The antidotal treatment of organophosphates (OP) nerve agents (NA) poisoning is based on anticholinergics (e.g. atropine) combined with oxime reactivators (e.g. 2PAM) of acetylcholinesterase (AChE). This treatment is symptomatic and does not degrade the OP. New small-molecule OP scavengers were developed as bifunctional hybrids. Their molecular design was based on combining a nucleophile that directly degrades OP with a moiety that reactivates OP-inhibited AChE. The OP degrading moiety is either benzhydroxamic acid (BHA) or 4-pyridinehydroxamic acid (4PHA) coupled via (CH2)n, (n = 1 or 3) to 2PAM. Three newly synthesized oxime-hydroxamate hybrids: 2PAMPr4PHA, 2PAMMeBHA and 2,4-DiPAMMeBHA were found to detoxify sarin, cyclosarin and soman in solution at 3-10-fold faster rate than 2PAM and to reactivate OP-AChE in vitro. 2PAMPr4PHA displayed 18-fold faster reactivation than 2-PAM of cyclosarin-inhibited HuAChE (kr = 3.6 x 102 vs. 0.2 x 102 M-1min-1, respectively, 37 degrees C). These hybrids inhibited AChE reversibly, IC50 = 16-48 muM, thereby decreasing the inhibition rates by OPs. The LD50 (im) of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA are >568, 508 and >506 mumol/kg in rats and 144, 203 and >506 mumol/kg in guinea pigs. The rate of blood ChE recovery by the hybrids administered either pre- or post-exposure to 0.8xLD50 sarin was comparable or faster than 2PAM. Antidotal efficacy of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA administered with atropine, as pre-treatment to sarin in rats (im), yielded protection ratios (PR) 11.6, 11.5 and 4.7, respectively, vs. 5.5 with 2PAM. Post-treatment against various OPs in rats and guinea-pigs yielded PRs higher or similar to that of 2 PAM. Our in vivo data indicates that some hybrids may serve as efficient small molecule scavengers for mitigating the toxicity of OP NAs.
Rivastigmine, a reversible cholinesterase inhibitor, approved as a remedy in Alzheimer's disease, was suggested as pretreatment against nerve agents poisoning. We evaluated the pharmacokinetic, pharmacodynamic, physiologic, cognitive and emotional effects of repeated rivastigmine in young healthy male adults, in a double blind, placebo controlled crossover trial. Three groups completed 3 treatment periods: 0, 1.5 and 3mg twice a day, for a total of 5 intakes. Parameters monitored were: vital signs, ECG, laboratory tests, sialometry, visual accommodation, inspiratory peak flow, and cognitive function tests. Adverse reactions were mild. Peak blood levels and peak cholinesterase inhibition increased with repeated intakes, and high variability and non-linear pharmacokinetics were demonstrated. In addition, two cognitive functions were affected (perceptual speed and dynamic tracking). The complicated pharmacological profile and the high inter-personal variability limit the potential use of rivastigmine as pretreatment for war fighters and first responders.
Title: Caramiphen edisylate: An optimal antidote against organophosphate poisoning Raveh L, Eisenkraft A, Weissman BA Ref: Toxicology, 325C:115, 2014 : PubMed
Potent cholinesterase inhibitors such as sarin, induce an array of harmful effects including hypersecretion, convulsions and ultimately death. Surviving subjects demonstrate damage in specific brain regions that lead to cognitive and neurological dysfunctions. An early accumulation of acetylcholine in the synaptic clefts was suggested as the trigger of a sequence of neurochemical events such as an excessive outpour of glutamate and activation of its receptors. Indeed, alterations in NMDA and AMPA central receptors' densities were detected in brains of poisoned animals. Attempts to improve the current cholinergic-based treatment by adding potent anticonvulsants or antiglutamatergic drugs produced unsatisfactory results. In light of recent events in Syria and the probability of various scenarios of military or terrorist attacks involving organophosphate (OP) nerve agent, research should focus on finding markedly improved countermeasures. Caramiphen, an antimuscarinic drug with antiglutamatergic and GABAergic facilitating properties, was evaluated in a wide range of animals and experimental protocols against OP poisoning. Its remarkable efficacy against OP exposure was established both in prophylactic and post-exposure therapies in both small and large animals. The present review will highlight the outstanding neuroprotective effect of caramiphen as the optimal candidate for the treatment of OP-exposed subjects.
Sarin, a potent organophosphate cholinesterase inhibitor, induces an array of toxic effects including convulsions. Many antidotal treatments contain anticonvulsants to block seizure activity and the ensuing brain damage. Magnesium sulfate (MGS) is used to suppress eclamptic seizures in pregnant women with hypertension and was shown to block kainate-induced convulsions. Magnesium sulfate was evaluated herein as an anticonvulsant against sarin poisoning and its efficacy was compared with the potent anticonvulsants midazolam (MDZ) and caramiphen (CRM). Rats were exposed to a convulsant dose of sarin (96 mug/kg, im) and 1 min later treated with the oxime TMB4 and atropine to increase survival. Five minutes after initiation of convulsions, MGS, CRM, or MDZ were administered. Attenuation of tonic-clonic convulsions was observed following all these treatments. However, radio-telemetric electro-corticography (ECoG) monitoring demonstrated sustained seizure activity in MGS-injected animals while this activity was completely blocked by MDZ and CRM. This disrupted brain activity was associated with marked increase in brain translocator protein levels, a marker for brain damage, measured 1 week following exposure. Additionally, histopathological analyses of MGS-treated group showed typical sarin-induced brain injury excluding the hippocampus that was partially protected. Our results clearly show that MGS demonstrated misleading features as an anticonvulsant against sarin-induced seizures. This stems from the dissociation observed between overt convulsions and seizure activity. Thus, the presence or absence of motor convulsions may be an unreliable indicator in the assessment of clinical status and in directing adequate antidotal treatments following exposure to nerve agents in battle field or terror attacks.
