Lily Raveh

Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity: In vitro and in vivo quantitative characterization

Butyrylcholinesterase purified from human plasma (HuBChE) was evaluated both in vitro and in vivo in mice and rats as a single prophylactic antidote against the lethal effects of highly toxic organophosphates (OP). The variation among the bimolecular rate constants for the inhibition of HuBChE by tabun, VX, sarin, and soman was 10-fold (0.47 to 5.12 x 10(7) M-1 min-1; pH 8.0, 26 degrees). The half-life of HuBChE in blood after its i.v. administration in mice and rats was 21 and 46 hr, respectively. The peak blood-enzyme level was obtained in both species approximately 9-13 hr following i.m. injection of HuBChE, and the fraction of the enzyme activity absorbed into the blood was 0.9 and 0.54 for rats and mice, respectively. The stoichiometry of the in vivo sequestration of the anti-cholinesterase toxicants was consistent with the HuBChE/OP ratio of the molar concentration required to inhibit 100% enzyme activity in vitro. Linear correlation was demonstrated between the blood level of HuBChE and the extent of protection conferred against the toxicity of nerve agents. Pretreatment with HuBChE alone was sufficient not only to increase survivability following exposure to multiple median lethal doses of a wide range of potent OPs, but also to alleviate manifestation of toxic symptoms in mice and rats without the need for additional post-exposure therapy. It appeared that in order to confer protection against lethality nerve agents had to be scavenged to a level below their median lethal dose LD50 within less than one blood circulation time. Since the high rate of sequestration of nerve agents by HuBChE is expected to underlie the activity of the scavenger in other species as well, a reliable extrapolation of its efficacy from experimental animals to humans can be made.

Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice

Human butyrylcholinesterase (BChE, EC 3.1.1.8) or acetylcholinesterase (AChE, EC 3.1.1.7) from fetal bovine serum (FBS), administered i.v. in mice, sequestered at approximately 1:1 stoichiometry the highly toxic anti-ChE organophosphate, 1,2,2-trimethylpropyl methyl-fluorophosphonate (soman). A quantitative linear correlation was demonstrated between blood-ChE levels and the protection conferred by exogeneously administered ChE. Results presented here demonstrate that either human BChE or FBS-AChE is an effective prophylactic measure sufficient to protect mice from multiple LD50S of soman without the administration of post-treatment supportive drugs.

Enzymes as pretreatment drugs for organophosphate toxicity

We have successfully demonstrated that exogenously administered acetyl- or butyrylcholinesterase (AChE, BChE respectively) will sequester organophosphates (OPs) before they reach their physiological targets. In addition, a third enzyme, endogenous carboxylesterase is known to be capable of scavenging OPs. In these studies, we have administered AChE and BChE to three different species of animals (mice, marmosets and monkeys) which were challenged with three different OPs (VX, MEPQ and soman). Results obtained from these systematic studies demonstrate that: (a) a quantitative linear correlation exists between blood AChE levels and the protection afforded by exogenously administered ChEs in animals challenged with OP, (b) approximately one mole of either AChE or BChE sequesters one mole of OP, (c) such prophylactic measures are sufficient to protect animals against OPs without the administration of any supportive drugs. Thus the OP dose, the blood-level of esterase, the ratio of the circulating enzyme to OP challenge, and the rate of reaction between them determine the overall efficacy of an enzyme as a pretreatment drug. The biochemical mechanism underlying the sequestration of various OPs by the use of exogenously administered scavenging esterases is the same in all species of animals studied. Therefore, the extrapolation of the results obtained by the use of ChE prophylaxis in animals to humans should be more reliable and effective than extrapolating the results from currently used multidrug antidotal modalities.

