2h for unpegylated rBuChE and 20h for plasma-derived huBuChE. Therapy delivered by the i.v. route would be expected to achieve improved protection because of the much shorter time to peak plasma concentration and the improved bioavailability of the scavenger enzyme (Mumford et al, 2011
; Yue-Jin Huang et al., 2007
In conclusion, the use of recombinant huBuChE (e.g. Protexia) as a therapeutic medical countermeasure is a promising treatment strategy against p.c. nerve agent poisoning. This study has demonstrated that bioscavengers can be beneficial when given as a post-exposure therapy. This suggests that it may be useful in both civilian and military exposure scenarios. In contrast to the military situation, some key features of therapy for nerve agent poisoning in a civilian setting are that neither pretreatment nor immediate self-medication are an option. Following a nerve agent release in a civilian context, the delay between exposure of casualties to nerve agent and the ability of medical first responders to deliver treatment will be influenced by several variables, including the timely identification of the intoxicating agent, the time taken to acquire the necessary therapeutic drugs from stockpiles and load them into emergency vehicles, the time taken to arrive at the incident and don protective equipment and the time taken for first responders to gain access to casualties at the scene of the release (Sheridan, R.D., pers. comm.). In this context, relatively little is known of the effectiveness of delayed pharmacological interventions in victims of nerve agent poisoning.
It is recommended that future work investigates the consequences of administration of therapy triggered by observed signs of systemic cholinesterase poisoning and the effect of route of administration on the outcome. For advanced development, it is recommended that this material be tested in an additional animal species (e.g. the minipig) to increase confidence in extrapolation to humans.