Title: Multifunctional drugs as novel antidotes for organophosphates' poisoning Weissman BA, Raveh L Ref: Toxicology, 290:149, 2011 : PubMed
Some organophosphorus compounds (OPs) are nerve agents that continue to concern military personnel and civilians as potential battlefield and terrorist threats. Additionally, OPs are used in agriculture where they are associated with numerous cases of intentional and accidental misuse. These toxicants induce an array of deleterious effects including respiratory distress, convulsions and ultimately death. A mechanism involving a rapid and potent inhibition of peripheral and central cholinesterases leading to a massive buildup of acetylcholine in synaptic clefts was suggested as the underlying trigger of the toxic events. Indeed, therapy comprised of an acetylcholinesterase reactivator (i.e., oxime) and a cholinergic antagonist (e.g., atropine) is the accepted major paradigm for protection. This approach yields a remarkable survival rate but fails to prevent neurological and behavioral deficits. Extensive research revealed a complex picture consisting of an early activation of several neurotransmitter systems, in which the glutamatergic plays a pivotal role., Data accumulated in recent years support the concept that multi-targeting of pathways including glutamatergic and cholinergic circuits is required for an effective treatment. Drugs that demonstrate the ability to interact with several systems (e.g., caramiphen) were found to afford a superior protection against OPs as compared to specific antimuscarinic ligands (e.g., scopolamine). Compounds that potently block muscarinic receptors, interact with the NMDA ion channel and in addition are able to modulate sigma(1) sites and/or GABAergic transmission seem to represent the emerging backbone for novel antidotes against OP poisoning. Several multifunctional drugs are already used for complex diseases e.g., cancer and depression.
Title: Next generation OP-bioscavengers: a circulatory long-lived 4-PEG hypolysine mutant of F338A-HuAChE with optimal pharmacokinetics and pseudo-catalytic characteristics Kronman C, Cohen O, Mazor O, Ordentlich A, Raveh L, Velan B, Shafferman A Ref: Chemico-Biological Interactions, 187:253, 2010 : PubMed
We have shown previously that conjugation of polyethylene glycol (PEG) chains to recombinant human acetylcholinesterase (rHuAChE) results in the extension of its residence time in the circulation of mice and monkeys [1,2]. By profiling the pharmacokinetic behavior of an array of well-defined hypolysine human mutant AChE molecules following PEGylation, we now determine that the duration of these enzyme forms in the circulation of rhesus macaques correlates with their number of appended PEG moieties, and is influenced by the actual location of the PEG chains at the molecule surface, as well. These findings, which concur with those we have previously established in mice, indicate that a common set of rules dictates the circulatory fate of PEGylated HuAChEs in rodents and non-human primates. In addition to its effect on circulatory residence, PEGylation reduces the ability of the rHuAChE bioscavenger to elicit an immune response in the heterologous mouse animal system. Thus, an inverse relationship between anti-AChE antibody production and PEG loading was observed following repeated administration of the different PEGylated hypolysine human AChEs to mice. We note however, that in rhesus macaques, the essentially homologous (human) AChE does not induce specific anti-AChE antibodies after repeated administration of high doses of the enzyme in its PEGylated form, and even in its non-PEGylated form. Taken together, these findings indicate that PEG acts by veiling enzyme-related epitopes, which would otherwise interact with host circulatory elimination pathways and immune system. The barring of such interactions by obstructive PEGs, confers the enzyme molecule with both extended circulatory residence and mitigated immunogenic properties. Further modulation by incorporation of the F338A mutation into the PEGylated hypolysine rHuAChE enzyme mold, resulted in the generation of an OP-bioscavenger that displayed reduced aging rates and could effectively protect mice against repeated exposure to CW agents. This selected 4-PEG F338A-AChE can serve as a paradigm for new generation OP-bioscavengers, specifically tailored for prophylactic treatment against toxic OP-agents.
Organophosphorus hydrolases (OPH) such as mammalian plama paraoxonase (PON1) detoxify asymmetric toxic organophosphorus (OP) nerve agents by preferentially hydrolyzing the less toxic P(+) optical isomer. In order to develop new OPHs with broader stereoselectivity we have prepared a series of asymmetric fluorogenic organophosphonates (Flu-OPs). Such Flu-OPs may serve as molecular probes for screening large libraries of OP hydrolases during directed evolution. Flu-OPs were prepared as methylphosphonates (MPs) diesters containing either ethyl (E), isopropyl (I), cyclohexyl (C) or pinacolyl (P) groups that are structural congeners of the nerve agents VX, sarin, cyclosarin and soman, respectively. The second ester bond was formed with fluorescent moieties that are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy 9,9-dimethyl-9H-acridin-2-one (DDAO). To further characterize the Flu-OPs as surrogates of their respective nerve agents, we have studied the reactivation of Flu-OP-inhibited AChE using 2-PAM and toxogonin (TOX). AChE was 90-95% inhibited by all Flu-OPs (0.36-0.9(M) and then was reactivated by either 2-PAM or TOX. TOX caused a more rapid reactivation than 2-PAM with the following rank order; EMP>IMP>CMP. TOX was also shown to be a better reactivator than 2-PAM for AChE inhibited by the nerve agents VX and cyclosarin. PMP-AChE could not be reactivated by either TOX or 2-PAM, similarly to aging of PMP-AChE formed by inhibition with soman. Racemic CMP-MeCyC was used for screening two new PON1 variants from a neutral library of PON1. These multiple mutation variants include replacement of active site amino acid residues. Neither mutation in these new variants appeared in PON1 variants previously discovered by directed evolution using symmetric Flu-OP. Detoxification rate of cylcosarin by these new PON1 variants was rather slow indicating the need to further screen PON1 clones using optically active Flu-OPs. Therefore, we have separated enzymatically the P(-) enantiomer of CMP-MeCyC and determined its 98% purity using chiral HPLC.