Sarin-induced neuropathology in rats

Sarin, a highly toxic cholinesterase (ChE) inhibitor, administered at near 1 LD50 dose causes severe signs of toxic cholinergic hyperactivity in both the peripheral and central nervous systems (CNS). The present study evaluated acute and long-term neuropathology following exposure to a single LD50 dose of sarin and compared it to lesions caused by equipotent doses of soman described previously. Rats surviving 1 LD50 dose of sarin (95 micrograms/kg; IM), were sacrificed at different time intervals post exposure (4 h-90 days) and their brains were taken for histological and morphometric study. Lesions of varying degrees of severity were found in about 70% of the animals, mainly in the hippocampus, piriform cortex, and thalamus. The damage was exacerbated with time and at three months post exposure, it extended to regions which were not initially affected. Morphometric analysis revealed a significant decline in the area of CA1 and CA3 hippocampal cells as well as in the number of CA1 cells. The neuropathological findings, although generally similar to those described following 1 LD50 soman, differed in some features, unique to each compound, for example, frontal cortex damage was specific to soman poisoning. It is concluded that sarin has a potent acute and long-term central neurotoxicity, which must be considered in the design of therapeutic regimes.

Development of the Bisquaternary Oxime HI-6 Toward Clinical Use in the Treatment of Organophosphate Nerve Agent Poisoning

The traditional therapeutic treatment of organophosphate cholinesterase inhibitor (nerve agents) poisoning consists of co-treatment with an antimuscarinic (atropine) and a reactivator of inhibited acetylcholinesterase (AChE), which contains a nucleophilic oxime function. Two oximes are presently widely available for clinical use, pralidoxime and obidoxime (toxogonin), but both offer little protection against important nerve agent threats. This has highlighted the real need for the development and availability of more effective oximes for human use, a search that has been going on for up to 30 years. However, despite the demonstration of more effective and safe oximes in animal experiments, no additional oximes have been licensed for human use. HI-6, (1-[[[4(aminocarbonyl)-pyridinio]methoxy]methyl]-2(hydroxyimino)pyridinium dichloride; CAS 34433-31-3) has been studied intensively and has been proved effective in a variety of species including non-human primates and appears from clinical experience to be safe in humans. These studies have led to the fielding of HI-6 for use against nerve agents by the militaries of the Czech republic, Sweden, Canada and under certain circumstances the Organisation for the Prohibition of Chemical Weapons. Nevertheless HI-6 has not been granted a license for clinical use, must be used only under restricted guidelines and is not available for civilian use as far as is known. This article will highlight those factors relating to HI-6 that pertain to the licensing of new compounds of this type, including the mechanism of action, the clinical and pre-clinical demonstration of safety and its efficacy against a variety of nerve agents particularly in non-human primates, since no relevant human population exists. This article also contains important data on the use of HI-6 in baboons, which has not been available previously. The article also discusses the possibility of successful therapy with HI-6 against poisoning in humans relative to doses used in non-human primates and relative to its ability to reactivate inhibited human AChE.

Prevention of soman-induced cognitive deficits by pretreatment with human butyrylcholinesterase in rats

This study examined the ability of pretreatment with human serum butyrylcholinesterase (HuBChE) to prevent soman-induced cognitive impairments. Behavioral testing was carried out using the Morris water maze task evaluating learning, memory, and reversal learning processes. Pretreatment with HuBChE significantly prevented the memory and reversal learning impairments induced by soman. A small deficiency in performance was observed only during part of the learning period in HuBChE-treated rats after administration of soman. Results support the contention that pretreatment alone with HuBChE is sufficient to increase survival and to prevent impairment in cognitive functioning following exposure to soman.

Large-scale purification and long-term stability of human butyrylcholinesterase: a potential bioscavenger drug

Butyrylcholinesterase from human plasma (HuBChE) is a potential drug candidate for detoxification of certain harmful chemicals that contain carboxylic or phosphoric acid ester bonds. Large quantities of purified HuBChE, displaying a high stability upon long-term storage, are required for the evaluation of its therapeutic capacity and its pharmaceutical properties. Several modifications of a previously reported procedure enabled us to purify the enzyme > 15,000-fold from pools of up to 100 1 of human plasma. The three-step procedure is based on precipitation of plasma proteins by ammonium sulfate (step I) and batch adsorption of HuBChE on procainamide-Sepharose 4B gel (step II). Ammonium sulfate was also employed in the third stage to fractionate the final product from procainamide-containing HuBChE solution. The overall yield (63%) of electrophoretically pure enzyme was significantly higher than that previously reported (34%) for the purification of HuBChE from 12.5 1 of plasma or from 5 kg of Cohn fraction IV-4. Purified HuBChE was stored at 5 degrees C in 10 mM phosphate buffer (pH 7.4) containing 1 mM EDTA and 0.02% NaN3. The specific activity, protein migration on gel electrophoresis, thermostability at 54 degrees C and the mean residence time in the circulation of mice remained essentially constant for at least 46 months. The modifications introduced can provide large quantities of purified enzyme that maintains its activity and bioavailability properties for several years.