The highly toxic organophosphorous compound VX [O-ethyl-S-(isoporopylaminoethyl) methyl phosphonothiolate] undergoes an incomplete decontamination by conventional chemicals and thus evaporates from urban surfaces, e.g., pavement, long after the initial insult. As a consequence to these characteristics of VX, even the expected low levels should be examined for their potential to induce functional impairments including those associated with neuronal changes. In the present study, we developed an animal model for subchronic, low-dose VX exposure and evaluated its effects in rats. Animals were exposed to VX (2.25 microg/kg/day, 0.05 LD(50)) for three months via implanted mini osmotic pumps. The rapidly attained continuous and marked whole-blood cholinesterase inhibition (approximately 60%), fully recovered 96 h post pump removal. Under these conditions, body weight, blood count and chemistry, water maze acquisition task, sensitivity to the muscarinic agonist oxotremorine, peripheral benzodiazepine receptors density and brain morphology as demonstrated by routine histopathology, remained unchanged. However, animals treated with VX showed abnormal initial response in an Open Field test and a reduction (approximately 30%) in the expression of the exocytotic synaptobrevin/vesicle associate membrane protein (VAMP) in hippocampal neurons. These changes could not be detected one month following termination of exposure. Our findings indicate that following a subchronic, low-level exposure to the chemical warfare agent VX some important processes might be considerably impaired. Further research should be addressed towards better understanding of its potential health ramifications and in search of optimal countermeasures.
The high reactivity of cholinesterases (ChEs) toward organophosphorus (OP) compounds has led to propose recombinant ChEs as bioscavengers of nerve agents. The bioscavenging potential of recombinant ChEs can be enhanced by conjugation of polyethylene glycol (PEG) moieties, to extend their circulatory residence. However, the ability of exogenously administered ChEs to confer long-term protection against repeated exposures to nerve agents is still limited due to the aging process, whereby organophosphate-ChE adducts undergo irreversible dealkylation, which precludes oxime-mediated reactivation of the enzyme. To generate an optimal acetylcholinesterase (AChE)-based OP bioscavenger, the F338A mutation, known to decelerate the rate of aging of AChE-OP conjugates, was incorporated into polyethylene glycol-conjugated (PEGylated) human AChE. The PEGylated F338A-AChE displayed unaltered rates of hydrolysis, inhibition, phosphylation, and reactivation and could effectively protect mice against exposure to soman (pinacolylmethyl phosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), or O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothioate (VX). Unlike PEGylated wild-type (WT)-AChE, the PEGylated F338A-AChE exhibits significantly reduced aging rates after soman inhibition and can be efficiently reactivated by the 1-[[[4(aminocarbonyl)-pyridinio]methoxy]methyl]-2(hydroxyimino)methyl]pyridinium dichloride (HI-6) oxime, both in vitro and in vivo. Accordingly, oxime administration to PEG-F338A-AChE-pretreated mice enabled them to withstand repeated soman exposure (5.4 and 4 LD(50)/dose), whereas same regime treatment of non-PEGylated F338A-AChE- or PEGylated WT-AChE-pretreated mice failed to protect against the second challenge, due to rapid clearance or irreversible aging of the latter enzymes. Thus, judicious incorporation of selected mutations into the AChE mold in conjunction with its chemical modification provides means to engineer an optimal ChE-based OP bioscavenger in terms of circulatory longevity, resistance to aging, and reduced doses required for protection, even against repeated exposures to nerve agents, such as soman.
Sarin, a potent cholinesterase inhibitor, induces an array of toxic effects including convulsions and behavioral impairments. We report here on the protection provided by post-exposure antidotal treatments against a lethal dose of sarin (1.2xLD50) by scopolamine, benactyzine, trihexyphenidyl or caramiphen, administered 5, 10 or 20 min after the initiation of convulsions. A mixture of the oxime TMB4 and atropine (TA) was injected 1 min following poisoning a paradigm that may represent a scenario reminiscent of a terror incident. Surviving TA-treated rats exhibited marked tonic-clonic convulsions, weight loss, poor clinical status and abnormal cognitive performance as assessed by the Morris water maze. Additionally, a dramatic increase in the density of peripheral benzodiazepine receptors (PBRs), a faithful marker for neuronal damage, was noted. Animals treated 5 min after the development of toxic signs with benactyzine, trihexyphenidyl or caramiphen demonstrated control levels of PBR values, whereas scopolamine produced binding densities significantly above basal levels. Examined at the 10-min time point, scopolamine and trihexyphenidyl afforded no protection against brain damage and did not differ from TA-injected rats. All four drugs failed to significantly prevent the alterations when applied 20 min after onset of convulsions. Assessment of learning processes yielded similar results, where caramiphen exibited some protection at the 20-min time point. Our results show that caramiphen and benactyzine, agents with combined anticholinergic and antiglutamatergic pharmacological profiles, offer considerable shielding against sarin, even when their administration is delayed.
Title: Therapy against organophosphate poisoning: the importance of anticholinergic drugs with antiglutamatergic properties Weissman BA, Raveh L Ref: Toxicol Appl Pharmacol, 232:351, 2008 : PubMed
Potent cholinesterase inhibitors (e.g., soman, sarin), induce a wide range of deleterious effects including convulsions, behavioral impairments and ultimately, death. Due to the likelihood of various scenarios of military or terrorist attacks by these and other chemical weapons, research has to be aimed at finding optimal therapies. Early accumulation of acetylcholine in synaptic clefts was suggested to trigger an array of toxic events including an excessive release of glutamate, culminating in the activation of its receptors. Stimulation of the N-Methyl-D-Aspartate (NMDA) subtype of these receptors was associated with the neuronal injury that initiates organophosphate-induced brain damage. The notion of a stepwise mechanism yielded treatments based on a combination of an immediate administration of enzyme reactivators and anticholinergic drugs. This strategy dramatically increased survival rates but did not abolish convulsions and failed to prevent the ensuing cognitive dysfunction. Efforts to improve this paradigm by adding anticonvulsants or antiglutamatergic drugs with anti-epileptic characteristics produced dubious results. Under these conditions, benactyzine and caramiphen, agents with anticholinergic and antiglutamatergic properties, provided improved protection when introduced as adjunct agents to oximes, reversible cholinesterase inhibitors and/or specific antimuscarinic drugs such as atropine. In contrast, the specific antimuscarinic drug scopolamine failed to block soman-induced changes in glutamatergic and behavioral parameters even when given prophylactically. These findings along with a large number of additional reports led towards the conclusion that the therapeutic advantage of drugs such as benactyzine and caramiphen could derive from their ability to modulate central cholinergic and glutamate neurotransmission.
Title: Polyethylene-glycol conjugated recombinant human acetylcholinesterase serves as an efficacious bioscavenger against soman intoxication Kronman C, Cohen O, Raveh L, Mazor O, Ordentlich A, Shafferman A Ref: Toxicology, 233:40, 2007 : PubMed
Extensive pharmacokinetic studies in both mice and rhesus macaques, with biochemically well defined forms of native and recombinant AChEs from bovine, rhesus and human origin, allowed us to determine an hierarchical pattern by which post-translation-related factors and specific amino-acid epitopes govern the pharmacokinetic performance of the enzyme molecule. In parallel, we demonstrated that controlled conjugation of polyethylene-glycol (PEG) side-chains to lysine residues of rHuAChE also results in the generation of active enzyme with improved pharmacokinetic performance. Here, we show that equally efficient extension of circulatory residence can be achieved by specific conditions of PEGylation, regardless of the post-translation-modification state of the enzyme. The masking effect of PEGylation, which is responsible for extending circulatory lifetime, also contributes to the elimination of immunological responses following repeated administration of AChE. Finally, in vivo protection studies in mice allowed us to determine that the PEGylated AChE protects the animal from a high lethal dose (2.5 LD(50)) of soman. On a mole basis, both the recombinant AChE and its PEGylated form provide higher levels of protection against soman poisoning than the native serum-derived HuBChE. The findings that circulatory long-lived PEGylated AChE can confer superior protection to mice against OP-compound poisoning while exhibiting reduced immunogenicity, suggest that this chemically modified version of rHuAChE may serve as a highly effective bioscavenger for prophylactic treatment against OP-poisoning.
Title: Comparison of polyethylene glycol-conjugated recombinant human acetylcholinesterase and serum human butyrylcholinesterase as bioscavengers of organophosphate compounds Cohen O, Kronman C, Raveh L, Mazor O, Ordentlich A, Shafferman A Ref: Molecular Pharmacology, 70:1121, 2006 : PubMed
Comparative protection studies in mice demonstrate that on a molar basis, recombinant human acetylcholinesterase (rHuAChE) confers higher levels of protection than native human butyrylcholinesterase (HuBChE) against organophosphate (OP) compound intoxication. For example, mice challenged with 2.5 LD50 of O-isopropyl methylphosphonofluoridate (sarin), pinacolylmethyl phosphonofluoridate (soman), and O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothiolate (VX) after treatment with equimolar amounts of the two cholinesterases displayed 80, 100, and 100% survival, respectively, when pre-treatment was carried out with rHuAChE and 0, 20, and 60% survival, respectively, when pretreatment was carried out with HuBChE. Kinetic studies and active site titration analyses of the tested OP compounds with acetylcholinesterases (AChEs) and butyrylcholinesterases (BChEs) from different mammalian species demonstrate that the superior in vivo efficacy of acetyl-cholinesterases is in accordance with the higher stereoselectivity of AChE versus BChE toward the toxic enantiomers comprising the racemic mixtures of the various OP agents. In addition, we show that polyethylene glycol-conjugated (PEGy-lated) rHuAChE, which is characterized by a significantly extended circulatory residence both in mice and monkeys ( Biochem J 357: 795-802, 2001 ; Biochem J 378: 117-128, 2004 ), retains full reactivity toward OP compounds both in vitro and in vivo and provides a higher level of protection to mice against OP poisoning, compared with native serum-derived HuBChE. Indeed, PEGylated rHuAChE also confers superior prophylactic protection when administered intravenously or intramuscularly over 20 h before exposure of mice to a lethal dose of VX (1.3-1.5 LD50). These findings together with the observations that the PEGylated rHuAChE exhibits unaltered biodistribution and high bioavailability present a case for using PEGylated rHuAChE as a very efficacious bioscavenger of OP agents.
Title: Development of the bisquaternary oxime HI-6 toward clinical use in the treatment of organophosphate nerve agent poisoning Lundy PM, Raveh L, Amitai G Ref: Toxicol Rev, 25:231, 2006 : PubMed
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.