Caramiphen and Scopolamine Prevent Soman-Induced Brain Damage and Cognitive Dysfunction

Exposure to soman, a toxic organophosphate nerve agent, causes severe adverse effects and long term changes in the peripheral and central nervous systems. The goal of this study was to evaluate the ability of prophylactic treatments to block the deleterious effects associated with soman poisoning. scopolamine, a classical anticholinergic agent, or caramiphen, an anticonvulsant anticholinergic drug with anti-glutamatergic properties, in conjunction with pyridostigmine, a reversible cholinesterase inhibitor, were administered prior to sbman (1 LD50). Both caramiphen and scopolamine dramatically attenuated the process of cell death as assessed by the binding of [3H]RoS-4864 to peripheral benzodiazepine receptors (omega3 sites) on microglia and astrocytes. In addition, caramiphen but not scopolamine, blocked the soman-evoked down-regulation of [3H]AMPA binding to forebrain membrane preparations. Moreover, cognitive tests utilizing the Morris water maze, examining learning and memory processes as well as reversal learning, demonstrated that caramiphen abolished the effects of soman intoxication on learning as early as the first trial day, while scopolamine exerted its effect commencing at the second day of training. Whereas the former drug completely prevented memory deficits, the latter exhibited partial protection. Both agents equally blocked the impairment of reversal learning. In addition, there is a significant correlation between behavioral parameters and [3H]RoS-4864 binding to forebrain membrane preparations of rats, which participated in these tests (r(21) = 0.66, P < 0.001; r(21) = 0.66, P < 0.001, -0.62, P < 0.002). These results demonstrate the beneficial use of drugs exhibiting both anti-cholinergic and anti-glutamatergic properties for the protection against changes in cognitive parameters caused by nerve agent poisoning. Moreover, agents such as caramiphen may eliminate the need for multiple drug therapy in organophosphate intoxications.

Huperzine A as a pretreatment candidate drug against nerve agent toxicity

Huperzine A (HUP) is a naturally-occurring, potent, reversible inhibitor of acetylcholinesterase (AChE) that crosses the blood-brain barrier. To examine its ability to protect against nerve agent poisoning, HUP was administered i.p. to mice, and the s.c. LD50 of soman was determined at various time intervals after pretreatment. Results were compared to those obtained for animals treated with physostigmine. A protective ratio of approximately 2 was maintained for at least 6 hr after a single injection of HUP, without the need for any post-challenge drug therapy. By contrast, pretreatment with physostigmine increased the LD50 of soman by 1.4- to 1.5-fold for only up to 90 min. The long-lasting antidotal efficacy displayed by HUP correlated with the time course of the blood-AChE inhibition. The results suggest that the protection of animals by HUP from soman poisoning was achieved by temporarily sequestering the active site region of the physiologically important AChE.

Prophylaxis against organophsphate poisoning by an enzyme hydrolysing organophosphorus compounds in mice

Parathion hydrolase purified from Pseudomonas sp. was injected i.v. into mice to demonstrate the feasibility of using organophosphorus acid anhydride (OPA) hydrolases as pretreatment against organophosphates (OP) poisoning. Results show that exogenous administration of as low as 7 to 26 micrograms of parathion hydrolase conferred protection against challenge with multiple median lethal doses (LD50) of diethyl p-nitrophenyl phosphate (paraoxon; 3.8-7.3 x LD50) and diethylfluorophosphate (DEFP; 2.9 x LD50) without administration of supportive drugs. The extent of protection observed was consistent with blood-parathion hydrolase levels and the kinetic constants of the enzymatic hydrolysis of paraoxon and DEFP by parathion hydrolase. OPA hydrolases not only appear to be potential prophylactic drugs capable of increasing survival ratio following OP intoxication but also to alleviate post-exposure symptoms.