Soman, a powerful inhibitor of acetylcholinesterase, causes an array of toxic effects in the central nervous system including convulsions, learning and memory impairments, and, ultimately, death. We report on the protection afforded by postexposure antidotal treatments, combined with pyridostigmine (0.1 mg/kg) pretreatment, against these consequences associated with soman poisoning. Scopolamine (0.1 mg/kg) or caramiphen (10 mg/kg) were administered 5 min after soman (1.2 LD50), whereas TAB (i.e., TMB4, atropine, and benactyzine, 7.5, 3, and 1 mg/kg, respectively) was injected in rats concomitant with the development of toxic signs. Atropine (4 mg/kg) was given to the two former groups at the onset of toxic symptoms. Caramiphen and TAB completely abolished electrographic seizure activity while scopolamine treatment exhibited only partial protection. Additionally, no significant alteration in the density of peripheral benzodiazepine receptors was noted following caramiphen or TAB administration, while scopolamine application resulted in a complex outcome: a portion of the animals demonstrated no change in the number of these sites whereas the others exhibited markedly higher densities. Cognitive functions (i.e., learning and memory processes) evaluated using the Morris water maze improved considerably by the three treatments when compared to soman-injected animals; the following rank order was observed: caramiphen > TAB > scopolamine. Additionally, statistically significant correlations (r = 0.72, r = 0.73) were demonstrated between two learning parameters and [3H]Ro5-4864 binding to brain membrane. These results show that drugs with a pharmacological profile consisting of anticholinergic and antiglutamatergic properties such as caramiphen and TAB, have a substantial potential as postexposure therapies against intoxication by organophosphates.
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.
This report presents a non-lethal method for estimating a range of therapeutic doses of bisquaternary oximes that serve as antidotes against organophosphorus poisoning. We have estimated therapeutic oxime doses that are equivalent in their relative toxicity rather than selecting arbitrary fractions of their LD(50). Thus, toxic signs of the oximes HI-6, HLo-7, Toxogonin, AB-8 and AB-13 were monitored quantitatively in baboon monkeys and beagle dogs. Using Toxogonin as a reference oxime, a calculated unit of equivalent dose (CED) was defined as the oxime dose equal to the ratio between its minimal toxic dose (MTD) and the therapeutic ratio (TR) of Toxogonin i.e. CED = MTD/TR. Assuming that the tails of dose-response curves of toxicity for bisquaternary oximes are shallow and similar to one another, one could substitute the ED(10) for the MTD. The ED(10) values for bisquaternary oximes were estimated using the log-log model following experimental observations and quantitative scoring of toxic signs in dogs and monkeys. The MTD values then were calculated using the ED(10) values and the experimental therapeutic dose of the reference oxime Toxogonin. The following CED values were obtained for AB-8, AB-13, Toxogonin, HI-6 and HLo-7 in dogs (d) and monkeys (m): 98.7, 74.2, 30.0, 14.5 and 12.1 (d) and 281.9, 232.1, 41.7, 192.9 and 92.9 (m) micromol kg(-1), respectively. The antidotal efficacy of these oximes against poisoning by the nerve agent tabun was determined in dogs and monkeys. These dose-dependent efficacy data were obtained at 0.3 x CED, 1 x CED and 3 x CED of oximes in combination with atropine. These data provide comparative therapeutic values using oxime doses based on their relative toxicity. The highest antidotal efficacy against tabun in dogs was obtained for toxogonin, whereas HLo-7 and AB-13 were most efficacious in monkeys.
Title: Stress does not enable pyridostigmine to inhibit brain cholinesterase after parenteral administration Grauer E, Alkalai D, Kapon J, Cohen G, Raveh L Ref: Toxicol Appl Pharmacol, 164:301, 2000 : PubMed
The peripherally acting cholinesterase inhibitor pyridostigmine was widely used during the Gulf War as a pretreatment against possible chemical warfare attack. Following consistent reports on long-term illness among Gulf War veterans, pyridostigmine was examined for its possible long-term effects. These effects were suggested to be induced by the combination of pyridostigmine administration and stress exposure that allowed this quaternary compound to enter the brain through stress induced changes in blood-brain barrier (BBB) permeability. Recently, pyridostigmine administration was demonstrated to inhibit brain cholinesterase following acute stress in mice. However, the effect was not replicated under similar conditions in guinea pigs. Because of the significant implication of these findings, we tested brain cholinesterase (ChE) inhibition following the administration of pyridostigmine, or the tertiary carbamate physostigmine, with or without stress in mice. Different experiments were performed to examine the contribution of gender, age (young and adults), stress (type and intensity), or strain (CD-1 and FVB/n) parameters. No inhibition of brain ChE was detected in any of these experiments. At the same time, physostigmine induced the expected decrease in brain ChE in all the experiments. Thus, we could not replicate the findings that suggest pyridostigmine can affect brain cholinesterase following stress.
Organophosphate poisoning is associated with adverse effects on the central nervous system such as seizure/convulsive activity and long term changes in neuronal networks. This study reports on investigations designed to assess the consequences of soman exposure on excitatory amino acids receptors in the rat brain. In addition, the protective effects of caramiphen which acts at these receptors, and scopolamine, which does not, was determined on soman-induced alteration in rat brain functions. Administration of soman (1xLD50) to pyridostigmine pretreated rats produced seizure activity (measured by EEG monitoring) in all animals tested. Estimation of [3H]MK-801 binding to brain membranes from intoxicated rats revealed a marked decrease in Bmax value 24 but not 2 hrs following soman administration. The specific nature of these effects of soman was demonstrated by the findings that [3H]flunitrazepam binding to central benzodiazepine receptors remained unchanged in soman-poisoned rat brain membranes. Both scopolamine and caramiphen, when used prophylactically prevented the lethal effect of soman and completely blocked the development of electrographic seizure activity (EGSA). In contrast, only caramiphen abolished soman-induced modifications in NMDA/ion channel characteristics. Caramiphen displaced [3H]MK-801 bound to the NMDA/ion channel complex, possibly by interacting with the Zn2+ site whereas scopolamine did not. Moreover, caramiphen, but not scopolamine, partially protected mice from NMDA-induced lethality. Thus, it is suggested that an important component of the protective efficacy of caramiphen against organophosphate poisoning might be attributed to its ability to modulate NMDA receptors in addition to its anticholinergic properties.
Title: Prophylaxis against soman inhalation toxicity in guinea pigs by pretreatment alone with human serum butyrylcholinesterase Allon N, Raveh L, Gilat E, Cohen E, Grunwald J, Ashani Y Ref: Toxicol Sci, 43:121, 1998 : PubMed
Human butyrylcholinesterase (HuBChE) has previously been shown to protect mice, rats, and monkeys against multiple lethal toxic doses of organophosphorus (OP) anticholinesterases that were challenged by i.v. bolus injections. This study examines the concept of using a cholinesterase scavenger as a prophylactic measure against inhalation toxicity, which is the more realistic simulation of exposure to volatile OPs. HuBChE-treated awake guinea pigs were exposed to controlled concentration of soman vapors ranging from 417 to 430 micrograms/liter, for 45 to 70 s. The correlation between the inhibition of circulating HuBChE and the dose of soman administered by sequential i.v. injections and by respiratory exposure indicated that the fraction of the inhaled dose of soman that reached the blood was 0.29. HuBChE to soman molar ratio of 0.11 was sufficient to prevent the manifestation of toxic signs in guinea pigs following exposure to 2.17x the inhaled LD50 dose of soman (ILD50, 101 micrograms/kg). A slight increase in HuBChE:soman ratio (0.15) produced sign-free animals after two sequential respiratory exposures with a cumulative dose of 4.5x ILD50. Protection was exceptionally high and far superior to the currently used traditional approach that consisted of pretreatment with pyridostigmine and postexposure combined administration of atropine, benactyzine, and an oxime reactivator. Quantitative analysis of the results suggests that in vivo sequestration of soman, and presumably other OPs, by exogenously administered HuBChE, is independent of the species used or the route of challenge entry. This assuring conclusion significantly expands the database of the bioscavenger strategy that now offers a dependable extrapolation from animals to human.
Title: Current Capabilities in Extrapolating from Animal to Human the Capacity of Human Butyrylcholinesterase to Detoxify Organophosphates Ashani Y, Grauer E, Grunwald J, Allon N, Raveh L Ref: In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases, (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp.:255, 1998 : PubMed
Title: The Role of Oligosaccharides in the Pharmacokinetics of Cholinesterases Saxena A, Ashani Y, Raveh L, Doctor BP Ref: In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases, (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp.:283, 1998 : PubMed
Title: Role of oligosaccharides in the pharmacokinetics of tissue-derived and genetically engineered cholinesterases Saxena A, Ashani Y, Raveh L, Stevenson D, Patel T, Doctor BP Ref: Molecular Pharmacology, 53:112, 1998 : PubMed
To understand the role of glycosylation in the circulation of cholinesterases, we compared the mean residence time of five tissue-derived and two recombinant cholinesterases (injected intravenously in mice) with their oligosaccharide profiles. Monosaccharide composition analysis revealed differences in the total carbohydrate, galactose, and sialic acid contents. The molar ratio of sialic acid to galactose residues on tetrameric human serum butyrylcholinesterase, recombinant human butyrylcholinesterase, and recombinant mouse acetylcholinesterase was found to be approximately 1.0. For Torpedo californica acetylcholinesterase, monomeric and tetrameric fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase, this ratio was approximately 0.5. However, the circulatory stability of cholinesterases could not be correlated with the sialic acid-to-galactose ratio. Fractionation of the total pool of oligosaccharides obtained after neuraminidase digestion revealed one major oligosaccharide for human serum butyrylcholinesterase and three or four major oligosaccharides in other cholinesterases. The glycans of tetrameric forms of plasma cholinesterases (human serum butyrylcholinesterase, fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase) clearly demonstrated a reduced heterogeneity and higher maturity compared with glycans of monomeric fetal bovine serum acetylcholinesterase, dimeric tissue-derived T. californica acetylcholinesterase, and recombinant cholinesterases. T. californica acetylcholinesterase, recombinant cholinesterases, and monomeric fetal bovine serum acetylcholinesterase showed a distinctive shorter mean residence time (44-304 min) compared with tetrameric forms of plasma cholinesterases (1902-3206 min). Differences in the pharmacokinetic parameters of cholinesterases seem to be due to the combined effect of the molecular weight and charge- and size-based heterogeneity in glycans.
Title: Large-scale purification and long-term stability of human butyrylcholinesterase: a potential bioscavenger drug Grunwald J, Marcus D, Papier Y, Raveh L, Pittel Z, Ashani Y Ref: Journal of Biochemical & Biophysical Methods, 34:123, 1997 : PubMed
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.
Title: The stoichiometry of protection against soman and VX toxicity in monkeys pretreated with human butyrylcholinesterase Raveh L, Grauer E, Grunwald J, Cohen E, Ashani Y Ref: Toxicol Appl Pharmacol, 145:43, 1997 : PubMed
Bioscavengers of organophophates (OP) have been examined as potential substitutes for the currently approved drug treatment against OP toxicity. The present work was designed to assess the ability of butyrylcholinesterase, purified from human serum (HuBChE), to prevent the toxicity induced by soman and VX in rhesus monkeys. The consistency of the data across species was then evaluated as the basis for the extrapolation of the data to humans. The average mean residence time of the enzyme in the circulation of monkeys following an intravenous loading was 34 hr. High bioavailability of HuBChE in blood (>80%) was demonstrated after intramuscular injection. A molar ratio of HuBChE:OP approximately 1.2 protected against an i.v. bolus injection of 2.1 x LD50 VX, while a ratio of 0.62 was sufficient to protect monkeys against an i.v. dose of 3.3 x LD50 of soman, with no additional postexposure therapy. A remarkable protection was also seen against soman-induced behavioral deficits detected in the performance of a spatial discrimination task. The consistency of the results across several species offers a reliable prediction of both the stoichiometry of the scavenging and the extent of prophylaxis with HuBChE against nerve agent toxicity in humans.
Title: Structure of glycan moieties responsible for the extended circulatory life time of fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase Saxena A, Raveh L, Ashani Y, Doctor BP Ref: Biochemistry, 36:7481, 1997 : PubMed
Cholinesterases are serine hydrolases that can potentially be used as pretreatment drugs for organophosphate toxicity, as drugs to alleviate succinylcholine-induced apnea, and as detoxification agents for environmental toxins such as heroin and cocaine. The successful application of serum-derived cholinesterases as bioscavengers stems from their relatively long residence time in the circulation. To better understand the relationship between carbohydrate structure and the stability of cholinesterases in circulation, we determined the monosaccharide composition, the distribution of various oligosaccharides, and the structure of the major asparagine-linked oligosaccharides units present in fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase. Our findings indicate that 70-80% of the oligosaccharides in both enzymes are negatively charged. This finding together with the molar ratio of galactose to sialic acid clearly suggests that the beta-galactose residues are only partially capped with sialic acid, yet they displayed a long duration in circulation. The structures of the two major oligosaccharides from fetal bovine serum acetylcholinesterase and one major oligosaccharide from equine serum butyrylcholinesterase were determined. The three carbohydrate structures were of the biantennary complex type, but only the ones from fetal bovine serum acetylcholinesterase were fucosylated on the innermost N-acetylglucosamine residue of the core. Pharmacokinetic studies with native, desialylated, and deglycosylated forms of both enzymes indicate that the microheterogeneity in carbohydrate structure may be responsible, in part, for the multiphasic clearance of cholinesterases from the circulation of mice.
Title: Protection of Guinea Pigs against Soman Inhalation by Pretreatment Alone with Human Butyrylcholinesterase Allon N, Raveh L, Gilat E, Grunwald J, Manistersky E, Cohen E, Ashani Y Ref: In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases, (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp.:398, 1995 : PubMed
Title: Bisquaternary Oximes as Antidotes against Tabun and Soman Poisoning Amitai G, Rabinovitz I, Zomber G, Chen R, Cohen G, Adani R, Raveh L Ref: In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases, (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp.:345, 1995 : PubMed
Title: Human Butyrylcholinesterase as Prophylaxis Treatment against Soman Grauer E, Raveh L, Kapon J, Grunwald J, Cohen E, Ashani Y Ref: In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases, (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp.:400, 1995 : PubMed
Title: Prevention of Brain Damage and Behavioral Performance Changes following an IV Injection of Soman and VX in Rats Pretreated with Human Butyrylcholinesterase Kadar T, Raveh L, Brandeis R, Grunwald J, Cohen E, Ashani Y Ref: In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases, (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp.:404, 1995 : PubMed
Title: Efficacy of Prophylaxis with Human Butyrylcholinesterase against Soman and VX Poisoning Raveh L, Grunwald J, Cohen E, Ashani Y Ref: In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases, (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp.:402, 1995 : PubMed
Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase (FBS AChE) provides complete protection against 5 LD50 of organophosphate (OP) without any signs of toxicity or performance decrements as measured by serial probe recognition tests or primate equilibrium platform performance (Maxwell et al., Toxicol Appl Pharmacol 115: 44-49, 1992; Wolfe et al., Toxicol Appl Pharmacol 117: 189-193, 1992). Although such use of enzyme as a single pretreatment drug for OP toxicity is sufficient to provide complete protection, a relatively large (stoichiometric) amount of enzyme was required in vivo to neutralize OP. To improve the efficacy of cholinesterases as pretreatment drugs, we have developed an approach in which the catalytic activity of OP-inhibited FBS AChE was rapidly and continuously restored, thus detoxifying the OP and minimizing enzyme aging by having sufficient amounts of appropriate oxime present. The efficacy of FBS AChE to detoxify several OPs was amplified by addition of bis-quaternary oximes, particularly 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxyaminopyridinium) -dimethyl ether hydrochloride (HI-6). When mice were pretreated with sufficient amounts of FBS AChE and HI-6 and challenged with repeated doses of O-isopropyl methylphosphonofluridate (sarin), the OP was continuously detoxified so long as the molar concentration of the sarin dose was less than the molar concentration of AChE in circulation. The in vitro experiments showed that the stoichiometry of sarin:FBS AChE was higher than 3200:1 and in vivo stoichiometry with mice was as high as 57:1. Addition of HI-6 to FBS AChE as a pretreatment drug amplified the efficacy of enzyme as a scavenger of nerve agents.
Title: Huperzine A as a pretreatment candidate drug against nerve agent toxicity Grunwald J, Raveh L, Doctor BP, Ashani Y Ref: Life Sciences, 54:991, 1994 : PubMed
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.
Title: Prevention of soman-induced cognitive deficits by pretreatment with human butyrylcholinesterase in rats Brandeis R, Raveh L, Grunwald J, Cohen E, Ashani Y Ref: Pharmacol Biochem Behav, 46:889, 1993 : PubMed
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.
Title: Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity. In vitro and in vivo quantitative characterization Raveh L, Grunwald J, Marcus D, Papier Y, Cohen E, Ashani Y Ref: Biochemical Pharmacology, 45:2465, 1993 : PubMed
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.
We demonstrate here the correlation between protection afforded by pretreatment alone with parathion hydrolase purified from Pseudomonas sp. against tabun toxicity in mice and the kinetic parameters which are assumed to determine the in vivo detoxification of tabun by the same enzyme. Results show that 15 and 22 micrograms of parathion hydrolase per animal conferred a protective ratio of 3.94 and 5.65 respectively, against tabun toxicity, without post-exposure treatment.
Title: Prophylaxis against organophosphate poisoning by an enzyme hydrolysing organophosphorus compounds in mice Ashani Y, Rothschild N, Segall Y, Levanon D, Raveh L Ref: Life Sciences, 49:367, 1991 : PubMed
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.
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.
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.
Title: Effects of CBDP and MEPQ on the toxicity and distribution of [3H]-soman in mice Shapira S, Kadar T, Cohen G, Chapman S, Raveh L Ref: Archives of Toxicology, 64:663, 1990 : PubMed
Soman poisoning presents a problem in terms of its detailed pathophysiology and its detoxification mechanism(s). The present study was designed to evaluate the role of carboxylesterases (CaE) and cholinesterase (ChE) in the distribution and detoxification of soman in vivo. Mice were injected (i.v.) with 0.06-1.0 LD50 of [3H]-soman, 60 min following pretreatment with either 2-O-cresyl-4H-1:2:3 benzodioxa-phosphorine-2-oxide (CBDP), which blocks CaE or 7-(methylethoxyphosphinyloxy)-1-methyl quinolinium iodide (MEPQ), which selectively inhibits intravascular ChE. One hour after [3']-soman administration animals were sacrificed and whole body autoradiography was performed. High concentrations of [3H]-soman were found in lung and kidney in control mice, and low concentrations were found in central nervous system. Pretreatment with CBDP caused a 93% decrease in radioactive labelling in the lung, and a minor decrease in overall labelling, whereas pretreatment with MEPQ did not change the distribution pattern of [3H]-soman. It is concluded that lung is a major target organ for soman detoxification and that it exerts this effect by means of enzymatic reaction with soman through the abundant amounts of CaE which are present in the lung. Intravascular ChE has little (if any) effect on the distribution and detoxification of soman in vivo.
Title: Acetylcholinesterase prophylaxis against organophosphate poisoning. Quantitative correlation between protection and blood-enzyme level in mice Raveh L, Ashani Y, Levy D, De La Hoz DM, Wolfe AD, Doctor BP Ref: Biochemical Pharmacology, 38:529, 1989 : PubMed
Fetal bovine serum acetylcholinesterase (FBS-AChE, EC 3.1.1.7) was titrated, both in vitro and in vivo, with a highly toxic anti-ChE organophosphate, 7-(methylethoxyphosphinyloxy)-1-methyl-quinolinium iodie (MEPQ). Approximately 1:1 stoichiometry was obtained for the sequestration of MEPQ by FBS-AChE in mice. A quantitative, linear correlation was demonstrated between blood-AChE levels and the protection afforded by exogenously administered AChE in mice when challenged with anti-ChE MEPQ. The results presented in this report demonstrate that such prophylactic measures are indeed sufficient to protect animals against poisoning by as high as an 8 x LD50 dose of organophosphate without the administration of any supportive drug. Despite the relatively large toxic dose, most of the mice that survived the challenge did not show any classical clinical signs of severe anti-ChE poisoning. MEPQ may be considered a suitable model compound for studying the quantitative aspects of the scavenger prophylactic approach described here.
Title: In vitro and in vivo protection of acetylcholinesterase against organophosphate poisoning by pretreatment with a novel derivative of 1,3,2-dioxaphosphorinane 2-oxide Ashani Y, Leader H, Raveh L, Bruckstein R, Spiegelstein M Ref: Journal of Medicinal Chemistry, 26:145, 1983 : PubMed
Covalent molecular combinations of a cyclic phosphate (dioxaphosphorinane) and a potential leaving group, such as 3-(trimethylammonio)phenol iodide (TMPH), suggested the synthesis of O-[3-(trimethylammonio)phenyl]-1,3,2-dioxaphosphorinane 2-oxide iodide (TDPI). TDPI inhibited acetylcholinesterase (AChE) (ki = 8.4 x 10(3) M-1 min-1) via the formation of an unstable covalent intermediate. TDPI-inhibited AChE hydrolyzed spontaneously with t1/2 approximately equal to 10 min. Butyrylcholinesterase (BuChE) was also inhibited by TDPI (ki = 1.8 x 10(4) M-1 min-1), but the inhibited BuChE was more stable (greater than 10 times) than the corresponding AchE-TDPI conjugate. Pretreatment of mice with TDPI conferred protection against 22 LD50's of paraoxon and 5 LD50's of soman, provided that treatment with anticholinergics and an oxime followed administration of these anticholinesterase poisons. Correlation between in vitro and in vivo observations suggests that the main protection of AChE conferred by TDPI results from temporary masking of the active site of the enzyme. The acute toxicity of TDPI was found to be 444 mg/kg (sc, mice), whereas analogous carbamates and a noncyclic phosphate also displaying antidotal properties are greater than 170 times more toxic.
